Search

Focused ultrasound stimulation (FUS) activates mechanosensitive ion channels and is emerging as a method of noninvasive neuromodulation. In preclinical studies, FUS of the spleen (sFUS) activates an anti-inflammatory neural pathway which suppresses acute and chronic inflammation. However, the relevance of sFUS for regulating inflammatory responses in humans is unknown. Here, we used a modified diagnostic ultrasound imaging system to target the spleen of healthy human subjects with 3 min of continuously swept or stationary focused pulsed ultrasound, delivered at three different energy levels within allowable safety exposure limits. Potential anti-inflammatory effects of sFUS were assessed by measuring sFUS-elicited changes in endotoxin-induced tumor necrosis factor (TNF) production in whole blood samples from insonified subjects. We found that stimulation with either continuously swept or focused pulsed ultrasound has an anti-inflammatory effect: sFUS lowers TNF production for >2 h, with TNF returning to baseline by 24 h following sFUS. This response is independent of anatomical target (i.e., spleen hilum or parenchyma) or ultrasound energy level. No clinical, biochemical, or hematological parameters are adversely impacted. This is the first demonstration that sFUS suppresses the normal inflammatory response in humans, with potential implications for noninvasive bioelectronic therapy of inflammatory disorders., (Copyright © 2023. Published by Elsevier Inc.)

Peptides, polymers of amino acids, comprise a vital and expanding therapeutic approach. Their rapid degradation by proteases, however, represents a major limitation to their therapeutic utility and chemical modifications to native peptides have been employed to mitigate this weakness. Herein, we describe functionalized thiocarbazate scaffolds as precursors of aza-amino acids, that, upon activation, can be integrated in a peptide sequence to generate azapeptides using conventional peptide synthetic methods. This methodology facilitates peptide editing-replacing targeted amino acid(s) with aza-amino acid(s) within a peptide-to form azapeptides with preferred therapeutic characteristics (extending half-life/bioavailability, while at the same time typically preserving structural features and biological activities). We demonstrate the convenience of this azapeptide synthesis platform in two well-studied peptides with short half-lives: FSSE/P5779, a tetrapeptide inhibitor of HMGB1/MD-2/TLR4 complex formation, and bradykinin, a nine-residue vasoactive peptide. This bench-stable thiocarbazate platform offers a robust and universal approach to optimize peptide-based therapeutics., (© 2022. The Author(s).)

Peripheral neurons that sense glucose relay signals of glucose availability to integrative clusters of neurons in the brain. However, the roles of such signalling pathways in the maintenance of glucose homoeostasis and their contribution to disease are unknown. Here we show that the selective activation of the nerve plexus of the hepatic portal system via peripheral focused ultrasound stimulation (pFUS) improves glucose homoeostasis in mice and rats with insulin-resistant diabetes and in swine subject to hyperinsulinemic-euglycaemic clamps. pFUS modulated the activity of sensory projections to the hypothalamus, altered the concentrations of metabolism-regulating neurotransmitters, and enhanced glucose tolerance and utilization in the three species, whereas physical transection or chemical blocking of the liver-brain nerve pathway abolished the effect of pFUS on glucose tolerance. Longitudinal multi-omic profiling of metabolic tissues from the treated animals confirmed pFUS-induced modifications of key metabolic functions in liver, pancreas, muscle, adipose, kidney and intestinal tissues. Non-invasive ultrasound activation of afferent autonomic nerves may represent a non-pharmacologic therapy for the restoration of glucose homoeostasis in type-2 diabetes and other metabolic diseases., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Obesity, a growing health concern, is associated with an increased risk of morbidity and mortality. Chronic low-grade inflammation is implicated in obesity-driven metabolic complications. Peripheral focused ultrasound stimulation (pFUS) is an emerging non-invasive technology that modulates inflammation. Here, we reasoned that focused ultrasound stimulation of the liver may alleviate obesity-related inflammation and other comorbidities. After 8 weeks on a high-fat high-carbohydrate "Western" diet, C57BL/6J mice were subjected to either sham stimulation or focused ultrasound stimulation at the porta hepatis. Daily liver-focused ultrasound stimulation for 8 weeks significantly decreased body weight, circulating lipids and mitigated dysregulation of adipokines. In addition, liver-focused ultrasound stimulation significantly reduced hepatic cytokine levels and leukocyte infiltration. Our findings demonstrate the efficacy of hepatic focused ultrasound for alleviating obesity and obesity-associated complications in mice. These findings suggest a previously unrecognized potential of hepatic focused ultrasound as a possible novel noninvasive approach in the context of obesity.

Tools for noninvasively modulating neural signaling in peripheral organs will advance the study of nerves and their effect on homeostasis and disease. Herein, we demonstrate a noninvasive method to modulate specific signaling pathways within organs using ultrasound (U/S). U/S is first applied to spleen to modulate the cholinergic anti-inflammatory pathway (CAP), and US stimulation is shown to reduce cytokine response to endotoxin to the same levels as implant-based vagus nerve stimulation (VNS). Next, hepatic U/S stimulation is shown to modulate pathways that regulate blood glucose and is as effective as VNS in suppressing the hyperglycemic effect of endotoxin exposure. This response to hepatic U/S is only found when targeting specific sub-organ locations known to contain glucose sensory neurons, and both molecular (i.e. neurotransmitter concentration and cFOS expression) and neuroimaging results indicate US induced signaling to metabolism-related hypothalamic sub-nuclei. These data demonstrate that U/S stimulation within organs provides a new method for site-selective neuromodulation to regulate specific physiological functions.

After publication of this article (He et al., 2018), the corresponding authors recognised an error in Scheme 1, in particular to section "A. HMGB1/TLR4/MD-2 complex formation". Above "Step 2: B box binding to MD-2", the text incorrectly read: "Low affinity / extremely slow off". In addition, some text was omitted below "TLR4/MD-2". The correct version of Scheme 1 is included in this Correction article. The original article (He et al., 2018) has been corrected.

