G6PDH Activity Research Articles

The investigation of the oxidative stress-related parameters in type2 diabetes mellitus.

Oxidative stress, defined as an imbalance between reactive oxygen species production and breakdown by endogenous antioxidants, is closely associated with diabetes mellitus. The diabetes is characterized by hyperglycemia together with biochemical alterations of glucose and lipid peroxidation. Oxidative stress has been implicated in the pathogenesis of type 2 diabetes and its complications. This study was conducted to investigate the variation in oxidative stress-related parameters in type 2 diabetes. Blood serum samples were collected from diabetes patients and nondiabetes healthy controls. Glucose concentrations, levels of glycated hemoglobin (A1C) and serum oxidative stress markers (glucose-6-phosphate dehydrogenase [G6PDH], malondialdehyde [MDA], glutathione [GSH], glutathione reductase [GR], glutathione peroxidase [GPx] and superoxide dismutase [SOD]) were estimated. Fasting serum glucose concentration in type 2 diabetes patients of both sexes was increased significantly as compared with the healthy controls. Level of A1C was greater than standards. Significant elevation in MDA level and depletion in GSH content were observed in diabetes patients in comparison with controls. The diminution in G6PDH activity was accompanied in part by a decrease in the antioxidative enzymes activities (GPx and GR), and in part by an increase in SOD activity in all diabetes patients as compared with the control group. The regression analysis showed no correlation between diabetes duration and severity of oxidative stress; however, there was a significant association between A1C and severity of oxidative stress. The present study shows that there is an oxidative stress state in type 2 diabetes patients compared with healthy subjects. Our data suggest that chronic hyperglycemia causes a significant change in oxidative stress markers.

Yearly growth and metabolic changes in earthen pond-cultured meagre <em>Argyrosomus regius</em>

Metabolic modifications associated with natural environmental conditions were assessed in the meagre Argyrosomus regius cultured in earthen ponds under natural photoperiod and temperature. Juvenile specimens (90-100 g initial weight) were sampled (plasma, liver and muscle) every two months for 18 months (between December 2004 and May 2006). Specimens showed seasonal variations in growth rate, with the highest values in spring and summer. Plasmatic, hepatic and muscular metabolite levels and hepatic and muscular metabolic enzymes also showed significant variations throughout the year. Enzymatic activity related to carbohydrate metabolism in the liver (HK, FBPase and G6PDH) showed great modifications in summer, increasing glycogenogenic pathways, while amino acid metabolism (GDH and GOT activity) was enhanced in spring and summer. However lipid-related (G3PDH activity) metabolic enzymes did not show a clear seasonal pattern. In muscle, enzymatic activity related to amino acid, lipid and lactate metabolism (LDH-O activity), but not carbohydrate metabolism, showed seasonal changes in parallel with changes in growth rate. Thus A. regius specimens showed a trend to grow in summer months and mobilize their energy reserves in winter. Differences in the hepatic level were observed between the first and the second year of the study, suggesting the possible existence of metabolic changes related to specimen age or size. Our results indicate that growth and metabolic responses in A. regius are environmentally dependent and that this species is a very good candidate for diversification in aquaculture.

Cyclin‐Dependent Kinase 9 Inhibition Protects Cartilage From the Catabolic Effects of Proinflammatory Cytokines

