Assessment of compensated advanced chronic liver disease based on serum bile acids in chronic hepatitis B patients.

Patients with chronic liver disease progressed to compensated advanced chronic liver disease (cACLD), the risk of liver-related decompensation increased significantly. This study aimed to develop prediction model based on individual bile acid (BA) profiles to identify cACLD. This study prospectively recruited 159 patients with hepatitis B virus (HBV) infection and 60 healthy volunteers undergoing liver stiffness measurement (LSM). With the value of LSM, patients were categorized as three groups: F1 [LSM ≤ 7.0 kilopascals (kPa)], F2 (7.1 < LSM ≤ 8.0 kPa), and cACLD group (LSM ≥ 8.1 kPa). Random forest (RF) and support vector machine (SVM) were applied to develop two classification models to distinguish patients with different degrees of fibrosis. The content of individual BA in the serum increased significantly with the degree of fibrosis, especially glycine-conjugated BA and taurine-conjugated BA. The Marco-Precise, Marco-Recall, and Marco-F1 score of the optimized RF model were all 0.82. For the optimized SVM model, corresponding score were 0.86, 0.84, and 0.85, respectively. RF and SVM models were applied to identify individual BA features that successfully distinguish patients with cACLD caused by HBV. This study provides a new tool for identifying cACLD that can enable clinicians to better manage patients with chronic liver disease.

Supplementing Glycine and N-Acetylcysteine (GlyNAC) in Older Adults Improves Glutathione Deficiency, Oxidative Stress, Mitochondrial Dysfunction, Inflammation, Physical Function, and Aging Hallmarks: A Randomized Clinical Trial.

BACKGROUND: Elevated oxidative stress (OxS), mitochondrial dysfunction, and hallmarks of aging are identified as key contributors to aging, but improving/reversing these defects in older adults (OA) is challenging. In prior studies, we identified that deficiency of the intracellular antioxidant glutathione (GSH) could play a role and reported that supplementing GlyNAC (combination of glycine and N-acetylcysteine [NAC]) in aged mice improved GSH deficiency, OxS, mitochondrial fatty-acid oxidation (MFO), and insulin resistance (IR). To test whether GlyNAC supplementation in OA could improve GSH deficiency, OxS, mitochondrial dysfunction, IR, physical function, and aging hallmarks, we conducted a placebo-controlled randomized clinical trial. METHODS: Twenty-four OA and 12 young adults (YA) were studied. OA was randomized to receive either GlyNAC (N = 12) or isonitrogenous alanine placebo (N = 12) for 16-weeks; YA (N = 12) received GlyNAC for 2-weeks. Participants were studied before, after 2-weeks, and after 16-weeks of supplementation to assess GSH concentrations, OxS, MFO, molecular regulators of energy metabolism, inflammation, endothelial function, IR, aging hallmarks, gait speed, muscle strength, 6-minute walk test, body composition, and blood pressure. RESULTS: Compared to YA, OA had GSH deficiency, OxS, mitochondrial dysfunction (with defective molecular regulation), inflammation, endothelial dysfunction, IR, multiple aging hallmarks, impaired physical function, increased waist circumference, and systolic blood pressure. GlyNAC (and not placebo) supplementation in OA improved/corrected these defects. CONCLUSION: GlyNAC supplementation in OA for 16-weeks was safe and well-tolerated. By combining the benefits of glycine, NAC and GSH, GlyNAC is an effective nutritional supplement that improves and reverses multiple age-associated abnormalities to promote health in aging humans. Clinical Trials Registration Number: NCT01870193.

Quantitative Assessment of Arsenite-Induced Perturbation of Ubiquitinated Proteome.

