Interaction of gamma-glutamyl transpeptidase with acivicin.

Inactivation of gamma-glutamyl transpeptidase by acivicin (L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazole acetic acid) is rapid, thought to be irreversible, and associated with binding of close to 1 mol of inhibitor/mol of enzyme. Previous studies with [3-14C]acivicin indicated binding (prevented by substrate) to a specific hydroxyl group (threonine 523) of the rat kidney enzyme. In the present work, we found that such inactivation can be reversed by treating the inhibited enzyme with hydroxylamine.

Active deglycosylated mammalian gamma-glutamyl transpeptidase.

gamma-Glutamyl transpeptidase, a highly glycosylated heterodimeric enzyme that is usually attached to the external surface of cell membranes, is of major importance in the metabolism of glutathione. The enzyme, which has been isolated from many animal sources, contains a large amount of carbohydrate, which is linked to both protein subunits. Previous work has not shown whether such carbohydrate is needed for enzyme activity nor indicated its functional role.

Utility of DNA amplified by degenerate oligonucleotide-primed PCR (DOP-PCR) from the total genome and defined chromosomal regions of field bean.

Degenerate oligonucleotide primed (DOP)-PCR has emerged as a simple and rapid method for representative amplification of highly complex genomic DNA from humans, mice and Drosophila. The present paper describes the adaptation of this method for use on a plant species, Vicia faba, with a large genome (2C = 30 pg). Specific low-copy-number sequences as well as highly repeated sequences were detectable among DOP-PCR products obtained from small samples of purified genomic DNA (100 pg), DNA from 10 prophase nuclei, 10 flow-sorted chromosomes or 15 microdissected chromosome segments (satellites) following reamplification with sequence-specific primers and/or Southern hybridization.

Nitrate tolerance in vivo is not associated with depletion of arterial or venous thiol levels.

Results from in vitro experiments suggest that development of nitrate tolerance is due to a depletion of vascular thiol compounds (ie, cysteine and glutathione [GSH]) necessary for the bioconversion of organic nitrates. However, it is unknown whether in vivo tolerance development is associated with changes in thiol levels. This study measures plasma and vessel tissue GSH and cysteine levels in nontolerant rats, nitrate-tolerant rats, and rats treated with the two characteristically different thiol donors N-acetyl-L-cysteine and L-2-oxothiazolidine-4-carboxylic acid (OXO).

Differentiation of field bean heterochromatin by in situ hybridization with a repeated FokI sequence.

The chromosomes of a field bean line with a reconstructed karyotype (ACB) were hybridized in situ with biotinylated probes of a repetitive Fok I sequence, of DOP-PCR (degenerate oligonucleotide primed polymerase chain reaction) amplified DNA from a chromosome that does not contain this sequence, and with probes containing dispersed repetitive sequences. The results were compared with Giemsa banding, DNA late replication and Fok I in situ digestion patterns. This allowed further differentiation between the chromatin types of this species.

Transport of glutathione diethyl ester into human cells.

Glutathione monoesters in which the carboxyl group of the glycine residue is esterified were previously found, in contrast to glutathione itself, to be effectively transported into various types of cells and to be converted intracellularly into glutathione. Glutathione monoesters are thus useful for prevention of oxidative stress, certain toxicities, and for treatment of glutathione deficiency. Glutathione diethyl ester is rapidly split to the glutathione monoethyl ester by mouse plasma glutathione diester alpha-esterase activity.

On the synthesis and characterization of N-formyl glutathione and N-acetyl glutathione.

Treatment of glutathione (GSH) with formic acid and acetic anhydride leads to preparation of crystalline N-formyl GSH. This is the first characterized preparation of N-formyl GSH; this product was previously erroneously thought to be N-acetyl GSH. Authentic N-acetyl GSH (crystalline) was prepared by treating GSH with acetic acid and acetic anhydride.

Amino acid sequence and function of the light subunit of rat kidney gamma-glutamylcysteine synthetase.

