Decreased Lactate Dehydrogenase Activity Research Articles

Disruption of the S41 Peptidase Gene in Mycoplasma mycoides capri Impacts Proteome Profile, H2O2 Production, and Sensitivity to Heat Shock

Members of the Mycoplasma mycoides cluster are among the most virulent of the mycoplasmas, causing worldwide economically significant diseases of cattle and goats. A distinguishing phenotype among the members of the cluster is the ability to degrade casein. The MMCAP2_0241 gene, an S41 peptidase, confers the proteolytic phenotype in Mycoplasma mycoides subsp. capri GM12. In order to determine the impact of disruption of the gene, we used differential proteome profiling to compare the M. mycoides subsp. capri wild type with a mutant lacking the proteolytic phenotype. Disruption of MMCAP2_0241 resulted in altered phenotypes reminiscent of M. mycoides subsp. mycoides SC and had significant impacts on the proteome profile of the microbe. The mutant exhibited increased production of hydrogen peroxide, decreased lactate dehydrogenase activity, and increased sensitivity to heat shock.

Quercetin supplementation: insight into the potentially harmful outcomes of neurodegenerative prevention

Dietary antioxidant supplements have been considered for the prevention of neuronal oxidative injury and death. Recent studies indicate that excessive antioxidants could exert adverse effects, thereby questioning the safety of prolonged supplementation. The aim of our study was to investigate the effects of quercetin (up to 150 μM), the ubiquitous plant-derived flavonoid and highly potent scavenger of reactive oxygen species (ROS) on healthy P19 neurons, in order to assess the efficacy and safety of its long-term use in neurodegenerative prevention. Although exposure for 24 h to quercetin did not compromise neuronal survival, morphological examination revealed diminished neuronal branching, a finding probably related to an observed decrease in lactate dehydrogenase activity. Using 2',7'-dichlorofluorescin diacetate and dot-blot analysis, we found reduced basal levels of ROS and 4-hydroxy-2-nonenal, a biomarker of lipid peroxidation, confirming the antioxidative mechanism of quercetin action. Unexpectedly, quercetin also depleted intracellular glutathione content. Reverse transcriptase PCR and western blot analysis showed depletion of total RNA amount and changes in the expression of cell survival regulating genes Bcl-2, p53, and c-fos. Nuclear condensation and caspase-3/7 activity, phenomena related to programmed cell death cascade, were not affected. The potential risk of observed changes indicates that quercetin-enriched supplements should be taken with caution. The diversity of quercetin effects and complexity of possible intracellular interactions between affected genes pointed out the necessity for additional pharmacological and toxicological studies in order to better elucidate the mechanisms of quercetin action and to recognize its potential side effects at higher doses and during long-term administration.

EFFECTS OF WEDELOLACTONE ON THE PROTEIN EXPRESSION OF APOPTOSIS-ASSOCIATED BCL-2, BAX AND PARP (89KD) IN THE PRIMARY CULTURED RAT CARDIOMYOCYTES SUBJECTED TO ANOXIA/REOXYGENATION INJURY

ObjectivesTo study the effects of Wedelolactone (Wed) on the protein expression of apoptosis-associated Bcl-2, Bax and PARP (89KD) in the primary cultured rat cardiomyocytes subjected to anoxia/reoxygenation injury.MethodsThe primary cultured...

Protective effect of 20-hydroxyeicosatetraenoic acid (20-HETE) on adriamycin-induced toxicity of human renal tubular epithelial cell (HK-2)

