Hepatocyte glutathione S-transferase mu 2 prevents non-alcoholic steatohepatitis by suppressing ASK1 signaling.

BACKGROUND & AIMS: Non-alcoholic fatty liver disease (NAFLD) has become the most common chronic liver disease worldwide. The advanced stage of NAFLD, non-alcoholic steatohepatitis (NASH), has been recognized as a leading cause of end-stage liver injury for which there are no FDA-approved therapeutic options. Glutathione S-transferase Mu 2 (GSTM2) is a phase II detoxification enzyme. However, the roles of GSTM2 in NASH have not been elucidated. METHODS: Multiple RNA-seq analyses were used to identify hepatic GSTM2 expression in NASH. In vitro and in vivo gain- or loss-of-function approaches were used to investigate the role and molecular mechanism of GSTM2 in NASH. RESULTS: We identified GSTM2 as a sensitive responder and effective suppressor of NASH progression. GSTM2 was significantly downregulated during NASH progression. Hepatocyte GSTM2 deficiency markedly aggravated insulin resistance, hepatic steatosis, inflammation and fibrosis induced by a high-fat diet and a high-fat/high-cholesterol diet. Mechanistically, GSTM2 sustained MAPK pathway signaling by directly interacting with apoptosis signal-regulating kinase 1 (ASK1). GSTM2 directly bound to the N-terminal region of ASK1 and inhibited ASK1 N-terminal dimerization to subsequently repress ASK1 phosphorylation and the activation of its downstream JNK/p38 signaling pathway under conditions of metabolic dysfunction. CONCLUSIONS: These data demonstrated that hepatocyte GSTM2 is an endogenous suppressor that protects against NASH progression by blocking ASK1 N-terminal dimerization and phosphorylation. Activating GSTM2 holds promise as a therapeutic strategy for NASH. CLINICAL TRIAL NUMBER: IIT-2021-277. LAY SUMMARY: New therapeutic strategies for non-alcoholic steatohepatitis are urgently needed. We identified that the protein GSTM2 exerts a protective effect in response to metabolic stress. Therapies that aim to increase the activity of GSTM2 could hold promise for the treatment of non-alcoholic steatohepatitis.

Diselenide-derivative of 3-pyridinol targets redox enzymes leading to cell cycle deregulation and apoptosis in A549 cells.

The aim of present study was to understand the mechanism of action of 2,2'-diselenobis(3-pyridinol) or DISPOL in human lung cancer (A549) cells. A549 cells were treated with 10 µM (∼IC50) of DISPOL for varying time points to corelate the intracellular redox changes with its cytotoxic effect. The results indicated that DISPOL treatment led to a time dependant decrease in the basal level of reactive oxygen species (ROS). Additionally, DISPOL treatment elevated the ratio of reduced (GSH) and oxidised (GSSG) glutathione by upregulating gamma-glutamylcysteine ligase (γ-GCL) involved in GSH biosynthesis and inhibiting the activities of redox enzymes responsible for GSH utilization and recycling, such as glutathione-S-transferase (GST) and glutathione reductase (GR). Molecular docking analysis suggests putative interactions of DISPOL with GST and GR which could account for its inhibitory effect on these enzymes. Further, DISPOL induced reductive environment preceded G1 arrest and apoptosis as evidenced by decreased expression of cell cycle genes (Cyclin D1 and Cyclin E1) and elevation of p21 and apoptotic markers (cleaved caspase 3 and cleaved PARP). The combinatorial experiments involving DISPOL and redox modulatory agents such as N-acetylcysteine (NAC) and buthionine sulfoximine (BSO) indeed confirmed the role of reductive stress in DISPOL-induced cell death. Finally, Lipinski's rule suggests attributes of drug likeness in DISPOL. Taken together, DISPOL exhibits a novel mechanism of reductive stress-mediated cell death in A549 cells that warrants future exploration as anticancer agent.

Protective effect of the Spirulina platensis against toxicity induced by Diuron exposure in Mytilus galloprovincialis.

