Combination of inhibitors for two glycolytic enzymes portrays high synergistic efficacy against Cryptosporidium parvum.

Cryptosporidium is an intracellular protozoan parasite that causes serious enteric disease in humans and in a wide range of animals worldwide. Despite its high prevalence, no effective therapeutic drugs are available against life-threatening cryptosporidiosis in at-risk populations including malnourished children, immunocompromised patients, and neonatal calves. Thus, new efficacious drugs are urgently needed to treat all susceptible populations with cryptosporidiosis. Unlike other apicomplexans, Cryptosporidium parvum lacks the tricarboxylic acid cycle and the oxidative phosphorylation steps, making it solely dependent on glycolysis for metabolic energy production. We have previously reported that individual inhibitors of two unique glycolytic enzymes, the plant-like pyruvate kinase (CpPyK) and the bacterial-type lactate dehydrogenase (CpLDH), are effective against C. parvum, both in vitro and in vivo. Herein, we have derived combinations of CpPyK and CpLDH inhibitors with strong synergistic effects against the growth and survival of C. parvum, both in vitro and in an infection mouse model. In infected immunocompromised mice, compound combinations of NSC303244 + NSC158011 and NSC252172 + NSC158011 depicted enhanced efficacy against C. parvum reproduction and ameliorated intestinal lesions of cryptosporidiosis at doses fourfold lower than the total effective doses of individual compounds. Importantly, unlike individual compounds, NSC303244 + NSC158011 combination was effective in clearing the infection completely without relapse in immunocompromised mice. Collectively, our study has unveiled compound combinations that simultaneously block two essential catalytic steps for metabolic energy production in C. parvum to achieve improved efficacy against the parasite. These combinations are, therefore, lead compounds for the development of a new generation of efficacious anti-cryptosporidial drugs.

A novel peptide suppresses adipogenic differentiation through activation of the AMPK pathway.

Obesity rates have risen rapidly over the past several decades and obesity is now a global public health challenge. The reduction of excessive adipogenesis is thought to be an effective intervention for obesity and obesity-related metabolic diseases such as type 2 diabetes. In this study, a novel peptide PDBSN was identified that functions to suppress adipogenesis. In both human preadipocytes and mouse adipose-derived stem cells (ADSCs), PDBSN exhibited a suppressive effect on the accumulation of lipids and the expression of genes as well as their corresponding proteins (CCAAT/enhancer binding protein (C/EBP)ß, C/EBPα and nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ)) relevant to adipogenic cell differentiation. Although adipogenesis decreased, the preadipocyte number and proliferation were not influenced by the PDBSN treatment. Apoptosis and the cell cycle were also determined to not have a role in the action of PDBSN. Mechanistically, the activity of the AMPK (adenosine 5'-monophosphate-activated protein kinase) pathway was markedly increased upon PDBSN treatment. Moreover, treatment of preadipocytes with compound C, a selective AMPK inhibitor, abolished the effect of PDBSN in anti-adipogenesis, suggesting that the function of PDBSN relied on the AMPK pathway. These results suggest an effective role for PDBSN in suppressing adipogenesis and show potential for anti-obesity drug discovery.

Development of antifertility vaccine using sperm specific proteins.

Sperm proteins are known to be associated with normal fertilization as auto- or iso-antibodies to these proteins may cause infertility. Therefore, sperm proteins have been considered to be the potential candidate for the development of antifertility vaccine. Some of the sperm proteins proved to be promising antigens for contraceptive vaccine includes lactate dehydrogenase (LDH-C4), protein hyaluronidase (PH-20), and Eppin. Immunization with LDH-C4 reduced fertility in female baboons but not in female cynomolgus macaques. Active immunization with PH-20 resulted in 100 per cent inhibition of fertility in male guinea pigs but it induced autoimmune orchitis. Immunization with Eppin elicited high antibody titres in 78 per cent of immunized monkeys and induced infertility but the immunopathological effect of immunization was not examined. Human sperm antigen (80 kDa HSA) is a sperm specific, highly immunogenic and conserved sperm protein. Active immunization with 80 kDa HSA induced immunological infertility in male and female rats. Partial N-terminal amino acid sequence of 80 kDa HSA (Peptide NT) and its peptides (Peptides 1, 2, 3 and 4) obtained by enzymatic digestion did not show homology with any of the known proteins in gene bank. Peptides NT, 1, 2 and 4 were found to mimic immunobiological activity of native protein. Passive administration of antibodies to peptides NT, 1, 2 and 4 induced infertility in male and female rats and peptide 1 was found to be most effective in suppressing fertility. Active immunization with keyhole limpet haemocynin (KLH) conjugated synthetic peptide 1 impaired fertility in all the male rabbits and six of the seven male marmosets. The fertility was restored following decline in antibody titre. All these findings on 80 kDA HAS suggest that the synthetic Peptide-1 of 80 kDa HSA is the promising candidate for development of male contraceptive vaccine.

