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Helicobacter pylori is one of the most common bacterial infections; over two‐thirds of the world's population is infected by early childhood. Persistent H. pylori infection results in gastric ulcers and cancers. Due to drug resistance, there is a need to develop alternative treatments to clear H. pylori. The Seattle Structural Genomics Center for Infectious Disease (SSGCID) conducts structure–function analysis of potential therapeutic targets from H. pylori. Glutamyl‐tRNA synthetase (GluRS) is essential for tRNA aminoacylation and is under investigation as a bacterial drug target. The SSGCID produced, crystallized and determined the apo structure of H. pylori GluRS (HpGluRS). HpGluRS has the prototypical bacterial GluRS topology and has similar binding sites and tertiary structures to other bacterial GluRS that are promising drug targets. Residues involved in glutamate binding are well conserved in comparison with Pseudomonas aeruginosa GluRS (PaGluRS), which has been studied to develop promising new inhibitors for P. aeruginosa. These structural similarities can be exploited for drug discovery and repurposing to generate new antibacterials to clear persistent H. pylori infection and reduce gastric ulcers and cancer. [ABSTRACT FROM AUTHOR]

Background and Aim: Hepatocellular carcinoma (HCC) is one of the most common and most lethal cancers worldwide. The objective of this study was to investigate the relationship between basal parameters and survival characteristics in patients with HCC. Materials and Methods: The records of 1447 HCC patients of a tertiary center during the period 2000-2017 were screened retrospectively. The demographic details; basal clinical, laboratory, and radiological characteristics; treatments; and survival time were recorded and prognostic scores were calculated. Results: A total of 788 patients with HCC (male/female: 623/165; mean age: 60.5±10.9 years) were included in the study. The median length of survival was 26.3 months (95% confidence interval [CI], 22.3-30.4 months). The 5-year survival rate was 28.1%. The number and diameter of the tumors; platelet count; platelet-to-lymphocyte ratio; level of aspartate aminotransferase, alanine aminotransferase, and gamma-glutamyl transferase; portal and hepatic vein involvement; and an alpha-fetoprotein level of <9.6 ng/mL were found to be independently related to survival. Conclusion: The positive predictive value of the prognostic index derived from independent survival-related parameters for 5- and 10-year survival or overall survival was approximately 86%. Integration of this prognostic index to the criteria used in making treatment decisions for patients with HCC should be considered. [ABSTRACT FROM AUTHOR]

Equine parvovirus-hepatitis (EqPV-H) causes equine hepatitis. The prevalence of EqPV-H in healthy horses has been reported in the United States, China, Germany, and Austria. The present study determined the prevalence of EqPV-H in the sera of clinically healthy horses in South Korea to identify the potential factors for infection and examine the genetic diversity of EqPV-H DNA sequences through comparison with foreign strains. Serum samples collected from 321 horses were tested for EqPV-H using non-structural protein 1 (NS1)-specific polymerase chain reaction. The associations of EqPV-H infection with sex, age, aspartate aminotransferase and -glutamyl transferase levels, and race performance were analyzed. Fourteen samples tested positive for EqPV-H (4.4%, 14/321), and EqPV-H infection was associated with sex (p = 0.006) and performance (p = 0.049). In both EqPV-H-positive and control horses, liver-specific biochemical analytes were within the normal ranges. Phylogenetic analyses based on the partial sequences of EqPV-H NS1 revealed that the Korean EqPV-H isolates shared approximately 98.7–100% similarity. Of these, 11 Korean isolates shared high similarity with strains from the United States, Germany, and China, and the remaining three strains were distinct in phylogenetic analyses. The present study describes the current molecular prevalence, potential risk factors, and genetic diversity of Korean EqPV-H. [ABSTRACT FROM AUTHOR]

Obstructive sleep apnea (OSA) can cause local and systemic inflammation due to hypoxia, asphyxia, hypercapnia, respiratory acidosis. Searching for new inflammatory markers in OSA may facilitate the prediction of OSA-related conditions. Therefore, we aimed to investigate the relationship between OSA severity and serum gamma-glutamyl transferase (GGT) levels. In the study, the GGT levels of the patients were compared according to the degree of OSA determined by polysomnography. The patients' demographic, polysomnographic, and laboratory parameters were evaluated with SPSS 25 program. A total of 500 patients, 299 (59.8%) male and 201 (40.2%) female, were included in the study. The mean age of the patients was 47.9±13.1, and body mass index (BMI) was 32.2±6.6. There were 98 (19.6%) patients in the control group, 100 (20%) in the mild OSA group, 103 (20.6%) in the moderate OSA group, and 199 (39.8%) in the severe OSA group. When the BMI and desaturation index of the groups were compared, there was a statistical difference (respectively, p<0.001, p<0.001). When the serum GGT values of the groups were compared, the GGT values of the moderate and severe OSA groups were higher than the control group (respectively, p=0.011, p=0.001), and there was a statistically significant difference between the groups (p=0.001). There was a positive correlation between GGT levels and BMI (r=0.151, p=0.010). There was a positive correlation between GGT levels and age (r=0.615, p=0.029). As a result of our study, a relationship was found between high GGT levels and the severity of OSA. Serum GGT level may be a promising biomarker to identify OSA patients at high risk. [ABSTRACT FROM AUTHOR]

Glutathione (GSH) is often upregulated in cancer, where it serves to mitigate oxidative stress. γ-glutamyl-transferase (GGT) is a key enzyme in GSH homeostasis, and compared to normal brain its expression is elevated in tumors, including in primary glioblastoma. GGT is therefore an attractive imaging target for detection of glioblastoma. The goal of our study was to assess the value of hyperpolarized (HP) γ-glutamyl-[1-13C]glycine for non-invasive imaging of glioblastoma. Nude rats bearing orthotopic U87 glioblastoma and healthy controls were investigated. Imaging was performed by injecting HP γ-glutamyl-[1-13C]glycine and acquiring dynamic 13C data on a preclinical 3T MR scanner. The signal-to-noise (SNR) ratios of γ-glutamyl-[1-13C]glycine and its product [1-13C]glycine were evaluated. Comparison of control and tumor-bearing rats showed no difference in γ-glutamyl-[1-13C]glycine SNR, pointing to similar delivery to tumor and normal brain. In contrast, [1-13C]glycine SNR was significantly higher in tumor-bearing rats compared to controls, and in tumor regions compared to normal-appearing brain. Importantly, higher [1-13C]glycine was associated with higher GGT expression and higher GSH levels in tumor tissue compared to normal brain. Collectively, this study demonstrates, to our knowledge for the first time, the feasibility of using HP γ-glutamyl-[1-13C]glycine to monitor GGT expression in the brain and thus to detect glioblastoma. [ABSTRACT FROM AUTHOR]

Intracranial artery calcification (IAC) is an important risk factor for cerebral infarction and a key biomarker for intracranial artery stenosis. Gamma-glutamyl transferase (GGT) has been independently associated with increased cardiovascular events and coronary calcification. Our study assessed whether GGT is an independent factor for IAC in acute ischemic stroke (AIS) patients. This cross-sectional study involved a total of 754 patients with AIS (mean age: 65 ± 13.2 years). All the patients had received brain computed tomography angiography (CTA) examination to evaluate IAC. Further, serum GGT levels and other biochemical parameters were analyzed. The average GGT level in patients who died was also significantly increased (37.0 ± 26.8 vs 29.0 ± 21.5 U/L, p = 0.012). Partial correlation analysis showed that serum GGT levels were associated with NIHSS score at admission after adjustment for age and gender was considered (r = 0.150, p = 0.001). Logistic regression analysis showed that serum GGT levels independently predicted all-cause mortality (OR = 1.036, 95% CI: 1.014–1.060, p = 0.002), NIHSS scores (β = 0.051, 95% CI: 0.020–0.082, p = 0.001) and IAC scores (β = 0.006, 95% CI: 0.003–0.014, p = 0.005) in male patients. Each SD (standard deviation) increase of serum GGT levels was also associated with risk of all-cause mortality (OR = 2.272, 95% CI: 1.364–3.787, P = 0.002). GGT levels in patients with severe IAC were significantly elevated (37.6 ± 33.6 vs 28.6 ± 19.2, p < 0.001). However, serum GGT levels could not independently predict the severity of IAC in AIS patients. Our study identified that serum GGT levels were significantly elevated in patients who died, and the GGT levels had a certain association with the risk of death and IAC in male patients. [ABSTRACT FROM AUTHOR]

