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Unstable transcripts have emerged as markers of active enhancers in vertebrates and shown to be involved in many cellular processes and medical disorders. However, their prevalence and role in plants is largely unexplored. Here, we comprehensively captured all actively initiating (nascent) transcripts across diverse crops and other plants using capped small (cs)RNA sequencing. We discovered that unstable transcripts are rare in plants, unlike in vertebrates, and when present, often originate from promoters. In addition, many 'distal' elements in plants initiate tissue-specific stable transcripts and are likely bona fide promoters of as-yet-unannotated genes or non-coding RNAs, cautioning against using reference genome annotations to infer putative enhancer sites. To investigate enhancer function, we integrated data from self-transcribing active regulatory region (STARR) sequencing. We found that annotated promoters and other regions that initiate stable transcripts, but not those marked by unstable or bidirectional unstable transcripts, showed stronger enhancer activity in this assay. Our findings underscore the blurred line between promoters and enhancers and suggest that cis-regulatory elements can encompass diverse structures and mechanisms in eukaryotes, including humans., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Next-Generation Sequencing (NGS) catalyzed breakthroughs across various scientific domains. Illumina's sequencing by synthesis method has long been essential for NGS but emerging technologies like Element Biosciences' sequencing by avidity (AVITI) represent a novel approach. It has been reported that AVITI offers improved signal-to-noise ratios and cost reductions. However, the method relies on rolling circle amplification which can be impacted by polymer size, raising questions about its efficacy sequencing small RNAs (sRNA) molecules including microRNAs (miRNAs), piwi-interacting RNAs (piRNAs), and others that are crucial regulators of gene expression and involved in various biological processes. In addition, capturing capped small RNAs (csRNA-seq) has emerged as a powerful method to map active or "nascent" RNA polymerase II transcription initiation in tissues and clinical samples. Here, we report a new protocol for seamlessly sequencing short DNA fragments on the AVITI and demonstrate that AVITI and Illumina sequencing technologies equivalently capture human, cattle ( Bos taurus ) and the bison ( Bison bison ) sRNA or csRNA sequencing libraries, augmenting the confidence in both approaches. Additionally, analysis of generated nascent transcription start sites (TSSs) data for cattle and bison revealed inaccuracies in their current genome annotations and highlighted the possibility and need to translate small RNA sequencing methodologies to livestock. Our accelerated and optimized protocol therefore bridges the advantages of AVITI sequencing and critical methods that rely on sequencing short DNA fragments., Competing Interests: COMPETING INTERESTS A.M.B, X.Q., and J.Z are employees of Element Biosciences. S.H.D. is leading nascent Transcriptomic Services (nTSS) at Washington State University.

Apolipoprotein B messenger RNA (mRNA) editing enzyme, catalytic polypeptide-like (APOBEC) cytidine deaminases cause genetic instability during cancer development. Elevated APOBEC3A (A3A) levels result in APOBEC signature mutations; however, mechanisms regulating A3A abundance in breast cancer are unknown. Here, we show that dysregulating the ubiquitin-proteasome system with proteasome inhibitors, including Food and Drug Administration-approved anticancer drugs, increased A3A abundance in breast cancer and multiple myeloma cell lines. Unexpectedly, elevated A3A occurs via an ∼100-fold increase in A3A mRNA levels, indicating that proteasome inhibition triggers a transcriptional response as opposed to or in addition to blocking A3A degradation. This transcriptional regulation is mediated in part through FBXO22, a protein that functions in SKP1-cullin-F-box ubiquitin ligase complexes and becomes dysregulated during carcinogenesis. Proteasome inhibitors increased cellular cytidine deaminase activity, decreased cellular proliferation and increased genomic DNA damage in an A3A-dependent manner. Our findings suggest that proteasome dysfunction, either acquired during cancer development or induced therapeutically, could increase A3A-induced genetic heterogeneity and thereby influence therapeutic responses in patients., (© The Author(s) 2023. Published by Oxford University Press on behalf of NAR Cancer.)