Chlorhexidine, ethanol, lipopolysaccharide and nicotine do not enhance the cytotoxicity of a calcium hydroxide pulp capping material.

AIM: To determine whether cells pre-stressed by known cytotoxic or inflammatory agents are more susceptible to the deleterious effects of a calcium hydroxide formulation used in pulp capping. METHODOLOGY: Adult human dermal fibroblasts were treated for 48 h with 0.001% chlorhexidine, 0.2% ethanol, 5 µg mL(-1) Escherichia coli lipopolysaccharide (LPS) or 0.05 mmol L(-1) nicotine. Cells were subsequently treated with the soluble materials extracted from Dycal pellets for an additional 24 h. Controls included cells cultured in medium only and cells exposed to Dycal only. Cytotoxicity was measured using colorimetric MTT, WST and secreted lactate dehydrogenase assays. In addition, mitotic activity was evaluated using a colorimetric histone H3 phosphorylation assay. Data were statistically analysed using anova with Tukey's multiple comparison post-test and significance at P ≤ 0.05. RESULTS: For all assays, measured values for cells treated with chlorhexidine, ethanol, LPS or nicotine plus the soluble materials extracted from Dycal pellets were significantly lower compared to control (P < 0.05) for all comparisons between experimental conditions. However, between treatments and for comparisons of treatments with Dycal, there were no differences observed for any assay. CONCLUSIONS: Calcium hydroxide in a formulation used in dental clinical procedures is highly cytotoxic to cultured cells, as evidenced by several cellular assays. However, other known toxic agents, including chlorhexidine, ethanol, bacterial LPS and nicotine, do not appear to function synergistically to increase the deleterious cellular effects of the calcium hydroxide in an in vitro model of cytotoxicity.

Lactate Dehydrogenase as a Potential Therapeutic Drug Target to Control Babesia bigemina.

Babesia bigemina is a tick-borne apicomplexan hemoprotozoan responsible for bovine babesiosis. The current drugs used for bovine babesiosis treatment have several drawbacks, including toxicity, the lack of effectiveness to clear the parasite, and potential to develop resistance. Identifying compounds that target essential and unique parasite metabolic pathways is a rational approach toward finding alternative drug treatments. Based on the genome sequence and transcriptomics analysis, it can be inferred that anaerobic glycolysis is the dominant adenosine triphosphate (ATP) supply for Babesia, and lactate dehydrogenase (LDH) is one of the essential enzymes in this pathway. Furthermore, the Babesia LDH sequence is distinct from its bovine homologue and thus a potential chemotherapeutic target that would result in decreasing the ATP supply to the parasite but not to the host. Gossypol is a known efficient specific inhibitor of LDH in the sensu stricto B. bovis and the sensu lato B. microti, among other related parasites, but no such data are currently available in the sensu stricto B. bigemina parasites. Hereby, we show that the LDH amino acid sequence is highly conserved among sensu stricto but not in sensu lato Babesia spp. A predictive structural analysis of B. bigemina LDH showed the conservation of the key amino acids involved in the binding to gossypol compared to B. bovis. Gossypol has a significant (P < 0.0001) inhibitory effect on the in vitro growth of B. bigemina, with IC50 of 43.97 mM after 72 h of treatment. The maximum IC (IC98) was observed at 60 mM gossypol. However, a significant effect on the viability of cattle PBMC was observed when the cells were cultured with 60 mM (IC98) gossypol compared with DMSO-exposed control cells. Interestingly, B. bigemina cultured at 3% oxygen expresses significantly higher levels of LDH and is more resistant to gossypol than the parasites maintained at ambient conditions containing ~20% oxygen. Altogether, the results suggest the potential of gossypol as an effective drug against B. bigemina infection, but the risk of host toxicity at therapeutic doses should be further evaluated in in vivo studies.

