Transpeptidase Activity Research Articles

Unraveling the Molecular Functions of Multifaced Plant‐Vacuolar Processing Enzymes

AbstractIn the plant development cycle, different types of proteases play essential functions. Plant vacuolar processing enzymes (VPEs) are a group of cysteine proteases localized in the cell vacuoles that process and regulate several larger proprotein precursors. VPEs activate the proteolytic cleavage of proproteins precursors within aspartic acid and asparagine residues to trigger the tightly controlled programmed cell death process induced by different stages of plant development and environmental cues, impacting myriad plant physiological processes. In plants, VPEs are categorized into embryo, vegetative, and seed coat‐types, in which embryo and seed coat‐type VPEs are mainly involved in processing the seed storage protein precursors to provide nutrients for early growth, while the vegetative type induces programmed cell death in a varied range of vegetative tissues. Recently, several plant VPEs exhibited transpeptidase activity, which is essential for the cyclization mechanism during the production of cyclotides. In this review, we explore and understand the complex functions of plant VPEs, which can lead to the production of stress‐tolerant and higher‐yield crops, and provide the recent advances in the application of VPEs in molecular pharming, which highlights the significant implications of VPEs in protein science and agriculture.

Peptidoglycan biosynthesis-associated enzymatic kinetic characteristics and β-lactam antibiotic inhibitory effects of different Streptococcus pneumoniae penicillin-binding proteins

Penicillin-binding proteins (PBPs) include transpeptidases, carboxypeptidases, and endopeptidases for biosynthesis of peptidoglycans in the cell wall to maintain bacterial morphology and survival in the environment. Streptococcus pneumoniae expresses six PBPs, but their enzymatic kinetic characteristics and inhibitory effects on different β-lactam antibiotics remain poorly understood. In this study, all the six recombinant PBPs of S. pneumoniae displayed transpeptidase activity with different substrate affinities (Km = 1.56–9.11 mM) in a concentration-dependent manner, and rPBP3 showed a greater catalytic efficiency (Kcat = 2.38 s−1) than the other rPBPs (Kcat = 3.20–7.49 × 10−2 s−1). However, only rPBP3 was identified as a carboxypeptidase (Km = 8.57 mM and Kcat = 2.57 s−1). None of the rPBPs exhibited endopeptidase activity. Penicillin and cefotaxime inhibited the transpeptidase and carboxypeptidase activity of all the rPBPs but imipenem did not inhibited the enzymatic activities of rPBP3. Except for the lack of binding of imipenem to rPBP3, penicillin, cefotaxime, and imipenem bound to all the other rPBPs (KD = 3.71–9.35 × 10−4 M). Sublethal concentrations of penicillin, cefotaxime, and imipenem induced a decrease of pneumococcal pbps-mRNA levels (p < 0.05). These results indicated that all six PBPs of S. pneumoniae are transpeptidases, while only PBP3 is a carboxypeptidase. Imipenem has no inhibitory effect on pneumococcal PBP3. The pneumococcal genes for encoding endopeptidases remain to be determined.

The effect of transfusion of erythrocyte mass on clinical and haematological indicators of dogs with hemolytic anaemia caused by babesiosis