It is now well-established that stress elicits brain- and body-wide changes in physiology and has significant impacts on many aspects of health. The hypothalamic-pituitary-adrenal (HPA) axis is the major neuroendocrine system mediating the integrated response to stress. Appropriate engagement and termination of HPA activity enhances survival and optimizes physiological and behavioral responses to stress, while dysfunction of this system is linked to negative health outcomes such as depression, anxiety, and post-traumatic stress disorder. Glutamate signaling plays a large role in the transmission of stress-related information throughout the brain. Furthermore, aberrant glutamate signaling has negative consequences for neural plasticity and synaptic function and is linked to stress-related pathology. However, the connection between HPA dysfunction and glutamate signaling is not fully understood. We tested how HPA axis dysfunction (using low dose chronic corticosterone in the drinking water) affects glutamate homeostasis and neural responses under baseline and acute stress in male C57BL/6N mice. Using laser microdissection and transcriptomic analyses, we show that chronic disruption of the HPA axis alters the expression of genes related to glutamate signaling in the medial prefrontal cortex (mPFC), hippocampus, and amygdala. While neural responses to stress (as measured by FOS) in the hippocampus and amygdala were not affected in our model of HPA dysfunction, we observed an exaggerated response to stress in the mPFC. To further probe this we undertook in vivo biosensor measurements of the dynamics of extracellular glutamate responses to stress in the mPFC in real-time, and found glutamate dynamics in the mPFC were significantly altered by chronic HPA dysfunction. Together, these findings support the hypothesis that chronic HPA axis dysfunction alters glutamatergic signaling in regions known to regulate emotional behavior, providing more evidence linking HPA dysfunction and stress vulnerability., (© 2022 The Authors.)

The basolateral amygdala (BLA) is a critical brain region for cocaine-memory reconsolidation. Corticotropin-releasing factor receptor type 1 (CRFR1) is densely expressed in the BLA, and CRFR1 stimulation can activate intra-cellular signaling cascades that mediate memory reconsolidation. Hence, we tested the hypothesis that BLA CRFR1 stimulation is necessary and sufficient for cocaine-memory reconsolidation. Using an instrumental model of drug relapse, male and female Sprague-Dawley rats received cocaine self-administration training in a distinct environmental context over 10 days followed by extinction training in a different context over 7 days. Next, rats were re-exposed to the cocaine-paired context for 15 min to initiate cocaine-memory retrieval and destabilization. Immediately or 6 h after this session, the rats received bilateral vehicle, antalarmin (CRFR1 antagonist; 500 ng/hemisphere), or corticotropin-releasing factor (CRF; 0.2, 30 or 500 ng/hemisphere) infusions into the BLA. Resulting changes in drug context-induced cocaine seeking (index of context-cocaine memory strength) were assessed three days later. Female rats self-administered more cocaine infusions and exhibited more extinction responding than males. Intra-BLA antalarmin treatment immediately after memory retrieval (i.e., when cocaine memories were labile), but not 6 h later (i.e., after memory reconsolidation), attenuated drug context-induced cocaine seeking at test independent of sex, relative to vehicle. Conversely, intra-BLA CRF treatment increased this behavior selectively in females, in a U-shaped dose-dependent fashion. In control experiments, a high (behaviorally ineffective) dose of CRF treatment did not reduce BLA CRFR1 cell-surface expression in females. Thus, BLA CRFR1 signaling is necessary and sufficient, in a sex-dependent manner, for regulating cocaine-memory strength., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Circulating blood glucocorticoid levels are dynamic and responsive to stimuli that impact autonomic function. In the brain stem, vagal afferent terminals release the excitatory neurotransmitter glutamate to neurons in the nucleus of the solitary tract (NTS). Vagal afferents integrate direct visceral signals and circulating hormones with ongoing NTS activity to control autonomic function and behavior. Here, we investigated the effects of corticosterone (CORT) on glutamate signaling in the NTS using patch-clamp electrophysiology on brain stem slices containing the NTS and central afferent terminals from male C57BL/6 mice. We found that CORT rapidly decreased both action potential-evoked and spontaneous glutamate signaling. The effects of CORT were phenocopied by dexamethasone and blocked by mifepristone, consistent with glucocorticoid receptor (GR)-mediated signaling. While mRNA for GR was present in both the NTS and vagal afferent neurons, selective intracellular quenching of G protein signaling in postsynaptic NTS neurons eliminated the effects of CORT. We then investigated the contribution of retrograde endocannabinoid signaling, which has been reported to transduce nongenomic GR effects. Pharmacological or genetic elimination of the cannabinoid type 1 receptor signaling blocked CORT suppression of glutamate release. Together, our results detail a mechanism, whereby the NTS integrates endocrine CORT signals with fast neurotransmission to control autonomic reflex pathways.

The BDNF gene contains a polymorphism (Val66Met) that influences sleep and may be associated with more flexible adaptation to circadian misalignment. Fifteen adult men (10 Val/Val homozygotes, 5 Val/Met heterozygotes) participated in a laboratory study involving two 5 d cycles of simulated night shifts. Circulating interleukin-6 (IL-6) was measured from plasma, sleep was recorded polysomnographically, and performance was measured using a psychomotor vigilance test. Compared to Val/Val homozygotes, heterozygotes exhibited a blunted IL-6 temporal (diurnal) pattern, less daytime sleep restriction, and less nighttime performance impairment after the first simulated night-shift cycle. These observations suggest that heterozygotes experienced more flexible circadian adaptation.