Pyrazole based Furanone Hybrids as Novel Antimalarial: A Combined Experimental, Pharmacological and Computational Study.

BACKGROUND: Malaria parasite strains are resistant to the therapeutic effect of prophylactics medicines presently available. This resistance now poses a significant challenge to researchers to beat malaria parasitic infections. Strategies such as investigating newer hybrid chemical entities and specified drug targets may help us spot new efficient derivatives that bind to the parasites in a more specific manner and inhibit their growth. OBJECTIVE: To scientifically perform the experimental, pharmacological, and computational studies of pyrazole-based furanone hybrids as novel antimalarial agents. METHODS: A series of new furanone-based pyrazole derivatives were synthesized and investigated as potential antimalarial agents by performing in vitro antimalarial activity. To get further optimization, these synthesized derivatives were virtually screened based on ADME-T filters, and molecular docking studies were also accomplished on the crystal structures of Plasmodium falciparum lactate dehydrogenase (PfLDH). Furthermore, the in-silico prediction was supported by performing an LDH assay. RESULTS: The docking data suggested that the designed hybrid of furanone-pyrazole may act as PfLDH inhibitors. It was found that the results of experimental in vitro antimalarial activity and in silico analysis correlate well to each other to a good extent. The compounds (7d), (7g), and (8e) were found to be the most potent derivatives with IC50 values of 1.968, 1.983, and 2.069 µg/ml, respectively. CONCLUSION: From the results, it may be concluded that compounds that are active in low doses might be adopted as a lead compound for the development of more active antimalarial agents. The synthesized compounds (7d), (7g), and (8e) exhibited good antimalarial activity with PfLDH inhibition. The best compounds can be explored further in the future for designing the potent inhibitors of PfLDH as new potent antimalarial agents.

Protein arginine methyltransferase 3 promotes glycolysis and hepatocellular carcinoma growth by enhancing arginine methylation of lactate dehydrogenase A.

BACKGROUND: Protein arginine methylation has emerged a pivotal role in cancer progression. However, the role of protein arginine methyltransferase 3 (PRMT3) in hepatocellular carcinoma (HCC) remains unknown. METHODS: The expression pattern of PRMT3 in HCC was analysed using quantitative real-time-polymerase chain reaction (qRT-PCR), Western blotting and immunohistochemistry assays. Loss- and gain-of-function experiments were carried out to determine the oncogenic role of PRMT3 in HCC. Glucose consumption and lactate production assays, seahorse bioscience, mass spectrometry, co-immunoprecipitation, metabonomic analysis and site-specific mutation experiments were used to explore the underlying molecular mechanisms. Furthermore, a xenograft mouse model was established to investigate the effects of PRMT3 and its inhibitor, SGC707, treatment on tumour growth in vivo. RESULTS: The expression of PRMT3 was significantly upregulated in HCC, with high expression of which correlated with poor prognosis. PRMT3 knockdown led to the decrease in proliferation, glycolysis of HCC cells and tumour growth, whilst its overexpression showed opposite results. The catalytic activity of PRMT3 was important in mediating these biological processes. Mechanistically, our data showed that PRMT3 interacted with and mediated asymmetric dimethylarginine (ADMA) modification of lactate dehydrogenase A (LDHA) at arginine 112 (R112). Compared with LDHA-wild-type (LDHA-WT) cells, LDHA-R112K-mutant-expressing HCC cells exhibited a decrease in lactate dehydrogenase (LDH) activity, HCC cell glycolysis and proliferation. Furthermore, the administration of SGC707, a selective inhibitor of PRMT3, disrupted the PRMT3-mediated LDHA methylation and abolished PRMT3-induced HCC glycolysis and tumour growth. CONCLUSIONS: Our results suggested a novel oncogenic role of PRMT3 in HCC, and it could be a promising therapeutic target for HCC by linking post-translational modification and cancer metabolism.

