Recent studies have shown that the inhibitory effects of disulfiram and diethyldithiocarbamate (DDTC) (to which disulfiram is rapidly reduced in vivo) on the liver mitochondrial low-Km form of aldehyde dehydrogenase (ALDH) may be mediated by a reactive metabolite(s) of these compounds. In order to investigate the nature of such electrophilic intermediates in vivo, the present study was carried out with the goal of detecting and identifying their respective glutathione (GSH) conjugates in the bile of rats dosed ip with either disulfiram (75 mg kg-1) or sodium DDTC (114 mg kg-1). By means of highly selective screening strategies based on coupled liquid chromatography-tandem mass spectrometry techniques, one major and four minor GSH adducts were identified as common biliary metabolites of disulfiram and DDTC. The major conjugate, whose excretion into bile over 4 h accounted for ca. 1% of the dose of either precursor, was identified as S-(N,N-diethylcarbamoyl)glutathione (SDEG). In vitro experiments with synthetic SDEG demonstrated that this carbamate thioester derivative is chemically stable in aqueous media under physiological conditions and does not carbamoylate nucleophiles such as cysteine. Consistent with these findings, SDEG failed to inhibit yeast ALDH in vitro. The minor GSH conjugates in bile were identified as S-(N,N-diethylthiocarbamoyl)glutathione, S-(N-ethyl-carbamoyl)glutathione, S-(N-ethylthiocarbamoyl)glutathione, and S-[N-(carboxymethyl)-N- ethylcarbamoyl]glutathione, the structures of which indicate that metabolic oxidation takes place at the thiono sulfur group and at each of the carbon atoms of disulfiram and DDTC.(ABSTRACT TRUNCATED AT 250 WORDS)

Aldehyde Dehydrogenase↗

PubMedDate not supplied

Leptin and leptin receptor in anterior pituitary function.

Leptin is a 16 kDa protein that exerts important effects on the regulation of food intake and energy expenditure by interacting with the leptin receptor in the brain and in many other tissues. Although leptin is produced mainly by white adipose tissue, several laboratories have shown low levels of leptin production by a growing number of tissues including the anterior pituitary gland. Many studies have implicated leptin in anterior pituitary function including the observation that homozygous mutations of the leptin receptor gene led to morbid obesity, lack of pubertal development and decreased GH and TSH secretion. In addition, leptin functions as a neuroendocrine hormone and regulates many metabolic activities. Leptin also interacts with and regulates the hypothalamic-pituitary-adrenal, the hypothalamic-pituitary-thyroid and the hypothalamic-pituitary-gonadal axes. All of the anterior pituitary cell types express the leptin receptor. However, leptin has been localized in specific subtypes of anterior pituitary cells indicating cell type-specific production of leptin in the anterior pituitary. Subcellular localization of leptin indicates co-storage with secretory granules and implicates hypothalamic releasing hormones in leptin secretion from anterior pituitary hormone cells. Leptin signal transduction in the anterior pituitary has been shown to involve the janus protein-tyrosine kinase (JAK)/signal transducer and activation of transcription (STAT) as well as suppressor of cytokine signalling (SOCS). These proteins are activated by tyrosine-phosphorylation in anterior pituitary cells. The various steps in pituitary leptin signal transduction remain to be elucidated.

Aging↗

PubMedDate not supplied

Galvanotactic response of mouse epididymal sperm: in vitro effects of zinc and diethyldithiocarbamate.

