Nutrient-specific proteomic analysis of the mucin degrading bacterium Akkermansia muciniphila

Akkermansia muciniphila is a prominent mucin-degrading bacterium that acts as a keystone species in regulating the human gut microbiota. Despite recently increasing research into this bacterium and its relevance to human health, a high-resolution database of its functional proteins remains scarce. Here, we provide a proteomic overview of A. muciniphila grown in different nutrient conditions ranging from defined to complex. Of 2318 protein-coding genes in the genome, we identified 841 (40%) that were expressed at the protein level. Overall, proteins involved in energy production and carbohydrate metabolism indicate that A. muciniphila relies mainly on the Embden-Meyerhof-Parnas pathway, and produces short-chain fatty acids through anaerobic fermentation in a nutrient-specific manner. Moreover, this bacterium possesses a broad repertoire of glycosyl hydrolases, together with putative peptidases and sulfatases, to cleave O-glycosylated mucin. Of them, putative mucin-degrading enzymes (Amuc_1220, Amuc_1120, Amuc_0052, Amuc_0480, and Amuc_0060) are highly abundant in the mucin-supplemented media. Furthermore, A. muciniphila uses mucin-derived monosaccharides as sources of energy and cell wall biogenesis. Our dataset provides nutrient-dependent global proteomes of A. muciniphila ATCC BAA-835 to offer insights into its metabolic functions that shape the composition of the human gut microbiota via mucin degradation.     

Insights into the Role of Erythrina corallodendron L. in Alzheimer's Disease: in Vitro and in Silico Approach

Alzheimer's disease (AD) is a major health problem. Cholinergic transmission is greatly affected in AD. Phytochemical investigation of the alkaloid rich fraction (AF) of Erythrina corallodendron L leaves resulted in isolation of five known alkaloids: erysodine, erythrinine, 8-oxoerythrinine, erysovine N-oxide and erythrinine N-oxide. In this study, eysovine N-oxide was reported for the second time in nature. AF was assayed for cholinesterase inhibition at the concentration of 100 μg mL⁻¹. AF showed a higher percent inhibition for butyrylcholinesterase enzyme (BuChE) (83.28 %) compared to acetylcholinesterase enzyme (AChE) (64.64 %). The isolated alkaloids were also assayed for their anti-BuChE effect. In-silico docking study was done for the isolated compounds at the binding sites of AChE and BuChE to determine their binding pattern and interactions, also molecular dynamics were estimated for the compound displaying the best fit for AChE and BuChE. In addition, ADME parameters and toxicity were predicted for the isolated alkaloids compared to donepezil.     

Glycosylation reactions — present status future directions

A short review of the present status of glycosylation reactions is presented. The reactivity of both proven and newer glycosylation methods are briefly discussed. Emphasis is placed on the control of stereochemistry and regiochemistry. As well, the identification and avoidance of side reactions is covered. Polymer-supported synthesis of oligosaccharides is noted as a promising direction for eliminating some of the problems associated with purification. It is suggested that a better understanding of the mechanism of glycosylation reactions is necessary for future improvements to stereoselectivity and regioselectivity. A key advance would be methods for enhancing the reactivity of weakly nucleophilic hydroxyls.Abbreviations BF₃ OEt₂ boron trifluoride diethyl ether complex - TMSOTf trimethylsilyl trifluorometh-anesulfonate - NIS NBS N-iodosuccinimide andN-bromosuccinimide - TfOH trifluoromethanesulfonic acid or triflic acid - AgOTf CuOTf₂ silver triflate and copper(II)triflate - Tf₂O triflic anhydride - IDCP iodonium dicollidine perchlorate - TEP triethyl phosphite - HfCp₂Cl₂ hafnium dicyclopentadienyl dichloride - Ac acetyl - Bz benzoyl - Bn benzyl - Ph phenyl - Me methyl - Et ethyl - Bu₄NOTf tetrabutylammonium triflate - Ph₂IOTf diphenyliodinium triflate - PhSeNPhth N-(phenylseleno)phthalimide - Pent 4-pentenyl - TCI trichloroactemidyl - TBDPS t-butyl diphenylsilyl - DTBP 2,6-di-t-butylpyridine - Tr trityl or triphenylmethyl Dedicated to Professor J.J. Krepinsky on the occasion of his 60th birthday     

Acid hydrolases and their Release in Food Vacuole-Less Mutants of Tetrahymena thermophila*

SYNOPSIS. Mutants (NP1 and PSJ5) of Tetrahymena thermophila strains B and D 1968 exist that are unable to construct a functional oral apparatus and form food vacuoles at 37 C but which do so normally at 30 C. Food vacuole-less cells starved in dilute salt solution released similar amounts of acid phosphatase, β-N-acetyl-glucosaminidase and ±-glucosidase activity into the medium as wildtype cells during an 8-h period. Actively growing, food vacuole-less cells had ?50% less total protein, acid phosphatase, β-N-acetyl-glucosamin-idase, and ±-glucosidase per cell than wildtype cells after 72-h growth. During this time food vacuole-less cells released significant amounts of the 3 acid hydrolases into the growth medium. For each hydrolase, the total activity released from growing, food vacuole-less cells was less, on a per cell basis, than the amount released from food vacuole formers. The proportion of the total activity secreted by the mutant and the wildtype cells was the same for acid phosphatase and β-N-acetyl-glucosaminidase and somewhat lower for ±-glucosidase. It is concluded that the release of a significant amount of acid hydrolase activity from Tetrahymena is independent of food vacuole formation and may be analogous to the secretory activity of other nonphagocytic eukaryotic cells.     

