Identification and characterization of bioactive metabolites of 12-hydroxyheptadecatrienoic acid, a ligand for leukotriene B4 receptor 2.

12(S)-hydroxyheptadecatrienoic acid (12-HHT) is a bioactive fatty acid synthesized from arachidonic acid via the cyclooxygenase pathway and serves as an endogenous ligand for the low-affinity leukotriene B4 receptor 2 (BLT2). Although the 12-HHT/BLT2 axis contributes to the maintenance of epithelial homeostasis, 12-HHT metabolism under physiological conditions is unclear. In this study, 12-keto-heptadecatrienoic acid (12-KHT) and 10,11-dihydro-12-KHT (10,11dh-12-KHT) were detected as 12-HHT metabolites in the human megakaryocytic cell line MEG01s. We found that 12-KHT and 10,11dh-12-KHT are produced from 12-HHT by 15-hydroxyprostaglandin dehydrogenase (15-PGDH) and prostaglandin reductase 1 (PTGR1), key enzymes in the degradation of prostaglandins, respectively. The 15-PGDH inhibitor SW033291 completely suppressed the production of 12-KHT and 10,11dh-12-KHT in MEG01s cells, resulting in a 9-fold accumulation of 12-HHT. 12-KHT and 10,11dh-12-KHT were produced in mouse skin wounds, and the levels were significantly suppressed by SW033291. Surprisingly, the agonistic activities of 12-KHT and 10,11dh-12-KHT on BLT2 were comparable to that of 12-HHT. Taken together, 12-HHT is metabolized into 12-KHT by 15-PGDH, and then 10,11dh-12-KHT by PTGR1 without losing the agonistic activity.

Kolaviron alleviates haematological abnormalities and hepato-renal damage in Naja nigricollis nigricollis venom-treated rats.

Objectives: To investigate the effect of kolaviron against haematological abnormalities and hepato-renal damage in Naja n. nigricollis (NNN) venom-treated rats. Methods: Twenty-four male rats were grouped into four (n = 6). A single intravenous dose of NNN venom (≈½LD50) was given to group B-D (excluding A). All the groups were immediately treated intraperitoneally as follows: A (Normal control) and B (Envenom) received 0.40 mL/kg of 0.1% Tween 80, while C and D (test groups), received 200 and 400 mg/kg of kolaviron respectively. After 6 h, they were anaesthetized, and sacrificed. Results: NNN-venom LD50 was estimated at 1.14 mg/kg. Injected half LD50, after 6 h, caused significant (p < 0.05) decreases in RBC, HGB and PCV, with increases in WBC and NEUT. Significantly (p < 0.05) increased AST, ALT, GGT, TB, CRE, URE, UA and K with concomitant decreases in Na and HCO3. Oxidant/antioxidant markers (MDA, CAT and SOD) were significantly (p < 0.05) increased in liver and kidney homogenates. Histological analysis confirmed liver and kidney injuries. All these alterations were alleviated dose-dependently, when cotreated with kolaviron at 200 and 400 mg/kg. Conclusions: Our study suggests that kolaviron could alleviates haematological abnormalities and hepato-renal damage in NNN venom-treated rats by depleting ROS and/or boasting the antioxidant system.

Biochemistry and toxicology of proteins and peptides purified from the venom of Vipera berus berus.

The isolation and characterization of individual snake venom components is important for a deeper understanding of the pathophysiology of envenomation and for improving the therapeutic procedures of patients. It also opens possibilities for the discovery of novel toxins that might be useful as tools for understanding cellular and molecular processes. The variable venom composition, toxicological and immunological properties of the common vipers (Vipera berus berus) have been reviewed. The combination of venom gland transcriptomics, bottom-up and top-down proteomics enabled comparison of common viper venom proteomes from multiple individuals. V. b. berus venom contains proteins and peptides belonging to 10-15 toxin families: snake venom metalloproteinase, phospholipases A2 (PLA2), snake venom serine proteinase, aspartic protease, L-amino acid oxidase (LAAO), hyaluronidase, 5'-nucleotidase, glutaminyl-peptide cyclotransferase, disintegrin, C-type lectin (snaclec), nerve growth factor, Kunitz type serine protease inhibitor, snake venom vascular endothelial growth factor, cysteine-rich secretory protein, bradykinin potentiating peptide, natriuretic peptides. PLA2 and LAAO from V. b. berus venom produce more pronounced cytotoxic effects in cancer cells than normal cells, via induction of apoptosis, cell cycle arrest and suppression of proliferation. Proteomic data of V. b. berus venoms from different parts of Russia and Slovakian Republic have been compared with analogous data for Vipera nikolskii venom. Proteomic studies demonstrated quantitative differences in the composition of V. b. berus venom from different geographical regions. Differences in the venom composition of V. berus were mainly driven by the age, sex, habitat and diet of the snakes. The venom variability of V. berus results in a loss of antivenom efficacy against snakebites. The effectiveness of antibodies is discussed. This review presents an overview with a special focus on different toxins that have been isolated and characterized from the venoms of V. b. berus. Their main biochemical properties and toxic actions are described.