Arsenic contamination in food and groundwater constitutes a public health concern for more than 200 million people worldwide. Individuals chronically exposed to arsenic through drinking and ingestion exhibit a higher risk of developing cancers and cardiovascular diseases. Nevertheless, the underlying mechanisms of arsenic toxicity are not fully understood. Arsenite is known to bind to and deactivate RING finger E3 ubiquitin ligases; thus, we reason that a systematic interrogation about how arsenite exposure modulates global protein ubiquitination may reveal novel molecular targets for arsenic toxicity. By employing liquid chromatography-tandem mass spectrometry, in combination with stable isotope labeling by amino acids in cell culture (SILAC) and immunoprecipitation of di-glycine-conjugated lysine-containing tryptic peptides, we assessed the alterations in protein ubiquitination in GM00637 human skin fibroblast cells upon arsenite exposure at the entire proteome level. We observed that arsenite exposure led to altered ubiquitination of many proteins, where the alterations in a large majority of ubiquitination events are negatively correlated with changes in expression of the corresponding proteins, suggesting their modulation by the ubiquitin-proteasomal pathway. Moreover, we observed that arsenite exposure confers diminished ubiquitination of a rate-limiting enzyme in cholesterol biosynthesis, HMGCR, at Lys248. We also revealed that TRC8 is the major E3 ubiquitin ligase for HMGCR ubiquitination in HEK293T cells, and the arsenite-induced diminution of HMGCR ubiquitination is abrogated upon genetic depletion of TRC8. In summary, we systematically characterized arsenite-induced perturbations in a ubiquitinated proteome in human cells and found that the arsenite-elicited attenuation of HMGCR ubiquitination in HEK293T cells involves TRC8.

Endpoints as human biomarkers in exposure assessment of triazoles fungicides.

Potential endpoint biomarkers were evaluated in the assessment of exposure to triazoles, in the southern region of Minas Gerais, Brazil. Volunteers were divided into three groups: occupationally exposed and rural residents (n = 21), non-occupationally exposed and rural residents (n = 35) and non-occupationally exposed and urban residents (n = 30). Of all endpoints evaluated, plasma concentration of androstenedione (p < 0.001) and glycine-conjugated bile acids presented statistical differences in the three studied groups (p < 0.05). However, our findings concerning oxidative stress and testosterone levels, plus that related to unconjugated and taurine conjugated bile acids, suggested that more studies are necessary to evaluate their potential as biomarkers for triazole exposure, as statistical significance was not attained between the groups. Our human population data contributes to the development of triazole exposure risk assessment with respect to these potential effect biomarkers, in potentially vulnerable groups and individuals.

Economical synthesis of 14C-labeled aminolevulinic acid for specific in situ labeling of plant tetrapyrroles.

The application of metabolic radiolabeling techniques to plant tetrapyrroles, i.e., chlorophyll and hemes, is complicated by the difficulty of obtaining sufficient quantities of radiolabeled aminolevulinic acid (ALA). ALA, the first committed intermediate in the tetrapyrrole biosynthetic pathway, is inconvenient to synthesize chemically and is generally not produced in significant quantities in biological systems. Radiolabeled ALA is therefore usually quite expensive and available only in limited quantities. Here, we describe bulk biosynthesis and purification of 14C-labeled ALA from 14C glycine. We first cloned ALA synthase (ALAS) from Rhodobacter sphaeroides into an expression vector for expression and purification as a fusion with maltose-binding protein. We then used the purified ALAS to synthesize ALA in vitro from 14C-labeled glycine and succinyl-coenzyme A. Finally, we used ion exchange chromatography to separate the ALA product from the crude reaction. We achieved conversion and recovery efficiencies of 80-90%, and chlorophyll radiolabeling experiments with the 14C ALA product revealed no detectable non-specific incorporation into proteins. The ability to economically produce robust quantities of 14C ALA using common methodologies provides a new tool for working with tetrapyrroles, which includes both hemes and chlorophylls and their respective binding proteins. This tool allows the specific detection and quantification of the tetrapyrrole of interest from standard acrylamide gels or hybridization transfer membranes via radiographic imaging, which enables a wide array of experiments involving spatial and temporal resolution of the movement of pigments as they are synthesized, incorporated into their target binding proteins, and eventually degraded.