Background: High Mobility Group Box 1 (HMGB1) was first identified as a nonhistone chromatin-binding protein that functions as a pro-inflammatory cytokine and a Damage-Associated Molecular Pattern molecule when released from necrotic cells or activated leukocytes. HMGB1 consists of two structurally similar HMG boxes that comprise the pro-inflammatory (B-box) and the anti-inflammatory (A-box) domains. Paradoxically, the A-box also contains the epitope for the well-characterized anti-HMGB1 monoclonal antibody "2G7", which also potently inhibits HMGB1-mediated inflammation in a wide variety of in vivo models. The molecular mechanisms through which the A-box domain inhibits the inflammatory activity of HMGB1 and 2G7 exerts anti-inflammatory activity after binding the A-box domain have been a mystery. Recently, we demonstrated that: 1) the TLR4/MD-2 receptor is required for HMGB1-mediated cytokine production and 2) the HMGB1-TLR4/MD-2 interaction is controlled by the redox state of HMGB1 isoforms., Methods: We investigated the interactions of HMGB1 isoforms (redox state) or HMGB1 fragments (A- and B-box) with TLR4/MD-2 complex using Surface Plasmon Resonance (SPR) studies., Results: Our results demonstrate that: 1) intact HMGB1 binds to TLR4 via the A-box domain with high affinity but an appreciable dissociation rate; 2) intact HMGB1 binds to MD-2 via the B-box domain with low affinity but a very slow dissociation rate; and 3) HMGB1 A-box domain alone binds to TLR4 more stably than the intact protein and thereby antagonizes HMGB1 by blocking HMGB1 from interacting with the TLR4/MD-2 complex., Conclusions: These findings not only suggest a model whereby HMGB1 interacts with TLR4/MD-2 in a two-stage process but also explain how the A-box domain and 2G7 inhibit HMGB1.

The immune and nervous systems are two major organ systems responsible for host defense and memory. Both systems achieve memory and learning that can be retained, retrieved, and utilized for decades. Here, we report the surprising discovery that peripheral sensory neurons of the dorsal root ganglia (DRGs) of immunized mice contain antigen-specific antibodies. Using a combination of rigorous molecular genetic analyses, transgenic mice, and adoptive transfer experiments, we demonstrate that DRGs do not synthesize these antigen-specific antibodies, but rather sequester primarily IgG 1 subtype antibodies. As revealed by RNA-seq and targeted quantitative PCR (qPCR), dorsal root ganglion (DRG) sensory neurons harvested from either naïve or immunized mice lack enzymes (i.e., RAG1, RAG2, AID, or UNG) required for generating antibody diversity and, therefore, cannot make antibodies. Additionally, transgenic mice that express a reporter fluorescent protein under the control of Igγ1 constant region fail to express Ighg1 transcripts in DRG sensory neurons. Furthermore, neural sequestration of antibodies occurs in mice rendered deficient in neuronal Rag2 , but antibody sequestration is not observed in DRG sensory neurons isolated from mice that lack mature B cells [e.g., Rag1 knock out (KO) or μMT mice]. Finally, adoptive transfer of Rag1 -deficient bone marrow (BM) into wild-type (WT) mice or WT BM into Rag1 KO mice revealed that antibody sequestration was observed in DRG sensory neurons of chimeric mice with WT BM but not with Rag1 -deficient BM. Together, these results indicate that DRG sensory neurons sequester and retain antigen-specific antibodies released by antibody-secreting plasma cells. Coupling this work with previous studies implicating DRG sensory neurons in regulating antigen trafficking during immunization raises the interesting possibility that the nervous system collaborates with the immune system to regulate antigen-mediated responses.

Autism spectrum disorder (ASD) occurs in 1 in 68 births, preferentially affecting males. It encompasses a group of neurodevelopmental abnormalities characterized by impaired social interaction and communication, stereotypic behaviors and motor dysfunction. Although recent advances implicate maternal brain-reactive antibodies in a causative role in ASD, a definitive assessment of their pathogenic potential requires cloning of such antibodies. Here, we describe the isolation and characterization of monoclonal brain-reactive antibodies from blood of women with brain-reactive serology and a child with ASD. We further demonstrate that male but not female mice exposed in utero to the C6 monoclonal antibody, binding to contactin-associated protein-like 2 (Caspr2), display abnormal cortical development, decreased dendritic complexity of excitatory neurons and reduced numbers of inhibitory neurons in the hippocampus, as well as impairments in sociability, flexible learning and repetitive behavior. Anti-Caspr2 antibodies are frequent in women with brain-reactive serology and a child with ASD. Together these studies provide a methodology for obtaining monclonal brain-reactive antibodies from blood B cells, demonstrate that ASD can result from in utero exposure to maternal brain-reactive antibodies of single specificity and point toward the exciting possibility of prognostic and protective strategies.

Pulmonary hypertension (PH) is a complex disease comprising a pathologic remodeling and thickening of the pulmonary vessels causing an after load on the right heart ventricle that can result in ventricular failure. Triggered by oxidative stress, episodes of hypoxia, and other undetermined causes, PH is associated with poor outcomes and a high rate of morbidity. In the neonate, this disease has a similar etiology but is further complicated by the transition to breathing after birth, which requires a reduction in vascular resistance. Persistent pulmonary hypertension of the newborn (PPHN) is one form of PH that is frequently unresponsive to current therapies including inhaled nitric oxide (due to lack of proper absorption and diffusion), and other therapeutics targeting signaling mediators in vascular endothelium and smooth muscle. The need for novel agents, which target distinct pathways in pulmonary hypertension, remains. Herein, we investigated the therapeutic effects of novel muscarinic receptor ligand C1213 in models of PH We demonstrated that via M3 muscarinic receptors, C1213 induced activating- eNOS phosphorylation (serine-1177), which is known to lead to nitric oxide (NO) production in endothelial cells. Using signaling pathway inhibitors, we discovered that AKT and calcium signaling contributed to eNOS phosphorylation induced by C1213. As expected for an eNOS-stimulating agent, in ex vivo and in vivo models, C1213 triggered pulmonary vasodilation and induced both pulmonary artery and systemic blood pressure reductions demonstrating its potential value in PH and PPHN In brief, this proof-of-concept study provides evidence that an M3 muscarinic receptor functionally selective ligand stimulates downstream pathways leading to antihypertensive effects using in vitro, ex vivo, and in vivo models of PH., (© 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