Aim: We aimed at investigating the effects of age on the predictive performances of noninvasive fibrosis scores for significant fibrosis in patients with chronic hepatitis B (CHB). Methods: A total of 496 CHB patients who underwent liver biopsy were stratified into four age groups: ≤30, 31 to 40, 41 to 50, and ≥51 years. Receiver operating characteristic curves were used to evaluate the diagnostic performance of aspartate aminotransferase to platelet ratio index (APRI), fibrosis score‐4 (Fib‐4) and γ‐glutamyl transpeptidase to platelet ratio (GPR) in different age groups. Results: The extent of fibrosis significantly increased with age, and the percentage of significant fibrosis (≥F2) was 21.3%, 29.0%, 38.5%, and 46.1%, respectively. All three scores displayed a moderate accuracy to diagnose significant fibrosis in overall patients. However, for patients with age ≤30 years, APRI, Fib‐4, and GPR performed poorly with the AUROC of 0.567, 0.627 and 0.596, respectively. Furthermore, using the established cut‐off values—1.45 for Fib‐4, the sensitivity for significant fibrosis increased with age, from 14.8%, 38.1%, 74.5% to 97.87% in above age groups, respectively. To improve the diagnostic accuracy for significant fibrosis, the proposed low and high cut‐off points for Fib‐4 were 0.41 and 1.15 in ≤30 years, 0.8 and 1.59 in 31 to 40 years, 1.17 and 1.94 in 41 to 50 years, 1.76 and 3.10 in ≥ 51 years, respectively. Conclusions: Age may influence the diagnostic thresholds and performance of APRI, Fib‐4, and GPR for significant fibrosis in patients with CHB. In particular, these scores performed poorly for identifying significant fibrosis in younger patients (≤30 years). Highlights: 1. APRI, Fib‐4 and GPR had the low accuracy to diagnose significant fibrosis in younger CHB patients (≤30 years).2.The cut‐off points of APRI, Fib‐4 and GPR should be adjusted by age to improve the diagnostic accuracy for significant fibrosis. Taking Fib‐4 as an example, the LC and HC were 0.41 and 1.15 in ≤30 years, 0.8 and 1.59 in 31‐40 years, 1.17 and 1.94 in 41‐50 years, 1.76 and 3.10 in ≥51 years, respectively. [ABSTRACT FROM AUTHOR]

In many cases protein assemblies are stabilized by covalent bonds, one example of which is the formation of intra- or intermolecular ε-(γ-glutamyl)lysil cross-links catalyzed by transglutaminases (TGases). Because of the potential for unwanted cross-linking reactions, the activities of many TGases have been shown to be tightly controlled. Bacterial endospores are highly resilient cells in part because they are surrounded by a complex protein coat. Proteins in the coat that surrounds Bacillus subtilis endospores are crosslinked by a TGase (Tgl). Unlike other TGases, however, Tgl is produced in an active form, and efficiently catalyzes amine incorporation and protein cross-linking in vitro with no known additional requirements. The absence of regulatory factors raises questions as to how the activity of Tgl is controlled during spore coat assembly. Here, we show that substrates assembled onto the spore coat prior to Tgl production govern the localization of Tgl to the surface of the developing spore. We also show that Tgl residues important for substrate recognition are crucial for its localization. We identified the glutamyl (Q) and lysil (K) substrate docking sites and we show that residues on the Q side of Tgl are more important for the assembly of Tgl than those on the K side. Thus, the first step in the reaction cycle, the interaction with Q-substrates and formation of an acyl-enzyme intermediate, is also the determinant step in the localization of Tgl. Consistent with the idea that Tg exerts a “spotwelding” activity, cross-linking pre-formed assemblies, we show that C30 is an oblong hexamer in solution that is cross-linked in vitro into high molecular weight forms. Moreover, during the reaction, Tgl becomes part of the cross-linked products. We suggest that the dependency of Tgl on its substrates is used to accurately control the time, location and extent of the enzyme´s activity, directed at the covalent fortification of pre-assembled complexes at the surface of the developing spore. [ABSTRACT FROM AUTHOR]

Aminoacyl-tRNA synthetases (aaRSs), essential components of the protein synthesizing machinery, have been often chosen for devising therapeutics against parasitic diseases. Due to their relevance in drug development, the current study was designed to explore functional and structural aspects of Leishmania donovani glutamyl-tRNA synthetase (Ld GluRS). Hence, Ld GluRS was cloned into an expression vector and purified to homogeneity using chromatographic techniques. Purified protein showed maximum enzymatic activity at physiological pH, with more binding capacity towards its cofactor (Adenosine triphosphate, 0.06 ± 0.01 mM) than the cognate substrate (L-glutamate, 9.5 ± 0.5 mM). Remarkably, salicylate inhibited Ld GluRS competitively with respect to L-glutamate and exhibited druglikeness with negligible effect on human macrophages. The protein possessed more α-helices (43 %) than β-sheets (12 %), whereas reductions in thermal stability and cofactor-binding affinity, along with variation in mode of inhibition after mutation signified the role of histidine (H60) as a catalytic residue. Ld GluRS could also generate a pro-inflammatory milieu in human macrophages by upregulating cytokines. The docking study demonstrated the placement of salicylate into Ld GluRS substrate-binding site, and the complex was found to be stable during molecular dynamics (MD) simulation. Altogether, our study highlights the understanding of molecular inhibition and structural features of glutamyl-tRNA synthetase from kinetoplastid parasites. • Ld GluRS is a dimeric enzyme with higher enzymatic affinity towards the cofactor than the substrate. • Mutation in ATP binding residue presents reduction in affinity for cofactor and alterations in secondary structure. • Ld GluRS stimulates the pro-inflammatory cytokines and nitric oxide production in macrophages. • Salicylate inhibits Ld GluRS competitively and exhibits higher binding along with druglikeness. • The stable complex between Ld GluRS and salicylate provides structural insights of inhibition. [ABSTRACT FROM AUTHOR]

The synthesis of 5-aminolevulinic acid (ALA) determines adequate amounts of metabolites for the tetrapyrrole biosynthetic pathway. Glutamyl-tRNA reductase (GluTR) catalyzes the rate-limiting step of ALA synthesis and was previously considered to be exclusively localized in the chloroplast stroma of light-exposed plants. To assess the intraplastidic localization of GluTR, we developed a fast separation protocol of soluble and membrane-bound proteins and reassessed the subplastidal allocation of GluTR in stroma and membrane fractions of Arabidopsis plants grown under different light regimes as well as during de-etiolation and dark incubations. Under the examined conditions, the amount of stroma-localized GluTR correlated with the ALA synthesis rate. The transfer to dark repression of ALA synthesis resulted in a loss of soluble GluTR. Arabidopsis mutants lacking one of the GluTR-interacting factors FLUORESCENT (FLU), the GluTR-binding protein (GBP) or ClpC, a chaperone of the Clp protease system, were applied to examine the amount of GluTR and its distribution to the stroma or membrane in darkness and light. Taking into consideration the different compartmental allocation of GluTR, its stability and ALA synthesis rates, the post-translational impact of these regulatory factors on GluTR activity and plastidic sublocalization is discussed. [ABSTRACT FROM AUTHOR]

Abstract Background There is a strong correlation between liver fibrosis and postoperative morbidity after hepatectomy in hepatocellular carcinoma (HCC) patients. The aim of this study was to evaluate which noninvasive fibrosis index (gamma-glutamyl transpeptidase to platelet ratio [GPR], aspartate aminotransferase to platelet ratio index, fibrosis-4 index, or Forns index) was best able to predict complications in patients undergoing hepatectomy for HCC. Materials and methods This retrospective analysis included 275 patients who underwent hepatectomy for HCC from January 2008 to December 2012. Postoperative mortality was defined as death within 90 d after surgery. Complications were grouped into seven grades on the basis of the modified Clavien classification, and major postoperative complications were defined as grade 3 or above. The influence of noninvasive fibrosis indices on postoperative outcomes was assessed by receiver operating characteristic analysis. The primary outcomes were overall complications and major complications, estimated by univariate and multivariate analysis. Results Patients with HCC undergoing anatomical liver resection in the authors' department were evaluated for this study. Finally, 275 patients who underwent minor liver resection (≤2 liver segments) were included. Of these, 231 (84%) were male. The multivariate analysis indicated that the GPR index was not only independently associated with overall complications (hazard ratio, 2.692; 95% confidence interval [CI], 1.626-4.250; P < 0.001) but also independently predictive of major complications (hazard ratio, 1.143; 95% CI, 1.046-1.249; P = 0.03). The areas under the receiver operating characteristic curve for predicting overall complications and major complications for the GPR index were 0.704 (95% CI, 0.643-0.765; P < 0.001) and 0.752 (95% CI, 0.638-0.865; P < 0.001), respectively. Conclusions The data suggested that the GPR index could be a promising predictor of overall postoperative complications and major complications after minor hepatectomy for HCC. [ABSTRACT FROM AUTHOR]

