HPLC-based assays for enzymes of glutathione biosynthesis.

Glutamate cysteine ligase and glutathione synthase carry out the two-step synthesis of glutathione. The fluorescent thiol-reactive compound monobromobimane is used to derivatize reaction products in an HPLC-based assay with fluorescence detection. The assay described in this unit can be adapted for tissue homogenates or cultured cells.

Enac degradation in A6 cells by the ubiquitin-proteosome proteolytic pathway.

Amiloride-sensitive epithelial Na(+) channels (ENaC) are responsible for trans-epithelial Na(+) transport in the kidney, lung, and colon. The channel consists of three subunits (alpha, beta, gamma) each containing a proline rich region (PPXY) in their carboxyl-terminal end. Mutations in this PPXY domain cause Liddle's syndrome, an autosomal dominant, salt-sensitive hypertension, by preventing the channel's interactions with the ubiquitin ligase Neural precursor cell-expressed developmentally down-regulated protein (Nedd4).

Expression of highly selective sodium channels in alveolar type II cells is determined by culture conditions.

Alveolar fluid clearance in the developing and mature lungs is believed to be mediated by some form of epithelial Na channels (ENaC). However, single-channel studies using isolated alveolar type II (ATII) cells have failed to demonstrate consistently the presence of highly selective Na+ channels that would be expected from ENaC expression. We postulated that in vitro culture conditions might be responsible for alterations in the biophysical properties of Na+ conductances observed in cultured ATII cells.

Attitudes towards transferable skills in medical undergraduates.

Context: Changes to the style of medical teaching will place a greater responsibility on individual medical students to manage their own learning, highlighting the need for students to develop good so-called 'transferable' skills at an early stage in their undergraduate career.

Objectives: To assess the attitudes of first year undergraduates towards transferable skills, and investigate the gender difference in these attitudes. To assess the contribution of their first year course to skills development.

Digital toxicology education tools: education, training, case studies, and tutorials.

This review presents a brief and non-comprehensive overview of a representative sampling of some of the broad array of toxicology-related learning, tutorial and information resources now becoming widely available to educators, health professionals, students and the general public in digital media and/or via the Internet. A broad variety of useful learning and reference resources in the general fields of toxicology and the environmental health sciences is provided to introduce the reader to the diverse types of information currently available. The sources and Internet links contained in this review will hopefully constitute a useful resource of basic toxicology information that should be readily accessible to most if not all readers.

Purification and characterization of hexahistidine-tagged cyclohexanone monooxygenase expressed in Saccharomyces cerevisiae and Escherichia coli.

Cyclohexanone monooxygenase (CMO) is a soluble flavoenzyme originally isolated from Acinetobacter spp. which carries out Baeyer-Villiger reactions with cyclic ketone substrates. In the present study we cloned the Acinetobacter CMO gene and modified it for facile purification from heterologous expression systems by incorporation of a His(6)-tag at its C-terminus.

Cytosolic phospholipase A2 is required for optimal ATP activation of BK channels in GH(3) cells.

To test the hypothesis that ATP activation of BK channels in GH(3) cells involves cytosolic phospholipase A(2) (cPLA(2)) as a potential protein target for phosphorylation, we first inhibited the activity of cPLA(2) by both pharmacologic and molecular biologic approaches. Both approaches resulted in a decrease rather than an increase in BK channel activity by ATP, suggesting that in the absence of cPLA(2), phosphorylation of other regulatory elements, possibly the BK channel protein itself, results in inactivation rather than activation of the channel. The absence of changes in activity in the presence of the non-substrate ATP analog 5'-adenylyl-beta,gamma-imidodiphosphate verified that ATP hydrolysis was required for channel activation by ATP.

The efficacy of recombinant TPO in murine And nonhuman primate models for myelosuppression and stem cell transplantation.

Radiation-induced pancytopenia proved to be a suitable model system in mice and rhesus monkeys to study thrombopoietin (TPO) target cell range and efficacy. TPO was highly effective in rhesus monkeys exposed to the midlethal dose of 5-Gy (300 kV x-rays) TBI, a model in which it alleviated thrombocytopenia, promoted red cell reconstitution, accelerated reconstitution of immature CD34+ bone marrow (BM) cells and potentiated the response to growth factors such as GM-CSF and G-CSF. The accelerated reconstitution of BM CD34+ cells appeared to be reflected by a similar rise in peripheral blood CD34+ cells, both being augmented by concomitant GM-CSF.