In mammals, one of the most salient outputs of the circadian (daily) clock is the timing of the sleep-wake cycle. Modern industrialized society has led to a fundamental breakdown in the relationship between our endogenous timekeeping systems and the solar day, disrupting normal circadian rhythms. We have argued that disrupted circadian rhythms could lead to changes in allostatic load, and the capacity of organisms to respond to other environmental challenges. In this set of studies, we apply a model of circadian disruption characterized in our lab in which mice are housed in a 20h long day, with 10h of light and 10h of darkness. We explored the effects of this environmental disruption on sleep patterns, to establish if this model results in marked sleep deprivation. Given the interaction between circadian, sleep, and immune systems, we further probed if our model of circadian disruption also alters the innate immune response to peripheral bacterial endotoxin challenge. Our results demonstrate that this model of circadian disruption does not lead to marked sleep deprivation, but instead affects the timing and quality of sleep. We also show that while circadian disruption does not lead to basal changes in the immune markers we explored, the immune response is affected, both in the brain and the periphery. Together, our findings further strengthen the important role of the circadian timing system in sleep regulation and immune responses, and provide evidence that disrupting the circadian clock increases vulnerability to further environmental stressors, including immunological challenges., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Mate preference behavior is an essential first step in sexual selection and is a critical determinant in evolutionary biology. Previously an environmental compound (the fungicide vinclozolin) was found to promote the epigenetic transgenerational inheritance of an altered sperm epigenome and modified mate preference characteristics for three generations after exposure of a gestating female., Results: The current study investigated gene networks involved in various regions of the brain that correlated with the altered mate preference behavior in the male and female. Statistically significant correlations of gene clusters and modules were identified to associate with specific mate preference behaviors. This novel systems biology approach identified gene networks (bionetworks) involved in sex-specific mate preference behavior. Observations demonstrate the ability of environmental factors to promote the epigenetic transgenerational inheritance of this altered evolutionary biology determinant., Conclusions: Combined observations elucidate the potential molecular control of mate preference behavior and suggests environmental epigenetics can have a role in evolutionary biology.

Background: Environmentally induced epigenetic transgenerational inheritance of adult onset disease involves a variety of phenotypic changes, suggesting a general alteration in genome activity., Results: Investigation of different tissue transcriptomes in male and female F3 generation vinclozolin versus control lineage rats demonstrated all tissues examined had transgenerational transcriptomes. The microarrays from 11 different tissues were compared with a gene bionetwork analysis. Although each tissue transgenerational transcriptome was unique, common cellular pathways and processes were identified between the tissues. A cluster analysis identified gene modules with coordinated gene expression and each had unique gene networks regulating tissue-specific gene expression and function. A large number of statistically significant over-represented clusters of genes were identified in the genome for both males and females. These gene clusters ranged from 2-5 megabases in size, and a number of them corresponded to the epimutations previously identified in sperm that transmit the epigenetic transgenerational inheritance of disease phenotypes., Conclusions: Combined observations demonstrate that all tissues derived from the epigenetically altered germ line develop transgenerational transcriptomes unique to the tissue, but common epigenetic control regions in the genome may coordinately regulate these tissue-specific transcriptomes. This systems biology approach provides insight into the molecular mechanisms involved in the epigenetic transgenerational inheritance of a variety of adult onset disease phenotypes.

Ancestral environmental exposures have previously been shown to promote epigenetic transgenerational inheritance and influence all aspects of an individual's life history. In addition, proximate life events such as chronic stress have documented effects on the development of physiological, neural, and behavioral phenotypes in adulthood. We used a systems biology approach to investigate in male rats the interaction of the ancestral modifications carried transgenerationally in the germ line and the proximate modifications involving chronic restraint stress during adolescence. We find that a single exposure to a common-use fungicide (vinclozolin) three generations removed alters the physiology, behavior, metabolic activity, and transcriptome in discrete brain nuclei in descendant males, causing them to respond differently to chronic restraint stress. This alteration of baseline brain development promotes a change in neural genomic activity that correlates with changes in physiology and behavior, revealing the interaction of genetics, environment, and epigenetic transgenerational inheritance in the shaping of the adult phenotype. This is an important demonstration in an animal that ancestral exposure to an environmental compound modifies how descendants of these progenitor individuals perceive and respond to a stress challenge experienced during their own life history.

