Search

Nonmethane hydrocarbons (NMHCs) are important precursors of ozone and other photo oxidants. We presented continuous hourly average concentrations of 45 C2–C10 NMHCs measured in urban area of Dallas, USA from 1996 to 2004. Most of the selected compounds are good variables with less noise. The top 10 species with high ozone-generating potential were identified according to their concentrations and reactivities. The ambient concentrations of abundant anthropogenic emission hydrocarbons measured in Dallas were about 2–4 times of the background values measured in the remote areas with adjacent latitude. The time series for anthropogenic emission hydrocarbons showed an obvious seasonal cycle with relatively high concentration in winter and low concentration in summer. The sinusoidal function with a linearly decreasing factor could well fit the time series of NMHCs. The phase of seasonal cycle for the aromatic hydrocarbons of toluene, m/p xylene and o-xylene that might come from both vehicle emission and solvent utilities evaporation was about 1 month earlier than that for alkanes and alkenes that mainly came from vehicle emission. Ambient NMHCs in Dallas decreased with a stable rate during 1996–2004. For most of compounds with high ozone-generating potential, the rate of ambient concentration decrease was higher or much higher than the rate of volatile organic compounds (VOCs) source emission reduction estimated by EPA''s National Emission Inventory. On weekdays, the morning hydrocarbon concentration peak was coincident with morning traffic rush time in Dallas. Another concentration peak was delayed to afternoon traffic rush time. The characteristics of VOCs sources, photochemical removal processes and atmospheric dilution could be interpreted by the diurnal variations of benzene/ethylbenzene (B/E), toluene/ethylbenzene (T/E) and xylene/ethylbenzene (X/E). The ratio of VOC/NO x measured in Dallas was substantially smaller than that calculated for USA cities. Ozone formation in Dallas was VOC sensitive. [Copyright &y& Elsevier]

Mutations in the SOD1 gene are associated with familial amyotrophic lateral sclerosis (fALS). The mechanisms by which these mutations lead to anterior horn cell loss are unknown, however, increased binding of Hsps on the demetallated mutant SOD1 has been described which would make the HSPs unavailable for other purposes, and reduce the SOD1 concentration in mitochondria, thereby creating a proapoptotic situation finally leading to motor neuron death. Here we report the recombinant expression of four human copper/zinc superoxide dismutase (CuZnSOD) variants, including the wild-type enzyme and mutant proteins associated with familial ALS. The bacterial expression level of soluble mutated proteins was influenced by the mutations leading to drastically reduced levels of soluble CuZnSOD. Simultaneously, increasing levels of insoluble and probably aggregated mutated CuZnSOD were identified in bacterial cell pellets. In addition, altered reactivation kinetics of the purified mutant apoproteins after expression in bacterial culture was shown. Biophysical and biochemical analysis showed that zinc incorporation is severely reduced in the CuZnSOD proteins associated with the most severely forms of fALS (A4V, G93A). These data indicate that a reduced holoenzyme formation rate of mutant enzymes may be a critical factor in the etiopathology of fALS. [ABSTRACT FROM AUTHOR]

Vitamin D is involved in brain physiology and lower-extremity function. We investigated spectroscopy in a cohort of older adults to explore the hypothesis that lower vitamin D status was associated with impaired neuronal function in caudal primary motor cortex (cPMC) measured by proton magnetic resonance spectroscopic imaging.Twenty Caucasian community-dwellers (mean±standard deviation, 74.6±6.2 years; 35.0% female) from the 'Gait and Brain Study' were included in this analysis. Ratio of N-acetyl-aspartate to creatine (NAA/Cr), a marker of neuronal function, was calculated in cPMC. Participants were categorized according to mean NAA/Cr. Lower vitamin D status was defined as serum 25-hydroxyvitamin D (25OHD) concentration