20-Hydroxyeicosatetraenoic acid is a cytochrome P4504A11 metabolite of arachidonic acid that plays an important role in the regulation of human renal functions. In the present study, we investigated the role of 20-hydroxyeicosatetraenoic acid on adriamycin induced toxicity in human renal tubular epithelial cells. Results showed that cell viability was decreased significantly and lactate dehydrogenase activity was increased significantly in a concentration-dependent manner when human renal tubular epithelial cells were incubated with adriamycin (10−7–10−3mol/l) for 24h. In contrast, 20-hydroxyeicosatetraenoic acid (0.1, 1, 10, 50μmol/l) increased cell survival and decreased lactate dehydrogenase activity concentration dependently in human renal tubular epithelial cells. When 20-hydroxyeicosatetraenoic acid (10, 50μmol/l) was co-administered with adriamycin (10−3mol/l), it significantly increased cell viability and decreased lactate dehydrogenase activity. On the other hand, N-hydroxy-N′-(4-butyl-2-methylphenyl)formamidine (HET-0016) (1μM), a selective inhibitor of 20-hydroxyeicosatetraenoic acid synthesizing enzyme exaggerated cell viability reduction and lactate dehydrogenase activity augmentation induced by adriamycin. Adriamycin suppressed the expression of cytochrome P4504A11 gene and its protein production in human renal tubular epithelial cells. Furthermore, adriamycin was more effective than N-hydroxy-N′-(4-butyl-2-methylphenyl)formamidine at lowering the expression of cytochrome P4504A11 gene and its protein. These results suggest that 20-hydroxyeicosatetraenoic acid may protect adriamycin-induced toxicity of human renal tubular epithelial cells, meanwhile, adriamycin-induced toxicity of human renal tubular epithelial cells possibly involves inhibiting cytochrome P4504A11 expression.

The role of PI3K-Akt signal pathway in protection of myocardium agalnst ischemia-reperfusion injury by diazoxide postconditioning in rats

Objective To investigate the role of phosphatidylinositol 3-kinase/protein-serine-threonine ki- nases （PI3K-Akt） signal pathway in the protection of myocardium against ischemia-repeffusion （I/R） injury by diazoxide postconditioning in rats. Methods Thirty-six male SD rats weighing 250-300 g were randomly divided into 4 groups using random number table （ n = 9 each） ： group I/R; group diazoxide （group D） ; group wortmannin （ PI3K inhibitor） （group W） and group wortmannin ＋ diazoxide （group WD）. The animals were anesthetized with intraperitoneal 10% chloral hydrate 4 ml/kg, intubated and mechanically ventilated. Myocardial I/R was produced by 30 min occlusion of left anterior descending coronary artery followed by 120 rain repeffusion. 0.1% dimethyl sulfoxide （the vehicle for diazoxide and wortmannin ）/diazoxide at 0.47 mg. kg-1 . min-I/wortmannin at 1 μg.kg-1 .min-1 was infused for 15 rain starting from 25 min of isehemia in groups I/R,D and W respectively. In group DW wortmannin was infused at 5 rain before diazoxide infusion. Blood samples were collected from left ventricle at the end of 120 min repeffusion for measurement of lactate dehydrogenase （LDH） activity. Myocardial in-farct size was measured. Myocardial specimens were obtained for determination of apoptosis index （the number of apoptotic cells/the total number of ceils examined） and expression of Bcl-2 and Bax. Bcl-2/Bax ratio was calculat- ed. Results Diazoxide postconditioning significantly decreased plasma LDH activity, myocardial infarct size, apoptosis index and Bax expression in myocardium and increased myocardial Bcl-2 expression and Bcl-2JBax ratio in group D compared with group I/R. Wortmannin partly counteracted the protective effects of diazoxide postconditioning against myocardiul infarct. There was no significant difference in the above variables between groups I/R and W. Conclusion PI3K-Akt signal pathway is involved in the protective effects of diazoxide postconditioning on myocardium against I/R injury. Key words: 1-Phosphatidylinositol 3-kinase; Protein-serine-threonine kinases; Diazoxide; Myocardial reperfusion injury

Role of dopamine D2 receptors in ischemia/reperfusion induced apoptosis of cultured neonatal rat cardiomyocytes