The actions of environmental toxicants and relevant mixtures in promoting the epigenetic transgenerational inheritance of ovarian disease was investigated with the use of a fungicide, a pesticide mixture, a plastic mixture, dioxin and a hydrocarbon mixture. After transient exposure of an F0 gestating female rat during embryonic gonadal sex determination, the F1 and F3 generation progeny adult onset ovarian disease was assessed. Transgenerational disease phenotypes observed included an increase in cysts resembling human polycystic ovarian disease (PCO) and a decrease in the ovarian primordial follicle pool size resembling primary ovarian insufficiency (POI). The F3 generation granulosa cells were isolated and found to have a transgenerational effect on the transcriptome and epigenome (differential DNA methylation). Epigenetic biomarkers for environmental exposure and associated gene networks were identified. Epigenetic transgenerational inheritance of ovarian disease states was induced by all the different classes of environmental compounds, suggesting a role of environmental epigenetics in ovarian disease etiology.

A complex of ExbB, ExbD, and TonB couples cytoplasmic membrane (CM) proton motive force (pmf) to the active transport of large, scarce, or important nutrients across the outer membrane (OM). TonB interacts with OM transporters to enable ligand transport. Several mechanical models and a shuttle model explain how TonB might work. In the mechanical models, TonB remains attached to the CM during energy transduction, while in the shuttle model the TonB N terminus leaves the CM to deliver conformationally stored potential energy to OM transporters. Previous studies suggested that TonB did not shuttle based on the activity of a GFP-TonB fusion that was anchored in the CM by the GFP moiety. When we recreated the GFP-TonB fusion to extend those studies, in our hands it was proteolytically unstable, giving rise to potentially shuttleable degradation products. Recently, we discovered that a fusion of the Vibrio cholerae ToxR cytoplasmic domain to the N terminus of TonB was proteolytically stable. ToxR-TonB was able to be completely converted into a proteinase K-resistant conformation in response to loss of pmf in spheroplasts and exhibited an ability to form a pmf-dependent formaldehyde crosslink to ExbD, both indicators of its location in the CM. Most importantly, ToxR-TonB had the same relative specific activity as wild-type TonB. Taken together, these results provide conclusive evidence that TonB does not shuttle during energy transduction. We had previously concluded that TonB shuttles based on the use of an Oregon Green(®) 488 maleimide probe to assess periplasmic accessibility of N-terminal TonB. Here we show that the probe was permeant to the CM, thus permitting the labeling of the TonB N-terminus. These former results are reinterpreted in the context that TonB does not shuttle, and suggest the existence of a signal transduction pathway from OM to cytoplasm.

The current study was designed to investigate the actions of Anti-Müllerian Hormone (AMH) on primordial follicle assembly. Ovarian primordial follicles develop from the breakdown of oocyte nests during fetal development for the human and immediately after birth in rodents. AMH was found to inhibit primordial follicle assembly and decrease the initial primordial follicle pool size in a rat ovarian organ culture. The AMH expression was found to be primarily in the stromal tissue of the ovaries at this period of development, suggesting a stromal-epithelial cell interaction for primordial follicle assembly. AMH was found to promote alterations in the ovarian transcriptome during primordial follicle assembly with over 200 genes with altered expression. A gene network was identified suggesting a potential central role for the Fgf2/Nudt6 antisense transcript in the follicle assembly process. A number of signal transduction pathways are regulated by AMH actions on the ovarian transcriptome, in particular the transforming growth factor-beta (TGFß) signaling process. AMH is the first hormone/protein shown to have an inhibitory action on primordial follicle assembly. Due to the critical role of the primordial follicle pool size for female reproduction, elucidation of factors, such as AMH, that regulate the assembly process will provide insights into potential therapeutics to manipulate the pool size and female reproduction.

The current study investigates the direct effects of in utero vinclozolin exposure on the developing F1 generation rat testis transcriptome. Previous studies have demonstrated that exposure to vinclozolin during embryonic gonadal sex determination induces epigenetic modifications of the germ line and transgenerational adult onset disease states. Microarray analyses were performed to compare control and vinclozolin treated testis transcriptomes at embryonic days 13, 14 and 16. A total of 576 differentially expressed genes were identified and the major cellular functions and pathways associated with these altered transcripts were examined. The sets of regulated genes at the different development periods were found to be transiently altered and distinct. Categorization by major known functions of altered genes was performed. Specific cellular process and pathway analyses suggest the involvement of Wnt and calcium signaling, vascular development and epigenetic mechanisms as potential mediators of the direct F1 generation actions of vinclozolin., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Ovarian primordial follicles are critical for female reproduction and comprise a finite pool of gametes arrested in development. A systems biology approach was used to identify regulatory gene networks essential for primordial follicle development. Transcriptional responses to eight different growth factors known to influence primordial follicles were used to construct a bionetwork of regulatory genes involved in rat primordial follicle development. Over 1,500 genes were found to be regulated by the various growth factors and a network analysis identified critical gene modules involved in a number of signaling pathways and cellular processes. A set of 55 genes was identified as potential critical regulators of these gene modules, and a sub-network associated with development was determined. Within the network two previously identified regulatory genes were confirmed (i.e., Pdgfa and Fgfr2) and a new factor was identified, connective tissue growth factor (CTGF). CTGF was tested in ovarian organ cultures and found to stimulate primordial follicle development. Therefore, the relevant gene network associated with primordial follicle development was validated and the critical genes and pathways involved in this process were identified. This is one of the first applications of network analysis to a normal developmental process. These observations provide insights into potential therapeutic targets for preventing ovarian disease and promoting female reproduction.