Infertility in female rabbits immunized with lactate dehydrogenase X.

Immunization of female rabbits with the sperm-specific lactate dehydrogenase (LDH-X) resulted in a highly significant reduction of pregnancies compared to nonimmunized controls. This is the first demonstration of immunosuppression of fertility by a crystalline protein shown to be strictly homogeneous by ultracentrifugation, polyacrylamide gel electrophoresis, immunodiffusion, and micro complement fixation.

Liposomes modulate docetaxel-induced lipid oxidization and membrane damage in human hepatoma cells.

The aim of this study was to investigate the effect of liposomes on docetaxel-induced lipid oxidization and membrane damage in human hepatoma cells. Cytotoxicity of free docetaxel and docetaxel-containing liposomes was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay in human hepatoma cell lines HepG2 and SMMC-7721. To the cell lines, blank liposomes prepared with soybean phosphatidylcholine (SPC), dimyristoylphosphocholine (DMPC), and dioleoylphosphocholine (DOPC) did not show any significant toxicity below a 0.02-mg/mL phospholipid concentration. On the other hand, free docetaxel showed IC(50) values of 9.13 x 10(-6) +/- 1.54 x 10(-5) and 1.58 x 10(-2) +/- 2.71 x 10(-2) mg/mL in HepG2 cells and SMMC-7721 cells, respectively, after of 24 hours of incubation. IC(50) values of docetaxel-encapsulating liposomes, measured in terms of total docetaxel concentration, were at least 1.5-fold higher than those of free docetaxel. SPC liposomes reduced cellular damage caused by free docetaxel, as evidenced by the attenuation of docetaxel-induced lactate dehydrogenase (LDH) leakage by over 11% after liposome encapsulation at each dosage. Docetaxel-induced oxidative membrane damage was monitored by the formation of the lipid peroxidation product, malondialdehyde (MDA), and the antioxidative property of SPC liposome was monitored by the suppression of superoxide dismutase (SOD). These data demonstrated that free docetaxel facilitated MDA formation and suppressed SOD, and that these membrane-damaging effects were reduced by SPC liposomes.

Necroptosis occurs in osteoblasts during tumor necrosis factor-α stimulation and caspase-8 inhibition.

Necroptosis is a regulated cell death mechanism. However, it is unknown whether necroptosis is involved in the death of tumor necrosis factor-α (TNF-α)-treated osteoblasts. Therefore, we conducted the study with TNF-α, Nec-1 (a specific inhibitor of necroptosis), and Z-IETD-FMK (a specific inhibitor of apoptosis) to determine whether necroptosis plays a role in the death of TNF-α-treated osteoblast cell line MC3T3-E1. Cell viability, cell death, and lactate dehydrogenase (LDH) release were assayed to evaluate cytotoxicity. Specific marker proteins receptor interacting protein kinase (RIPK3) and phosphorylated mixed lineage kinase domain-like protein (p-MLKL) for necroptosis, and cleaved caspase 3 for apoptosis were detected by western blot, and mRNA was measured by quantitative real-time polymerase chain reaction (qRT-PCR). We found that TNF-α inhibited cell proliferation in a dose- and time-dependent manner. Nec-1 plus Z-IETD-FMK restored cell viability and significantly decreased LDH release. In addition, TNF-α alone increased the cell population of AV+PI-, while Z-IETD-FMK caused a shift in the cell population from AV+PI- to AV+PI+. Furthermore, TNF-α significantly increased protein cleaved caspase 3. TNF-α plus Z-IETD-FMK significantly increased the proteins RIPK3 and MLKL phosphorylation in MC3T3-E1 cells, while the changes in mRNA levels of RIPK3, MLKL, and caspase 3 were not consistent with the changes in the corresponding protein expression levels. In conclusion, TNF-α induced preferentially apoptosis in osteoblast cell line and necroptosis played a decisive role when TNF-α-induced death was inhibited by the inhibitor of apoptosis. Combined treatment with Nec-1 and Z-IETD-FMK protected mouse osteoblasts from death induced by TNF-α.