Cyclin-dependent kinase 9 (CDK-9) controls the activation of primary inflammatory response genes. The purpose of this study was to determine whether CDK-9 inhibition protects cartilage from the catabolic effects of proinflammatory cytokines. Human chondrocytes were challenged with different proinflammatory stimuli (interleukin-1β [IL-1β], lipopolysaccharides, and tumor necrosis factor α) in the presence or absence of either the CDK-9 inhibitor flavopiridol or small interfering RNA (siRNA). The expression of messenger RNA (mRNA) for inflammatory mediator genes, catabolic genes, and anabolic genes were determined by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. Cartilage explants were incubated for 6 days with IL-1β in the presence or absence of flavopiridol. Cartilage matrix degradation was assessed by the release of glycosaminoglycan (GAG) and cleaved type II collagen (COL2A) peptides. CDK-9 inhibition by flavopiridol or knockdown by siRNA effectively suppressed the induction of mRNA for inducible nitric oxide synthase by all 3 proinflammatory stimuli. Results from NF-κB-targeted PCR array analysis showed that flavopiridol suppressed IL-1β induction of a broad range of inflammatory mediator genes (59 of 67 tested). CDK-9 inhibition also suppressed the induction of catabolic genes (matrix metalloproteinase 1 [MMP-1], MMP-3, MMP-9, MMP-13, ADAMTS-4, and ADAMTS-5), but did not affect the basal expression of anabolic genes (COL2A, aggrecan, and cartilage oligomeric matrix protein) and housekeeping genes. Flavopiridol had no apparent short-term cytotoxicity, as assessed by G6PDH activity. Finally, in IL-1β-treated cartilage explants, flavopiridol reduced the release of the matrix degradation product GAG and cleaved COL2A peptides, but did not affect long-term chondrocyte viability. CDK-9 activity is required for the primary inflammatory response in chondrocytes. Flavopiridol suppresses the induction of inflammatory mediator genes and catabolic genes to protect cartilage from the deleterious effects of proinflammatory cytokines, without affecting cell viability and functions.

The root extract of Angelica gigas Nakai promotes adipogenic differentiation via activation of the insulin signaling pathway in 3T3‐L1 cells (854.6)

Type 2 diabetes (T2D) is a complex metabolic disorder characterized by insulin resistance and hyperglycemia. Peroxisome proliferator‐activated receptor gamma (PPARγ) is a key transcription factor and plays an important role in the regulation of genes involved in adipogenic differentiation, glucose metabolism and insulin signal transduction. In this study, the effects of the root extract of Angelica gigas Nakai (AGN) on the differentiation of 3T3‐L1 preadipocytes and the possible mechanism of glucose transport were investigated. Post‐confluent 3T3‐L1 cells were differentiated in the presence or absence of AGN (up to 400μg). Oil red O staining showed that AGN extract significantly enhanced adipocyte differentiation in a dose dependent manner. GPDH activity as well as intracellular lipid accumulation was significantly increased by the AGN treatment. RT‐PCR and Western Blot analysis showed that AGN increased cell differentiation by upregulating the expression of PPARγ dose dependently. AGN also promoted glucose transport by increasing the expression of glucose transporter 4 (GLUT‐4), phosphatidylinositol 3‐kinase (PI3K) and insulin receptor substrates‐1 (IRS‐1). Our results suggest that AGN extract may be a potential therapeutic agent for managing T2D by promoting the differentiation of adipocytes via the upregulation of PPARγ levels and the activation of the insulin signaling pathway.

Effect of high hydrostatic pressure extract of Korean ginseng on adipogenesis in 3T3‐L1 adipocytes (1045.4)

Ginseng, especially steam‐and‐dry processed red ginseng, has been shown to decrease adipogenesis in adipose tissue. In the food processing industry, high hydrostatic pressure extraction technique has become an alternative to heat processing. The aim of this study was to comparatively investigate the anti‐adipogenic effects of water extract of red ginseng (WRG) and high hydrostatic pressure extract of fresh ginseng (HPG) in 3T3‐L1 adipocytes. Intracellular lipid and triglyceride were decreased in both WRG and HPG treated cells, while the cell viability remained unaffected. This observed decrease was consistent with the reduction of GPDH activity, a key enzyme in triglyceride biosynthesis. The mRNA expressions of adipogenic genes, including peroxisome proliferator‐activated receptor‐gamma, CCAAT/enhancer‐binding protein‐α, sterol regulatory element‐binding protein‐1c, and adipocyte fatty acid binding protein, were significantly decreased in both WEG and HPG treated cells. The activity of AMPK, a key regulator of energy metabolism, was significantly increased in the HPG treated cells compared to the WRG. From these results, it is assumed that anti‐adipogenic mechanisms of the WRG and HPG in adipocytes might be associated with inhibition of GPDH activity and adipogenic gene expression as well as AMPK activation. Moreover, efficiency of the HPG might be comparable to that of the WRG.Grant Funding Source: This work was supported by the Brain Korea 21 plus project funding (No. 22A20130012143)

