45 results on '"Volloch"'
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2. ACH2.0/E, the Consolidated Theory of Conventional and Unconventional Alzheimer’s Disease: Origins, Progression, and Therapeutic Strategies
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Vladimir Volloch and Sophia Rits-Volloch
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Conventional and unconventional Alzheimer’s disease ,Alzheimer’s disease-like dementia (ADLD) and Alzheimer’s disease-related dementia (ADRD) ,Neuronal integrated stress response (ISR) ,Intraneuronal Aβ (iAβ) and AβPP-independent generation of iAβ ,Amyloid cascade hypothesis 2.0 (ACH2.0) and expanded amyloid cascade hypothesis 2.0 (ACH2.0/E) ,Therapeutic strategies for conventional and unconventional Alzheimer’s disease and for aging-associated cognitive decline ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
The centrality of amyloid-beta (Aβ) is an indisputable tenet of Alzheimer’s disease (AD). It was initially indicated by the detection (1991) of a mutation within Aβ protein precursor (AβPP) segregating with the disease, which served as a basis for the long-standing Amyloid Cascade Hypothesis (ACH) theory of AD. In the intervening three decades, this notion was affirmed and substantiated by the discovery of numerous AD-causing and AD-protective mutations with all, without an exception, affecting the structure, production, and intraneuronal degradation of Aβ. The ACH postulated that the disease is caused and driven by extracellular Aβ. When it became clear that this is not the case, and the ACH was largely discredited, a new theory of AD, dubbed ACH2.0 to re-emphasize the centrality of Aβ, was formulated. In the ACH2.0, AD is caused by physiologically accumulated intraneuronal Aβ (iAβ) derived from AβPP. Upon reaching the critical threshold, it triggers activation of the autonomous AβPP-independent iAβ generation pathway; its output is retained intraneuronally and drives the AD pathology. The bridge between iAβ derived from AβPP and that generated independently of AβPP is the neuronal integrated stress response (ISR) elicited by the former. The ISR severely suppresses cellular protein synthesis; concurrently, it activates the production of a small subset of proteins, which apparently includes components necessary for operation of the AβPP-independent iAβ generation pathway that are absent under regular circumstances. The above sequence of events defines “conventional” AD, which is both caused and driven by differentially derived iAβ. Since the ISR can be elicited by a multitude of stressors, the logic of the ACH2.0 mandates that another class of AD, referred to as “unconventional”, has to occur. Unconventional AD is defined as a disease where a stressor distinct from AβPP-derived iAβ elicits the neuronal ISR. Thus, the essence of both, conventional and unconventional, forms of AD is one and the same, namely autonomous, self-sustainable, AβPP-independent production of iAβ. What distinguishes them is the manner of activation of this pathway, i.e., the mode of causation of the disease. In unconventional AD, processes occurring at locations as distant from and seemingly as unrelated to the brain as, say, the knee can potentially trigger the disease. The present study asserts that these processes include traumatic brain injury (TBI), chronic traumatic encephalopathy, viral and bacterial infections, and a wide array of inflammatory conditions. It considers the pathways which are common to all these occurrences and culminate in the elicitation of the neuronal ISR, analyzes the dynamics of conventional versus unconventional AD, shows how the former can morph into the latter, explains how a single TBI can hasten the occurrence of AD and why it takes multiple TBIs to trigger the disease, and proposes the appropriate therapeutic strategies. It posits that yet another class of unconventional AD may occur where the autonomous AβPP-independent iAβ production pathway is initiated by an ISR-unrelated activator, and consolidates the above notions in a theory of AD, designated ACH2.0/E (for expanded ACH2.0), which incorporates the ACH2.0 as its special case and retains the centrality of iAβ produced independently of AβPP as the driving agent of the disease.
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- 2024
- Full Text
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3. On the Inadequacy of the Current Transgenic Animal Models of Alzheimer’s Disease: The Path Forward
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Vladimir Volloch and Sophia Rits-Volloch
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Alzheimer’s disease (AD) ,conventional AD ,unconventional AD ,amyloid cascade hypothesis (ACH) ,ACH-based models of AD ,ACH-based AD drugs ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
For at least two reasons, the current transgenic animal models of Alzheimer’s disease (AD) appear to be patently inadequate. They may be useful in many respects, the AD models; however, they are not. First, they are incapable of developing the full spectrum of the AD pathology. Second, they respond spectacularly well to drugs that are completely ineffective in the treatment of symptomatic AD. These observations indicate that both the transgenic animal models and the drugs faithfully reflect the theory that guided the design and development of both, the amyloid cascade hypothesis (ACH), and that both are inadequate because their underlying theory is. This conclusion necessitated the formulation of a new, all-encompassing theory of conventional AD—the ACH2.0. The two principal attributes of the ACH2.0 are the following. One, in conventional AD, the agent that causes the disease and drives its pathology is the intraneuronal amyloid-β (iAβ) produced in two distinctly different pathways. Two, following the commencement of AD, the bulk of Aβ is generated independently of Aβ protein precursor (AβPP) and is retained inside the neuron as iAβ. Within the framework of the ACH2.0, AβPP-derived iAβ accumulates physiologically in a lifelong process. It cannot reach levels required to support the progression of AD; it does, however, cause the disease. Indeed, conventional AD occurs if and when the levels of AβPP-derived iAβ cross the critical threshold, elicit the neuronal integrated stress response (ISR), and trigger the activation of the AβPP-independent iAβ generation pathway; the disease commences only when this pathway is operational. The iAβ produced in this pathway reaches levels sufficient to drive the AD pathology; it also propagates its own production and thus sustains the activity of the pathway and perpetuates its operation. The present study analyzes the reason underlying the evident inadequacy of the current transgenic animal models of AD. It concludes that they model, in fact, not Alzheimer’s disease but rather the effects of the neuronal ISR sustained by AβPP-derived iAβ, that this is due to the lack of the operational AβPP-independent iAβ production pathway, and that this mechanism must be incorporated into any successful AD model faithfully emulating the disease. The study dissects the plausible molecular mechanisms of the AβPP-independent iAβ production and the pathways leading to their activation, and introduces the concept of conventional versus unconventional Alzheimer’s disease. It also proposes the path forward, posits the principles of design of productive transgenic animal models of the disease, and describes the molecular details of their construction.
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- 2024
- Full Text
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4. Next Generation Therapeutic Strategy for Treatment and Prevention of Alzheimer’s Disease and Aging-Associated Cognitive Decline: Transient, Once-in-a-Lifetime-Only Depletion of Intraneuronal Aβ (iAβ) by Its Targeted Degradation via Augmentation of Intra-iAβ-Cleaving Activities of BACE1 and/or BACE2
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Vladimir Volloch and Sophia Rits-Volloch
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Alzheimer’s disease (AD) ,Aging-Associated Cognitive Decline (AACD) ,Amyloid Cascade Hypothesis 2.0 (ACH2.0) ,intraneuronal Aβ (iAβ) ,Aβ protein precursor (AβPP) ,AβPP-independent iAβ production pathway ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
Although the long-standing Amyloid Cascade Hypothesis (ACH) has been largely discredited, its main attribute, the centrality of amyloid-beta (Aβ) in Alzheimer’s disease (AD), remains the cornerstone of any potential interpretation of the disease: All known AD-causing mutations, without a single exception, affect, in one way or another, Aβ. The ACH2.0, a recently introduced theory of AD, preserves this attribute but otherwise differs fundamentally from the ACH. It posits that AD is a two-stage disorder where both stages are driven by intraneuronal (rather than extracellular) Aβ (iAβ) albeit of two distinctly different origins. The first asymptomatic stage is the decades-long accumulation of Aβ protein precursor (AβPP)-derived iAβ to the critical threshold. This triggers the activation of the self-sustaining AβPP-independent iAβ production pathway and the commencement of the second, symptomatic AD stage. Importantly, Aβ produced independently of AβPP is retained intraneuronally. It drives the AD pathology and perpetuates the operation of the pathway; continuous cycles of the iAβ-stimulated propagation of its own AβPP-independent production constitute an engine that drives AD, the AD Engine. It appears that the dynamics of AβPP-derived iAβ accumulation is the determining factor that either drives Aging-Associated Cognitive Decline (AACD) and triggers AD or confers the resistance to both. Within the ACH2.0 framework, the ACH-based drugs, designed to lower levels of extracellular Aβ, could be applicable in the prevention of AD and treatment of AACD because they reduce the rate of accumulation of AβPP-derived iAβ. The present study analyzes their utility and concludes that it is severely limited. Indeed, their short-term employment is ineffective, their long-term engagement is highly problematic, their implementation at the symptomatic stages of AD is futile, and their evaluation in conventional clinical trials for the prevention of AD is impractical at best, impossible at worst, and misleading in between. In contrast, the ACH2.0-guided Next Generation Therapeutic Strategy for the treatment and prevention of both AD and AACD, namely the depletion of iAβ via its transient, short-duration, targeted degradation by the novel ACH2.0-based drugs, has none of the shortcomings of the ACH-based drugs. It is potentially highly effective, easily evaluable in clinical trials, and opens up the possibility of once-in-a-lifetime-only therapeutic intervention for prevention and treatment of both conditions. It also identifies two plausible ACH2.0-based drugs: activators of physiologically occurring intra-iAβ-cleaving capabilities of BACE1 and/or BACE2.
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- 2023
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5. The Amyloid Cascade Hypothesis 2.0 for Alzheimer’s Disease and Aging-Associated Cognitive Decline: From Molecular Basis to Effective Therapy
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Vladimir Volloch and Sophia Rits-Volloch
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the amyloid cascade hypothesis 2.0 (ACH2.0) ,intraneuronal Aβ (iAβ) ,Aβ protein precursor (AβPP)-independent generation of iAβ ,aging-related cognitive dysfunction (AACD) ,iAβ depletion therapy for AD and AACD ,BACE1 and BACE2 activators as AD and AACD drugs ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
With the long-standing amyloid cascade hypothesis (ACH) largely discredited, there is an acute need for a new all-encompassing interpretation of Alzheimer’s disease (AD). Whereas such a recently proposed theory of AD is designated ACH2.0, its commonality with the ACH is limited to the recognition of the centrality of amyloid-β (Aβ) in the disease, necessitated by the observation that all AD-causing mutations affect, in one way or another, Aβ. Yet, even this narrow commonality is superficial since AD-causing Aβ of the ACH differs distinctly from that specified in the ACH2.0: Whereas in the former, the disease is caused by secreted extracellular Aβ, in the latter, it is triggered by Aβ-protein-precursor (AβPP)-derived intraneuronal Aβ (iAβ) and driven by iAβ generated independently of AβPP. The ACH2.0 envisions AD as a two-stage disorder. The first, asymptomatic stage is a decades-long accumulation of AβPP-derived iAβ, which occurs via internalization of secreted Aβ and through intracellular retention of a fraction of Aβ produced by AβPP proteolysis. When AβPP-derived iAβ reaches critical levels, it activates a self-perpetuating AβPP-independent production of iAβ that drives the second, devastating AD stage, a cascade that includes tau pathology and culminates in neuronal loss. The present study analyzes the dynamics of iAβ accumulation in health and disease and concludes that it is the prime factor driving both AD and aging-associated cognitive decline (AACD). It discusses mechanisms potentially involved in AβPP-independent generation of iAβ, provides mechanistic interpretations for all principal aspects of AD and AACD including the protective effect of the Icelandic AβPP mutation, the early onset of FAD and the sequential manifestation of AD pathology in defined regions of the affected brain, and explains why current mouse AD models are neither adequate nor suitable. It posits that while drugs affecting the accumulation of AβPP-derived iAβ can be effective only protectively for AD, the targeted degradation of iAβ is the best therapeutic strategy for both prevention and effective treatment of AD and AACD. It also proposes potential iAβ-degrading drugs.
