Search Results (13)

Two enzymatic assays, based on release of p-nitroaniline and its spectrophotometric detection at 405 nm, were used to screen lager and ale brewing yeasts for carboxypeptidase and γ-glutamyltranspeptidase activity. Both activities were found in all the investigated yeasts and did not significantly distinguish Saccharomyces cerevisiae from S. pastorianus species. Large between-strain differences were measured for both carboxypeptidase (from 1.61 A/h for BRAS-45 to 41.71 A/h for E-30) and γ-glutamyltranspeptidase (from 1.26 A/h for US-05 to 48.72 A/h for S-33). No correlation was found between either enzymatic activity and the previously published ability of Saccharomyces yeasts to degrade glutathionyl or γ-GluCys- precursors to free polyfunctional thiols. Only for fermentation at lower temperatures does carboxypeptidase activity seem relevant for identifying the most interesting candidates. Measuring transport efficiency and β-lyase activities individually on the three possible intermediates emerges here as more promising for future flavor potential screening. Full article

The design and development of fluorescent materials for detecting cancer-related enzymes are crucial for cancer diagnosis and treatment. Herein, we present a substituted rhodamine derivative for the chromogenic and fluorogenic detection of the cancer-relevant enzyme γ-glutamyltranspeptidase (GGT). Initially, the probe is non-chromic and non-emissive due to its spirolactam form, which hinders extensive electronic delocalization over broader pathway. However, selective enzymatic cleavage of the side-coupled group triggers spirolactam ring opening, resulting in electronic flow across the rhodamine skeleton, and reduces the band gap for low-energy electronic transitions. This transformation turns the reaction mixture from colorless to intense pink, with prominent UV and fluorescence bands. The sensor’s selectivity was tested against various human enzymes, including urease, alkaline phosphatase, acetylcholinesterase, tyrosinase, and cyclooxygenase, and showed no response. Absorption and fluorescence titration analyses of the probe upon incremental addition of GGT into the probe solution revealed a consistent increase in both absorption and emission spectra, along with intensified pink coloration. The cellular toxicity of the receptor was evaluated using the MTT assay, and bioimaging analysis was performed on BHK-21 cells, which produced bright red fluorescence, demonstrating the probe’s excellent cell penetration and digestion capabilities for intracellular analytical detection. Molecular docking results supported the fact that probe-4 made stable interactions with the GGT active site residues. Full article

Lentinula edodes (L. edodes) is a globally popular edible mushroom because of its characteristic sulfur-containing flavor compounds. However, the formation of the volatile sulfur-containing compounds in the mycelium of L. edodes has not been studied. We found that there were also sulfur-containing aroma compounds in the mycelium of L. edodes, and the content and composition varied at different stages of mycelial growth and development. The γ-glutamyl-transpeptidase (GGT) and cysteine sulfoxide lyase (C-S lyase) related to the generation of sulfur compounds showed the highest activities in the 15-day sample. Candidate genes for the metabolism of volatile sulfur compounds in mycelium were screened using transcriptome analysis, including encoding the GGT enzyme, C-S lyase, fatty acid oxidase, HSP20, and P450 genes. The expression patterns of Leggt3 and Leccsl3 genes were consistent with the measured activities of GGT and C-S lyase during the cultivation of mycelium and molecular dynamics simulations showed that they could stably bind to the substrate. Our findings provide insights into the formation of sulfur-containing flavor compounds in L. edodes. The mycelium of L. edodes is suggested for use as material for the production of sulfur-containing flavor compounds. Full article