Changes in thyroid hormone (TH) levels in rat brain at early developmental stages are correlated with adverse effects on offspring development. To characterize the ability of substances to interfere with the TH concentrations in, e.g., rat brain, it is essential to know the mean TH concentrations in this tissue under control conditions. In this publication, an online solid-phase extraction (SPE) liquid chromatography (LC) tandem mass spectrometry (MS/MS) method was validated and used to measure TH metabolites (T4, T3, rT3, T2 and T1) in the brains of untreated rats. Data on TH concentrations in the whole brain and separate data from the cerebellum and the cortex are shown. The corresponding samples were gathered from young rats at postnatal days (PND) 4 and 21/22 and from adult rats. The results show inter alia the high accuracy and precision of the method, and LOQs of 0.02 ng/mL were determined for T1, T2 and rT3 and of 0.15 ng/mL for T3 and T4. Technical variability is low, as shown by the relative standard deviations of 7.5-20%. For our rat model, we found that T4, T3 and T2 concentrations rise from PND4 to PND21, whereas the rT3 concentration decreases; as well as there is no statistical difference between TH concentrations in the male and female rat brain. This method is suitable to analyze TH metabolites in the brain and build up a database of historical TH concentrations in control rats. Together, this yields a robust diagnostic tool to detect potentially adverse disturbances of TH homeostasis in the most vulnerable anatomic structure.

Since the focus in regulatory toxicology has drifted toward the identification of endocrine disruptors, the improvement in determination of alterations in the thyroid hormone system has become more important. THs are involved in several molecular processes important for a proper pre- and postnatal development so that disturbances can inter alia lead to incorrect brain maturation and/or disturbed metabolic processes (thermogenesis or lipolysis). In this publication, a new automated online solid-phase extraction (SPE)-liquid chromatography (LC)-tandem mass spectrometry (MS/MS, xLC-MS/MS) is introduced which simultaneously analyzes total T4, T3, rT3, T2, and T1. Method validation parameters are presented, and the method was positively verified by analyzing control and PTU-treated rat plasma samples (time points day 7, 14, and 28) for their total TH content. The obtained results were compared to published results by using a radioimmunoassay method. The automated SPE system ensures a consistent unified sample preparation, and this method overall showed sufficient specificity and accuracy to detect the given analytes in rat plasma. For the preparation of 50 μL of rat plasma, the following LOQs were established: 0.020 nM for T1, 0.029 nM for T2, 0.023 nM for rT3 and T3, and 3.22 nM for T4. This method is suitable to assess the identification of mechanisms leading to adverse effects, such as disturbed TH metabolism and regulation.

Molecules metabolized to para-tert-butyl-benzoic acid (p-TBBA) affect male reproduction in rats through effects on spermatogenesis. This toxicity is specific to p-TBBA and not observed in meta-substituted analogues. The underlying mode of action was evaluated by comparing effects of p-TBBA and the position isomer m-TBBA (2-50 µM) in an ex vivo 3D primary seminiferous tubule cell culture system from juvenile Sprague Dawley rats (Bio-AlteR ® ). Treated cultures were evaluated for CoA-conjugate formation, cytotoxicity, blood-testis barrier functionality and different germ cell populations to assess effects on spermatogenesis. In addition, an evaluation of the metabolome of treated cultures was performed by using MxP ® Broad Profiling via a LC-MS/MS and GC-MS platform. Para-TBBA decreased germ cell populations of late stages of spermatogenesis and led to the formation of CoA-conjugates in the ex vivo tissue. In addition, p-TBBA had a pronounced effect on the metabolome by affecting lipid balance and other CoA-dependent pathways contributing to energy production and the redox system. Meta-TBBA did not affect germ cell populations and no m-TBBA related CoA-conjugates were detectable. The metabolic profile of m-TBBA treated cells was comparable to vehicle control treated cultures, indicating that formation of CoA-conjugates, inhibition of spermatogenesis, and effects on the metabolome are mechanistically linked events. Thus, for this specific chemical group an adverse outcome pathway can be postulated, including the formation of benzoic acid metabolites, accumulation of CoA-conjugates to a certain threshold and CoA depletion, which affects the metabolic and lipid profile and leads to tissue specific effects with impaired functionalities such as spermatogenesis., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

To elucidate if artificial sweeteners modify fecal bacterial composition and the fecal and plasma metabolomes, Wistar rats from both sexes were treated for 28 days with acesulfame potassium (40 and 120 mg/kg body weight) and saccharin (20 and 100 mg/kg body weight). Targeted MS-based metabolome profiling (plasma and feces) and fecal 16S gene sequencing were conducted. Both sweeteners exhibited only minor effects on the fecal metabolome and microbiota. Saccharin treatment significantly altered amino acids, lipids, energy metabolism and specifically, bile acids in the plasma metabolome. Additionally, sex-specific differences were observed for conjugated primary and secondary bile acids. Acesulfame potassium treated male rats showed larger alterations in glycine conjugated primary and secondary bile-acids than females. Other changes in the plasma metabolome were more profound for saccharin than acesulfame potassium, for both sexes. Changes in conjugated bile-acids in plasma, which are often associated with microbiome changes, and the absence of similarly large changes in microbiota suggest an adaptative change of the latter, rather than toxicity. Further studies with a high resolution 16S sequencing data and/or metagenomics approach, with particular emphasis on bile acids, will be required to explore the mechanisms driving this metabolic outcome of saccharin in Wistar rats., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

