Investigation on Intestinal Proteins and Drug Metabolizing Enzymes in Simulated Microgravity Rats by a Proteomics Method.

The present study aimed to investigate the change of intestinal mucosa proteins, especially the alteration of intestinal drug metabolizing enzymes (IDMEs) following 14-day simulated microgravity. Morey-Holton tail-suspension analog was used to simulate microgravity. Intestinal mucosa proteins of rats were determined by label-free quantitative proteomic strategy. A total of 335 differentially expressed proteins (DEPs) were identified, 190 DEPs were upregulated, and 145 DEPs were downregulated. According to bioinformatic analysis, most of DEPs exhibited hydrolase, oxidoreductase, transferase, ligase, or lyase catalytic activity. DEPs were mainly enriched in metabolic pathways, including metabolism of amino acid, glucose, and carbon. Moreover, 11 of DEPs were involved in exogenous drug and xenobiotics metabolism. Owing to the importance of IDMEs for the efficacy and safety of oral drugs, the expression of cytochrome P450 1A2 (CYP1A2), CYP2D1, CYP3A2, CYP2E1, alcohol dehydrogenase 1 (ADH1), and glutathione S-transferase mu 5 (GSTM5) in rat intestine mucosa was determined by Western-blot. The activity of ADH, aldehyde dehydrogenase (ALDH) and GST was evaluated. Compared with control rats, the expression of CYP1A2, CYP2D1, CYP3A2, and ADH1 in the simulated microgravity (SMG) group of rats were dramatically decreased by 33.16%, 21.93%, 48.49%, and 22.83%, respectively. GSTM5 was significantly upregulated by 53.14% and CYP2E1 expression did not show a dramatical change in SMG group rats. Moreover, 14-day SMG reduced ADH activity, while ALDH and GST activities was not altered remarkably. It could be concluded that SMG dramatically affected the expression and activity of some IDMEs, which might alter the efficacy or safety of their substrate drugs under microgravity. The present study provided some preliminary information on IDMEs under microgravity. It revealed the potential effect of SMG on intestinal metabolism, which may be helpful to understand the intestinal health of astronauts and medication use.

Cloning, characterization and expression analysis of glutathione S-transferase from the Antarctic yeast Rhodotorula mucilaginosa AN5.

The gene for glutathione S-transferase (GST) in Antarctic sea-ice yeast Rhodotorula mucilaginosa AN5 was cloned and expressed in Escherichia coli and named RmGST. Sequence analysis showed that the RmGST gene contained a 843 bp open reading frame, which encoded 280 amino acid residues with a calculated molecular mass of 30.4 kDa and isoelectric point of 5.40. RmGST has the typical C- and N-terminal double domains of glutathione S-transferase. Recombinant RmGST (rRmGST) was expressed in E. coli to produce heterologous protein that had a high specific activity of 60.2 U/mg after purification. The apparent Km values of rRmGST for glutathione and 1-chloro-2,4-dinitrobenzene were 0.35 mM and 0.40 mM, respectively. Optimum enzyme activity was measured at 35 °C and at pH 7.0 and complete inactivation was observed after incubation at 55 °C for 60 min rRmGST tolerated high salt concentrations (1.0 M NaCl) and was stable at pH 3.0. Additionally, the recombinant protein nearly kept whole activity in Hg2+ and Mn2+, and could tolerate Ca2+, Cu2+, Mg2+, Cd2+, EDTA, thiourea, urea, Tween-80, H2O2 and Triton X-100. Real-time quantitative PCR showed that relative expression of the GST gene was significantly increased under Cu2+ and low temperature stress. These results indicate that rRmGST is a typical low thermostable enzyme, while its other characteristics, heavy metal and low temperature tolerance, might be related to its Antarctic home environment.

Changes in the content of thiol compounds and the activity of glutathione s-transferase in maize seedlings in response to a rose-grass aphid infestation.

The rose-grass aphid (Methopolophium dirhodum Walk.) is a major pest of maize (Zea mays L.), but little is known about the biochemical interactions between M. dirhodum and its host plant. Thiol compounds and glutathione S-transferase (GST) play a crucial role in the defense responses of maize to biotic stress factors, including aphids. The purpose of this research was to evaluate the impact of M. dirhodum herbivory on the total thiol (TT), protein bound thiol (PT), reduced glutathione (GSH) and oxidized glutathione (GSSG) contents as well as the activity of GST in three varieties of Z. mays (Zlota Karlowa, Ambrozja and Plomyk), that were classified as aphid-susceptible, aphid-relatively resistant and aphid-resistant, respectively. The earliest and strongest aphid-triggered alterations in the levels of TT, PT and GSH, and the greatest induction of GST activity, were recorded in the resistant Plomyk seedlings in relation to the relatively resistant Ambrozja and the susceptible Zlota Karlowa.