BackgroundMyocardial ischemia/reperfusion injury is the major cause of morbidity and mortality for cardiovascular diseases. Dopamine D2 receptors are expressed in cardiac tissues. However, the roles of dopamine D2 receptors in myocardial ischemia/reperfusion injury and cardiomyocyte apoptosis are unclear. Here we investigated the effects of both dopamine D2 receptors agonist (bromocriptine) and antagonist (haloperidol) on apoptosis of cultured neonatal rat ventricular myocytes induced by ischemia/reperfusion injury.MethodsMyocardial ischemia/reperfusion injury was simulated by incubating primarily cultured neonatal rat cardiomyocytes in ischemic (hypoxic) buffer solution for 2 h. Thereafter, these cells were incubated for 24 h in normal culture medium.ResultsTreatment of the cardiomyocytes with 10 μM bromocriptine significantly decreased lactate dehydrogenase activity, increased superoxide dismutase activity, and decreased malondialdehyde content in the culture medium. Bromocriptine significantly inhibited the release of cytochrome c, accumulation of [Ca2+]i, and apoptosis induced by ischemia/reperfusion injury. Bromocriptine also down-regulated the expression of caspase-3 and -9, Fas and Fas ligand, and up-regulated Bcl-2 expression. In contrast, haloperidol (10 μM) had no significant effects on the apoptosis of cultured cardiomyocytes under the aforementioned conditions.ConclusionsThese data suggest that activation of dopamine D2 receptors can inhibit apoptosis of cardiomyocytes encountered during ischemia/reperfusion damage through various pathways.

Effects of Caulophine on Caffeine‐induced Cellular Injury and Calcium Homeostasis in Rat Cardiomyocytes

Caulophine is a novel fluorenone alkaloid isolated from the radix of Caulophyllum robustum Maxim. Caulophine showed high affinity for the rat myocardial cell membrane as assessed by cell membrane chromatography, suggesting that the compound may exert bioactivity in the heart. It is known that calcium plays an important role in the pathogenesis of ischaemic heart disease, and caffeine can cause calcium overload in cardiomyocytes by inducing calcium release from the sarcoplasmic reticulum. Therefore, the present study evaluated the effects of caulophine on caffeine-induced injury and calcium homeostasis in cardiomyocytes. Cardiomyocytes were pre-treated with caulophine before exposure to caffeine or potassium chloride (KCl). Cell viability was assayed using the MTT method, and lactate dehydrogenase (LDH) and malondialdehyde (MDA) were measured spectrophotometrically. Caulophine-pre-treated cardiomyocytes were incubated with Fluo-3/AM, and then caffeine or KCl was used to induce Ca(2+) overload. The total intracellular Ca(2+) concentration was measured by flow cytometry. Fluorescence densities of single cardiomyocytes were detected using a confocal microscope. Caulophine increased the viability of caffeine-injured cardiomyocytes and decreased LDH activity and MDA level in cardiomyocytes. Furthermore, caulophine significantly decreased the total intracellular free Ca(2+) concentration and intracellular calcium release in cardiomyocytes in response to caffeine. However, the same concentrations of caulophine did not affect KCl-induced calcium influx. Our results suggest that caulophine protects cardiomyocytes from caffeine-induced injury as a result of calcium antagonism. This finding provides a basis for further study and development of caulophine as a new calcium antagonist for treating ischaemic cardiovascular diseases.

Cyclophosphamide and ascorbic acid-mediated ultrastructural and biochemical changes in Dalton's lymphoma cells in vivo

Cyclophosphamide, an antineoplastic drug effective against a wide variety of cancers is cytotoxic to normal cells also. Ascorbic acid (vitamin C) at higher concentrations possesses cytotoxic effects and it can also enhance the cytotoxicity of 5-fluorouracil in a dose-dependent manner in mouse lymphoma. In the present study, effect of cyclophosphamide treatment alone and in combination with ascorbic acid in vivo on the ultrastructure and some biochemical changes in Dalton's lymphoma tumor cells were investigated . Cyclophosphamide treatment causes disappearance of cell membrane processes, thickening and reduction in the number of mitochondrial cristae as well as the manifestation of rounded shape of mitochondria. The combination treatment with ascorbic acid plus cyclophosphamide caused further changes in tumor cells showing disintegration in the cell surface membrane, disruption in the nuclear membrane and roundish mitochondria with reduction and disruption in the mitochondrial cristae. The observed ascorbic acid plus cyclophosphamide-mediated decrease in reduced glutathione (GSH) in tumor cells may play an important role in the antitumor activity of cyclophosphamide by weakening cellular antioxidant-mediated defense mechanism, thereby increasing tumor cell's susceptibility to cell death. The cyclophosphamide-mediated decrease in lactate dehydrogenase activity in tumor cells and simultaneous increase in ascites supernatant may possibly indicate alteration in the membrane permeability of tumor cells for lactate dehydrogenase as well as tumor cell injury. Further investigation should determine detailed mechanism(s) involved in cyclophosphamide-induced ultrastructural and biochemical changes in tumor cells.