A healthy immune system results from a balance of stimulatory and inhibitory pathways that allow effective responses to acute insults, without descending into chronic inflammation. Failed homeostasis is characteristic of autoimmune diseases such as systemic lupus erythematosus. Although HMGB1 induces proinflammatory M1-like macrophage differentiation, we describe a mechanism by which C1q modulates this activity and collaborates with HMGB1 to induce the differentiation of monocytes to anti-inflammatory M2-like macrophages. These anti-inflammatory macrophages are unresponsive to dendritic cell induction factors, effectively removing them from participation in an adaptive immune response. This pathway is mediated through a complex with RAGE and LAIR-1 and depends on relative levels of C1q and HMGB1. Importantly, these data provide insight into a homeostatic mechanism in which C1q and HMGB1 can cooperate to terminate inflammation, and which may be impaired in C1q-deficient patients with autoimmune disease., (© 2016 by The American Society of Hematology.)

Systemic lupus erythematosus is an autoimmune disease that can affect numerous tissues and is characterized by the production of nuclear antigen-directed autoantibodies (e.g., anti-dsDNA). Using a combination of virtual and ELISA-based screens, we made the intriguing discovery that several HIV-protease inhibitors can function as decoy antigens to specifically inhibit the binding of anti-dsDNA antibodies to target antigens such as dsDNA and pentapeptide DWEYS. Computational modeling revealed that HIV-protease inhibitors comprised structural features present in DWEYS and predicted that analogues containing more flexible backbones would possess preferred binding characteristics. To address this, we reduced the internal amide backbone to improve flexibility, producing new small-molecule decoy antigens, which neutralize anti-dsDNA antibodies in vitro, in situ, and in vivo. Pharmacokinetic and SLE model studies demonstrated that peptidomimetic FISLE-412,1 a reduced HIV protease inhibitor analogue, was well-tolerated, altered serum reactivity to DWEYS, reduced glomeruli IgG deposition, preserved kidney histology, and delayed SLE onset in NZB/W F1 mice.

A growing body of evidence implicates macrophage migration inhibitory factor (MIF) in tumorigenesis and metastasis. In this study, we investigated whether MIF expression was associated with clinicopathologic features of colorectal carcinoma (CRC), especially in tumors with hepatic metastasis, and whether neutralization of endogenous MIF using anti-MIF therapeutics would inhibit tumor growth and/or decrease the frequency of colorectal hepatic metastases in a mouse colon carcinoma model. The concentration of serum MIF was positively correlated with an increased risk of hepatic metastasis in human patients with CRC (R = 1.25, 95% confidence internal = 1.02-1.52, P = 0.03). MIF was also dramatically upregulated in human colorectal tissue, with 20-40 times as many MIF-positive cells found in the mucosa of patients with CRC than in normal tissue (P < 0.001 ANOVA). Moreover, in those patients with metastatic colorectal cancer in the liver, MIF-positive cells were similarly increased in the diseased hepatic tissue. This increased MIF expression was restricted to diseased tissue and not found in areas of the liver with normal morphology. In subsequent in vitro experiments, we found that addition of recombinant MIF to colonic cell lines significantly increased their invasive properties and the expression of several genes (for example, matrix metalloproteinase 9 and vascular endothelial growth factor) known to be upregulated in cancerous tissue. Finally, we treated mice that had been given CT26 colon carcinoma cell transplants with anti-MIF therapeutics--either the MIF-specific inhibitor ISO-1 or neutralizing anti-MIF antibodies--and observed a significant reduction in tumor burden relative to vehicle-treated animals. Taken together, these data demonstrate that MIF expression was not only correlated with the presence of colorectal cancer but also may play a direct role in cancer development.

Ischemia and chronic hypoxia (CH) trigger a variety of adverse effects arising from metabolic stress that injures cells. In response to reduced O2, hypoxia-inducible factor 1alpha (HIF-1alpha) activates erythropoietin (Epo) as well as many other target genes that counteract the effects of O2 deficiency. Epo produced by the kidney stimulates erythrocyte production, leading to decreased HIF-1alpha production by improved tissue O2 delivery. However, Epo is produced by many other tissues, and it is currently unclear to what extent, if any, locally produced Epo modulates HIF-1alpha expression. Derivatives of Epo that possess tissue-protective activities but do not stimulate erythropoiesis [e.g., carbamylated Epo (CEpo)] are useful tools with which to determine whether exogenous Epo modulates HIF-1alpha in the absence of changes in hemoglobin concentration. We compared the effects of CH (6.5% O2 for 10 days) with or without CEpo administered by daily s.c. injection (10 microg/kg of body weight). CEpo administration did not alter the survival rate, weight loss, or increased hemoglobin concentration associated with CH. Therefore, CEpo does not directly suppress HIF-mediated erythropoiesis. CEpo does, however, prevent CH-induced neuronal increases of HIF-1alpha and Epo receptor-associated immunoreactivity (a measure of stress) while reducing the apoptotic index. In contrast, the myocardium did not exhibit increased HIF-1alpha expression during CH, although CEpo did reduce the apoptotic index. These observations therefore demonstrate that CEpo administration reduces the metabolic stress caused by severe CH, resulting in improved cellular survival independent of erythrocyte production.