Recombinant human interferon regulatory factor-1 (IRF-1) protein expression and solubilisation study in Escherichia coli.

Interferon regulatory factor-1 (IRF-1) is a tumor suppressor gene, which encodes a mammalian transcription factor that serves various vital functions in a cell, such as cell cycle regulation, immunomodulation, and antiviral response. We report full-length human IRF-1 cDNA cloning and expression in E. coli/BL21 cells with complete solubilisation of recombinant protein. We cloned the gene by the RT-PCR technique using ORF-specific primers followed by expression of recombinant IRF-1 in E. coli under GST fusion system. The profound expression of recombinant protein was observed after inducing with 0.5 mM IPTG for 3 h at 37 °C. We observed few degradation products of low molecular mass along with full-length fusion protein. We successfully minimized the formation of low molecular mass degradation products of GST-huIRF-1 protein at 16 °C. Simultaneously, we achieved the expression of recombinant protein in soluble fraction of E. coli/BL21 cells at 20 °C with higher yield, which is crucial to the study of the biological functions of any protein. We further confirmed it by the immunoblotting technique using anti-IRF-1 and anti-GST antibodies under the induction of E. coli cells harboring the IRF-1 recombinant plasmid after sonicated and fractioned fractions. This work will serve as a platform for characterizing the recombinant protein that may pave the way to understand molecular mechanism of tumour suppression caused by this molecule.

The regulatory role of Nrf2 in antioxidants phase2 enzymes and IL-17A expression in patients with ulcerative colitis.

BACKGROUND: Reactive oxygen species (ROS) is one of the pathogenic factors responsible for intestinal injury in Ulcerative colitis (UC). Nuclear factor erythroid-2 related factor 2 (Nrf2) plays a critical role against ROS factors to conserve epithelial integrity. This study aimed to localize Nrf2 and IL-17A protein in the inflamed mucosa of patients with ulcerative colitis. The gene expression of Nrf2 was also correlated with GST-A4 and PRDX1. MATERIALS AND METHODS: A total of 20 patients and 20 healthy controls with definite UC based on the clinical criteria were enrolled for this study. The expression pattern of Nrf2 and IL-17A protein was compared in inflamed and non-inflamed colonic biopsies by immunohistochemical staining. Nrf2, GST-A4 and PRDX1 gene expression were determined by real-time polymerase chain reaction (RT-PCR). RESULTS: In inflamed colonic biopsies, an increased level of Nrf2 protein factor was detected in epithelial cells. Conversely, IL-17A protein was presented more in mononuclear cells in mucosa and lamina propria regions. A significant increase of Nrf2, GST-A4 gene expression was observed in both mild and severe patients with ulcerative colitis. GST-A4 gene expression indicated a high exponential rate in logistic regression. CONCLUSION: Oxidative stress in inflamed colonic tissue can induce Nrf2 gene expression. The performance of Nrf2 transcription factor may lead to the induction of GST-A4 and PRDX1. IL-17A is less detected in intestinal inflammation, presenting Nrf2 factor. The present findings suggest that Nrf2 function in the gut plays a role in arresting both inflammatory response and oxidative damages of UC.

Development of bicistronic expression system for the enhanced and reliable production of recombinant proteins in Leuconostoc citreum.

The lactic acid bacteria (LAB) Leuconostoc citreum are non-sporulating hetero-fermentative bacteria that play an important role in the fermented food industry. In this study, for the enhanced and reliable production of recombinant proteins in L. citreum, we developed a bicistronic design (BCD) expression system which includes a short leader peptide (1st cistron) followed by target genes (2nd cistron) under the control of a single promoter. Using superfolder green fluorescent protein (sfGFP) as a reporter, the functionality of BCD in L. citreum was verified. Further, to improve the expression in BCD, we tried to engineer a Shine-Dalgarno sequence (SD2) for the 2nd cistron and a promoter by FACS screening of random libraries, and both strong SD2 (eSD2) and promoter (P710V4) were successfully isolated. The usefulness of the engineered BCD with P710V4 and eSD2 was further validated using three model proteins-glutathione-s-transferase, human growth hormone, and α-amylase. All examined proteins were successfully produced with levels highly increased compared with those in the original BCD as well as the monocistronic design (MCD) expression system.

