Polyethylene Terephthalate Hydrolases in Human Gut Microbiota and Their Implications for Human Health.

Polyethylene terephthalate (PET), primarily utilized for food and beverage packaging, consistently finds its way into the human gut, thereby exerting adverse effects on human health. PET hydrolases, critical for the degradation of PET, have been predominantly sourced from environmental microbial communities. Given the fact that the human gut harbors a vast and intricate consortium of microorganisms, inquiry into the presence of potential PET hydrolases within the human gut microbiota becomes imperative. In this investigation, we meticulously screened 22,156 homologous sequences that could potentially encode PET hydrolases using the hidden Markov model (HMM) paradigm, drawing from 4984 cultivated genomes of healthy human gut bacteria. Subsequently, we methodically validated the hydrolytic efficacy of five selected candidate PET hydrolases on both PET films and powders composed of micro-plastics (MPs). Notably, our study also unveiled the influence of both diverse PET MP powders and their resultant hydrolysates on the modulation of cytokine expression in macrophages. In summary, our research underscores the ubiquitous prevalence and considerable potential of the human gut microbiota in PET hydrolysis. Furthermore, our study significantly contributes to the holistic evaluation of the potential health hazards posed by PET MPs to human well-being.

Plant Polyphenols as Inhibitors of Hydrolases are Regulators of Digestion.

BACKGROUND: There are numerous reports on the use of polyphenol-containing foods and various medicinal plant preparations for the prophylaxis and therapy of metabolic diseases, such as metabolic syndrome and diabetes mellitus, respectively. One unifying aspect to the effect of these natural compounds is their ability to inhibit digestive enzymes, which is the focus of this review. SUMMARY: Polyphenols inhibit nonspecifically hydrolytic enzymes included in the digestion process, e.g., amylases, proteases, lipases. By that, the digestion process is protracted with different consequences as result of the incomplete absorption of monosaccharides, fatty acids, and amino acids as well as for the enhanced availability of substrates for the microbiome in ileum and colon. The resulting postprandial blood concentration of monosaccharides, fatty, and amino acids is lowered and by that different metabolic pathways proceed more slowly. As another positive result, polyphenols can also modulate the intestinal microbiome and thus mediate additional beneficial health effects. KEY MESSAGES: Many medicinal plants possess a broad spectrum of different polyphenols, thereby mediating the nonspecific inhibition of all hydrolytic enzyme activities in the gastrointestinal digestive process. As a consequence of the slowing down of digestive processes, risk factors for the development of metabolic disorders are reduced and the health of the patients with metabolic syndrome improves.Es gibt zahlreiche Berichte über die Verwendung von polyphenolhaltigen Lebensmitteln und verschiedenen Arzneilpflanzenpräparaten zur Prophylaxe und Therapie von Stoffwechselkrankheiten wie dem metabolischen Syndrom und Diabetes mellitus. Ein übergreifender Aspekt der Wirkung dieser Naturstoffe ist ihre Fähigkeit, Verdauungsenzyme zu hemmen, was im Mittelpunkt dieser Übersicht steht.Polyphenole hemmen unspezifisch hydrolytische Enzyme, die am Verdauungsprozess beteiligt sind, z.B. Amylasen, Proteasen, Lipasen. Dadurch wird der Verdauungsprozess verzögert, was sich in einer unvollständigen Resorption von Monosacchariden, Fettsäuren und Aminosäuren sowie in einer erhöhten Verfügbarkeit von Substraten für das Mikrobiom im Ileum und Kolon äußert. Dadurch wird die postprandiale Blutkonzentration von Monosacchariden, Fettsäuren und Aminosäuren gesenkt und verschiedene Stoffwechselwege laufen langsamer ab. Ein weiteres positives Ergebnis ist, dass Polyphenole auch das intestinale Mikrobiom modulieren können und damit zusätzliche positive Gesundheitseffekte vermitteln.Viele Arzneipflanzen verfügen über ein breites Spektrum verschiedener Polyphenole, die eine unspezifische Hemmung aller hydrolytischen Enzymaktivitäten im gastrointestinalen Verdauungsprozess bewirken. Durch die Verlangsamung der Verdauungsprozesse werden Risikofaktoren für die Entwicklung von Stoffwechselstörungen reduziert und der Gesundheitszustand von Patienten mit metabolischem Syndrom verbessert.