Coordinated interactions between ovarian granulosa and theca cells are required for female endocrine function and fertility. To elucidate these interactions the regulation of the granulosa and theca cell transcriptomes during bovine antral follicle development were investigated. Granulosa cells and theca cells were isolated from small (<5 mm), medium (5-10 mm), and large (>10 mm) antral bovine follicles. A microarray analysis of 24,000 bovine genes revealed that granulosa cells and theca cells each had gene sets specific to small, medium and large follicle cells. Transcripts regulated (i.e., minimally changed 1.5-fold) during antral follicle development for the granulosa cells involved 446 genes and for theca cells 248 genes. Only 28 regulated genes were common to both granulosa and theca cells. Regulated genes were functionally categorized with a focus on growth factors and cytokines expressed and regulated by the two cell types. Candidate regulatory growth factor proteins mediating both paracrine and autocrine cell-cell interactions include macrophage inflammatory protein (MIP1 beta), teratocarcinoma-derived growth factor 1 (TDGF1), stromal derived growth factor 1 (SDF1; i.e., CXCL12), growth differentiation factor 8 (GDF8), glia maturation factor gamma (GMFG), osteopontin (SPP1), angiopoietin 4 (ANGPT4), and chemokine ligands (CCL 2, 3, 5, and 8). The current study examined granulosa cell and theca cell regulated genes associated with bovine antral follicle development and identified candidate growth factors potentially involved in the regulation of cell-cell interactions required for ovarian function.

Embryonic exposure to the endocrine disruptor vinclozolin during gonadal sex determination promotes an epigenetic reprogramming of the male germ-line that is associated with transgenerational adult onset disease states. Further analysis of this transgenerational phenotype on the brain demonstrated reproducible changes in the brain transcriptome three generations (F3) removed from the exposure. The transgenerational alterations in the male and female brain transcriptomes were distinct. In the males, the expression of 92 genes in the hippocampus and 276 genes in the amygdala were transgenerationally altered. In the females, the expression of 1,301 genes in the hippocampus and 172 genes in the amygdala were transgenerationally altered. Analysis of specific gene sets demonstrated that several brain signaling pathways were influenced including those involved in axon guidance and long-term potentiation. An investigation of behavior demonstrated that the vinclozolin F3 generation males had a decrease in anxiety-like behavior, while the females had an increase in anxiety-like behavior. These observations demonstrate that an embryonic exposure to an environmental compound appears to promote a reprogramming of brain development that correlates with transgenerational sex-specific alterations in the brain transcriptomes and behavior. Observations are discussed in regards to environmental and transgenerational influences on the etiology of brain disease.

The active transport of iron siderophores and vitamin B(12) across the outer membrane (OM) of Escherichia coli requires OM transporters and the potential energy of the cytoplasmic membrane (CM) proton gradient and CM proteins TonB, ExbB, and ExbD. A region at the amino terminus of the transporter, called the TonB box, directly interacts with TonB Q160 region residues. R158 and R166 in the TonB Q160 region were proposed to play important roles in cocrystal structures of the TonB carboxy terminus with OM transporters BtuB and FhuA. In contrast to predictions based on the crystal structures, none of the single, double, or triple alanyl substitutions at arginyl residues significantly decreased TonB activity. Even the quadruple R154A R158A R166A R171A mutant TonB still retained 30% of wild-type activity. Up to five residues centered on TonB Q160 could be deleted without inactivating TonB or preventing its association with the OM. TonB mutant proteins with nested deletions of 7, 9, or 11 residues centered on TonB Q160 were inactive and appeared never to have associated with the OM. Because the 7-residue-deletion mutant protein (TonBDelta7, lacking residues S157 to Y163) could still form disulfide-linked dimers when combined with W213C or F202C in the TonB carboxy terminus, the TonBDelta7 deletion did not prevent necessary energy-dependent conformational changes that occur in the CM. Thus, it appeared that initial contact with the OM is made through TonB residues S157 to Y163. It is hypothesized that the TonB Q160 region may be part of a large disordered region required to span the periplasm and contact an OM transporter.