Sublethal effects of chlorfenapyr on the life table parameters, nutritional physiology and enzymatic properties of Bradysia odoriphaga (Diptera: Sciaridae).

Bradysia odoriphaga (Diptera: Sciaridae) is the major pest affecting Chinese chive production. Chlorfenapyr is a halogenated pyrrole-based pro-insecticide that is currently used to control insects and mites on a variety of crops. In the present study, fourth-instar larvae of B. odoriphaga were exposed to chlorfenapyr at LC1, LC20 and LC50 concentrations. The developmental duration of the treated larvae was not significantly different, but fecundity was significantly increased in the LC1 and LC20 treatment groups compared with the control group. The population parameters of the LC1 treatment group were increased significantly, whereas those of the LC50 treatment group were reduced significantly compared with the control. The food consumption by larvae and pupal weight were significantly increased under the LC1 treatment and decreased under the LC50 treatment compared with the control. Moreover, chlorfenapyr decreased the lipid, carbohydrate and trehalose contents significantly, whereas the total protein content was increased compared with the control. Additionally, the activities of protease, lipase and trehalase were significantly decreased. Chlorfenapyr treatment for 24 h also induced the activities of glutathione S-transferase (GST), carboxylesterase (CarE) and O-demethylation. The results of this study suggest that low lethal concentrations of chlorfenapyr can affect oviposition, population development, the activities of digestion and detoxification enzymes, and nutrient accumulation in B. odoriphaga. This study provides valuable information for the assessment and rational application of chlorfenapyr for effective control of this pest.

Benzyl isothiocyanate attenuates the hydrogen peroxide-induced interleukin-13 expression through glutathione S-transferase P induction in T lymphocytic leukemia cells.

We investigated the effect of benzyl isothiocyanate (BITC) on the hydrogen peroxide-induced gene expression of a T-helper-2 cytokine, interleukin (IL)-13, in T lymphocytic leukemia Jurkat cells. The 24-h pretreatment of BITC significantly inhibited the IL-13 expression enhanced by hydrogen peroxide. Although the BITC pretreatment did not change the enhanced level of the phosphorylated c-Jun N-terminal kinase (JNK), it significantly inhibited the nuclear translocation of c-Jun induced by hydrogen peroxide. BITC also increased the protein expression of glutathione S-transferase (GST) isozymes, GSTP1/2, as well as the total GST activity. A GSTP1/2-specific inhibitor, 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX), significantly counteracted the inhibitory effect of BITC on the hydrogen peroxide-enhanced IL-13 upregulation as well as the c-Jun nuclear translocation. Taken together, these results suggested that BITC inhibits the oxidative stress-mediated IL-13 mRNA expression, possibly through interference of the c-Jun phosphorylation by GSTP.

Microcystin induction small Maf protein involve in transcriptional regulation of GST from freshwater mussel Cristaria plicata.

The small Mafs, MafF, MafG and MafK play critical roles in morphogenesis and homeostasis through associating with Cap "n" Collar family of transcription factors. In this study, we tried to identify a small Maf protein in the freshwater mussel Cristaria plicata. The MafK cDNA of C. plicata, designated as CpMafK, was cloned from the hemocytes using degenerate primers by the rapid amplification of cDNA ends PCR. The full length cDNA of CpMafK is 2170 bp, which includes an open reading frame of 570 bp, encoding 189 amino acids. CpMafK possesses four conserved domains and shows a low level (54-63%) of sequence similarity to small Mafs from other species. The results of Real-time quantitative PCR revealed that CpMafK mRNA was constitutively expressed in tissues, and the highest expression level was in hepatopancreas. After microcystin challenge, the expression levels of CpMafK mRNA were up-regulated in hemocytes and hepatopancreas. The cDNA of CpMafK was cloned into the plasmid pET-32, and the recombinant protein was expressed in Escherichia coli BL21(DE3). CpMafK could combine to the promoters of CpGST1 and CpGST2 with high-affinity in vitro. Therefore, CpMafK could regulate the expression of detoxification.

Inducible glutathione S-transferase (IrGST1) from the tick Ixodes ricinus is a haem-binding protein.