A novel, orally active LPA(1) receptor antagonist inhibits lung fibrosis in the mouse bleomycin model.

The aim of this study was to assess the potential of an antagonist selective for the lysophosphatidic acid receptor, LPA(1), in treating lung fibrosis We evaluated the in vitro and in vivo pharmacological properties of the high affinity, selective, oral LPA(1)-antagonist (4'-{4-[(R)-1-(2-chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid (AM966). The potency and selectivity of AM966 for LPA(1) receptors was determined in vitro by calcium flux and cell chemotaxis assays using recombinant and native cell cultures. The in vivo efficacy of AM966 to reduce tissue injury, vascular leakage, inflammation and fibrosis was assessed at several time points in the mouse bleomycin model. AM966 was a potent antagonist of LPA(1) receptors, with selectivity for this receptor over the other LPA receptors. In vitro, AM966 inhibited LPA-stimulated intracellular calcium release (IC(50)= 17 nM) from Chinese hamster ovary cells stably expressing human LPA(1) receptors and inhibited LPA-induced chemotaxis (IC(50)= 181 nM) of human IMR-90 lung fibroblasts expressing LPA(1) receptors. AM966 demonstrated a good pharmacokinetic profile following oral dosing in mice. In the mouse, AM966 reduced lung injury, vascular leakage, inflammation and fibrosis at multiple time points following intratracheal bleomycin instillation. AM966 also decreased lactate dehydrogenase activity and tissue inhibitor of metalloproteinase-1, transforming growth factor beta1, hyaluronan and matrix metalloproteinase-7, in bronchoalveolar lavage fluid. These findings demonstrate that AM966 is a potent, selective, orally bioavailable LPA(1) receptor antagonist that may be beneficial in treating lung injury and fibrosis, as well as other diseases that are characterized by pathological inflammation, oedema and fibrosis.

The protective effects of Polygonum multiflorum stilbeneglycoside preconditioning in an ischemia/reperfusion model of HUVECs

To investigate the protective effects of preconditioning human umbilical vein endothelial cells (HUVECs) with Polygonum multiflorum stilbeneglycoside (PMS) under anoxia/reoxygenation (A/R), and the mechanism of protection. Prior to A/R, HUVECs were incubated with PMS (0.6 x 10(-11), 1.2 x 10(-11), or 2.4 x 10(-11) mol/L) for 3 h. Cell injury was subsequently evaluated by measuring cell viability with an MTT assay and lactate dehydrogenase (LDH) release, whereas lipid peroxidation was assayed by measuring malondialdehyde (MDA) content. Antioxidant capacity was quantified by superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity. Nitric oxide (NO) production was determined by nitrite accumulation. Endothelial NO synthase (eNOS) and inducible NOS (iNOS) protein expression was detected by Western blotting. Guanylate cyclase activity and cyclic GMP (cGMP) activity were assessed by an enzyme immunoassay kit. PMS incubation attenuated A/R-induced injury in a concentration-dependent manner, as evidenced by a decrease in LDH activity and an increase in cell viability. PMS exerted its protective effect by inhibiting the A/R-mediated elevation of MDA content, as well as by promoting the recovery of SOD and GSH-Px activities. Additionally, PMS incubation enhanced NO and cGMP formation by increasing iNOS expression and guanylate cyclase activity. The protective effects of PMS were markedly attenuated by NOS inhibitor L-NAME, soluble guanylate cyclase inhibitor ODQ or PKG inhibitor KT5823. PMS preincubation resulted in the enhancement of antioxidant activity and anti-lipid peroxidation. The NO/cGMP/cGMP-dependent protein kinase (PKG) signaling pathway was involved in the effect of PMS on HUVECs.