Aminoacyl-tRNA synthetases are ubiquitous, evolutionarily conserved enzymes catalyzing the conjugation of amino acids onto cognate tRNAs. During eukaryotic evolution, tRNA synthetases have been the targets of persistent structural modifications. These modifications can be additive, as in the evolutionary acquisition of noncatalytic domains, or subtractive, as in the generation of truncated variants through regulated mechanisms such as proteolytic processing, alternative splicing, or coding region polyadenylation. A unique variant is the human glutamyl-prolyl-tRNA synthetase (EPRS) consisting of two fused synthetases joined by a linker containing three copies of the WHEP domain (termed by its presence in tryptophanyl-, histidyl-, and glutamyl-prolyl-tRNA synthetases). Here, we identify site-selective proteolysis as a mechanism that severs the linkage between the EPRS synthetases in vitro and in vivo. Caspase action targeted Asp-929 in the third WHEP domain, thereby separating the two synthetases. Using a neoepitope antibody directed against the newly exposed C terminus, we demonstrate EPRS cleavage at Asp-929 in vitro and in vivo. Biochemical and biophysical characterizations of the N-terminally generated EPRS proteoform containing the glutamyl-tRNA synthetase and most of the linker, including two WHEP domains, combined with structural analysis by small-angle neutron scattering, revealed a role for the WHEP domains in modulating conformations of the catalytic core and GSH-S-transferase-C-terminal-like (GST-C) domain. WHEP-driven conformational rearrangement altered GST-C domain interactions and conferred distinct oligomeric states in solution. Collectively, our results reveal long-range conformational changes imposed by the WHEP domains and illustrate how noncatalytic domains can modulate the global structure of tRNA synthetases in complex eukaryotic systems. [ABSTRACT FROM AUTHOR]

Higher eukaryotes have developed extensive compartmentalization of amino acid (aa) - tRNA coupling through the formation of a multi-synthetase complex (MSC) that is composed of eight aa-tRNA synthetases (ARS) and three scaffold proteins: aminoacyl tRNA synthetase complex interacting multifunctional proteins (AIMP1, 2 and 3). Lower eukaryotes have a much smaller complex while yeast MSC consists of only two ARS (MetRS and GluRS) and one ARS cofactor 1 protein, Arc1p ( Simos et al., 1996 ), the homolog of the mammalian AIMP1. Arc1p is reported to form a tripartite complex with GluRS and MetRS through association of the N-terminus GST-like domains (GST-L) of the three proteins ( Koehler et al., 2013 ). Mammalian AIMP1 has no GST-L domain corresponding to Arc1p N-terminus. Instead, AIMP3, another scaffold protein of 18 kDa composed entirely of a GST-L domain, interacts with Methionyl-tRNA synthetase (MARS) ( Quevillon et al., 1999 ) and Glutamyl-Prolyl-tRNA Synthetase (EPRS) ( Cho et al., 2015 ). Here we report two new interactions between MSC members: AIMP1 binds to EPRS and AIMP1 binds to AIMP3. Interestingly, the interaction between AIMP1 and AIMP3 complex makes it the functional equivalent of a single Arc1p polypeptide in yeast. This interaction is not mapped to AIMP1 N-terminal coiled-coil domain, but rather requires an intact tertiary structure of the entire protein. Since AIMP1 also interacts with AIMP2, all three proteins appear to compose a core docking structure for the eight ARS in the MSC complex. [ABSTRACT FROM AUTHOR]

Background/Objectives: Gamma-glutamyltranspeptidase (GGT) levels are an independent risk marker for the development of type 2 diabetes (T2DM). We investigated the relationship between the newly identified serum GGT fractions and glucose metabolism in obese subjects before and after bariatric surgery.Subjects/Methods: Twenty-nine T2DM subjects, wait-listed for Roux-en-Y gastric bypass (RYGB; n = 21) or laparoscopic sleeve gastrectomy (LSG; n = 8), received a 5-h mixed meal test before (T0), 15 days (T15), and 1 year after surgery (T365). Insulin sensitivity was assessed by the OGIS index and β-cell function by C-peptide analysis; fractional GGT (b-, s-, m-, and f-GGT) analysis was performed by gel-filtration chromatography.Results: At T15, total GGT activity decreased by 40% after LSG (p = 0.007) but remained unchanged after RYGB. At T365, all patients showed a reduction in total GGT, in particular b-GGT (≥ 60%) and m-GGT (≥ 50%). In patients with biopsy-proven steatohepatitis (n = 10), total, b-, s-, and m-GGT fractions at T0 were significantly higher than in patients with low-grade steatosis (p = 0.016, 0.0003, and 0.005, respectively); at T365, there was a significant fall in total GGT as well as in each fraction in both groups. In a multiple regression model, b-GGT was the only fraction related to insulin sensitivity (p = 0.016; β coeff. = − 14.0) independently of BMI, fasting glucose, and triglycerides.Conclusions: While GGT activity is generally associated with impaired glucose metabolism, fractional GGT analysis showed that the b-GGT fraction specifically and independently tracks with insulin resistance. [ABSTRACT FROM AUTHOR]

Objective High grade serous ovarian cancer (HGSC) remains one of the most lethal malignancies in females. We previously reported that γ-glutamyl cyclotransferase (GGCT) was significantly upregulated in serous ovarian cancer. The current study was aimed to explore the function and underlying mechanism of GGCT in HGSC. Methods GGCT expression was assessed by immunohistochemistry in 128 HGSC patients. Stable cell lines with GGCT gene overexpression or knockdown were established to investigate the function of GGCT in HGSC in vitro and in vivo . Results GGCT is highly upregulated in HGSC tissues and associated with FIGO stage, lymph node metastasis and ascitic fluid volume. High expression of GGCT is associated with poor survival in HGSC patients. The Harrell's c-indexes of the prognostic models for overall survival and progression-free survival prediction were 0.758 and 0.726, respectively. GGCT knockdown suppresses proliferation, clone formation, migration, and invasion of tumor cells in vitro while forced GGCT overexpression presents opposite results. Furthermore, GGCT silencing inhibits tumor growth and spread in vivo . Epithelial-mesenchymal transition (EMT) and PI3K/AKT/mTOR signaling pathway are suppressed in GGCT silenced cells and enhanced in GGCT overexpressed cells. Inactivation of PI3K/AKT/mTOR signaling pathway in GGCT overexpressed cells induces EMT inhibition. Conclusions Our data reveals an important role of GGCT in regulating EMT and progression of HGSC, providing a valuable prognostic marker and potential target for treatment of HGSC patients. [ABSTRACT FROM AUTHOR]

Introduction Serum gamma glutamyl transferase (GGT), produced and released mostly from the liver and bile duct, is an enzyme involved in response to oxidative stress, and has been used as a maker for prediction of cardiovascular events. Umbilical artery blood flow resistance index, e.g., the systolic/diastolic ratio (S/D ratio) as determined by ultrasound, has been used to assess the fetal intrauterine conditions. While changes of GGT and S/D ratio in preeclampsia are found to be associated with the risk for adverse perinatal outcome, the potential value of combined use of the two measurements for the prediction of adverse perinatal outcome has not been determined. Materials and methods This study included severe preeclampsia patients in late pregnancy and determined their serum GGT levels and ultrasonic flow resistance index of umbilical artery within a week before delivery. Demographic data and perinatal outcomes including perinatal death, five-minute Apgar score, admission to neonatal intensive care unit, respiratory distress syndrome, and intrauterine growth restriction, are documented and analyzed. Results It was found that serum GGT combined with umbilical artery S/D ratio predicted perinatal adverse outcomes in severe preeclampsia patients with a sensitivity of 94.30% and a specificity of 80.00%. Moreover, absent or reversed UA diastolic blood flow was found to be an independent risk factor for intrauterine growth restriction. Conclusion GGT in combination with umbilical artery S/D ratio is a potentially useful marker for the prediction of adverse outcome in severe preeclampsia patients. Future studies in a larger cohort of patients should be performed to verify the efficacy of the strategy. Early and accurate prediction of adverse perinatal events can facilitate the efforts to improve the perinatal outcomes of neonates associated with preeclamptic pregnancies. [ABSTRACT FROM AUTHOR]