Assessment of the Biotransformation of Low-Turnover Drugs in the HµREL Human Hepatocyte Coculture Model.

Metabolic profiles of four drugs possessing diverse metabolic pathways (timolol, meloxicam, linezolid, and XK469) were compared following incubations in both suspended cryopreserved human hepatocytes and the HµREL hepatocyte coculture model. In general, minimal metabolism was observed following 4-hour incubations in both suspended hepatocytes and the HµREL model, whereas incubations conducted up to 7 days in the HµREL coculture model resulted in more robust metabolic turnover. In the case of timolol, in vivo human data suggest that 22% of the dose is transformed via multistep oxidative opening of the morpholine moiety. Only the first-step oxidation was detected in suspended hepatocytes, whereas the relevant downstream metabolites were produced in the HµREL model. For meloxicam, both the hydroxymethyl and subsequent carboxylic acid metabolites were abundant following incubation in the HµREL model, while only a trace amount of the hydroxymethyl metabolite was observed in suspension. Similar to timolol, linezolid generated substantially higher levels of morpholine ring-opened carboxylic acid metabolites in the HµREL model. Finally, while the major aldehyde oxidase-mediated mono-oxidative metabolite of XK469 was minimally produced in hepatocyte suspension, the HµREL model robustly produced this metabolite, consistent with a pathway reported to account for 54% of the total urinary excretion in human. In addition, low-level taurine and glycine conjugates were identified in the HµREL model. In summary, continuous metabolite production was observed for up to 7 days of incubation in the HµREL model, covering cytochrome P450, aldehyde oxidase, and numerous conjugative pathways, while predominant metabolites correlated with relevant metabolites reported in human in vivo studies.

Molecular assessment of glyphosate-degradation pathway via sarcosine intermediate in Lysinibacillus sphaericus.

The widespread use of glyphosate has permeated not only small- and large-scale agriculture, but also the fight against drug trafficking and illicit crops. Health, alimentary security, and the rights of peasant and indigenous communities have been compromised in countries with intensive use of glyphosate-based herbicides. In 2015, the International Agency for Research on Cancer classified this substance as probably carcinogenic to humans, leading to the suspension of aerial glyphosate spraying the same year in countries like Colombia, where glyphosate has been extensively used in illicit crop eradication. Notwithstanding, according to a study of the U.S. Geological Survey, traces of glyphosate and its main degradation product, AMPA, remain in soil year after year. This underscores the urgency and importance of assessing new technologies to degrade glyphosate present in soils and waterbodies without leaving persistent byproducts. The aim of this study was to evaluate Lysinibacillus sphaericus' glyphosate uptake as a carbon and phosphorous source by a sarcosine-mediated metabolic pathway that releases glycine as final degradation product. To accomplish this, molecular and analytic evidence were collected in vitro from sarcosine oxidase activity, a key enzyme of a degradation pathway which releases byproducts that are easy to incorporate into natural biosynthesis routes.

Roles of dietary glycine, proline, and hydroxyproline in collagen synthesis and animal growth.