We describe a strategy using an in vitro metabolomics assay with tubular rat NRK-52E cells to investigate the Modes of Action (MoAs) of nephrotoxic compounds. Chemicals were selected according to their MoAs based on literature information: acetaminophen, 4-aminophenol and S-(trichlorovinyl-)L-cysteine (TCVC), (covalent protein binding); gentamycin, vancomycin, polymycin B and CdCl 2 (lysosomal overload) and tenofovir and cidofovir (mitochondrial DNA-interaction). After treatment and harvesting of the cells, intracellular endogenous metabolites were quantified relative to vehicle control. Metabolite patterns were evaluated in a purely data-driven pattern generation process excluding published information. This strategy confirmed the assignment of the chemicals to the respective MoA except for TCVC and CdCl 2 . Finally, TCVC was defined as unidentified and CdCl 2 was reclassified to the MoA "covalent protein binding". Hierarchical cluster analysis of 58 distinct metabolites from the patterns enabled a clear visual separation of chemicals in each MoA. The assay reproducibility was very good and metabolic responses were consistent. These results support the use of metabolome analysis in NRK-52E cells as a suitable tool for understanding and investigating the MoA of nephrotoxicants. This assay could enable the early identification of nephrotoxic compounds and finally reduce animal testing., Competing Interests: Declaration of Competing Interest BASF SE might use this approach also in the future for registration of their products., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Various environmental factors can alter the gut microbiome's composition and functionality, and modulate host health. In this study, the effects of oral and parenteral administration of two poorly bioavailable antibiotics (i.e., vancomycin and streptomycin) on male Wistar Crl/Wi(Han) rats for 28 days were compared to distinguish between microbiome-derived or -associated and systemic changes in the plasma metabolome. The resulting changes in the plasma metabolome were compared to the effects of a third reference compound, roxithromycin, which is readily bioavailable. A community analysis revealed that the oral administration of vancomycin and roxithromycin in particular leads to an altered microbial population. Antibiotic-induced changes depending on the administration routes were observed in plasma metabolite levels. Indole-3-acetic acid (IAA) and hippuric acid (HA) were identified as key metabolites of microbiome modulation, with HA being the most sensitive. Even though large variations in the plasma bile acid pool between and within rats were observed, the change in microbiome community was observed to alter the composition of the bile acid pool, especially by an accumulation of taurine-conjugated primary bile acids. In-depth investigation of the relationship between microbiome variability and their functionality, with emphasis on the bile acid pool, will be necessary to better assess the potential adverseness of environmentally induced microbiome changes.

Metabolomics is a widely used technology in academic research, yet its application to regulatory science has been limited. The most commonly cited barrier to its translation is lack of performance and reporting standards. The MEtabolomics standaRds Initiative in Toxicology (MERIT) project brings together international experts from multiple sectors to address this need. Here, we identify the most relevant applications for metabolomics in regulatory toxicology and develop best practice guidelines, performance and reporting standards for acquiring and analysing untargeted metabolomics and targeted metabolite data. We recommend that these guidelines are evaluated and implemented for several regulatory use cases.

Common soil fungi, Aspergillus flavus and Aspergillus parasiticus, are opportunistic pathogens that invade preharvest peanut seeds. These fungi often produce carcinogenic aflatoxins that pose a threat to human and animal health through food chains and cause significant economic losses worldwide. Detection of aflatoxins and further processing of crops are mandated to ensure that contaminated agricultural products do not enter food channels. Under favorable conditions, the fungus-challenged peanut seeds produce phytoalexins, structurally related stilbenoids, capable of retarding fungal development. The purpose of the present study was to evaluate the potential influence of peanut phytoalexins on fungal development and aflatoxin formation in the course of peanut-fungus interaction. The present research revealed that during such interaction, aflatoxin formation was completely suppressed in A. flavus and A. parasiticus strains tested, when low concentrations of spores were introduced to wounded preincubated peanuts. In most of the experiments, when fungal spore concentrations were 2 orders of magnitude higher, the spores germinated and produced aflatoxins. Of all experimental seeds that showed fungal growth, 57.7% were aflatoxin-free after 72 h of incubation. The research provided new knowledge on the aflatoxin/phytoalexin formation in the course of peanut-fungus interaction.