The relevance of the research is due to the spread of protozoal diseases of animals, which, in the absence of the necessary veterinary care, lead to death. In this regard, the aim of the study was to investigate changes in clinical and haematological parameters in dogs under complex treatment of babesiosis with simultaneous red blood cell transfusion. Transfusions were performed in five animals infected with Babesia canis by intravenous injection of red blood cells, the volume of which was calculated using the Sackmen formula. The diagnosis of babesiosis was made based on anamnesis, clinical symptoms (weakness, refusal to food, pallor of visible mucous membranes, fever up to 40°C), and confirmed by microscopic detection of babesiae in blood smears. Before transfusion, rapid tests were performed to exclude heartworm disease in donor dogs. The compatibility of the blood of the donor and recipient animal was determined using a large cross-test. The condition of the abdominal organs of the patients was assessed using ultrasound diagnostics. Clinical examination of the dogs’ blood before treatment indicated the development of anaemia. The results of biochemical blood tests indicated an increase in the activity of alanine aminotransferase and gamma-glutamyl transpeptidase, which indicates a load on the hepatobiliary system. There were no contraindications to blood transfusion. After haemotransfusion, the clinical condition of the dogs was monitored based on the results of the examination of the animals, taking into account the anamnesis and haematological studies on the second, fifth, and tenth days of treatment. During the second and fifth days, a gradual increase in blood haemoglobin content and haematocrit value was noted. On the tenth day, there was a significant positive trend in the number of red blood cells, haemoglobin content and haematocrit to the reference values. It was established that the use of red blood cell transfusion in dogs with babesiosis as part of therapy contributed to the fastest recovery of the general condition of the animal. The material of the article is of practical value for the use of red blood cell transfusion in the treatment of dogs with babesiosis

Localization of the Remnant of a Cell Wall Sorting Signal and Its Interaction with a Sensor Kinase.

Sortases are highly conserved enzymes with endopeptidase and transpeptidase activities in Gram-positive bacteria. Sortase A cleaves within an LPXTG-motif and covalently crosslinks cell wall proteins to become anchored to the peptidoglycan of the cell wall. We showed that a peptide cleaved by sortase A from the C-terminus (C-pep) of the LPXTG-adhesin SspA intercalates in the cell membrane. Nested in the membrane, this C-pep docks with the intramembrane sensor histidine kinase, SraS, to activate the response regulator, SraR. SraR signals that the C-pep has been cleaved as an indicator of the fidelity of sortase A processing. SraSR also signals that key LPXTG-proteins in concert with lipoteichoic acid engage the mucin, MUC5B, which elicits a different transcriptional response than the binding of other salivary constituents. To visualize the C-pep intercalating in the cell membrane in vivo, we used Structured Illumination Microscopy (SIM). And to show that the C-pep complexes with SraS, we used bimolecular fluorescence experiments. The C-pep and SraS were each expressed with one or the other half of yellow fluorescence protein (YFP). Reconstitution of the complete YFP signal indicated that the C-pep and SraS interacted at molecular distances within the cell membrane in vivo. Using these imaging protocols, we learned that the C-pep functions as a signaling molecule within the cell membrane of the streptococcal cell.

Are Selected Cytokines and Epstein-Barr Virus DNA Load Predictors of Hepatological Complications of Epstein-Barr Virus Infection in Children?

The aim of the study was to evaluate tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6), soluble intracellular adhesion molecules 1 (s-ICAM-1) and Epstein-Barr virus (EBV) DNA load levels as predictors of hepatological complications of EBV infection in children. The study group consisted of 54 children aged one to eighteen years, who were hospitalised from 1 December 2018 to 31 December 2020 in the Department of Paediatrics, Infectious Diseases and Hepatology and who had hepatological complications in the course of serologically and molecularly confirmed EBV infection. It was shown that IL-6, TNF-α, and s-ICAM-1 concentrations were the highest in patients with hepatitis and biliary pole damage. Higher EBV DNA viremia positively correlated with increased C-reactive protein (CRP) and TNF-α levels and increased leukocyte, lymphocyte, and monocyte counts. Increases in lymphocyte counts and TNF-α concentrations were observed along with increases in gamma-glutamyl transpeptidase (GGTP) activity. Increased concentrations of IL-6, TNF-α, and s-ICAM-1 may indicate the risk of hepatitis with concomitant biliary pole damage during EBV infection.

Investigation of the inhibitory effect and mechanism of EGCG against Streptococcus suis sortase A.