Remodeling of white adipose tissue metabolism by physical training prevents insulin resistance

AimThis study sought to determine the role of white adipose tissue (WAT) metabolism in the prevention of insulin resistance (IR) by physical training (PT). Main methodsMale C57BL/6J mice were assigned into groups CHOW-SED (chow diet, sedentary; n=15), CHOW-TR (chow diet, trained; n=18), CAF-SED (cafeteria diet, sedentary; n=15) and CAF-TR (cafeteria diet, trained; n=18). PT consisted of running sessions of 60min at 60% of maximal speed conducted five days per week for eight weeks. Key findingsPT prevented body weight and fat mass accretion in trained groups and prevented hyperglycemia, hyperinsulinemia, glucose intolerance and IR in the CAF-TR. The CAF-SED group presented higher leptin and free fatty acid and lower adiponectin serum levels compared with other groups. Lipolytic activity (in mmol/106 adipose cells) stimulated by isoproterenol increased in CHOW-TR (16347±3005), CAF-SED (18110±3788) and CAF-TR (15837±2845) compared with CHOW-SED (8377±2284). The CAF-SED group reduced FAS activity compared with CHOW-SED and CHOW-TR, reduced citrate synthase activity and increased DGAT2 content compared with other groups. Both trained groups reduced G6PDH activity and increased the expression of p-AMPK (Thr172) compared with sedentary groups. CAF-SED group had lower levels of AMPK, p-AMPK (Thr172), ACC and p-ACC (Ser79) compared with other groups. SignificanceThe prevention of IR by PT is mediated by adaptations in WAT metabolism by improving lipolysis, preventing an increase in enzymes responsible for fatty acid esterification and by activating enzymes that improve fat oxidation instead of fat storage.

Comparison of multipotency and molecular profile of MSCs between CKD and healthy rats

We previously showed that mesenchymal stem cells (MSCs) can differentiate into a functional miniature kidney, suggesting that MSCs may be a cell source for kidney regeneration. However, MSCs from long-term dialysis patients, which have been exposed to uremic toxin, can exhibit reduced viability. Therefore, the aim of this study was to examine the gene expression profiles and differentiation capabilities of bone marrow- and adipose-derived MSCs from chronic kidney disease (CKD) model rats. CKD was induced in rats by adenine feeding, and then MSCs were isolated from bone marrow (BMSCs) and adipose tissue (ASCs). After confirming MSC surface marker expression, comprehensive gene expression profiles were obtained by RT-PCR array. MSCs were differentiated into adipocytes, osteoblasts, and chondrocytes, and histological and/or functional assays were performed. Tgfb3 expression was up-regulated, while Bmp6, Gdf15, Mmp2, and Vegfa were down-regulated in CKD-ASCs compared with Control-ASCs. There were no significant differences in the gene expression of stemness markers, and the morphology of cells that underwent adipogenesis, osteogenesis, and chondrogenesis, or GPDH activity between CKD and control groups. Comparing BMSCs with ASCs, gene expression of Bglap, Bmp4, Igf1, Itgax, Pparg, Ptprc, and Tnf were up-regulated, while Col1a1, Mmp2, Sox9, and Vegfa were down-regulated in both CKD and control groups. Uremic toxin in CKD rats had a small effect on the gene expression and differentiation of MSCs. However, long-term exposure to uremic toxin and the differences in gene expression of MSCs derived from bone marrow or adipose tissue may affect renal regeneration.