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- 2023
- Full Text
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6. News from Mars: Two-Tier Paradox, Intracellular PCR, Chimeric Junction Shift, Dark Matter mRNA and Other Remarkable Features of Mammalian RNA-Dependent mRNA Amplification. Implications for Alzheimer’s Disease, RNA-Based Vaccines and mRNA Therapeutics
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Vladimir Volloch and Sophia Rits-Volloch
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Messenger RNA ,RNA ,Translation (biology) ,Biology ,Article ,Reverse transcriptase ,Cell biology ,Antisense RNA ,Psychiatry and Mental health ,chemistry.chemical_compound ,Neuropsychology and Physiological Psychology ,chemistry ,Chimeric RNA ,RNA polymerase ,Transcription factor - Abstract
Molecular Biology, a branch of science established to examine the flow of information from “letters” encrypted into DNA structure to functional proteins, was initially defined by a concept of DNA-to-RNA-to-Protein information movement, a notion termed the Central Dogma of Molecular Biology. RNA-dependent mRNA amplification, a novel mode of eukaryotic protein-encoding RNA-to-RNA-to-Protein genomic information transfer, constitutes the extension of the Central Dogma in the context of mammalian cells. It was shown to occur in cellular circumstances requiring exceptionally high levels of production of specific polypeptides, e.g. globin chains during erythroid differentiation or defined secreted proteins in the context of extracellular matrix deposition. Its potency is reflected in the observed cellular levels of the resulting amplified mRNA product: At the peak of the erythroid differentiation, for example, the amount of globin mRNA produced in the amplification pathway is about 1500-fold higher than the amount of its conventionally generated counterpart in the same cells. The cellular enzymatic machinery at the core of this process, RNA-dependent RNA polymerase activity (RdRp), albeit in a non-conventional form, was shown to be constitutively and ubiquitously present, and RNA-dependent RNA synthesis (RdRs) appeared to regularly occur, in mammalian cells. Under most circumstances, the mammalian RdRp activity produces only short antisense RNA transcripts. Generation of complete antisense RNA transcripts and amplification of mRNA molecules require the activation of inducible components of the mammalian RdRp complex. The mechanism of such activation is not clear. The present article suggests that it is triggered by a variety of cellular stresses and occurs in the context of stress responses in general and within the framework of the integrated stress response (ISR) in particular. In this process, various cellular stresses activate, in a stress type-specific manner, defined members of the mammalian translation initiation factor 2α, eIF2α, kinase family: PKR, GCN2, PERK and HRI. Any of these kinases, in an activated form, phosphorylates eIF2α. This results in suppression of global cellular protein synthesis but also in activation of expression of select group of transcription factors including ATF4, ATF5 and CHOP. These transcription factors either function as inducible components of the RdRp complex or enable their expression. The assembly of the competent RdRp complex activates mammalian RNA-dependent mRNA amplification, which appears to be a two-tier process. Tier One is a “chimeric” pathway, named so because it results in an amplified chimeric mRNA molecule containing a fragment of the antisense RNA strand at its 5’ terminus. Tier Two further amplifies one of the two RNA end products of the chimeric pathway and constitutes the physiologically occurring intracellular polymerase chain reaction, iPCR. Depending on the structure of the initial mRNA amplification progenitor, the chimeric pathway, Tier One, may result in multiple outcomes including chimeric mRNA that produces either a polypeptide identical to the original, conventional mRNA progenitor-encoded protein or only its C-terminal fragment, CTF. The chimeric RNA end product of Tier One may also produce a polypeptide that is non-contiguously encoded in the genome, activate translation from an open reading frame, which is “silent” in a conventionally transcribed mRNA, or initiate an abortive translation. In sharp contrast, regardless of the outcome of Tier One, the mRNA end product of Tier Two of mammalian mRNA amplification, the iPCR pathway, always produces a polypeptide identical to a conventional mRNA progenitor-encoded protein. This discordance is referred to as the Two-Tier Paradox and discussed in detail in the present article. On the other hand, both Tiers are similar in that they result in heavily modified mRNA molecules resistant to reverse transcription, undetectable by reverse transcription-based methods of sequencing and therefore constituting a proverbial “Dark Matter” mRNA, despite being highly ubiquitous. It appears that in addition to their other functions, the modifications of the amplified mRNA render it compatible, unlike the bulk of cellular mRNA, with phosphorylated eIF2α in translation, implying that in addition to being extraordinarily abundant due to the method of its generation, amplified mRNA is also preferentially translated under the ISR conditions, thus augmenting the efficiency of the amplification process. The vital importance of powerful mechanisms of amplification of protein-encoding genomic information in normal physiology is self-evident. Their malfunctions or misuse appear to be associated with two types of abnormalities, the deficiency of a protein normally produced by these mechanisms and the mRNA amplification-mediated overproduction of a protein normally not generated by such a process. Certain classes of beta-thalassemia exemplify the first type, whereas the second type is represented by overproduction of beta-amyloid in Alzheimer’s disease. Moreover, the proposed mechanism of Alzheimer’s disease allows a crucial and verifiable prediction, namely that the disease-causing intraneuronally retained variant of beta-amyloid differs from that produced conventionally by βAPP proteolysis in that it contains the additional methionine or acetylated methionine at its N-terminus. Because of its extraordinary evidential value as a natural reporter of the mRNA amplification pathway, this feature, if proven, would, arguably, constitute the proverbial Holy Grail not only for Alzheimer’s disease but also for the mammalian RNA-dependent mRNA amplification field in general. Both examples are discussed in detail in the present article, which summarizes and systematizes our current understanding of the field and describes two categories of reporter constructs, one for the chimeric Tier of mRNA amplification, another for the iPCR pathway; both reporter types are essential for elucidating underlying molecular mechanisms. It also suggests, in light of the recently demonstrated feasibility of RNA-based vaccines, that the targeted intracellular amplification of exogenously introduced amplification-eligible antigen-encoding mRNAs via the induced or naturally occurring RNA-dependent mRNA amplification pathway could be of substantial benefit in triggering a fast and potent immune response and instrumental in the development of future vaccines. Similar approaches can also be effective in achieving efficient and sustained expression of exogenous mRNA in mRNA therapeutics.
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- 2019
7. Alzheimer’s Disease is Driven by Beta-Amyloid Generated in the Amyloid Precursor Protein-Independent Pathway and Retained Intraneuronally: Research and Therapeutic Strategies in a New AD Paradigm
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Vladimir Volloch and Sophia Rits-Volloch
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Psychiatry and Mental health ,Neuropsychology and Physiological Psychology ,biology ,Amyloid ,Chemistry ,Amyloid precursor protein ,biology.protein ,Cancer research ,Disease ,Beta (finance) - Abstract
The present article describes a New Paradigm of Alzheimer’s disease (AD). In the Old Paradigm, formalized in the Amyloid Cascade Hypothesis (ACH) theory of AD, beta amyloid (Aβ) is produced, both in health and disease, solely in the amyloid precursor protein (βAPP) proteolytic/secretory pathway. Two βAPP cleavages are involved. First cleavage, by beta-secretase (Beta-site APP Cleaving Enzyme, BACE) between Met671 and Asp672 (numbering according to the βAPP770 isoform), generates the C-terminal fragment of βAPP (C99, reflecting the number of its amino acid residues) and forms the N-terminus of Aβ. Subsequent second cleavage of C99 by gamma-secretase (gamma-site βAPP cleaving enzyme) forms the C-terminus of Aβ, completes its production, and coincides with its secretion. The overproduction of Aβ results in its extracellular accumulation commencing early in life. This triggers a cascade of molecular and cellular events, including formation of neurofibrillary tangles, which results in neurodegeneration. When the extent of neurodegeneration reaches critical levels, symptoms of the disease are manifested. In this Paradigm, Alzheimer’s disorder is a quintessential “slow” disease. The ACH clearly defined therapeutic targets, which included key events of βAPP proteolysis as well as secreted extracellular Aβ. Eventually, a number of candidate AD drugs, highly effective in animal model systems, was developed. Of those, especially successful were inhibitors of beta-sectretase that not only prevented the emergence of AD symptoms, but also reversed them when administered after symptomatic manifestation of the disease in animal models. At this point, there was every reason to hope that a solution to the Alzheimer’s problem is at hand; this, however proved not to be the case. Both the Old and the New Paradigms share the common point of departure, namely that the overproduction of beta-amyloid is the causative basis of AD. The rest of the notions of the New Paradigm are distinctly different from those of the Old one. Formulation of the New Paradigm theory of Alzheimer’s disease was necessitated by the analysis of results of massive human clinical trials of candidate AD drugs that performed outstandingly in animal studies. They all failed in human trials as spectacularly as they succeeded in animal studies. Or did they? Whereas they indeed showed no efficacy whatsoever, they performed perfectly within confines of their design and purpose. For example, a BACE inhibitor verubecestat penetrated the brain of AD patients, greatly inhibited βAPP cleavage, and strongly suppressed extracellular levels of Aβ. It did all this with the same efficiency it exhibited in animal studies, where it indisputably succeeded in mitigating symptoms of the disease. Why did it fail to do so in human clinical trials? This failure, apparently inexplicable within the confines of the ACH, seems as good an occasion as any to apply the central dictum of Sherlock Holmes: “... when you eliminated the impossible, whatever remains, however improbable, must be the truth”. In the case under discussion, after the elimination of the “impossible”, including elements of the ACH, and provided that Alzheimer’s disease is indeed caused by the overproduction of Aβ, a notion strongly supported by experimental data, the “however improbable”, which is consistent with the outcomes of human clinical trials of AD candidate drugs, is the following. 1. In addition to the βAPP proteolytic/secretory process, in Alzheimer’s disease in humans, Aβ is also produced in the βAPP-independent pathway. This pathway is active only in AD patients. It does not operate in animals and healthy humans. 2. The output of the βAPP-independent Aβ generation pathway is retained intraneuronally, and it is this pool of intraneuronal beta-amyloid that causes and sustains Alzheimer’s disease. These notions constitute the core of the New Paradigm theory of AD. Several mechanisms are capable of achieving the above. They include: RNA-dependent βAPP mRNA amplification, a process implicated in overproduction of specific proteins in mammalian cells; the internal initiation of transcription within the human βAPP gene; cleavage within βAPP mRNA; the internal initiation of translation within βAPP mRNA. Conceptually, in the context of the present article, the nature of the mechanism generating Aβ independently of βAPP is not important; in every case, identical therapeutic strategies would be indicated. All potential mechanisms of βAPP-independent Aβ generation share several common features. (a) In each case, the expression of a crucial component(s) required for activation of a specific mechanism is induced by the integrated stress response (ISR) elicited via OMA1-DELE1- HRI signaling pathway activated by mitochondrial dysfunction triggered by over-the-threshold levels of βAPP-derived Aβ accumulated intracellularly through cellular uptake of secreted Aβ as well as by retention of a fraction of beta-amyloid produced in the βAPP proteolytic pathway. (b) In every potential mechanism of βAPP-independent Aβ generation, translation initiates at the AUG normally encoding Met671 of βAPP and results in C100, i.e. N-terminal Met-containing C99, which is subsequently cleaved by gamma-secretase to produce Aβ (or Met-Aβ). (c) N-terminal Met of C100 is removed post- rather than co-translationally. Therefore a steady-state population of C100 and, possibly, of N-terminal Met-containing Aβ should occur within human neuronal cells with the activated βAPP-independent Aβ production pathway; their detection would provide irrefutable proof of operation of the pathway. (d) The Aβ (or Met-Aβ) output of every potential mechanism is retained intraneuronally. (e) Once activated, every potential βAPP-independent mechanism would, through generation of intraneuronally retained Aβ, sustain mitochondrial dysfunction and support the activity of the OMA1-DELE1-HRI signaling pathway, which, in turn, will promote, via elicitation of the ISR, the operation of the βAPP-independent Aβ production pathway. These self-perpetuating {βAPP-independent generation of intracellularly retained Aβ}/{mitochondrial dysfunction} mutual feedback cycles constitute the “Engine” that drives Alzheimer’s disease. The life- long accumulation of intraneuronal βAPP-derived Aβ to critical levels, sufficient to trigger mitochondrial dysfunction, plays the role of a starter motor in getting car engine moving in a self-sustainable manner. Only when the AD “Engine” is activated does the disease commence. In the New Paradigm, therefore, Alzheimer’s disorder is a “fast” disease that can be treated and cured at the symptomatic stages. The present article proposes conceptually novel research and therapeutic strategies and suggests that BACE activation (yes, activation!) could be a valid approach in AD therapy.