Diuron herbicide is widely used for weeds control in many kinds of cultivations. It reaches the waterbodies through various fate routes and can adversely threaten non-target organism. The current study was carried out to evaluate the antioxidant activity of Spirulina as feed additive against the toxicity of Diuron concentrations (40 and 80 µg/L) on the edible mollusk Mytilus galloprovincialis during seven days of exposure. Oxidative stress biomarkers were applied on mussel gills and digestive gland, investigating changes in enzymes activities such as catalase (CAT), Glutathione-S-transferase (GST) and Acetylcholinesterase (AChE) and the Malondialdehyde level (MDA). The obtained results show that diuron altered oxidative stress biomarkers in both organs, gills and digestive gland. Performed principle component analysis (PCA) highlighted relationship between biomarkers involved in functional response. Spirulina platensis supplemented diet (1 mg/L), completely ameliorated diuron-induced oxidative stress in mussel tissues. Thus, Spirulina seems to be a promising microalgae and eco-friendly tool helping the health recovery of aquatic animals subjected to environmental stressors.

This study provided recent and new data on the impact of Diuron in marine bivalve and the protective effect of Spirulina against Diuron-induced oxidative stress. The results of our study suggest that the antioxidant potential of Spirulina should be strongly candidate for the phytoremediation of Diuron-aquatic contaminated.

Cold-Adapted Glutathione S-Transferases from Antarctic Psychrophilic Bacterium Halomonas sp. ANT108: Heterologous Expression, Characterization, and Oxidative Resistance.

Glutathione S-transferases are one of the most important antioxidant enzymes to protect against oxidative damage induced by reactive oxygen species. In this study, a novel gst gene, designated as hsgst, was derived from Antarctic sea ice bacterium Halomonas sp. ANT108 and expressed in Escherichia coli (E. coli) BL21. The hsgst gene was 603 bp in length and encoded a protein of 200 amino acids. Compared with the mesophilic EcGST, homology modeling indicated HsGST had some structural characteristics of cold-adapted enzymes, such as higher frequency of glycine residues, lower frequency of proline and arginine residues, and reduced electrostatic interactions, which might be in relation to the high catalytic efficiency at low temperature. The recombinant HsGST (rHsGST) was purified to apparent homogeneity with Ni-affinity chromatography and its biochemical properties were investigated. The specific activity of the purified rHsGST was 254.20 nmol/min/mg. The optimum temperature and pH of enzyme were 25 °C and 7.5, respectively. Most importantly, rHsGST retained 41.67% of its maximal activity at 0 °C. 2.0 M NaCl and 0.2% H2O2 had no effect on the enzyme activity. Moreover, rHsGST exhibited its protective effects against oxidative stresses in E. coli cells. Due to its high catalytic efficiency and oxidative resistance at low temperature, rHsGST may be a potential candidate as antioxidant in low temperature health foods.

Leukotriene E4 elicits respiratory epithelial cell mucin release through the G-protein-coupled receptor, GPR99.

Cysteinyl leukotrienes (cysLTs), leukotriene C4 (LTC4), LTD4, and LTE4 are proinflammatory lipid mediators with pathobiologic function in asthma. LTE4, the stable cysLT, is a weak agonist for the type 1 and type 2 cysLT receptors (CysLTRs), which constrict airway smooth muscle, but elicits airflow obstruction and pulmonary inflammation in patients with asthma. We recently identified GPR99 as a high-affinity receptor for LTE4 that mediates cutaneous vascular permeability. Here we demonstrate that a single intranasal exposure to extract from the respiratory pathogen Alternaria alternata elicits profound epithelial cell (EpC) mucin release and submucosal swelling in the nasal mucosa of mice that depends on cysLTs, as it is absent in mice deficient in the terminal enzyme for cysLT biosynthesis, LTC4 synthase (LTC4S). These mucosal changes are associated with mast cell (MC) activation and absent in MC-deficient mice, suggesting a role for MCs in control of EpC function. Of the three CysLTRs, only GPR99-deficient mice are fully protected from EpC mucin release and swelling elicited by Alternaria or by intranasal LTE4 GPR99 expression is detected on lung and nasal EpCs, which release mucin to doses of LTE4 one log lower than that required to elicit submucosal swelling. Finally, mice deficient in MCs, LTC4S, or GPR99 have reduced baseline numbers of goblet cells, indicating an additional function in regulating EpC homeostasis. These results demonstrate a novel role for GPR99 among CysLTRs in control of respiratory EpC function and suggest that inhibition of LTE4 and of GPR99 may have therapeutic benefits in asthma.