Introduction: Chronic hepatitis C (CHC) is a clinical and pathological syndrome with various causes and is characterized by varying degrees of hepatocellular necrosis and inflammation. It is a significant cause of liver transplantation and liver-related death worldwide. The hepatic manifestations of CHC are typically characterized by slowly progressing liver fibrosis, which is a non-specific and often disproportionate response to tissue damage. A large majority of HCV patients have extrahepatic manifestations with varying degrees of severity. HCV infection is a risk factor for cardiovascular disease and diabetes mellitus, which increases insulin resistance, oxidative stress, and iron overload and causes chronic systemic inflammation. HCV infection is treated using direct-acting antivirals (DAAs) with cure rates of over 95 percent, minimal side effects, and shorter therapeutic courses. Despite the effective elimination of the virus, it seemed pertinent to understand to what extent HCV clearance eliminates or attenuates all the systemic alterations already induced by the virus during infection and chronicity. Objectives: Our study aimed to determine whether eliminating HCV with DAAs alters the severity of liver disease (liver stiffness and liver fibrosis stage by TE) and the metabolic/cellular profile of patients with CHC. Materials and methods: A group of 329 CHC patients from a Gastroenterology and Hepatology outpatient department were prospectively studied. Of these, 134 were also studied with DAAs. The liver fibrosis stage was evaluated by transient elastography (TE) using a FibroScan^® device, and two groups were established for the analysis of liver stiffness (LS): mild and moderate stiffness (fibrosis F1 and F2; F1/2) and severe stiffness (fibrosis and cirrhosis F3 and F4; F3/4). Metabolic/cellular parameters were evaluated before and after antiviral treatment using standard methods: alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), γ-glutamyl-transpeptidase (γ-GT), haptoglobin (Hp), total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoprotein (LDL), triglycerides (TG), free iron (Fe), transferrin saturation (TS), total iron binding capacity (TIBC), ferritin (Ft), glycemia, insulin, Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) and platelets count. The results were statistically analyzed using SPSS 24.0 for Windows. Results: Comparing the fibrosis stage before and after DAAs treatment, we verify a reduction in LS in 85.7% of patients and an improvement in liver fibrosis stage in 22.2% of them after DAAs treatment. Before DAAs treatment, patients showed a 2.410 risk for higher fibrosis stages (F3/4). Comparing metabolic/cellular parameters before and after DAAs treatment, patients showed lower ALP, AST, ALT, γGT, TG, Fe, TIBC, and Ft values and higher TC, LDL, and Hp values after treatment. As such, HCV elimination reduces iron overload and insulin resistance. On the other hand, it caused dyslipidemia, raising total cholesterol and LDL to levels outside the reference values. The improvement in the liver fibrosis stage by TE was mainly associated with higher baseline platelet count and HDL values and lower insulin resistance. Conclusions: With this study, we were able to contribute to the knowledge of the effects of HCV elimination with DAAs on liver disease and metabolic profile to improve the quality of treatment and follow-up of these patients after HCV elimination. Full article

The goal of this study was to produce a sufficient amount of glutathione in the fermentation medium without the addition of cysteine. This would simplify and reduce the cost of its purification. In addition to reducing the cost of cysteine, it also avoids the inhibition of bacterial growth by cysteine. The gshA, gshB, and cysE genes of Escherichia coli were cloned under the control of the strong T5 promoter of the pQE-80L plasmid and introduced into an E. coli strain knocked out for the genes encoding γ-glutamyltranspeptidase and the GsiABCD glutathione transporter, which are responsible for the recycling of excreted glutathione. The overexpression of the gshA and gshB genes, genes for γ-glutamylcysteine synthetase and glutathione synthetase, and the cysE^{V95R D96P} gene, a gene for serine acetyltransferase with the V95R D96P mutation that makes it insensitive to cysteine, were effective on glutathione production. Na₂S₂O₃ was a good sulfur source for glutathione production, while the addition of Na₂SO₄ did not affect the glutathione production. With the addition of 50 mM glutamic acid and 75 mM glycine, but without the addition of cysteine, to the simplified SM1 medium, 4.6 mM and 0.56 mM of the reduced and oxidized glutathione, respectively, were accumulated in the extracellular space after 36 h of batch culture. This can eliminate the need to extract glutathione from the bacterial cells for purification. Full article