Blood-feeding parasites are inadvertently exposed to high doses of potentially cytotoxic haem liberated upon host blood digestion. Detoxification of free haem is a special challenge for ticks, which digest haemoglobin intracellularly. Ticks lack a haem catabolic mechanism, mediated by haem oxygenase, and need to dispose of vast majority of acquired haem via its accumulation in haemosomes. The knowledge of individual molecules involved in the maintenance of haem homeostasis in ticks is still rather limited. RNA-seq analyses of the Ixodes ricinus midguts from blood- and serum-fed females identified an abundant transcript of glutathione S-transferase (gst) to be substantially up-regulated in the presence of red blood cells in the diet. Here, we have determined the full sequence of this encoding gene, ir-gst1, and found that it is homologous to the delta-/epsilon-class of GSTs. Phylogenetic analyses across related chelicerates revealed that only one clear IrGST1 orthologue could be found in each available transcriptome from hard and soft ticks. These orthologues create a well-supported clade clearly separated from other ticks' or mites' delta-/epsilon-class GSTs and most likely evolved as an adaptation to tick blood-feeding life style. We have confirmed that IrGST1 expression is induced by dietary haem(oglobin), and not by iron or other components of host blood. Kinetic properties of recombinant IrGST1 were evaluated by model and natural GST substrates. The enzyme was also shown to bind haemin in vitro as evidenced by inhibition assay, VIS spectrophotometry, gel filtration, and affinity chromatography. In the native state, IrGST1 forms a dimer which further polymerises upon binding of excessive amount of haemin molecules. Due to susceptibility of ticks to haem as a signalling molecule, we speculate that the expression of IrGST1 in tick midgut functions as intracellular buffer of labile haem pool to ameliorate its cytotoxic effects upon haemoglobin intracellular hydrolysis.

Genome-wide profiling of expression and biochemical functions of the Medicago glutathione S-transferase gene family.

Glutathione S-transferases are ubiquitous enzyme in plants, playing vital roles in several physiological and developmental processes. In this study we identified 73 GST genes from the genome of Medicago truncatula. The Medicago GSTs were divided to eight classes with tau and phi being the most numerous. Six clusters were found on four Medicago chromosomes. The local gene duplication mainly contributed to the expansion of this large gene family. Functional divergence was found in their gene structures, gene expression patterns, and enzyme properties. A genomic comparative analysis revealed lineage-specific loss/gain events between Medicago and Glycine. This study offered new insights into the evolution of gene family between closely related species.

Melatonin Improves Heat Tolerance in Kiwifruit Seedlings through Promoting Antioxidant Enzymatic Activity and Glutathione S-Transferase Transcription.

Evidence exists to suggest that melatonin (MT) is important to abiotic stress tolerance in plants. Here, we investigated whether exogenous MT reduces heat damage on biological parameters and gene expression in kiwifruit (Actinidia deliciosa) seedlings. Pretreatment with MT alleviates heat-induced oxidative harm through reducing H2O2 content and increasing proline content. Moreover, MT application raised ascorbic acid (AsA) levels and the activity of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD). We also observed elevation in the activity of enzymes related to the AsA-GSH cycle, such as ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GR). Furthermore, MT application increased the expression of 28/31 glutathione S-transferase (GST) genes, reducing oxidative stress. These results clearly indicate that in kiwifruit, MT exerts a protective effect against heat-related damage through regulating antioxidant pathways.

Identification and characterization of two distinct sigma-class glutathione-S-transferase from freshwater bivalve Cristaria plicata.

Glutathione-S-transferases (GSTs) are multifunctional phase II detoxification enzymes that catalyze the attachment of electrophilic substrates to glutathione, and play an important role in protecting organisms against the toxicity of reactive oxygen species. In this study, two distinct sigma-class GST (CpGSTσ1 and 2) cDNA sequences were cloned from freshwater bivalve Cristaria plicata. The full length cDNA of CpGSTσ1 and 2 was 826 bp and 1609 bp, which encoded 213 and 248 amino acid residues, respectively. Their transcripts were expressed in all detected tissues and the highest expression level was in hepatopancreas from C. plicata. The expression level of CpGSTσ1 and 2 in hepatopancreas and hemocytes showed a significantly increased trend after bacterial challenge. The recombinant CpGSTσ1 was successfully expressed as a soluble form in Escherichia coli DE3. The specific activity of recombinase toward CDNB was 46.965 ±â€¯0.082 µmol/min/mg, and its optimum temperature and pH was 37 °C and 9.0, respectively. The recombinant of CpGSTσ1 could bear 6 M urea and 8% SDS, when the concentration of urea was 8 M and its activity was only below 20%. The results might provide a better perspective on the mechanisms of resistance to bacterial infection in molluscs.