A rapid increase in lysophospholipids after geranylgeranoic acid treatment in human hepatoma-derived HuH-7 cells revealed by metabolomics analysis.

Geranylgeranoic acid (GGA) was developed as a preventative agent against second primary hepatoma, and was reported to induce cell death in human hepatoma cells via Toll-like receptor 4 (TLR4)-mediated pyroptosis. We recently reported that GGA is enzymatically biosynthesized from mevalonic acid in human hepatoma-derived HuH-7 cells and that endogenous GGA is found in most rat organs including the liver. An unbiased metabolomics analysis of ice-cold 50% acetonitrile extracts from control and GGA-treated cells was performed in this study to characterize the intracellular metabolic changes in GGA-induced pyroptosis and to analyze their relationship with the mechanism of GGA-induced cell death. The total positive ion chromatograms of the cellular extracts in ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry were apparently unchanged after GGA treatment, but an orthogonal partial least squares-discriminant analysis score plot clearly discriminated the intracellular metabolite profiles of GGA-treated cells from that of control cells. S-plot analysis revealed 15 potential biomarkers up-regulated by 24-h GGA treatment according to their variable importance in the projection value of more than 1, and the subsequent metabolomics analysis identified nine of these metabolites as a group of lysophospholipids containing lysophosphatidylcholine with C16:0, C20:4, or C20:3 fatty acids. The possible roles of these lysophospholipids in GGA-induced pyroptosis are discussed.

Stability of lipids in plasma and serum: Effects of temperature-related storage conditions on the human lipidome.

Large epidemiological studies often require sample transportation and storage, presenting unique considerations when applying advanced lipidomics techniques. The goal of this study was to acquire lipidomics data on plasma and serum samples stored at potential preanalytical conditions (e.g., thawing, extracting, evaporating), systematically monitoring lipid species for a period of one month. Split aliquots of 10 plasma samples and 10 serum samples from healthy individuals were kept in three temperature-related environments: refrigerator, laboratory benchtop, or heated incubator. Samples were analyzed at six different time points over 28 days using a Bligh & Dyer lipid extraction protocol followed by direct infusion into a lipidomics platform using differential mobility with tandem mass spectrometry. The observed concentration changes over time were evaluated relative to method and inter-individual biological variability. In addition, to evaluate the effect of lipase enzyme levels on concentration changes during storage, we compared corresponding fasting and post-prandial plasma samples collected from 5 individuals. Based on our data, a series of low abundance free fatty acid (FFA), diacylglycerol (DAG), and cholesteryl ester (CE) species were identified as potential analytical markers for degradation. These FFA and DAG species are typically produced by endogenous lipases from numerous triacylglycerols (TAGs), and certain high abundance phosphatidylcholines (PCs). The low concentration CEs, which appeared to increase several fold, were likely mass-isobars from oxidation of other high concentration CEs. Although the concentration changes of the high abundant TAG, PC, and CE precursors remained within method variability, the concentration trends of FFA, DAG, and oxidized CE products should be systematically monitored over time to inform analysts about possible pre-analytical biases due to degradation in the study sample sets.

Comprehensive lipidomics reveals phenotypic differences in hepatic lipid turnover in ALD and NAFLD during alcohol intoxication.