Suitable extracellular oxidoreduction potential inhibit

Polyketides, such as spinosad, are mainly synthesized in the stationary phase of the fermentation. The synthesis of these compounds requires many primary metabolites, such as acetyl-CoA, propinyl-CoA, NADPH, and succinyl-CoA. Their synthesis is also significantly influenced by NADH/NAD+. Rex is the sensor of NADH/NAD+ redox state, whose structure is under the control of NADH/NAD+ ratio. The structure of rex controls the expression of many NADH dehydrogenases genes and cytochrome bd genes. Intracellular redox state can be influenced by adding extracellular electron acceptor H2O2. The effect of extracellular oxidoreduction potential on spinosad production has not been studied. Although extracellular oxidoreduction potential is an important environment effect in polyketides production, it has always been overlooked. Thus, it is important to study the effect of extracellular oxidoreduction potential on Saccharopolyspora spinosa growth and spinosad production. During stationary phase, S. spinosa was cultured under oxidative (H2O2) and reductive (dithiothreitol) conditions. The results show that the yield of spinosad and pseudoaglycone increased 3.11 fold under oxidative condition. As H2O2 can be served as extracellular electron acceptor, the ratios of NADH/NAD+ were measured. We found that the ratio of NADH/NAD+ under oxidative condition was much lower than that in the control group. The expression of cytA and cytB in the rex mutant indicated that the expression of these two genes was controlled by rex, and it was not activated under oxidative condition. Enzyme activities of PFK, ICDH, and G6PDH and metabolites results indicated that more metabolic flux flow through spinosad synthesis. The regulation function of rex was inhibited by adding extracellular electron acceptor-H2O2 in the stationary phase. Under this condition, many NADH dehydrogenases which were used to balance NADH/NAD+ by converting useful metabolites to useless metabolites and unefficient terminal oxidases (cytochrome bd) were not expressed. So lots of metabolites were not waste to balance. As a result, un-wasted metabolites related to spinosad and PSA synthesis resulted in a high production of spinosad and PSA under oxidative condition.

Histochemical Investigation of Cyanogenic Toxicity in the Thyroid Epithelial Cells and Follicles of Thyroid Gland in Adult Female Wistar Rats

Aim: This study was aimed at understanding the earlier findings involving chronic, lowlevel cyanide exposure resulting from eating poorly processed cassava products associated with the development of goitre as seen in cassava endemic regions of Nigeria. Study Design: 30F1 female adult Wistar rats were divided into five (5) groups of 6 animals each. Groups 1 to 4 represented the treatment groups while group 5 was the Original Research Article British Journal of Medicine & Medical Research, 4(8): 1750-1762, 2014 1751 control of the experiment. The cyanide treatment dose were; group1: 20mg/KgBW, group 2: 12mg/KgBW, group 3: 6mg/KgBW and group 4: 2mg/KgBW while the control group received 0.25M Sucrose. Place and Duration of Study: The animal facility of College of Health Sciences, Osun State University, Osogbo, Osun State, Nigeria. The treatment duration was 30days. Methodology: Animals were sacrificed by cervical dislocation. The blood samples were collected to determine Serum FT3, FT4 and TSH concentration. The thyroid gland was excised and processed for light microscopic examination; while the activities of G6PDH, LDH, ALP, MDA and SOD were assayed from the thyroid tissue homogenates. Results: Histological observation of thyroid gland of rats from the experimental treated groups revealed markedly distended follicles and diffusely hyperplastic thyroid follicles lined with tall columnar epithelial cells. These thyroid epithelial cells are crowded and enlarged projecting into the lumens of their respective follicles. Their interstitial tissue all had dilated blood vessels. Application of one-way ANOVA statistical analytical method showed that there were highly significant differences P˂0.05 in the activities of G6PDH, LDH, ALP, MDA, SOD, FT3, FT4 and TSH when compared with those of the control group. Conclusion: The results obtained from this study showed hyperthyroidism was effectively induced by cyanide.

Differences in Cytoplasmic Maturation Between the BCB+ and Control Porcine Oocytes Do Not Justify Application of the BCB Test for a Standard IVM Protocol

