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Plant roots navigate in the soil environment following the gravity vector. Cell divisions in the meristem and rapid cell growth in the elongation zone propel the root tips through the soil. Actively elongating cells acidify their apoplast to enable cell wall extension by the activity of plasma membrane AHA H+-ATPases. The phytohormone auxin, central regulator of gravitropic response and root development, inhibits root cell growth, likely by rising the pH of the apoplast. However, the role of auxin in the regulation of the apoplastic pH gradient along the root tip is unclear. Here, we show, by using an improved method for visualization and quantification of root surface pH, that the Arabidopsis thaliana root surface pH shows distinct acidic and alkaline zones, which are not primarily determined by the activity of AHA H+-ATPases. Instead, the distinct domain of alkaline pH in the root transition zone is controlled by a rapid auxin response module, consisting of the AUX1 auxin influx carrier, the AFB1 auxin co-receptor, and the CNCG14 calcium channel. We demonstrate that the rapid auxin response pathway is required for an efficient navigation of the root tip. [ABSTRACT FROM AUTHOR]

Methylation patterns established and maintained at CpG sites may be altered by single nucleotide polymorphisms (SNPs) within these sites and may affect the regulation of nearby genes. Our aims were to: 1) identify and generate a database of SNPs potentially subject to epigenetic control by DNA methylation via their involvement in creating, removing or displacing CpG sites (meSNPs), and; 2) investigate the association of these meSNPs with CpG islands (CGIs), and with methylation profiles of DNA extracted from tissues from cattle with divergent feed efficiencies detected using MIRA-Seq. Using the variant annotation for 56,969,697 SNPs identified in Run5 of the 1000 Bull Genomes Project and the UMD3.1.1 bovine reference genome sequence assembly, we identified and classified 12,836,763 meSNPs according to the nature of variation created at CpGs. The majority of the meSNPs were located in intergenic regions (68%) or introns (26.3%). We found an enrichment (p<0.01) of meSNPs located in CGIs relative to the genome as a whole, and also in differentially methylated sequences in tissues from animals divergent for feed efficiency. Seven meSNPs, located in differentially methylated regions, were fixed for methylation site creating (MSC) or destroying (MSD) alleles in the differentially methylated genomic sequences of animals differing in feed efficiency. These meSNPs may be mechanistically responsible for creating or deleting methylation targets responsible for the differential expression of genes underlying differences in feed efficiency. Our methyl SNP database (dbmeSNP) is useful for identifying potentially functional "epigenetic polymorphisms" underlying variation in bovine phenotypes. [ABSTRACT FROM AUTHOR]

Protein methylation is a very diverse, widespread, and important post-translational modification affecting all aspects of cellular biology in eukaryotes. Methylation on the side-chain of lysine residues in histones has received considerable attention due to its major role in determining chromatin structure and the epigenetic regulation of gene expression. Over the last 20 years, lysine methylation of non-histone proteins has been recognized as a very common modification that contributes to the fine-tuned regulation of protein function. In plants, our knowledge in this field is much more fragmentary than in yeast and animal cells. In this review, we describe the plant enzymes involved in the methylation of non-histone substrates, and we consider historical and recent advances in the identification of non-histone lysine-methylated proteins in photosynthetic organisms. Finally, we discuss our current knowledge about the role of protein lysine methylation in regulating molecular and cellular functions in plants, and consider challenges for future research. [ABSTRACT FROM AUTHOR]

Diffusion tensor imaging (DTI) is an MRI-based technique that delineates white matter tracts in the brain by tracking the diffusion of water in neural tissue. This methodology, known as "tractography", has been extensively applied in clinical neuroscience to explore nervous system architecture and diseases. More recently, tractography has been used to assist with neurosurgical targeting in functional neurosurgery. This review provides an overview of DTI principles, and discusses current applications of tractography for improving and helping develop novel deep brain stimulation (DBS) targets. [ABSTRACT FROM AUTHOR]

The northern elephant seal, Mirounga angustirostris, was heavily hunted and declared extinct in the 19th century. However, a colony remained on remote Guadalupe Island, Mexico and the species has since repopulated most of its historical distribution. Here, we present a comprehensive evaluation of genetic variation in the species. First, we assess the effect of the demographic bottleneck on microsatellite variability and compare it with that found in other pinnipeds, demonstrating levels of variation similar to that in species that continue to be threatened with extinction. Next, we use sequence data from these markers to demonstrate that some of the limited polymorphism predates the bottleneck. However, most contemporary variation appears to have arisen recently and persisted due to exponential growth. We also describe how we use the range in allele size of microsatellites to estimate ancestral effective population size before the bottleneck, demonstrating a large reduction in effective size. We then employ a classical method for bacteria to estimate the microsatellite mutation rate in the species, deriving an estimate that is extremely similar to that estimated for a similar set of loci in humans, indicating consistency of microsatellite mutation rates in mammals. Finally, we find slight significant structure between some geographically separated colonies, although its biological significance is unclear. This work demonstrates that genetic analysis can be useful for evaluating the population biology of the northern elephant seal, in spite of the bottleneck that removed most genetic variation from the species. [ABSTRACT FROM AUTHOR]

We report 1212 radial-velocity (RV) measurements obtained in the years 2009-2013 using an iodine cell for the spectroscopic binary ν Octantis (K1 III/IV). This system (abin ~ 2.6 au, P ~ 1050 d) is conjectured to have a Jovian planet with a semimajor axis half that of the binary host. The extreme geometry only permits long-term stability if the planet is in a retrograde orbit. Whilst the reality of the planet (P ~ 415 d) remains uncertain, other scenarios (stellar variability or apsidal motion caused by a yet unobserved third star) continue to appear substantially less credible based on cross-correlation function bisectors, line-depth ratios and many other independent details. If this evidence is validated but the planet is disproved, the claims of other planets using RVs will be seriously challenged. We also describe a significant revision to the previously published RVs and the full set of 1437 RVs now encompasses nearly 13 yr. The sensitive orbital dynamics allow us to constrain the 3D architecture with a broad prior probability distribution on the mutual inclination, which with posterior samples obtained from an N-body Markov chain Monte Carlo is found to be 152:5±0.70.6. None of these samples are dynamically stable beyond 106 yr. However, a grid search around the best-fitting solution finds a region that has many models stable for 107 yr, and includes one model within 1σ that is stable for at least 108 yr. The planet's exceptional nature demands robust independent verification and makes the theoretical understanding of its formation a worthy challenge. [ABSTRACT FROM AUTHOR]