Reduced diagnostic value of lactate dehydrogenase (LDH) in the presence of radiographic contrast media

Isoforms of the enzyme lactate dehydrogenase (LDH) were found in almost all cells of the organism and an elevated activity of LDH in the circulation is thought to be a clear indicator of elevated cell destruction coinciding with an increased release of components from the cellular cytoplasm, e.g. LDH. Here, we report on an in-vitro examination to test whether radiographic contrast media (RCM) could induce cell destruction followed by an increase in LDH release. The RCM were tested in non-flow cultures of human umbilical venous endothelial cells (HUVEC) of the fourth passage seeded on extracellular matrix and the results were compared to those from control cultures not exposed to contrast media. The examination revealed that the addition of contrast media to the cell culture media supplemented with pooled human serum (HSP) as source of exogenous LDH was followed by a strong decrease in LDH activity both in the absence and presence of HUVEC. Within 1.5 min after the addition of contrast media to the culture medium supplemented with HSP (30% vol of the culture medium were replaced by either of two contrast media, Iodixanol or Iopromide) the LDH activity decreased about 80% compared to the initial values. In contrast, the LDH activity did not change in cell culture media not supplemented with RCM. The partial replacement of HSP supplemented cell culture medium by RCM will cause a dilution of cell culture medium constituents. The decrease of LDH activity, however, was much stronger than the decrease thought to be attributable to the effects of dilution of cell culture medium, so that the role of dilution seems to be a minor one in this case. It has to be assumed that the RCM could interact with the LDH available in the culture medium as well as with the substrates delivered with the measurement system for the assessment of LDH activity, so that both, the amount of LDH and the activities of enzymes involved might be influenced. In the presence of HUVEC a similar effect was observed. Here, a little less strong decrease of LDH activity occurred compared to the decrease in cell culture medium without HUVEC. This was unexpected because a considerable amount of HUVEC were detached after the addition of contrast media and many of these cells were damaged seriously so that a significant amount of endogenous LDH should have been released. These unexpected results make it necessary to re-evaluate those past time examinations focussed on cell damage/destruction in the presence of contrast media, where the measurement of LDH activity was used as indicator or cell vitality and where cell decease rates were correlated to questionable toxic influences. According to the results of the examination reported here it is difficult to uphold the interpretation of recently published findings that contrast media almost exclusively induce cellular apoptosis and not necrosis.

Possible roles of myostatin and PGC-1α in the increase of skeletal muscle and transformation of fiber type in cold-exposed chicks: Expression of myostatin and PGC-1α in chicks exposed to cold

This study examined the hypothesis that myostatin and PGC-1α are involved in the increase in skeletal muscle mass and transformation of fiber type in cold-exposed chicks. One-week-old chicks were exposed to acute (24 h) or long-term (8 d) cold at 4 °C or kept warm at 30 °C. Acute cold exposure induced a significant increase in the skeletal muscle weight and the ratio of slow- to fast-fiber specific troponin I expression (sTnI/fTnI), accompanied by a significant decrease in lactate dehydrogenase activity. Expression of myostatin mRNA in the muscle was significantly lower in cold-exposed chicks than in the controls, whereas PGC-1α mRNA expression was significantly enhanced. These changes in the gene expression rapidly returned to the levels of the control chicks after the end of cold exposure, whereas the changes in fiber type and enzymatic activity were not resumed within 24 h after removal of cold exposure. On the other hand, long-term exposure to cold resulted in a remarkable increase in skeletal muscle weight, accompanied by a significant increase in the ratio of sTnI/fTnI and the enzymatic activities of cytochrome oxidase and lactate dehydrogenase. However, the expression level of myostatin mRNA in cold-exposed chicks was not different from that in their age-matched control chicks and that of PGC-1α mRNA was significantly lower than in the controls. These results indicate that myostatin and PGC-1α expression in the skeletal muscle rapidly change in response to acute cold, suggesting the possibility that these two genes could be involved in the increase in muscle mass and transformation of fiber type, respectively, at the initial stage of adaptation in cold-exposed chicks.