Characterization and expression analysis of a newly identified glutathione S-transferase of the hard tick Haemaphysalis longicornis during blood-feeding.

BACKGROUND: Ticks are obligate hematophagous parasites important economically and to health. Ticks consume large amounts of blood for their survival and reproduction; however, large amounts of iron in blood could lead to oxidative stress. Ticks use several molecules such as glutathione S-transferases (GSTs), ferritins, and peroxiredoxins to cope with oxidative stress. This study aimed to identify and characterize the GSTs of the hard tick Haemaphysalis longicornis in order to determine if they have a role in coping with oxidative stress. METHODS: Genes encoding GSTs of H. longicornis were isolated from the midgut CDNA library. Genes have been cloned and recombinant GSTs have been expressed. The enzymatic activities, enzyme kinetic constants, and optimal pH of the recombinant GSTs toward 1-chloro-2,4-dinitrobenzene (CDNB) were determined. The gene transcription and protein expression profiles were determined in the whole ticks and internal organs, and developmental stages using real time RT-PCR and Western blotting during blood feeding. The localization of GST proteins in organs was also observed using immunofluorescent antibody test (IFAT). RESULTS: We have isolated two genes encoding GSTs (HlGST and HlGST2). The enzymatic activity toward CDNB is 9.75 ± 3.04 units/mg protein for recombinant HlGST and 11.63 ± 4.08 units/mg protein for recombinant HlGST2. Kinetic analysis of recombinant HlGST showed K m values of 0.82 ± 0.14 mM and 0.64 ± 0.32 mM for the function of CDNB and GSH, respectively. Meanwhile, recombinant HlGST2 has K m values of 0.61 ± 0.20 mM and 0.53 ± 0.02 mM for the function of CDNB and GSH, respectively. The optimum pH of recombinant HlGST and recombinant HlGST2 activity was 7.5-8.0. Transcription of both GSTs increases in different developmental stages and organs during blood-feeding. GST proteins are upregulated during blood-feeding but decreased upon engorgement in whole ticks and in some organs, such as the midgut and hemocytes. Interestingly, salivary glands, ovaries, and fat bodies showed decreasing protein expression during blood-feeding to engorgement. Varying localization of GSTs in the midgut, salivary glands, fat bodies, ovaries, and hemocytes was observed depending on the feeding state, especially in the midgut and salivary glands. CONCLUSIONS: In summary, a novel GST of H. longicornis has been identified. Characterization of the GSTs showed that GSTs have positive correlation with the degree and localization of oxidative stress during blood-feeding. This could indicate their protective role during oxidative stress.

Role of induced glutathione-S-transferase from Helicoverpa armigera (Lepidoptera: Noctuidae) HaGST-8 in detoxification of pesticides.

The present study deals with glutathione-S-transferase (GST) based detoxification of pesticides in Helicoverpa armigera and its potential application in eliminating pesticides from the environment. Dietary exposure of a pesticide mixture (organophosphates - chlorpyrifos and dichlorvos, pyrethroid - cypermethrin; 2-15ppm each) to H. armigera larvae resulted in a dose dependant up-regulation of GST activity and gene expression. A variant GST from H. armigera (HaGST-8) was isolated from larvae fed with 10ppm pesticide mixture and it was recombinantly expressed in yeast (Pichia pastoris HaGST-8). HaGST-8 had a molecular mass of 29kDa and was most active at pH 9 at 30°C. GC-MS and LC-HRMS analysis validated that HaGST-8 was effective in eliminating organophosphate type of pesticides and partially reduced the cypermethrin content (53%) from aqueous solutions. Unlike the untransformed yeast, P. pastoris HaGST-8 grew efficiently in media supplemented with pesticide mixtures (200 and 400ppm each pesticide) signifying the detoxification ability of HaGST-8. The amino acid sequence of HaGST-8 and the already reported sequence of HaGST-7 had just 2 mismatches. The studies on molecular interaction strengths revealed that HaGST-8 had stronger binding affinities with organophosphate, pyrethroid, organochloride, carbamate and neonicotinoid type of pesticides. The abilities of recombinant HaGST-8 to eliminate pesticides and P. pastoris HaGST-8 to grow profusely in the presence of high level of pesticide content can be applied for removal of such residues from food, water resources and bioremediation.