Recombinant human erythropoietin (rhEPO) is receiving increasing attention as a potential therapy for prevention of injury and restoration of function in nonhematopoietic tissues. However, the minimum effective dose required to mimic and augment these normal paracrine functions of erythropoietin (EPO) in some organs (e.g., the brain) is higher than for treatment of anemia. Notably, a dose-dependent risk of adverse effects has been associated with rhEPO administration, especially in high-risk groups, including polycythemia-hyperviscosity syndrome, hypertension, and vascular thrombosis. Of note, several clinical trials employing relatively high dosages of rhEPO in oncology patients were recently halted after an increase in mortality and morbidity, primarily because of thrombotic events. We recently identified a heteromeric EPO receptor complex that mediates tissue protection and is distinct from the homodimeric receptor responsible for the support of erythropoiesis. Moreover, we developed receptor-selective ligands that provide tools to assess which receptor isoform mediates which biological consequence of rhEPO therapy. Here, we demonstrate that rhEPO administration in the rat increases systemic blood pressure, reduces regional renal blood flow, and increases platelet counts and procoagulant activities. In contrast, carbamylated rhEPO, a heteromeric receptor-specific ligand that is fully tissue protective, increases renal blood flow, promotes sodium excretion, reduces injury-induced elevation in procoagulant activity, and does not effect platelet production. These preclinical findings suggest that nonerythropoietic tissue-protective ligands, which appear to elicit fewer adverse effects, may be especially useful in clinical settings for tissue protection.

Gamma knife radiosurgery is an attractive noninvasive treatment of brain tumors and vascular malformations that minimizes collateral tissue damage. However, exposure of normal tissue to even low-dose radiation triggers a cascade of acute and chronic injury and potentially significant morbidity and mortality. Because many irradiated patients now survive for years, identifying methods to prevent radiotherapy-induced collateral tissue damage is a major focus of current research. Erythropoietin (EPO), a cytokine produced locally by many tissues in response to injury, antagonizes apoptosis, reduces inflammation, and promotes healing. Systemic administration of recombinant EPO, widely used for treatment of anemia, provides robust protection from numerous insults in a variety of tissues, including the brain. Although irradiation injury is likely sensitive to EPO, the hematopoietic activity of EPO is undesirable in this setting, increasing erythrocyte number and predisposing to thrombosis. To avoid these potential adverse effects, we developed carbamylated EPO (CEPO) which does not stimulate the bone marrow. In this study, we show that CEPO (50 microg kg(-1) intraperitoneally) improves functional outcome when administered to adult rats just before, and then once daily for 10 d after, a necrotizing dose of radiation (100 Gy) to the right striatum. Immediately following irradiation, use and reflex movements of the contralateral forelimb to vibrissae stimulation were abnormal but rapidly improved in animals receiving CEPO. Moreover, histological examination revealed that the extent of brain necrosis after 90 days was reduced by approximately 50%. These findings further extend the kinds of injury for which administration of a tissue-protective cytokine provides benefit.

Tuberous sclerosis complex is a tumor suppressor gene syndrome whose manifestations can include seizures, mental retardation, and benign tumors of the brain, skin, heart, and kidneys. Hamartin and tuberin, the products of the TSC1 and TSC2 genes, respectively, form a complex and inhibit signaling by the mammalian target of rapamycin. Here, we demonstrate that endogenous hamartin is threonine-phosphorylated during nocodazole-induced G2/M arrest and during the G2/M phase of a normal cell cycle. In vitro assays showed that cyclin-dependent kinase 1 phosphorylates hamartin at three sites, one of which (Thr417) is in the hamartin-tuberin interaction domain. Tuberin interacts with phosphohamartin, and tuberin expression attenuates the phosphorylation of exogenous hamartin. Hamartin with alanine mutations in the three cyclin-dependent kinase 1 phosphorylation sites increased the inhibition of p70S6 kinase by the hamartin-tuberin complex. These findings support a model in which phosphorylation of hamartin regulates the function of the hamartin-tuberin complex during the G2/M phase of the cell cycle.

Ischemic brain injury resulting from stroke arises from primary neuronal losses and by inflammatory responses. Previous studies suggest that erythropoietin (EPO) attenuates both processes. Although EPO is clearly antiapoptotic for neurons after experimental stroke, it is unknown whether EPO also directly modulates EPO receptor (EPO-R)-expressing glia, microglia, and other inflammatory cells. In these experiments, we show that recombinant human EPO (rhEPO; 5,000 U/kg body weight, i.p.) markedly reduces astrocyte activation and the recruitment of leukocytes and microglia into an infarction produced by middle cerebral artery occlusion in rats. In addition, ischemia-induced production of the proinflammatory cytokines tumor necrosis factor, interleukin 6, and monocyte chemoattractant protein 1 concentration is reduced by >50% after rhEPO administration. Similar results were also observed in mixed neuronal-glial cocultures exposed to the neuronal-selective toxin trimethyl tin. In contrast, rhEPO did not inhibit cytokine production by astrocyte cultures exposed to neuronal homogenates or modulate the response of human peripheral blood mononuclear cells, rat glial cells, or the brain to lipopolysaccharide. These findings suggest that rhEPO attenuates ischemia-induced inflammation by reducing neuronal death rather than by direct effects upon EPO-R-expressing inflammatory cells.

Xenopus laevis early development is characterized by rapid and synchronous cleavage cycles, which consist of alternating S and M phases. At midblastula transition, zygotic transcription begins and these cleavage cycles are replaced by longer cell division cycles that include gap phases and checkpoints. Herein, we demonstrate developmentally regulated Cdc6 isoform switching that contributes to this developmental cell cycle remodeling. Cdc6 is an essential component of the eukaryotic DNA replication machine that licenses each origin to one round of DNA replication each cell division cycle. The originally characterized Xenopus Cdc6 isoform (here termed Xcdc6A) and a novel isoform (Xcdc6B) have divergent N-terminal regulatory regions and different temporal patterns of expression. Although abundant in the early embryo, Xcdc6A becomes undetectable following midblastula transition. In contrast, while Xcdc6B is present in the early embryo, it is nonfunctional, as judged by lack of chromatin binding. In somatic tissue, however, Xcdc6B binds chromatin and its inhibition blocks entry into S phase. This is the first example of developmental regulation of Cdc6, raising intriguing implications for cell cycle remodeling during embryogenesis.