Glycine, proline, and hydroxyproline (Hyp) contribute to 57% of total amino acids (AAs) in collagen, which accounts for one-third of proteins in animals. As the most abundant protein in the body, collagen is essential to maintain the normal structure and strength of connective tissue, such as bones, skin, cartilage, and blood vessels. Mammals, birds, and fish can synthesize: (1) glycine from threonine, serine, choline, and Hyp; (2) proline from arginine; and (3) Hyp from proline residues in collagen, in a cell- and tissue-specific manner. In addition, livestock (e.g., pigs, cattle, and sheep) produces proline from glutamine and glutamate in the small intestine, but this pathway is absent from birds and possibly most fish species. Results of the recent studies indicate that endogenous synthesis of glycine, proline, and Hyp is inadequate for maximal growth, collagen production, or feed efficiency in pigs, chickens, and fish. Although glycine, proline and Hyp, and gelatin can be used as feed additives in animal diets, these ingredients except for glycine are relatively expensive, which precludes their inclusion in practical rations. Alternatively, hydrolyzed feather meal (HFM), which contains 9% glycine, 5% Hyp, and 12% proline, holds great promise as a low cost but abundant dietary source of glycine, Hyp, and proline for ruminants and nonruminants. Because HFM is deficient in most AAs, future research efforts should be directed at improving the bioavailability of its AAs and the balance of AAs in HFM-supplemented diets. Finally, HFM may be used as a feed additive to prevent or ameliorate connective tissue disorders in domestic and aquatic animals.

Ecotoxicological risk assessment for the herbicide glyphosate to non-target aquatic species: A case study with the mussel Mytilus galloprovincialis.

Glyphosate (GLY) is one of the most used herbicide worldwide. Considering that information concerning the impact of GLY on bivalves is scarce, in this study we evaluated for the first time the effects of environmentally realistic concentrations of GLY (10, 100 and 1000 µg/L) to the mussel Mytilus galloprovincialis. Mussels were exposed for 7, 14 and 21 days and several biomarkers were measured in haemocytes/haemolymph (total haemocyte counts, haemocyte diameter and volume, haemolymph pH, haemolymph lactate dehydrogenase activity, haemocyte lysate lysozyme and acid phosphatase activities), as well as in gills and digestive gland (antioxidant enzyme and acetylcholinesterase activities). The concentrations of GLY and its main metabolite aminomethylphosphonic acid in the experimental tanks were also measured. The MANOVA analysis demonstrated that the experimental variables considered (exposure concentration, exposure duration, and their interaction) affected significantly biomarker responses. In addition, the two-way ANOVA analysis indicated that GLY was able to affect most of the cellular parameters measured, whereas antioxidant enzyme activities resulted to be influenced moderately. Interestingly, exposure to GLY reduced significantly acetylcholinesterase activity in gills. Although preliminary, the results of this study demonstrated that GLY can affect both cellular and biochemical parameters in mussels, highlighting a potential risk for aquatic invertebrates.

Ligand-Mediated Folding of the OmpA Periplasmic Domain from Acinetobacter baumannii.

The periplasmic domain of OmpA from Acinetobacter baumannii (AbOmpA-PD) binds to diaminopimelate and anchors the outer membrane to the peptidoglycan layer in the cell wall. Although the crystal structure of AbOmpA-PD with its ligands has been reported, the mechanism of ligand-mediated folding of AbOmpA remains elusive. Here, we report that in vitro refolded apo-AbOmpA-PD in the absence of ligand exists as a mixture of two partially folded forms in solution: mostly unfolded (apo-state I) and hololike (apo-state II) states. Binding of the diaminopimelate or glycine ligand induced complete folding of AbOmpA-PD. The apo-state I was highly flexible and contained some secondary structural elements, whereas the apo-state II closely resembled the holo-state in terms of both structure and backbone dynamics, except for the ligand-binding region. 15N-relaxation-dispersion analyses for apo-state II revealed substantial motion on a millisecond timescale of residues in the H3 helix near the ligand-binding site, with this motion disappearing upon ligand binding. These results provide an insight into the ligand-mediated folding mechanism of AbOmpA-PD in solution.

Nonketotic hyperglycinemia: Functional assessment of missense variants in GLDC to understand phenotypes of the disease.