The Brilliant Cresyl Blue (BCB) test relies on G6PDH activity and a simple protocol for the selection of higher quality oocytes. Although the BCB+ oocytes of all the species that have been investigated are characterized by superior quality when compared to BCB- counterparts, application of the test for embryo production still remains an open issue. The aim of our study was to compare BCB+ and the control oocytes (not subjected to the BCB test) in terms of selected aspects of cytoplasmic maturation (mtDNA copy number, mitochondria distribution, relative transcript abundance of six marker genes). The results of our study revealed more relevant differences within the BCB+ and the control oocytes (before and after IVM) than between the two categories of oocytes. There was no difference in the transcript abundance of the BCB+ and the control oocytes in 5 out of 6 analyzed genes (BMP15, GDF9, ATP5A1, EEF1A, ZAR1) and in mtDNA content (pre-IVM 179609 vs. 176595 and post-IVM 187243 vs. 246984, respectively). With regard to mitochondria distribution in pre- and post-IVM oocytes, there was nonsignificant tendency for a more frequent occurrence of the expected patterns in the BCB+ group. The results of the present study do not support the application of BCB staining in a routine IVM protocol due to relatively high similarity in selected parameters characterizing cytoplasmic maturation of BCB+ and control oocytes. This high similarity may results from the limited amount of less competent BCB- oocytes (10%) still present among nonselected oocytes of proper morphology.

Suppression of mitochondrial dehydrogenases accompanying post-glyoxylate cycle activation of gluconeogenesis and reduced lipid peroxidation events during dormancy breakage of walnut kernels by moist chilling

Cold stratification supposedly allows dormant seeds to germinate by removing their metabolic blocks. Nothing is known about the role of mitochondrial enzymes in metabolic regulation of seeds during dormancy release. As cold stratification removes walnut (Juglans regia L.) kernel dormancy, the activities of three mitochondrial enzymes i.e. pyruvate dehydrogenase complex (PDH), NAD+-isocitrate dehydrogenase (NAD+-IDH; EC 1.1.1.41), succinate dehydrogenase (SDH; EC 1.3.99.1) and the extra-mitochondrial glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) were compared in both cold-stratified (5°C; 60days) and warm-incubated (27°C; 20days) kernels. The kernel gluconeogenic competence was also assessed by measuring the activity of phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.49). These were then correlated to kernel lipid hydroperoxide contents and lipoxygenase (EC 1.13.11.12) activity. Lipid hydroperoxides and lipoxygenase activity were significantly greater in warm-incubated kernels. The mitochondrial NAD+-IDH activity was indifferent between cold-stratified and warm-incubated kernels. The activities of PDH and SDH though undetectable in cold-stratified kernels, were relatively high under warm conditions. Succinate oxidation in cold stratified kernels however, was achieved by a glyoxysomal succinate oxidase activity formerly reported in monocotyledons. Furthermore, the kernel succinate oxidase, PEPCK and G6PDH activities were significantly greater at cold conditions. It was concluded that cold induced dormancy release of walnut kernels is associated with suppression of kernel mitochondrial respiration and oxidative stress and meanwhile it allows activation of oxidative pentose phosphate (OPP) and post-glyoxylate cycle gluconeogenesis pathways. The build-up of oxidative stress and toxic lipid hydroperoxide derivatives however, probably result in the compromised germination of warm-incubated walnut kernels.

Inhibitory Effects of S-Allylmercaptocysteine Derived from Aged Garlic on Cholesterol Biosynthesis in Hepatocytes

마늘 숙성 중 생성되는 S-allylmercaptocysteine의 콜레스테롤 생합성 억제 효과에 대하여 Hepatocytes를 이용하여 조사하였다. HepG2 cells을 Dulbecco`s modified Eagle`s medium(DMEM)에 배양하여 S-allylmercaptocysteine의 농도를 20, 40, 60, 80 및 100 mL 씩 각각 첨가하여 cell viability를 살펴본 결과 20∼40 μg/mL에서는 높았으며, 60 μg/mL 농도에서 약 50%가 유지되었다. S-allylmercaptocysteine을 5, 10, 15 및 20 μg/mL 농도로 [14C]-acetatecholesterol에서 처리하였을 경우 15 μg/mL 농도에서 cholesterol 생합성이 79%로 억제되었다. Fatty acid synthase의 활성은 0.95 nmol에서 19%의 억제효과를 나타내었으나, Glucose 6-phosphate dehydrogenase (G6PDH)의 활성에는 거의 영향을 미치지 않았다. S-allylmercaptocysteine의 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase의 활성은 용량 의존형으로 감소하였다. 이상의 결과로 보아 마늘 숙성 과정에서 생성되는 주요 성분인 S-allylmercaptocysteine은 간 세포에서 cholesterol의 생합성을 억제하는데 기여하는 것으로 나타났다.