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- 2019
8. RNA-dependent Amplification of Mammalian mRNA Encoding Extracellullar Matrix Proteins: Identification of Chimeric RNA Intermediates for α1, β1, and γ1 Chains of Laminin
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Sophia Rits, Bjorn R. Olsen, and Vladimir Volloch
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Messenger RNA ,chemistry.chemical_compound ,Chemistry ,RNA interference ,Chimeric RNA ,RNA polymerase ,Sense (molecular biology) ,RNA-dependent RNA polymerase ,RNA ,Antisense RNA ,Cell biology - Abstract
De novo production of RNA on RNA template, a process known as RNA-dependent RNA synthesis, RdRs, and the enzymatic activity conducting it, RNA-dependent RNA polymerase, RdRp, were initially considered to be exclusively virus-specific. Eventually, however, the occurrence of RdRs and the ubiquitous presence of conventional RdRp were demonstrated in numerous eukaryotic organisms. The evidence that the enzymatic machinery capable of RdRs is present in mammalian cells was derived from studies of viruses, such as hepatitis delta virus, HDV, that do not encode RdRp yet undergo a robust RNA replication once inside the mammalian host; thus firmly establishing its occurrence and functionality. Moreover, it became clear that RdRp activity, apparently in a non-conventional form, is constitutively present in most, if not in all, mammalian cells. Because such activity was shown to produce short transcripts, given its apparent involvement in RNA interference phenomena, and because double-stranded RNA is known to trigger cellular responses leading to its degradation, it was generally assumed that its role in mammalian cells is restricted to a regulatory function. However, at the same time, an enzymatic activity capable of generating complete antisense RNA complements of mRNAs was discovered in mammalian cells undergoing terminal differentiation. Moreover, observations of widespread synthesis of antisense RNAs initiating at the 3'poly(A) of mRNAs in human cells suggested an extensive cellular utilization of mammalian RdRp. These results led to the development of a model of RdRp-facilitated and antisense RNA-mediated amplification of mammalian mRNA. Recent detection of the major model-predicted identifiers, chimeric RNA intermediates containing both sense and antisense RNA strands covalently joined in a rigorously predicted and uniquely defined manner, as well as the identification of a putative chimeric RNA end product of this process, validated the proposed model. The results corroborating mammalian RNA-dependent mRNA amplification were obtained in vivo with cells undergoing terminal erythroid differentiation and programmed for only a short survival span. This raises a question of whether mammalian RNA-dependent mRNA amplification is a specialized occurrence limited to extreme circumstances of terminal differentiation or a general physiological phenomenon. The present study addresses this question by testing for the occurrence of RNA-dependent amplification of mRNA encoding extracellular matrix proteins abundantly produced throughout the tissue and organ development and homeostasis, an exceptionally revealing indicator of the range and scope of this phenomenon. We report here the detection of major identifiers of RNA-dependent amplification of mRNA encoding α1, β1, and γ1 chains of laminin in mouse tissues producing large quantities of extracellular matrix proteins. The results obtained warrant reinterpretation of the mechanisms involved in ubiquitous and abundant production and deposition of extracellular matrix proteins, confirm the occurrence of mammalian RNA-dependent mRNA amplification as a new mode of genomic protein-encoding information transfer, and establish it as a general physiological phenomenon.
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- 2019
9. Protein-Encoding RNA to RNA Information Transfer in Mammalian Cells: RNA-dependent mRNA Amplification. Identification of Chimeric RNA Intermediates and Putative RNA End Products
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Bjorn R. Olsen, Vladimir Volloch, and Sophia Rits
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chemistry.chemical_compound ,chemistry ,Transcription (biology) ,RNA interference ,Chimeric RNA ,RNA polymerase ,Sense (molecular biology) ,Protein biosynthesis ,RNA ,Biology ,Antisense RNA ,Cell biology - Abstract
Our initial unidirectional understanding of the flow of protein-encoding genetic information, DNA to RNA to protein, a process defined as the "Central Dogma of Molecular Biology" and usually depicted as a downward arrow, was eventually amended to account for the "vertical" information back-flow from RNA to DNA, reverse transcription, and for its "horizontal" side-flow from RNA to RNA, RNA-dependent RNA synthesis, RdRs. These processes, both potentially leading to protein production, were assumed to be strictly virus-specific. However, whereas this presumption might be true for the former, it became apparent that the cellular enzymatic machinery for the later, a conventional RNA-dependent RNA polymerase activity, RdRp, is ubiquitously present and RdRs regularly occurs in eukaryotes. The strongest evidence for the occurrence and functionality of RdRp activity in mammalian cells comes from viruses, such as hepatitis delta virus, HDV, that do not encode RdRp yet undergo a robust RNA replication once inside the host. Eventually, it became clear that RdRp activity, apparently in a non-conventional form, is constitutively present in most, if not in all, mammalian cells. Because such activity was shown to produce short transcripts, because of its apparent involvement in RNA interference phenomena, and because double-stranded RNA is known to trigger cellular responses leading to its degradation, it was generally assumed that its role in mammalian cells is restricted to a regulatory function. However, at the same time, an enzymatic activity capable of generating complete antisense RNA complements of mRNAs was discovered in mammalian cells undergoing terminal differentiation. Moreover, observations of widespread synthesis of antisense RNA initiating at the 3'poly(A) of mRNAs in human cells suggested an extensive cellular utilization of mammalian RdRp. These results led to the development of a model of RdRp-facilitated and antisense RNA-mediated amplification of mammalian mRNA. Here, we report the in vivo detection in cells undergoing terminal erythroid differentiation of the major model-predicted identifiers of such a process, a chimeric double-stranded/pinhead-structured intermediates containing both sense and antisense RNA strands covalently joined in a rigorously predicted and uniquely defined manner. We also report the identification of the putative chimeric RNA end product of mRNA amplification. It is heavily modified, uniformly truncated, yet retains the intact coding region, and terminates with the OH group at both ends; its massive cellular amount is unprecedented for a conventional mRNA transcription product and it translates into polypeptides indistinguishable from the translation product of conventional mRNA. Moreover, we describe the occurrence of the second Tier of mammalian RNA-dependent mRNA amplification, a physiologically occurring, RdRp-driven intracellular PCR process, "iPCR", and report the detection of its distinct RNA end products. Whether mammalian mRNA amplification is a specialized occurrence limited to extreme circumstances of terminal differentiation in cells programmed for only a short survival span or a general physiological phenomenon was answered in the companion article Volloch et al. Ann Integr Mol Med. 2019;1(1):1004. by the detection of major identifiers of this process for mRNA encoding α1, β1, and γ1 chains of laminin, a major extracellular matrix protein abundantly produced throughout the tissue and organ development and homeostasis and an exceptionally revealing indicator of the range and scope of this phenomenon. The results obtained introduce the occurrence of RNA-dependent mRNA amplification as a new mode of genomic protein-encoding information transfer in mammalian cells and establish it as a general physiological phenomenon.
- Published
- 2019
10. A trimeric human angiotensin-converting enzyme 2 as an anti-SARS-CoV-2 agent
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Anthony Griffiths, Rebecca I. Johnson, Sophia Rits-Volloch, Michael Farzan, Nadia Storm, Hanqin Peng, Brian D. Quinlan, Bing Chen, Jianming Lu, Tianshu Xiao, Shen Lu, Jun Zhang, Michael S. Seaman, Lindsay G. A. McKay, Christy L. Lavine, and Yongfei Cai
- Subjects
chemistry.chemical_classification ,0303 health sciences ,Angiotensin II receptor type 1 ,biology ,Chemistry ,Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ,Dimer ,Protein engineering ,Carboxypeptidase ,Cell biology ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Enzyme ,Ectodomain ,Structural Biology ,Angiotensin-converting enzyme 2 ,biology.protein ,Molecular Biology ,hormones, hormone substitutes, and hormone antagonists ,030217 neurology & neurosurgery ,030304 developmental biology - Abstract
Effective intervention strategies are urgently needed to control the COVID-19 pandemic. Human angiotensin-converting enzyme 2 (ACE2) is a membrane-bound carboxypeptidase that forms a dimer and serves as the cellular receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). ACE2 is also a key negative regulator of the renin-angiotensin system that modulates vascular functions. We report here the properties of a trimeric ACE2 ectodomain variant, engineered using a structure-based approach. The trimeric ACE2 variant has a binding affinity of ~60 pM for the spike protein of SARSCoV2 (compared with 77 nM for monomeric ACE2 and 12-22 nM for dimeric ACE2 constructs), and its peptidase activity and the ability to block activation of angiotensin II receptor type 1 in the renin-angiotensin system are preserved. Moreover, the engineered ACE2 potently inhibits SARSCoV2 infection in cell culture. These results suggest that engineered, trimeric ACE2 may be a promising anti-SARS-CoV-2 agent for treating COVID-19.
- Published
- 2021
- Full Text
- View/download PDF
11. Structural impact on SARS-CoV-2 spike protein by D614G substitution
- Author
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Sarah M. Sterling, Tianshu Xiao, Hanqin Peng, Yongfei Cai, Bing Chen, Jun Zhang, Jianming Lu, Piotr Sliz, Richard M. Walsh, and Sophia Rits-Volloch
- Subjects
Infectivity ,Immunogen ,Viral entry ,Chemistry ,Aspartic acid ,Lipid bilayer fusion ,Trimer ,Protomer ,Article ,Virus ,Cell biology - Abstract
Substitution for aspartic acid by glycine at position 614 in the spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the ongoing pandemic, appears to facilitate rapid viral spread. The G614 variant has now replaced the D614-carrying virus as the dominant circulating strain. We report here cryo-EM structures of a full-length S trimer carrying G614, which adopts three distinct prefusion conformations differing primarily by the position of one receptor-binding domain (RBD). A loop disordered in the D614 S trimer wedges between domains within a protomer in the G614 spike. This added interaction appears to prevent premature dissociation of the G614 trimer, effectively increasing the number of functional spikes and enhancing infectivity. The loop transition may also modulate structural rearrangements of S protein required for membrane fusion. These findings extend our understanding of viral entry and suggest an improved immunogen for vaccine development.
- Published
- 2020
12. A trimeric human angiotensin-converting enzyme 2 as an anti-SARS-CoV-2 agent in vitro
- Author
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Tengfei Xiao, Michael S. Seaman, Michael Farzan, Shen Lu, Sophia Rits-Volloch, Jianming Lu, Anthony Griffiths, Jessica Zhang, Bing Chen, Hanqin Peng, Brian D. Quinlan, Nadia Storm, Christy L. Lavine, Rebecca I. Johnson, Yongfei Cai, and Lindsay G. A. McKay
- Subjects
chemistry.chemical_classification ,Angiotensin II receptor type 1 ,biology ,Dimer ,Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ,Wild type ,Carboxypeptidase ,Article ,In vitro ,Cell biology ,chemistry.chemical_compound ,Enzyme ,chemistry ,Angiotensin-converting enzyme 2 ,biology.protein ,hormones, hormone substitutes, and hormone antagonists - Abstract
Effective intervention strategies are urgently needed to control the COVID-19 pandemic. Human angiotensin-converting enzyme 2 (ACE2) is a membrane-bound carboxypeptidase that forms a dimer and serves as the cellular receptor for SARS‑CoV‑2. ACE2 is also a key negative regulator of the renin-angiotensin system (RAS) that modulates vascular functions. We report here the properties of a trimeric ACE2 ectodomain variant, engineered using a structure-based approach. The trimeric ACE2 variant has a binding affinity of ~60 pM for the spike (S) protein of SARS‑CoV‑2 (compared to 77 nM for monomeric ACE2 and 12–22 nM for dimeric ACE2 constructs), while preserving its peptidase activity and the ability to block activation of angiotensin II receptor type 1 in the RAS. Moreover, the engineered ACE2 potently inhibits infection of SARS‑CoV‑2 in cell culture. These results suggest that engineered, trimeric ACE2 may be a promising anti-SARS-CoV-2 agent for treating COVID-19.
- Published
- 2020
13. Distinct conformational states of SARS-CoV-2 spike protein
- Author
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Richard M. Walsh, Yongfei Cai, Sophia Rits-Volloch, Bing Chen, Jun Zhang, Shaun Rawson, Tianshu Xiao, Sarah M. Sterling, and Hanqin Peng
- Subjects
0301 basic medicine ,Glycan ,Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ,Protein domain ,Cell ,Trimer ,Peptidyl-Dipeptidase A ,Protein Structure, Secondary ,Article ,Virus ,03 medical and health sciences ,0302 clinical medicine ,Immune system ,Protein structure ,Protein Domains ,medicine ,Humans ,Structural transition ,Receptor ,Research Articles ,Multidisciplinary ,biology ,Chemistry ,R-Articles ,Cryoelectron Microscopy ,HEK 293 cells ,Biochem ,Spike Protein ,Microbio ,Virus Internalization ,Cell biology ,HEK293 Cells ,030104 developmental biology ,medicine.anatomical_structure ,Ectodomain ,Host-Pathogen Interactions ,Spike Glycoprotein, Coronavirus ,biology.protein ,Receptors, Virus ,Angiotensin-Converting Enzyme 2 ,Protein Multimerization ,030217 neurology & neurosurgery ,Fusion peptide ,Research Article - Abstract
The ongoing SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) pandemic has created urgent needs for intervention strategies to control the crisis. The spike (S) protein of the virus forms a trimer and catalyzes fusion between viral and target cell membranes - the first key step of viral infection. Here we report two cryo-EM structures, both derived from a single preparation of the full-length S protein, representing the prefusion (3.1Å resolution) and postfusion (3.3Å resolution) conformations, respectively. The spontaneous structural transition to the postfusion state under mild conditions is independent of target cells. The prefusion trimer forms a tightly packed structure with three receptor-binding domains clamped down by a segment adjacent to the fusion peptide, significantly different from recently published structures of a stabilized S ectodomain trimer. The postfusion conformation is a rigid tower-like trimer, but decorated by N-linked glycans along its long axis with almost even spacing, suggesting possible involvement in a mechanism protecting the virus from host immune responses and harsh external conditions. These findings advance our understanding of how SARS-CoV-2 enters a host cell and may guide development of vaccines and therapeutics.