The effect of prostacyclin on the morphological and enzymatic properties of CNS cultures exposed to anoxia.

Organotypic cultures of rat cerebella were exposed to anoxia for 30 min. Prostacyclin (0.5 micrograms/ml medium) or 0.05 M Tris buffer pH 7.5 were added to the cultures immediately before exposure to anoxia. Cultures for histological, histoenzymatic and electron microscopic examination were taken immediately, 30 min and one, three, 24 and 72 h following anoxia. Cultures pretreated with prostacyclin showed a marked decrease in lactate dehydrogenase activity. Electron microscopic examination revealed that severe swelling and degenerative changes of neurons and glia induced by anoxia were much less pronounced in cultures treated with PGI2. Prostacyclin pretreatment also resulted in the appearance of morphological features of activation in glial cells. The results indicate that prostacyclin exerts a cytoprotective effect on the nerve tissue in vitro, predominantly reducing cell swelling and acidosis. This direct protection, besides vascular action of prostacyclin, may provide a basis for its beneficial clinical effect in ischemic stroke.

Enhanced hyaluronic acid production of Streptococcus zooepidemicus by an intermittent alkaline-stress strategy

Enhanced hyaluronic acid (HA) production of Streptococcus zooepidemicus by redirecting carbon flux through an intermittent alkaline-stress strategy. pH value was kept at 7.0 for the first 6 h, and then intermittently switched to 8.5 for 1 h and back to 7.0 for 1 h until the end of fermentation at 16 h (one pH switch cycle every 2 h). With this intermittent alkaline-stress strategy, HA production was increased to 6.5 +/- 0.2 g l(-1) from 5.0 +/- 0.1 g l(-1) of the control, in which pH was always kept at 7.0. In addition, biomass and lactic acid concentration decreased by 24% and 14%, respectively, while acetic acid concentration increased by 10% under intermittent alkaline stress. The redirection of carbon flux from lactic acid to acetic acid was further supported by the decreased lactate dehydrogenase activity and the increased acetate kinase activity. As indicated by the increased NADH oxidase (NOX) activity, intermittent alkaline-stress induced a more oxidative intracellular environment which would facilitate HA synthesis. Overproduction of HA was realized by redirecting carbon flux through the proposed intermittent alkaline-stress strategy. This study clearly demonstrated the importance of metabolic-pathway-analysis based fermentation strategy in industrial processes and provided an alternative optimization approach for high viscosity fermentation.

Baseline blood biochemistry of Australian green turtles (Chelonia mydas) and effects of exposure to the toxic cyanobacterium Lyngbya majuscula

Quantifying health in wild marine turtles is challenging because reptiles have characteristically wide-ranging normal reference values for many indicators of health and because of the shortage of population-specific baseline data for wild animals. We measured blood biochemistry profiles (calcium, magnesium, sodium, lactate dehydrogenase (LDH), urea, cholesterol, triglycerides, and glucose) of green turtles (Chelonia mydas) in Moreton and Shoalwater Bays, Australia, and compared them in relation to capture site, age, sex and exposure to harmful algal blooms of the toxic cyanobacteria Lyngbya majuscula. Turtles were considered to be clinically healthy when no external injuries or lesions were observed and there was no evidence of disease or emaciation. Differences in blood profiles were detected between sites, but not between age groups or sexes. Turtles that were exposed to L. majuscula generally had lower plasma glucose concentrations and decreased LDH activity, which may represent a metabolic downregulation resulting from food limitation. This study provides the first blood biochemistry reference values for green turtles in Queensland, Australia, that can be used in future assessments of green turtles in these foraging habitats.