Streptococcus suis (S. suis) seriously harms people and animals, and importantly, causes great economic losses in the pig industry. Similar to most gram-positive pathogenic bacteria, sortase A (SrtA) of S. suis can mediate the anchoring of a variety of virulence factors that contain specific sorting sequences to the surface of the bacterial cell wall envelope and participate in pathogenicity. The purpose of this study is to clarify the molecular mechanism of epigallocatechin-3-gallate (EGCG) inhibiting Streptococcus suis SrtA and provide more evidence for the development of novel anti- Streptococcus suis infections drugs. Through the SrtA substrate cleavage experiment, we found that the main component of green tea, EGCG, can effectively inhibit the enzyme activity of Streptococcus suis SrtA. Further, molecular docking and molecular dynamics simulation were used to clarify the molecular mechanism of its inhibitory effect, demonstrating that EGCG mainly interacts with amino acids at 113 and 115 to exert its inhibitory function. It was previously found that EGCG can inhibit the growth of S. suis and reduce the activity of suilysin and inhibit its expression. Our research reveals a new function of EGCG in S. suis infection. Our research proves that EGCG can effectively inhibit the transpeptidase activity of SrtA. We also clarify the accompanying molecular mechanism, providing more sufficient evidence for the use of EGCG as a potential lead compound against S. suis infection.

Synergistic Protective Effect of Fermented Schizandrae Fructus Pomace and Hoveniae Semen cum Fructus Extracts Mixture in the Ethanol-Induced Hepatotoxicity.

Schizandrae Fructus (SF), fruits of Schisandra chinensis (Turcz.) Baill. and Hoveniae Semen cum Fructus (HSCF), the dried peduncle of Hovenia dulcis Thunb., have long been used for alcohol detoxification in the traditional medicine of Korea and China. In the current study, we aimed to evaluate the potential synergistic hepatoprotective effect of a combination mixture (MSH) comprising fermented SF pomace (fSFP) and HSCF hot water extracts at a 1:1 (w:w) ratio against ethanol-induced liver toxicity. Subacute ethanol-mediated hepatotoxicity was induced by the oral administration of ethanol (5 g/kg) in C57BL/6J mice once daily for 14 consecutive days. One hour after each ethanol administration, MSH (50, 100, and 200 mg/kg) was also orally administered daily. MSH administration significantly reduced the serum activities of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and γ-glutamyl transpeptidase. Histological observation indicated that MSH administration synergistically and significantly decreased the fatty changed region of hepatic parenchyma and the formation of lipid droplet in hepatocytes. Moreover, MSH significantly attenuated the hepatic triglyceride accumulation through reducing lipogenesis genes expression and increasing fatty acid oxidation genes expression. In addition, MSH significantly inhibited protein nitrosylation and lipid peroxidation by lowering cytochrome P450 2E1 enzyme activity and restoring the glutathione level, superoxide dismutase and catalase activity in liver. Furthermore, MSH synergistically decreased the mRNA level of tumor necrosis factor-α in the hepatic tissue. These findings indicate that MSH has potential for preventing alcoholic liver disease through inhibiting hepatic steatosis, oxidative stress, and inflammation.

A Mixture of Fermented Schizandrae Fructus Pomace and Hoveniae Semen cum Fructus Extracts Synergistically Protects against Oxidative Stress-Mediated Liver Injury.

Schizandrae Fructus (SF) and Hoveniae Semen cum Fructus (HSCF) have long been used as medicinal herbs for treating various diseases in Asian traditional medicine. In the current study, we investigated the protective effect of fermented SF pomace and HSCF extract 1:1 (w:w) combination mixture (MSH) against carbon tetrachloride (CCl4)-induced acute liver injury mice. After MSH (50-200 mg/kg) oral administration for 7 consecutive days, animals were injected intraperitoneally with CCl4 (0.5 mL/kg). Histopathological observation revealed that administration of MSH synergistically decreased the degeneration of hepatocytes and the infiltration of inflammatory cells induced by CCl4. Moreover, MSH administration reduced the activities of alanine aminotransferase, aspartate aminotransferase, and γ-glutamyl transpeptidase in serum, and mitigated apoptotic cell death in hepatic parenchyma. In addition, MSH alleviated CCl4-mediated lipid peroxidation by restoring endogenous antioxidants capacities including glutathione contents, superoxide dismutase, and catalase activities. In vitro assessments using tert-butyl hydroperoxide-induced oxidative stress in HepG2 cells revealed that MSH protects hepatocytes by lowering ROS generation and lipid peroxidation via upregulating the transcriptional activity of nuclear factor erythroid-2-related factor 2 and the expression of antioxidant genes. Furthermore, MSH synergistically attenuated the expression of proinflammatory cytokines in CCl4-injured liver and lipopolysaccharide-stimulated RAW 264.7 cells. Taken together, these findings suggest that MSH has the potential to prevent acute liver damage by effectively suppressing oxidative stress and inflammation.