Critical assessment of extracellular polymeric substances extraction methods from mixed culture biomass

Extracellular polymeric substances (EPS) have a presumed determinant role in the structure, architecture, strength, filterability, and settling behaviour of microbial solids in biological wastewater treatment processes. Consequently, numerous EPS extraction protocols have recently been published that aim to optimize the trade off between high EPS recovery and low cell lysis. Despite extensive efforts, the obtained results are often contradictory, even when analysing similar biomass samples and using similar experimental conditions, which greatly complicates the selection of an extraction protocol. This study presents a rigorous and critical assessment of existing physical and chemical EPS extraction methods applied to mixed-culture biomass samples (nitrifying, nitritation-anammox, and activated sludge biomass). A novel fluorescence-based method was developed and calibrated to quantify the lysis potential of different EPS extraction protocols. We concluded that commonly used methods to assess cell lysis (DNA concentrations or G6PDH activities in EPS extracts) do not correlate with cell viability. Furthermore, we discovered that the presence of certain chemicals in EPS extracts results in severe underestimation of protein and carbohydrate concentrations by using standard analytical methods. Keeping both maximum EPS extraction yields and minimal biomass lysis as criteria, it was identified a sonication-based extraction method as the best to determine and compare tightly-bound EPS fractions in different biomass samples. Protein was consistently the main EPS component in all analysed samples. However, EPS from nitrifying enrichments was richer in DNA, the activated sludge EPS had a higher content in humic acids and carbohydrates, and the nitritation-anammox EPS, while similar in composition to the nitrifier EPS, had a lower fraction of hydrophobic biopolymers. In general, the easily-extractable EPS fraction was more abundant in carbohydrates and humic substances, while DNA could only be found in tightly bound EPS fractions. In conclusion, the methodology presented herein supports the rational selection of analytical tools and EPS extraction protocols in further EPS characterization studies.

Characterization of a novel proinflammatory effect mediated by BK and the kinin B2 receptor in human preadipocytes

Obesity and adipose tissue contribute to local and systemic inflammation. However the role of the inflammatory mediator bradykinin (BK) in this context is not known. We therefore evaluated the effect of BK on adipokines secretion in human preadipocytes during the course of differentiation and characterized the receptors involved. Results obtained from antibody array and ELISA experiments showed that several adipokines are released by human preadipocytes under basal conditions while BK specifically stimulated the production of interleukin(IL)-6 and IL-8. The effect of BK diminished with the progression of differentiation, being almost inactive on adipocytes. In preadipocytes, BK also induced a rapid and transient [Ca2+]i mobilization, a rapid and sustained increase in ERK1/2 activation and enhanced forskolin-stimulated cAMP accumulation. BK was without effect on cell proliferation and viability as assessed by bromodeoxyuridine incorporation, WST-1 conversion, or lactate dehydrogenase leakage and was without effect on adipogenesis as measured by triglyceride accumulation, GPDH activity and leptin release. The B1 receptor agonist, Lys-[des-Arg9]-BK, displayed poor activity or was without effect while overall BK effects were prevented by the selective B2 receptor antagonist, fasitibant chloride, but not by the B1 selective antagonist, Lys-[Leu8][des-Arg9]-BK. Immunoblot analysis and immunofluorescence studies showed that the kinin B2 receptor was essentially expressed at the beginning of the differentiation program. In conclusion, human preadipocytes expressed kinin B2 receptors linked to multiple signaling pathways, IL-6 and IL-8 production, and BK proinflammatory response in adipose tissue could be prevented by fasitibant chloride.