Chronic hepatitis C (CHC) progression is highly variable and can be influenced by lipid metabolism. The ATP-binding cassette transporter A1 (ABCA1) is involved in lipid metabolism and mediates cholesterol efflux from liver cells. ABCA1 gene polymorphism rs2230808 (R1587K) modulates lipid levels as it is located in an ABCA1 protein domain, which is essential for cholesterol efflux. We aimed to analyze the role of ABCA1 polymorphism R1587K (rs2230808) in modulating the biochemical parameters of lipid metabolism and liver function and its association with liver disease severity, according to gender. A total of 161 CHC patients were clinically, histologically, and biochemically evaluated. Genotyping was performed by melting-curve analysis and statistical analysis by SPSS 24.0. There were significant differences between ABCA1_rs2230808 genotypes and total cholesterol, γGT (γ-glutamyl-transpeptidase), and HCV-RNA. Gender differences: in females, ABCA1_rs2230808 (GG or GA) was associated with higher HCV-RNA serum levels; in males, ABCA1_rs2230808 (GG or GA) was associated with higher γGT, lower total cholesterol, increased risk for γGT ≥ 38 UI/L, and total cholesterol < 4.92 mmol/L. Only in the case of males were higher γGT and lower total cholesterol associated with severe fibrosis and steatosis. Total cholesterol < 4.92 mmol/L also associates with severe necroinflammation. We conclude that ABCA1_rs2230808 is gender-specific. ABCA1_rs2230808 Allele G was associated with different clinical and biochemical parameters, which are related to more severe liver disease. Full article

Chinese chives is a popular herb vegetable and medicine in Asian countries. Southwest China is one of the centers of origin, and the mountainous areas in this region are rich in wild germplasm. In this study, we collected four samples of germplasm from different altitudes: a land race of cultivated Chinese chives (Allium tuberosum), wide-leaf chives and extra-wide-leaf chives (Allium hookeri), and ovoid-leaf chives (Allium funckiaefolium). Leaf metabolites were detected and compared between A. tuberosum and A. hookeri. A total of 158 differentially accumulated metabolites (DAM) were identified by Gas Chromatography—Mass Spectrometry (GC-MS) and Liquid Chromatography—Mass Spectrometry (LC-MS), among which there was a wide range of garlic odor compounds, free amino acids, and sugars. A. hookeri contains a higher content of fructose, garlic odor compounds, and amino acids than A. tuberosum, which is supported by the higher expression level of biosynthetic genes revealed by transcriptome analysis. A. hookeri accumulates the same garlic odor compound precursors that A. tuberosum does (mainly methiin and alliin). We isolated full-length gene sequences of phytochelatin synthase (PCS), γ-glutamyltranspeptidases (GGT), flavin-containing monooxygenase (FMO), and alliinase (ALN). These sequences showed closer relations in phylogenetic analysis between A. hookeri and A. tuberosum (with sequence identities ranging from 86% to 90%) than with Allium cepa or Allium sativum (which had a lower sequence identity ranging from 76% to 88%). Among these assayed genes, ALN, the critical gene controlling the conversion of odorless precursors into odor compounds, was undetected in leaves, bulbs, and roots of A. tuberosum, which could account for its weaker garlic smell. Moreover, we identified a distinct FMO1 gene in extra-wide-leaf A. hookeri that is due to a CDS-deletion and frameshift mutation. These results above reveal the molecular and metabolomic basis of impressive strong odor in wild Chinese chives. Full article

Background: This study aimed to compare the diagnostic performance of carbohydrate-deficient transferrin (CDT) and gamma-glutamyltranspeptidase (γ-GTP) to assess the single and combined benefits of these biological markers for the detection of chronic excessive alcohol consumption in patients with alcoholic cirrhosis. Methods: Biological markers were determined in blood samples from patients with alcoholic cirrhosis (drinking group, n = 35; nondrinking group, n = 81). The prediction accuracy of %CDT alone, γ-GTP alone, and their combination for the detection of excessive alcohol consumption was determined in patients with alcoholic cirrhosis. Results: Serum total bilirubin, alanine aminotransferase, aspartate aminotransferase, γ-GTP, and alkaline phosphatase levels and %CDT were significantly higher and serum albumin levels were significantly lower in the drinking group than in the nondrinking group. The combination of %CDT and γ-GTP compared with %CDT or γ-GTP alone showed a higher prediction accuracy. The combination of %CDT and γ-GTP exhibited a higher specificity than γ-GTP alone. However, in terms of sensitivity, no significant difference was found between single or combined markers. Conclusions: The combination of %CDT and γ-GTP is considered a useful biomarker of chronic excessive alcohol consumption in patients with alcoholic cirrhosis. Full article