We describe here the agreed upon first development steps and priority objectives of a community engagement effort to address current challenges in quality assurance (QA) and quality control (QC) in untargeted metabolomic studies. This has included (1) a QA and QC questionnaire responded to by the metabolomics community in 2015 which recommended education of the metabolomics community, development of appropriate standard reference materials and providing incentives for laboratories to apply QA and QC; (2) a 2-day 'Think Tank on Quality Assurance and Quality Control for Untargeted Metabolomic Studies' held at the National Cancer Institute's Shady Grove Campus and (3) establishment of the Metabolomics Quality Assurance and Quality Control Consortium (mQACC) to drive forward developments in a coordinated manner.

Drought is known to limit carbon assimilation in plants. However, it has been debated whether photosynthesis is primarily inhibited by stomatal or non-stomatal factors. This research assessed the underlying limitations to photosynthesis in peanuts (Arachis hypogaea L.) grown under progressive drought. Specifically, field-grown peanut plants were exposed to either well-watered or drought-stressed conditions during flowering. Measurements included survey measurements of gas exchange, chlorophyll fluorescence, PSII thermotolerance, pigment content, and rapid A-C i response (RACiR) assessments. Drought significantly decreased stomatal conductance with consequent declines in photosynthesis (A N ), actual quantum yield of PSII, and electron transport rate (ETR). Pigment contents were variable and depended on stress severity. Stomatal closure on stressed plants resulted in higher leaf temperatures, but F v /F m and PSII thermotolerance were only slightly affected by drought. A strong, hyperbolic relationship was observed between stomatal conductance, A N , and ETR. However, when RACiR analysis was conducted, drought significantly decreased A N at C i values comparable to drought-stressed plants, indicating non-stomatal limitations to A N . The maximum rate of carboxylation and maximum electron transport rate were severely limited by drought, and chloroplast CO 2 concentration (C C ) declined substantially under drought along with a comparable increase in partitioning of electron flow to photorespiration. Thus, while stomatal conductance may be a viable reference indicator of water deficit stress in peanut, we conclude that declines in A N were largely due to non-stomatal (diffusional and metabolic) limitations. Additionally, this is the first study to apply the rapid A-C i response method to peanut, with comparable results to traditional A-C i methods., (Published by Elsevier GmbH.)

Liver toxicity is a leading systemic toxicity of drugs and chemicals demanding more human-relevant, high throughput, cost effective in vitro solutions. In addition to contributing to animal welfare, in vitro techniques facilitate exploring and understanding the molecular mechanisms underlying toxicity. New 'omics technologies can provide comprehensive information on the toxicological mode of action of compounds, as well as quantitative information about the multi-parametric metabolic response of cellular systems in normal and patho-physiological conditions. Here, we combined mass-spectroscopy metabolomics with an in vitro liver toxicity model. Metabolite profiles of HepG2 cells treated with 35 test substances resulted in 1114 cell supernatants and 3556 intracellular samples analyzed by metabolomics. Control samples showed relative standard deviations of about 10-15%, while the technical replicates were at 5-10%. Importantly, this procedure revealed concentration-response effects and patterns of metabolome changes that are consistent for different liver toxicity mechanisms (liver enzyme induction/inhibition, liver toxicity and peroxisome proliferation). Our findings provide evidence that identifying organ toxicity can be achieved in a robust, reliable, human-relevant system, representing a non-animal alternative for systemic toxicology.

Aspergillus flavus is a soil fungus that commonly invades peanut seeds and often produces carcinogenic aflatoxins. Under favorable conditions, the fungus-challenged peanut plant produces and accumulates resveratrol and its prenylated derivatives in response to such an invasion. These prenylated stilbenoids are considered peanut antifungal phytoalexins. However, the mechanism of peanut-fungus interaction has not been sufficiently studied. We used pure peanut stilbenoids arachidin-1, arachidin-3, and chiricanine A to study their effects on the viability of and metabolite production by several important toxigenic Aspergillus species. Significant reduction or virtually complete suppression of aflatoxin production was revealed in feeding experiments in A. flavus, Aspergillus parasiticus, and Aspergillus nomius. Changes in morphology, spore germination, and growth rate were observed in A. flavus exposed to the selected peanut stilbenoids. Elucidation of the mechanism of aflatoxin suppression by peanut stilbenoids could provide strategies for preventing plant invasion by the fungi that produce aflatoxins.