Mechanisms of Copper Ion Mediated Huntington's Disease Progression

Huntington's disease (HD) is caused by a dominant polyglutamine expansion within the N-terminus of huntingtin protein and results in oxidative stress, energetic insufficiency and striatal degeneration. Copper and iron are increased in the striata of HD patients, but the role of these metals in HD pathogenesis is unknown. We found, using inductively-coupled-plasma mass spectroscopy, that elevations of copper and iron found in human HD brain are reiterated in the brains of affected HD transgenic mice. Increased brain copper correlated with decreased levels of the copper export protein, amyloid precursor protein. We hypothesized that increased amounts of copper bound to low affinity sites could contribute to pro-oxidant activities and neurodegeneration. We focused on two proteins: huntingtin, because of its centrality to HD, and lactate dehydrogenase (LDH), because of its documented sensitivity to copper, necessity for normoxic brain energy metabolism and evidence for altered lactate metabolism in HD brain. The first 171 amino acids of wild-type huntingtin, and its glutamine expanded mutant form, interacted with copper, but not iron. N171 reduced Cu2+ in vitro in a 1∶1 copper∶protein stoichiometry indicating that this fragment is very redox active. Further, copper promoted and metal chelation inhibited aggregation of cell-free huntingtin. We found decreased LDH activity, but not protein, and increased lactate levels in HD transgenic mouse brain. The LDH inhibitor oxamate resulted in neurodegeneration when delivered intra-striatially to healthy mice, indicating that LDH inhibition is relevant to neurodegeneration in HD. Our findings support a role of pro-oxidant copper-protein interactions in HD progression and offer a novel target for pharmacotherapeutics.

Biocompatible, biodegradable and sterically stabilized phospholipid nanomicelles improve cryopreservation of oral keratinocytes: A preliminary investigation

In this proof-of principle study, we determined whether biocompatible, biodegradable and sterically stabilized phospholipid nanomicelles (SSNMs) improve viability and membrane integrity of cryopreserved oral keratinocytes. Cultured chemically transformed hamster oral keratinocytes were frozen gradually with and stored in liquid nitrogen in the presence of 10% dimethylsulfoxide (DMSO) or SSNMs composed of distearoylphosphatidyl-ethanolamine- N-poly(ethylene glycol) 2000 (size, 17 ± 1 nm; 0.1 and 1.0 nmol). Forty-eight hours later, cells were thawed and their viability was determined. Lactate dehydrogenase (LDH) activity in cell lysates and supernatants was quantified as well. SSNMs evoked a significant, concentration-dependant increase in cell viability in comparison to 10% DMSO ( p < 0.05). There was also a significant decrease in LDH activity in the supernatant of cells cryopreserved with SSNMs in comparison to 10% DMSO ( p < 0.05). These data indicate that SSNMs improve cryopreservation of oral keratinocyte by promoting cell viability and plasma membrane integrity. We suggest that SSNMs should be further developed as a novel nanocryopreservative for keratinocytes.

A peroxynitrite decomposition catalyst: FeTPPS confers cardioprotection during reperfusion after cardioplegic arrest in a working isolated rat heart model

Heart transplant is considered to be an extremely severe ischemia-reperfusion sequence. Post-ischemic dysfunction triggers multiple processes especially oxidative stress, but the mechanisms remain unclear. Free radical interactions lead to peroxynitrite generation, which seems to be involved in early post-transplant heart failure. The aim of this study was to evaluate the potential impact of a peroxynitrite decomposition catalyst: FeTPPS (5,10,15,20-tetrakis-[4-sulfonatophenyl]-porphyrinato-fer[III]) and pyruvate on myocardial functional recovery after cardioplegic arrest using an experimental protocol in rat hearts. Isolated working rat hearts were subjected to ischemia (4 h at 4 degrees C in cardioplegic solutions), followed by 45 min of reperfusion. Four groups were constituted: control, pyruvate: (2 mm) added to cardioplegic and Ringer-lactate solutions, FeTPPS: (10 microm) perfused during the reperfusion, and a combination of both treatments. Lactate dehydrogenase (LDH) activity was assessed during the reperfusion to evaluate the level of cardiac injury. Oxidative stress was evaluated on heart slices using a fluorescent probe: dihydroethidium, and the collagen content was assessed using picro-Sirius coloration. Global post-ischemic recovery in the control group was about 35% of pre-ischemic values. Results showed that addition of pyruvate led to an increase in myocardial function and to a decrease in LDH activity released during the reperfusion. FeTPPS protected against injury after cardioplegic arrest during reperfusion. No additive effect of the two treatments (pyruvate + FeTPPS) was observed. The collagen content was better preserved in the FeTPPS group than in the control and pyruvate groups. In conclusion, this study shows that peroxynitrite plays an important role in the functional and cellular alterations associated with cardiac ischemia-reperfusion sequences and confirms that pyruvate helped to preserve myocardial function. The use of the peroxynitrite decomposition catalyst (FeTPPS) may help to improve myocardial preservation during a prolonged ischemia sequence.