- Published
- 2020
14. Precursor-Independent Overproduction of Beta-Amyloid in AD: Mitochondrial Dysfunction as Possible Initiator of Asymmetric RNA-Dependent βAPP mRNA Amplification. An Engine that Drives Alzheimer’s Disease
- Author
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Volloch, Bjorn R. Olsen, and Rits S
- Subjects
Messenger RNA ,chemistry.chemical_compound ,Eukaryotic translation ,medicine.diagnostic_test ,Chemistry ,Proteolysis ,RNA polymerase ,Sense (molecular biology) ,medicine ,Unfolded protein response ,RNA ,Antisense RNA ,Cell biology - Abstract
The present study defines RNA-dependent amplification of βAPP mRNA as a molecular basis of beta-amyloid overproduction in Alzheimer’s disease. In this process, βAPP mRNA serves as a template for RNA-dependent RNA polymerase, RdRp complex. The resulting antisense RNA self-primes its extension utilizing two complementary elements: 3’-terminal and internal, located within an antisense segment corresponding to the coding portion of βAPP mRNA. The extension produces 3’-terminal fragment of βAPP mRNA, a part of a hairpin-structured antisense/sense RNA molecule. Cleavage at the 3’ end of the hairpin loop produces RNA end product encoding a C-terminal fragment of βAPP. Since each conventional βAPP mRNA can be used repeatedly as a template, the process constitutes an asymmetric mRNA amplification. The 5’-most translation initiation codon of the amplified mRNA is the AUG preceding immediately and in-frame the Aβ-coding segment. Translation from this codon overproduces Aβ independently of βAPP. Such process can occur in humans but not in mice and other animals where segments of βAPP antisense RNA required for self-priming have little, if any, complementarity. This explains why Alzheimer’s disease occurs exclusively in humans and implies that βAPP mRNA amplification is requisite in AD. In AD, therefore, there are two pathways of beta-amyloid production: βAPP proteolytic pathway and βAPP mRNA amplification pathway independent of βAPP and insensitive to beta-secretase inhibition. This implies that in healthy humans, where only the proteolytic pathway is in operation, Aβ production should be suppressed by the BACE inhibition, and indeed it is. However, since βAPP-independent pathway operating in AD is by far the predominant one, BACE inhibition has no effect in Alzheimer’s disease. It appears that, physiologically, the extent of beta-amyloid overproduction sufficient to trigger amyloid cascade culminating in AD requires asymmetric RNA-dependent amplification of βAPP mRNA and cannot be reached without it. In turn, the occurrence of mRNA amplification process depends on the activation of inducible components of RdRp complex by certain stresses, for example the ER stress in case of amplification of mRNA encoding extracellular matrix proteins. In case of Alzheimer’s disease, such an induction appears to be triggered by stresses associated with mitochondrial dysfunction, a phenomenon closely linked to AD. The cause-and-effect relationships between mitochondrial dysfunction and AD appear to be very different in familial, FAD, and sporadic, SAD cases. In FAD, increased levels or more toxic species of Aβ resulting from the abnormal proteolysis of βAPP trigger mitochondrial dysfunction, activate mRNA amplification and increase the production of Aβ, reinforcing the cycle. Thus in FAD, mitochondrial dysfunction is an intrinsic component of the amyloid cascade. The reverse sequence is true in SAD where aging-related mitochondrial dysfunction activates amplification of βAPP mRNA and enhances the production of Aβ. This causes further mitochondrial dysfunction, the cycle repeats and degeneration increases. Thus in SAD, the initial mitochondrial dysfunction arises prior to the disease, independently of and upstream from the increased Aβ production, i.e. in SAD, mitochondrial pathology hierarchically supersedes Aβ pathology. This is the primary reason for the formulation of the Mitochondrial Cascade Hypothesis. But even in terms of the MCH, the core of the disease is the amyloid cascade as defined in the amyloid cascade hypothesis, ACH. The role of mitochondrial dysfunction in relation to this core is causative in SAD and auxiliary in FAD. In FAD, the initial increase in the production of Aβ is mutations-based and occurs relatively early in life, whereas in SAD it is coerced by an aging-contingent component, but both lead to mechanistically identical self-perpetuating mutual Aβ/mitochondrial dysfunction feedback cycles, an engine that drives, via RNA-dependent βAPP mRNA amplification, overproduction of beta-amyloid and, consequently, AD; hence drastic difference in the age of onset, yet profound pathological and symptomatic similarity in the progression, of familial and sporadic forms of Alzheimer’s disease. Interestingly, the recent findings that mitochondrial microprotein PIGBOS interacts with the ER in mitigating the unfolded protein response indicate a possible connection between mitochondrial dysfunction and ER stress, implicated in activation of RNA-dependent mRNA amplification pathway. The possible involvement of mitochondrial dysfunction in βAPP mRNA amplification makes it a promising therapeutic target. Recent successes in mitigating, and even reversing, Aβ-induced metabolic defects with anti-diabetes drug metformin are encouraging in this respect.
- Published
- 2019
15. Protein-Encoding RNA-to-RNA Information Transfer in Mammalian Cells: Principles of RNA-Dependent mRNA Amplification
- Author
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Vladimir Volloch
- Subjects
Messenger RNA ,molecular_biology ,RNA ,RNA-dependent RNA polymerase ,Central dogma of molecular biology ,Biology ,Article ,Reverse transcriptase ,Cell biology ,chemistry.chemical_compound ,chemistry ,Chimeric RNA ,RNA polymerase ,Protein biosynthesis ,DNA - Abstract
The transfer of protein-encoding genetic information from DNA to RNA to protein, a process formalized as the “Central Dogma of Molecular Biology”, has undergone a significant evolution since its inception. It was amended to account for the information flow from RNA to DNA, the reverse transcription, and for the information transfer from RNA to RNA, the RNA-dependent RNA synthesis. These processes, both potentially leading to protein production, were initially described only in viral systems, and although RNA-dependent RNA polymerase activity was shown to be present, and RNA-dependent RNA synthesisfound to occur, in mammalian cells, its function was presumed to be restricted to regulatory. However, recent results, obtained with multiple mRNA species in several mammalian systems, strongly indicate the occurrence of protein-encoding RNA to RNA information transfer in mammalian cells. It can result in the rapid production of the extraordinary quantities of specific proteins as was seen in cases of terminal cellular differentiation and during cellular deposition of extracellular matrix molecules. A malfunction of this process may be involved in pathologies associated either with the deficiency of a protein normally produced by this mechanism or with the abnormal abundanceof a protein or of its C-terminal fragment. It seems to be responsible for some types of familial thalassemia and may underlie the overproduction of beta amyloid in sporadic Alzheimer’s disease. The aim of the present article is to systematize the current knowledge and understanding of this pathway. The outlined framework introduces unexpected features of the mRNA amplification such as its ability to generate polypeptides non-contiguously encoded in the genome, its second Tier, a physiologically occurring intracellular polymerase chain reaction, iPCR, a Two-Tier Paradox and RNA Dark Matter. RNA-dependent mRNA amplification represents a new mode of genomic protein-encoding information transfer in mammalian cells. Its potential physiological impact is substantial, it appears relevant to multiple pathologies and its understanding opens new venues of therapeutic interference, it suggests powerful novel bioengineering approaches and its further rigorous investigations are highly warranted.
- Published
- 2019
16. Structure of the membrane proximal external region of HIV-1 envelope glycoprotein
- Author
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Hanqin Peng, Qingshan Fu, James J. Chou, Sophia Rits-Volloch, Munan Shaik, Michael S. Seaman, Stephen C. Harrison, Bing Chen, Fadi Ghantous, Yongfei Cai, Zhijun Liu, and Alessandro Piai
- Subjects
0301 basic medicine ,Magnetic Resonance Spectroscopy ,Viral protein ,HIV Antigens ,Protein domain ,Lipid Bilayers ,medicine.disease_cause ,Membrane Fusion ,Epitope ,03 medical and health sciences ,Immunoglobulin Fab Fragments ,Protein Domains ,medicine ,Lipid bilayer ,chemistry.chemical_classification ,Multidisciplinary ,030102 biochemistry & molecular biology ,biology ,Chemistry ,Virion ,env Gene Products, Human Immunodeficiency Virus ,Lipid bilayer fusion ,Cell biology ,Transmembrane domain ,030104 developmental biology ,PNAS Plus ,biology.protein ,HIV-1 ,Antibody ,Glycoprotein - Abstract
The membrane-proximal external region (MPER) of the HIV-1 envelope glycoprotein (Env) bears epitopes of broadly neutralizing antibodies (bnAbs) from infected individuals; it is thus a potential vaccine target. We report an NMR structure of the MPER and its adjacent transmembrane domain in bicelles that mimic a lipid-bilayer membrane. The MPER lies largely outside the lipid bilayer. It folds into a threefold cluster, stabilized mainly by conserved hydrophobic residues and potentially by interaction with phospholipid headgroups. Antigenic analysis and comparison with published images from electron cryotomography of HIV-1 Env on the virion surface suggest that the structure may represent a prefusion conformation of the MPER, distinct from the fusion-intermediate state targeted by several well-studied bnAbs. Very slow bnAb binding indicates that infrequent fluctuations of the MPER structure give these antibodies occasional access to alternative conformations of MPER epitopes. Mutations in the MPER not only impede membrane fusion but also influence presentation of bnAb epitopes in other regions. These results suggest strategies for developing MPER-based vaccine candidates.
- Published
- 2018
17. RNA-Dependent Amplification of Mammalian mRNA Encoding Extracellular Matrix Components: Identification of Chimeric RNA Intermediates for α1, β1, and γ1 Chains of Laminin
- Author
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Vladimir Volloch, Bjorn R. Olsen, and Sophia Rits
- Subjects
Messenger RNA ,Matrigel ,biology ,Laminin ,Chimeric RNA ,Chemistry ,Sense (molecular biology) ,biology.protein ,Extracellular ,RNA ,Cleavage (embryo) ,Cell biology - Abstract
The aim of the present study was to test for the occurrence of key elements predicted by the previously postulated mammalian RNA-dependent mRNA amplification model in a tissue producing massive amounts of extracellular matrix proteins. At the core of RNA-dependent mRNA amplification, until now only described in one mammalian system, is the self-priming of an antisense strand and extension of its 3’ terminus into a sense-oriented RNA containing the protein-coding information of a conventional mRNA. The resulting product constitutes a new type of biomolecule. It is chimeric in that it contains covalently connected antisense and sense sequences in a hairpin configuration. Cleavage of this chimeric intermediate in the loop region of a hairpin structure releases mRNA which contains an antisense segment in its 5’UTR; depending on the position of self-priming, the chimeric end product may encode the entire protein or its C-terminal fragment. The occurrence of such composite chimeric molecules is unique for this type of mRNA amplification and represents a conclusive “identifier” of this process. We report here the detection, by next generation sequencing, of such chimeric junction sequences for mRNAs molecules encoding αl, β1, and γ1 chains of laminin in cells of the extracellular matrix-generating Engelbreth-Holm-Swarm (EHS) mouse tumor, best known for producing extraordinarily large amounts of “Matrigel”.