To overcome the ethical and technical limitations of in vivo human disease models, the broader scientific community frequently employs model organism-derived cell lines to investigate disease mechanisms, pathways, and therapeutic strategies. Despite the widespread use of certain in vitro models, many still lack contemporary genomic analysis supporting their use as a proxy for the affected human cells and tissues. Consequently, it is imperative to determine how accurately and effectively any proposed biological surrogate may reflect the biological processes it is assumed to model. One such cellular surrogate of human disease is the established mouse neural precursor cell line, SN4741, which has been used to elucidate mechanisms of neurotoxicity in Parkinson disease for over 25 years. Here, we are using a combination of classic and contemporary genomic techniques – karyotyping, RT-qPCR, single cell RNA-seq, bulk RNA-seq, and ATAC-seq – to characterize the transcriptional landscape, chromatin landscape, and genomic architecture of this cell line, and evaluate its suitability as a proxy for midbrain dopaminergic neurons in the study of Parkinson disease. We find that SN4741 cells possess an unstable triploidy and consistently exhibits low expression of dopaminergic neuron markers across assays, even when the cell line is shifted to the non-permissive temperature that drives differentiation. The transcriptional signatures of SN4741 cells suggest that they are maintained in an undifferentiated state at the permissive temperature and differentiate into immature neurons at the non-permissive temperature; however, they may not be dopaminergic neuron precursors, as previously suggested. Additionally, the chromatin landscapes of SN4741 cells, in both the differentiated and undifferentiated states, are not concordant with the open chromatin profiles of ex vivo, mouse E15.5 forebrain- or midbrain-derived dopaminergic neurons. Overall, our data suggest that SN4741 cells may reflect early aspects of neuronal differentiation but are likely not a suitable proxy for dopaminergic neurons as previously thought. The implications of this study extend broadly, illuminating the need for robust biological and genomic rationale underpinning the use of in vitro models of molecular processes. [ABSTRACT FROM AUTHOR]

Crystallographic studies of insulin bound to receptor domains have defined the primary hormone-receptor interface. Weinvestigated the role of TyrB26, a conserved aromatic residue at this interface. To probe the evolutionary basis for such conservation, we constructed 18 variants at B26. Surprisingly, nonaromatic polar or charged side chains (such as Glu, Ser, or ornithine (Orn)) conferred high activity, whereas the weakestbinding analogs contained Val, Ile, and Leu substitutions. Modeling of variant complexes suggested that the B26 side chains pack within a shallow depression at the solvent-exposed periphery of the interface. This interface would disfavor large aliphatic side chains. The analogs with highest activity exhibited reduced thermodynamic stability and heightened susceptibility to fibrillation. Perturbed self-assembly was also demonstrated in studies of the charged variants (Orn and Glu); indeed, the GluB26 analog exhibited aberrant aggregation in either the presence or absence of zinc ions. Thus, although TyrB26 is part of insulin's receptor-binding surface, our results suggest that its conservation has been enjoined by the aromatic ring's contributions to native stabilityand self-assembly. We envisage that such classical structural relationships reflect the implicit threat of toxic misfolding (rather than hormonal function at the receptor level) as a general evolutionary determinant of extant protein sequences. [ABSTRACT FROM AUTHOR]

We discuss an on-line tool that facilitates access to the large collection of climate impacts on crop yields produced by the Agricultural Model Intercomparison and Improvement Project. This collection comprises the output of seven crop models which were run on a global grid using climate data from five different general circulation models under the current set of representative pathways. The output of this modeling endeavor consists of more than 36,000 publicly available global grids at a spatial resolution of one half degree. We offer flexible ways to aggregate these data while reducing the technical barriers implied by learning new download platforms and specialized formats. The tool is accessed trough any standard web browser without any special bandwidth requirement. [ABSTRACT FROM AUTHOR]

The effect of administration of nanoliposome encapsulated malunggay (Moringa oleifera) phenolic extract in dogs given cholesterol on body fat and hepatic and renal ultrasonograms was investigated in this study. Six adult male dogs were given cholesterol without (T1) or with non-encapsulated (T2) and nanoliposome encapsulated (T3) malunggay (M. oleifera) phenolic extract. Data on the back fat thickness, body condition score and hepatic and renal ultrasonograms were collected at weeks 0, 1 and 5. Dogs in T3 showed inconsistent changes in the back fat thickness and body condition score. Moreover, T3 dogs exhibited decrease in both hepatic and renal echo mean values. The initial findings suggest that monitoring of the liver and kidney in cases of dyslipidemia or hyperlipidemia should be considered since ultrasonogram results indicate probable development of liver and kidney problems. Further studies are recommended to verify these results. [ABSTRACT FROM AUTHOR]

The hematologic profile and biochemical values in adult dogs given nanoliposome-encapsulated malunggay (Moringa oleifera) phenolic extract were determined in this study. Six adult dogs were divided into three groups: Group A: cholesterol only; Group B: cholesterol with malunggay phenolic extract; and Group C: cholesterol with nanoliposome-encapsulated malunggay phenolic extract, with two dogs per group. Each dog was subjected to a complete blood count at weeks 0, 1 and 5 and serum biochemistry tests at weeks 0 and 5. The results showed that thrombocytopenia and a decrease in the elevated blood urea nitrogen level were observed after administration of the nanoliposome-encapsulated malunggay phenolic extract. It also showed below normal cholesterol and low density lipoprotein levels and within normal triglyceride and high density lipoprotein levels after the experiment. These preliminary findings suggest that nanoliposome-encapsulated malunggay phenolic extract may have an effect on the platelets, kidneys, and lipid profile. However, further studies should be done to verify this association. [ABSTRACT FROM AUTHOR]

Negamycin is a natural product with broad-spectrum antibacterial activity and efficacy in animal models of infection. Although its precise mechanism of action has yet to be delineated, negamycin inhibits cellular protein synthesis and causes cell death. Here, we show that single point mutations within 16S rRNA that confer resistance to negamycin are in close proximity of the tetracycline binding site within helix 34 of the small subunit head domain. As expected from its direct interaction with this region of the ribosome, negamycin was shown to displace tetracycline. However, in contrast to tetracycline-class antibiotics, which serve to prevent cognate tRNA from entering the translating ribosome, single-molecule fluorescence resonance energy transfer investigations revealed that negamycin specifically stabilizes near-cognate ternary complexes within the A site during the normally transient initial selection process to promote miscoding. The crystal structure of the 70S ribosome in complex with negamycin, determined at 3.1 A resolution, sheds light on this finding by showing that negamycin occupies a site that partially overlaps that of tetracycline-class antibiotics. Collectively, these data suggest that the small subunit head domain contributes to the decoding mechanism and that small-molecule binding to this domain may either prevent or promote tRNA entry by altering the initial selection mechanism after codon recognition and before GTPase activation. [ABSTRACT FROM AUTHOR]