BACKGROUND & AIMS: In experimental models, alcohol induces acute changes in lipid metabolism that cause hepatocyte lipoapoptosis and inflammation. Here we study human hepatic lipid turnover during controlled alcohol intoxication. METHODS: We studied 39 participants with 3 distinct hepatic phenotypes: alcohol-related liver disease (ALD), non-alcoholic fatty liver disease (NAFLD), and healthy controls. Alcohol was administrated via nasogastric tube over 30 min. Hepatic and systemic venous blood was sampled simultaneously at 3 time points: baseline, 60, and 180 min after alcohol intervention. Liver biopsies were sampled 240 min after alcohol intervention. We used ultra-high performance liquid chromatography mass spectrometry to measure levels of more than 250 lipid species from the blood and liver samples. RESULTS: After alcohol intervention, the levels of blood free fatty acid (FFA) and lysophosphatidylcholine (LPC) decreased, while triglyceride (TG) increased. FFA was the only lipid class to decrease in NAFLD after alcohol intervention, whereas LPC and FFA decreased and TG increased after intervention in ALD and healthy controls. Fatty acid chain uptake preference in FFAs and LPCs were oleic acid, linoleic acid, arachidonic acid, and docosahexaenoic acid. Hepatic venous blood FFA and LPC levels were lower when compared with systemic venous blood levels throughout the intervention. After alcohol intoxication, liver lipidome in ALD was similar to that in NAFLD. CONCLUSIONS: Alcohol intoxication induces rapid changes in circulating lipids including hepatic turnaround from FFA and LPC, potentially leading to lipoapoptosis and steatohepatitis. TG clearance was suppressed in NAFLD, possibly explaining why alcohol and NAFLD are synergistic risk factors for disease progression. These effects may be central to the pathogenesis of ALD. CLINICAL TRIALS REGISTRATION: The study is registered at Clinicaltrials.gov (NCT03018990). LAY SUMMARY: We report that alcohol induces hepatic extraction of free unsaturated fatty acids and lysophosphatidylcholines, hepatotoxic lipids which have not been previously associated with alcohol-induced liver injury. We also found that individuals with non-alcoholic fatty liver disease have reduced lipid turnover during alcohol intoxication when compared with people with alcohol-related fatty liver disease. This may explain why alcohol is particularly more harmful in people with non-alcoholic fatty liver and why elevated BMI and alcohol have a synergistic effect on the risk of liver-related death.

Stability of human salivary extracellular vesicles containing dipeptidyl peptidase IV under simulated gastrointestinal tract conditions.

BACKGROUND: Extracellular vesicles (EVs) have been isolated from various sources, including primary and cultured cell lines and body fluids. Previous studies, including those conducted in our laboratory, have reported the stability of EVs under various storage conditions. METHODS: EVs from human whole saliva were separated via size-exclusion chromatography. To simulate the effects of gastric or intestinal fluids on the stability of EVs, pepsin or pancreatin was added to the samples. Additionally, to determine the effect of bile acids, sodium cholate was added. The samples were then subjected to western blotting, dynamic light scattering, and transmission electron microscopy analyses. In addition, the activity of dipeptidyl peptidase (DPP) IV retained in the samples was examined to monitor the stability of EVs. RESULTS: Under acidic conditions, with pepsin mimicking the milieu of the stomach, the EVs remained stable. However, they partially lost their membrane integrity in the presence of pancreatin and sodium cholate, indicating that they may be destabilized after passing through the duodenum. Although several associated proteins, such as mucin 5B and CD9 were degraded, DPP IV was stable, and its activity was retained under the simulated gastrointestinal conditions. CONCLUSION: Our data indicate that although EVs can pass through the stomach without undergoing significant damage, they may be disrupted in the intestine to release their contents. The consistent delivery of active components such as DPP IV from EVs into the intestine might play a role in the efficient modulation of homeostasis of the signal transduction pathways occurring in the gastrointestinal tract.

Neutralising effects of small molecule toxin inhibitors on nanofractionated coagulopathic Crotalinae snake venoms.