A highly conserved ⁴⁵⁸PLSSMXP⁴⁶⁴ sequence in the small subunit (S-subunit) of an industrially important Bacillus licheniformis γ-glutamyltranspeptidase (BlGGT) was identified by sequence alignment. Molecular structures of the precursor mimic and the mature form of BlGGT clearly reveal that this peptide sequence is in close spatial proximity to the self-processing and catalytic sites of the enzyme. To probe the role of this conserved sequence, ten mutant enzymes of BlGGT were created through a series of deletion and alanine-scanning mutagenesis. SDS-PAGE and densitometric analyses showed that the intrinsic ability of BlGGT to undergo autocatalytic processing was detrimentally affected by the deletion-associated mutations. However, loss of self-activating capacity was not obviously observed in most of the Ala-replacement mutants. The Ala-replacement mutants had a specific activity comparable to or greater than that of the wild-type enzyme; conversely, all deletion mutants completely lost their enzymatic activity. As compared with BlGGT, S460A and S461S showed greatly enhanced k_cat/K_m values by 2.73- and 2.67-fold, respectively. The intrinsic tryptophan fluorescence and circular dichroism spectral profiles of Ala-replacement and deletion mutants were typically similar to those of BlGGT. However, heat and guanidine hydrochloride-induced unfolding transitions of the deletion-associated mutant proteins were severely reduced as compared with the wild-type enzyme. The predictive mutant models suggest that the microenvironments required for both self-activation and catalytic reaction of BlGGT can be altered upon mutations. Full article

Peroxisome proliferator activated receptor α (PPARα) has the most relevant biological functions among PPARs. Activation by drugs and dietary components lead to major metabolic changes, from reduced triglyceridemia to improvement in the metabolic syndrome. Polymorphisms of PPARα are of interest in order to improve our understanding of metabolic disorders associated with a raised or reduced risk of diseases. PPARα polymorphisms are mainly characterized by two sequence changes, L162V and V227A, with the latter occurring only in Eastern nations, and by numerous SNPs (Single nucleotide polymorphisms) with a less clear biological role. The minor allele of L162V associates with raised total cholesterol, LDL-C (low-density lipoprotein cholesterol), and triglycerides, reduced HDL-C (high-density lipoprotein metabolism), and elevated lipoprotein (a). An increased cardiovascular risk is not clear, whereas a raised risk of diabetes or of liver steatosis are not well supported. The minor allele of the V227A polymorphism is instead linked to a reduction of steatosis and raised γ-glutamyltranspeptidase levels in non-drinking Orientals, the latter being reduced in drinkers. Lastly, the minor allele of rs4353747 is associated with a raised high-altitude appetite loss. These and other associations indicate the predictive potential of PPARα polymorphisms for an improved understanding of human disease, which also explain variability in the clinical response to specific drug treatments or dietary approaches. Full article