The silkworm GSTe4 is sensitive to phoxim and protects HEK293 cells against UV-induced cell apoptosis.

Glutathione S-transferases (GSTs, EC 2.5.1.18) are a family of super enzymes with multiple functions that play a major role in the detoxification of endogenous and xenobiotic compounds. In our previous study, we have predicted 23 putative cytosolic GSTs in the silkworm genome using bioinformatic methods. In this study, we cloned and studied the insect-specific epsilon-class GST gene GSTe4 from the silkworm, Bombyx mori. The recombinant BmGSTe4 (Bac-BmGSTe4) was overexpressed in SF-9 cell lines, and it was found to have effective GST activity. We also found that the expression of BmGSTe4 was especially down-regulated after the silkworms were fumigated with or ingested phoxim. Moreover, BmGSTe4 protected HEK293 cells against UV-induced cell apoptosis. These results demonstrated that BmGSTe4 has GST activity, is sensitive to phoxim, and plays a role in inhibition of UV-induced cell apoptosis.

Activated α2-macroglobulin induces Müller glial cell migration by regulating MT1-MMP activity through LRP1.

In retinal proliferative diseases, Müller glial cells (MGCs) acquire migratory abilities. However, the mechanisms that regulate this migration remain poorly understood. In addition, proliferative disorders associated with enhanced activities of matrix metalloprotease 2 (MMP-2) and MMP-9 also present increased levels of the protease inhibitor α2-macroglobulin (α2M) and its receptor, the low-density lipoprotein receptor-related protein 1 (LRP1). In the present work, we investigated whether the protease activated form of α2M, α2M*, and LRP1 are involved with the MGC migratory process. By performing wound-scratch migration and zymography assays, we demonstrated that α2M* induced cell migration and proMMP-2 activation in the human Müller glial cell line, MIO-M1. This induction was blocked when LRP1 and MT1-MMP were knocked down with siRNA techniques. Using fluorescence microscopy and biochemical procedures, we found that α2M* induced an increase in LRP1 and MT1-MMP accumulation in early endosomes, followed by endocytic recycling and intracellular distribution of MT1-MMP toward cellular protrusions. Moreover, Rab11-dominant negative mutant abrogated MT1-MMP recycling pathway, cell migration, and proMMP-2 activation induced by α2M*. In conclusion, α2M*, through its receptor LRP1, induces cellular migration of Müller glial cells by a mechanism that involves MT1-MMP intracellular traffic to the plasma membrane by a Rab11-dependent recycling pathway.

A study to assess the health effects of an anticancer drug (cyclophosphamide) in zebrafish (Danio rerio): eco-toxicity of emerging contaminants.