Insulin provides a model for the therapeutic application of protein engineering. A paradigm in molecular pharmacology was defined by design of rapid-acting insulin analogs for the prandial control of glycemia. Such analogs, a cornerstone of current diabetes regimens, exhibit accelerated subcutaneous absorption due to more rapid disassembly of oligomeric species relative to wild-type insulin. This strategy is limited by a molecular trade-off between accelerated disassembly and enhanced susceptibility to degradation. Here, we demonstrate that this trade-off may be circumvented by nonstandard mutagenesis. Our studies employed LysB28, ProB29-insulin ("lispro") as a model prandial analog that is less thermodynamically stable and more susceptible to fibrillation than is wild-type insulin. We have discovered that substitution of an invariant tyrosine adjoining the engineered sites in lispro (TyrB26) by 3-iodo-Tyr (i) augments its thermodynamic stability (ΔΔGu 0.5 ±0.2 kcal/ mol), (ii) delays onset of fibrillation (lag time on gentle agitation at 37 °C was prolonged by 4-fold), (iii) enhances affinity for the insulin receptor (1.5 ± 0.1-fold), and (iv) preserves biological activity in a rat model of diabetes mellitus. 1HNMRstudies suggest that the bulky iodo-substituent packs within a nonpolar interchain crevice. Remarkably, the 3-iodo-TyrB26 modification stabilizes an oligomeric form of insulin pertinent to pharmaceutical formulation (the R6 zinc hexamer) but preserves rapid disassembly of the oligomeric form pertinent to subcutaneous absorption (T6 hexamer). By exploiting this allosteric switch, 3-iodo-TyrB26-lispro thus illustrates how a nonstandard amino acid substitution can mitigate the unfavorable biophysical properties of an engineered protein while retaining its advantages. [ABSTRACT FROM AUTHOR]

Proteins targeted to the plasma membrane (PM) of cells are degraded at different rates. Sorting motifs contained within the cytoplasmic domains of transmembrane proteins, post-translational modifications (e.g. ubiquitination), and assembly into multiprotein or protein-lipid complexes all may affect the efficiency of endocytosis and recycling and influence the delivery to degradative compartments. Using the SNAP-tag labeling system, we examined the turnover of a model PM protein, the α chain of the interleukin-2 receptor (Tac). The surface lifetimes of SNAP-Tac fusions were influenced by their mode of entry into cells (clathrin-dependent versus clathrin-independent), their orientation in the PM (transmembrane versus glycosylphosphatidylinositol- anchored), and ubiquitination in their cytosolic domains. In addition, shedding of SNAP-Tac into the medium was greatly influenced by its O-linked glycosylation status. For a number of PM proteins, delivery to lysosomes and ectodomain shedding represent distinct parallel mechanisms to determine protein half-life. [ABSTRACT FROM AUTHOR]

This study marks the first floristic inventory of Phillips and Valley counties on the glaciated plains of northeastern Montana. The 23,191 sq km (8,954 sq mi) area was surveyed for all vascular plant taxa on lands managed by the Bureau of Land Management, U.S. Fish and Wildlife Service, State of Montana, American Prairie Reserve, and The Nature Conservancy. In the summers of 2010 and 2011, 12,768 voucher specimens were collected from 308 sites documenting 762 unique taxa, 718 species, and 358 genera from 86 families. Among these are 108 taxa exotic to Montana, nine noxious weed species, and 15 taxa of conservation concern. Approximately 30 percent of the taxa collected are newly documented within the area. An additional 70 taxa previously collected by other workers and housed at MONT, MONTU, or RM/ USFS raised the total number of unique taxa to 832. Results are enumerated in an annotated checklist and vegetation types are described. Analyses of the study's sampling adequacy are also discussed. [ABSTRACT FROM AUTHOR]

The COVID-19 pandemic has resulted in a predominantly global quarantine response that has been associated with social isolation, loneliness, and anxiety. The foregoing experiences have been amply documented to have profound impacts on health, morbidity, and mortality. This narrative review uses the extant neurobiological and theoretical literature to explore the association between social isolation, loneliness, and anxiety in the context of quarantine during the COVID-19 pandemic. Emerging evidence suggests that distinct health issues (e.g., a sedentary lifestyle, a diminished overall sense of well-being) are associated with social isolation and loneliness. The health implications of social isolation and loneliness during quarantine have a heterogenous and comorbid nature and, as a result, form a link to anxiety. The limbic system plays a role in fear and anxiety response; the bed nucleus of the stria terminalis, amygdala, HPA axis, hippocampus, prefrontal cortex, insula, and locus coeruleus have an impact in a prolonged anxious state. In the conclusion, possible solutions are considered and remarks are made on future areas of exploration. [ABSTRACT FROM AUTHOR]

Many plasma membrane (PM) proteins enter cells nonselectively through clathrin-independent endocytosis (CIE). Here, we present evidence that cytoplasmic sequences in three CIE cargo proteins--CD44, CD98, and CD147--were responsible for the rapid sorting of these proteins into endosomal tubules away from endosomes associated with early endosomal antigen 1 (EEA1). We found that Hook1, a microtubule- and cargo-tethering protein, recognized the cytoplasmic tail of CD147 to help sort it and CD98 into Rab22a-dependent tubules associated with recycling. Depletion of Hook1 from cells altered trafficking of CD44, CD98, and CD147 toward EEA1 compartments and impaired the recycling of CD98 back to the PM. In contrast, another CIE cargo protein, major histocompatibility complex class I, which normally traffics to EEA1 compartments, was not affected by depletion of Hook1. Loss of Hook1 also led to an inhibition of cell spreading, implicating a role for Hook1 sorting of specific CIE cargo proteins away from bulk membrane and back to the PM. [ABSTRACT FROM AUTHOR]

This inventory expands the floristic coverage of the Medicine Bow Mountains (Medicine Bow National Forest proper) located in southeastern Wyoming. The Forest, covering 2,150 sq km (830 sq mi) and ranging in elevation from 2,400-3,650 m (7,900-12,013 ft), was surveyed for all species of vascular plants. Most of the lands inventoried are in the Medicine Bow-Routt National Forest but small parcels are under the jurisdiction of the Bureau of Land Management or the State of Wyoming. These parcels are within three miles of the Forest boundary. Over three summers, 9,837 numbered specimens were collected at 168 sites. Two unnamed putative hybrids were documented. A summary of taxa follows (numbers in parentheses are taxa documented by other workers): 88 families, 376 genera, 835 (201) species, 910 (212) unique taxa for a combined total of 1,122 unique or terminal taxa. Alchemilla filicaulis ssp. filicaulis and Carex arcta are species new to Wyoming. Also documented were 51 (18) exotics as well as seven noxious weeds. Twenty-two species of conservation concern were also found at 51 locations. [ABSTRACT FROM AUTHOR]