Modulation of DNA polymerases alpha, delta and epsilon by lactate dehydrogenase and 3-phosphoglycerate kinase.

Literature documents that glycolytic enzymes (among them lactate dehydrogenase and 3-phosphoglycerate kinase) can reside in nuclei of mammalian cells and exert functions in DNA replication, transcription and DNA repair, in addition to their role as catalysts in the cytoplasm. Transfer of glycolytic enzymes to cell nuclei requires modification, for example phosphorylation. We studied the effects of phosphorylated lactate dehydrogenase and 3-phosphoglycerate kinase on (i) UV-induced DNA repair, using permeabilized human fibroblasts, and (ii) in vitro DNA synthesis catalyzed by purified DNA polymerases alpha, delta, and epsilon from proliferating rat liver. (i) Phosphorylated lactate dehydrogenase stimulated UV-induced DNA repair synthesis in normal fibroblasts in a dose-dependent manner; the unphosphorylated enzyme slightly inhibited. In repair-deficient xeroderma pigmentosum fibroblasts reparative synthesis was not enhanced whether lactate dehydrogenase was phosphorylated or not, indicating that reparative DNA synthesis must be possible in order to be stimulated. (ii) Activity of purified DNA polymerases alpha, delta, and epsilon was differentially stimulated or inhibited, according to the phosphorylation status of lactate dehydrogenase. DNA polymerases were also modulated by 3-phosphoglycerate kinase, depending on the primer-templates used which were gapped DNA (mimicking a repair mode of DNA synthesis) or single-stranded M13 DNA (representing the replicative mode of DNA synthesis). Since glycolytic enzymes in cell nuclei retain binding ability for their cofactors, cytoplasmic substrates and inhibitors, a regulatory linkage might exist between the energy state of a cell and its replicative and reparative functions.

A methodology to study intracellular distribution of nanoparticles in brain endothelial cells.

Cell internalisation and intracellular distribution of PEG-coated polyhexadecylcyanoacrylate (PEG-PHDCA) nanoparticles in rat brain endothelial cells (RBEC) have been investigated. A cell fractionation method has been developed based on the selective permeabilisation of RBEC plasma membrane by digitonin. By interacting with membrane cholesterol, digitonin creates pores allowing the release of soluble and diffusible species outside the cell. The selectivity of plasma membrane permeabilisation was controlled by using compartment markers such as lactate dehydrogenase (LDH) for cytoplasm and cathepsin B for lysosomes. An optimal digitonin concentration of 0.003% (w/v) has been identified to induce a pattern of membrane permeabilisation corresponding to the extraction of 72% LDH and less than 15% of Cathepsin B. Membrane permeabilisation at this digitonin concentration allows one to distinguish between the cell cytoplasm and its endo/lysosomal fraction. This methodology was applied to investigate the intracellular distribution of the nanoparticles after their incubation with the RBEC. The results showed that PEG-PHDCA nanoparticles were able to be internalised to a higher extent than PHDCA nanoparticles (after 20 min incubation). Additionally, these nanoparticles displayed different patterns of intracellular capture, depending on their specific surface composition: PEG-PHDCA nanoparticles were 48% in the plasma membrane, 24% in the cytoplasm, 20% in vesicular compartments and 8% associated with the fraction of the nucleus, the cytoskeleton and caveolae suggesting that PEG-PHDCA nanoparticle uptake by RBEC is specific and presumably due to endocytosis. Confocal microscopy studies confirmed the cellular uptake of PEG-PHDCA nanoparticles.

A mutation detected in DNA polymerase delta cDNA from Novikoff hepatoma cells correlates with abnormal catalytic properties of the enzyme.