The rapid analysis of genomic data is providing effective mutational confirmation in patients with clinical and biochemical hallmarks of a specific disease. This is the case for nonketotic hyperglycinemia (NKH), a Mendelian disorder causing seizures in neonates and early-infants, primarily due to mutations in the GLDC gene. However, understanding the impact of missense variants identified in this gene is a major challenge for the application of genomics into clinical practice. Herein, a comprehensive functional and structural analysis of 19 GLDC missense variants identified in a cohort of 26 NKH patients was performed. Mutant cDNA constructs were expressed in COS7 cells followed by enzymatic assays and Western blot analysis of the GCS P-protein to assess the residual activity and mutant protein stability. Structural analysis, based on molecular modeling of the 3D structure of GCS P-protein, was also performed. We identify hypomorphic variants that produce attenuated phenotypes with improved prognosis of the disease. Structural analysis allows us to interpret the effects of mutations on protein stability and catalytic activity, providing molecular evidence for clinical outcome and disease severity. Moreover, we identify an important number of mutants whose loss-of-functionality is associated with instability and, thus, are potential targets for rescue using folding therapeutic approaches.

Assessment of a glycine uptake inhibitor in animal models of effort-related choice behavior: implications for motivational dysfunctions.

RATIONALE: Motivated behavior can be characterized by a substantial exertion of effort, and organisms often make effort-related decisions based upon analyses of work-related response costs and reinforcement preference. Moreover, alterations in effort-based choice can be seen in people with major depression and schizophrenia. Effort-related decision making is studied using tasks offering choices between high effort options leading to highly valued reinforces vs low effort/low reward options. Interference with dopamine (DA) transmission by administration of the DA D2 family antagonist haloperidol biases behavior towards the lower effort option that can be obtained with minimal work, and previous research has shown that DA interacts with other transmitters, including adenosine and GABA, to regulate effort-based choice. OBJECTIVES: The present studies focused upon the ability of the glycine transport inhibitor bitopertin to attenuate haloperidol-induced shifts in effort-related choice behavior. METHODS: Effort-based choice in rats was assessed using the concurrent fixed ratio (FR) 5/chow feeding choice task and the T-maze barrier choice procedure. RESULTS: Haloperidol shifted effort-based choice, biasing animals towards the low effort option in each task. Co-administration of bitopertin (1.0-10.0 mg/kg) significantly attenuated haloperidol-induced shifts in choice behavior, but the same doses of bitopertin had no effect when administered alone. CONCLUSIONS: These results indicated that elevation of extracellular glycine via inhibition of glycine uptake was able to reverse the effects of D2 antagonism. Increases in extracellular glycine, possibly through actions on the glycine allosteric site on the NMDA receptor, may be a useful strategy for treating motivational dysfunctions in humans.

From laboratory to environmental conditions: a new approach for chemical's biodegradability assessment.

With thousands of organic chemicals released every day into our environment, Europe and other continents are confronted with increased risk of health and environmental problems. Even if a strict regulation such as REgistration, Authorization and restriction of CHemicals (REACH) is imposed and followed by industry to ensure that they prove the harmlessness of their substances, not all testing procedures are designed to cope with the complexity of the environment. This is especially true for the evaluation of persistence through biodegradability assessment guidelines. Our new approach has been to adapt "in the lab" biodegradability assessment to the environmental conditions and model the probability for a biodegradation test to be positive in the form of a logistic function of both the temperature and the viable cell density. Here, a proof of this new concept is proposed with the establishment of tri-dimensional biodegradability profiles of six chemicals (sodium benzoate, 4-nitrophenol, diethylene glycol, 2,4,5-trichlorophenol, atrazine, and glyphosate) between 4 to 30 °C and 10(4) to 10(8) cells ml(-1) as can be found in environmental compartments in time and space. The results show a significant increase of the predictive power of existing screening lab-scale tests designed for soluble substances. This strategy can be complementary to those current testing strategies with the creation of new indicators to quantify environmental persistence using lab-scale tests.

Neurotransmitter Specific, Cellular-Resolution Functional Brain Mapping Using Receptor Coated Nanoparticles: Assessment of the Possibility.