Dairy food wastewater disposal represents a major environmental problem. This review discusses micro-organisms associated with anaerobic digestion of dairy food wastewater, biochemistry of the process, factors affecting anaerobic digestion, and efforts to develop defined cultures. Anaerobic digestion of dairy food waste-water offers many advantages over other treatments in that a high level of waste stabilization is achieved with much lower levels of sludge. In addition, the process produces readily usable methane with low nutrient requirements and no oxygen. Anaerobic digestion is a series of complex reactions that broadly involve 2 groups of anaerobic or facultative anaerobic microorganisms: acidogens and methanogens. The first group of microorganisms breaks down organic compounds into CO2 and volatile fatty acids. Some of these organisms are acetogenic, which convert long-chain fatty acids to acetate, CO2, and hydrogen. Methanogens convert the acidogens' products to methane. The imbalance among the different microbial groups can lead not only to less methane production, but also to process failure. This is due to accumulation of intermediate compounds, such as volatile fatty acids, that inhibit methanogens. The criteria used for evaluation of the anaerobic digestion include levels of hydrogen and volatile fatty acids, methane:carbon ratio, and the gas production rate. A steady state is achieved in an anaerobic digester when the pH, chemical oxygen demand of the effluent, the suspended solids of the effluent, and the daily gas production remain constant. Factors affecting efficiency and stability of the process are types of microorganisms, feed C:N ratio, hydraulic retention time, reactor design, temperature, pH control, hydrogen pressure, and additives such as manure and surfactants. As anaerobic digesters become increasingly used in dairy plants, more research should be directed toward selecting the best cultures that maximize methane production from dairy food waste. [ABSTRACT FROM AUTHOR]

Abstract: It is well established that exposure of mice to perfluorooctanoate (PFOA) or perfluorooctane sulfonate (PFOS) exerts adverse effects on the thymus and spleen. Here, we characterize the effects of a 10-day dietary treatment with these compounds (0.001–0.02%, w/w) on the bone marrow (BM) of mice. At a dose of 0.02%, both compounds reduced food consumption and caused atrophy of the thymus and spleen. At this same dose, histopathological and flow cytometric analysis revealed that (i) the total numbers of BM as well as the numbers of myeloid, pro/pre B, immature B and early mature B cells were all reduced significantly; and (ii) these adverse effects were reversed either partially or completely 10days after withdrawal of these compounds. At the lower dose of 0.002%, only PFOA reduced the B-lymphoid cell population. Finally, mice fed an amount of diet equivalent to that consumed by the animals exposed to 0.02% PFOA also exhibited atrophy of the thymus and spleen, and a reduction in the number of B-lymphoid population, without affecting myeloid cells. Thus, in mice, immunotoxic doses of PFOA or PFOS induce adverse effects on the myeloid and B-lymphoid cells in the BM, in part as a consequence of reduced food consumption. [Copyright &y& Elsevier]

Mammalian testis-determining factor SRY contains a high mobility group box, a conserved eukaryotic motif of DNA bending. Mutations in SRY cause XY gonadal dysgenesis and somatic sex reversal. Although such mutations usually arise de novo in spermatogenesis, some are inherited and so specify male development in one genetic background (the father) but not another (the daughter). Here, we describe the biophysical properties of a representative inherited mutation, V60L, within the minor wing of the L-shaped domain (box position 5). Although the stability and DNA binding properties of the mutant domain are similar to those of wild type, studies of SRY-induced DNA bending by subnanosecond time-resolved fluorescence resonance energy transfer (FRET) revealed enhanced conformational fluctuations leading to long range variation in bend angle. 1H NMR studies of the variant protein-DNA complex demonstrated only local perturbations near the mutation site. Because the minor wing of SRY folds on DNA binding, the inherited mutation presumably hinders induced fit. Stopped-flow FRET studies indicated that such frustrated packing leads to accelerated dissociation of the bent complex. Studies of SRY-directed transcriptional regulation in an embryonic gonadal cell line demonstrated partial activation of downstream target Sox9. Our results have demonstrated a nonlocal coupling between DNA-directed protein folding and protein-directed DNA bending. Perturbation of this coupling is associated with a genetic switch poised at the threshold of activity. [ABSTRACT FROM AUTHOR]

Methionine sulfoxide reductase A is an essential enzyme in the antioxidant system, which scavenges reactive oxygen species through cyclic oxidation and reduction of methionine and methionine sulfoxide. In mammals, one gone encodes two forms of the reductase, one targeted to the cytosol and the other to mitochondria. The cytosolic form displays faster mobility than the mitochondrial form, suggesting a lower molecular weight for the former. The apparent size difference and targeting to two cellular compartments had been proposed to result from differential splicing of mRNA. We now show that differential targeting is effected by use of two initiation sites, one of which includes a mitochondrial targeting sequence, whereas the other does not. We also demonstrate that the mass of the cytosolic form is not less than that of the mitochondrial form; the faster mobility of cytosolic form is due to its myristoylation. Lipidation of methionine sulfoxide reductase A occurs in the mouse, in transfected tissue culture cells, and even in a cell-free protein synthesis system. The physiologic role of myristoylation of MsrA remains to he elucidated. [ABSTRACT FROM AUTHOR]

The carbohydrate 2,4-diacetamido-2,4,6-trideoxy-α-D-glucopyranose (BacAc2) is found in a variety of eubacterial pathogens. In Campylobacter jejuni, PglD acetylates the C4 amino group on UDP-2-acetamido-4-amino-2,4,6-trideoxy-α-D-glucopyranose (UDP-4-amino-sugar) to form UDP-BacAc2. Sequence analysis predicts PglD to be a member of the left-handed β helix family of enzymes. However, poor sequence homology between PglD and left-handed β helix enzymes with existing structural data precludes unambiguous identification of the active site. The co-crystal structures of PglD in the presence of citrate, acetyl coenzyme A, or the UDP-4-amino-sugar were solved. The biological assembly is a trimer with one active site formed between two protomers. Residues lining the active site were identified, and results from functional assays on alanine mutants suggest His-125 is critical for catalysis, whereas His-15 and His-134 are involved in substrate binding. These results are discussed in the context of implications for proteins homologous to PglD in other pathogens. [ABSTRACT FROM AUTHOR]