Tumor development is characterized by accumulation of mutations. Such mutations, if induced by carcinogens in DNA polymerase genes, would confer mutator properties on the DNA replication machinery, even at later stages of development. To investigate whether DNA polymerase delta can be mutated, we compared these enzymes from highly malignant Novikoff hepatoma cells and from regenerating normal rat liver. We sequenced the DNA polymerase delta cDNA from both sources and investigated the physico-chemical properties, inhibition characteristics, and copying fidelity of the purified enzymes. The cDNA sequences examined included the entire reading frame encoding the catalytic subunit (subunit I) of DNA polymerase delta. First-strand cDNAs were prepared from total RNA of both normal rat liver and Novikoff cells by reverse transcription, and the polymerase delta sequences were amplified by the polymerase chain reaction. cDNA (3325 bp) were sequenced. A single heterozygous mutation (CGG --> CAG) has been detected in nucleotide position 1948 (codon 648) of the polymerase delta gene from Novikoff cells, resulting in an Arg to Gln change. Position 648 lies just proximal to the conserved region VI, which is part of the "fingers" subdomain of alpha-like polymerases. This subdomain is involved in dNTP binding. Upon comparison of biochemical characteristics of partially purified DNA polymerase delta from both Novikoff cells and rat liver, the following properties of the enzyme from Novikoff cells were found to be altered: (i) K(50) values for nucleotide analogs (e.g. butylphenyl-dGTP) were lower, (ii) sensitivity to various antineoplastic drugs (e.g. doxorubicin, topotecan and distamycin) was enhanced, (iii) copying fidelity was decreased when primer templates containing O(6)-methylguanine were used, and (iv) the activity of DNA polymerase delta from Novikoff tumor cells was less stimulated by lactate dehydrogenase than the enzyme from normal cells. The altered biochemical characteristics of DNA polymerase delta from Novikoff cells suggest mutator properties. We conclude that the point mutation detected in the cDNA might be causally related to the observed changes in inhibition characteristics and copying fidelity.

Lactate dehydrogenase in human cervical mucus: correlation with ovulation, influence of ovarian steroid hormones, and isozyme pattern.

The glycolytic enzymes, lactate dehydrogenase (LDH), glucose-6-phosphate dehydrogenase, and hexokinase in cervical mucus of 96 ovulatory women were serially measured daily during the menstrual cycle. Among all of the enzymes, the cyclic changes in LDH activity were the most significant, being high during the proliferative phase, gradually decreasing to the lowest level around ovulation, and then increasing markedly again. Estrogen inhibited cervical mucus LDH activity; progestrone accelerated it. Examination of the LDH isozyme pattern showed that H-LDH (H-subunit of LDH), being predominant throughout the cycle, apparently carries some responsibility for the cyclic change in LDH activity. In a comparison of LDH activity with the serum luteinizing hormone (LH) peak, the activity level nadir of LDH usually appeared between 1 day before and 1 day after the serum LH peak, pointing to the advantage of daily measurement of cervical mucus LDH activity for predicting the potential day of conception.

PIP: Approximately 20% of all causes of inability to conceive by married couples originate in the uterine cervix. This paper examines: 1) cyclic changes in LDH (lactate dehydrogenase) activity in cervical mucus during the menstrual cycle and the effect of hormone treatment on LDH; 2) the effects of ovarian steroid hormones on LDH activity in the cervical mucus; and 3) changes in LDH isozyme patterns in cervical mucus during the menstrual cycle. Multiple cervical mucus samples were obtained from 96 ovulatory women during their menstrual cycle. The women were divided into: 1) untreated group consisting of 46 infertile women; 2) gonadotropin-treated group of 20 women; 3) clomiphene citrate-treated group of 15 patients, and 4) estriol-treated group of 15 patients. In addition to LDH, the following enzymes were serially measured: glycolytic enzymes, glucose-6-phosphate dehydrogenase, and hexokinase. The cyclic changes in LDH activity proved to be the most significant, being high during the proliferative phase, gradually diminishing to the lowest level around ovulation, and then increasing markedly again. Cervical mucus LDH activity was inhibited by estrogen and accelerated by progesterone. Analysis of the LDH isozyme pattern revealed that H-LDH (a subunit of LDH) predominated throughout the cycle and appeared to be responsible for the cyclic changes in LDH activity. The lowest values for cervical mucus LDH usually appeared between 1 day before and 1 day after the serum LH peak. Daily measurement of cervical mucus LDH activity may be of help in predicting potential day of conception.