Metabolites and metagenomic analysis reveals the quality of Pu-erh "tea head".

Tea head, a derivative product of Pu-erh tea, are tight tea lumps formed during pile-fermentation. The aim of this study was to reveal the differences of quality-related metabolites and microbial communities between ripened Pu-erh tea (PE-21) and tea heads (CT-21). Compared with PE-21, CT-21 showed a more mellow and smooth taste with slight bitterness and astringency, and can withstand multiple infusions. Metabolites analysis indicated CT-21 had more abundant water-soluble substances (47.39%) and showed significant differences with PE-21 in the main compositions of amino acids, catechins and saccharides which contributed to the viscosity of tea liquor, mellow taste and the tight tea lumps formation. Microbial communities and COG annotation analysis revealed CT-21 had lower abundance of Bacteria (84.05%), and higher abundance of Eukaryota (15.10%), carbohydrate transport and metabolism (8.28%) and glycoside hydrolases (37.36%) compared with PE-21. The different microbial communities may cause metabolites changes, forming distinct flavor of Pu-erh.

Alteration of Community Metabolism by Prebiotics and Medicinal Herbs.

Several studies have examined the impact of prebiotics on gut microbiota and associated changes in host physiology. Here, we used the in vitro cultivation of human fecal samples stimulated with a series of chemically related prebiotics and medicinal herbs commonly used in Ayurvedic medicine, followed by 16S rRNA sequencing. We applied a genome-wide metabolic reconstruction of enumerated communities to compare and contrast the structural and functional impact of prebiotics and medicinal herbs. In doings so, we examined the relationships between discrete variations in sugar composition and sugar linkages associated with each prebiotic to drive changes in microbiota composition. The restructuring of microbial communities with glycan substrates alters community metabolism and its potential impact on host physiology. We analyzed sugar fermentation pathways and products predicted to be formed and prebiotic-induced changes in vitamin and amino acid biosynthesis and degradation. These results highlight the utility of combining a genome-wide metabolic reconstruction methodology with 16S rRNA sequence-based community profiles to provide insights pertaining to community metabolism. This process also provides a rational means for prioritizing in vivo analysis of prebiotics and medicinal herbs in vivo to test hypotheses related to therapeutic potential in specific diseases of interest.

It's time to go glyco in cell wall bioengineering.

Tailoring the structure of cellulose, hemicellulose or pectin in plant cell walls can modulate growth, disease resistance, biomass yield and other important agronomic traits. Recent advances in the biosynthesis of microfibrils and matrix polysaccharides force us to re-examine old assumptions about the assembly and functions of cell wall components. The engineering of living or hybrid materials in microorganisms could be adapted to plant biopolymers or to inspire the development of new plant-based composites. High-throughput cellular factories and synthetic biology toolkits could unveil the biological roles and biotechnological potential of the large, unexplored space of carbohydrate-active enzymes. Increasing automation and enhanced carbohydrate detection methods are unlocking new routes to design plant glycans for a sustainable bioeconomy.

Molecular and biochemical modifications of suspension-cultured tobacco cell walls after exposure to alternative gravity.