Thioester-containing proteins (TEPs), characterized by a unique intrachain β-cysteinyl-γ-glutamyl thioester bond, form an ancient and diverse family of secreted proteins that play central roles in the innate immune response. But the existence form and immune protection mechanism of TEP in invertebrates still remain unclear, especially in the mollusks. The fragmentation and the immune-protective effect of thioester bond in Cf TEP, a previously identified thioester-containing protein in scallop Chlamys farreri , were characterized in the present study. During the early embryonic development of scallop, the mRNA transcript of Cf TEP could be detected in all the stages, and its expression levels in D-larvae, veliger larvae and eye-spot larvae were significantly higher than that in eggs. The Cf TEP protein was also detected in peripheral of D-larvae, veliger larvae and eye-spot larva by immunofluorescence. In the adult scallop, the Cf TEP protein was mainly distributed in the hepatopancreas, gill, kidney, gonad, and mantle. The expression of Cf TEP mRNA in the hemocytes of adult scallop was significantly up-regulated when the scallops were stimulated by LPS, PGN or β-glucan. Two bands (100 and 55 kDa) were detected using anti- Cf TEP-R1 (spanned the C-terminal portion of the thioester, A2M-comp and A2M-recep domain, 942–1472), and a single band (46 kDa) was detected by using anti- Cf TEP-R2 (the N-terminal portion of the following A2M-N-2 domain, 452–496) in the serum of scallop at 12 h after LPS stimulation. When the thioester bond of Cf TEP protein was inactivated by injecting methylamine, the survival rate of scallop was significantly decreased after challenged by Vibrio angulillarum . All these results suggested that Cf TEP protein existed as fragments similar to vertebrate C3, and played central roles in the immune response against pathogen in the innate immunity of scallops. [ABSTRACT FROM AUTHOR]

γ-Glutamyl transpeptidase (GGT) is a cell-membrane-bound enzyme that is involved in various physiological and pathological processes and is regarded as a potential biomarker for many malignant tumors, precise detection of which is useful for early cancer diagnosis. Herein, a new GGT-activatable near-infrared (NIR) fluorescence imaging probe (GANP) by linking of a GGT-recognitive substrate γ-glutamate (γ-Glu) and a NIR merocyanine fluorophore (mCy-Cl) with a self-immolative linker p-aminobenzyl alcohol (PABA) is reported. GANP was stable under physiological conditions, but could be efficiently activated by GGT to generate ≈100-fold enhanced fluorescence, enabling high sensitivity (detection limit of ≈3.6 mU L−1) and specificity for the real-time imaging of GGT activity as well as rapid evaluation of the inhibition efficacy of GGT inhibitors in living tumor cells. Notably, the deep tissue penetration ability of NIR fluorescence could further allow GANP to image GGT in frozen tumor tissue slices with large penetration depth (>100 μm) and in xenograft tumors in living mice. This GGT activatable NIR fluorescence imaging probe could facilitate the study and diagnosis of other GGT-correlated diseases in vivo. [ABSTRACT FROM AUTHOR]

Introduction: Previous studies have examined the correlation between hyperandrogenemia and non-alcoholic fatty liver disease (NAFLD) in women and showed contradictory results. Therefore, we aimed to evaluate the relationship between testosterone level and Fatty Liver Index (FLI), as a surrogate marker for NAFLD, in a cohort of postmenopausal women. Material and methods: A total of 150 postmenopausal women were included in this cross-sectional study. Anthropometric and biochemical parameters, as well as blood pressure, were obtained. Non-alcoholic fatty liver disease is assessed by FLI, an algorithm based on body mass index, waist circumference, triglycerides and g-glutamyl transferase, as a simple and accurate predictor of hepatic steatosis. Women were divided into three groups (FLI < 30, n = 80; 30 ≥ FLI < 60, n = 44; FLI ≤ 60, n = 26). Homeostasis model assessment of insulin resistance (HOMA-IR) as a surrogate marker of insulin resistance was calculated. Results: Multiple linear regression analysis revealed that the best model consisted of 4 parameters (e.g., bioavailable testosterone (β = 0.288, p = 0.001), log HOMA-IR (β = 0.227, p = 0.005), log high-sensitivity C-reactive protein (β = 0.322, p < 0.001), and retinol-binding protein 4 (β = 0.226, p < 0.001)). Adjusted R2 for the best model was 0.550, which means that as much as 55.0% of variation in FLI could be explained with this model. Conclusions: Bioavailable testosterone is independently associated with FLI in postmenopausal women. [ABSTRACT FROM AUTHOR]

Xanthomonas oryzae pv. oryzae (Xoo) is the causal agent of bacterial blight of rice, one of the most devastating rice diseases. We analysed the time-resolved transcriptional expression of aminoacyl-tRNA synthetase (aaRS) genes in Xoo cells treated with rice-leaf extract. Most aaRS genes showed decreased expression in the initial 30 min and recovered or increased expression in the later 30 min. The protein-synthetic machinery of bacterial cells is an important target for developing antibiotic agents; aaRSs play an essential role in peptide synthesis by attaching amino acids onto the corresponding tRNA. In bacteria, glutaminyl-tRNA (Gln-tRNAGln) is synthesised in two steps by glutamyl-tRNA synthetase (GluRS) and tRNA-dependent aminotransferase, the indirect biosynthetic mechanism of which is not present in eukaryotes. We determined the crystal structure of GluRS from Xoo (XoGluRS) at resolution of 3.0 Å, this being the first GluRS structure from a plant pathogen such as Xoo. The XoGluRS structure consists of five domains, which are conserved in other bacterial GluRS structures. In the bacterial GluRS structures, the Rossmann-fold catalytic domain and the stem-contact domain are most conserved in both sequence and structure. The anticodon-binding domain 1 is less conserved in sequence but overall structure is conserved. The connective-polypeptide domain and the anticodon-binding domain 2 show various conformations in structure. The XoGluRS structure could provide useful information to develop a new pesticide against Xoo and bacterial blight. [ABSTRACT FROM AUTHOR]

Background: Mercury is a toxic heavy metal and is known to affect many diseases. However, few studies have examined the effects of mercury exposure on liver function in the general population. We examined the association between blood mercury concentrations and liver enzyme levels in the elderly.Methods: We included 560 elderly participants (60 years or older) who were recruited from 2008 to 2010 and followed up to 2014. Subjects visited a community welfare center and underwent a medical examination and measurement of mercury levels up to five times. Analyses using generalized estimating equations model were performed after adjusting for age, sex, education, overweight, alcohol consumption, smoking, regular exercise, high-density lipoproteins cholesterol, and total calorie intake. Additionally, we estimated interaction effects of alcohol consumption with mercury and mediation effect of oxidative stress in the relationship between mercury levels and liver function.Results: The geometric mean (95% confidence interval (CI)) of blood mercury concentrations was 2.81 μg/L (2.73, 2.89). Significant relationships were observed between blood mercury concentrations and the level of liver enzymes, including aspartate aminotransferase (AST), alanine aminotransferase (ALT), and gamma glutamyl transferase (GGT), after adjusting for potential confounders (P < 0.05). The odds ratios of having abnormal ALT levels were statistically significant in the highest mercury quartile compared to those with the lowest quartile. Particularly, regular alcohol drinkers showed greater effect estimates of mercury on the liver function than non-drinkers groups. There was no mediation effect of oxidative stress in the relationship between blood mercury concentrations and liver function.Conclusions: Our results suggest that blood mercury levels are associated with elevated liver enzymes and interact with alcohol consumption for the association in the elderly. [ABSTRACT FROM AUTHOR]

Background:Microbial γ-glutamyl transpeptidases (GGTs) have been exploited in biotechnological, pharmaceutical, and food sectors for the synthesis of various γ-glutamyl compounds. But, till date, no bacterial GGTs are commercially available in the market because of lower levels of production from various sources. In the current study, production of GGT fromBacillus licheniformisER15 was investigated to achieve high GGT titers.Results:Hyperproduction of GGT fromB. licheniformisER15 was achieved with 6.4-fold enhancement (7921.2 ± 198.7 U/L) by optimization of culture medium following one-variable-at-a-time strategy and statistical approaches. Medium consisting of Na2HPO4: 0.32% (w/v); KH2PO4: 0.15% (w/v); starch: 0.1% (w/v); soybean meal: 0.5% (w/v); NaCl: 4.0% (w/v), and MgCl2: 5 mM was found to be optimal for maximum GGT titers. Maximum GGT titers were obtained, in the optimized medium at 37°C and 200 rpm, after 40 h. It was noteworthy that GGT production was a linear function of sodium chloride concentration, as observed during response surface methodology. While investigating the role of NaCl on GGT production, it was found that NaCl drastically decreased subtilisin concentration and indirectly increasing GGT recovery.Conclusion: B. licheniformisER15 is proved to be a potential candidate for large-scale production of GGT enzyme and its commercialization. [ABSTRACT FROM PUBLISHER]