- Published
- 2018
- Full Text
- View/download PDF
18. Effect of the cytoplasmic domain on antigenic characteristics of HIV-1 envelope glycoprotein
- Author
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Michael S. Seaman, Jianming Lu, Elise Zablowsky, Jia Chen, James M. Kovacs, Bing Chen, Sophia Rits-Volloch, Donghyun Park, and Hanqin Peng
- Subjects
chemistry.chemical_classification ,Multidisciplinary ,Immunogen ,Lipid bilayer fusion ,Biology ,Virology ,Epitope ,Protein structure ,Antigen ,chemistry ,Cytoplasm ,biology.protein ,Antibody ,Glycoprotein - Abstract
Steps in the right direction HIV-1 mutates rapidly, making it difficult to design a vaccine that will protect people against all of the virus' iterations. A potential successful vaccine design might protect by eliciting broadly neutralizing antibodies (bNAbs), which target specific regions on HIV-1's trimeric envelope glycoprotein (Env) (see the Perspective by Mascola). Jardine et al. used mice engineered to express germline-reverted heavy chains of a particular bNAb and immunized them with an Env-based immunogen designed to bind to precursors of that bNAb. Sanders et al. compared rabbits and monkeys immunized with Env trimers that adopt a nativelike conformation. In both cases, immunized animals produced antibodies that shared similarities with bNAbs. Boosting these animals with other immunogens may drive these antibodies to further mutate into the longsought bNAbs. Chen et al. report that retaining the cytoplasmic domain of Env proteins may be important to attract bNAbs. Removing the cytoplasmic domain may distract the immune response and instead generate antibodies that target epitopes on Env that would not lead to protection. Science , this issue p. 139 , 10.1126/science.aac4223 , p. 156 ; see also p. 191
- Published
- 2015
19. Results of Beta Secretase-Inhibitor Clinical Trials Support Amyloid Precursor Protein-Independent Generation of Beta Amyloid in Sporadic Alzheimer’s Disease
- Author
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Sophia Rits and Vladimir Volloch
- Subjects
0301 basic medicine ,Amyloid ,lcsh:Medicine ,amyloid precursor protein-independent generation of β amyloid ,Review ,amyloid precursor protein ,sporadic Alzheimer’s disease ,Disease ,Pharmacology ,β-site amyloid precursor protein-cleaving enzyme 1 inhibitors ,03 medical and health sciences ,0302 clinical medicine ,mental disorders ,familial Alzheimer’s disease ,Amyloid precursor protein ,Medicine ,Beta (finance) ,chemistry.chemical_classification ,biology ,business.industry ,lcsh:R ,Healthy subjects ,Clinical trial ,030104 developmental biology ,Enzyme ,chemistry ,biology.protein ,business ,Alzheimer’s disease ,Amyloid precursor protein secretase ,030217 neurology & neurosurgery - Abstract
The present review analyzes the results of recent clinical trials of β secretase inhibition in sporadic Alzheimer’s disease (SAD), considers the striking dichotomy between successes in tests of β-site Amyloid Precursor Protein-Cleaving Enzyme (BACE) inhibitors in healthy subjects and familial Alzheimer’s disease (FAD) models versus persistent failures of clinical trials and interprets it as a confirmation of key predictions for a mechanism of amyloid precursor protein (APP)-independent, β secretase inhibition-resistant production of β amyloid in SAD, previously proposed by us. In light of this concept, FAD and SAD should be regarded as distinctly different diseases as far as β-amyloid generation mechanisms are concerned, and whereas β secretase inhibition would be neither applicable nor effective in the treatment of SAD, the β-site APP-Cleaving Enzyme (BACE) inhibitor(s) deemed failed in SAD trials could be perfectly suitable for the treatment of FAD. Moreover, targeting the aspects of Alzheimer’s disease (AD) other than cleavages of the APP by β and α secretases should have analogous impacts in both FAD and SAD.
- Published
- 2018
20. Human bone marrow-derived stromal cells show highly efficient stress-resistant adipogenesis on denatured collagen IV matrix but not on its native counterpart: Implications for obesity
- Author
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Joshua R. Mauney and Vladimir Volloch
- Subjects
MAPK/ERK pathway ,medicine.medical_specialty ,Stromal cell ,Chemistry ,p38 mitogen-activated protein kinases ,Adipose tissue ,Matrix metalloproteinase ,Endocrinology ,medicine.anatomical_structure ,Adipogenesis ,Internal medicine ,medicine ,Denaturation (biochemistry) ,Bone marrow ,Molecular Biology - Abstract
Collagen IV is the major matrix component associated with differentiating adipocytes in adipose tissues, and the understanding of its contribution in adipogenic differentiation could be important for elucidation of mechanisms and processes driving the obesity. Therefore, in the light of our previous findings of differential effects of structural conformation of collagen I matrix on differentiation of bone marrow stromal cells, we investigated whether similar phenomenon occurs on collagen IV matrix in native and denatured structural states. The results of the present study show that native collagen IV is unsupportive of adipogenic differentiation and very little if any adipogenesis occurs on this matrix in the presence of adipogenic stimuli. In sharp contrast to native collagen IV, the same matrix in denatured structural state drives highly efficient adipogenic differentiation suggesting that it might be the major driver of adipogenesis in adipose tissues and that the ratio of native to denatured matrix might regulate the intensity of adipogenesis and possibly underlies the obesity. In contrast to observations that adipogenesis on denatured collagen I (collagen I is the major matrix component in musculoskeletal tissues) is suppressed by stress, adipogenesis on denatured collagen IV appears to be stress-resistant suggesting an explanation for the observed ineffectiveness of physical exercise, i.e. mechanical stress, in the reduction of adipose tissues. The obesity was shown to be associated with overproduction of MMPs and decline in levels of TIMPs. Such a shift in MMP/TIMP balance was considered a consequence of the pathology. In the light of the present study, however, this shift might constitute the primary source of the decease. The findings of the present study suggest strategies for the treatment of obesity, raise significant questions and indicate directions for further experimentation.
- Published
- 2010
21. Role of HIV membrane in neutralization by two broadly neutralizing antibodies
- Author
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Bing Chen, Sophia Rits-Volloch, Hua-Xin Liao, Shi-Mao Xia, Ruijun Zhang, S. Moses Dennison, Stephen C. Harrison, Li Sun, S. Munir Alam, Marco Morelli, and Barton F. Haynes
- Subjects
Models, Molecular ,medicine.drug_class ,HIV Infections ,Monoclonal antibody ,Gp41 ,Neutralization ,Epitope ,Neutralization Tests ,Viral entry ,medicine ,Humans ,Multidisciplinary ,biology ,Chemistry ,Viral Vaccine ,Antibodies, Monoclonal ,Membranes, Artificial ,Viral Vaccines ,Biological Sciences ,Viral membrane ,Antibodies, Neutralizing ,Virology ,HIV Envelope Protein gp41 ,Mutation ,HIV-1 ,biology.protein ,Antibody ,Protein Binding - Abstract
Induction of effective antibody responses against HIV-1 infection remains an elusive goal for vaccine development. Progress may require in-depth understanding of the molecular mechanisms of neutralization by monoclonal antibodies. We have analyzed the molecular actions of two rare, broadly neutralizing, human monoclonal antibodies, 4E10 and 2F5, which target the transiently exposed epitopes in the membrane proximal external region (MPER) of HIV-1 gp41 envelope during viral entry. Both have long CDR H3 loops with a hydrophobic surface facing away from the peptide epitope. We find that the hydrophobic residues of 4E10 mediate a reversible attachment to the viral membrane and that they are essential for neutralization, but not for interaction with gp41. We propose that these antibodies associate with the viral membrane in a required first step and are thereby poised to capture the transient gp41 fusion intermediate. These results bear directly on strategies for rational design of HIV-1 envelope immunogens.
- Published
- 2009
22. Denatured Collagen Modulates the Phenotype of Normal and Wounded Human Skin Equivalents
- Author
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Joshua R. Mauney, Christophe Egles, Yulia Shamis, Vladimir Volloch, Jonathan A. Garlick, and David L. Kaplan
- Subjects
Keratinocytes ,Cell signaling ,Protein Denaturation ,Human skin ,Cell Communication ,Dermatology ,Matrix (biology) ,Biochemistry ,Article ,Extracellular matrix ,Tissue culture ,Cell Movement ,medicine ,Humans ,Fibroblast ,Molecular Biology ,Cells, Cultured ,Cell Proliferation ,Skin ,Wound Healing ,integumentary system ,Cell growth ,Chemistry ,Cell Biology ,Fibroblasts ,Cell biology ,Extracellular Matrix ,medicine.anatomical_structure ,Phenotype ,Immunology ,Collagen ,Wound healing - Abstract
Epithelial–mesenchymal interactions are known to play an important role in modulating homeostasis and repair. However, it remains unclear how the composition of the extracellular matrix may regulate the ability of dermal fibroblasts to engage in such cross talk. To address this, we studied how fibroblast phenotype was linked to the behavior of normal and wounded human skin equivalents (HSE) by comparing human dermal fibroblasts (HDF) incorporated into the three-dimensional tissues to those extensively cultivated in two-dimensional (2D) monolayer culture on denatured collagen (DC) matrix, native collagen, or tissue culture plastic before incorporation into HSEs. We first established that prolonged passage and growth of HDF on DC increased their migratory potential in a 2D monolayer culture. When HDF variants were grown in HSEs, we found that extended passage on DC and incorporation of DC directly into the collagen gel enhanced proliferation of both HDF and basal keratinocytes in HSEs. By adapting HSEs to study wound reepithelialization, we found that the extended passage of HDF on DC accelerated the rate of wound healing by 38%. Thus, extensive ex vivo expansion on DC was able to modify the phenotype of skin fibroblasts by augmenting their reparative properties in skin-like HSEs.
- Published
- 2008
- Full Text
- View/download PDF
23. Restraining the conformation of HIV-1 gp120 by removing a flexible loop
- Author
-
Sophia Rits-Volloch, Stephen C. Harrison, Gary Frey, and Bing Chen
- Subjects
Models, Molecular ,Conformational change ,Molecular model ,viruses ,Plasma protein binding ,HIV Envelope Protein gp120 ,Biology ,Gp41 ,Article ,Protein Structure, Secondary ,General Biochemistry, Genetics and Molecular Biology ,Epitope ,Structure-Activity Relationship ,Humans ,Structure–activity relationship ,Protein Structure, Quaternary ,Receptor ,Molecular Biology ,chemistry.chemical_classification ,General Immunology and Microbiology ,General Neuroscience ,Antibodies, Monoclonal ,Gene Products, env ,virus diseases ,Molecular biology ,HIV Envelope Protein gp41 ,chemistry ,Multiprotein Complexes ,CD4 Antigens ,HIV-1 ,Biophysics ,Glycoprotein ,Protein Binding - Abstract
The trimeric HIV/SIV envelope glycoprotein, gp160, is cleaved to noncovalently associated fragments, gp120 and gp41. Binding of gp120 to viral receptors leads to large structural rearrangements in both fragments. The unliganded gp120 core has a disordered beta3-beta5 loop, which reconfigures upon CD4 binding into an ordered, extended strand. Molecular modeling suggests that residues in this loop may contact gp41. We show here that deletions in the beta3-beta5 loop of HIV-1 gp120 weaken the binding of CD4 and prevent formation of the epitope for monoclonal antibody (mAb) 17b (which recognizes the coreceptor site). Formation of an encounter complex with CD4 binding and interactions of gp120 with mAbs b12 and 2G12 are not affected by these deletions. Thus, deleting the beta3-beta5 loop blocks the gp120 conformational change and may offer a strategy for design of restrained immunogens. Moreover, mutations in the SIV beta3-beta5 loop lead to greater spontaneous dissociation of gp120 from cell-associated trimers. We suggest that the CD4-induced rearrangement of this loop releases structural constraints on gp41 and thus potentiates its fusion activity.
- Published
- 2006
24. Stable, uncleaved HIV-1 envelope glycoprotein gp140 forms a tightly folded trimer with a native-like structure
- Author
-
James M. Kovacs, Erik Noeldeke, Heather Jiwon Ha, Sophia Rits-Volloch, Bing Chen, Stephen C. Harrison, and Hanqin Peng
- Subjects
Models, Molecular ,Antigenicity ,Protein Folding ,Stereochemistry ,viruses ,Trimer ,CHO Cells ,Gp41 ,law.invention ,Cricetulus ,law ,Viral entry ,Cricetinae ,Animals ,Humans ,Protein Structure, Quaternary ,Multidisciplinary ,Chemistry ,env Gene Products, Human Immunodeficiency Virus ,virus diseases ,Biological Sciences ,Crystallography ,Ectodomain ,Recombinant DNA ,HIV-1 ,Protein folding ,Protein Multimerization ,Linker - Abstract
The HIV-1 envelope spike [trimeric (gp160) 3 , cleaved to (gp120/gp41) 3 ] is the mediator of viral entry and the principal target of humoral immune response to the virus. Production of a recombinant preparation that represents the functional spike poses a challenge for vaccine development, because the (gp120/gp41) 3 complex is prone to dissociation. We have reported previously that stable HIV-1 gp140 trimers, the uncleaved ectodomains of (gp160) 3 , have nearly all of the antigenic properties expected for native viral spikes. Because of recent claims that uncleaved gp140 proteins may adopt a nonnative structure with three gp120 moieties “dangling” from a trimeric gp41 ectodomain in its postfusion conformation, we have inserted a long, flexible linker between gp120 and gp41 in our stable gp140 trimers to assess their stability and to analyze their conformation in solution. The modified trimer has biochemical and antigenic properties virtually identical to those of its unmodified counterpart. Both forms bind a single CD4 per trimer, suggesting that the trimeric conformation occludes two of the three CD4 sites even when a flexible linker has relieved the covalent constraint between gp120 and gp41. In contrast, an artificial trimer containing three gp120s flexibly tethered to a trimerization tag binds three CD4s and has antigenicity nearly identical to that of monomeric gp120. Moreover, the gp41 part of both modified and unmodified gp140 trimers has a structure very different from that of postfusion gp41. These results show that uncleaved gp140 trimers from suitable isolates have compact, native-like structures and support their use as candidate vaccine immunogens.