Background: Phosphorus (P) plays important roles in plant growth and development. MicroRNAs involved in P signaling have been identified in Arabidopsis and rice, but P-responsive microRNAs and their targets in soybean leaves and roots are poorly understood., Results: Using high-throughput sequencing-by-synthesis (SBS) technology, we sequenced four small RNA libraries from leaves and roots grown under phosphate (Pi)-sufficient (+Pi) and Pi-depleted (-Pi) conditions, respectively, and one RNA degradome library from Pi-depleted roots at the genome-wide level. Each library generated ~21.45-28.63 million short sequences, resulting in ~20.56-27.08 million clean reads. From those sequences, a total of 126 miRNAs, with 154 gene targets were computationally predicted. This included 92 new miRNA candidates with 20-23 nucleotides that were perfectly matched to the Glycine max genome 1.0, 70 of which belong to 21 miRNA families and the remaining 22 miRNA unassigned into any existing miRNA family in miRBase 18.0. Under both +Pi and -Pi conditions, 112 of 126 total miRNAs (89%) were expressed in both leaves and roots. Under +Pi conditions, 12 leaf- and 2 root-specific miRNAs were detected; while under -Pi conditions, 10 leaf- and 4 root-specific miRNAs were identified. Collectively, 25 miRNAs were induced and 11 miRNAs were repressed by Pi starvation in soybean. Then, stem-loop real-time PCR confirmed expression of four selected P-responsive miRNAs, and RLM-5' RACE confirmed that a PHO2 and GmPT5, a kelch-domain containing protein, and a Myb transcription factor, respectively are targets of miR399, miR2111, and miR159e-3p. Finally, P-responsive cis-elements in the promoter regions of soybean miRNA genes were analyzed at the genome-wide scale., Conclusions: Leaf- and root-specific miRNAs, and P-responsive miRNAs in soybean were identified genome-wide. A total of 154 target genes of miRNAs were predicted via degradome sequencing and computational analyses. The targets of miR399, miR2111, and miR159e-3p were confirmed. Taken together, our study implies the important roles of miRNAs in P signaling and provides clues for deciphering the functions for microRNA/target modules in soybean.

Background: Phosphorus (P) is essential for plant growth and development. Phosphate (Pi) transporter genes in the Pht1 family play important roles in Pi uptake and translocation in plants. Although Pht1 family genes have been well studied in model plants, little is known about their functions in soybean, an important legume crop worldwide., Principal Findings: We identified and isolated a complete set of 14 Pi transporter genes (GmPT1-14) in the soybean genome and categorized them into two subfamilies based on phylogenetic analysis. Then, an experiment to elucidate Pi transport activity of the GmPTs was carried out using a yeast mutant defective in high-affinity Pi transport. Results showed that 12 of the 14 GmPTs were able to complement Pi uptake of the yeast mutant with Km values ranging from 25.7 to 116.3 µM, demonstrating that most of the GmPTs are high-affinity Pi transporters. Further results from qRT-PCR showed that the expressions of the 14 GmPTs differed not only in response to P availability in different tissues, but also to other nutrient stresses, including N, K and Fe deficiency, suggesting that besides functioning in Pi uptake and translocation, GmPTs might be involved in synergistic regulation of mineral nutrient homeostasis in soybean., Conclusions: The comprehensive analysis of Pi transporter function in yeast and expression responses to nutrition starvation of Pht1 family genes in soybean revealed their involvement in other nutrient homeostasis besides P, which could help to better understand the regulation network among ion homeostasis in plants.