The plant cell wall is a flexible physical barrier surrounding the cell which functions in growth and differentiation, signaling, and response to environmental stimuli including the Earth gravity force. In the present study, structural and molecular modifications of cell wall components of cultured tobacco (Nicotiana tabacum cv. Burley 21) cells under alternative gravity conditions induced by 7 days exposure to 2-D clinostat have been investigated. In comparison with the control group, clinorotation significantly increased biomass but reduced the total amounts of wall and the contents of cellulose, pectin, uronic acidic, and xyloglucan. Gene expression of H+-ATPase was not changed but of expansin A reduced in clinostat-treated cells. However, the gene expression and activity of xyloglucan endotransglycosylase/hydrolases (XTH; EC 2.4.1.207) and endo-(1,4)-ß-D-glucanase (EGase; EC 3.2.1.4), the amount of arabinogalactan proteins (AGP), and the expression of wall-associated kinase (WAK) gene significantly increased by clinorotation. Altered gravity also reduced the activity of polyphenol oxidase and covalently bound peroxidase. The results suggest that altered gravity promoted orchestrated changes of wall-modifying genes and proteins which reduced its stiffness and enhanced cell expansion and division potential.

Flavonoids and proanthocyanidins-rich fractions from Eugenia dysenterica fruits and leaves inhibit the formation of advanced glycation end-products and the activities of α-amylase and α-glucosidase.

ETHNOPHARMACOLOGICAL RELEVANCE: Different parts of Eugenia dysenterica have been popularly used in Brazil for treating diabetes mellitus and its complications. The present study aimed to screen extracts from E. dysenterica fruit pulp, peel, seed and leaf for carbohydrate digestive enzymes inhibitors with antioxidant and anti-glycation capacities. MATERIALS AND METHODS: Ethanol extracts of E. dysenterica were subjected to a liquid-liquid fractionation and the fractions were used to evaluate their antioxidant properties and inhibitory potential against the formation of advanced glycation end-products (AGEs) and α-amylase and α-glucosidase. RESULTS: The ethyl acetate fraction (EtOAcF) from seed and the dichloromethane fraction (CH2Cl2F) and EtOAcF from leaf had high antioxidant capacities (ORAC >5500 µmol trolox eq g-1, FRAP >1500 µmol trolox eq g-1 and DPPH IC50 < 35 µg mL-1) and showed exceptional inhibitory activities against AGEs formation (glycation inhibition above 80% at 10 µg mL-1) and α-amylase and α-glucosidase (inhibition above 50% at 10 µg mL-1). The gallated B-types proanthocyanidins were the most active ingredients found in the leaf of E. dysenterica (CH2Cl2 and EtOAcF), being responsible for the notorious inhibitory effects against glycation and glycoside hydrolases due to their ortho-hydroxyl groups, which play role in scavenge and quench free radicals and glycated products, and may occupy the enzymes' substrate binding pocket. Furthermore, gallic acid, quercetin and its glycoside derivatives were detected by the first time in the E. dysenterica fruit seed (EtOAcF). CONCLUSIONS: The results strongly contribute to the understanding of the antidiabetic potential of seeds and leaves from E. dysenterica, a species from a global biodiversity hotspot, which appears to be linked to the prevention of oxidative stress, AGEs production and postprandial hyperglycemia.

Leaf Apoplast of Field-Grown Potato Analyzed by Quantitative Proteomics and Activity-Based Protein Profiling.

Multiple biotic and abiotic stresses challenge plants growing in agricultural fields. Most molecular studies have aimed to understand plant responses to challenges under controlled conditions. However, studies on field-grown plants are scarce, limiting application of the findings in agricultural conditions. In this study, we investigated the composition of apoplastic proteomes of potato cultivar Bintje grown under field conditions, i.e., two field sites in June-August across two years and fungicide treated and untreated, using quantitative proteomics, as well as its activity using activity-based protein profiling (ABPP). Samples were clustered and some proteins showed significant intensity and activity differences, based on their field site and sampling time (June-August), indicating differential regulation of certain proteins in response to environmental or developmental factors. Peroxidases, class II chitinases, pectinesterases, and osmotins were among the proteins more abundant later in the growing season (July-August) as compared to early in the season (June). We did not detect significant differences between fungicide Shirlan treated and untreated field samples in two growing seasons. Using ABPP, we showed differential activity of serine hydrolases and ß-glycosidases under greenhouse and field conditions and across a growing season. Furthermore, the activity of serine hydrolases and ß-glycosidases, including proteins related to biotic stress tolerance, decreased as the season progressed. The generated proteomics data would facilitate further studies aiming at understanding mechanisms of molecular plant physiology in agricultural fields and help applying effective strategies to mitigate biotic and abiotic stresses.