Cyclophosphamide (CP) is widely used for treating various kinds of cancer. Because of its high intake, metabolism and excretion, these anticancer medications have been detected in the aquatic environment. There is very limited data on the toxicity and effects of CP on aquatic organisms. The present study aims to assess the toxic effect of CP on certain oxidative stress biomarkers (superoxide dismutase-SOD, catalase-CAT, glutathione peroxidase-GPx, glutathione-GSH, glutathione S-transferases-GST and lipid peroxidation-LPO), protein, glucose, metabolising enzymes (aspartate aminotransferase-AST, alanine aminotransferase-ALT), and ion-regulatory markers (sodium ions-Na+, potassium ions-K+, and chloride ions-Cl-), and histology in the gills and liver of Danio rerio at environmentally relevant concentrations (10, 100 and 1000 ng L-1). Exposure to CP for 42 days led to a significant decrease in SOD, CAT, GST, GPx and GSH levels in the gills and liver tissues of zebrafish. The level of lipid peroxidation in the gills and liver tissues of zebrafish was significantly increased compared to the control group. Chronic exposure significantly changes protein, glucose, AST, ALT, Na+, K+ and Cl- biomarkers. Fish exposed to different levels of CP showed necrosis, inflammation, degeneration and hemorrhage in the gills and hepatic tissues. The observed changes in the studied tissue biomarkers were proportional to both dose and time. In conclusion, CP at environmentally relevant concentrations causes oxidative stress, energy demand, homeostasis disturbances, and enzyme and histological alterations in the vital tissues of zebrafish. These alterations were similar to the toxic effects reported in mammalian models.

Recombinant human beta 2-defensin fusion proteins as a tool to investigate defensin structure and function in small human intestinal tissue samples.

OBJECTIVE: Effects of immune cells on the beta 2 (ß2)-defensin (HBD2) expression and its antibacterial activity in the intestinal mucosa of patients with inflammatory bowel diseases remains unclear. The small size of these proteins presents a major challenge in localizing antibacterial activities in human intestinal tissue. In this study, we evaluated the detection limits at mRNA and protein level by approaching HBD2 from small tissue samples. METHODS: HT-29 colonic epithelial cells were incubated with proinflammatory cytokines before HBD2 mRNA was investigated by quantitative polymerase chain reaction. The HBD2 protein was assessed by Western blot analysis using HBD2 fused with enhanced green fluorescent protein (HBD2-EGFP). Purified HBD2 fused with the glutathione-S-transferase (GST-HBD2) was used to detect antibacterial activity in a densitometric assay. RESULTS: Interleukin (IL)-1ß induced HBD2 mRNA in HT-29 cells; however, tumor necrosis factor-α, IL-6 and IL-17 did not. The Western blot had a sensitivity of 1.5 pmol to detect recombinant HBD2, but did not detect HBD2 in either human intestinal or IL-1ß-treated HT-29 cells. HBD2-EGFP was detected by HBD2-specific Western blot within cell lysates and culture supernants of transfected HT-29 and primary cells. In nanomolar ranges, GST-HBD2 reduced bacterial growth. The HBD2 bioactivity depended on solution conditions, but not on the size of the fusion partner. CONCLUSION: The established fusion proteins provide excellent tools to evaluate expression patterns and antibacterial effects of HBD2 in human intestinal tissue samples.

Maternal safflower oil consumption improve reflex maturation, memory and reduces hippocampal oxidative stress in the offspring rats treated during pregnancy and lactation.

OBJECTIVE: Evaluate the influence of maternal consumption of safflower oil on reflex maturation, memory and offspring hippocampal oxidative stress. METHODOLOGY: Two groups were formed: control group (C), whose mothers received a standard diet, and Safflower group (SF), whose mothers received a normolipidic diet with safflower oil as lipid source. Treatment was given from the 14th day of gestation and throughout lactation. To evaluate newborn development, the reflex ontogeny indicators between the 1st and the 21st days of life were evaluated; to assess memory, from the 42nd day of life on these animals were examined on open field habituation and novel object recognition test. Following behavioral analysis, the animals were anesthetized and decapitated. Hippocampus was rapidly dissected. In the hippocampal tissues, we evaluated the levels of malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), glutathione S transferase (GST) and reduced glutathione (GSH). RESULTS: SF offspring showed delayed maturation of reflexes and improvement of novel object recognition in short-term and long-term (p < 0.05). Safflower oil decreases lipid peroxidation evaluated by MDA levels (p < 0.001) and increases antioxidant defenses as shown by SOD, CAT, GST and GSH levels (p < 0.05). In our study, the composition of flavonoids present in the oil was not evaluated. Furthermore, in a future study, the effect of maternal consumption on female offspring should be verified. CONCLUSION: Maternal intake of safflower oil could: (1) change neonate reflex parameters, (2) promote improvement of cognitive development in adolescence (3) improve antioxidant enzymatic and non-enzymatic defenses in the hippocampus.