Repurposing small molecule drugs and drug candidates is considered as a promising approach to revolutionise the treatment of snakebite envenoming. In this study, we investigated the inhibiting effects of the small molecules varespladib (nonspecific phospholipase A2 inhibitor), marimastat (broad spectrum matrix metalloprotease inhibitor) and dimercaprol (metal ion chelator) against coagulopathic toxins found in Crotalinae (pit vipers) snake venoms. Venoms from Bothrops asper, Bothrops jararaca, Calloselasma rhodostoma and Deinagkistrodon acutus were separated by liquid chromatography, followed by nanofractionation and mass spectrometry identification undertaken in parallel. Nanofractions of the venom toxins were then subjected to a high-throughput coagulation assay in the presence of different concentrations of the small molecules under study. Anticoagulant venom toxins were mostly identified as phospholipases A2, while procoagulant venom activities were mainly associated with snake venom metalloproteinases and snake venom serine proteases. Varespladib was found to effectively inhibit most anticoagulant venom effects, and also showed some inhibition against procoagulant toxins. Contrastingly, marimastat and dimercaprol were both effective inhibitors of procoagulant venom activities but showed little inhibitory capability against anticoagulant toxins. The information obtained from this study aids our understanding of the mechanisms of action of toxin inhibitor drug candidates, and highlights their potential as future snakebite treatments.

Anti-platelet role of Korean ginseng and ginsenosides in cardiovascular diseases.

Cardiovascular diseases prevail among modern societies and underdeveloped countries, and a high mortality rate has also been reported by the World Health Organization affecting millions of people worldwide. Hyperactive platelets are the major culprits in thrombotic disorders. A group of drugs is available to deal with such platelet-related disorders; however, sometimes, side effects and complications caused by these drugs outweigh their benefits. Ginseng and its nutraceuticals have been reported to reduce the impact of thrombotic conditions and improve cardiovascular health by antiplatelet mechanisms. This review provides (1) a comprehensive insight into the available pharmacological options from ginseng and ginsenosides (saponin and nonsaponin fractions) for platelet-originated cardiovascular disorders; (2) a discussion on the impact of specific functional groups on the modulation of platelet functions and how structural modifications among ginsenosides affect platelet activation, which may further provide a basis for drug design, optimization, and the development of ginsenoside scaffolds as pharmacological antiplatelet agents; (3) an insight into the synergistic effects of ginsenosides on platelet functions; and (4) a perspective on future research and the development of ginseng and ginsenosides as super nutraceuticals.

Xenobiotic receptors in mediating the effect of sepsis on drug metabolism.

Sepsis is an infection-induced systemic inflammatory syndrome. The immune response in sepsis is characterized by the activation of both proinflammatory and anti-inflammatory pathways. When sepsis occurs, the expression and activity of many inflammatory cytokines are markedly affected. Xenobiotic receptors are chemical-sensing transcription factors that play essential roles in the transcriptional regulation of drug-metabolizing enzymes (DMEs). Xenobiotic receptors mediate the functional crosstalk between sepsis and drug metabolism because the inflammatory cytokines released during sepsis can affect the expression and activity of xenobiotic receptors and thus impact the expression and activity of DMEs. Xenobiotic receptors in turn may affect the clinical outcomes of sepsis. This review focuses on the sepsis-induced inflammatory response and xenobiotic receptors such as pregnane X receptor (PXR), aryl hydrocarbon receptor (AHR), glucocorticoid receptor (GR), and constitutive androstane receptor (CAR), DMEs such as CYP1A, CYP2B6, CYP2C9, and CYP3A4, and drug transporters such as p-glycoprotein (P-gp), and multidrug resistance-associated protein (MRPs) that are affected by sepsis. Understanding the xenobiotic receptor-mediated effect of sepsis on drug metabolism will help to improve the safe use of drugs in sepsis patients and the development of new xenobiotic receptor-based therapeutic strategies for sepsis.

In vitro synthesis of phospholipids with yeast phospholipase B, a phospholipid deacylating enzyme.

The gene encoding the Saccharomyces cerevisiae phospholipid deacylation enzyme, phospholipase B (ScPLB1), was successfully expressed in E. coli. The enzyme (Scplb1p) was engineered to have a histidine-tag at the C-terminal end and was purified by metal (Ni) affinity chromatography. Enzymatic properties, optimal pH, and substrate specificity were similar to those reported previously. For example, deacylation activity was observed in acidic pH in the absence of Ca2+ and was additive in neutral pH in the presence of Ca2+, and the enzyme had the same substrate priority as reported previously, with the exception of PE, suggesting that yeast phospholipase B could be produced in its native structure in bacterial cells. Scplb1p retained transacylation activity in aqueous medium, and esterified lysophosphatidylcholine with free fatty acid to form phosphatidylcholine in a non-aqueous, glycerin medium. We propose that phospholipase B could serve as an additional tool for in vitro enzyme-mediated phospholipid synthesis.