Identification and response analysis of xyloglucan endotransglycosylase/hydrolases (XTH) family to fluoride and aluminum treatment in Camellia sinensis.

BACKGROUND: Xyloglucan endotransglycosylase/hydrolases (XTH) can disrupt and reconnect the xyloglucan chains, modify the cellulose-xyloglucan complex structure in the cell wall to reconstruct the cell wall. Previous studies have reported that XTH plays a key role in the aluminum (Al) tolerance of tea plants (Camellia sinensis), which is a typical plant that accumulates Al and fluoride (F), but its role in F resistance has not been reported. RESULTS: Here, 14 CsXTH genes were identified from C. sinensis and named as CsXTH1-14. The phylogenetic analysis revealed that CsXTH members were divided into 3 subclasses, and conserved motif analysis showed that all these members included catalytic active region. Furthermore, the expressions of all CsXTH genes showed tissue-specific and were regulated by Al3+ and F- treatments. CsXTH1, CsXTH4, CsXTH6-8 and CsXTH11-14 were up-regulated under Al3+ treatments; CsXTH1-10 and CsXTH12-14 responded to different concentrations of F- treatments. The content of xyloglucan oligosaccharide determined by immunofluorescence labeling increased to the highest level at low concentrations of Al3+ or F- treatments (0.4 mM Al3+ or 8 mg/L F-), accompanying by the activity of XET (Xyloglucan endotransglucosylase) peaked. CONCLUSION: In conclusion, CsXTH activities were regulated by Al or F via controlling the expressions of CsXTH genes and the content of xyloglucan oligosaccharide in C. sinensis roots was affected by Al or F, which might finally influence the elongation of roots and the growth of plants.

Investigation of cell wall proteins of C. sinensis leaves by combining cell wall proteomics and N-glycoproteomics.

BACKGROUND: C. sinensis is an important economic crop with fluoride over-accumulation in its leaves, which poses a serious threat to human health due to its leaf consumption as tea. Recently, our study has indicated that cell wall proteins (CWPs) probably play a vital role in fluoride accumulation/detoxification in C. sinensis. However, there has been a lack in CWP identification and characterization up to now. This study is aimed to characterize cell wall proteome of C. sinensis leaves and to develop more CWPs related to stress response. A strategy of combined cell wall proteomics and N-glycoproteomics was employed to investigate CWPs. CWPs were extracted by sequential salt buffers, while N-glycoproteins were enriched by hydrophilic interaction chromatography method using C. sinensis leaves as a material. Afterwards all the proteins were subjected to UPLC-MS/MS analysis. RESULTS: A total of 501 CWPs and 195 CWPs were identified respectively by cell wall proteomics and N-glycoproteomics profiling with 118 CWPs in common. Notably, N-glycoproteomics is a feasible method for CWP identification, and it can enhance CWP coverage. Among identified CWPs, proteins acting on cell wall polysaccharides constitute the largest functional class, most of which might be involved in cell wall structure remodeling. The second largest functional class mainly encompass various proteases related to CWP turnover and maturation. Oxidoreductases represent the third largest functional class, most of which (especially Class III peroxidases) participate in defense response. As expected, identified CWPs are mainly related to plant cell wall formation and defense response. CONCLUSION: This was the first large-scale investigation of CWPs in C. sinensis through cell wall proteomics and N-glycoproteomics. Our results not only provide a database for further research on CWPs, but also an insight into cell wall formation and defense response in C. sinensis.