Experimental evaluation of the influence of electromagnetic radiation and acute alcohol intoxication on the indicators of mineral metabolism and antioxidant protection in erythrocytes and blood plasma of rats

The aim of the research was a comparative analysis of the effect of electromagnetic radiation and acute alcohol intoxication on the parameters of mineral metabolism and antioxidant protection in erythrocytes and blood plasma of rats. Materials and methods. The research was performed on 128 white non-linear rats. The study was conducted on five experimental models: acute ethanol poisoning, acute ethanol poisoning against the background of preliminary alcoholization, exposure to electromagnetic radiation for 1, 2 and 3 months. Spectrophotometrically determined the content of iron, copper, transferrin, ceruloplasmin and the activity of gamma-glutamyl transpeptidase in the blood plasma of rats, the content of glutathione in blood erythrocytes. Results and discussion. A unidirectional nature of changes in the indicators of antioxidant protection and mineral metabolism in acute alcohol intoxication and electromagnetic irradiation was established, which consists in a decrease in the content of iron, transferrin, an increase in the content of ceruloplasmin, glutathione, and gamma-glutamyl transpeptidase activity. At the same time, a decrease in the iron content and a change in the parameters of antioxidant protection in the blood plasma are more pronounced in acute ethanol poisoning than in electromagnetic irradiation. Conclusions. Ceruloplasmin, transferrin, glutathione and gamma-glutamyl transpeptidase are targets for the action of electromagnetic radiation and ethanol. Determination of the activity of gamma-glutamyl transpeptidase in blood plasma has a prognostic value for determining the degree of liver damage not only with alcohol intoxication, but also with electromagnetic irradiation of the decimeter range.

SAR of L-ABBA analogs for GGT1 inhibitory activity and L-ABBA's effect on plasma cysteine and GSH species

Gamma-glutamyl transferase 1 (GGT1) is a critical enzyme involved in the hydrolysis and/or transfer of gamma-glutamyl groups of glutathione, which helps maintain cysteine levels in plasma. In this study, we synthesized L-ABBA analogs to investigate their inhibitory effect on GGT1 hydrolysis and transpeptidase activity, with the goal of defining the pharmacophore of L-ABBA. Our structure–activity relationship (SAR) study revealed that an α-COO- and α-NH3+ group, as well as a two-CH2 unit distance between α-C and boronic acid, are essential for the activity. The addition of an R (alkyl) group at the α-C reduced the activity of GGT1 inhibition, with L-ABBA being the most potent inhibitor among the analogs. Next, we investigated the impact of L-ABBA on plasma levels of cysteine and GSH species, with the expectation of observing reduced cysteine levels and enhanced GSH levels due to its GGT1 inhibition. We administered L-ABBA intraperitoneally and determined the plasma levels of cysteine, cystine, GSH, and GSSG using LCMS. Our results showed time- and dose-dependent L-ABBA changes in total plasma cysteine and GSH levels. This study is the first to demonstrate the regulation of plasma thiol species upon GGT1 inhibition, with plasma cystine levels reduced by up to ∼ 75 % with L-ABBA (0.3 mg/dose). Cancer cells are highly dependent on the uptake of cysteine from plasma for maintaining high levels of intracellular glutathione. Thus, our findings suggest that GGT1 inhibitors, such as L-ABBA, have the potential to be used in GSH reduction thereby inducing oxidative stress in cancer cells and reducing their resistance to many chemotherapeutic agents.