GSTA1 Expression Is Correlated With Aldosterone Level in KCNJ5-Mutated Adrenal Aldosterone-Producing Adenoma.

Context: KCNJ5 mutation is a major cause of aldosterone-producing adenomas (APAs). The development of APA apart from KCNJ5 mutation is less investigated. Objective: To investigate other mechanisms affecting aldosterone secretion apart from KCNJ5. Patients and Methods: Six pairs of KCNJ5-mutated, high and low aldosterone-secreting APAs, five non-KCNJ5-mutated APAs, and four normal adrenal glands were assayed by Affymetrix GeneChip Human Transcriptome Array 2.0. A total of 113 APA samples were investigated to explore the expression of glutathione-S-transferase A1 (GSTA1). H295R cells were used to verify the function of GSTA1. Results: GSTA1 was the top gene downregulated in high-aldosterone KCNJ5-mutated APAs. GSTA1 was also downregulated in KCNJ5-mutated APAs compared with wild-type KCNJ5 APAs. Accordingly, mutant KCNJ5 decreased GSTA1 messenger RNA and protein expression levels. GSTA1 overexpression suppressed aldosterone secretion whether in wild-type or mutant KCNJ5 H295R cells. Adding ethacrynic acid or silencing of GSTA1 increased aldosterone secretion by increasing reactive oxygen species (ROS), superoxide, H2O2 levels, and Ca2+ influx. The expression of the transcription factors NR4A1, NR4A2, and CAMK1 and intracellular Ca2+ were significantly upregulated by GSTA1 inhibition. The reduced form of NAD phosphate oxidase inhibitor or H2O2 scavenger or blocking calmodulin or calcium channels could significantly reduce aldosterone secretion in GSTA1-inhibited cells. Conclusions: (1) GSTA1 expression is reversely correlated with aldosterone level in KCNJ5-mutated APAs, (2) GSTA1 regulates aldosterone secretion by ROS and Ca2+ signaling, and (3) KCNJ5 mutation downregulates GSTA1 expression, and overexpression of GSTA1 reverses increased aldosterone in KCNJ5-mutated adrenal cells.

Association of Impaired Reactive Aldehyde Metabolism with Delayed Graft Function in Human Kidney Transplantation.

Delayed graft function is an early complication following kidney transplantation with an unclear molecular mechanism. Here we determined whether impaired reactive aldehyde metabolism is associated with delayed graft function. Human kidney biopsies from grafts with delayed graft function were compared with grafts that did not develop delayed graft function by Ingenuity gene pathway analysis. A second series of grafts with delayed graft function (n = 10) were compared to grafts that did not develop delayed graft function (n = 10) by measuring reactive aldehyde metabolism, reactive aldehyde-induced protein adduct formation, and aldehyde dehydrogenase (ALDH) gene and protein expression. In the first series of kidney biopsies, several gene families known for metabolizing reactive aldehydes, such as aldehyde dehydrogenase (ALDH), aldo-keto reductase (AKR), and glutathione-S transferase (GSTA), were upregulated in kidneys that did not develop delayed graft function versus those that did. In the second series of kidney grafts, we focused on measuring aldehyde-induced protein adducts and ALDH enzymatic activity. The reactive aldehyde metabolism by ALDH enzymes was reduced in kidneys with delayed graft function compared to those that did not (37 ± 12∗ vs. 79 ± 5 µg/min/mg tissue, ∗ P < 0.005, respectively). ALDH enzymatic activity was also negatively correlated with length of hospital stay after a kidney transplant. Together, our study identifies a reduced ALDH enzymatic activity with kidneys developing delayed graft function compared to those that did not. Measuring ALDH enzymatic activity and reactive aldehyde-induced protein adducts can potentially be further developed as a biomarker to assess for delayed graft function and recovery from a kidney transplant.

A 6-bp Deletion Variant in a Novel Canine Glutathione-S-Transferase Gene (GSTT5) Leads to Loss of Enzyme Function.