- Published
- 2014
25. Osteogenic Differentiation of Human Bone Marrow Stromal Cells on Partially Demineralized Bone Scaffoldsin Vitro
- Author
-
Joshua R. Mauney, Vladimir Volloch, Jeff Blumberg, David L. Kaplan, Gordana Vunjak-Novakovic, and Mono Pirun
- Subjects
Adult ,Male ,Scaffold ,Stromal cell ,Cell ,Bone Marrow Cells ,Bone healing ,Dexamethasone ,Tissue engineering ,medicine ,Animals ,Humans ,Glucocorticoids ,Chemistry ,General Engineering ,Cell Differentiation ,Alkaline Phosphatase ,medicine.disease ,In vitro ,Cell biology ,medicine.anatomical_structure ,Bone Substitutes ,Microscopy, Electron, Scanning ,Alkaline phosphatase ,Cattle ,Stromal Cells ,Infiltration (medical) ,Biomedical engineering - Abstract
Tissue engineering has been used to enhance the utility of biomaterials for clinical bone repair by the incorporation of an osteogenic cell source into a scaffold followed by the in vitro promotion of osteogenic differentiation before host implantation. In this study, three-dimensional, partially demineralized bone scaffolds were investigated for their ability to support osteogenic differentiation of human bone marrow stromal cells (BMSCs) in vitro. Dynamic cell seeding resulted in homogeneous cell attachment and infiltration within the matrix and produced significantly higher seeding efficiencies when compared with a conventional static seeding method. Dynamically seeded scaffolds were cultured for 7 and 14 days in the presence of dexamethasone and evaluated on biochemical, molecular, and morphological levels for osteogenic differentiation. Significant elevation in alkaline phosphatase activity was observed versus controls over the 14-day culture, with a transient peak indicative of early mineralization on day 7. On the basis of RT-PCR, dexamethasone-treated samples showed elevations in alkaline phosphatase and osteocalcin expression levels at 7 and 14 days over nontreated controls, while bone sialoprotein was produced only in the presence of dexamethasone at 14 days. Scanning electron microscopy evaluation of dexamethasone-treated samples at 14 days revealed primarily cuboidal cells indicative of mature osteoblasts, in contrast to nontreated controls displaying a majority of cells with a fibroblastic cell morphology. These results demonstrate that partially demineralized bone can be successfully used with human BMSCs to support osteogenic differentiation in vitro. This osseous biomaterial may offer new potential benefits as a tool for clinical bone replacement.
- Published
- 2004
26. Matrix-mediated cellular rejuvenation
- Author
-
David L. Kaplan and Vladimir Volloch
- Subjects
Time Factors ,Chemistry ,medicine.medical_treatment ,Cell ,Fibroblasts ,Matrix (biology) ,beta-Galactosidase ,Protein oxidation ,Cell Line ,Extracellular Matrix ,Cell biology ,Extracellular matrix ,Cytokine ,medicine.anatomical_structure ,Tissue engineering ,medicine ,Humans ,HSP70 Heat-Shock Proteins ,Collagen ,Stem cell ,Cytoskeleton ,Oxidation-Reduction ,Molecular Biology ,Cellular Senescence - Abstract
Biomaterial surface morphology and chemistry influence cell responses mediated via signaling cascades that regulate a wide range of metabolic processes. These responses may range from changes in surface adhesion and remodeling of the extracellular matrix to activation of cytokine, cytoskeletal and other biochemical pathways regulating or modulating cellular morphology and function. The present study has focused on collagen Type I, a key extracellular matrix protein, and its potential impact on the process of cellular aging. This study was undertaken for several reasons. First, several investigators reported that growth of cells on a collagen matrix markedly enhanced the resistance of cells to stresses. Second, a large body of accumulated data strongly indicated a relationship between the potential to respond to stresses and cellular aging with the former strongly influencing the rate of the latter. Finally, it has been recently demonstrated that in aged cells one of the key aging-related processes previously considered irreversible, attenuation of the expression of a major stress response protein, Hsp70, can be reversed. This fact together with a probable regulatory role of the stress response potential in cellular aging suggested a possibility that the cellular aging process as a whole can be altered. Indeed, in the present study, growth on a denatured collagen matrices reversed in aged cells not only the attenuation of Hsp70 expression but also other aging-related processes, such as beta-galactosidase expression, increase in protein oxidation and changes in cell morphology. Moreover, it appeared to reduce the rate of aging in young cells. Understanding the nature of collagen matrix-mediated cellular rejuvenation might suggest approaches for interfering with organismic aging. Some immediate applications include cell rejuvenation for purposes of cloning and reduction of the rate of aging during expansion of stem cells for purposes of tissue engineering.
- Published
- 2002
27. Mechanism of HIV-1 neutralization by antibodies targeting a membrane-proximal region of gp41
- Author
-
Hanqin Peng, Jetta Garrity, Sophia Rits-Volloch, Jia Chen, Michael S. Seaman, Gary Frey, and Bing Chen
- Subjects
Immunology ,Amino Acid Motifs ,HIV Infections ,Complementarity determining region ,Biology ,HIV Antibodies ,Gp41 ,Microbiology ,Neutralization ,Cell Line ,Cell membrane ,Neutralization Tests ,Virology ,Vaccines and Antiviral Agents ,medicine ,Humans ,Lipid bilayer ,chemistry.chemical_classification ,Cell Membrane ,Viral membrane ,Antibodies, Neutralizing ,Complementarity Determining Regions ,HIV Envelope Protein gp41 ,Cell biology ,medicine.anatomical_structure ,chemistry ,Insect Science ,biology.protein ,HIV-1 ,Antibody ,Glycoprotein - Abstract
Induction of broadly neutralizing antibodies (bNAbs) is an important goal for HIV-1 vaccine development. Two autoreactive bNAbs, 2F5 and 4E10, recognize a conserved region on the HIV-1 envelope glycoprotein gp41 adjacent to the viral membrane known as the membrane-proximal external region (MPER). They block viral infection by targeting a fusion-intermediate conformation of gp41, assisted by an additional interaction with the viral membrane. Another MPER-specific antibody, 10E8, has recently been reported to neutralize HIV-1 with potency and breadth much greater than those of 2F5 or 4E10, but it appeared not to bind phospholipids and might target the untriggered envelope spikes, raising the hope that the MPER could be harnessed for vaccine design without major immunological concerns. Here, we show by three independent approaches that 10E8 indeed binds lipid bilayers through two hydrophobic residues in its CDR H3 (third heavy-chain complementarity-determining region). Its weak affinity for membranes in general and preference for cholesterol-rich membranes may account for its great neutralization potency, as it is less likely than other MPER-specific antibodies to bind cellular membranes nonspecifically. 10E8 binds with high affinity to a construct mimicking the fusion intermediate of gp41 but fails to recognize the envelope trimers representing the untriggered conformation. Moreover, we can improve the potency of 4E10 without affecting its binding to gp41 by a modification of its lipid-interacting CDR H3. These results reveal a general mechanism of HIV-1 neutralization by MPER-specific antibodies that involves interactions with viral lipids.
- Published
- 2013
28. ATPase activity of the heat shock protein Hsp72 is dispensable for its effects on dephosphorylation of stress kinase JNK and on heat-induced apoptosis
- Author
-
Vladimir L. Gabai, Sophia Rits, Vladimir Volloch, and Michael Y. Sherman
- Subjects
Time Factors ,ATPase ,Immunoblotting ,Biophysics ,HSP72 Heat-Shock Proteins ,Apoptosis ,Hsp70 ATPase ,Biochemistry ,Cell Line ,Dephosphorylation ,Structural Biology ,Heat shock protein ,Genetics ,Animals ,Humans ,Phosphorylation ,Molecular Biology ,Heat-Shock Proteins ,Adenosine Triphosphatases ,biology ,Kinase ,Chemistry ,JNK Mitogen-Activated Protein Kinases ,Temperature ,Cell Biology ,Fibroblasts ,JNK stress kinase ,Molecular biology ,Recombinant Proteins ,Rats ,Cell biology ,Cell culture ,biology.protein ,Mitogen-Activated Protein Kinases ,Signal transduction ,Signal Transduction - Abstract
A major inducible heat shock protein, Hsp72, has previously been found to stimulate dephosphorylation (inactivation) of stress kinase JNK in heat-shocked cells and protect them from apoptosis. Using Rat-1 fibroblasts with constitutive expression of a human Hsp72 or its deletion mutant lacking an ATPase domain (C-terminal fragment (CTF)), we tested whether ATPase activity of Hsp72 is necessary for these effects. We found that expression of CTF markedly increased, similarly to the intact protein, JNK dephosphorylation in heat-shocked cells. As a result, JNK inactivation following heat shock occurred much faster in cells expressing either full-length or mutant Hsp72 than in parental cells and this was accompanied by suppression of heat-induced apoptosis. Thus, protein refolding activity of Hsp72 appears to be dispensable for its effect on JNK inactivation and apoptosis.
- Published
- 1999
29. Matrix remodeling as stem cell recruitment event: a novel in vitro model for homing of human bone marrow stromal cells to the site of injury shows crucial role of extracellular collagen matrix
- Author
-
Bjorn R. Olsen, Joshua R. Mauney, and Vladimir Volloch
- Subjects
Collagen Type IV ,Pathology ,medicine.medical_specialty ,Proteases ,Stromal cell ,Serine Proteinase Inhibitors ,Bone Marrow Cells ,Biology ,Matrix metalloproteinase ,Cysteine Proteinase Inhibitors ,Antibodies ,Collagen Type I ,Article ,chemistry.chemical_compound ,Cell Movement ,medicine ,Humans ,Immunoprecipitation ,Regeneration ,Molecular Biology ,Cells, Cultured ,Mesenchymal stem cell ,Leupeptin ,Serine Endopeptidases ,Mesenchymal Stem Cells ,High-Temperature Requirement A Serine Peptidase 1 ,Fibroblasts ,Cell biology ,Extracellular Matrix ,Adult Stem Cells ,medicine.anatomical_structure ,chemistry ,Culture Media, Conditioned ,Wounds and Injuries ,Bone marrow ,Collagen ,Stem cell ,Stromal Cells ,Heat-Shock Response ,Homing (hematopoietic) - Abstract
The goal of the present study was to devise an in vitro model suitable for investigations of the homing of mesenchymal stem cells to sites of injury. Such a model was designed on the basis of a "transwell" assay, with an insert seeded with human bone marrow stromal cells and a well with a desired cell type. To mimic physiological environment and to simulate "injury", cells in a well were maintained not only on tissue culture plastic but also on collagens I and IV, major matrix components in musculoskeletal and adipose tissues respectively, and subjected to a severe thermal stress. The results obtained showed a massive translocation of bone marrow stromal cells through the inserts' membrane toward the "injury" site. Unexpectedly, it emerged that collagen matrix is essential in producing such a migration. The results obtained suggest that upon injury cells secrete a substance which interacts with collagen matrix to produce a homing agent. The substance in question appears to be a protease and its interaction with the collagen matrix appears to be a digestion of the latter into fragments shown to be chemotactic. Both AEBSF, an inhibitor of serine proteases, and leupeptin, an inhibitor of cysteine proteases as well as of trypsin-like serine proteases, but not the broad spectrum MMP inhibitor marimastat, significantly inhibit the observed homing effect and this inhibition is not due to cytotoxicity. Moreover, immunoprecipitation of HTRA1, a trypsin-like serine protease known to be secreted by cells differentiating into all three major mesenchymal lineages and by stressed cells in general and shown to degrade a number of matrix proteins including collagen, significantly diminished the homing effect. The data suggest that this protease is a major contributor to the observed chemotaxis of bone marrow stromal cells. The present study indicates that collagen fragments can mediate the migration of bone marrow stromal cells. The results also suggest that, at least in musculoskeletal and in adipose tissues, matrix remodeling occurrences, usually closely associated with tissue remodeling, should also be regarded as potential stem cells recruitment events.