New Insight into the Substrate Selectivity of Bovine Milk γ-glutamyl Transferase via Structural and Molecular Dynamics Predictions.

Bovine milk γ-glutamyltransferase (BoGGT) can produce γ-glutamyl peptides using L-glutamine as a donor substrate, and the transpeptidase activity is highly dependent on both γ-glutamyl donors and acceptors. To explore the molecular mechanism behind the donor and acceptor substrate preferences for BoGGT, molecular docking and molecular dynamic simulations were performed with L-glutamine and L-γ-glutamyl-p-nitroanilide (γ-GpNA) as donors. Ser450 is a crucial residue for the interactions between BoGGT and donors. BoGGT forms more hydrogen bonds with L-glutamine than γ-GpNA, promoting the binding affinity between BoGGT and L-glutamine. Gly379, Ile399, and Asn400 are crucial residues for the interactions between the BoGGT intermediate and acceptors. The BoGGT intermediate forms more hydrogen bonds with Val-Gly than L-methionine and L-leucine, which can promote the transfer of the γ-glutamyl group from the intermediate to Val-Gly. This study reveals the critical residues responsible for the interactions of donors and acceptors with the BoGGT and provides a new understanding of the substrate selectivity and catalytic mechanism of GGT.

Activatable Raman Probes Utilizing Enzyme-Induced Aggregate Formation for Selective Ex Vivo Imaging

Detecting multiple enzyme activities simultaneously with high spatial specificity is a promising strategy to investigate complex biological phenomena, and Raman imaging would be an excellent tool for this purpose due to its high multiplexing capabilities. We previously developed activatable Raman probes based on 9CN-pyronins, but specific visualization of cells with target enzyme activities proved difficult due to leakage of the hydrolysis products from the target cells after activation. Here, focusing on rhodol bearing a nitrile group at the position of 9 (9CN-rhodol), we established a novel mechanism for Raman signal activation based on a combination of aggregate formation (to increase local dye concentration) and the resonant Raman effect along with the bathochromic shift of the absorption, and utilized it to develop Raman probes. We selected the 9CN-rhodol derivative 9CN-JCR as offering a suitable combination of increased stimulated Raman scattering (SRS) signal intensity and high aggregate-forming ability, resulting in good retention in target cells after probe activation. By using isotope-edited 9CN-JCR-based probes, we could simultaneously detect β-galactosidase, γ-glutamyl transpeptidase, and dipeptidyl peptidase-4 activities in live cultured cells and distinguish cell regions expressing target enzyme activity in Drosophila wing disc and fat body ex vivo.

Improvement in salt-tolerance of Aspergillus oryzae γ-glutamyl transpeptidase via protein chimerization with Aspergillus sydowii homolog

γ-Glutamyl transpeptidase is one of the key enzymes involved in glutamate production during high-salt fermentation of soy sauce and miso by koji mold, Aspergillus oryzae. However, the activity of γ-glutamyl transpeptidase from A. oryzae (AOggtA) is markedly reduced in the presence of NaCl, thus classifying it as a non-salt-tolerant enzyme. In contrast, the homologous protein from the xerophilic mold, A. sydowii (ASggtA) maintains its activity under high-salt conditions. Therefore, in this study, a chimeric enzyme, ASAOggtA, was designed and engineered to improve salt-tolerance in AOggtA by swapping the N-terminal region, based on sequence and structure comparisons between salt-tolerant ASggtA and non-salt-tolerant AOggtA. The parental AOggtA and ASggtA and their chimera, ASAOggtA, were heterologously expressed in A. oryzae and purified. The chimeric enzyme inherited the superior activity and stability from each of the two parent enzymes. ASAOggtA showed > 2-fold greater tolerance than AOggtA in the presence of 18% NaCl. In addition, the chimera showed a broader range of pH stability and greater thermostability than ASggtA. AOggtA and ASAOggtA were sy over the range pH 3.0 to pH 10.5. Thermal stability was found to be in the order AOggtA (57.5 °C, t1/2 = 32.5 min) > ASAOggtA (55 °C, t1/2 = 20.5 min) > ASggtA (50 °C, t1/2 = 12.5 min). The catalytic and structural characteristics indicated that non-salt-tolerant AOggtA would not undergo irreversible structural changes in the presence of NaCl, but rather a temporary conformational change, which might result in reducing the substrate binding and catalytic activity, on the basis of kinetic properties. In addition, the chimeric enzyme showed hydrolytic activity toward L-glutamine that was as high as that of AOggtA. The newly-designed chimeric ASAOggtA might have potential applications in high-salt fermentation, such as miso and shoyu, to increase the content of the umami-flavor amino acid, L-glutamate.