Histological, histochemical and immunohistochemical evaluation of the effects of seed and pulp of Carica papaya on the visual relay centres in animal model

The effects of the pulp and seeds of Carica papaya (Honey dew variety) on the visual relay centres in rats were investigated. Thirty rats were randomly grouped into three groups (A, B, and C) of ten rats each. Each of the rats in groups A, B and C were fed with 8.50 g of standard rat pellet, 8.50 g of 1:1 mixture of C. papaya pulp and the standard rat chow, and 8.50 g of 1:1 mixture of C. papaya seeds and standard rats’ chow, twice daily, respectively. Twenty-four hours after the last administration, the rats were sacrificed by cervical dislocation, the brain was removed, superior colliculus (SC) and lateral geniculate body (LGB) were carefully excised. Fraction of the SC and LGB meant for quantitative enzyme histochemistry were homogenized in sucrose solution and processed accordingly for the activities of lactate dehydrogenase (LDH), glucose-6-phosphate dehydrogenase (G6PD) and acetylcholinesterase (AChE) while the fraction of the SC and LGB meant for histological and immunohistochemical analysis were fixed in 10% formol-calcium solution and were processed for paraffin embedding, respectively. The histological, histochemical and immunohistochemical studies revealed that C. papaya seeds caused neuronal degeneration and disruption in the visual relay centres of the treated rats. The activity of G6PDH was increased in the SC and LGB of rats in groups B and C, and there was statistically significant (P<0.05) inhibition in the activity of AChE in both the SC and LGB of rats in group C. Key words: Quantitative histochemistry, histology, Carica papaya, superior colliculus, lateral geniculate body, 10% formol calcium.

Why are chlorinated pollutants so difficult to degrade aerobically? Redox stress limits 1,3-dichloprop-1-ene metabolism byPseudomonas pavonaceae

Chlorinated pollutants are hardly biodegradable under oxic conditions, but they can often be metabolized by anaerobic bacteria through organohalide respiration reactions. In an attempt to identify bottlenecks limiting aerobic catabolism of 1,3-dichloroprop-1-ene (1,3-DCP; a widely used organohalide) in Pseudomonas pavonaceae, the possible physiological restrictions for this process were surveyed. Flow cytometry and a bioluminescence reporter of metabolic state revealed that cells treated with 1,3-DCP experienced an intense stress that could be traced to the endogenous production of reactive oxygen species (ROS) during the metabolism of the compound. Cells exposed to 1,3-DCP also manifested increased levels of D-glucose-6-P 1-dehydrogenase activity (G6PDH, an enzyme key to the synthesis of reduced NADPH), observed under both glycolytic and gluconeogenic growth regimes. The increase in G6PDH activity, as well as cellular hydroperoxide levels, correlated with the generation of ROS. Additionally, the high G6PDH activity was paralleled by the accumulation of D-glucose-6-P, suggesting a metabolic flux shift that favours the production of NADPH. Thus, G6PDH and its cognate substrate seem to play an important role in P. pavonaceae under redox stress caused by 1,3-DCP, probably by increasing the rate of NADPH turnover. The data suggest that oxidative stress associated with the biodegradation of 1,3-DCP reflects a significant barrier for the evolution of aerobic pathways for chlorinated compounds, thereby allowing for the emergence of anaerobic counterparts.

Effect of 1,5-Anhydro-D-Fructose on the Inhibition of Adipogenesis in 3T3-L1 Adipocytes

1,5-Anhydro-D-fructose (1,5-AF) is a monosaccharide that shares a structural similarity to glucose. 1,5-AF is found in fungi, algae, Escherichia coli and rat liver and is produced by the degradation of starch and glycogen, which is catalyzed by the enzyme alpha-1,4-glucan lyase. However, the physiological role of 1,5-AF in mammalian tissues is not well understood. Here, we investigated the anti-obesity potential of 1,5-AF on adipogenesis in 3T3-L1 adipocytes. 1,5-AF caused a significant decrease in GPDH activity in 3T3-L1 preadipocytes and mature adipocytes without eliciting cytotoxicity, and inhibited cellular lipid accumulation through down-regulation of transcription factors such as PPARgamma and C/EBPalpha. 1,5-AF also induced dose-dependent phosphorylation of AMP-activated protein kinase (AMPK), a cellular energy sensor. However, the total AMPK protein content remained unchanged. Furthermore, 1,5-AF increased the levels of reactive oxygen species, an important upstream signal for AMPK activation in 3T3-L1 adipocytes. Our results show that 1,5-AF exerts anti-obesity action in vitro and suggest that 1,5-AF is potentially a novel preventative agent for obesity and other metabolic diseases.