Comparative proton NMR studies have been carried out on high-spin and low-spin forms of recombinant native cytochrome c peroxidase (rCcP) and its His52 --> Leu variant. Proton NMR spectra of rCcP(H52L) (high spin) and rCcP(H52L)CN (low spin) reveal the presence of multiple enzyme forms in solution, whereas only single enzyme forms are found in spectra of wild-type and recombinant wild-type CcP and CcPCN near neutral pH. The spectroscopic behaviors of these forms have been studied in detail when pH, temperature, and solvent isotope composition were varied. For resting-state rCcP(H52L) the comparatively large NMR line widths compromise resolution, but two specific enzyme forms were found. They were interconvertible on the basis of varying temperature. For rCcP(H52L)CN four magnetically distinct enzyme forms were identified by NMR. It was found that these forms dynamically interconvert with changing pH, temperature, and solvent isotope composition (percent D(2)O). These studies have identified the alkaline titration of His52 and essentially identical alkaline enzyme forms for natWTCcPCN and rCcP(H52L)CN. From this work we interpret an essential role of His52 in CcP function to be preservation of a single active site structure in addition to the critical role of general base catalysis.

The heme-containing PAS domain of the direct oxygen-sensor protein (DOS(H)), a bona fide oxygen-sensor protein, has been cloned from Escherichia coli strain K12 and successfully purified. The oxidized form of this protein was crystallized by the hanging-drop method with a PEG 8000-based precipitant. Preliminary X-ray diffraction studies of the PAS-domain crystal show that it belongs to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 46.1, b = 68.1, c = 82.6 A. A complete diffraction data set was collected to 1.9 A for MAD phasing. The electron-density map shows two molecules in an asymmetric unit and a unique six-coordination of the heme iron.

Two forms of extensively deuterated S. cerevisiae cytochrome c peroxidase (CcP; EC 1.11.1.5) have been overexpressed in E. coli by growth in highly deuterated medium. One of these ferriheme enzyme forms (recDCcP) was produced using >97% deuterated growth medium and was determined to be approximately 84% deuterated. The second form [recD(His)CcP] was grown in the same highly deuterated medium that had been supplemented with excess histidine (at natural hydrogen isotope abundance) and was also approximately 84% deuterated. This resulted in direct histidine incorporation without isotope scrambling. Both of these enzymes along with the corresponding recombinant native CcP (recNATCcP), which was expressed in a standard medium with normal hydrogen isotope abundance, consisted of 294 amino acid polypeptide chains having the identical sequence to the yeast-isolated enzyme, without any N-terminal modifications. Comparative characterizations of all three enzymes have been carried out for the resting-state, high-spin forms and in the cyanide-ligated, low-spin forms. The primary physical methods employed were electrophoresis, UV-visible spectroscopy, hydrogen peroxide reaction kinetics, mass spectrometry, and (1)H NMR spectroscopy. The results indicate that high-level deuteration does not significantly alter CcP's reactivity or spectroscopy. As an example of potential NMR uses, recDCcPCN and recD(His)CcPCN have been used to achieve complete, unambiguous, stereospecific (1)H resonance assignments for the heme hyperfine-shifted protons, which also allows the heme side chain conformations to be assessed. Assigning these important active-site protons has been an elusive goal since the first NMR spectra on this enzyme were reported 18 years ago, due to a combination of the enzyme's comparatively large size, paramagnetism, and limited thermal stability.

A more efficient 2-day isolation and purification method for recombinant yeast cytochrome c peroxidase produced in Escherichia coli is presented. Two types of recombinant "wild-type" CcP have been produced and characterized, the recombinant nuclear gene sequence and the 294-amino-acid original protein sequence. These two sequences constitute the majority of the recombinant "native" or wild-type CcP currently in production and from which all recombinant variants now derive. The enzymes have been subjected to extensive physical characterizations, including sequencing, UV-visible spectroscopy, HPLC, gel electrophoresis, kinetic measurements, NMR spectroscopy, and mass spectrometry. Less extensive characterization data are also presented for recombinant, perdeuterated CcP, an enzyme produced in >95% deuterated medium. All of these results indicate that the purified recombinant wild-type enzymes are functionally and spectroscopically identical to the native, yeast-isolated wild-type enzyme. This improved method uses standard chromatography to produce highly purified holoenzyme in a more efficient manner than previously achieved. Two methods for assembling the holoenzyme are described. In one, exogenous heme is added at lysis, while in the other heme biosynthesis is stimulated in E. coli. A primary reason for developing this method has been the need to minimize loss of precious, isotope-labeled enzyme and, so, this method has also been used to produce both the perdeuterated and the (15)N-labeled enzyme, as well as several variants., (Copyright 2000 Academic Press.)