Mycoparasitism as a mechanism of Trichoderma-mediated suppression of plant diseases

Trichoderma spp. are widely used as plant disease biocontrol agents in agriculture. Mycoparasitism, which is an ancestral trait of Trichoderma, is one of the most important mechanisms of reducing the pathogen inocula. Mycoparasitism is a complex physiological process that should be viewed in the broad perspective of microbial competition, and involves the production of enzymes and secondary metabolites. Trichoderma spp. have traditionally been viewed as necrotrophic mycoparasites; however, there are evidences that, at least in some instances, they behave as hemibiotrophs, causing minor damage to the host cell wall and having an intracellular existence in the host cell for a significant period. In this review, we cover different aspects of Trichoderma as mycoparasites, ranging from evolution to genomics and interactions with “non-target” fungi.     

Biotransformations of α,β-Epoxyalcohols Catalyzed by Epoxide Hydrolases

Microsomal and cytosolic fractions of mammalian livers were screened for their capacity to resolve racemic mixtures of trans -2,3-epoxy-l-alkanols. The epoxide hydrolase activities showed some specificity for the 2S, 3S enantiomers which were attacked at the proximate carbon atom. The best resolutions were observed with guinea pig liver microsomal enzymes.     

Elevated Levels of the Endosomal-Lysosomal Proteinase Cathepsin D in Cerebrospinal Fluid in Alzheimer Disease

Abstract: Lysosomal hydrolases are normally intracellular enzymes but are abundant extracellularly within senile plaques in Alzheimer disease and in other conditions where β-amyloid accumulates. To examine whether acid hydrolases released from abnormal hydrolase-laden neurons are detectable in CSF, we measured levels of the major aspartic proteinase of lysosomes, cathepsin D (Cat D), in ventricular CSF collected after death from 30 patients with Alzheimer disease, 14 patients with Huntington disease, and seven patients with other neurodegenerative diseases. The levels of Cat D-immunoreactive protein, expressed as micrograms per milliliter of protein, determined by western blot immunoassay using a polyclonal antiserum against human brain Cat D, were more than fourfold higher in the Alzheimer patients than in the other patient groups ( p < 0.0005). Cat D activity, assayed separately against [¹⁴C]methemoglobin at pH 3.2, was also significantly elevated but less than Cat D content. The lower specific activity of Cat D in Alzheimer CSF therefore indicated that the abnormally accumulated Cat D included a high proportion of inactive enzyme. These results indicate that abnormal Cat D release from affected neurons into the extracellular space is an active, ongoing process in Alzheimer brain. In addition, the levels of this enzyme and possibly other lysosomal hydrolases in CSF may prove to be useful biological markers of Alzheimer disease.     

Ultrastructural and cytochemical studies of acid phosphatase and trimetaphosphatase in rat peritoneal mast cells developing in vivo

Summary The ultrastructural and cytochemical features of peritoneal mast cells of the rat were studied. Immature mast cells show specific cytoplasmic granules of different sizes, the smaller ones localized in the Golgi region. The rough endoplasmic reticulum and Golgi apparatus are well developed, and mitochondria are numerous. Nuclei show deep indentations. Acid phosphatase is present in the Golgi saccules, in GERL (Golgi apparatus-endoplasmic reticulumlysosome) and in some small granules. It is not present in mature granules. Trimetaphosphatase is present in the Golgi saccules, in GERL, in most immature granules and in some mature granules. These enzymes appear to be transported and packaged into granules by the Golgi apparatus, suggesting that the specific mast cell granules may be a form of lysosome. The results of this study are consistent with the hypothesis that peritoneal mast cells may be derived from macrophage-like precursors.     

Lysosomal hydrolases in reproductive organs during estrous cycle of the hamster

Changes in the total protein content and the activities of lysosomal hydrolases (arylsulfatase, acid phosphatases, β-glucuronidase, β-N-acetylhexosaminidase, α-L-fucosidase, and β-galactosidase) of the hamster genital tract during the 4 days of estrous cycle and in hormonally superovulated hamsters were measured. Levels of lysosomal hydrolases in uteri and uterine fluid changed significantly during the cycle. Similar changes were observed in uterine wet weight and uterine proteins. The pattern of enzyme activities in both the ovary and the oviduct were different from those in uteri. In the ovary, most enzyme activities and the total protein concentration remained elevated after ovulation. Protein concentration and enzyme activities were significantly higher in the ovary, oviduct, and uteri of superovulated hamsters as compared to controls.     

A critical survey of average distances between catalytic carboxyl groups in glycoside hydrolases

Published X‐ray crystallographic structures for glycoside hydrolases (GHs) from 39 different families are surveyed according to some rigorous selection criteria, and the distances separating 208 pairs of catalytic carboxyl groups (20 α‐retaining, 87 β‐retaining, 38 α‐inverting, and 63 β‐inverting) are analyzed. First, the average of all four inter‐carboxyl O^…O distances for each pair is determined; second, the mean of all the pair‐averages within each GH family is determined; third, means are determined for groups of GH families. No significant differences are found for free structures compared with those complexed with a ligand in the active site of the enzyme, nor for α‐GHs as compared with β‐GHs. The mean and standard deviation (1σ) of the unimodal distribution of average O^…O distances for all families of inverting GHs is 8 ± 2Å, with a very wide range from 5Å (GH82) to nearly 13Å (GH46). The distribution of average O^…O distances for all families of retaining GHs appears to be bimodal: the means and standard deviations of the two groups are 4.8 ± 0.3Å and 6.4 ± 0.6Å. These average values are more representative, and more likely to be meaningful, than the often‐quoted literature values, which are based on a very small sample of structures. The newly‐updated average values proposed here may alter perceptions about what separations between catalytic residues are “normal” or “abnormal” for GHs. Proteins 2014; 82:1747–1755. © 2014 Wiley Periodicals, Inc.