For a wide variety of microbial pathogens, the outcome of the infection is indeterminate. In some individuals the microbe is cleared, but in others it establishes a chronic infection, and the factors that tip this balance are often unknown. In a widely used model of chronic viral infection, C57BL/6 mice clear the Armstrong strain of lymphocytic choriomeningitis virus (LCMV), but the clone 13 strain persists. Here we show that the Armstrong strain induces a profound lymphopenia at days 1–3 after infection, but the clone 13 strain does not. If we transiently augment lymphopenia by treating the clone-13-infected mice with the drug FTY720 at days 0–2 after infection, the mice successfully clear the infection by day 30. Clearance does not occur when CD4 T cells are absent at the time of treatment, indicating that the drug is not exerting direct antiviral effects. Notably, FTY720 treatment of an already established persistent infection also leads to viral clearance. In both models, FTY720 treatment preserves or augments LCMV-specific CD4 and CD8 T-cell responses, a result that is counter-intuitive because FTY720 is generally regarded as a new immunosuppressive agent. Because FTY720 targets host pathways that are completely evolutionarily conserved, our results may be translatable into new immunotherapies for the treatment of chronic microbial infections in humans. [ABSTRACT FROM AUTHOR]

Phytophthora root rot, caused by Phytophthora sojae, is the most important disease of soybean (Glycine max) in North Dakota. Because of the expansion of soybean hectares and appearance of disease on cultivars with resistance genes, we investigated the pathotypes, distribution, and metalaxyl sensitivity of P. sojae in North Dakota. Soil from 347 soybean fields in 20 counties in eastern North Dakota was collected between 2002 and 2004, and P. sojae was baited from the soil with the susceptible cultivar McCall. The virulence phenotype of each isolate was determined on eight differentials, and all isolates were tested for sensitivity to metalaxyl incorporated into V8 agar. The pathogen was recovered from 80 fields located in five counties. Sixteen pathotypes, which included 14 known races and two previously reported pathotypes that had not been assigned a race, were identified out of 157 isolates. A single pathotype was recovered from 61 fields, 2 pathotypes from 14 fields, 3 pathotypes from 4 fields, and 4 pathotypes from 1 field. Pathotypes with virulence phenotypes 1a,1c,7 (race 4; 39%) and 1a,7 (race 3; 28%) were the most common, representing 67% of the total isolates. One or both of these pathotypes was found in 79% of the fields where P. sojae was recovered. Seven of the 157 isolates showed limited growth on metalaxyl after 14 days of incubation. In the past 10 years, the number of pathotypes of P. sojae in North Dakota has increased from 4 to 16, and pathotypes have developed that can attack the three most common resistance genes found in soybean cultivars for the region. [ABSTRACT FROM AUTHOR]

Activation of cyclic nucleotide-modulated channels such as CNG and HCN channels is promoted by ligand-induced conformational changes in their C-terminal regions. The primary intersubunit interface of these C termini includes two salt bridges per subunit, formed between three residues (one positively charged and two negatively charged amino acids) that we term the SB triad. We previously hypothesized that the SB triad is formed in the closed channel and breaks when the channel opens. Here we tested this hypothesis by dynamically manipulating the SB triad in functioning CNGA1 channels. Reversing the charge at positions Arg-431 and Glu-462, two of the SB triad residues, by either mutation or application of charged reagents increased the favorability of channel opening. To determine how a charge reversal mutation in the SB triad structurally affects the channel, we solved the crystal structure of the HCN2 C-terminal region with the equivalent E462R mutation. The backbone structure of this mutant was very similar to that of wild type, but the SB triad was rearranged such that both salt bridges did not always form simultaneously, suggesting a mechanism for the increased ease of opening of the mutant channels. To prevent movement in the SB triad, we tethered two components of the SB triad region together with cysteine-reactive cross-linkers. Preventing normal movement of the SB triad region with short cross-linkers inhibited channel opening, whereas longer cross-linkers did not. These results support our hypothesis that the SB triad forms in the closed channel and indicate that this region expands as the channel opens. [ABSTRACT FROM AUTHOR]

Non-human primates (NHP) provide crucial research models. Their strong similarities to humans make them particularly valuable for understanding complex behavioral traits and brain structure and function. We report here the genetic mapping of an NHP nervous system biologic trait, the cerebrospinal fluid (CSF) concentration of the dopamine metabolite homovanillic acid (HVA), in an extended inbred vervet monkey (Chlorocebus aethiops sabaeus) pedigree. CSF HVA is an index of CNS dopamine activity, which is hypothesized to contribute substantially to behavioral variations in NHP and humans. For quantitative trait locus (QTL) mapping, we carried out a two-stage procedure. We first scanned the genome using a first-generation genetic map of short tandem repeat markers. Subsequently, using >100 SNPs within the most promising region identified by the genome scan, we mapped a QTL for CSF HVA at a genome-wide level of significance (peak logarithm of odds score >4) to a narrow well delineated interval (<10 Mb). The SNP discovery exploited conserved segments between human and rhesus macaque reference genome sequences. Our findings demonstrate the potential of using existing primate reference genome sequences for designing high-resolution genetic analyses applicable across a wide range of NHP species, including the many for which full genome sequences are not yet available. Leveraging genomic information from sequenced to nonsequenced species should enable the utilization of the full range of NHP diversity in behavior and disease susceptibility to determine the genetic basis of specific biological and behavioral traits. [ABSTRACT FROM AUTHOR]

Whey proteins in general and specifically β-lactoglobulin, α-lactalbumin, and immunoglobulins have been thought to decrease proteolysis in cheeses manufactured from concentrated retentates from ultrafiltration. The proteins found in whey are called whey proteins and are called milk serum proteins (SP) when they are in milk. The experiment included 3 treatments; low milk SP (0.18%), control (0.52%), and high milk SP (0.63%), and was replicated 3 times. The standardized milk for cheese making of the low milk SP treatment contained more casein as a percentage of true protein and more calcium as a percentage of crude protein, whereas the nonprotein nitrogen and total calcium content was not different from the control and high SP treatments. The nonprotein nitrogen and total calcium content of the milks did not differ because of the process used to remove the milk SP from skim milk. The low milk SP milk contained less free fatty acids (FFA) than the control and high milk SP treatment; however, no differences in FFA content of the cheeses was detected. Approximately 40 to 45% of the FFA found in the milk before cheese making was lost into the whey during cheese making. Decreasing the milk SP content of milk by 65% and increasing the content by 21% did not significantly influence general Cheddar cheese composition. Higher fat recovery and cheese yield were detected in the low milk SP treatment cheeses. There was more proteolysis in the low milk SP cheese and this may be due to the lower concentration of undenatured β-lactoglobulin, α-lactalbumin, and other high molecular weight SP retained in the cheeses made from milk with low milk SP content. [ABSTRACT FROM AUTHOR]