Resveratrol is a polyphenol found in different plant species and having numerous health-promoting properties in animals and humans. However, its protective action against deleterious effects of ethanol is poorly elucidated. In the present study, the influence of resveratrol (10 mg/kg/day) on some hormones and metabolic parameters was determined in rats ingesting 10 % ethanol solution for two weeks. Blood levels of insulin, glucagon and adiponectin were affected by ethanol, however, resveratrol partially ameliorated these changes. Moreover, in ethanol drinking rats, liver lipid accumulation was increased, whereas resveratrol was capable of reducing liver lipid content, probably due to decrease in fatty acid synthesis. Resveratrol decreased also blood levels of triglycerides and free fatty acids and reduced γ-glutamyl transferase activity in animals ingesting ethanol. These results show that resveratrol, already at low dose, alleviates hormonal and metabolic changes induced by ethanol in the rat and may be useful in preventing and treating some consequences of alcohol consumption. [ABSTRACT FROM AUTHOR]

Tetrapyrroles, such as chlorophyll and heme, are integral to the metabolism of all living organisms. Glutamyl-tRNA reductase (GluTR) is the first unique enzyme in the tetrapyrrole biosynthetic pathway in plants. We firstly cloned the GluTR complete sequences (1733 bp) from Chinese jujube (Ziziphus jujuba Mill., belonging to the family Rhamnaceae), named as ZjGluTR (GenBank accession no. KF530842). ZjGluTR shares high similarity (90%) to those of Malus domestica and Prunus mume through BLASTX analysis. It contains a 1659-bp ORF and encodes a predicted polypeptide of 552 amino acids, with an estimated molecular mass of 60.0 kDa and a theoretical pI of 8.55. The speculated formula of ZjGluTR is C2615H4318N756 O804S25. ZjGluTR has a chloroplast transit peptide which contains 61 amino acids in the N terminal. The subcellular localization result showed that ZjGluTR protein exists in chloroplasts. ZjGluTR has three typical functional domains, i.e. GluTR N-terminal domain (101-259 aa), NAD(P)-binding Rossmann-fold domain (266-422 aa) and GluTR dimerization domain (425-534 aa). The molecular phylogenetic tree of GluTR indicated that the family Rhamnaceae has a close genetic relationship with the family Rosaceae. Our studies on the GluTR using molecular biology and bioinformatics approaches would play an important role in chlorophyll metabolic research of Chinese jujube. [ABSTRACT FROM AUTHOR]

The use of organ-on-a-chip (OOC) platforms enables improved simulation of the human kidney's response to nephrotoxic drugs. The standard method of analyzing nephrotoxicity from existing OOC has majorly consisted of invasively collecting samples (cells, lysates, media, etc. ) from an OOC. Such disruptive analyses potentiate contamination, disrupt the replicated in vivo environment, and require expertize to execute. Moreover, traditional analyses, including immunofluorescence microscopy, immunoblot, and microplate immunoassay are essentially not in situ and require substantial time, resources, and costs. In the present work, the incorporation of fluorescence nanoparticle immunocapture/immunoagglutination assay into an OOC enabled dual-mode monitoring of drug-induced nephrotoxicity in situ . A smartphone-based fluorescence microscope was fabricated as a handheld in situ monitoring device attached to an OOC. Both the presence of γ-glutamyl transpeptidase (GGT) on the apical brush-border membrane of 786-O proximal tubule cells within the OOC surface, and the release of GGT to the outflow of the OOC were evaluated with the fluorescence scatter detection of captured and immunoagglutinated anti-GGT conjugated nanoparticles. This dual-mode assay method provides a novel groundbreaking tool to enable the internal and external in situ monitoring of the OOC, which may be integrated into any existing OOCs to facilitate their subsequent analyses. [ABSTRACT FROM AUTHOR]

γ-Glutamyltranspetidase (GGT) is a cell-membrane-bound enzyme which selectively catalyzes cleavage of the γ-glutamyl bond of glutathione (GSH). It has been identified to be overexposed in a number of malignant tumor cells. Therefore, fluorescent probes for fast and selective detection of GGT activities are greatly needed. However, the majority of currently available GGT fluorescent probes based on direct conjugation of a γ-glutamyl group to a specific fluorophore generally has slow enzymatic kinetics due to bulky fluorophore too close to the enzyme's active site. Moreover, the uncaged fluorophore with a free amine group might undergo oxidation or other enzymatic transformation and resulted in a complicated time-dependent fluorescence response. Herein, we reported design of a novel fluorescent GGT probe NM-GSH (2), which incorporated a fast intramolecular transcyclization cascade for rapid detection of GGT activities after enzymatic cleavage of the γ-glutamyl group. This design strategy allows introduction of bulky 1,8-naphthalimide fluorophore with improved enzymatic kinetics and lowered detection limit. The transcyclized product 4 gives more than 200-fold fluorescence increment The probe NM-GSH showed both good selectivity and fast detection of GGT activities with the detection limit as low as 0.21 mU/mL. In addition, the fluorescent product 4 contains no free amine group and is more stable for detection. Most importantly, cell imaging studies showed that the transcyclized product 4 was enriched in lysosomes for selectively lighting up GGT-overexpressed ovarian cancer cells (OVCAR5) but not normal cells (HUVEC), indicating NM-GSHs potentials as an imaging agent in cancer diagnosis and treatment. [ABSTRACT FROM AUTHOR]

5-Aminolevulinic acid (ALA), the committed intermediate of the heme biosynthetic pathway, attracts close attention among researchers because of its potential applications to cancer treatment and agriculture. Overexpression of heterologous hemA and hemL , which encode glutamyl-tRNA reductase and glutamate-1-semialdehyde aminotransferase, respectively, in Corynebacterium glutamicum produces ALA, although whether ALA accumulation causes unintended effects on the host is unknown. Here we used an integrated systems approach to compare global transcriptional changes induced by the expression of hemA and hemL . Metabolic pathway such as glycolysis was inhibited, but tricarboxylic acid cycle, pentose phosphate pathway, and respiratory metabolism were stimulated. Moreover, the transcriptional levels of certain genes involved in heme biosynthesis were up-regulated, and the data implicate the two-component system (TCS) HrrSA was involved in the regulation of heme synthesis. With these understandings, it is proposed that ALA accumulation stimulates heme synthesis pathway and respiratory metabolism. Our study illuminates the physiological effects of overexpressing hemA and hemL on the phenotype of C. glutamicum and contributes important insights into the regulatory mechanisms of the heme biosynthetic pathways. [ABSTRACT FROM AUTHOR]

Mitochondrial glutamyl-tRNA synthetase is a major component of protein biosynthesis that loads tRNAs with cognate amino acids. Mutations in the gene encoding this enzyme have been associated with a variety of disorders related to oxidative phosphorylation. Here, we present a case of leukoencephalopathy with thalamus and brainstem involvement and high lactate (LTBL) presenting a biphasic clinical course characterized by delayed psychomotor development and seizure. High-throughput sequencing revealed a novel compound heterozygous mutation in mitochondrial glutamyl-tRNA synthetase 2 (EARS2), which appears to be causative of disease symptoms. [ABSTRACT FROM AUTHOR]

The γ-glutamyl transpeptidase (GGT) enzyme plays a central role in glutathione homeostasis. Direct detection of GGT activity could provide critical information for the diagnosis of several pathologies. We propose a new molecular probe, γ-Glu-[1-13C]Gly, for monitoring GGT activity in vivo by hyperpolarized (HP) 13C magnetic resonance (MR). The properties of γ-Glu-[1-13C]Gly are suitable for in vivo HP 13C metabolic analysis since the chemical shift between γ-Glu-[1-13C]Gly and its metabolic product, [1-13C]Gly, is large (4.3 ppm) and the T1 of both compounds is relatively long (30 s and 45 s, respectively, in H2O at 9.4 T). We also demonstrate that γ-Glu-[1-13C]Gly is highly sensitive to in vivo modulation of GGT activity induced by the inhibitor acivicin. [ABSTRACT FROM AUTHOR]

Arc1p is a yeast-specific tRNA-binding protein that forms a ternary complex with glutamyl-tRNA synthetase (GluRSc) and methionyl-tRNA synthetase (MetRS) in the cytoplasm to regulate their catalytic activities and subcellular distributions. Despite Arc1p not being involved in any known biotin-dependent reaction, it is a natural target of biotin modification. Results presented herein show that biotin modification had no obvious effect on the growth-supporting activity, subcellular distribution, tRNA binding, or interactions of Arc1p with GluRSc and MetRS. Nevertheless, biotinylation of Arc1p was temperature dependent; raising the growth temperature from 30 to 37 °C drastically reduced its biotinylation level. As a result, Arc1p purified from a yeast culture that had been grown overnight at 37 °C was essentially biotin free. Non-biotinylated Arc1p was more heat stable, more flexible in structure, and more effective than its biotinylated counterpart in promoting glutamylation activity of the otherwise inactive GluRSc at 37 °C in vitro. Our study suggests that the structure and function of Arc1p can be modulated via biotinylation in response to temperature changes. [ABSTRACT FROM AUTHOR]