Two genes related to apoptosis in the hepatopancreas of juvenile prawn, Macrobrachium nipponense: Molecular characterization and transcriptional response to nanoplastic exposure

Nanoplastics have been widely found in the global water environment, causing plastic pollution and affecting human beings and numerous organisms. Studies involving freshwater crustacean exposure to nanoplastics, however, are limited. In this study, juvenile prawns (Macrobrachium nipponense) were exposed to 75 nm polystyrene nanoplastics at different concentrations (0, 5, 10, 20, or 40 mg/L) for a 28-d chronic exposure experiment. To study the effects of exposure to nanoplastics on hepatopancreas cell apoptosis, C-Jun N-terminal kinase (JNK) and phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) genes were selected, and hepatotoxic enzyme activities and Toll pathway- and apoptosis-related gene expression were determined. For the first time, full-length Mn-JNK and Mn-PIK3CA cDNAs were cloned from M. nipponense. Homologous comparisons showed that JNK and PIK3CA had conserved functional sequences. The apoptosis rate in the high-concentration nanoplastic group (40 mg/L) was significantly higher than in the low-concentration nanoplastic (5 mg/L) and control groups (0 mg/L). The alanine aminotransferase (ALT), aspartate aminotransferase (AST), glutamyl transpeptidase (GGT) and xanthine oxidase (XOD) enzyme activities in the hepatopancreas increased with exposure to higher concentrations of nanoplastics. In addition, the levels of apoptosis- and Toll pathway-related gene expression and JNK and PIK3CA gene expression were initially increased, then decreased with exposure to higher concentrations of nanoplastics. This study showed that polystyrene nanoplastics activate toll-related pathways leading to apoptosis and hepatopancreas damage, which provides theoretical support for future aquatic toxicological research.

Dietary patterns and fertility status in men: Mediterranean diet does make a difference in ameliorating the rise in male infertility problems due to changing lifestyle

Dietary patterns and fertility status in men: Mediterranean diet does make a difference in ameliorating the rise in male infertility problems due to changing lifestyle

Biochemical parameters of blood in cats with cardiogenic arterial thromboembolism and acute heart failure