Transcriptional dysregulation as a result of sequestration of essential transcription factors into protein aggregates formed by polyglutamine (polyQ) expansions can lead to late-onset progressive neurodegeneration. DNA microarray analysis of Drosophila expressing polyQ in the compound eye over time revealed large numbers of transcriptional changes at the earliest stages of the disease including repression of the transient receptor potential calcium channels in a polyQ-induced cell death specific manner. While significant differences in expression profiles were found between the Drosophila compound eye and polyQ-sensitive neural cells, a number of possible key overlapping regulators were extracted. Among these, PDK1 was shown to act as a mediator for polyQ-toxicity, suggesting the involvement of the TOR pathway in polyQ-induced neurodegeneration.Cell Death and Differentiation (2005) 12, 1115–1123. doi:10.1038/sj.cdd.4401635; published online 29 April 2005 [ABSTRACT FROM AUTHOR]

Microfiltration (MF) is a membrane process that can separate casein micelles from milk serum proteins (SP), mainly β-lactoglobulin and α-lactalbumin. Our objective was to develop a multistage MF process to remove a high percentage of SP from skim milk while producing a low concentration factor retentate from microfiltration (RMF) with concentrations of soluble minerals, nonprotein nitrogen (NPN), and lactose similar to the original skim milk. The RMF could be blended with cream to standardize milk for traditional Cheddar cheese making. Permeate from ultrafiltration (PUF) obtained from the ultrafiltration (UF) of permeate from MF (PMF) of skim milk was successfully used as a diafiltrant to remove SP from skim milk before cheese making, while maintaining the concentration of lactose, NPN, and nonmicellar calcium. About 95% of the SP originally in skim milk was removed by combining one 3x MF stage and two 3x PUF diafiltration stages. The final 3x RMF can be diluted with PUF to the desired concentration of casein for traditional cheese making. The PMF from the skim milk was concentrated in a UF system to yield an SP concentrate with protein content similar to a whey protein concentrate, but without residuals from cheese making (i.e., rennet, culture, color, and lactic acid) that can produce undesirable functional and sensory characteristics in whey products. Additional processing steps to this 3-stage MF process for SP removal are discussed to produce an MF skim retentate for a continuous cottage cheese manufacturing process. [ABSTRACT FROM AUTHOR]

Nine isolates of binucleate Rhizoctonia (BNR) from soybean were screened in the greenhouse for control of Rhizoctonia solani anastomosis groups AG-4 and AG-2-2. Eight of nine BNR isolates, when combined with AG-4 or AG-2-2, significantly increased emergence and survival of soybean (cv. Ozzie) and reduced disease severity compared with AG-4 or AG-2-2 alone. The interaction of soybean cultivar and BNR isolates in the presence of AG-4 and AG-2-2 was also studied using three isolates of BNR, BNR-4, BNR-8-2, and BNR-8-3, and seven soybean cultivars. There was no BNR x cultivar interaction. With AG-4, BNRs significantly increased emergence and survival of cultivars and reduced disease severity, whereas with AG-2-2, BNRs reduced disease severity. Control of R. solani by BNRs was achieved in both a potting soil mix and natural soil. In the initial screening experiments, two BNR isolates reduced emergence, but in all subsequent experiments using three BNR isolates alone, there were no negative effects on germination, survival, or height of soybean plants, and there was no evidence of pathogenicity. In several experiments, BNRs alone significantly increased height of plants compared with the noninoculated controls. BNRs were consistently isolated from hypocotyls and roots, indicating colonization of tissues was associated with control. These BNR isolates may have potential use in management of R. solani in soybean, but will require rigorous testing under field conditions and more extensive studies of their biology. [ABSTRACT FROM AUTHOR]

Oxidative stress is implicated in a number of neurodegenerative diseases and is associated with the selecrive loss of dopaminergic neurons of the substantia nigra in Parkinson's disease. The role of α-synuclein as a potential target of intracellular oxidants has been demonstrated by the identification of posttranslational modifications of synuclein within intracellular aggregates that accumulate in Parkinson's disease brains, as well as the ability of a number of oxidative insults to induce synuclein oligomerization. The relationship between these relatively small soluble oligomers, potentially neurotoxic synuclein protofibrils, and synuclein filaments remains unclear. We have found that metal-catalyzed oxidation of α-synuclein inhibited formation of synuclein filaments with a concomitant accumulation of β sheet-rich oligomers that may represent synuclein protofibrils. Similar results with a number of oxidative and enzymatic treatments suggest that the covalent association of synuclein into higher molecular mass oligomers/protofibrils represents an alternate pathway from filament formation and renders synuclein less prone to proteasomal degradation. [ABSTRACT FROM AUTHOR]

Cyclic nucleotides directly enhance the opening of the tetrameric CNG and HCN channels, although the mechanism remains unclear. We examined why HCN and certain CNG subunits form functional homomeric channels, whereas other CNG subunits only function in heteromeric channels. The “defect” in the CNGA4 subunit that prevents its homomeric expression was localized to its C-linker, which connects the transmembrane domain to the binding domain and contains a tripeptide that decreases the efficacy of ligand gating. Remarkably, replacement of the homologous HCN tripeptide with the CNGA4 sequence transformed cAMP into an inverse agonist that inhibits HCN channel opening. Using analytical ultracentrifugation, we identified the structural basis for this gating switch: whereas cAMP normally enhances the assembly of HCN C-terminal domains into a tetrameric gating ring, inclusion of the CNGA4 tripeptide reversed this action so that cAMP now causes gating ring disassembly. Thus, ligand gating depends on the dynamic oligomerization of C-terminal binding domains. [Copyright &y& Elsevier]

To determine the influence of milk preacidification with CO2 on Cheddar cheese aging and proteolysis, cheese was manufactured from milk with and without added CO2. The experiment was replicated 3 times. Carbon dioxide (approximately 1600 ppm) was added to the cold milk, resulting in a milk pH of 5.9 at 31°C in the cheese vat. The starter and coagulant usage rates were equal for the control and CO2 treatment cheeses. The calcium content of the CO2 treatment cheese was lower, but no difference in moisture content was detected. The higher CO2 content of the treatment cheeses (337 vs. 124 ppm) was maintained throughout 6 mo of aging. In spite of having almost one and a half times the salt-in-moisture, proteolysis as measured by pH 4.6 and 12% trichloroacetic acid soluble nitrogen expressed as percentages of total nitrogen, was higher in the CO2 treatment cheeses throughout aging. The ratio of αs-casein (CN) to para-κ-CN decreased faster in the CO2 treatment cheeses than in the control cheeses, especially before refrigerated storage. No difference was detected in the ratio of β-CN to para-κ-CN between the control and CO2 treatment cheeses. Intact αs- and β-CN were found in the expressible serum (ES) from the CO2 treatment cheese as well as αs1-I-CN, but they were not detected in the ES from the control cheese. No CN was detected in the ES from the curd before the salting of either the control or CO2 treatment cheese. Higher proteolysis in the cheese made from milk preacidified with CO2 may have been due to increased substrate availability in the water phase or increased chymosin activity or retention in the cheese. [ABSTRACT FROM AUTHOR]