Recently, a large body of evidences indicates the existence in the mitochondrial matrix of foci that contain different proteins involved in mitochondrial RNA metabolism. Some of these proteins have a pentatricopeptide repeat motif that constitutes their RNA-binding structures. Here we report that MSC6, a mitochondrial pentatricopeptide protein of unknown function, is a multi copy suppressor of mutations in QRS1/HER2 a component of the trimeric complex that catalyzes the transamidation of glutamyl-tRNAQ to glutaminyl-tRNAQ. This is an essential step in mitochondrial translation because of the lack of a specific mitochondrial aminoacyl glutaminyl-tRNA synthetase. MSC6 over-expression did not abolish translation of an aberrant variant form of Cox2p detected in QRS1/HER2 mutants, arguing against a suppression mechanism that bypasses Qrs1p function. A slight decrement of the mitochondrial translation capacity as well as diminished growth on respiratory carbon sources media for respiratory activity was observed in the msc6 null mutant. Additionally, the msc6 null mutant did not display any impairment in RNA transcription, processing or turnover. We concluded that Msc6p is a mitochondrial matrix protein and further studies are required to indicate the specific function of Msc6p in mitochondrial translation. [ABSTRACT FROM AUTHOR]

Leukoencephalopathy with thalamus and brainstem involvement, and high lactate (LTBL) is a recently identified disease related to mutations in the EARS2 gene encoding glutamyl-tRNA synthetase. We report clinical and radiological findings for two siblings with new pathogenic mutations in the EARS2 gene. Both patients showed symptoms of mild-type disease, but there were clinical differences between the two siblings. While the older brother had hypotonia and delayed developmental milestones, the younger brother had seizures and spasticity in the lower extremities. Brain magnetic resonance imaging (MRI) findings were quite similar for the two siblings. MRI findings were specific to LTBL. MRI lesions of the older sibling had regressed over time. Clinical and radiological improvement, as in the previously reported patients with LTBL, may be an important clue for diagnosis. [ABSTRACT FROM AUTHOR]

In the integrated stress response, phosphorylation of eIF2α (eIF2α-P) reduces protein synthesis while concomitantly promoting preferential translation of specific transcripts associated with stress adaptation. Translation of the glutamyl-prolyl-tRNA synthetase gene EPRS is enhanced in response to eIF2α-P. To identify the underlying mechanism of translation control, we employed biochemical approaches to determine the regulatory features by which upstream ORFs (uORFs) direct downstream translation control and expression of the EPRS coding region. Our findings reveal that translation of two inhibitory uORFs encoded by noncanonical CUG and UUG initiation codons in the EPRS mRNA 5'-leader serve to dampen levels of translation initiation at the EPRS coding region. By a mechanism suggested to involve increased translation initiation stringency during stress-induced eIF2α-P, we observed facilitated ribosome bypass of these uORFs, allowing for increased translation of the EPRS coding region. Importantly, EPRS protein expression is enhanced through this preferential translation mechanism in response to multiple known activators of eIF2α-P and likely serves to facilitate stress adaptation in response to a variety of cellular stresses. The rules presented here for the regulated ribosome bypass of noncanonical initiation codons in the EPRS 5α-leader add complexity into the nature of uORF-mediated translation control mechanisms during eIF2α-P and additionally illustrate the roles that previously unexamined uORFs with noncanonical initiation codons can play in modulating gene expression. [ABSTRACT FROM AUTHOR]

Aminoacyl-tRNA synthetases (aaRSs) are essential enzymes in protein translation machinery that provide the charged tRNAs needed for protein synthesis. Over the past decades, aaRSs have been studied as anti-parasitic, anti-bacterial, and anti-fungal drug targets. This study focused on the cytoplasmic glutamyl-tRNA synthetase (GluRS) from Plasmodium falciparum , which belongs to class Ib in aaRSs. GluRS unlike most other aaRSs requires tRNA to activate its cognate amino acid substrate L -Glutamate (L -Glu), and fails to form an intermediate adenylate complex in the absence of tRNA. The crystal structures of the Apo, ATP, and ADP-bound forms of Plasmodium falciparum glutamyl-tRNA synthetase (Pf GluRS) were solved at 2.1 Å, 2.2 Å, and 2.8 Å respectively. The structural comparison of the Apo- and ATP-bound holo-forms of Pf GluRS showed considerable conformational changes in the loop regions around the ATP-binding pocket of the enzyme. Biophysical characterization of the Pf GluRS showed binding of the enzyme substrates L -Gluand ATP.. The sequence and structural conservation were evident across GluRS compared to other species. The structural dissection of the Pf GluRS gives insight into the critical residues involved in the binding of ATP substrate, which can be harvested to develop new antimalarial drugs. • Cytoplasmic glutamyl-tRNA synthetase crystal structures from Plasmodium falciparum (Pf GluRS). • Conformational changes in loop regions may facilitate the entry of ATP molecule into the active site pocket of the enzyme. • Insight into the critical residues in the ATP-binding site, which can be exploited for structure-based drug design. [ABSTRACT FROM AUTHOR]

Insulin-like growth factor 1 (IGF-1), an insulin sensitivity and vasculoprotective factor, associates negatively with the metabolic syndrome. However, IGF-1 is reduced by factors such as inflammation, oxidative stress and liver dysfunction. We investigated the relationship between bioavailable IGF-1 and the number of metabolic syndrome components and determined whether this relationship is independent of inflammation, oxidative stress and gamma glutamyl transferase (γ-GT; a marker of liver dysfunction). This study included 907 black and white participants stratified by sex (aged 43.0 ± 11.8 years). Among them 63 participants had fasting glucose levels of ≥ 7.0 mmol/l and/or used diabetes medication. Via standard methods we determined waist circumference, fasting glucose, triglycerides, high-density lipoprotein cholesterol and blood pressure. We also determined high-sensitivity C-reactive protein (CRP), reactive oxygen species (ROS), γ-GT, IGF-1 and insulin-like growth factor binding protein 3 (IGFBP-3). IGF-1/IGFBP-3 was used as an estimate of bioavailable IGF-1. Total IGF-1 was similar between men and women (p = 0.10), however, bioavailable IGF-1 was lower in women (p < 0.001). In multivariate-adjusted analyses, IGF-1/IGFBP-3 was inversely associated with the number of metabolic syndrome components in both sexes (men: β = ? 0.11; p = 0.013 and women: β = ? 0.17; p = 0.003). Upon inclusion of ROS, γ-GT and CRP, significance was lost. In patients without diabetes, the results for men changed marginally, but were consistent for women. We found an inverse association between bioavailable IGF-1 and the number of metabolic syndrome components. But the relationship was dependent on oxidative stress, liver dysfunction and inflammation, suggesting underlying processes by which the metabolic syndrome attenuates IGF-1. [ABSTRACT FROM AUTHOR]

EgtB from Mycobacterium thermoresistibile catalyzes O2-dependent sulfur–carbon bond formation between the side chains of Nα-trimethyl histidine and γ-glutamyl cysteine as a central step in ergothioneine biosynthesis. A single point mutation converts this enzyme into a γ-glutamyl cysteine dioxygenase with an efficiency that rivals naturally evolved thiol dioxygenases. [ABSTRACT FROM AUTHOR]

Synthetic small interfering RNA (siRNA) is a class of therapeutic entities that allow for specific silencing of target genes via RNA interference (RNAi) and comprise an enormous clinical potential for a variety of diseases, including cancer. However, efficient tissue-specific delivery of siRNA remains the major limitation in the development of RNAi-based cancer therapeutics. To achieve this, we have synthesized a series of sequence-defined oligomers, which include a cationic (oligoethanamino)amide core (for nanoparticle formation with siRNA), cysteines (as bioreversible disulfide units), and a polyethylene glycol chain (for shielding of surface charges) coupled to a terminal targeting ligand. The antifolate drug methotrexate (MTX), a well-established chemotherapeutic agent, serves as both targeting ligand and anticancer agent. The oligomers form homogeneous spherical siRNA polyplexes with a hydrodynamic diameter of approximately 6 nm. These polyplexes access KB cells by binding to the folate receptor in a MTX-dependent manner and induce efficient gene silencing activity in vitro . Impressively, in the in vivo studies, MTX-conjugated polyplexes significantly increase the intratumoral retention (168 h) of the siRNA, as compared to alanine-substituted non-targeted control polyplexes (48 h). The combination of MTX-conjugated polyplexes and eglin 5 (EG5) siRNA provides enhanced antitumoral potency with 50% of recurrence-free survival of KB tumor-bearing mice. The design of such siRNA carrier systems with a dual-functional ligand for cellular delivery and augmented tumor suppression could be a valuable strategy for translating RNAi-based cancer therapeutics to the clinics. [ABSTRACT FROM AUTHOR]