Background and Aims: Purple acid phosphatases (PAPs) are members of the metallo-phosphoesterase family and have been known to play important roles in phosphorus (P) acquisition and recycling in plants. Low P availability is a major constraint to growth and production of soybean, Glycine max. Comparative studies on structure, transcription regulation and responses to phosphate (Pi) deprivation of the soybean PAP gene family should facilitate further insights into the potential physiological roles of GmPAPs., Methods: BLAST searches were performed to identify soybean PAP genes at the phytozome website. Bioinformatic analyses were carried out to investigate their gene structure, conserve motifs and phylogenetic relationships. Hydroponics and sand-culture experiments were carried out to obtain the plant materials. Quantitative real-time PCR was employed to analyse the expression patterns of PAP genes in response to P deficiency and symbiosis., Key Results: In total, 35 PAP genes were identified from soybean genomes, which can be classified into three distinct groups including six subgroups in the phylogenetic tree. The expression pattern analysis showed flowers possessed the largest number of tissue-specific GmPAP genes under normal P conditions. The expression of 23 GmPAPs was induced or enhanced by Pi starvation in different tissues. Among them, nine GmPAP genes were highly expressed in the Pi-deprived nodules, whereas only two GmPAP genes showed significantly increased expression in the arbuscular mycorrhizal roots under low-P conditions., Conclusions: Most GmPAP genes are probably involved in P acquisition and recycling in plants. Also we provide the first evidence that some members of the GmPAP gene family are possibly involved in the response of plants to symbiosis with rhizobia or arbuscular mycorrhizal fungi under P-limited conditions.

PlantMetabolomics.org (PM) is a web portal and database for exploring, visualizing, and downloading plant metabolomics data. Widespread public access to well-annotated metabolomics datasets is essential for establishing metabolomics as a functional genomics tool. PM integrates metabolomics data generated from different analytical platforms from multiple laboratories along with the key visualization tools such as ratio and error plots. Visualization tools can quickly show how one condition compares to another and which analytical platforms show the largest changes. The database tries to capture a complete annotation of the experiment metadata along with the metabolite abundance databased on the evolving Metabolomics Standards Initiative. PM can be used as a platform for deriving hypotheses by enabling metabolomic comparisons between genetically unique Arabidopsis (Arabidopsis thaliana) populations subjected to different environmental conditions. Each metabolite is linked to relevant experimental data and information from various annotation databases. The portal also provides detailed protocols and tutorials on conducting plant metabolomics experiments to promote metabolomics in the community. PM currently houses Arabidopsis metabolomics data generated by a consortium of laboratories utilizing metabolomics to help elucidate the functions of uncharacterized genes. PM is publicly available at http://www.plantmetabolomics.org.

Polypeptide constructs containing up to four copies of the T cell epitope 306-318 of influenza virus haemagglutinin have been synthesized on solid phase. Between the copies, a non-natural PEG-based spacer amino acid has been introduced. The oligomeric epitopes were analysed by RP-HPLC and ES-MS. The arrangement of the epitopes within the peptide constructs was either linear or comb-like. The proliferative response in a T helper cell assay induced by these oligomerized epitopes has been tested, showing that the linearly arranged epitopes are more effective than the comb-like oligomers.

Specific and major histocompatibility complex (MHC)-restricted T-cell recognition of antigenic peptides is based on interactions of the T-cell receptor (TCR) with the MHC alpha helices and solvent exposed peptide residues termed TCR contacts. In the case of MHC class II-presented peptides, the latter are located in the positions p2/3, p5 and p7/8 between MHC anchor residues. For numerous epitopes, peptide substitution studies have identified the central residue p5 as primary TCR contact characterized by very low permissiveness for peptide substitution, while the more peripheral positions generally represent auxiliary TCR contacts. In structural studies of TCR/peptide/MHC complexes, this has been shown to be due to intimate contact between the TCR complementarity determining region (CDR) three loops and the central peptide residue. We asked whether this model also applied to two HLA-DR presented epitopes derived from an antigen targeted in type 1 diabetes. Large panels of epitope variants with mainly conservative single substitutions were tested for human leukocyte antigen (HLA) class II binding affinity and T cell stimulation. Both epitopes bind with high affinity to the presenting HLA-DR molecules. However, in striking contrast to the standard distribution of TCR contacts, recognition of the central p5 residue displayed high permissiveness even for non-conservative substitutions, while the more peripheral p2 and p8 TCR contacts showed very low permissiveness for substitution. This suggests that intimate TCR interaction with the central peptide residue is not always required for specific antigen recognition and can be compensated by interactions with positions normally acting as auxiliary contacts.