Budding yeast (Saccharomyces cerevisiae) origin recognition complex (ORC) requires ATP to bind specific DNA sequences, whereas fission yeast (Schizosaccharomyces pombe) ORC binds to specific, asymmetric A:T-rich sites within replication origins, independently of ATP, and frog (Xenopus laevis) ORC seems to bind DNA non-specifically. Here we show that despite these differences, ORCs are functionally conserved. Firstly, SpOrc1, SpOrc4 and SpOrc5, like those from other eukaryotes, bound ATP and exhibited ATPase activity, suggesting that ATP is required for pre-replication complex (pre-RC) assembly rather than origin specificity. Secondly, SpOrc4, which is solely responsible for binding SpORC to DNA, inhibited up to 70% of XlORC-dependent DNA replication in Xenopus egg extract by preventing XlORC from binding to chromatin and assembling pre-RCs. Chromatin-bound SpOrc4 was located at AT-rich sequences. XlORC in egg extract bound preferentially to asymmetric A:T-sequences in either bare DNA or in sperm chromatin, and it recruited XlCdc6 and XlMcm proteins to these sequences. These results reveal that XlORC initiates DNA replication preferentially at the same or similar sites to those targeted in S.pombe.

The process of cellular morphogenesis is highly conserved in eukaryotes and is dependent upon the function of proteins that are centrally involved in specification of the cell cycle. The human enhancer of invasion clone 10 (HEI10) protein was identified from a HeLa cell library based on its ability to promote yeast agar invasion and filamentation. Through two-hybrid screening, the mitotic cyclin B1 and an E2 ubiquitin-conjugating enzyme were isolated as HEI10-interacting proteins. Mutation of the HEI10 divergent RING finger motif (characteristic of E3 ubiquitin ligases) and Cdc2/cyclin binding and phosphorylation sites alter HEI10-dependent yeast phenotypes, including delay in G(2)/M transition. In vertebrates, the addition of HEI10 inhibits nuclear envelope breakdown and mitotic entry in Xenopus egg extracts. Mechanistically, HEI10 expression reduces cyclin B levels in cycling Xenopus eggs and reduces levels of the cyclin B ortholog Clb2p in yeast. HEI10 is itself a specific in vitro substrate of purified cyclin B/cdc2, with a TPVR motif as primary phosphorylation site. Finally, HEI10 is itself ubiquitinated in egg extracts and is also autoubiquitinated in vitro. These and other points lead to a model in which HEI10 defines a divergent class of E3 ubiquitin ligase, functioning in progression through G(2)/M.

Mammalian p21-activated kinase 1 (Pak1) is a highly conserved effector for the small GTPases Cdc42 and Rac1. In lower eukaryotes, Pak1 homologs are regulated during the cell cycle by phosphorylation. Here, we show that Pak1 is phosphorylated during mitosis in mammalian fibroblasts. This phosphorylation occurs at a single site, Thr 212, within a domain that is unique to Pak1. Cdc2 phosphorylates Pak1 at the identical site in vitro, and inhibition of Cdc2 abolishes Pak1 mitotic phosphorylation in vivo, indicating that Cdc2 is the kinase responsible for phosphorylating Pak1 in mitotic cells. Expression of a Pak1 mutant in which Thr 212 is replaced with a phosphomimic (aspartic acid) has marked effects on the rate and extent of postmitotic spreading of fibroblasts. The mitotic phosphorylation of Pak1 does not alter the basal or Rac-stimulated activity of this kinase, but it does affect the coimmunoprecipitation of at least three proteins with Pak1. These findings are the first to implicate a mammalian Pak in cell cycle regulation and suggest that Pakl, as a result of phosphorylation by Cdc2, alters its association with binding partners and/or substrates that are relevant to the morphologic changes associated with cell division.

Cdc6 performs an essential role in the initiation of eukaryotic DNA replication by recruiting the minichromosome maintenance (MCM) complex onto DNA. Using immunodepletion/add-back experiments in Xenopus egg extracts, we have determined that both Walker A (ATP binding) and Walker B (ATP hydrolysis) motifs of Xenopus Cdc6 (Xcdc6) are essential, but have distinct functional roles. Although Walker B mutant protein binds chromatin well, Walker A mutant protein binds chromatin poorly. Neither Walker A nor Walker B mutant protein, however, load appreciable MCM onto DNA. Herein, we provide evidence that Cdc6 functions as a multimer: 1) mutant and wild-type Xcdc6 form multimers; 2) either mutant protein is dominant negative when added before wild-type Xcdc6, but stimulates DNA replication when added simultaneously with wild-type Xcdc6; and 3) the two mutants restore DNA replication when added together, in the absence of wild-type Xcdc6. Our findings suggest that ATP may play a key regulatory role within this multimer: its binding to Cdc6 promotes chromatin association and its hydrolysis facilitates MCM loading. Moreover, ATP binding and hydrolysis may occur in trans between Cdc6 subunits within the complex.

Previous studies have suggested that cell cycle-dependent changes in the affinity of the origin recognition complex (ORC) for chromatin are involved in regulating initiation of DNA replication. To test this hypothesis, chromatin lacking functional ORCs was isolated from metaphase hamster cells and incubated in Xenopus egg extracts to initiate DNA replication. Intriguingly, Xenopus ORC rapidly bound to hamster somatic chromatin in a Cdc6-dependent manner and was then released, concomitant with initiation of DNA replication. Once pre-replication complexes (pre-RCs) were assembled either in vitro or in vivo, further binding of XlORC was inhibited. Neither binding nor release of XlORC was affected by inhibitors of either cyclin-dependent protein kinase activity or DNA synthesis. In contrast, inhibition of pre-RC assembly, either by addition of Xenopus geminin or by depletion of XlMcm proteins, augmented ORC binding by inhibiting ORC release. These results demonstrate a programmed release of XlORC from somatic cell chromatin as it enters S phase, consistent with the proposed role for ORC in preventing re-initiation of DNA replication during S phase.