Background & Aims: It remains unclear whether the respective dose-response relationships between serum alanine aminotransferase (ALT) and gamma-glutamyl transferase (GGT) levels and risk of mortality are consistent by age. Methods: We used sampled cohort data from the National Health Insurance Corporation to conduct a retrospective cohort study. A total of 313 252 participants who received medical health check-ups from 2002 to 2008 were assessed for risk of death according to serum ALT and GGT levels over an average of 6 years. The hazard ratios (HRs) for mortality were analysed with Cox proportional hazard model. Results: The crude mortality rate increased linearly with increasing serum ALT and GGT levels in adults aged <60 years. However, the all-cause mortality rate showed a J-shaped relationship with increasing serum ALT levels whereas all-cause mortality rate showed a linear relationship with increasing serum GGT levels in adults aged ≥60 years. The HR of death showed U-shaped relationships with increasing serum ALT levels in adults aged ≥60 years. On the contrary, the HR of death from any cause had a linear association with increasing serum GGT levels among all age groups. Conclusions: In this study, U-shaped relationship patterns were demonstrated between serum ALT levels and risk for all-cause mortality in adults aged ≥60 years while serum GGT levels showed a linear relationship with risk for all-cause death. Very low levels of serum ALT in elderly patients suggest that they are at high risk of mortality. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa glutamyl-tRNA synthetase (GluRS) was overexpressed in Escherichia coli. Sequence analysis indicated that P. aeruginosa GluRS is a discriminating GluRS and, similar to other GluRS proteins, requires the presence of tRNAGlu to produce a glutamyl-AMP intermediate. Kinetic parameters for interaction with tRNA were determined and the kcat and KM were 0.8 s–1 and 0.68 µM, respectively, resulting in a kcat/KM of 1.18 s–1 µM–1. A robust aminoacylation-based scintillation proximity assay (SPA) assay was developed and 800 natural products and 890 synthetic compounds were screened for inhibitory activity against P. aeruginosa GluRS. Fourteen compounds with inhibitory activity were identified. IC50s were in the low micromolar range. The minimum inhibitory concentration (MIC) was determined for each of the compounds against a panel of pathogenic bacteria. Two compounds, BT_03F04 and BT_04B09, inhibited GluRS with IC50s of 21.9 and 24.9 µM, respectively, and both exhibited promising MICs against Gram-positive bacteria. Time-kill studies indicated that one compound was bactericidal and one was bacteriostatic against Gram-positive bacteria. BT_03F04 was found to be noncompetitive with both ATP and glutamic acid, and BT_04B09 was competitive with glutamic acid but noncompetitive with ATP. The compounds were not observed to be toxic to mammalian cells in MTT assays. [ABSTRACT FROM AUTHOR]

Background: Glutathione (GSH), a pivotal non-protein thiol, can be biosynthesized through three pathways in different organisms: (1) two consecutive enzymatic reactions catalyzed by γ-glutamylcysteine synthetase (Gsh1 or GshA) and glutathione synthetase (Gsh2 or GshB); (2) a bifunctional γ-glutamylcysteine synthetase/glutathione synthetase (GshF); (3) an alternative condensation of γ-glutamyl phosphate synthesized by γ-glutamyl kinase (Pro1 or ProB) with cysteine to form γ-glutamylcysteine which was further conjugated to glycine by glutathione synthetase. The Gsh1 and Gsh2 of conventional GSH biosynthetic pathway or the bifunctional GshF reported previously have been independently modulated for GSH production. This study developed a novel three-pathway combination method to improve GSH production in Saccharomyces cerevisiae. Results: A bifunctional enzyme GshF of Actinobacillus pleuropneumoniae was functionally expressed in S. cerevisiae and Pro1 in proline biosynthetic pathway was exploited for improving GSH yield. Moreover, two fusion proteins Gsh2- Gsh1 and Pro1-GshB were constructed to increase the two-step coupling efficiency of GSH synthesis by mimicking the native domain fusion of GshF. The engineered strain W303-1b/FGP with three biosynthetic pathways presented the highest GSH concentration (216.50 mg/L) and GSH production of W303-1b/FGP was further improved by 61.37 % when amino acid precursors (5 mM glutamic acid, 5 mM cysteine and 5 mM glycine) were fed in shake flask cultures. In batch culture process, the recombinant strain W303-1b/FGP also kept high efficiency in GSH production and reached an intracellular GSH content of 2.27 % after 24-h fermentation. Conclusions: The engineered strains harbouring three GSH pathways displayed higher GSH producing capacity than those with individually modulated pathways. Three-pathway combinatorial biosynthesis of GSH promises more effective industrial production of GSH using S. cerevisiae. [ABSTRACT FROM AUTHOR]

Recently, it has been demonstrated that kokumi substances such as glutathione are perceived through the calcium-sensing receptor (CaSR), and screening by CaSR assay and sensory evaluation has shown that γ-glutamyl-valyl-glycine (γ-Glu-Val-Gly) is a potent kokumi peptide. In this study, γ-Glu-Val-Gly contents in various brewed alcoholic beverages were investigated. Contents of γ-Glu-Val-Gly in four brands of wine, four brands of rice wine (sake) and eight brands of beer were analyzed by high performance liquid chromatography-tandem mass spectrometry followed by derivatization with 6-aminoquinoyl- N -hydroxysuccinimidyl-carbamate. The analyses indicated that γ-Glu-Val-Gly was present in all of eight beer samples at concentrations in the range of 0.08–0.18 mg/L, although the peptide was not detected in any wine or rice wine samples. These results suggest that amongst the brewed beverages tested, beer contains γ-Glu-Val-Gly, and that γ-Glu-Val-Gly is widely distributed in beer. [ABSTRACT FROM AUTHOR]

Despite its biological importance, iron is a pro-oxidant element and its accumulation results in tissue injury. Iron overload diseases such as thalassemia and hereditary hemochromatosis are commonly associated with liver tissue injury. Glutamyl cysteine (GC) is a dipeptide with antioxidant properties owing to its cysteine residue. The aim of the current work was to investigate the hepatoprotective effect of GC against iron overload-induced liver injury. Rats were distributed into five groups; normal control, GC control, iron-treated (150 mg/kg ip injection) and both iron and GC-treated (total iron: 150 mg/kg ip and GC: 50 mg or 100 mg/kg/day ip for 30 days). Our results showed that treatment with GC at the two-dose levels attenuated iron-induced liver tissue injury as evidenced by significant reduction in serum activity of liver enzymes ALT and AST, amelioration of iron-induced histopathological alteration, suppression of iron-induced oxidative stress as demonstrated by significant reduction of malondialdehyde and protein carbonyl content beside elevation of total antioxidant capacity, reduced glutathione and the antioxidant enzymes GPx and SOD in liver tissue. In addition, GC significantly reduced levels of the proinflammatory cytokines TNF-α, IL-6 and IL-1β and activity of the apoptotic marker caspase-3 in liver tissues. To our surprise, GC reduced liver iron content and ferritin expression, denoting the possible iron chelation competency. Collectively our results highlight evidence for the hepatoprotective effect of GC against iron overload-induced liver injury that is potentially mediated through suppression of oxidative tissue injury, attenuation of inflammatory response, amelioration of hepatocellular apoptosis and possibly through iron chelation. [ABSTRACT FROM AUTHOR]