IL-3 does not contribute to platelet production in c-Mpl-deficient mice.

Thrombopoietin is a lineage-dominant cytokine involved primarily in the control of platelet production. The physiological importance of thrombopoietin (TPO) in the regulation of megakaryocyte and platelet production was demonstrated by the production of mice deficient in TPO or its receptor, c-Mpl. Even though these mice are profoundly thrombocytopenic they maintain a basal level of approximately 10% of the normal count of fully functional platelets.

Effects of fatty acids on BK channels in GH(3) cells.

Ca(2+)-activated K(+) (BK) channels in GH(3) cells are activated by arachidonic acid (AA). Because cytosolic phospholipase A(2) can produce other unsaturated free fatty acids (FFA), we examined the effects of FFA on BK channels in excised patches. Control recordings were made at several holding potentials.

Modulation of glutathione and glutamate-L-cysteine ligase by methylmercury during mouse development.

The antioxidant tripeptide glutathione has been proposed to be important in defense against oxidative stress and heavy metal toxicity. We evaluated alterations in glutathione regulation and synthesis associated with low-level chronic methylmercury (MeHg) exposure in the developing mouse fetus. Female C57Bl/6 mice were given 0, 3, or 10 ppm MeHg in the drinking water for 2 weeks prior to breeding and throughout pregnancy.

Aldosterone induces ras methylation in A6 epithelia.

Aldosterone increases Na(+) reabsorption by renal epithelial cells: the acute actions (<4 h) appear to be promoted by protein methylation. This paper describes the relationship between protein methylation and aldosterone's action and describes aldosterone-mediated targets for methylation in cultured renal cells (A6). Aldosterone increases protein methylation from 7.

The effect of rapamycin on single ENaC channel activity and phosphorylation in A6 cells.

Rapamycin and FK-506 are immunosuppressive drugs that bind a ubiquitous immunophilin, FKBP12, but immunosuppressive mechanisms and side effects appear to be different. Rapamycin binds renal FKBP12 to change renal transport. We used cell-attached patch clamp to examine rapamycin's effect on Na(+) channels in A6 cells.

Mu-class GSTs are responsible for aflatoxin B(1)-8, 9-epoxide-conjugating activity in the nonhuman primate macaca fascicularis liver.

Mice are resistant to the carcinogenic effects of the mycotoxin aflatoxin B(1) (AFB(1)) because they constitutively express an alpha-class glutathione S-transferase (mGSTA3-3) that has high (approximately 200,000 pmol/min/mg) activity toward aflatoxin B(1)-8, 9-epoxide (AFBO). Rats do not constitutively express a GST with high AFBO-conjugating activity and are sensitive to AFB(1)-induced hepatocarcinogenesis. Constitutively expressed human hepatic alpha-class GSTs (hGSTA1-1 and hGSTA2-2) possess little or no AFBO-detoxifying activity (<2 pmol/min/mg).

Differential effects of protein kinase C on the levels of epithelial Na+ channel subunit proteins.

Regulation of epithelial Na(+) channel (ENaC) subunit levels by protein kinase C (PKC) was investigated in A6 cells. PKC activation altered ENaC subunit levels, differentially decreasing the levels of both beta and gamma, but not alphaENaC. Temporal regulation of beta and gammaENaC by PKC differed; gammaENaC decreased with a time constant of 3.

Glutathione S-transferase M1, T1, and P1 polymorphisms as risk factors for renal cell carcinoma: a case-control study.

Renal cell carcinoma (RCC) has known environmental risk factors, notably smoking, and enzymes that biotransform carcinogens have high levels of activity in the kidney. However, a possible role of polymorphisms in these enzymes in RCC etiology has received little study. We investigated glutathione S-transferase (GST) polymorphisms in a population-based case-control study of RCC.

Biotransformation enzyme polymorphism and pesticide susceptibility.