Cytotoxic effect and role of exogenous antioxidants in carpet dust mediated toxicity in rat hepatocytes in vitro.

Carpet industries bear a great deal of economic and commercial significance in India. In order to safe guard the workers against the health hazards caused by dust in their occupational environment; it necessitates studying the biological importance of these dusts. The present study was designed to investigate the toxicity of carpet dust (knotted and tuffted) on isolated rat hepatocytes. The hepatocytes were isolated by collagenase perfusion method and cells were incubated with different concentration of carpet dust (100-5000 microg/10(6) cells) with various time (30-180 min) intervals. An exogenous antioxidant vitamin-E also used to find out the role of antioxidants and free radical production in carpet dust mediated toxicity. Cell viability by trypan blue exclusion and leakage of enzyme lactate dehydrogenase (LDH) were determined. Reduced glutathione (GSH), formation of thiobarbituric acid reactive substance (TBARS) were also measured. A significant decrease in the cell viability was observed after 60, 180 min upon incubation with tuffted carpet dust, while knotted carpet dust caused a significant decrease in the viability after 180 min. LDH leakage was parallel to the cell viability. Thiobarbituric acid reactive substance was significantly increased at 30 and 60 min with carpet dust treated hepatocytes. Dust at 1000 and 5000 microg dose level showed significantly increased formation of TBARS at 30 min incubation. However, when hepatocytes were co-incubated with carpet dust and Vit-E (10, 15 microM), a significant decrease in LDH release and TBARS production was observed while 15 microM Vit-E showed an enhanced protection than 10 microM Vit-E treated hepatocytes. The effect of carpet dust on cell viability, LDH leakage, TBARS production, GSH depletion was time and dose-dependent. Moreover, we observed that tuffted carpet dust causes greater effect than knotted one on the above mentioned parameters. Our studies also revealed that Vit-E in culture media diminishes the carpet dust mediated toxicity.

Viability assessment in sandwich-cultured rat hepatocytes after xenobiotic exposure.

Troglitazone, bosentan and glibenclamide inhibit the bile salt export pump (Bsep) which transports taurocholate into bile. Sandwich-cultured rat hepatocytes maintain functional sodium taurocholate co-transporting polypeptide and Bsep transport proteins, and may be useful to study inhibition of transport by xenobiotics at concentrations below the lowest observable adverse effect level (LOAEL). The purpose of this study was to compare viability assessments determined with the neutral red, lactate dehydrogenase (LDH), alamar blue, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and propidium iodide assays in sandwich-cultured rat hepatocytes following exposure to xenobiotics known to inhibit Bsep, and to define the LOAEL for these xenobiotics in this system. The neutral red assay was not amenable to use in this model due to crystal formation on the collagen. Troglitazone decreased viability in every assay examined, with a LOAEL approximately 100 microM. Bosentan also decreased viability as measured by the LDH, MTT and propidium iodide assays, with a LOAEL approximately 200 microM; however, a significant decrease in viability was not observed with the alamar blue assay. Glibenclamide did not decrease viability with any assay at the xenobiotic concentrations examined in this study. Based on the results of this study, the LDH or propidium iodide assays would be the methods of choice to assess viability in sandwich-cultured rat hepatocytes after xenobiotic exposure.

An enzyme-assessed microplate-assay for neutrophil adherence. I. IgA-induced adherence of human PMNs.

The binding of PMNs to extracellular matrix and cells is crucial to PMN host defense. Adherence mechanisms and the many families of molecules involved are major areas of study. We present here details of an enzyme-assessed microtiter plate assay for neutrophil adherence. This assay uses low numbers of cells (50,000/well) and permits analysis of several hundred wells in a short period of time, by using an ELISA reader. With this assay we observed 5- to 10-fold increases in the number of adherent human PMNs in response to nanogram amounts of LPS or as little as 5.0 micrograms/ml of aggregated IgA. Although fluoride blocked the LPS-induced adherence response, IgA-induced cell binding was largely unaffected.