Investigating the impact of vascular blockage on blood biochemical parameters, particularly in the context of acute heart failure, is crucial for understanding the underlying mechanisms of arterial thromboembolism in animals. The purpose of the study is to investigate the effect of vascular occlusion on the biochemical parameters of blood in cats with cardiogenic arterial thromboembolism. The study involved 12 cats and formed two groups: the first – animals with acute heart failure and the second – animals with arterial thromboembolism. Anamnesis data were collected for experimental animals, clinical examination, echocardiography, and biochemical examination of blood samples were performed. The activity of alanine aminotransferase, aspartate aminotransferase and gamma-glutamyl transpeptidase, the concentration of glucose, creatinine, urea, potassium, calcium, total and inorganic phosphorus were determined spectrophotometrically in blood serum. It was found that in cats with acute heart failure, the average value of serum aspartate aminotransferase activity increased by 2.8 times, and the creatinine concentration by 1.9 times compared to the upper limit of reference values. This indicates functional changes in the myocardium and kidneys in this pathology. In cats affected by arterial thromboembolism, the blood serum shows a significant elevation in various biochemical parameters compared to the upper limit of the corresponding reference intervals. Specifically, there is a 4.4-fold increase in alanine aminotransferase activity, a 4.3-fold increase in aspartate aminotransferase activity, a 1.4- fold increase in glucose concentration, a 1.8-fold increase in creatinine levels, a 2.0-fold increase in urea levels, and a 1.3-fold increase in inorganic phosphorus levels. The changes in biochemical parameters for feline thromboembolism may be a consequence of functional renal failure, ischemia, and muscle necrosis. Moreover, it was established that in most cats with acute heart failure, the urinary system is primarily affected, and with arterial thromboembolism, changes in muscle tissue also occur. The results obtained are of practical value for veterinary doctors when choosing a therapeutic strategy and prescribing additional diagnostic examinations

Effects of Sodium Dodecyl Sulfate on the Enzyme Catalysis and Conformation of a Recombinant γ-Glutamyltranspeptidase from Bacillus licheniformis.

The study of interactions between proteins and surfactants is of relevance in a diverse range of applications including food, enzymatic detergent formulation, and drug delivery. In spite of sodium dodecyl sulfate(SDS)-induced unfolding has been studied in detail at the protein level, deciphering the conformation-activity relationship of a recombinant γ-glutamyltranspeptidase (BlrGGT) from Bacillus licheniformis remains important to understand how the transpeptidase activity is related to its conformation. In this study, we examined the enzyme catalysis and conformational transition of BlrGGT in the presence of SDS. Enzymatic assays showed that the transpeptidase activity of BlrGGT was greatly affected by SDS in a concentration-dependent manner with approximately 90% inactivation at 6mM. Native polyacrylamide gel electrophoresis of SDS-treated samples clearly revealed that the heterodimeric enzyme was apparently dissociated into two different subunits at concentrations above 2mM. The study of enzyme kinetics showed that SDS can act as a mixed-type inhibitor to reduce the catalytic efficiency of BlrGGT. Moreover, the t1/2 value of the enzyme at 55°C was greatly reduced from 495.1min to 7.4min in the presence of 1mM SDS. The I3/I1 ratio ofpyreneexcimerfluorescence emission changed around 3.7mMSDSin the absence ofBlrGGT and the inflection point of enzyme samples was reduced to less than 2.7mM. The Far-UV CD spectrum of the native enzyme had two negative peaks at 208 and 222nm, respectively; however, both negative peaks increased in magnitude with increasing SDS concentration and reached maximal values at above 4.0mM. Theintrinsic fluorescence spectraof tryptophan further demonstrated that the SDS-induced enzyme conformational transition occurred at approximately 5.1mM. Tween 20 significantly suppressed the interaction of BlrGGT with SDS by forming mixed micelles at a molar ratio of 1.0. Taken together, this study definitely promotes our better understanding of the relationship between the conformation and catalysis of BlrGGT.

Elucidation of the Molecular Mechanism of Bovine Milk γ-Glutamyltransferase Catalyzed Formation of γ-Glutamyl-Valyl-Glycine.

γ-Glu-Val-Gly (γ-EVG) is a potent kokumi peptide that can be synthesized through the transpeptidase reaction catalyzed by γ-glutamyl transferase from bovine milk (BoGGT). To explore the molecular mechanism between BoGGT and l-glutamine, γ-glutamyl peptides were generated through the transpeptidase reaction catalyzed by BoGGT at various reaction conditions. Quantitation of γ-glutamyl peptides, structure prediction of BoGGT, molecular docking, and molecular dynamic simulations were performed. Membrane-free BoGGT had a higher transpeptidase activity with Val-Gly as an acceptor than membrane BoGGT. The suitable conditions for γ-EVG production using BoGGT were 100 mM Val-Gly, 20 mM Gln, 1.2 U/mL BoGGT, pH 8.5, and 37 °C, and 13.1 mM γ-EVG was produced. The hydrogen bonds are mainly formed between residues from the light subunit of BoGGT (Thr380, Thr398, Ser450, Ser451, Met452, and Gly473) and the l-glutamine donor. NaCl might inhibit the transpeptidase activity by destroying the hydrogen bonds between BoGGT and l-glutamine, thereby increasing the distance between the hydroxyl oxygen atom on Thr380 of BoGGT and the amide carbon atom on l-glutamine.