Curzerene suppresses progression of human glioblastoma through inhibition of glutathione S-transferase A4.

AIMS: Glioblastoma is the central nervous system tumor with the highest mortality rate, and the clinical effectiveness of chemotherapy is low. Curzerene can inhibit the progression of non-small-cell lung cancer, but its role in glioma has not been reported. The purpose of this study was to clarify the effect of curzerene on glioma progression and further explore its potential mechanism. METHODS: The expression of glutathione S-transferase A4 (GSTA4) in glioblastoma and the effect of curzerene on the expression of GSTA4 and matrix metalloproteinase 9 and the activation of the mTOR pathway were detected by Western blotting and RT-PCR, and the effects of curzerene treatment on glioma malignant character were detected by cell biological assays. The in vivo antitumor effects of curzerene were analyzed in a nude mouse xenograft model. RESULTS: Curzerene was found to inhibit the expression of GSTA4 mRNA and protein in U251 and U87 glioma cells, and this effect correlated with a downregulation of the proliferation of these cells in a time- and dose-dependent manner. Invasion and migration were also inhibited, and curzerene treatment correlated with induction of apoptosis. Curzerene inhibited the activation of the mTOR pathway and the expression of matrix metalloproteinase 9, and it correlated with increased 4-hydroxynonenal levels. In vivo, curzerene was found to significantly inhibit tumor growth in nude mice and to prolong the survival time of tumor-bearing nude mice. CONCLUSION: In conclusion, inhibition of GSTA4 correlates with positive outcomes in glioma models, and thus, this molecule is a candidate drug for the treatment of glioma.

[Animal models for bone and joint disease. RANKL-injected bone loss model].

Discovery of the osteoclast differentiation factor RANKL (receptor activator of NF-κB ligand) has opened a new era in the understanding of mechanisms in osteoclast differentiation over the last decade. The discovery results in the development of a fully human anti-RANKL neutralizing monoclonal antibody (called denosumab) and denosumab has been approved for the treatment of osteoporosis in Europe and the US. Here I review a novel rapid bone loss model with GST (glutathione-S-transferase) -RANKL. Pharmacologic studies of candidates for the treatment of osteoporosis with this model can be done in short periods such as 3 days and a couple of weeks although it took several months in the conventional methods with ovariectomized (OVX) -rats. This model also is useful for the rapid analyses in the functions of osteoclasts in vivo . I am expecting the RANKL-induced bone loss model to be the gold standard in the animal osteoporosis model as well as OVX-rats in the near future.

Glutathione-S-transferase A3 protein suppresses thiram-induced tibial dyschondroplasia by regulating prostaglandin-related genes expression.

Tibial dyschondroplasia (TD) is an intractable avian cartilage disease in which proximal growth plates of tibia lack blood vessels and contain nonviable cells, and it leads to the inflammatory response. Prostaglandins (PGs) genes have not been studied yet in TD chicken, and they might play role in skeletal metabolism, therefore we planned to explore the role of recombinant glutathione-S-transferase A3 (rGSTA3) protein and PG-related genes. In this study, qRT-PCR, enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry (IHC) analysis were used to identify the expression patterns of eight PG-related genes in the tibial growth plate of broiler chicken. The results showed that the expression of PG-related genes glutathione-S-transferase A3 (GSTA3), cyclooxygenase 2 (COX-2), prostaglandin D2 synthase (PTGDS), prostaglandin E synthase (PTGES), prostaglandin E2 receptor (PTGER) 3, PTGER4, prostaglandin reductase 1 (PTGR1) and hematopoietic prostaglandin D synthases (HPGDS) expression were identified and could significantly respond to thiram-induced TD chicken. Interestingly, the expression of rate-limiting enzyme COX-2 and PGE2 were induced after the treatment of rGSTA3 protein. These findings demonstrated that the occurrence of TD is closely related to the inhibition of PGs. Moreover, rGSTA3 protein participated in the recovery of TD by strengthening the expression of PG-related genes.