Numerous specific genetic polymorphisms (PM) in the multi-gene families of cytochromes P450 (CYPs) and glutathione S-transferases (GSTs) have been described in the human population in the past decade. For example, one or more PM have been identified in human CYP1A1, CYP1B1, CYP2C9, CYP2C18, CYP2D6, and CYP2E1. Recent studies using cDNA expressed human CYPs have suggested that CYP3A4 is the principal human CYP involved in the oxidation of parathion and probably other organo(thio)phosphate (OP) insecticides and thus PM in this CYP might influence susceptibility to OP.

Responses of seventh grade students to "do you have a partner with whom you would like to have a baby?".

This study investigated differences in demographic data, self-esteem, and coping skills for 225 students in Grade 7 who reported having a partner with whom they wanted to have a baby and 946 students who did not. Data were collected on the Rosenberg Self-esteem Scale, the Family Crisis-oriented Personal Evaluation Scale, and questions related to attitudes towards teen pregnancy and demographic data. The sample included 548 (46.

Methylation increases the open probability of the epithelial sodium channel in A6 epithelia.

We used single channel methods on A6 renal cells to study the regulation by methylation reactions of epithelial sodium channels. 3-Deazaadenosine (3-DZA), a methyltransferase blocker, produced a 5-fold decrease in sodium transport and a 6-fold decrease in apical sodium channel activity by decreasing channel open probability (P(o)). 3-Deazaadenosine also blocked the increase in channel open probability associated with addition of aldosterone.

Effects of dietary oltipraz and ethoxyquin on aflatoxin B1 biotransformation in non-human primates.

Following aflatoxin B1 (AFB) exposure, rats readily develop liver tumors. However, treatment of rats with a variety of compounds, including the synthetic dithiolthione oltipraz and the antioxidant ethoxyquin, protects these rodents from AFB-induced hepatocarcinogenesis. Several epidemiological studies strongly suggest that AFB is also a causative agent of liver cancer in humans.

Use of phage display for the generation of human antibodies that neutralize factor IXa function.

The use of libraries of phage-displayed human single-chain antibody fragments (scFv) has become a new, powerful tool in rapidly obtaining therapeutically useful antibodies. Here, we describe the generation of human scFv and F(ab')2 directed against the gamma-carboxyglutamic acid (Gla) domain of coagulation factor IX. A large library of human scFv, displayed either on M13 phage or expressed as soluble proteins, was screened for binding to human Gla-domain peptide (Tyr1-Lys43).

Comparison of oxidative stress response parameters in newborn mouse liver versus simian virus 40 (SV40)-transformed hepatocyte cell lines.

Induction of approximately one dozen genes and/or enzyme activities in liver of the untreated newborn c(14CoS)/c(14CoS) mouse-when compared with the c(ch)/c(14CoS) heterozygote or the c(ch)/c(ch) wild-type-is the result of enhanced levels of reactive oxygenated metabolites originating from a block in the tyrosine degradation pathway. Oxidative stress activates genes via the electrophile response element, whereas dioxin activates genes via the receptor-mediated aromatic hydrocarbon response element. Here, we compared several parameters in 14CoS/14CoS versus ch/ch newborn mouse liver with that in simian virus 40 (SV40)-transformed hepatocyte lines that had been derived from newborn liver.

Alcohol attitudes and beliefs related to actual alcohol experience in a fifth-grade cohort.

Despite a gradual overall decrease in alcohol consumption in the United States, alcohol remains the most widely used and abused drug in society. This study assessed lifetime and recent alcohol use in a cohort of fifth graders, and examined attitudes and beliefs held by these youth concerning alcohol. An instrument addressing alcohol use, other substance use, and other health-related problem behaviors was completed by 813 fifth graders in a west central Florida school district.

Induction of glutamate-cysteine ligase (gamma-glutamylcysteine synthetase) in the brains of adult female mice subchronically exposed to methylmercury.

Methylmercury (MeHg) is widely known for its potent neurotoxic properties. One proposed mechanism of action of MeHg relates to its high affinity for sulfhydryl groups, especially those found on glutathione (GSH) and proteins. Previous studies have shown that acute MeHg exposure results in an increase in the mRNA for the rate-limiting enzyme in GSH synthesis, glutamate-cysteine ligase (GLCL) (also known as gamma-glutamylcysteine synthetase).