Evaluation of Cottonseed Meal as an Alternative to Fish Meal in Diet for Juvenile Asian Red-Tailed Catfish Hemibagrus wyckioides.

A 10-week trial was performed to investigate the effects of replacing fishmeal with cottonseed meal (CSM) on the growth rate, protein metabolism, and antioxidant response of Asian red-tailed catfish Hemibagrus wyckioides. Five isonitrogenous and isocaloric diets (C0, C8.5, C17.2, C25.7, and C34.4) were prepared to contain 0%, 8.5%, 17.2%, 25.7%, and 34.4% CSM replacing fishmeal, respectively. The weight gain, daily growth coefficient, pepsin, and intestinal amylase activities initially increased and then decreased with the raising dietary CSM levels; the highest values were observed in the C17.2 group (P < 0.05). However, feed cost exhibited the opposite trend. With the increasing dietary CSM levels, the protein efficiency ratio and intestinal trypsin activity decreased but feed conversion rate increased gradually; while no differences were observed among the C0, C8.5, and C17.2 groups (P > 0.05). Dietary CSM inclusion regardless of levels increased the plasma growth hormone level as well as hepatic aspartate aminotransferase (AST) and γ-glutamyl transpeptidase activities but decreased the plasma glutamate dehydrogenase and AST activities (P < 0.05). With the increasing dietary CSM levels, the plasma alkaline phosphatase (AKP) and hepatic superoxide dismutase activities decreased but malondialdehyde content increased gradually, while no differences were observed among the C0, C8.5, and C17.2 groups (P > 0.05). The plasma immunoglobulin M content and hepatic glutathione reductase activity initially increased but then decreased with the raising dietary CSM levels; the highest values were found in the C17.2 group. These results indicated that dietary CSM inclusion level up to 17.2% improved the growth rate, feed cost, digestive enzyme activity, and protein metabolism without compromising antioxidant capacity of H. wyckioide, whereas these parameters were depressed by further inclusion of CSM. CSM is a potentially cost-effective alternative plant protein source in diet of H. wyckioide.

Significance of expression of selected miRNAs in the diagnosis of hepatological complications of Epstein-Barr virus infection in children.

To assess the expression of selected miRNAs as predictive factors of hepatological complications in Epstein-Barr virus (EBV)-infected children. This study included 68 children infected with EBV aged 1 to 18 years hospitalised between 01.12.2018 and 31.12.2020 in the Department of Paediatrics, Infectious Diseases and Hepatology. The expression of 5 miRNAs (miR-122-5p, miR-21-3p, miR-34a-5p, miR-26b-5p, miR-199a-5p) was analysed by real-time PCR using the TaqMan Fast Advanced Master Mix kit. In the group of EBV-infected children, statistically significantly higher expression for miR-21-3p, miR-122-5p, miR-26b-5p and miR-34a-5p was confirmed. In a group of 54 children diagnosed with hepatitis, along with an increase in alanine aminotransferase (ALT) and g-glutamyl transpeptidase (GGTP) activity, an increase in expression of miR-21-3p, miR-122-5p, miR-26b-5p, miR-34a-5p and miR-199a-5p was observed. Increased expression of miR-21-3p, miR-122-5p, miR-26b-5p, miR-34a-5p and miR-199a-5p in EBV-infected children appears to be a risk factor for hepatological complications progressing to biliary pole defects.