We have cloned a Xenopus Cdc6 homolog (Xcdc6) and characterized its role in DNA replication with Xenopus egg extracts. Immunodepletion of Xcdc6 abolishes chromosomal replication but not elongation on single-stranded DNA templates. Xcdc6 binds to chromatin at the beginning of interphase but disappears from chromatin upon initiation of replication. Immunodepletion studies indicate that binding of Xcdc6 to chromatin requires Xorc2, a component of the origin recognition complex. Moreover, Xmcm3 cannot bind to chromatin lacking Xcdc6, suggesting that Xorc2, Xcdc6, and Xmcm3 associate with the DNA sequentially. In postreplicative nuclei, Xcdc6 is associated with the nuclear envelope. These studies indicate that Xcdc6, is essential for initiation of replication in vertebrates and that interaction with the nuclear envelope may regulate its function.

Cdc2 is the cyclin-dependent kinase that controls entry of cells into mitosis. Phosphorylation of Cdc2 on threonine-14 and tyrosine-15 inhibits the activity of the enzyme and prevents premature initiation of mitosis. Although Wee1 has been identified as the kinase that phosphorylates tyrosine-15 in various organisms, the threonine-14-specific kinase has not been isolated. A complementary DNA was cloned from Xenopus that encodes Myt1, a member of the Wee1 family that was discovered to phosphorylate Cdc2 efficiently on both threonine-14 and tyrosine-15. Myt1 is a membrane-associated protein that contains a putative transmembrane segment. Immunodepletion studies suggested that Myt1 is the predominant threonine-14-specific kinase in Xenopus egg extracts. Myt1 activity is highly regulated during the cell cycle, suggesting that this relative of Wee1 plays a role in mitotic control.

Using a polymerase chain reaction-based strategy, we have isolated a gene encoding a Wee1-like kinase from Xenopus eggs. The recombinant Xenopus Wee1 protein efficiently phosphorylates Cdc2 exclusively on Tyr-15 in a cyclin-dependent manner. The addition of exogenous Wee1 protein to Xenopus cell cycle extracts results in a dose-dependent delay of mitotic initiation that is accompanied by enhanced tyrosine phosphorylation of Cdc2. The activity of the Wee1 protein is highly regulated during the cell cycle: the interphase, underphosphorylated form of Wee1 (68 kDa) phosphorylates Cdc2 very efficiently, whereas the mitotic, hyperphosphorylated version (75 kDa) is weakly active as a Cdc2-specific tyrosine kinase. The down-modulation of Wee1 at mitosis is directly attributable to phosphorylation, since dephosphorylation with protein phosphatase 2A restores its kinase activity. During interphase, the activity of this Wee1 homolog does not vary in response to the presence of unreplicated DNA. The mitosis-specific phosphorylation of Wee1 is due to at least two distinct kinases: the Cdc2 protein and another activity (kinase X) that may correspond to an MPM-2 epitope kinase. These studies indicate that the down-regulation of Wee1-like kinase activity at mitosis is a multistep process that occurs after other biochemical reactions have signaled the successful completion of S phase.

A growing family of kinases and phosphatases controls the activity of the cyclin-dependent kinase cdc2. The past year has seen the identification of the cdk activating kinase as well as considerable elucidation of the cdc25/wee1 regulatory pathways. Both cdc25 and wee1 appear to be regulated by upstream kinase/phosphatase networks. In addition, it is likely that other regulatory mechanisms cooperate with the wee1/cdc25 phosphorylation systems to control the action of cdc2. Together, these elaborate checks and balances ensure that cdc2 triggers mitosis at the appropriate time.

The Wee1 protein kinase negatively regulates the entry into mitosis by catalyzing the inhibitory tyrosine phosphorylation of the Cdc2 protein. To examine the potential mechanisms for Wee1 regulation during the cell cycle, we have introduced a recombinant form of the fission yeast Wee1 protein kinase into Xenopus egg extracts. We find that the Wee1 protein undergoes dramatic changes in its phosphorylation state and kinase activity during the cell cycle. The Wee1 protein oscillates between an underphosphorylated 107 kDa form during interphase and a hyperphosphorylated 170 kDa version at mitosis. The mitosis-specific hyperphosphorylation of the Wee1 protein results in a substantial reduction in its activity as a Cdc2-specific tyrosine kinase. This phosphorylation occurs in the N-terminal region of the protein that lies outside the C-terminal catalytic domain, which was recently shown to be a substrate for the fission yeast Nim1 protein kinase. These experiments demonstrate the existence of a Wee1 regulatory system, consisting of both a Wee1-inhibitory kinase and a Wee1-stimulatory phosphatase, which controls the phosphorylation of the N-terminal region of the Wee1 protein. Moreover, these findings indicate that there are apparently two potential mechanisms for negative regulation of the Wee1 protein, one involving phosphorylation of its C-terminal domain by the Nim1 protein and the other involving phosphorylation of its N-terminal region by a different kinase.

The wee1 protein kinase suppresses the entry into mitosis by mediating the inhibitory tyrosine phosphorylation of p34cdc2. Genetic studies have suggested that the nim1 protein kinase (also known as cdr1) acts as a positive regulator of mitosis by down-regulating the wee1 pathway in yeast cells. We have overexpressed the nim1 protein in both bacteria and insect cells. The recombinant nim1 protein autophosphorylates on both tyrosine and serine residues and can phosphorylate the isolated wee1 protein directly in a cell-free system. The nim1-catalyzed phosphorylation of the wee1 protein occurs in its C-terminal region and leads to a substantial drop in its activity as a cdc2-specific tyrosine kinase. This nim1-dependent inhibition of the wee1 protein kinase can be reversed readily in vitro by treatment with a protein phosphatase. These experiments provide direct biochemical evidence that the wee1 protein is subject to negative regulation by phosphorylation and indicate that the nim1 protein acts as an inhibitory, wee1-specific kinase.