To examine the role of Arg38 in the peroxidative and peroxygenative activity of horseradish peroxidase (HRP), we have expressed the R38A, R38H, and R38H/H42V mutants. The R38A HRP mutant gives a normal compound I species with H2O2 that is reduced by ferrocyanide to the ferric state without the detectable formation of a compound II species. In the case of the R38H and R38H/H42V mutants, compound I itself is only detected by stopped flow methods. The rates of compound I formation at 4 degrees C are 8.0 x 10(4), 1.3 x 10(6), and 1.6 x 10(3) M-1 s-1 for the R38A, R38H, and R38H/H42V mutants, respectively. The R38A, R38H, and R38H/H42V mutants oxidize guaiacol 10-, 2-, and 55-fold, respectively, more slowly than the wild-type enzyme and oxidize ABTS 6-, 3-, and 32-fold more slowly than the wild-type enzyme. The apparent kcat/K(m) values for thioanisole sulfoxidation and styrene epoxidation indicate that the reaction efficiencies of the R38H and wild-type enzymes are comparable. However, the R38A and R38H/H42V mutants are 190- and 1400-fold more efficient as sulfoxidation catalysts, and 25- and 26-fold more efficient as styrene epoxidation catalysts, respectively, than the wild-type enzyme. Thus, even though Arg38 plays a role in the formation and stabilization of compounds I and II, its replacement by other residues can be used to improve peroxygenative catalysis.

In horseradish peroxidase (HRP), hydrogen bonding of Asn-70 to His-42 enhances the catalytic activity of the histidine, and mutation of His-42 to a neutral residue greatly decreases peroxidase activity. The N70D/H42A HRP mutant is compared here to the previously characterized H42A mutant to determine if the Asp-70 substitution can rescue the catalytic activity. The N70D/H42A and H42A mutants give Compound I species with a high ratio of H2O2 at the low rates of 37 and 81 M-1 s-1 at 4 degrees C, respectively. The kcat values for the oxidation of guaiacol and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) by N70D/H42A HRP are 2.7 and 143 s-1, respectively, compared to values of 0.015 and 0.41 s-1 for the H42A mutant. The kcat values for thioanisole sulfoxidation by the N70D/H42A and H42A mutants are 0.18 and 0.03 s-1, respectively, and the corresponding values for styrene epoxidation are 0.005 and 0.007 s-1. Due to changes in the substrate Km values, the efficiencies of the N70D/H42A and H42A mutants defined by kcat/Km are guaiacol, 5 vs 4; ABTS, 286 vs 68; thioanisole, 3 vs 0.1; and styrene, 0.025 vs 0.002, respectively. The N70D mutation in N70D/H42A HRP thus increases the activity versus the H42A mutant with respect to all four substrates. The increased efficiency is due to enhancements in catalytic steps other than the formation of Compound I., (Copyright 1998 Academic Press.)

Formation of the ferryl (FeIV=O) porphyrin radical cation known as Compound I in the reaction of horseradish peroxidase (HRP) with H2O2 is catalyzed by His-42, a residue that facilitates the binding of H2O2 to the iron and subsequent rupture of the dioxygen bond. An H42A mutation was shown earlier to decrease the rate of Compound I formation by a factor of approximately 10(6) and of guaiacol oxidation by a factor of approximately 10(4). In contrast, an F41A mutation has little effect on peroxidative catalysis (Newmyer, S. L., and Ortiz de Montellano, P. R. (1995) J. Biol. Chem. 270, 19430-19438). We report here construction, expression, and characterization of the F41H/H42A double mutant. The pH profile for guaiacol oxidation by this double mutant has a broad maximum at approximately pH 6.3. Addition of H2O2 produces a Compound I species (lambdamax = 406 nm) that is reduced by 1 eq of K4Fe(CN)6 to the ferric state (lambdamax = 407 nm) without the detectable formation of Compound II. A fraction of the heme chromophore is lost in the process. The rate of Compound I formation for the F41H/H42A double mutant is 3.0 x 10(4) M-1 s-1. This is to be compared with 0.9 x 10(7) M-1 s-1 for wild-type HRP and 19 M-1 s-1 for the H42A mutant. The kcat values for guaiacol oxidation by wild-type, H42A, and F41H/H42A HRP are 300, 0.015, and 1.8 s-1. The corresponding kcat values for ABTS oxidation are 4900, 0.41, and 100 s-1, respectively. These results show that a histidine at position 41 substitutes, albeit imperfectly, for His-42 in peroxidative turnover of the enzyme. The F41H/H42A double mutant has peroxidative properties intermediate between those of the native enzyme and the H42A mutant. The F41H/H42A double mutant, however, is a considerably better thioanisole sulfoxidation and styrene epoxidation catalyst than native or H42A HRP. The surrogate catalytic residue introduced by the F41H mutation thus partially compensates for the H42A substitution used to increase access to the ferryl oxygen.