Gamma Glutamyl Transferase (GGT) is an important biomarker in malignant cancers. The redox processes ensuing from GGT-mediated metabolism of extracellular GSH are implicated in critical aspects of tumor cell biology. Reportedly, Glutathione monoethyl ester (GSHMe) is a substrate of GGT, which has been used for its rapid transport over glutathione. Exploring GGT to be an important target, a homobivalent peptide system, DT(GSHMe)2 was designed to target GGT-over expressing tumors for diagnostic purposes. DT(GSHMe)2 was synthesized, characterized and preclinically evaluated in vitro using toxicity, cell binding assays and time dependent experiments. Stable and defined radiochemistry with 99mTc and 68Ga was optimized for high radiochemical yield. In vivo biodistribution studies were conducted for different time points along with scintigraphic studies of radiolabeled DT(GSHMe)2 on xenografted tumor models. For further validation, in silico docking studies were performed on GGT (hGGT1, P19440). Preclinical in vitro evaluations on cell lines suggested minimal toxicity of DT(GSHMe)2 at 100 μM concentration. Kinetic analysis revealed transport of 99mTc-DT(GSHMe)2 occurs via a saturable high-affinity carrier with Michaelis constant (Km) of 2.25 μM and maximal transport rate velocity (Vmax) of 0.478 μM/min. Quantitative estimation of GGT expression from western blot experiments showed substantial expression with 41.6 ± 7.07 % IDV for tumor. Small animal micro PET (Positron Emission Tomography)/CT(Computed Tomography) coregistered images depicted significantly high uptake of DT(GSHMe)2 at the BMG-1 tumor site. ROI analysis showed high tumor to contra lateral muscle ratio of 9.33 in PET imaging studies. Avid accumulation of radiotracer was observed at tumor versus inflammation site at 2 h post i.v. injection in an Ehrlich Ascites tumor (EAT) mice model, showing evident specificity for tumor. We propose DT(GSHMe)2 to be an excellent candidate for prognostication and tumor imaging using PET/SPECT. [ABSTRACT FROM AUTHOR]

The tetratricopeptide repeat (TPR)-containing protein FLU is a negative regulator of chlorophyll biosynthesis in plants. It directly interacts through its TPR domain with glutamyl-tRNA reductase (GluTR), the rate-limiting enzyme in the formation of δ-aminolevulinic acid (ALA). Delineation of how FLU binds to GluTR is important for understanding the molecular basis for FLU-mediated repression of synthesis of ALA, the universal tetrapyrrole precursor. Here, we characterize the FLU-GluTR interaction by solving the crystal structures of the uncomplexed TPR domain of FLU (FLUTPR) at 1.45-Å resolution and the complex of the dimeric domain of GluTR bound to FLUTPR at 2.4-Å resolution. Three non-canonical TPR motifs of each FLUTPR form a concave surface and clamp the helix bundle in the C-terminal dimeric domain of GluTR. We demonstrate that a 2:2 FLUTPR-GluTR complex is the functional unit for FLU-mediated GluTR regulation and suggest that the formation of the FLU-GluTR complex prevents glutamyl-tRNA, the GluTR substrate, from binding with this enzyme. These results also provide insights into the spatial regulation of ALA synthesis by the membrane-located FLU protein. [ABSTRACT FROM AUTHOR]

For tRNA-dependent protein biosynthesis, amino acids are first activated by aminoacyl-tRNA synthetases (aaRSs) yielding the reaction intermediates aminoacyl-AMP (aa-AMP). Stable analogues of aa-AMP, such as aminoacyl-sulfamoyl-adenosines, inhibit their cognate aaRSs. Glutamyl-sulfamoyl-adenosine (Glu-AMS) is the best known inhibitor of Escherichia coli glutamyl-tRNA synthetase (GluRS). Thermodynamic parameters of the interactions between Glu-AMS and E. coli GluRS were measured in the presence and in the absence of tRNA by isothermal titration microcalorimetry. A significant entropic contribution for the interactions between Glu-AMS and GluRS in the absence of tRNA or in the presence of the cognate tRNAGlu or of the non-cognate tRNAPhe is indicated by the negative values of –TΔSb, and by the negative value of ΔCp. On the other hand, the large negative enthalpy is the dominant contribution to ΔGb in the absence of tRNA. The affinity of GluRS for Glu-AMS is not altered in the presence of the non-cognate tRNAPhe, but the dissociation constant Kd is decreased 50-fold in the presence of tRNAGlu; this result is consistent with molecular dynamics results indicating the presence of an H-bond between Glu-AMS and the 3’-OH oxygen of the 3’-terminal ribose of tRNAGlu in the Glu-AMS•GluRS•tRNAGlu complex. Glu-AMS being a very close structural analogue of Glu-AMP, its weak binding to free GluRS suggests that the unstable Glu-AMP reaction intermediate binds weakly to GluRS; these results could explain why all the known GluRSs evolved to activate glutamate only in the presence of tRNAGlu, the coupling of glutamate activation to its transfer to tRNA preventing unproductive cleavage of ATP. [ABSTRACT FROM AUTHOR]

The putative zinc-binding domain (pZBD) in Escherichia coli glutamyl-tRNA synthetase (GluRS) is known to correctly position the tRNA acceptor arm and modulate the amino acid-binding site. However, its functional role in other bacterial species is not clear since many bacterial GluRSs lack a zinc-binding motif in the pZBD. From experimental studies on pZBD-swapped E. coli GluRS, with Thermosynechoccus elongatus GluRS, Burkholderia thailandensis GluRS and E. coli glutamyl-queuosine-tRNAAsp synthetase (Glu-Q-RS), we show that E. coli GluRS, containing the zinc-free pZBD of B. thailandensis, is as functional as the zinc-bound wild-type E. coli GluRS, whereas the other constructs, all zinc-bound, show impaired function. A pZBD-tinkered version of E. coli GluRS that still retained Zn-binding capacity, also showed reduced activity. This suggests that zinc is not essential for the pZBD to be functional. From extensive structural and sequence analyses from whole genome database of bacterial GluRS, we further show that in addition to many bacterial GluRS lacking a zinc-binding motif, the pZBD is actually deleted in some bacteria, all containing either glutaminyl-tRNA synthetase (GlnRS) or a second copy of GluRS (GluRS2). Correlation between the absence of pZBD and the occurrence of glutamine amidotransferase CAB (GatCAB) in the genome suggests that the primordial role of the pZBD was to facilitate transamidation of misacylated Glu-tRNAGln via interaction with GatCAB, whereas its role in tRNAGlu interaction may be a consequence of the presence of pZBD. [ABSTRACT FROM AUTHOR]

Glutamyl-tRNA (Glu-tRNAGlu) is the common substrate for both protein translation and heme biosynthesis via the C5 pathway. Under normal conditions, an adequate supply of this aminoacyl-tRNA is available to both pathways. However, under certain circumstances, Glu-tRNAGlu can become scarce, resulting in competition between the two pathways for this aminoacyl-tRNA. In Acidithiobacillus ferrooxidans, glutamyl-tRNA synthetase 1 (GluRS1) is the main enzyme that synthesizes Glu-tRNAGlu. Previous studies have shown that GluRS1 is inactivated in vitro by hydrogen peroxide (H2O2). This raises the question as to whether H2O2 negatively affects in vivo GluRS1 activity in A. ferrooxidans and whether Glu-tRNAGlu distribution between the heme and protein biosynthesis processes may be affected by these conditions. To address this issue, we measured GluRS1 activity. We determined that GluRS1 is inactivated when cells are exposed to H2O2, with a concomitant reduction in intracellular heme level. The effects of H2O2 on the activity of purified glutamyl-tRNA reductase (GluTR), the key enzyme for heme biosynthesis, and on the elongation factor Tu (EF-Tu) were also measured. While exposing purified GluTR, the first enzyme of heme biosynthesis, to H2O2 resulted in its inactivation, the binding of glutamyl-tRNA to EF-Tu was not affected. Taken together, these data suggest that in A. ferrooxidans, the flow of glutamyl-tRNA is diverted from heme biosynthesis towards protein synthesis under oxidative stress conditions. [ABSTRACT FROM AUTHOR]

The nature of interaction between glutamyl-tRNA synthetase (GluRS) and its tRNA substrate is unique in bacteria in that many bacterial GluRS are capable of recognizing two tRNA substrates: tRNAGlu and tRNAGln. To properly understand this distinctive GluRS-tRNA interaction it is important to pursue detailed structure-function studies; however, because of the fact that tRNA-GluRS interaction in bacteria is also associated with phylum-specific idiosyncrasies, the structure-function correlation studies must also be phylum-specific. GluRS from Thermus thermophilus and Escherichia coli, which belong to evolutionarily distant phyla, are the biochemically best characterized. Of these, only the structure of T. thermophilus GluRS is available. To fully unravel the subtleties of tRNAGlu-GluRS interaction in E. coli, a model bacterium that can also be pathogenic, determination of the E. coli GluRS structure is essential. However, previous attempts have failed to crystallize E. coli GluRS. By mapping crystal contacts of a homologous GluRS onto the E. coli GluRS sequence, two surface residues were identified that might have been hindering crystallization attempts. Accordingly, these two residues were mutated and crystallization of the double mutant was attempted. Here, the design, expression, purification and crystallization of an engineered E. coli GluRS in which two surface residues were mutated to optimize crystal contacts are reported. [ABSTRACT FROM AUTHOR]