Dietary phosphatidylcholine impacts on growth performance and lipid metabolism in adult Genetically Improved Farmed Tilapia (GIFT) strain of Nile tilapia Oreochromis niloticus.

This study aimed to determine the effects of supplementing the diet of adult Nile tilapia Oreochromis niloticus with phosphatidylcholine (PC) on growth performance, body composition, fatty acid composition and gene expression. Genetically Improved Farmed Tilapia fish with an initial body weight of 83·1 (sd 2·9) g were divided into six groups. Each group was hand-fed a semi-purified diet containing 1·7 (control diet), 4·0, 6·5, 11·5, 21·3 or 41·0 g PC/kg diet for 68 d. Supplemental PC improved the feed efficiency rate, which was highest in the 11·5 g PC/kg diet. Weight gain and specific growth rate were unaffected. Dietary PC increased PC content in the liver and decreased crude fat content in the liver, viscera and body. SFA and MUFA increased and PUFA decreased in muscle with increasing dietary PC. Cytoplasmic phospholipase A 2 and secreted phospholipase A 2 mRNA expression were up-regulated in the brain and heart in PC-supplemented fish. PC reduced fatty acid synthase mRNA expression in the liver and visceral tissue but increased expression in muscle. Hormone-sensitive lipase and lipoprotein lipase expression increased in the liver with increasing dietary PC. Growth hormone mRNA expression was reduced in the brain and insulin-like growth factor-1 mRNA expression in liver reduced with PC above 6·5 g/kg. Our results demonstrate that dietary supplementation with PC improves feed efficiency and reduces liver fat in adult Nile tilapia, without increasing weight gain, representing a novel dietary approach to reduce feed requirements and improve the health of Nile tilapia.

Do Animal Models of Acute Pancreatitis Reproduce Human Disease?

Acute pancreatitis is currently the most common cause of hospital admission among all nonmalignant gastrointestinal diseases. To understand the pathophysiology of the disease and as a potential step toward developing targeted therapies, attempts to induce the disease experimentally began more than 100 years ago. Recent decades have seen progress in developing new experimental pancreatitis models as well as elucidating many underlying cell biological and pathophysiological disease mechanisms. Some models have been developed to reflect specific causes of acute pancreatitis in human beings. However, the paucity of data relating to the molecular mechanisms of human disease, the likelihood that multiple genetic and environmental factors affect the risk of disease development and its severity, and the limited information regarding the natural history of disease in human beings make it difficult to evaluate the value of disease models. Here, we provide an overview of key models and discuss our views on their strengths for characterizing cell biological disease mechanisms or for identifying potential therapeutic targets. We also acknowledge their limitations.

A comprehensive lipidomic screen of pancreatic ß-cells using mass spectroscopy defines novel features of glucose-stimulated turnover of neutral lipids, sphingolipids and plasmalogens.

OBJECTIVE: Glucose promotes lipid remodelling in pancreatic ß-cells, and this is thought to contribute to the regulation of insulin secretion, but the metabolic pathways and potential signalling intermediates have not been fully elaborated. METHODS: Using mass spectrometry (MS) we quantified changes in approximately 300 lipid metabolites in MIN6 ß-cells and isolated mouse islets following 1 h stimulation with glucose. Flux through sphingolipid pathways was also assessed in (3)H-sphinganine-labelled cells using TLC. RESULTS: Glucose specifically activates the conversion of triacylglycerol (TAG) to diacylglycerol (DAG). This leads indirectly to the formation of 18:1 monoacylglycerol (MAG), via degradation of saturated/monounsaturated DAG species, such as 16:0_18:1 DAG, which are the most abundant, immediate products of glucose-stimulated TAG hydrolysis. However, 16:0-containing, di-saturated DAG species are a better direct marker of TAG hydrolysis since, unlike the 18:1-containing DAGs, they are predominately formed via this route. Using multiple reaction monitoring, we confirmed that in islets under basal conditions, 18:1 MAG is the most abundant species. We further demonstrated a novel site of glucose to enhance the conversion of ceramide to sphingomyelin (SM) and galactosylceramide (GalCer). Flux and product:precursor analyses suggest regulation of the enzyme SM synthase, which would constitute a separate mechanism for localized generation of DAG in response to glucose. Phosphatidylcholine (PC) plasmalogen (P) species, specifically those containing 20:4, 22:5 and 22:6 side chains, were also diminished in the presence of glucose, whereas the more abundant phosphatidylethanolamine plasmalogens were unchanged. CONCLUSION: Our results highlight 18:1 MAG, GalCer, PC(P) and DAG/SM as potential contributors to metabolic stimulus-secretion coupling.