A Combination of Lactoplantibacillus plantarum Strains CECT7527, CECT7528, and CECT7529 Plus Monacolin K Reduces Blood Cholesterol: Results from a Randomized, Double-Blind, Placebo-Controlled Study.

BACKGROUND: Dietary supplements have been proposed to help manage blood cholesterol, including red yeast rice (RYR) extracts, plant sterols and stanols, beta-glucans, and some probiotics. This study was conducted to evaluate the efficacy of RYR (containing 10 mg of monacolin K) combined with 109 CFU of three Lactoplantibacillus plantarum strains (CECT7527, CECT7528, and CECT7529). METHODS: A 12-week randomized, double-blinded, placebo-controlled clinical trial was conducted. In total, 39 adult patients were enrolled, having total cholesterol (TC) ≥200 mg/dL, and being statin-naïve or having recently stopped statin treatment because of intolerance. Active product or placebo were taken once daily, and subjects were evaluated at baseline, 6, and 12 weeks. RESULTS: Study groups were comparable at baseline, except for history of recent hypercholesterolemia treatment (81% in active vs. 22% in placebo). Changes in LDL cholesterol and TC became significant compared to placebo (mean difference between groups and standard error of the mean = 23.6 ± 1.5 mg/dL, p = 0.023 and 31.4 ± 1.9 mg/dL, p = 0.011, respectively) upon adjusting for the baseline imbalance in hypercholesterolemia treatment. No adverse effects were noted during the study. CONCLUSION: This combination of 10 mg of monacolin K and L. plantarum strains was well tolerated and achieved a statistically significant greater reduction in LDL-C and TC in the intervention group compared to the placebo, once adjusting for recent history of hypercholesterolemia treatment.

Identification of α-Amylase and α-Glucosidase Inhibitors and Ligularoside A, a New Triterpenoid Saponin from Passiflora ligularis Juss (Sweet Granadilla) Leaves, by a Nuclear Magnetic Resonance-Based Metabolomic Study.

The leaves of Passiflora ligularis Juss (known as sweet granadilla for its edible fruits) are a crop byproduct that is discarded. With the aim of contributing to give value-added products from these crop by-side products to farmers of Colombian Andes, we carried out a 1H-NMR-metabolomics analysis of polar extracts from leaves collected in three locations and stored in two conditions in order to identify glucosyl-hydrolase inhibitors. Variations in the metabolic profile and the bioactivity among samples were analyzed by orthogonal partial least square discriminant analysis. Thus, 1H-NMR signals related to polyphenolic compounds, saponins, and amino acids were correlated with higher inhibitory activities. Moreover, a targeted NMR and HPLC-MS/MS analysis allowed the identification of 14 polyphenolic compounds and the structural characterization of a new triterpenoid saponin, ligularoside A. The measurements of IC50 values for α-amylase and α-glycosidase inhibitors allowed the identification of quercetin-3-O-ß-glucoside, kaempferol-3-O-ß-glucoside, and ligularoside A as the most active compounds. These results suggest that P. ligularis leaves are a source of glucosyl-hydrolase inhibitors and lay the foundation for exploring additional applications.

Targeting Hidden Pathogens: Cell-Penetrating Enzybiotics Eradicate Intracellular Drug-Resistant Staphylococcus aureus.