Preacidification of milk for cheese making may have a beneficial impact on increasing proteolysis during cheese aging. Unlike other acids, CO2 can easily be removed from whey. The objectives of this work were to determine the effect of milk preacidification on Cheddar cheese composition, the recovery of individual milk components, and yield. Carbon dioxide was injected inline after the cooling section of the pasteurizer. Cheeses with and without added CO2 were made simultaneously from the same batch of milk. This procedure was replicated 3 times. Carbon dioxide in the cheese milk was about 1600 ppm, which resulted in a milk pH of about 5.9 at 31°C. The starter culture and coagulant addition rates were the same for both the CO2 treatment and the control. The whey pH at draining of the CO2 treatment was lower than the control. Total make time was shorter for the CO2 treatment compared with the control. Cheese manufactured from milk acidified with CO2 retained less of the total calcium and fat than the control cheese. The higher fat loss was primarily in the whey at draining. Preacidification with CO2 did not alter the crude protein recovery in the cheese. The CO2 treatment resulted in a higher added salt recovery in the cheese and produced a cheese that contained too much salt. Considering the higher added salt retention, the salt application rate could be lowered to achieve a typical cheese salt content. Cheese yield efficiency of the CO2 treated milk was 4.4% lower than the control due to fat loss. Future work will focus on modifying the make procedure to achieve a normal fat loss into the whey when CO2 is added to milk. [ABSTRACT FROM AUTHOR]

Normally, reduced-fat Cheddar cheese is made by removal of fat from milk prior to cheese making. Typical aged flavor may not develop when 50% reduced-fat Cheddar cheese is produced by this approach. Moreover, the texture of the reduced-fat cheeses produced by the current method may often be hard and rubbery. Previous researchers have demonstrated that aged Cheddar cheese flavor intensity resides in the watersoluble fraction. Therefore, we investigated the feasibility of fat removal after the aging of Cheddar cheese. We hypothesized the typical aged cheese flavor would remain with the cheese following fat removal. A physical process for the removal of fat from full-fat aged Cheddar cheese was developed. The efficiency of fat removal at various temperatures, gravitational forces, and for various durations of applied forces was determined. Temperature had the greatest effect on the removal of fat. Gravitational force and the duration of applied force were less important at higher temperatures. A positive linear relationship between temperature and fat removal was observed from 20 to 33°C. Conditions of 30°C and 23,500 x g for 5 min removed 50% of the fat. The removed fat had some aroma but little or no taste. The fatty acid composition, triglyceride molecular weight distribution, and melting profile of the fat retained in the reduced-fat cheeses were all consistent with a slight increase in the proportion of saturated fat relative to the full-fat cheeses. The process of fat removal decreased the grams of saturated fat per serving of cheese from 6.30 to 3.11 g. The flavor intensity of the reduced-fat cheeses were at least as intense as the full-fat cheeses. [ABSTRACT FROM AUTHOR]

The effects of Sclerotinia stem rot, caused by Scierotinia scierotiorum, on yield of soybean were evaluated in the field with two cultivars in maturity group 0. Plants were inoculated at two growth stages, R3 and R5, using two inoculation methods. Seed weight, seed and pod numbers, seed protein, and oil content were measured. The effects of disease on yield were variable. Disease resulted in significant seed weight loss, with reductions per diseased plant ranging from 18.8 to 38.6%. The estimated yield loss per 10% disease incidence ranged from 83.2 to 229.0 kg/ha, with an average loss of 136.6 kg/ha for four field experiments. A reduction in the number of seeds and pods per plant and seed oil content occurred in some, but not all, experiments Seed protein was not affected. When disease reduced seed weight, seed and pod numbers, or oil content, there was no growth stage x treatment interaction in the experiments, indicating that inoculation at R5 compared with R3 had a similar effect on yield. [ABSTRACT FROM AUTHOR]

The family of hyperpolarization-activated, cyclic nucleotide-modulated (HCN) channels are crucial for a range of electrical signalling, including cardiac and neuronal pacemaker activity, setting resting membrane electrical properties and dendritic integration. These nonselective cation channels, underlying the If, Ih and Iq currents of heart and nerve cells, are activated by membrane hyperpolarization and modulated by the binding of cyclic nucleotides such as cAMP and cGMP. The cAMP-mediated enhancement of channel activity is largely responsible for the increase in heart rate caused by ß-adrenergic agonists. Here we have investigated the mechanism underlying this modulation by studying a carboxy-terminal fragment of HCN2 containing the cyclic nucleotide-binding domain (CNBD) and the C-linker region that connects the CNBD to the pore. X-ray crystallographic structures of this C-terminal fragment bound to cAMP or cGMP, together with equilibrium sedimentation analysis, identify a tetramerization domain and the mechanism for cyclic nucleotide specificity, and suggest a model for ligand-dependent channel modulation. On the basis of amino acid sequence similarity to HCN channels, the cyclic nucleotide-gated, and eag- and KAT1-related families of channels are probably related to HCN channels in structure and mechanism. [ABSTRACT FROM AUTHOR]

Presents an obituary to Lodewijk A. Sandkuiji, a statistical geneticist who died on December 4, 2002. Contributions of Sandkuiji in setting database for registration of DNA samples in Netherlands; Details of Sandkuiji's early career; Role of Sandkuiji in successfully mapping a minimum of 30 Mendelian disorders; Assignments undertaken in Dutch medical schools.

Potassium ion (K+) plays a critical role as an essential electrolyte in all biological systems. Genetically encoded fluorescent K+ biosensors are promising tools to further improve our understanding of K+-dependent processes under normal and pathological conditions. Here, we report the crystal structure of a previously reported genetically encoded fluorescent K+ biosensor, GINKO1, in the K+-bound state. Using structure-guided optimization and directed evolution, we have engineered an improved K+ biosensor, designated GINKO2, with higher sensitivity and specificity. We have demonstrated the utility of GINKO2 for in vivo detection and imaging of K+ dynamics in multiple model organisms, including bacteria, plants, and mice. Potassium ions play a critical role as an essential electrolyte in all biological systems. This study describes high performance genetically encoded potassium ion sensors to enable in vivo measurement of potassium ion concentrations across multiple model organisms. [ABSTRACT FROM AUTHOR]

Reports on a study which investigated the hypothesis which stated that the neurotrophin brain-derived neurotrophic factor (BDNF) influenced estrogen and the gamma-aminobutyric acid (GABA)ergic interneurons' control. Importance of dendritic spines in the central neurons' information processing and memory formation; In-depth look at the BDNF; Methodology used to conduct the study; Results of the study.