Receptor coated resonant nanoparticles and quantum dots are proposed to provide a cellular-level resolution image of neural activities inside the brain. The functionalized nanoparticles and quantum dots in this approach will selectively bind to different neurotransmitters in the extra-synaptic regions of neurons. This allows us to detect neural activities in real time by monitoring the nanoparticles and quantum dots optically. Gold nanoparticles (GNPs) with two different geometries (sphere and rod) and quantum dots (QDs) with different sizes were studied along with three different neurotransmitters: dopamine, gamma-Aminobutyric acid (GABA), and glycine. The absorption/emission spectra of GNPs and QDs before and after binding of neurotransmitters and their corresponding receptors are reported. The results using QDs and nanorods with diameter 25nm and aspect rations larger than three were promising for the development of the proposed functional brain mapping approach.

Application of NMR Spectroscopy in the Assessment of Radiation Dose in Human Primary Cells.

We employed the primary cell model system as a first step toward establishing a method to assess the influence of ionizing radiation by using a combination of common and abundant metabolites. We applied X-ray irradiation amounts of 0, 1, and 5 Gy to the cells that were harvested 24, 48, or 72 h later, and profiled metabolites by 2D-NMR spectroscopy to sort out candidate molecules that could be used to distinguish the samples under different irradiation conditions. We traced metabolites stemming from the input ¹³C-glucose, identified twelve of them from the cell extracts, and applied statistical analysis to find out that all the metabolites, including glycine, alanine, and gluatamic acid, increased upon irradiation. The combinatorial use of the selected metabolites showed promising results where the product of signal intensities of alanine and lactate could differentiate samples according to the dose of X-ray irradiation. We hope that this work can form a base for treating radiation-poisoned patients in the future.

Metabolomics strategy reveals therapeutical assessment of limonin on nonbacterial prostatitis.

Limonin has been found to possess significant anti-inflammatory properties in animal tests and with, human cells, however, its precise metabolism mechanism has not been well explored. The aim of this study was to investigate the anti-inflammatory effects of limonin in a nonbacterial prostatitis (NBP) animal model. Global metabolite profiling was performed by ultra-high-performance liquid chromatography combined with time-of-flight mass spectrometry (UPLC/ESI-TOFMS) and in conjunction with multivariate data analysis and pathway analysis which were integrated to explore differentiating metabolites and clarify the mechanism of limonin against capsaicin-induced NBP. Limonin has a potential protective function revealed by the metabolic profiling of limonin-treated rats located closer to the normal group. Twenty potential biomarker candidates and several key metabolic pathways contributing to the treatment of NBP were discovered and identified. Among the pathways, the related glycine, serine and threonine metabolism, glycerophospholipid metabolism were acutely perturbed. The changes in metabolites were restored to their base-line levels after limonin treatment, which might be through regulating the perturbed pathways to the normal state. The results indicate that changed biomarkers and pathways may provide evidence and insight into limonin action mechanisms and enable us to increase research productivity toward metabolomics in therapeutical assessment and drug discovery.

Functional assessment of mycosporine-like amino acids in Microcystis aeruginosa strain PCC 7806.

The biological role of the widespread mycosporine-like amino acids (MAAs) in cyanobacteria is under debate. Here, we have constructed and characterized two mutants impaired in MAA biosynthesis in the bloom-forming cyanobacterium Microcystis aeruginosa PCC 7806. We could identify shinorine as the sole MAA type of the strain, which is exclusively located in the extracellular matrix. Bioinformatic studies as wells as polymerase chain reaction screening revealed that the ability to produce MAAs is sporadically distributed within the genus. Growth experiments and reactive oxygen species quantification with wild-type and mutant strains did not support a role of shinorine in protection against UV or other stress conditions in M. aeruginosa PCC 7806. The shinorine content per dry weight of cells as well as transcription of the mys gene cluster was not significantly elevated in response to UV-A, UV-B or any other stress condition tested. Remarkably, both mutants exhibited pronounced morphological changes compared with the wild type. We observed an increased accumulation and an enhanced hydrophobicity of the extracellular matrix. Our study suggests that MAAs in Microcystis play a negligible role in protection against UV radiation but might be a strain-specific trait involved in extracellular matrix formation and cell-cell interaction.