The human plasma protein patterns obtained by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is a good model system for post-translational modifications because of the existence of several "ladders" of protein spots [Anderson, N. L., Anderson, N. G., Electrophoresis 1991, 12, 883-906], so-called "trains" of spots. Our investigation of several proteins, amongst others beta2-microglobulin and the haptoglobin chains, found the differences in isoelectric points (p/) to be due to deamidation of asparagines. After enzymatic cleavage with endopeptidases in the 2-D polyacrylamide gel, the asparagine and deamidated asparagine containing peptides were separated and quantified by reversed-phase HPLC. In order to separate these peptides, a neutral pH system was established and, as a result, the differences in hydrophobicity of asparagine-containing and deamidated asparagine-containing peptides increased. But how do deamidated asparagines contribute to the observed spot pattern? One spot in the 2-D gel consists of a mixture of protein species with the same number of deamidated asparagines but on different sequence position sites. The difference between the spots in the "ladder" is a growing number of negative charges introduced in the protein by an increasing number of deamidated asparagines. As a consequence, the mass difference between two spots is exactly 1 Da, which is shown in this paper for intact protein masses and the corresponding deamidated peptides.

Linear biosynthesis products from balhimycin, a tricyclic glycopeptide antibiotic of the vancomycin type, were isolated from mutants of Amycolatopsis mediterranei. The structures of these intermediates, determined by NMR spectroscopy, give new impulses for the understanding of the vancomycin biosynthesis., (© 1999 WILEY‐VCH Verlag GmbH, Weinheim, Fed. Rep. of Germany.)

The determination of the primary structure of peptides and proteins is routine in many laboratories; however, many of the obtained sequences are incomplete or can be misinterpreted when the samples contain unusual amino acids. Here we report the development of an automated peptide sequenator coupled to an electrospray-ionization (ESI) mass spectrometer (MS) that, in conjunction with minor modifications to the sequencing conditions and, in some cases, prior derivatization of amino acids, allows the detection of the phenylthiohydantoin (PTH) derivatives of a number of unusual amino acids. Using the coupled sequenator-ESI-MS system we were able to determine the complete sequence of the lantibiotic gallidermin, a partial sequence of the calcium-dependent peptide antibiotic CDA2 as well as the pool sequence of a mixture of synthetic peptides containing nonproteinogenic amino acids. In addition to the 20 proteinogenic amino acids, the procedure was able to detect PTH derivatives of hydroxyphenylglycine, 2,3-didehydroasparagine, 3-methylglutamic acid, oxytryptophan, ornithine, N-methylglycine, dihydroxyphenylalanine, and alpha-aminoisobutyric acid. Similarly, after a simple derivatization procedure, we were also able to correctly identify educts of 2,3-didehydroalanine, 2,3-didehydrobutyrine, lanthionine, and 3-methyllanthionine.

A novel polypeptide containing the unusual posttranslationally modified amino acids L-3,4,5-trihydroxyphenylalanine (TOPA) and L-6-bromotryptophan (6-BrW) has been isolated from the morula cells of the vanadium-accumulating ascidian, Phallusia mammillata. The polypeptide, designated Morulin Pm, has a molecular weight of 3825 +/- 0.6 and has a simple amino acid composition consisting mainly of TOPA and 6-BrW as well as Ser, Leu, Phe, and Ala. To our knowledge, this is the first reported example of multiple sites of brominated tryptophan in a polypeptide of this size. Edman degradation revealed the N-terminal sequence to be BrW-Leu-Phe-BrW before sequencing was blocked. While the N-terminal tripeptide could be isolated from chymotrypsin digests of Morulin Pm, the rest of the polypeptide resisted further cleavage by the proteases, a feature common among this class of peptides. However, unlike other ascidian blood cell peptides examined to date, microheterogeneity was minimal. For the first time a detailed NMR investigation could be undertaken on a member of this class of polypeptides. In addition to signals assignable to the constituent amino acids by extensive 2D experiments, resonances were present both in the 13C and 1H spectra not typical of a simple linear peptide. Two proton resonances were identified with a cross peak in the correlation spectrum strongly indicative of a C-terminal decarboxy-delta 2,3-unsaturated TOPA residue as observed in certain tunichromes and clionamide. Chemical degradation experiments were undertaken in an effort to produce identifiable fragments to which these signals could be assigned, including full and partial acid hydrolysis and tryptophan-targeted BNPS-skatole treatment. However, the nature of the modification remains unknown. Possible structures for the modification, which may represent the source of the difficulties encountered in the structural elucidation of this and related peptides, are assessed. Conjecture is made as to the biological relevance of Morulin Pm, based on its localization and chemical characteristics.