Staphylococcus aureus is a major concern in human health care, mostly due to the increasing prevalence of antibiotic resistance. Intracellular localization of S. aureus plays a key role in recurrent infections by protecting the pathogens from antibiotics and immune responses. Peptidoglycan hydrolases (PGHs) are highly specific bactericidal enzymes active against both drug-sensitive and -resistant bacteria. However, PGHs able to effectively target intracellular S. aureus are not yet available. To overcome this limitation, we first screened 322 recombineered PGHs for staphylolytic activity under conditions found inside eukaryotic intracellular compartments. The most active constructs were modified by fusion to different cell-penetrating peptides (CPPs), resulting in increased uptake and enhanced intracellular killing (reduction by up to 4.5 log units) of various S. aureus strains (including methicillin-resistant S. aureus [MRSA]) in different tissue culture infection models. The combined application of synergistic PGH-CPP constructs further enhanced their intracellular efficacy. Finally, synergistically active PGH-CPP cocktails reduced the total S. aureus by more than 2.2 log units in a murine abscess model after peripheral injection. Significantly more intracellular bacteria were killed by the PGH-CPPs than by the PGHs alone. Collectively, our findings show that CPP-fused PGHs are effective novel protein therapeutics against both intracellular and drug-resistant S. aureusIMPORTANCE The increasing prevalence of antibiotic-resistant bacteria is one of the most urgent problems of our time. Staphylococcus aureus is an important human pathogen that has acquired several mechanisms to evade antibiotic treatment. In addition, S. aureus is able to invade and persist within human cells, hiding from the immune response and antibiotic therapies. For these reasons, novel antibacterial strategies against these pathogens are needed. Here, we developed lytic enzymes which are able to effectively target drug-resistant and intracellular S. aureus Fusion of these so-called enzybiotics to cell-penetrating peptides enhanced their uptake and intracellular bactericidal activity in cell culture and in an abscess mouse model. Our results suggest that cell-penetrating enzybiotics are a promising new class of therapeutics against staphylococcal infections.

Exploration and Characterization of Novel Glycoside Hydrolases from the Whole Genome of Lactobacillus ginsenosidimutans and Enriched Production of Minor Ginsenoside Rg3(S) by a Recombinant Enzymatic Process.

BACKGROUND: Several studies have reported that ginsenoside Rg3(S) is effective in treating metastatic diseases, obesity, and various cancers, however, its presence in white ginseng cannot be estimated, and only a limited amount is present in red ginseng. Therefore, the use of recombinant glycosidases from a Generally Recognized As Safe (GRAS) host strain is a promising approach to enhance production of Rg3(S), which may improve nutritional activity, human health, and quality of life. METHOD: Lactobacillus ginsenosidimutans EMML 3041T, which was isolated from Korean fermented pickle (kimchi), presents ginsenoside-converting abilities. The strain was used to enrich the production of Rg3(S) by fermenting protopanaxadiol (PPD)-mix-type major ginsenosides (Rb1, Rb2, Rc, and Rd) in four different types of food-grade media (1, MRS; 2, Basel Food-Grade medium; 3, Basel Food-Grade medium-I, and 4, Basel Food-Grade medium-II). Due to its tendency to produce Rg3(S), the presence of glycoside hydrolase in Lactobacillus ginsenosidimutans was proposed, the whole genome was sequenced, and the probable glycoside hydrolase gene for ginsenoside conversion was cloned. RESULTS: The L. ginsenosidimutans EMML 3041T strain was whole genome sequenced to identify the target genes. After genome sequencing, 12 sets of glycoside hydrolases were identified, of which seven sets (α,ß-glucosidase and α,ß-galactosidase) were cloned in Escherichia coli BL21 (DE3) using the pGEX4T-1 vector system. Among the sets of clones, only one clone (BglL.gin-952) showed ginsenoside-transforming abilities. The recombinant BglL.gin-952 comprised 952 amino acid residues and belonged to glycoside hydrolase family 3. The enzyme exhibited optimal activity at 55 °C and a pH of 7.5 and showed a promising conversion ability of major ginsenoside Rb1→Rd→Rg3(S). The recombinant enzyme (GST-BglL.gin-952) was used to mass produce Rg3(S) from major ginsenoside Rb1. Scale-up of production using 50 g of Rb1 resulted in 30 g of Rg3(S) with 74.3% chromatography purity. CONCLUSION: Our preliminary data demonstrated that this enzyme would be beneficial in the preparation of pharmacologically active minor ginsenoside Rg3(S) in the functional food and pharmaceutical industries.