Dynamics and environmental risk assessment of the herbicide glyphosate and its metabolite AMPA in a small vineyard river of the Lake Geneva catchment.

The use of pesticides may lead to environmental problems, such as surface water pollution, with a risk for aquatic organisms. In the present study, a typical vineyard river of western Switzerland was first monitored to measure discharged loads, identify sources, and assess the dynamic of the herbicide glyphosate and its metabolite aminomethylphosphonic acid (AMPA). Second, based on river concentrations, an associated environmental risk was calculated using laboratory tests and ecotoxicity data from the literature. Measured concentrations confirmed the mobility of these molecules with elevated peaks during flood events, up to 4970 ng/L. From April 2011 to September 2011, a total load of 7.1 kg was calculated, with 85% coming from vineyards and minor urban sources and 15% from arable crops. Compared with the existing literature, this load represents an important fraction (6-12%) of the estimated amount applied because of the steep vineyard slopes (∼10%). The associated risk of these compounds toward aquatic species was found to be negligible in the present study, as well as for other rivers in Switzerland. A growth stimulation was nevertheless observed for the algae Scenedesmus vacuolatus with low concentrations of glyphosate, which could indicate a risk of perturbation in aquatic ecosystems, such as eutrophication. The combination of field and ecotoxicity data allowed the performance of a realistic risk assessment for glyphosate and AMPA, which should be applied to other pesticide molecules.

A new strategy for improved glutathione production from Saccharomyces cerevisiae: use of cysteine- and glycine-rich chicken feather protein hydrolysate as a new cheap substrate.

BACKGROUND: Glutathione (GSH) is composed of the amino acids glutamic acid, cysteine and glycine. This study investigated the usability of chicken feather protein hydrolysate (chicken feather peptone, CFP) as a substrate for GSH production from Saccharomyces cerevisiae. RESULTS: CFP was found to be rich in ash (36.7 g per 100 g), protein (61.1 g per 100 g) and minerals (S, P, K, Ca, Fe, Na and Mg). It also had high contents of cysteine and glycine. CFP augmented biomass and GSH production by 53 and 115% respectively compared with the control medium. The highest biomass (17.4 g l(-1)) and GSH (271 mg L(-1)) concentrations were attained in CFP medium. The second highest biomass (16.8 g l(-1)) and GSH (255 mg L(-1)) concentrations were obtained in fish peptone medium. It was assumed that the high mineral, cysteine and glycine contents of CFP were related to cell growth and GSH synthesis in S. cerevisiae. CONCLUSION: This is the first report on the effect of cysteine- and glycine-rich protein hydrolysates on GSH production from S. cerevisiae. In this regard, CFP was tested for the first time as a GSH production substrate. As an additional contribution, a new hydrolysis process was developed for the preparation of protein hydrolysates.

Construction and assessment of reaction models of Class I EPSP synthase. Part II: investigation of the EPSP ketal.

Although the proposed mechanisms are reasonable, there are still many questions about the 5-enolpyruvyl shikimate-3-phosphate (EPSP) synthase mechanism that are difficult to answer by experimental means alone. EPSP synthase is a key enzyme in the shikimic acid pathway, which is found only in plants and some micro-organisms and is also molecular target of glyphosate, active component of one of the top-selling herbicides. In the study of reaction mechanism of EPSP synthase, in addition to inorganic phosphate and EPSP products, after long time at equilibrium, it was shown that a side product is formed, the EPSP ketal. In this line, studies using density functional theory (DFT) techniques were performed to investigate the reaction mechanism of formation of EPSP and the corresponding ketal. Our findings indicate some key amino acid residues in the EPSP synthase mechanism and a possible route for the formation of the EPSP ketal.