- Published
- 2010
30. Adult human bone marrow stromal cells regulate expression of their MMPs and TIMPs in differentiation type-specific manner
- Author
-
Joshua R. Mauney and Vladimir Volloch
- Subjects
Adult ,Stromal cell ,Human bone ,Stimulation ,Bone Marrow Cells ,Matrix metalloproteinase ,Article ,Osteogenesis ,medicine ,Animals ,Humans ,Molecular Biology ,Cells, Cultured ,Adipogenesis ,Chemistry ,Cell Differentiation ,Tissue Inhibitor of Metalloproteinases ,Phenotype ,Matrix Metalloproteinases ,Cell biology ,Extracellular Matrix ,Isoenzymes ,medicine.anatomical_structure ,Immunology ,Bone marrow ,Collagen ,Stromal Cells ,Adult stem cell - Abstract
Previously, we described a profound impact of structural conformation of collagen matrix on osteogenic and adipogenic differentiation of bone marrow stromal cells. Thus, a marginal p38-independent adipogenesis on native collagen I matrix contrasts with an efficient p38-dependent differentiation on denatured collagen I. An efficient Hsp90-dependent osteogenesis occurs on native collagen I matrix but not on its denatured counterpart where it is insignificant and proceeds in an Hsp90-independent manner. Whereas only marginal osteogenesis and no detectable adipogenesis of bone marrow stromal cells occur on native collagen IV, the same matrix supports a highly efficient adipogenesis in denatured structural state. The present study addresses the opposite direction in the flow of cell-matrix interaction, namely the cells' influence on structural state of collagen matrix, and tests the possibility that differentiating bone marrow stromal cells may adjust the expression phenotype of MMP and TIMP in such a way that, if translated into matrix modification, would facilitate the maintenance of collagen matrix in or its modification into structural state optimal for the ongoing differentiation process. The results obtained indicate that this is indeed the case. In bone marrow stromal cells stimulated to undergo adipogenesis the expression of MMP increases and that of TIMP decreases. In cells induced to undergo osteogenesis the opposite is true: MMP/TIMP expression is adjusted in a manner that, if translated into matrix modification, could promote the native structural conformation optimal for this type of differentiation. The results obtained also indicate that the observed adjustment in MMP/TIMP expression phenotype might be an early differentiation event and that differentiation stimulation alone might be sufficient to trigger it even on matrices not favorable to a given type of differentiation. The findings of the present study raise significant questions and indicate directions for further experimentation.
- Published
- 2009
31. Identification of negative-strand complements to cytochrome oxidase subunit III RNA in Trypanosoma brucei
- Author
-
Bruce Schweitzer, Xun Zhang, Sophia Rits, and Vladimir Volloch
- Subjects
Transcription, Genetic ,Macromolecular Substances ,Molecular Sequence Data ,Trypanosoma brucei brucei ,RNA-dependent RNA polymerase ,Biology ,Polymerase Chain Reaction ,Electron Transport Complex IV ,chemistry.chemical_compound ,RNA polymerase ,RNA polymerase I ,Animals ,RNA, Messenger ,Cloning, Molecular ,Multidisciplinary ,Base Sequence ,Intron ,Nucleic Acid Hybridization ,RNA ,DNA ,Blotting, Northern ,Non-coding RNA ,Molecular biology ,Oligodeoxyribonucleotides ,Biochemistry ,chemistry ,RNA editing ,Dactinomycin ,Nucleic Acid Conformation ,Small nuclear RNA ,Research Article - Abstract
A substantial amount of cytochrome oxidase subunit III (COIII) mRNA continues to be synthesized de novo in Trypanosoma brucei in the presence of actinomycin D, presumably by a DNA-independent transcription process. We describe the identification of negative-strand COIII RNA molecules, characterization of their termini, and the detection of RNA-dependent RNA polymerase activity. Three lines of evidence for the existence of negative-strand COIII RNA are presented: (i) hybridization with oligonucleotide probes with the same polarity as mRNA after preliminary enrichment for putative negative-strand RNA by affinity purification; (ii) cloning and sequencing of negative-strand complements for the unedited, edited, and partially edited COIII RNA; and (iii) exact correspondence of the terminal sequences of the putative negative-strand RNA molecules to the ends of COIII RNA. The presence of negative-strand complements of COIII RNA is consistent with the notion that a significant amount of mRNA in T. brucei is synthesized by an RNA-dependent RNA polymerase with negative-strand RNA as an intermediate template.
- Published
- 1991
32. Mechanism for β-amyloid Overproduction in Alzheimer Disease: Possible Antisense RNA-mediated Generation of a 5'-truncated βAPP MRNA Encoding 12-kDa C-terminal Fragment of βAPP, the Immediate Precursor Aβ
- Author
-
Vladimir Volloch
- Subjects
Messenger RNA ,Fragment (logic) ,Mechanism (biology) ,Chemistry ,β amyloid ,medicine ,Alzheimer's disease ,medicine.disease ,Overproduction ,Antisense RNA ,Cell biology - Published
- 2003
33. Reduced thermotolerance in aged cells results from a loss of an hsp72-mediated control of JNK signaling pathway
- Author
-
Michael Y. Sherman, Bernard Massie, Dick D. Mosser, and Vladimir Volloch
- Subjects
Cell Biology ,Biology ,Biochemistry ,Hsp70 ,Cell biology ,chemistry.chemical_compound ,chemistry ,Apoptosis ,Cell culture ,Heat shock protein ,MG132 ,Proteasome inhibitor ,medicine ,pharmaceutical ,Signal transduction ,Cell aging ,medicine.drug - Abstract
Aged organisms exhibit a greatly decreased ability to induce the major heat shock protein, Hsp72, in response to stresses, a phenomenon that can also be observed in cell cultures (Heydari AR, Takahashi R, Gutsmann A, You S and Richardson A (1994) Hsp70 and aging. Experientia 50: 1092-1098). Hsp72 was shown to protect cells from a variety of stresses. The protective function of Hsp72 has been commonly ascribed to its chaperoning ability. However, recently we showed that Hsp72 protects cells from heat shock by suppression of a stress-kinase JNK, an essential component of the heat-induced apoptotic pathway (Gabai VL, Meriin AB, Mosser DD, Caron AW, Rits S, Shifrin VI and Sherman MY (1997) Hsp70 prevents activation of stress kinases. A novel pathway of cellular thermotolerance. J Biol Chem 272: 18033-18037). Here we demonstrate that because of the diminished inducibility of Hsp72 in aged cells, Hsp72-mediated control of JNK signaling pathway is compromised. This results in increased rate of apoptotic cell death following heat shock. We show that forced expression of Hsp72 in aged cells from an adenovirus-based vector completely suppresses activation of JNK by heat shock and consequently protects from heat-induced apoptosis. We also demonstrate for the first time that it is possible to restore endogenous expression of Hsp72 in aged cells. This can be achieved by treatment with the proteasome inhibitor MG132. Induction of Hsp72 in aged cells under these conditions leads to suppression of JNK activation by a heat shock and restoration of thermotolerance manifested in a lower rate of apoptosis.
- Published
- 1998
34. Mechanism for β-amyloid Overproduction in Alzheimer Disease
- Author
-
Vladimir Volloch
- Subjects
Messenger RNA ,medicine.diagnostic_test ,β amyloid ,Chemistry ,Proteolysis ,Normal tissue ,medicine ,Globin mrna ,Alzheimer's disease ,Overproduction ,medicine.disease ,Cleavage (embryo) ,Cell biology - Abstract
The overproduction of p-amyloid (AP) is associated with and appears to be a primary cause of Alzheimer’s disease (AD). Aβ can be generated by proteolysis of p-amyloid precursor protein (βAPP) in both AD-affected and normal cells. There is no evidence, however, that proteolytic cleavage leading to the production of AP in sporadic AD-affected and in normal tissues differs qualitatively or quantitatively to account for the overproduction of Aβ in AD. Therefore, an additional pathway for the enhanced production of Aβ may be involved in sporadic AD. A mechanism is proposed that may be responsible for the overproduction of Aβ in sporadic AD, which constitutes the majority of all AD cases. The proposed mechanism, which may be activated or enhanced in sporadic AD-affected tissues, is based on a model for cellular mRNA replication developed in the author’s laboratory and proposes the antisense RNA-mediated generation of a 5’-truncated βAPP mRNA encoding 12-kDa C-terminal fragment of βAPP, the immediate precursor of Aβ, followed by initiation of translation at met596 contiguously preceding Ap. The proposed model makes several verifiable predictions and suggests new directions of experimentation that may lead to a better understanding of the mechanisms involved in AD. It also sheds a new light on some previously unexplained results in the field of AD.
- Published
- 1997
35. Monitoring Mesenchymal Stromal Cell Developmental Stage to Apply On-Time Mechanical Stimulation for Ligament Tissue Engineering
- Author
-
Laura R. Geuss, Jodie E. Moreau, Yongzhong Wang, Gregory H. Altman, Vladimir Volloch, Diah S. Bramono, Adam L. Collette, Rebecca L. Horan, and Jingsong Chen
- Subjects
Integrins ,Stromal cell ,Time Factors ,Transcription, Genetic ,Surface Properties ,Cellular differentiation ,Integrin ,Gene Expression ,Collagen Type I ,Extracellular matrix ,Bioreactors ,Tissue engineering ,Animals ,HSP70 Heat-Shock Proteins ,Cells, Cultured ,Ligaments ,biology ,Tissue Engineering ,Cell growth ,Chemistry ,Mesenchymal stem cell ,General Engineering ,Cell Differentiation ,Mesenchymal Stem Cells ,Bombyx ,Cell biology ,Biomechanical Phenomena ,Extracellular Matrix ,Fibronectins ,Fibronectin ,Collagen Type III ,Immunology ,biology.protein ,Stromal Cells ,Fibroins - Abstract
To evaluate the appropriate time frame for applying mechanical stimuli to induce mesenchymal stromal cell (MSC) differentiation for ligament tissue engineering, developmental cell phenotypes were monitored during a period of in vitro culture. MSCs were seeded onto surface-modified silk fibroin fiber matrices and cultured in Petri dishes for 15 days. Cell metabolic activity, morphology, and gene expression of extracellular matrix (ECM) proteins (collagen type I and III and fibronectin), ECM receptors (integrins alpha-2, alpha-5, and beta-1), and heat-shock protein 70 (HSP-70) were monitored during the culture of MSC. MSCs showed fluctuations in cell metabolic activity, ECM, integrin, and HSP-70 transcription potentially correlating to innate developmental processes. Cellular response to mechanical stimulation was dependent on the stage of cell development. At day 9, when levels of cell metabolic activity, ECM, integrin, and HSP-70 transcription peaked, mechanical stimulation increased MSC metabolic activity, alignment, and collagen production. Mechanical stimulation applied at day 1 and 3 showed detrimental effects on MSCs seeded on silk matrices. The results presented in this study identify a unique correlation between innate MSC development processes on a surface-modified silk matrix and dynamic environmental signaling.
- Published
- 2006
36. HSP72 can protect cells from heat-induced apoptosis by accelerating the inactivation of stress kinase JNK
- Author
-
Sophia Rits, Vladimir Volloch, Vladimir L. Gabai, Michael Y. Sherman, and Thomas Force
- Subjects
Phosphatase ,Apoptosis ,HSP72 Heat-Shock Proteins ,Biochemistry ,Cell Line ,Dephosphorylation ,Heat shock protein ,Animals ,Phosphorylation ,Heat shock ,Heat-Shock Proteins ,biology ,Kinase ,Chemistry ,JNK Mitogen-Activated Protein Kinases ,Regular Article ,Cell Biology ,Fibroblasts ,Rats ,Cell biology ,Enzyme Activation ,Mitogen-activated protein kinase ,biology.protein ,Mitogen-Activated Protein Kinases ,Heat-Shock Response - Abstract
The major heat shock protein Hsp72 prevents heat-induced apoptosis. We have previously demonstrated that transiently expressed Hsp72 exerts its anti-apoptotic effect by suppressing the activity of stress-kinase JNK, an early component of the apoptotic pathway initiated by heat shock. On the other hand, constitutive expression of Hsp72 does not lead to suppression of heat-induced JNK activation, yet still efficiently prevents apoptosis. To address this apparent contradiction, we studied the effects of constitutively expressed Hsp72 on activation of JNK and apoptosis in Rat-1 fibroblasts. We found that the level of heat-induced apoptosis directly correlated with the duration rather than the magnitude of JNK activity following heat shock. Constitutively expressed Hsp72 strongly reduced the duration of JNK while it did not suppress initial JNK activation. These effects were due to Hsp72-mediated acceleration of JNK dephosphorylation. Addition of vanadate to inhibit JNK phosphatase activity completely prevented the anti-apoptotic action of Hsp72. Therefore, suppression of heat-induced apoptosis by Hsp72 could be fully accounted for by its effects on JNK activity.
- Published
- 2000
37. Pyrophosphate-condensing activity linked to nucleic acid synthesis
- Author
-
S. Rits, L. Tumerman, and V.Z. Volloch
- Subjects
Oligonucleotide ,RNA-Directed DNA Polymerase ,DNA-Directed DNA Polymerase ,DNA-Directed RNA Polymerases ,Biology ,DNA Polymerase I ,Pyrophosphate ,Diphosphates ,DNA/RNA non-specific endonuclease ,Nucleic acid thermodynamics ,chemistry.chemical_compound ,chemistry ,Biochemistry ,Escherichia coli ,Genetics ,Nucleic acid ,biology.protein ,Chromatography, Thin Layer ,Pyrophosphatases ,Nucleic acid structure ,Nucleic acid analogue ,Polymerase - Abstract
In some preparations of DNA dependent RNA polymerase a new enzymatic activity has been found which catalyzes the condensation of two pyrophosphate molecules, liberated in the process of RNA synthesis, to one molecule of orthophosphate and one molecule of Mg (or Mn) - chelate complex with trimetaphosphate. This activity can also cooperate with DNA-polymerase, on condition that both enzymes originate from the same cells. These results point to two general conclusions. First, energy is conserved in the overall process of nucleic acid synthesis and turnover, so that the process does not require an energy influx from the cell's general resources. Second, the synthesis of nucleic acids is catalyzed by a complex enzyme system which contains at least two separate enzymes, one responsible for nucleic acid polymerization and the other for energy conservation via pyrophosphate condensation.