Using solid phase enzyme-linked immunoassay (ELISA), rate constants for the formation and dissociation as well as equilibrium dissociation constants for strophanthin complexes interaction with specific antibodies (both soluble and immobilized on activated polystyrene beads and polyamide membranes) have been determined. A satisfactory correlation was found between dissociation constants for immune complexes determined by ELISA (both soluble and immobilized ones) and associations constants for these complexes determined by the Scatchard method. The experimental values of kinetic and equilibrium constants for strophanthin K and its peroxidase complexes interaction reflect the difference in the nature of interacting particles and their environment. Strophanthin complexes with antibodies appeared to be more stable when formed on adsorbents (in comparison with those formed in solution). Strophanthin interaction with immobilized antibodies occurred at a slower rate than that with soluble antibodies. Strophanthin reacts with antibodies more readily than its complexes both in solution and on solid matrices of various polymeric nature.

Catalase was immobilized on an immunosorbent prepared by anticatalase adsorption on an activated carbon fabric (ACF), and its kinetic parameters were determined. The immobilized catalase activity depended on the binding capacity of anticatalase. Under the optimum conditions (6 micrograms/mg anticatalase, 5.24 nM catalase) the immobilized catalase activity was 1.5-ford higher as compared to soluble catalase. Antibodies stabilized soluble catalase, but decreased its thermostability on immobilization of immunocomplexes on ACF. Noncovalent immobilization of catalase on adsorbed antibodies opens up the way to the use of this approach for immobilization of other oligomeric enzymes.

The kinetics of peroxidase-dependent cooxidation for two substrate pairs [p-iodophenol + 4-aminoantipyrine (AAP) and p-iodophenol + luminol was studied both in the absence and presence of polyclonal antibodies (polyAB), three types of peroxidase-specific monoclonal antibodies (monoAB) and their double or triple mixtures in a wide range of H2O2 concentrations (0.01-10.0 mM). MonoAB 2C, 3E and 9D at concentrations of 0.05-500 nM inhibited the cooxidation of p-iodophenol + AAP at H2O2 concentration above 1.0 mM but activated the cooxidation of p-iodophenol + luminol. The double and triple mixtures of monoAB activated the cooxidation of p-iodophenol + AAP at the same H2O2 concentrations without any effect on the p-iodophenol + luminol cooxidation. PolyAB activated the cooxidation of p-iodophenol + AAP more effectively and only slightly activated (or inhibited) that of p-iodophenol + luminol. PolyAB diminished the values of rate constants for the interaction of the peroxidase active intermediates, E1 and E2, with p-iodophenol, AAP or luminol. Possible modes of monoAB and polyAB effects on the two substrate pair cooxidation are discussed.

The alterations in the catalytic activity of the horseradish peroxidase after its interaction with antibodies against this enzyme have been studied in buffered solution and in reversed Aerosol OT (AOT) micelles in heptane. The antibodies were obtained by immunizing the rabbits with electrophoretically homogeneous enzyme and were purified by affinity chromatography. In the AOT micelles and mixed micelles containing AOT and Triton X-45, the enzyme interacted with antibodies very rapidly (in less than 5 min), i.e. the micelles did not hinder effective interaction between the enzyme and antibodies. The decrease in the peroxidase catalytic activity upon its interaction with antibodies in a micellar medium was determined by [H2O]/[AOT] ratio, pH and molarity of polar nucleus, as well as by the initial concentration of antibody. In buffered solutions, the decrease n the peroxidase activity of the enzyme--antibody complex was only weakly dependent on pH and molarity of a buffer solution.

The steady-state kinetics of peroxidation of 8 aromatic amines was studied. p-Phenylenediamine, o-dianisidine (o-DA) and 3,5,3',5'-tetramethylbenzidine were found to be optimal substrates of horse-radish peroxidase. The kinetics of oxidation of these substrates by horseradish peroxidase modified with three molecules of Strophanthin K was studied as well. Within the temperature range from 37 to 53 degrees C the inactivation rate constants were determined for peroxidase and its conjugate with Strophanthin K. The effect of sugars and polyols on thermal stability of the conjugate peroxidase-Strophanthin K was investigated. A comparative kinetic study was performed of oxidation of o-DA and its conjugate with dextran. The results obtained made a basis for an enzyme immunoassay of cardiac glycosides during their isolation from plant raw material.