Deciphering of The Effect of Chemotherapeutic Agents on Human Glutathione S-Transferase Enzyme and MCF-7 Cell Line.

BACKGROUND: Cancer is the disease that causes the most death after cardiovascular diseases all over the world these days. Breast cancer is the most common type of cancer among women and ranks the second among cancer-related deaths after lung cancer. Chemotherapeutics act by killing cancer cells, preventing their spread and slowing their growth. Recent studies focus on the effects of chemotherapeutics on cancer cells and new chemotherapy approaches that targeting enzymes that catalyze important metabolic reactions in the cell. OBJECTIVE: The aim of this study was to investigate the effects of chemotherapeutic agents, Tamoxifen and 5-FU, on MCF-7 cell line and human erythrocyte GST, an important enzyme of intracellular antioxidant metabolism. METHODS: In this study, it was investigated that the effect of chemotherapeutic agents, Tamoxifen and 5-FU, on MCF-7 breast cancer cell line and performed ROS analyzes. In addition, it was purified glutathione S-transferase (GST), one of the important enzymes of intracellular antioxidant mechanism, from human erythrocytes by using ammonium sulfate precipitation and glutathione agarose affinity chromatography, and investigated in vitro effects of chemotherapeutic agents, 5 - FU and Tamoxifen, on the activity of this enzyme for the first time. RESULTS: it was determined that Tamoxifen and 5-FU inhibited cellular viability and 5-FU increased intracellular levels of ROS, whereas Tamoxifen reduced intracellular levels of ROS. In addition, human erythrocyte GST enzyme with 16.2 EU/mg specific activity was purified 265.97-fold with a yield of 35% using ammonium sulfate precipitation and glutathione agarose affinity chromatography. The purity of the enzyme was checked by the SDS-PAGE method. In vitro effects of chemotherapeutics, 5-FU and Tamoxifen, on GST activity purified from human erythrocytes were investigated. The results showed that 5-FU increased the activity of GST in the concentration range of 77 to 1155 µM and that Tamoxifen increased the activity of GST in the concentration range of 0.54 to 2.70 µM. CONCLUSION: In this study, the effects of tamoxifen and 5-FU chemotherapeutic agents on both MCF-7 cell line and human GST enzyme were examined together for the first time. Our study showed that chemotherapeutic agents (5-FU and Tamoxifen) inhibited cellular viability and Tamoxifen reduced intracellular levels of ROS whereas 5-FU increased intracellular levels of ROS. In addition, 5-FU and Tamoxifen were found to increase the activity of GST enzyme purified from the human erythrocyte.

Recombinant glutathione-S-transferase A3 protein regulates the angiogenesis-related genes of erythrocytes in thiram induced tibial lesions.