Inactivation of lactate dehydrogenase by several chemicals: Implications for in vitro toxicology studies

Lactate dehydrogenase (LDH) release is frequently used as an end-point for cytotoxicity studies. We have been unable to measure LDH release during studies using para-aminophenol (PAP) in LLC-PK 1 cells. When LLC-PK 1 cells were incubated with either PAP (0–10 mM) or menadione (0–1000 μM), viability was markedly reduced when assessed by alamar Blue or total LDH activity but not by release of LDH into the incubation medium. In addition, we incubated cells with PAP or menadione and compared LDH activity using two different assays. Both assays confirmed our observation of decreased LDH activity in cell lysates without corresponding increases in LDH activity in incubation media. Using purified LDH and 10 mM PAP, we found that PAP produced loss of LDH activity that was inversely proportional to the amount of LDH initially added. In additional experiments, we incubated 0.5 units of LDH for 1 h with varying concentrations of PAP, menadione, hydrogen peroxide (H 2O 2) or cisplatin. All four chemicals produced concentration-dependent decreases in LDH activity. In previous experiments, inclusion of antioxidants such as reduced glutathione (GSH) and ascorbate protected cells from PAP toxicity. GSH (1 mM) preserved LDH activity in the presence of toxicants while ascorbate (1 mM) only prevented LDH loss induced by PAP. These studies suggest that LDH that is released into the incubation medium is susceptible to degradation when reactive chemicals are present.

Effect of the silk protein sericin on the production of adenovirus‐based gene‐therapy vectors

Adenoviral vectors are extensively used as gene-delivery vehicles in gene therapy. They are usually produced by HEK-293 cell (human embryonic kidney-293 cell) culture, which requires specially formulated serum-free medium, the cost of which is considerable or by supplementation with FBS (fetal bovine serum). The risk of infectious diseases such as BSE (bovine spongiform encephalopathy) and endogenous retrovirus derived from cattle is a serious concern. The present study reports the use of sericin protein derived from silkworm (Bombyx mori) as an effective supplement instead of FBS. Without FBS, HEK-293 cells significantly proliferated in the presence of 0.025-0.4% sericin, especially at 0.1%, but the effect was inferior to that of FBS. When a lower titre [MOI (multiplicity of infection) 0.03] of adenoviral vector pAxCAiLacZ was used as the inoculum, HEK-293 cells in the presence of 0.1% sericin produced a nearly 3-fold higher vector titre than culture in the presence of 5% (v/v) FBS. However, when a higher vector titre (MOI 3.7) was used as the inoculum, HEK-293 cells in the presence of sericin produced a slightly higher vector titre than in the presence of FBS, which might suggest that HEK-293 cells produce a maximum amount when a higher vector titre is used as the inoculum. These increases in vector production with sericin were confirmed by LacZ (beta-galactosidase reporter gene) activity assay. Supplementation with sericin decreased lactate dehydrogenase activity, an indicator of cell death, suggesting that sericin improved cell survival; hence, prolonging the culture period might be one of the reasons for increased vector production. On the basis of these results, sericin peptide seems to be a potent and effective alternative supplement for production of adenoviral vectors without such risks as BSE and retrovirus.