We have investigated the dynamics of intermediate filament assembly in vivo by following the fate of heterologous chicken vimentin subunits expressed under the control of an inducible promoter in transfected mouse fibroblasts. Using RNase protection, metabolic protein pulse-chase and immunofluorescence microscopy, we have examined the fate of newly assembled subunits under physiological conditions in situ. Following induction and subsequent removal of inducer, chicken vimentin mRNA had a half-life of approximately 6 h while both chicken and mouse vimentin protein polymer had long half-lives--roughly equivalent to the cell generation time. Moreover, following deinduction, chicken vimentin immunolocalization progressed from a continuous (8-10 h chase) to a discontinuous (> or = 20 h chase) pattern. The continuous chicken vimentin staining reflects the uniform incorporation of chicken vimentin throughout the endogenous mouse vimentin network while the discontinuous or punctate chicken vimentin staining represents short interspersed segments of assembled chicken vimentin superimposed on the endogenous polymer. This punctate staining pattern of chicken vimentin was present throughout the entire array of intermediate filaments, with no bias toward the perinuclear region. These results are consistent with a continuous growth model of intermediate filament assembly, wherein subunit addition occurs at discrete sites located throughout the cytoskeleton.

Background: Since 1985, donors of organs or tissues for transplantation in the United States have been screened for human immunodeficiency virus type 1 (HIV-1), and more than 60,000 organs and 1 million tissues have been transplanted. We describe a case of transmission of HIV-1 by transplantation of organs and tissues procured between the time the donor became infected and the appearance of antibodies. The donor was a 22-year-old man who died 32 hours after a gunshot wound; he had no known risk factors for HIV-1 infection and was seronegative., Methods: We reviewed the processing and distribution of all the transplanted organs and tissues, reviewed the medical histories of the donor and HIV-1-infected recipients, tested stored donor lymphocytes for HIV-1 by viral culture and the polymerase chain reaction, and tested stored serum samples from four organ recipients for HIV-1 antigen and antibody., Results: HIV-1 was detected in cultured lymphocytes from the donor. Of 58 tissues and organs obtained from the donor, 52 could be accounted for by the hospitals that received them. Of the 48 identified recipients, 41 were tested for HIV-1 antibody. All four recipients of organs and all three recipients of unprocessed fresh-frozen bone were infected with HIV-1. However, 34 recipients of other tissues--2 receiving corneas, 3 receiving lyophilized soft tissue, 25 receiving ethanol-treated bone, 3 receiving dura mater treated with gamma radiation, and 1 receiving marrow-evacuated, fresh-frozen bone--tested negative for HIV-1 antibody. Despite immunosuppressive chemotherapy, HIV-1 antibody appeared between 26 and 54 days after transplantation in the three organ recipients who survived more than four weeks., Conclusions: Although rare, transmission of HIV-1 by seronegative organ and tissue donors can occur. Improvements in the methods used to screen donors for HIV-1, advances in techniques of virus inactivation, prompt reporting of HIV infection in recipients, and accurate accounting of distributed allografts would help to reduce further this already exceedingly low risk.

We have assessed the mechanism of intermediate filament assembly by assaying the sites of incorporation of chicken vimentin subunits expressed under the control of an inducible promoter in transfected mouse fibroblasts. The localization of newly synthesized vimentin was determined by immunofluorescence and immunoelectron microscopy at short time periods of induced synthesis, using antibodies specific for chicken vimentin. Under conditions where neither the soluble subunit pools nor the steady-state distribution of endogenous filaments are affected, newly synthesized vimentin incorporates into the vimentin filament network at numerous and discrete sites throughout the cell. Over time, the pattern of newly assembled vimentin converts to a continuous array coincident with preexisting vimentin filaments. These results are consistent with a novel mechanism of intermediate filament assembly, whereby growth of intermediate filaments occurs by topographically restricted and localized subunit addition, necessitating a transient disruption of filament integrity.

The actin bundle within each microvillus of the intestinal brush border (BB) is tethered laterally to the membrane by bridges composed of BB myosin I. Avian BB myosin I, formerly termed 110K-calmodulin, consists of a heavy chain with an apparent Mr of 110 kD and three to four molecules of calmodulin "light chains." Recent studies have shown that this complex shares many properties with myosin including mechanochemical activity. In this report, the isolation and characterization of a membrane fraction enriched in bound BB myosin I is described. This membrane fraction, termed microvillar membrane disks, was purified from ATP extracts of nonionic detergent-treated microvilli prepared from avian intestinal BBs. Ultrastructural analysis revealed that these membranes are flat, disk-shaped sheets with protrusions which are identical in morphology to purified BB myosin I. The disks exhibit actin-activated Mg-ATPase activity and bind and cross-link actin filaments in an ATP-dependent fashion. The mechanochemical activity of the membrane disks was assessed using the Nitella bead movement assay (Sheetz, M. P., and J. A. Spudich. 1983. Nature [Lond.]. 303:31-35). These preparations were shown to be free of significant contamination by conventional BB myosin. Latex beads coated with microvillar membrane disks move in a myosin-like fashion along Nitella actin cables at rates of 12-60 nm/s (average rate of 33 nm/s); unlike purified BB myosin I, the movement of membrane disk-coated beads was most reproducibly observed in buffers containing low Ca2+.

Calcium plays a central role in the regulation of cytoskeletal assembly, structure and contractility. In the case of actin there are a number of functional classes of actin-binding proteins which confer on a given actin filament its specific function in the cell. Among these various classes of actin-binding proteins are a subset of proteins whose activity is either regulated directly or indirectly (for example, through calmodulin) by Ca2+. This includes the regulation of actin-myosin interaction, actin assembly, actin filament interaction and the formation of supramolecular cytoskeletal networks, and the interaction of actin with membranes. Examples of these various modes of Ca2+-dependent regulation of cytoskeletal structure and contractility are discussed.