Objective: To measure the urea and creatinine kinetics in a pediatric population., Patients and Methods: In 19 children treated with peritoneal dialysis (PD) KT/V, urea and creatinine clearances (Ccr) were measured. Thirteen children were on continuous ambulatory peritoneal dialysis (CAPD) and 6 on highly intermittent peritoneal dialysis (NIPD)., Results: Mean KT/V per week was 2.31 +/- 0.78 and mean creatinine clearance 74 +/- 47 L/week/1.73 m2. There was no difference in dialytic KT/V between patients treated with CAPD and NIPD (1.75 +/- 0.21 vs 1.76 +/- 0.50). The correlation between KT/V urea and creatinine clearance was 0.9 (p < 0.001). There was a clear relationship of these parameters with residual renal function, but not with age or blood urea level. A weak positive correlation was found with serum albumin and protein intake., Conclusions: Mean KT/V in this patient group was higher than the values reported for most adult patient groups. Residual renal function considerably contributes to this high KT/V. It is not clearly defined which KT/V should be aimed for, since criteria for adequate dialysis are multifactorially determined and therefore difficult to interpret.

A nondestructive technique was developed to characterize and separate eggs of soybean cyst nematode, Heterodera glycines, by developmental stage using flow cytometry. Eggs from cysts cultured on susceptible soybean roots were suspended in 0.1% xanthan gum or 59% sucrose and loaded into either a Coulter EPICS 752 or EPICS 753 flow cytometer. Eggs were analyzed and sorted according to forward angle and 90 degrees light scatter, flow cytometric parameters that are relative measures of object size and granularity, respectively. Mature eggs containing vermiform juveniles were less granular and slightly larger than eggs in earlier stages of embryogeny, allowing for separation of mature eggs from immature eggs. The effectiveness of flow cytometric sorting was evaluated by comparing the developmental stages of subpopulations of unsorted and sorted eggs. Of a subpopulation of unsorted eggs, 62% contained vermiform juveniles, whereas 85 to 95% of sorted subpopulations of larger, less granular eggs contained vermiform juveniles. Suspending H. glycines eggs in 0.1% xanthan gum or 59% sucrose for flow cytometric analysis had no effect on subsequent egg hatch in vitro. This technique is an efficient and effective means to collect large, relatively homogeneous quantities of H. glycines eggs in early or late embryogeny, and would likely be useful for analyzing and sorting eggs of other nematode species for use in developmental, genetic, or physiological research, or for identification and collection of parasitized eggs.

From 157 plasma samples taken randomly throughout normal pregnancy and from 42 plasma samples of nonpregnant women, total plasma dehydroepiandrosterone was measured by a method using Amberlite XAD-2 column chromatography at 45degreesC, enzyme hydrolysis, radioactive internal standard, thin-layer chromatography and gas-liquid chromatography after trimethylsilyl ether derivative formation. The following values for dehydroepiandrosterone were obtained: from individual, nonpregnant samples, (n = 25) 69.6 +/- 10.6 mug/100 ml (S.E.M.); from the pool of nonpregnant samples (n = 17) 67.7 mug/100 ml; from individual samples, 6-12 weeks of gestation (n = 32) 48.5 +/- 5.7 mug/100 ml (S.E.M.); from individual samples, 13-18 weeks of gestation (n = 13) 45.9 +/- 7.7 mug/100 ml (S.E.M.); from individual samples, 19-24 weeks of gestation (n = 20) 42.9 +/- 6.9 mug/100 ml (S.E.M.); from individual samples, 25-30 weeks of gestation (n = 22) 41.7 +/- 6.8 mug/100 ml (S.E.M.); from individual samples, 31-36 weeks of gestation (n = 31) 39.5 +/- 6.1 mug/100 ml (S.E.M.); from individual samples, 37-43 weeks of gestation (n = 29) 37.6 +/- 3.6 mug/100 ml (S.E.M.); and from the pool sample, 37-43 weeks of gestation (n = 10) 25.4 mug/100 ml. This study demonstrates a significant decrease of total plasma dehydroepiandrosterone throughout the course of normal pregnancy in individual and pooled plasma samples, thus confirming previous reports. These plasma hormone changes are discussed in relation to production and utilization of this steroid in pregnancy.