Prediction and analysis of GH14 family ß-amylases in oat seedling extract: Structure and function insights using in silico approaches.

Oat (Avena sativa L.) seedling extract exhibited a high degree of catalytic activities. Bioinformatics were used to identify ß-amylases as abundant enzymes in the oat seedling extract. These identified oat enzymes are a member of the GH14 family. Proteins in the Avena sativa seedling extract were separated by SDS-PAGE and 2 major protein bands with an apparent molecular weights of 53 and 42 kDa were the subject of this study. These materials were digested with trypsin and the amino acid sequences of the tryptic peptides were determined by LC/ESI/MS/MS and database searches. These sequences were used to identify cDNAs from expressed sequence tags (EST) and Transcriptome Shotgun Assembly (TSA) of Avena sativa. Based upon EST and TSA sequences, at least 6 predicted different sequences were identified and assigned as ß-amylases. Insights into structural characterization of the oat predicted ß-amylases were analyzed using in silico approaches. The identified ß-amylases conserved the two Glu residues assigned as the "putative" catalytic residues, which would act as an acid and base pair in the catalytic process. A similar core (ß/α)8-barrel architecture was found in the predicted oat ß-amylases with a specific location of the active site in a pocket-like cavity structure made at one end of this core (ß/α)8-barrel domain. This suggests an accessibility of the non-reducing end of the substrate towards the oat ß-amylases and thus confirming that are exo-acting hydrolases. The results provide a detailed view of the main residues involved in catalysis in this kind of enzyme.

Exploring Solanum tuberosum Epoxide Hydrolase Internal Architecture by Water Molecules Tracking.

Several different approaches are used to describe the role of protein compartments and residues in catalysis and to identify key residues suitable for the modification of the activity or selectivity of the desired enzyme. In our research, we applied a combination of molecular dynamics simulations and a water tracking approach to describe the water accessible volume of Solanum tuberosum epoxide hydrolase. Using water as a molecular probe, we were able to identify small cavities linked with the active site: (i) one made up of conserved amino acids and indispensable for the proper positioning of catalytic water and (ii) two others in which modification can potentially contribute to enzyme selectivity and activity. Additionally, we identified regions suitable for de novo tunnel design that could also modify the catalytic properties of the enzyme. The identified hot-spots extend the list of the previously targeted residues used for modification of the regioselectivity of the enzyme. Finally, we have provided an example of a simple and elegant process for the detailed description of the network of cavities and tunnels, which can be used in the planning of enzyme modifications and can be easily adapted to the study of any other protein.

[Chemical study on Cassiae Semen processing procedure].

References and our previous experiment showed that the contents of glycosides were significantly decreased，while the contents of aglycones were significantly increased after processing of Cassiae Semen.It may be related to its glycosidases or the heating process. In order to investigate the reasons, high performance liquid chromatographic (HPLC) was used to study the effects of these two factors on contents of Cassiae Semen's main chemical components in processing. The results showed that glycoside hydrolases was present in Cassiae Semen and could rapidly hydrolyze glycosides from Cassiae Semen into aglycones in suitable temperature with sufficient water.However，it didn't show effect on contents change of main constituents in the procedure of Cassiae Semen processing.The reason for content decrease of glycosides and content increase of aglycones in processed Cassiae Semen was glycoside bond cracking to produce corresponding aglycone at high temperature.This study further provides basis for further revealing of the processing mechanism of Cassiae Semen.

Autodigestion by migrated trypsin is a major factor in small intestinal ischemia-reperfusion injury.