The comparison of spontaneous LDH release activity from cultured PBMC with sera LDH activity in non-Hodgkin's lymphoma patients.

Based on the fact that lactate dehydrogenase (LDH) enzyme is a very sensitive indicator of the cellular metabolic state, aerobic or anaerobic direction of glycolysis, activation status, and malignant transformation, in this study we compared values of the spontaneous LDH release from circulating PBMC with sera LDH activity in 53 different subtypes of non-Hodgkin's lymphoma (NHL) patients. Results shows that serum LDH was significantly (p < 0.05) elevated in comparison to the range values only in the advance clinical stage (III and IV) in all investigated subtypes of NHL according to The Working and REAL classification. On the other hand, the spontaneous LDH release from cultures PBMC is significantly (p < 0.01) elevated in early and advanced stage in all investigated forms of NHL in comparison to healthy controls. Based on consideration that an increase in spontaneous LDH release appears before elevated sera LDH activity, we conclude that determination of spontaneous LDH release by microassay from cultured cells together with other findings may help in the diagnosis of NHL patients, especially in patients with early stage of disease.

Lactate dehydrogenase-induced conformational changes of F-actin in myosin-free ghost single fibres.

The changes in conformation of F-actin induced by the binding of the glycolytic enzyme lactate dehydrogenase were studied in myosin-free single ghost muscle fibres. The formation of the lactate dehydrogenase-F-actin complex was accompanied by changes in the parameters of intrinsic (tryptophan) and extrinsic (rhodaminyl-phalloin) polarized fluorescence of ghost muscle fibre F-actin. Lactate dehydrogenase stimulated actin-activated Mg2+-ATPase of myosin subfragment 1 by 30%. F-actin of ghost fibres depressed lactate dehydrogenase activity to 20% of the initial values. It is suggested that the energy-providing mechanism is coupled with that of muscle contraction through conformational changes in F-actin.

Restorative effects of zinc and selenium on cadmium-induced kidney oxidative damage in rats.

OBJECTIVE: To investigate whether cadmium-induced oxidative stress in the kidney is influenced by zinc and selenium. METHODS: Five groups of rats were maintained: (A) Cd (CdCl2, 400 micrograms.kg-1.day-1 intraperitoneal injection); (B) Cd + Zn (ZnCl2, 20 mg.kg-1.day-1 hypodermic injection); (C) Cd + Se (Na2SeO3, 350 micrograms.kg-1.day-1 via a stomach tube); (D) Cd + Zn + Se; (E) treated with physiological saline as a sham-handled control. The rats were given treatment for a period of 4 weeks. The activities of superoxide dismutase (SOD), glutathione peroxidase (GH-Px), catalase (CAT), and the level of malondialdehyde (MDA) in the kidney tissue were measured to assess the oxidative stress. Urinary lactate dehydrogenase (LDH) activity was used as an indicator of tubular cell damage caused by lipid peroxidation. RESULTS: In group C and D, activities of SOD (110.5 +/- 5.2, 126.8 +/- 7.0; P < 0.05) and GSH-Px (85.7 +/- 4.9, 94.6 +/- 7.3; P < 0.05) were higher than those in group A (84.7 +/- 3.3; 56.9 +/- 3.8); and in group B, only the activity of GSH-Px (80.0 +/- 4.3, P < 0.01) increased in comparison with that in group A (56.9 +/- 3.8). Significant increase of MDA (P < 0.05) was seen in group B (31.1 +/- 4.7) and C (35.0 +/- 4.1) when compared with control values (17.2 +/- 1.8). No difference was found in the level of MDA between group D (18.9 +/- 2.6) and control. The activity of LDH in urine of control group (0.06 +/- 0.02) was lower than that of group A (0.46 +/- 0.19, P < 0.05), B (0.10 +/- 0.05, P < 0.05) and C (0.14 +/- 0.07, P < 0.05), and there was no significant change between control (0.06 +/- 0.02) and group D (0.08 +/- 0.02). CONCLUSION: Zinc or selenium could partially alleviate the oxidative stress induced by cadmium in kidney, but administration cadmium in combination with zinc and selenium efficiently protects kidney from cadmium-induced oxidative damage.