Neuroinflammation in hepatic encephalopathy: mechanistic aspects.

Hepatic encephalopathy (HE) is a major neurological complication of severe liver disease that presents in acute and chronic forms. While elevated brain ammonia level is known to be a major etiological factor in this disorder, recent studies have shown a significant role of neuroinflammation in the pathogenesis of both acute and chronic HE. This review summarizes the involvement of ammonia in the activation of microglia, as well as the means by which ammonia triggers inflammatory responses in these cells. Additionally, the role of ammonia in stimulating inflammatory events in brain endothelial cells (ECs), likely through the activation of the toll-like receptor-4 and the associated production of cytokines, as well as the stimulation of various inflammatory factors in ECs and in astrocytes, are discussed. This review also summarizes the inflammatory mechanisms by which activation of ECs and microglia impact on astrocytes leading to their dysfunction, ultimately contributing to astrocyte swelling/brain edema in acute HE. The role of microglial activation and its contribution to the progression of neurobehavioral abnormalities in chronic HE are also briefly presented. We posit that a better understanding of the inflammatory events associated with acute and chronic HE will uncover novel therapeutic targets useful in the treatment of patients afflicted with HE.

Modulation of adrenocorticotrophin hormone (ACTH)-induced expression of stress-related genes by PUFA in inter-renal cells from European sea bass (Dicentrarchus labrax).

Dietary fatty acids have been shown to exert a clear effect on the stress response, modulating the release of cortisol. The role of fatty acids on the expression of steroidogenic genes has been described in mammals, but little is known in fish. The effect of different fatty acids on the release of cortisol and expression of stress-related genes of European sea bass (Dicentrarchus labrax) head kidney, induced by a pulse of adenocorticotrophin hormone (ACTH), was studied. Tissue was maintained in superfusion with 60 min of incubation with EPA, DHA, arachidonic acid (ARA), linoleic acid or α-linolenic acid (ALA) during 490 min. Cortisol was measured by RIA. The quantification of stress-related genes transcripts was conducted by One-Step TaqMan real-time RT-PCR. There was an effect of the type of fatty acid on the ACTH-induced release of cortisol, values from ALA treatment being elevated within all of the experimental period. The expression of some steroidogenic genes, such as the steroidogenic acute regulatory protein (StAR) and c-fos, were affected by fatty acids, ALA increasing the expression of StAR after 1 h of ACTH stimulation whereas DHA, ARA and ALA increased the expression of c-fos after 20 min. ARA increased expression of the 11ß-hydroxylase gene. Expression of heat shock protein 70 (HSP70) was increased in all the experimental treatments except for ARA. Results corroborate previous studies of the effect of different fatty acids on the release of cortisol in marine fish and demonstrate that those effects are mediated by alteration of the expression of steroidogenic genes.

AU4S: a novel synthetic peptide to measure the activity of ATG4 in living cells.