The three avian spectrins that have been characterized consist of a common alpha-subunit (240 kD) paired with an isoform-specific beta-subunit from either erythrocyte (220 or 230 kD), brain (235 kD), or intestinal brush border (260 kD). Analysis of avian spectrins, with their naturally occurring "subunit replacement" has proved useful in assessing the relative contribution of each subunit to spectrin function. In this study we have completed a survey of avian spectrin binding properties and present morphometric analysis of the relative flexibility and linearity of various avian and human spectrin isoforms. Evidence is presented that, like its mammalian counterpart, avian brain spectrin binds human erythroid ankyrin with low affinity. Cosedimentation analysis demonstrates that 1) avian erythroid protein 4.1 stimulates spectrin-actin binding of both mammalian and avian erythrocyte and brain spectrins, but not the TW 260/240 isoform, 2) calpactin I does not potentiate actin binding of either TW 260/240 or brain spectrin, and 3) erythrocyte adducin does not stimulate the interaction of TW 260/240 with actin. In addition, a morphometric analysis of rotary-shadow images of spectrin isoforms, individual subunits, and reconstituted complexes from isolated subunits was performed. This analysis revealed that the overall flexibility and linearity of a given spectrin heterodimer and tetramer is largely determined by the intrinsic rigidity and linearity of its beta-spectrin subunit. No additional rigidity appears to be imparted by noncovalent associations between the subunits. The scaled flexural rigidity of the most rigid spectrin analyzed (human brain) is similar to that reported for F-actin.

We have used two actin-binding proteins of the intestinal brush border, TW 260/240 and villin, to examine the effects of filament cross-linking and filament length on myosin-actin interactions. TW 260/240 is a nonerythroid spectrin that is a potent cross-linker of actin filaments. In the presence of this cross-linker we observed a concentration-dependent enhancement of skeletal muscle actomyosin ATPase activity (150-560% of control; maximum enhancement at a 1:70-80 TW 260/240:actin molar ratio). TW 260/240 did not cause a similar enhancement of either acto-heavy meromyosin (HMM) ATPase or acto-myosin subfragment-one (S1) ATPase. Villin, a Ca2+-dependent filament capping and severing protein of the intestinal microvillus, was used to generate populations of actin filaments of various lengths from less than 20 nm to 2.0 microns; (villin:actin ratios of 1:2 to 1:4,000). The effect of filament length on actomyosin ATPase was biphasic. At villin:actin molar ratios of 1:2-25 actin-activated myosin ATPase activity was inhibited to 20-80% of control values, with maximum inhibition observed at the highest villin:actin ratio. The ATPase activities of acto-HMM and acto-S1 were also inhibited at these short filament lengths. At intermediate filament lengths generated at villin:actin ratios of 1:40-400 (average lengths 0.26-1.1 micron) an enhancement of actomyosin ATPase was observed (130-260% of controls), with a maximum enhancement at average filament lengths of 0.5 micron. The levels of actomyosin ATPase fell off to control values at low concentrations of villin where filament length distributions were almost those of controls. Unlike intact myosin, the actin-activated ATPase of neither HMM nor S1 showed an enhancement at these intermediate actin filament lengths.

The ability of protein 4.1 to stimulate the binding of spectrin to F-actin has been compared by cosedimentation analysis for three avian (erythrocyte, brain, and brush border) and two mammalian (erythrocyte and brain) spectrin isoforms. Human erythroid protein 4.1 stimulated actin binding of all spectrins except the brush border isoform (TW 260/240). These results suggested that the beta subunit determined the protein 4.1 sensitivity of the heterodimer, since all avian alpha subunits are encoded by a single gene. Tissue-specific posttranslational modification of the alpha subunit was excluded by examining the properties of hybrid spectrins composed of the purified alpha subunit from avian erythrocyte or brush border spectrin and the beta subunit of human erythrocyte spectrin. A hybrid composed of avian brush border alpha and human erythroid beta spectrin ran on nondenaturing gels as a discrete band, migrating near human erythroid spectrin tetramers. The actin-binding activity of this hybrid was stimulated by protein 4.1, while either chain alone was devoid of activity. Therefore, although both subunits were required for actin binding, the sensitivity of the spectrin-actin interaction to protein 4.1 is a property uniquely bestowed on the heterodimer by the beta subunit. The singular insensitivity of brush border spectrin to stimulation by erythroid protein 4.1 was also consistent with the absence of proteins in avian intestinal epithelial cells which were immunoreactive with polyclonal antisera sensitive to all of the known avian and human erythroid 4.1 isoforms.

The purpose of this review on spectrin is to examine the functional properties of this ubiquitous family of membrane skeletal proteins. Major topics include spectrin-membrane linkages, spectrin-filament linkages, the subcellular localization of spectrins in various cell types and a discussion of major functional differences between erythroid and nonerythroid spectrins. This includes a summary of studies from our own laboratories on the functional and structural comparison of avian spectrin isoforms which are comprised of a common alpha subunit and a tissue-specific beta subunit. Consequently, the observed differences among these spectrins can be assigned to differences in the properties of the beta subunits.

The 110-kD protein-calmodulin complex (110K-CM) of the intestinal brush border serves to laterally tether microvillar actin filaments to the plasma membrane. Results from several laboratories have demonstrated that this complex shares many enzymatic and structural properties with myosin. The mechanochemical potential of purified avian 110K-CM was assessed using the Nitella bead motility assay (Sheetz, M. P., and J. A. Spudich. 1983. Nature (Lond.). 303:31-35). Under low Ca2+ conditions, 110K-CM-coated beads bound to actin cables, but no movement was observed. Using EGTA/calcium buffers (approximately 5-10 microM free Ca2+) movement of 110K-CM-coated beads along actin cables (average rate of approximately 8 nm/s) was observed. The movement was in the same direction as that for beads coated with skeletal muscle myosin. The motile preparations of 110K-CM were shown to be free of detectable contamination by conventional brush border myosin. Based on these and other observations demonstrating the myosin-like properties of 110K-CM, we propose that this complex be named "brush border myosin I."