BACKGROUND: The pathophysiological role of pancreatic digestive hydrolases in intestinal ischemia-reperfusion (I/R) injury is still not clear. Here, we studied whether ischemia-induced injury to the small intestine can be explained by the autodigestion hypothesis. MATERIALS AND METHODS: Mesenteric I/R was induced in rats by superior mesenteric artery occlusion (90 min) and reopening (120 min). Thirty minutes before superior mesenteric artery occlusion, aprotinin (14.7 mg/kg), orlistat (5 mg/kg), and their combination or α1-proteinase inhibitor (60 mg/kg) were injected into the lumen of the small intestine. Systemic and vital parameters, intestinal microcirculation, and mucosal barrier function were monitored during the observation phase; markers of small intestinal injury, as well as trypsin-, chymotrypsin-, elastase-, and lipase-like activities in intestinal effluates were assessed at the end. RESULTS: The pattern of small intestinal injury correlated inversely with the local alterations in microvascular tissue perfusion and corresponded with the intestinal distribution of trypsin-like activity. Aprotinin almost completely inhibited trypsin-like activity (P < 0.05) and significantly reduced intestinal tissue injury. Combined with orlistat, it also increased the postischemic blood pressure (P < 0.05) but not the intestinal barrier function. Macroscopic as well as the histologic alterations were decreased by α1-proteinase inhibitor, which significantly improved postischemic blood pressure (P < 0.05). CONCLUSIONS: The I/R-induced pattern of small intestinal injury is likely to result from both local differences in tissue ischemia and the digestive activity of migrated pancreatic trypsin. Therefore, administration of aprotinin and orlistat into ischemic small intestines may be a therapeutic option in patients with a poor diagnosis.

Growth, hydrolases and ultrastructure of Fusarium oxysporum as affected by phenolic rich extracts from several xerophytic plants.

Fusarium oxysporum, the causal agent of rot and wilt diseases, is one of the most detrimental phytopathogens for the productivity of many economic crops. The present study was conducted to evaluate the potentiality of some xerophytic plants as eco-friendly approach for management of F. oxysporum. Phenolic rich extracts from five plants namely: Horwoodia dicksoniae, Citrullus colocynthis, Gypsophila capillaris, Pulicaria incisa and Rhanterium epapposum were examined in vitro. The different extracts showed high variability in their phenolic and flavonoid contents as well as total antioxidant capacity. A strong positive correlation existed between the antifungal activity of the tested extracts and their contents of both total phenolics and flavonoids (r values are 0.91 and 0.82, respectively). Extract of P. incisa was the most effective in reducing the mycelial growth (IC50=0.92mg/ml) and inhibiting the activities of CMCase, pectinase, amylase and protease by 36, 42, 58 and 55%, respectively. The high performance liquid chromatography analysis of P. incisa extract revealed the presence of eight phenolic acids along with five polyphenolic compounds. The flavonol, quercetin and its glycosides rutin and quercetrin were the most abundant followed by the phenolic acids, t-cinnamic, caffeic, ferulic and vanillic. P. incisa extract not only affects the growth and hydrolases of F. oxysporum but also induces ultrastructure changes in the mycelium, as revealed by transmission electron microscopy. To our knowledge, this is the first study to investigate the mechanisms underlying the antifungal activity of P. incisa.

Ester-prodrugs of ethambutol control its antibacterial activity and provide rapid screening for mycobacterial hydrolase activity.

M. tuberculosis contains an unusually high number of serine hydrolases by proteome percentage compared to other common bacteria or humans. This letter describes a method to probe the global substrate specificity of mycobacterial serine hydrolases with ester-protected prodrugs of ethambutol, a first-line antibiotic treatment for TB. These compounds were synthesized directly from ethambutol using a selective o-acylation to yield products in high yield and purity with minimal workup. A library of derivatives was screened against M. smegmatis, a non-infectious model for M. tuberculosis, which displayed significantly lowered biological activity compared to ethambutol. Incubation with a general serine hydrolase reactivated each derivative to near-ethambutol levels, demonstrating that esterification of ethambutol should provide a simple screen for mycobacterial hydrolase activity.