This study was conducted to evaluate the galvanotactic response of mouse epididymal sperm as well as the in vitro effects of Zn-acetate and diethyldithiocarbamate (DEDTC) on the galvanotaxis of sperm. The galvanotaxis of sperm was observed in a direct current (DC) electric field between 0 and 10 V/cm. The sperm were treated with Zn-acetate or DEDTC at concentrations of 0.02, 0.20, and 2.00 mM before the observations. The sperm exhibited galvanotaxis toward both cathode and anode in the fields between 1 and 9 V/cm. The number of sperm that accumulated at both electrodes was found to reach the highest level between 5 and 7 V/cm. In comparison with the results from untreated sperm, the number of accumulated sperm at the electrodes increased with the addition of 0.20 and 2.00 mM Zn-acetate, but decreased with the addition of DEDTC at all 3 concentrations. The galvanotaxis of mouse epididymal sperm is related to the field strengths, and the guidance of sperm migration may be influenced by the sex chromosomes. The galvanotaxis of the sperm may be improved by addition of Zn-acetate but depressed by DEDTC in the presence of DC electric fields.

Animals↗

PubMedDate not supplied

Endothelial cell dysfunction in a model of oxidative stress.

BACKGROUND: We have investigated the role of L-arginine in hyperhomocysteinemia (HHCY). L-arginine is the substrate required for NO production by endothelial NOS (eNOS). When L-arginine is limited, NOS acts principally upon O2 to form superoxide (O2.-). Because HHCY causes formation of reactive oxygen species and reduced endothelial-dependent vasodilation, we hypothesized that HHCY decreases NO formation by limiting the cellular supply of L-arginine. MATERIAL AND METHODS: Studies with cultured bovine aortic endothelial cells (ECs) determined effects of HCY on transport of [3H] L-arginine. Effects on L-arginine transporter protein CAT-1 and eNOS protein were assessed by immunoblotting. Peroxynitrite formation was evaluated by an immunoassay for nitrotyrosine levels. eNOS activity in forming NO was determined by assay for 3H-L-arginine to 3H-citrulline conversion. RESULTS: HCY had a depressive effect on arginine transport in ECs. HCY treatment for a 24 hr period decreased arginine uptake by 27%. HCY treatment for 24 hr significantly reduced cellular levels of the CAT-1 arginine transporter protein ( approximately 30%) and increased nitrotyrosine formation, whereas levels of eNOS protein and basal NOS activity were not altered. Nevertheless, total NO production as indicated by citrulline conversion was significantly decreased. Treatment with the antioxidant N-acetylcysteine reversed the HCY effect on arginine transport, suggesting that transporter oxidation may contribute to the endothelial dysfunction. CONCLUSIONS: The association of HCY-induced decreases in NO formation with decreases in function and expression of the arginine transporter in the absence of alterations in eNOS expression or activity suggests a primary role for arginine transport alterations in HHCY. The action of HCY to reduce arginine uptake may accentuate endothelial dysfunction due to generation of O2.- and peroxynitrite formation, which may cause further oxidative injury.

Animals↗

PubMedDate not supplied

Differences in C/EBPs in normal tissue and papillomas of the larynx.

Transcription factors belonging to the family of CAAT enhancer binding proteins (C/EBPs) programme differentiation in a wide variety of cells. We asked about the expression of C/EBPs in squamous epithelium. Using immunohistochemistry, C/EBPalpha and C/EBPbeta were easily detected in tissue sections from normal larynx or laryngeal papillomas, benign tumours with a papillomavirus aetiology and characterized by abnormal differentiation. A temporal expression of these C/EBPs occured as keratinocytes differentiated. In both tissues, C/EBPbeta appeared to be exclusively nuclear. In normal tissue, the greatest amount of C/EBPbeta was present in the spinous layer, and much less occurred in the granular layer. The basal layer of the laryngeal papillomas contained the most C/EBPbeta. Less was present in the spinous layer. Little C/EBPbeta was in the granular layer. Much C/EBPalpha was in the cytoplasm in both tissues. In normal tissue, nuclear C/EBPalpha staining was virtually absent in the basal layer, and became present in the spinous layer. Nuclear C/EBPalpha was randomly distributed in all layers in papillomas. Using immunoblots and Southwestern blots, we detected abundant truncated isoforms of C/EBPalpha in the papillomas. Since differentiation of many tissues is determined by the relative amounts of different C/EBPs, our data supports a role for C/EBPs in the differentiation of squamous epithelium.

Blotting, Western↗