OBJECTIVES: Glutathione-S-transferases (GSTs) detoxify reactive xenobiotics, and defective GST gene polymorphisms increase cancer risk in humans. A low activity GST-theta variant was previously found in research beagles. The purpose of our study was to determine the molecular basis for this phenotype and its allele frequency in pet dogs. METHODS: Banked livers from 45 dogs of various breeds were screened for low GST-theta activity by the substrate 1,2-dichloro-4-nitrobenzene (DCNB), and were genotyped for variants in a novel canine GST gene, GSTT5. Whole-genome sequences from 266 dogs were genotyped at one discovered variant GSTT5 locus. RESULTS: Canine livers ranged 190-fold in GST-theta activities, and a GSTT5 exon coding variant 385_390delGACCAG (Asp129_Gln130del) was significantly associated with low activity (P < 0.0001) and a marked decrease in hepatic protein expression (P = 0.0026). Recombinant expression of variant GSTT5 led to a 92% decrease in Vmax for DCNB (P = 0.0095). The minor allele frequency (MAF) for 385_390delGACCAG was 0.144 in 45 dog livers, but was significantly higher in beagles (0.444) versus nonbeagles (0.007; P = 0.0004). The homozygous genotype was significantly over-represented in Pembroke Welsh corgis (P < 0.0001) based on available whole-genome sequence data. CONCLUSIONS: An Asp129_Gln130del variant in canine GSTT5 is responsible for marked loss of GST-theta enzyme activity. This variant is significantly over-represented in purpose-bred laboratory beagles and in Pembroke Welsh corgis. Additional work will determine the prevalence of this variant among other purebred dogs, and will establish the substrate range of this polymorphic canine enzyme with respect to common environmental carcinogens.

Characterization of xenobiotic metabolizing enzymes of a reconstructed human epidermal model from adult hair follicles.

In this study, a comprehensive characterization of xenobiotic metabolizing enzymes (XMEs) based on gene expression and enzyme functionality was made in a reconstructed skin epidermal model derived from the outer root sheath (ORS) of hair follicles (ORS-RHE). The ORS-RHE model XME gene profile was consistent with native human skin. Cytochromes P450 (CYPs) consistently reported to be detected in native human skin were also present at the gene level in the ORS-RHE model. The highest Phase I XME gene expression levels were observed for alcohol/aldehyde dehydrogenases and (carboxyl) esterases. The model was responsive to the CYP inducers, 3-methylcholanthrene (3-MC) and ß-naphthoflavone (ßNF) after topical and systemic applications, evident at the gene and enzyme activity level. Phase II XME levels were generally higher than those of Phase I XMEs, the highest levels were GSTs and transferases, including NAT1. The presence of functional CYPs, UGTs and SULTs was confirmed by incubating the models with 7-ethoxycoumarin, testosterone, benzo(a)pyrene and 3-MC, all of which were rapidly metabolized within 24h after topical application. The extent of metabolism was dependent on saturable and non-saturable metabolism by the XMEs and on the residence time within the model. In conclusion, the ORS-RHE model expresses a number of Phase I and II XMEs, some of which may be induced by AhR ligands. Functional XME activities were also demonstrated using systemic or topical application routes, supporting their use in cutaneous metabolism studies. Such a reproducible model will be of interest when evaluating the cutaneous metabolism and potential toxicity of innovative dermo-cosmetic ingredients.

Human cytosolic glutathione-S-transferases: quantitative analysis of expression, comparative analysis of structures and inhibition strategies of isozymes involved in drug resistance.

Glutathione-S-transferase (GST) inhibition is a strategy to overcome drug resistance. Several isoforms of human GSTs are present and they are expressed in almost all the organs. Specific expression levels of GSTs in various organs are collected from the human transcriptome data and analysis of the organ-specific expression of GST isoforms is carried out. The variations in the level of expressions of GST isoforms are statistically significant. The GST expression differs in diseased conditions as reported by many investigators and some of the isoforms of GSTs are disease markers or drug targets. Structure analysis of various isoforms is carried out and literature mining has been performed to identify the differences in the active sites of the GSTs. The xenobiotic binding H site is classified into H1, H2, and H3 and the differences in the amino acid composition, the hydrophobicity and other structural features of H site of GSTs are discussed. The existing inhibition strategies are compared. The advent of rational drug design, mechanism-based inhibition strategies, availability of high-throughput screening, target specific, and selective inhibition of GST isoforms involved in drug resistance could be achieved for the reversal of drug resistance and aid in the treatment of diseases.