Tibial dyschondroplasia (TD) is a skeletal deformity disease in broilers that occurs when vascularization in the growth plate (GP) is below normal. Although, blood vessels have been reported to contribute significantly in bone formation. Therefore, in the current study, we have examined the mRNA expression of angiogenesis-related genes in erythrocytes of thiram induced TD chickens by qRT-PCR and performed histopathological analysis to determine regulatory effect of recombinant Glutathione-S-Transferase A3 (rGSTA3) protein in response to the destructive effect of thiram following the injection of rGSTA3 protein. Histopathology results suggested that, blood vessels of GPs were damaged in thiram induced TD chicken group (D), it also affected the area and density of blood vessels. In the 20 and 50 µg·kg-1 of rGSTA3 protein-administered groups, E and F vessels appeared to be normal and improved on day 6 and 15. Furthermore, qRT-PCR results showed that rGSTA3 protein significantly (P < .05) up-regulated the expression of the most important angiogenesis-related integrin family genes ITGA2, ITGA5, ITGB2, ITGB3, ITGAV. The expression level of other genes including TBXA2R, FYN, IQGAP2, IL1R1, GIT1, RAP1B, RPL17, RAC2, MAML3, PTPN11, VAV1, PTCH1, NCOR2, CLU and ITGB3 up-regulated on dosage of rGSTA3 protein. In conclusion, angiogenesis is destroyed in thiram induced TD broilers, and rGSTA3 protein injection improved the vascularization of GPs by upregulating the angiogenesis related genes most importantly integrin family genes ITGAV, ITGA2, ITGB2, ITGB3, ITGA5.

Genetically engineered yeasts as a new delivery vehicle of active compounds to the digestive tract: in vivo validation of the concept in the rat.

An innovative "biodrug" concept based on oral administration of living recombinant microorganisms as a vehicle to deliver active compounds directly into the digestive tract has recently been developed. To validate this concept, we studied a recombinant Saccharomyces cerevisiae strain in order to investigate its viability and its ability to produce a protein (glutathione-S-transferase (GST)-V(5)H(6)) in the rat. Following oral administration, the recombinant yeast showed a survival rate of around 40% in the upper parts of the digestive tract, but was more sensitive to the conditions in the large intestinal, where viability dropped to 1%. Western blot analysis was able to detect the model protein throughout the digestive tract, including stomach, duodenum, jejunum (proximal, median and distal), ileum, cecum and colon. The gastrointestinal sac technique was employed to quantify GST-V(5)H(6) in all the digestive compartments. These results suggest that S. cerevisiae may represent a useful host for producing compounds of interest directly in the digestive tract.

Calcium release at fertilization in starfish eggs is mediated by phospholipase Cgamma.

Although inositol trisphosphate (IP3) functions in releasing Ca2+ in eggs at fertilization, it is not known how fertilization activates the phospholipase C that produces IP3. To distinguish between a role for PLCgamma, which is activated when its two src homology-2 (SH2) domains bind to an activated tyrosine kinase, and PLCbeta, which is activated by a G protein, we injected starfish eggs with a PLCgamma SH2 domain fusion protein that inhibits activation of PLCgamma. In these eggs, Ca2+ release at fertilization was delayed, or with a high concentration of protein and a low concentration of sperm, completely inhibited. The PLCgammaSH2 protein is a specific inhibitor of PLCgamma in the egg, since it did not inhibit PLCbeta activation of Ca2+ release initiated by the serotonin 2c receptor, or activation of Ca2+ release by IP3 injection. Furthermore, injection of a PLCgamma SH2 domain protein mutated at its phosphotyrosine binding site, or the SH2 domains of another protein (the phosphatase SHP2), did not inhibit Ca2+ release at fertilization. These results indicate that during fertilization of starfish eggs, activation of phospholipase Cgamma by an SH2 domain-mediated process stimulates the production of IP3 that causes intracellular Ca2+ release.

Involvement of Rabphilin-3A in cortical granule exocytosis in mouse eggs.

Rabphilin-3A is a putative target protein for Rab3A, a member of the small GTP-binding protein superfamily that has been suggested to play a role in regulated exocytosis in presynapses. In this study we determined the expression and the function of Rabphilin-3A in mouse eggs at fertilization. Rabphilin-3A mRNA and protein were detected by reverse transcriptase-PCR and immunoblot analysis, respectively, in metaphase II mouse eggs. Immunofluorescence analysis showed that Rabphilin-3A protein was distributed in the cortical region in eggs. Sperm induces cortical granule (CG) exocytosis via an increase in cytosolic Ca2+ at fertilization. We microinjected the NH2- or COOH-terminal fragment of recombinant Rabphilin-3A into metaphase II eggs. Neither treatments altered the sperm-induced cytosolic Ca2+ increase, but both inhibited CG exocytosis in a dose-dependent manner. The NH2-terminal fragment was more effective than the COOH-terminal fragment. Full-length Rabphilin-3A did not affect CG exocytosis, but it attenuated the inhibition of CG exocytosis by the NH2-terminal fragment. These results show that Rabphilin-3A is involved in Ca(2+)-dependent CG exocytosis at fertilization in mouse eggs.