- Published
- 1979
38. Hemin does not cause commitment of murine erythroleukemia (MEL) cells to terminal differentiation
- Author
-
Vladimir Volloch, Susan C. Weil, Asterios S. Tsiftsoglou, James F. Gusella, Jeffrey R. Neumann, Cheryl Keys, and David E. Housman
- Subjects
Cellular differentiation ,Immunology ,Cell Biology ,Hematology ,Cytidine deaminase ,Biology ,medicine.disease ,Biochemistry ,Molecular biology ,Leukemia ,chemistry.chemical_compound ,chemistry ,Cell culture ,hemic and lymphatic diseases ,polycyclic compounds ,medicine ,Globin ,Nuclear protein ,Reprogramming ,Hemin - Abstract
The effect of hemin on the differentiation program of murine erythroleukemia (MEL) cells has been investigated. While hemin treatment does induce increased levels of globin mRNA and hemoglobin, it fails to lead to other biochemical changes associated with MEL cell differentiation induced by DMSO and thioguanine. These include increased levels of the nuclear protein IP25 and of the enzyme cytidine deaminase. Clonal analysis of hemin-treated cells revealed that unlike other inducers, hemin does not cause a reprogramming of MEL cells to a specific limitation of proliferative capacity. These observations suggest that hemin differs from DMSO and thioguanine in that it exerts specific effects on globin expression in MEL cells without triggering commitment to the terminal differentiation program.
- Published
- 1980
39. Cytoplasmic synthesis of globin RNA in differentiated murine erythroleukemia cells: possible involvement of RNA-dependent RNA polymerase
- Author
-
Vladimir Volloch
- Subjects
Cytoplasm ,RNA-dependent RNA polymerase ,Biology ,Cell Line ,Mice ,chemistry.chemical_compound ,hemic and lymphatic diseases ,RNA polymerase ,Acetamides ,Animals ,RNA, Messenger ,Globin ,Multidisciplinary ,RNA ,Cell Differentiation ,RNA Nucleotidyltransferases ,Nuclease protection assay ,Templates, Genetic ,RNA-Dependent RNA Polymerase ,Non-coding RNA ,Molecular biology ,Globins ,Gene Expression Regulation ,chemistry ,RNA editing ,Dactinomycin ,Leukemia, Erythroblastic, Acute ,Small nuclear RNA ,Research Article - Abstract
Three lines of evidence indicate that RNA-dependent RNA synthesis occurs in mouse erythroleukemia cells. The first involves labeling studies with [3H]uridine and shows a greater initial labeling rate of globin RNA in the cytoplasm than in the nucleus. Labeled globin RNA found in the cytoplasm after a very short pulse with tritiated uridine is of the "mature" 9S size while labeled globin RNA in the nuclei is exclusively in the form of 15S precursor molecules, suggesting that cytoplasmic globin RNA is not of nuclear origin. A high concentration of actinomycin D has no effect on the initial rate of labeling of cytoplasmic globin RNA, supporting this conclusion. Other experiments showed that the labeling of cytoplasmic globin RNA does not involve end addition to preexisting globin RNA. The second line of evidence is the identification of globin RNA minus strand in the cytoplasm of differentiated murine erythroleukemia cells by hybridization with single-stranded DNA probes containing the strand of the same sense as globin mRNA. This material has the same electrophoretic mobility as globin RNA and hybridizes with probes containing only the 5' part or only the 3' part of the gene suggesting that it is a full size copy of globin RNA. Finally, in murine erythroleukemia cells an RNA-dependent RNA polymerase activity is detected by using poly(A) . oligo(U) as a template-primer combination. This activity increases significantly after induction, suggesting that it is differentiation specific.
- Published
- 1986
40. Synthesis of globin RNA in enucleated differentiating murine erythroleukemia cells
- Author
-
Vladimir Volloch, Sophia Rits, and Bruce Schweitzer
- Subjects
Cytoplasm ,Cell Membrane Permeability ,Transcription, Genetic ,Cations, Divalent ,RNA-dependent RNA polymerase ,Biology ,Cell Line ,chemistry.chemical_compound ,Mice ,Transcription (biology) ,Aurintricarboxylic acid ,medicine ,Animals ,Erythropoiesis ,Globin ,RNA, Messenger ,Cytochalasin B ,Messenger RNA ,Dactinomycin ,RNA ,Cell Differentiation ,RNA Nucleotidyltransferases ,Cell Biology ,Articles ,RNA-Dependent RNA Polymerase ,Molecular biology ,Friend murine leukemia virus ,Globins ,chemistry ,Depression, Chemical ,Leukemia, Erythroblastic, Acute ,medicine.drug - Abstract
In an earlier report (Volloch, V., 1986, Proc. Natl. Acad. Sci. USA., 83:1208-1212) we had presented evidence for the occurrence of the cytoplasmic synthesis of globin mRNA and of RNA complementary to globin mRNA which differed from DNA-dependent transcription by its insensitivity to actinomycin D. In this paper, we describe the use of enucleated differentiating mouse erythroleukemia cells to demonstrate directly the occurrence of cytoplasmic synthesis of both positive- and negative-strand globin RNA. For this purpose, we developed an enucleation procedure which yielded pure cytoplasts from differentiated mouse erythroleukemia cells in the absence of cytochalasin B and selectively permeabilized the cytoplasts to small molecules by treatment with dextran sulfate and saponin. The permeabilized cytoplasts incorporated [3H]dUTP into positive- and negative-strand globin RNA and experiments with mercurated nucleotide substrate suggested that this process involved de novo RNA synthesis rather than limited terminal nucleotide addition. Globin RNA synthesis required Mg++, was inhibited by Mn++, and was unaffected by the addition of Zn++. Studies of its response to inhibitors of DNA-dependent RNA synthesis showed that it differed from that process in its insensitivity to actinomycin D and alpha-amanitin, but that like many other macromolecular biosynthetic reactions it was inhibited by rifamycin AF/ABDP and aurintricarboxylic acid. These observations provide additional evidence for the occurrence of cytoplasmic RNA-dependent RNA synthesis in differentiated cells and show permeabilized enucleated cells to be a useful experimental system for further studies of the characteristics of that process.
- Published
- 1987
41. Messenger RNA population analysis during erythroid differentiation: A kinetical approach
- Author
-
Zeev Volloch, Haim Aviv, and Roberto N. Bastos
- Subjects
Erythrocytes ,Reticulocytes ,Population ,Cell ,Spleen ,Biology ,Mice ,chemistry.chemical_compound ,Structural Biology ,medicine ,Animals ,RNA, Messenger ,Globin ,education ,Molecular Biology ,Cells, Cultured ,Messenger RNA ,education.field_of_study ,Nucleic Acid Hybridization ,RNA ,Anemia ,Cell Differentiation ,Metabolism ,Molecular biology ,Uridine ,Globins ,Kinetics ,medicine.anatomical_structure ,Biochemistry ,chemistry ,Poly A - Abstract
The poly(A) + RNA of cultured spleen cells (derived from anaemic mice), pulse-labelled with [ 3 H]uridine, decays with the characteristics of a population composed of two major kinetical groups, one with a half-life of three hours, the other with one of 35 hours. The poly(A) + RNA also contains globin RNA, with a half-life of 17 hours. After a 30-hour labelling period, a steady-state level of globin RNA is reached within the poly(A) + population corresponding to about 10%. On the other hand, the poly(A) + RNA population of circulating mature reticulocytes of anaemic mice labelled in vivo for 24 hours contains more than 98% globin RNA. The half-life of globin RNA in these cells, however, is not changed from the value observed in the cultured spleen cells (17 h). Possible mechanisms to account for the population-shift between these two cell species are discussed.
- Published
- 1977
42. Dissociation of hemoglobin accumulation and commitment during murine erythroleukemia cell differentiation by treatment with imidazole
- Author
-
James F. Gusella, Susan C. Weil, Asterios S. Tsiftsoglou, Vladimir Volloch, David E. Housman, and Jeffrey R. Neumann
- Subjects
Physiology ,Chromosomal Proteins, Non-Histone ,Cellular differentiation ,Clinical Biochemistry ,Heme ,Biology ,Cell Line ,chemistry.chemical_compound ,Hemoglobins ,Mice ,hemic and lymphatic diseases ,Cytidine Deaminase ,Animals ,Dimethyl Sulfoxide ,Erythropoiesis ,Nuclear protein ,neoplasms ,chemistry.chemical_classification ,Imidazoles ,Cell Differentiation ,Cell Biology ,Cytidine deaminase ,Neoplasms, Experimental ,Molecular biology ,In vitro ,Globins ,Enzyme ,chemistry ,Gene Expression Regulation ,Specific activity ,Hemoglobin ,Leukemia, Erythroblastic, Acute ,Cell Division - Abstract
The effect of imidazole on DMSO-induced murine erythroleukemia (MEL) cell differentiation has been examined. While imidazole does inhibit heme, globin mRNA, and hemoglobin accumulation in DMSO-induced MEL cells, it does not affect the commitment of MEL cells to the specific limitation of proliferative capacity associated with the in vitro differentiation program. Furthermore, imidazole treatment does not affect DMSO-induced changes in cell volume, in the relative proportion of nuclear protein IP25, and in the specific activity of the enzyme cytidine deaminase. A clonal analysis in the presence of imidazole indicated that the drug prevents heme accumulation even in MEL cells already committed to terminal differentiation. These observations suggest that imidazole effectively dissociates two aspects of the erythroid differentiation program of MEL cells: globin gene expression and commitment to loss of proliferative capacity.
- Published
- 1982
43. Control of proliferation and differentiation in cells transformed by Friend virus
- Author
-
David E. Housman, D. Parker, James F. Gusella, Vladimir Volloch, Joseph Kernen, Robert Levenson, Tsiftsoglou As, P. Besmer, Alberto A. Mitrani, V. Weeks, and O. Witte
- Subjects
biology ,Chemistry ,Friend virus ,Cell Differentiation ,Heme ,biology.organism_classification ,Cell Transformation, Viral ,Biochemistry ,Virology ,Cell Line ,Friend murine leukemia virus ,Globins ,Butyrates ,Viral Proteins ,Hypoxanthines ,Genetics ,Dimethyl Sulfoxide ,Erythropoiesis ,RNA, Messenger ,Molecular Biology ,Cell Division ,Glycoproteins - Published
- 1980
44. A possible mechanism responsible for the correction of transcription errors
- Author
-
V.Z. Volloch, S. Rits, and L. Tumerman
- Subjects
biology ,Transcription, Genetic ,viruses ,RNA-dependent RNA polymerase ,RNA ,RNA polymerase II ,DNA-Directed RNA Polymerases ,Ribonucleotides ,Ribonucleoside ,Molecular biology ,Phosphoric Monoester Hydrolases ,Substrate Specificity ,chemistry.chemical_compound ,Kinetics ,chemistry ,Biochemistry ,Transcription (biology) ,RNA polymerase ,Genetics ,biology.protein ,Nucleoside triphosphate ,Escherichia coli ,Polymerase - Abstract
Nucleoside triphosphate phosphohydrolase (NTPase) activity was found in a preparation of E. Coli RNA polymerase. This enzymatic activity is capable of hydrolysing all four ribonucleoside triphosphates to the nucleoside diphosphates. However, during in vitro RNA synthesis directed by poly(dC) or poly(dT), only the non-complementary nucleoside triphosphate of the same heterocyclic class was hydrolysed. No incorporation of the non-complementary precursor into RNA could be detected in these experiments. When another RNA polymerase preparation, devoid of NTPase activity, was employed, there was no hydrolysis of any nucleoside triphosphate and significant incorporation of non-complemtary precursor into RNA was observed. These observations lead us to the conclusion that NTPase, acting in conjunction with RNA polymerase, has the function of correcting errors in transcription.
- Published
- 1979
45. Imaging of intrinsic optical stem cell changes in engineered tissues
- Author
-
Irene Georgakoudi, Joshua R. Mauney, William L. Rice, David L. Kaplan, Vladimir Volloch, and Shamaraz Firdous
- Subjects
Multiphoton fluorescence microscope ,Optical contrast ,Chemistry ,Confocal microscopy ,law ,Biophysics ,Nanotechnology ,Matrix (biology) ,Stem cell ,law.invention - Abstract
Interactions between stem cells and their surrounding matrix are essential in the development of engineered tissues. Spectroscopic imaging of endogenous sources of optical contrast provides a non-invasive means for monitoring such interactions.
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