Presents information pertaining to the biological aspects involving the endoplasmic reticulum (ER) while focusing on the plasma membrane. Information on the biological properties of the ER; Reference to the study of the retrieval of proteins back to the ER; Fusing of the lumenal domain of the temperature-sensitive viral glycoprotein.

Describes the functions of the membrane protein kinesin as the motor for microtubule-mediated Golgi apparatus-to-endoplasmic reticulum (ER) traffic. Dependence of distribution and dynamics of both ER and Golgi complex on microtubules; Kinesin as the motor powering peripherally directed movements of membrane within the system; Structure-activity relationship of kinesin.

Reports on a new technique for tagging proteins fluorescently which has made it possible for scientists to follow the complete life history of single transport intermediates in living cells, including their formation, path and velocity en route to the Golgi complex from the endoplasmic reticulum. Methods used in the technique's testing.

Details a study to uncover the mechanism by which Golgi membrane proteins are retained within the Golgi complex in the midst of a continuous flow of protein and lipid. Examination of the diffusional mobility of Golgi membrane components using fluorescence photobleaching recovery (FPR); What the diffusion coefficients were; Mobility and diffusion of the chimeric proteins; Conclusion about protein immobilization and Golgi targeting and retention.

Antibodies against isolated beef-heart ubiquinol- cytochrome c reductase (complex III) have been characterized. Antibodies to complex III react strongly with isolated beef heart complex III and intact beef heart mitochondria, as shown by immunodiffusion and rocket electrophoresis experiments. The complex III content of intact mitochondria can be quantitated with rocket electrophoresis using isolated complex III as a standard. Antibodies to complex III also react with beef liver mitochondria and with both heart and liver mitochondria from rats. The latter are very weak antigens compared to beef heart material. Antibodies to complex III do not react with respiratory chain complexes I and IV, or F1-ATPase from beef heart mitochondria, but gives a slight, but variable, reaction with complex II and the membrane fraction isolated from complex V (oligomycin-sensitive ATPase). Antigenic sites are located on at least five of the seven peptides of complex III. These peptides are presumably lacking in respiratory chain complexes which do not react with antibodies to complex III, and are assumed to be uniquely located in complex HI. Antiserum against complex III inhibits duroquinol-cytochrome c reductase activity in isolated complex III and in complex III incorporated into phospholipid vesicles. Oxidation of NADH and succinate is not affected in submitochondrial particles treated with 6-times more antibody than required for complete inhibition of enzyme activity in free complex III or in complex III-phospholipid vesicles. [ABSTRACT FROM AUTHOR]

The arrangement of the peptides of free complex III and complex III incorporated into phospholipid vesicles was investigated by labeling with 125I + lactoperoxidase and with p-diazoniumbenzene [35S]sulfonate (N=N-PhSO3H). The peptides were separated by sodium dodecylsulfate acrylamide gel electrophoresis and scanned for radioactivity. In free complex III, the most extensive iodination occurred in peptides II and IV, whereas all polypeptides (I -VI), except peptide VII, could be coupled to N=N-PhSO3H. Peptide VII was, however, labeled in the sodium-dodecylsulfate- dissociated enzyme. These results show that, with the possible exception of peptide VII, all peptides are at least partially exposed on the surface of the isolated complex. Complex III incorporated into phospholipid vesicles showed a labeling pattern which differed from the free enzyme, both with respect to the total incorporation and to the relative incorporation between the peptides. Total incorporation of N=N-Ph35SO3H and 125I was decreased by 60 - 70% in complex III=vesicles, suggesting that a large number of the binding sites on the free enzyme become buried within the membrane. In addition, peptides I, II, IV and V were relatively less labeled in complex III-vesicles than were peptides III and VI, suggesting a deeper location of the former peptides within the membrane. This is supported by the finding that labeling of deoxycholate-treated complex III-vesicles with N = N - Ph35SO3H results in an incorporation pattern similar to that in free complex III. Binding of N = N -PhSO3H and its effects on enzymatic activity was also studied in free and membrane-bound complex III. Both the binding of N = N -Ph35SO3H and inactivation of duroquinol -cytochrome c reductase were significantly slower in complex Ill-vesicles. These results indicate that many of the N = N -PhCO3H-binding sites on the free complex are inaccessible when the enzyme is incorporated into a vesicle. This approach was also used to ascertain intactness of the vesicles, and to show that N = N -PhSO3H labeled only those peptides exposed on the outer surface. [ABSTRACT FROM AUTHOR]

The peptides of purified complex III from beef heart mitochondria have been studied by electrophoresis on dodecylsulfate gels. Of the 12 peptides consistently observed, only eight appear to be integral peptides of the functional complex. Attempts to identify these peptides have been made through co-electrophoresis of complex [I and fractions in which the individual peptides were either purified or greatly enriched. Electrophoresis of complex III preparations which were not reduced by mercaptoethanol indicates that intermolecular disulfide bonds play no significant role in stabilizing the complex. [ABSTRACT FROM AUTHOR]

The nature of mitochondrial PBC-related antigens has been investigated with radioimmunoassay (RIA) and immunoblotting methods. The major antigen(s) was located by RIA in beef heart mitochondria, submitochondrial particles, chloroform-extracted F1-ATPase and Complex III. Cross-competition RIA experiments showed that the same antigen is present in all the above samples but at different concentrations. The antigen is not present in purified F1-ATPase, cytochrome oxidase, the oligomycin sensitivity conferring protein (OSCP), Factor6, or the Transhydrogenase. Immunoblot analysis of the above mitochondrial proteins revealed two PBC-related antigens (apparent molecular weights of 70 KD and 60 KD) whose distribution in the various proteins and protein complexes correlated well with the antigens determined by RIA. Immunoblot analysis of mitochondrial antigens was carried out using sera from normal subjects and from patients with PBC and with different autoimmune diseases (AID). Only PBC sera reacted with the 70 KD and 60 KD antigens. The PBC antigen detected by RIA in submitochondrial particles and the chloroform-F1-ATPase could be blocked by Mersalyl, suggesting its relationship to the mitochondrial 'M2' antigen. Furthermore, the antigenicity of the 70 KD peptide was shown by immunoblotting to be dependent upon mercaptoethanol. Thus, not only is the antigenicity of the 70 KD component dependent on a sulphur group, but the sulphur must be in the reduced form. [ABSTRACT FROM AUTHOR]