Adipocyte fatty acid-binding protein (A-FABP) is abundantly found in mature adipocytes and is involved in cardiovascular disease. Our aim is to investigate the association between serum A-FABP levels and endothelial function among kidney transplant (KT) patients. Fasting blood samples were obtained from 80 KT patients. Serum A-FABP levels were measured using a commercially available enzyme immunoassay kit. Endothelial function and vascular reactivity index (VRI) were measured using digital thermal monitoring test. In this study, VRI < 1.0, VRI 1.0–1.9, and VRI ≥ 2.0 were defined as poor, intermediate, and good vascular reactivity, respectively. There were 12 (15.0%), 30 (37.5%), and 38 (47.5%) KT patients categorized as having poor, intermediate, and good vascular reactivity, respectively. Increased serum levels of alkaline phosphatase (p = 0.012), γ-glutamyltranspeptidase (GGT; p = 0.032), and A-FABP (p < 0.001) were associated with decreased vascular reactivity. Multivariable forward stepwise linear regression analysis revealed that age (β = −0.283, adjusted R² change = 0.072; p = 0.003) and serum log-A-FABP level (β = −0.514, adjusted R² change = 0.268; p < 0.001) were significantly associated with VRI values in KT patients. We concluded that serum fasting A-FABP level is negatively associated with VRI values and plays a role in endothelial dysfunction of KT patients. Full article

The purpose of the current study was to examine the influence of single-nucleotide polymorphisms (SNPs) in the peroxisome proliferator-activated receptor-δ (PPAR-δ), PPAR-γ, and α2 isoforms of the catalytic subunit of AMP-activated protein kinase (PRKAA2) on the extent of changes in anthropometric indices and blood measurements through exercise-centered lifestyle intervention in middle-aged men. A total of 109 Japanese middle-aged male subjects (47.0 ± 0.4 years) participated in the baseline health checkup, 6-month exercise-centered lifestyle intervention, and second checkup conducted several months after the subject completed the intervention. The body mass index (BMI), waist circumference, and clinical measurements, including hemoglobin A_lc (HbA_1c), triglyceride (TG), alanine aminotransferase (ALT), and γ-glutamyl-transpeptidase (γ-GTP), were measured at the baseline and second checkup. The three SNPs of PPAR-δ A/G (rs2267668), PPAR-γ C/G (rs1801282), and PRKAA2 A/G (rs1418442) were determined. Blunted responses in the reduction in the BMI and waist circumference were observed in A/A carriers of PPAR-δ SNP compared with G allele carriers (all p < 0.05). The A/A carriers also displayed less-marked improvements in HbA_1c, TG, ALT, and γ-GTP (all p < 0.05). The current results suggest that A/A carriers of PPAR-δ SNP (rs2267668) may enjoy fewer beneficial effects of exercise-centered lifestyle intervention on anthropometric indices and blood measurements. Full article

In this study, we synthesized water-soluble hyperbranched poly(amido acid)s (HBPAAs) featuring multiple terminal CO₂H units and internal tertiary amino and amido moieties and then used them in conjunction with an in situ Fe^2+/Fe³⁺ co-precipitation process to prepare organic/magnetic nanocarriers comprising uniformly small magnetic iron oxide nanoparticles (NP) incorporated within the globular HBPAAs. Transmission electron microscopy revealed that the HBPAA-γ-Fe₂O₃ NPs had dimensions of 6–11 nm, significantly smaller than those of the pristine γ-Fe₂O₃ (20–30 nm). Subsequently, we covalently immobilized a bacterial γ-glutamyltranspeptidase (BlGGT) upon the HBPAA-γ-Fe₂O₃ nanocarriers through the formation of amide linkages in the presence of a coupling agent. Magnetization curves of the HBPAA-γ-Fe₂O₃/BlGGT composites measured at 300 K suggested superparamagnetic characteristics, with a saturation magnetization of 52 emu g⁻¹. The loading capacity of BlGGT on the HBPAA-γ-Fe₂O₃ nanocarriers was 16 mg g⁻¹ support; this sample provided a 48% recovery of the initial activity. The immobilized enzyme could be recycled 10 times with 32% retention of the initial activity; it had stability comparable with that of the free enzyme during a storage period of 63 days. The covalent immobilization and stability of the enzyme and the magnetization provided by the HBPAA-γ-Fe₂O₃ NPs suggests that this approach could be an economical means of depositing bioactive enzymes upon nanocarriers for BlGGT-mediated bio-catalysis. Full article