ATG4 plays a key role in autophagy induction, but the methods for monitoring ATG4 activity in living cells are limited. Here we designed a novel fluorescent peptide named AU4S for noninvasive detection of ATG4 activity in living cells, which consists of the cell-penetrating peptide (CPP), ATG4-recognized sequence "GTFG," and the fluorophore FITC. Additionally, an ATG4-resistant peptide AG4R was used as a control. CPP can help AU4S or AG4R to penetrate cell membrane efficiently. AU4S but not AG4R can be recognized and cleaved by ATG4, leading to the change of fluorescence intensity. Therefore, the difference between AU4S- and AG4R-measured fluorescence values in the same sample, defined as "F-D value," can reflect ATG4 activity. By detecting the F-D values, we found that ATG4 activity paralleled LC3B-II levels in rapamycin-treated cells, but neither paralleled LC3B-II levels in starved cells nor presented a correlation with LC3B-II accumulation in WBCs from healthy donors or leukemia patients. However, when DTT was added to the system, ATG4 activity not only paralleled LC3B-II levels in starved cells in the presence or absence of autophagy inhibitors, but also presented a positive correlation with LC3B-II accumulation in WBCs from leukemia patients (R(2) = 0.5288). In conclusion, this study provides a convenient, rapid, and quantitative method to monitor ATG4 activity in living cells, which may be beneficial to basic and clinical research on autophagy.

COX-2 dependent regulation of mechanotransduction in human breast cancer cells.

The ability of living cells to exert physical forces upon their surrounding is a necessary prerequisite for diverse biological processes, such as local cellular migrations in wound healing to metastatic-invasion of cancer. How forces are coopted in metastasis has remained unclear, however, because the mechanical interplay between cancer cells and the various stromal components has not been experimentally accessible. Current dogma implicates inflammation in these mechanical processes. Using Fourier transform traction microscopy, we measured the force-generating capacity of human breast cancer cells occupying a spectrum of invasiveness as well as basal and inducible COX-2 expression (MCF-7

Viperatoxin-II: A novel viper venom protein as an effective bactericidal agent.

Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) have become a rising threat to public health. There is an urgent need for development of promising new therapeutic agents against drug resistant bacteria like S. aureus. This report discusses purification and characterization of proteins from Indian Russell's viper snake venom. Novel 15-kDa proteins called "Viperatoxin" (VipTx-I and VipTx-II) were extracted from the whole venom and evaluated using in vitro antimicrobial experiments. The N-terminal amino acid sequence of "Viperatoxin" showed high sequence homology to daboiatoxin isolated from the same venom and also matched phospholipase A2 (PLA2) enzymes isolated from other snake venoms. In an in vitro plate assay, VipTx-II but not VipTx-I showed strong antimicrobial effects against S. aureus and Burkholderia pseudomallei (KHW & TES), Proteus vulgaris and P. mirabilis. The VipTx-II was further tested by a broth-dilution assay at 100-3.1 µg/ml concentrations. The most potent bactericidal effect was found at the lowest dilutions (MICs of 6.25 µg/ml) against B. pseudomallei, S. aureus and P. vulgaris (MICs of 12.25 µg/ml). Electron microscopic investigation revealed that the protein-induced bactericidal potency was closely associated with pore formation and membrane damage, even at the lowest concentrations (<20 µg/ml). The toxin caused a low level of cytotoxic effects as observed in human (THP-1) cells at higher concentrations. Molecular weight determinations of VipTx-II by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed one major, along with a few minor bands. The results indicate that VipTx-II plays a significant role in bactericidal and membrane damaging effects in vitro. Non-cytotoxic properties on human cells highlight it as a promising candidate for further evaluation of antimicrobial potential in vivo.

Reduced kidney levels of lysophosphatidic acids in rats after chronic administration of aristolochic acid: Its possible protective role in renal fibrosis.

Aristolochic acid (AA) is considered to be a causative agent for progressive interstitial renal fibrosis, leading to AA nephropathy. Lysophosphatidic acid (LPA) is a mediator in the onset of renal fibrosis. In this study, we analyzed the molecular species of LPA and its precursor lysophospholipids in kidney tissue from rats exposed to AA. Daily intraperitoneal injections of AA for 35 days to rats gave rise to fibrosis in kidney, decreased the kidney levels of LPA, lysophosphatidylserine and lysophosphatidylinositol. In rat renal cell lines (NRK52E and NRK49F), AA-induced cytotoxicity was potentiated by Ki16425, LPA1,3 receptor antagonist. The level of mRNA encording α-smooth muscle actin was significantly increased by AA-treatment only in NRK52E cells, while the mRNA level of collagen III was decreased in both NRK52E and NRK49F cells. These results suggest that endogenous LPA in rat kidney prevents AA-induced renal fibrosis.