Regulation of GST-MDA-7 toxicity in human glioblastoma cells by ERBB1, ERK1/2, PI3K, and JNK1-3 pathway signaling.

The present studies defined the biological effects of a GST fusion protein of melanoma differentiation-associated gene-7 (mda-7), GST-MDA-7 (1 and 30 nmol/L), on cell survival and cell signaling in primary human glioma cells in vitro. GST-MDA-7, in a dose- and time-dependent fashion killed glioma cells with diverse genetic characteristics; 1 nmol/L caused arrest without death, whereas 30 nmol/L caused arrest and killing after exposure. Combined inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2) and AKT function was required to enhance 1 nmol/L GST-MDA-7 lethality in all cell types, whereas combined activation of MEK1 and AKT was required to suppress 30 nmol/L GST-MDA-7 lethality; both effects are mediated in part by modulating c-Jun NH(2)-terminal kinase (JNK) 1-3 activity. The geldanamycin 17AAG inhibited AKT and ERK1/2 in GBM cells and enhanced GST-MDA-7 lethality. JNK1-3 signaling promoted BAX activation and mitochondrial dysfunction. In GBM6 cells, GST-MDA-7 (30 nmol/L) transiently activated p38 mitogen-activated protein kinase, which was modestly protective against JNK1-3-induced toxicity, whereas GST-MDA-7 (300 nmol/L) caused prolonged intense p38 mitogen-activated protein kinase activation, which promoted cell death. In GBM12 cells that express full-length mutant activated ERBB1, inhibition of ERBB1 did not modify GST-MDA-7 lethality; however, in U118 established glioma cells, stable overexpression of wild-type ERBB1 and/or truncated active ERBB1vIII suppressed GST-MDA-7 lethality. Our data argue that combined inhibition of ERK1/2 and AKT function, regardless of genetic background, promotes MDA-7 lethality in human primary human glioma cells via JNK1-3 signaling and is likely to represent a more ubiquitous approach to enhancing MDA-7 toxicity in this cell type than inhibition of ERBB1 function.

The expression of prostaglandins-related genes in erythrocytes of broiler chicken responds to thiram-induced tibial dyschondroplasia and recombinant glutathione-S-transferase A3 protein.

Tibial dyschondroplasia (TD) is a type of bone deformity found in fast-growing chickens, which induce inflammatory responses. Prostaglandins (PGs) implicate in bone formation and bone resorption, associated with inflammation in an autocrine/paracrine manner. This study used qRT-PCR and immunohistochemistry analysis to identify the expression patterns of PG-related genes in the erythrocytes of broiler chickens and explore the effects of thiram-induced TD and the recombinant glutathione-S-transferase A3 (rGSTA3) protein on the expression of PG-related genes: GSTA3, cyclooxygenase 2 (COX-2), prostaglandin D2 synthase (PTGDS), prostaglandin E synthase (PTGES), prostaglandin E2 receptor (PTGER) 3, PTGER4 and prostaglandin reductase 1 (PTGR1). Interestingly, the results showed that these seven PG-related genes expression was identified in the erythrocytes of broiler chicken, and thiram-induced TD suppressed the expression of these PG-related genes in the initial stage of TD and promoted their expression in TD recovery. These findings demonstrated that the immunoregulatory function of erythrocytes can be inhibited in the early stage of TD and promoted in the recovery stage by modulating the expression of PG-related genes. Further, the rGSTA3 protein can modulate the expression of PG-related genes in erythrocytes and participate in the recovery of TD.