Progesterone through progesterone receptors affects survival and metabolism of pig sperm

Progesterone receptors (PR) through interaction with the specific ligand progesterone (PRG), play a central coordinate role in different reproductive events. In this study conventional PR were identified in boar spermatozoa by Western blotting. Immunofluorescence techniques indicate that PR are located at sperm acrosomal region, suggesting a possible role in the oocyte fertilization events. Treatment with 17-hydroxyprogesterone (17-OHP) enhanced viability and induced cholesterol efflux, serine and tyrosine phosphorylation, p-Bcl2, p-Akt that are known hallmarks of capacitation in sperm. The analysis of the triglyceride contents, lipase and acyl-CoA dehydrogenase activities, as well as the G6PDH activity, was conducted so as to address whether there was an increase in energy expenditure via 17-OHP through the PR.Taken together these results, particularly the 17-OHP action on boar sperm lipid and glucose metabolism, give emphasis to the role of PR in sperm physiology within the oviductal environment. Moreover the present study highlights, the effect of PRG via PR on boar sperm survival, renewing the role of this hormone in male reproduction as candidate for boar fertility. Thus the present research contributes to further elucidating the role of progesterone on the physiological regulation of the most relevant spermatozoa functions for a successful fertilization.

Epigallocatechin gallate affects survival and metabolism of human sperm

Green tea and its major constituent epigallocatechin gallate (EGCG) have been extensively studied as potential treatment for a variety of diseases. We assessed the influence of EGCG on male fertilizing potential by analyzing different features of human sperm involved in capacitation process. Using human normozoospermic samples, we evaluated the effect of EGCG (2 μM, 20 μM, 60 μM) on sperm activities. Our results showed that lower doses of EGCG (from 2 to 20 μM) increased cholesterol efflux and tyrosine phosphorylation through the estrogen receptor (ER), since ICI 182,780, a specific ER antagonist, abrogated 20 μM EGCG effects. Besides, we evidenced that EGCG at similar concentrations, increased sperm motility, viability, and phosphorylation of proteins controlling cell survival such as Bcl2, Akt, and Src, via ER. Furthermore, we observed reduction of triglycerides content, induction of lipase, as well as the G6PDH activity. These results address to an increase in energy expenditure. On the contrary, treatment of 60 μM EGCG produced opposite effects that still appear after ICI cotreatment. These results provide a novel mechanism involving ERs through which low doses of EGCG exerted benefits to sperm physiology, also detected data evidence the adverse action of high EGCG concentrations probably related to its prooxidant and antiestrogenic potential.

Carbonyl stress and a combination of astaxanthin/vitamin C induce biochemical changes in human neutrophils

The purpose of the present study was to find out whether co-treatment of human neutrophils with high glucose and methylglyoxal (MGO) can alter the biochemical parameters of human neutrophils. We also examined if astaxanthin associated with vitamin C can improve those biochemical parameters. Neutrophils from healthy subjects were treated with 20mM of glucose and 30μM MGO followed or not by the addition of the antioxidants astaxanthin (2μM) and vitamin C (100μM). MGO/high glucose treatment reduced the phagocytic capacity and the G6PDH, total/SOD and GR activities. Additionally, there was an increase in the activity of myeloperoxidase (MPO) with consequent increase in the hypochlorous acid production, CAT activity and in the release of IL-6 cytokine without changes in intracellular calcium mobilization. Our study also shows that the association of astaxanthin with vitamin C greatly improved neutrophil phagocytic capacity, decreasing all reactive oxygen species measured, pro-inflammatory IL-1β and TNF-α release, MPO activity and HClO production. The combination of astaxanthin with vitamin C alone has more antioxidant and anti-inflammatory effects than when they were in the presence of MGO/high glucose. Injury to the function of neutrophils due to high glucose and methylglyoxal appears not to involve oxidative stress or calcium release. The association of antioxidants astaxanthin and vitamin C promoted a significant improvement in the function of neutrophils and in the redox status.