The heavy subunit (M(r), 72,614) of rat kidney gamma-glutamylcysteine synthetase, the enzyme that catalyzes the first step of glutathione (GSH) synthesis, mediates the catalytic activity of this enzyme and its feedback inhibition by GSH. There is evidence that the light subunit has a regulatory function (Huang, C.-S.

Catalytic and regulatory properties of the heavy subunit of rat kidney gamma-glutamylcysteine synthetase.

gamma-Glutamylcysteine synthetase (rat kidney), which catalyzes the first step of GSH synthesis, can be dissociated into subunits (M(r) 73,000 and 27,700) by native gel electrophoresis after treatment with dithiothreitol (DTT); the heavy subunit, which exhibits catalytic activity and feedback inhibition by GSH (Seelig, G. F., Simondsen, R.

Localization of seed protein genes on flow-sorted field bean chromosomes.

Chromosomes from reconstructed field bean (Vicia faba L.) karyotypes were flow-sorted and the DNA was used for the physical localization of seed storage and nonstorage (USP) protein genes using PCR with sequence specific primers. The data were confirmed and refined by using DNA of microisolated chromosomes of other karyotypes as the target for PCR.

[Curative and palliative therapy of esophageal carcinoma; role of radiotherapy].

Oesophageal carcinoma continues to represent a major problem for surgeons, radiation oncologists and medical oncologists. More aggressive surgery and more sophisticated radiation therapy with intraluminal afterloading techniques with radioactive material have failed to substantially improve the outcome of this dismal disease. The present paper evaluates the current position of the radiotherapeutic treatment, based on a review of published data and with emphasis on combined treatments with surgery and cytotoxic drugs.

Acute effects of nitroglycerin depend on both plasma and intracellular sulfhydryl compound levels in vivo. Effect of agents with different sulfhydryl-modulating properties.

Background: Changes in sulfhydryl (SH) compound availability may alter the hemodynamic effect of nitroglycerin (NTG). Data on the relation between NTG effect and thiol levels are, however, limited to in vitro experiments. The present study investigates how intracellular and extracellular changes in SH group concentrations (cysteine and glutathione [GSH]) affect the responsiveness to NTG in vivo.

Glutathione ester delays the onset of scurvy in ascorbate-deficient guinea pigs.

Previous studies showed that administration of ascorbate to glutathione (GSH)-deficient newborn rats and guinea pigs prevented toxicity and mortality and led to increased tissue and mitochondrial GSH levels; ascorbate thus spares GSH. In the present work, we tried to answer the converse question: Does administration of GSH spare ascorbate? Because administered GSH is not well transported into most cells, we gave GSH monoethyl ester (which is readily transported and converted into GSH intracellularly) to guinea pigs fed an ascorbate-deficient diet. We found that treatment with GSH ester significantly delays appearance of the signs of scurvy and that this treatment spares ascorbate; thus, the decrease of tissue levels of ascorbate was delayed.

Glutathione deficiency increases hepatic ascorbic acid synthesis in adult mice.

Glutathione deficiency, induced in adult mice by administering buthionine sulfoximine (an inhibitor of glutathione synthesis), led to a rapid and substantial increase in ascorbate in the liver. This effect was apparent 2-4 hr after giving the inhibitor; subsequently, the level of ascorbate decreased and that of dehydroascorbate increased markedly, supporting the conclusion that glutathione functions physiologically to keep ascorbate in its reduced form. In kidney and lung also, ascorbate levels decreased, and dehydroascorbate increased.

Ascorbic acid prevents oxidative stress in glutathione-deficient mice: effects on lung type 2 cell lamellar bodies, lung surfactant, and skeletal muscle.

Glutathione deficiency in adult mice leads to lung type 2 cell lamellar body and mitochondrial damage; as reported here, these effects are associated with marked decrease of the levels of phosphatidylcholine (the main component of lung surfactant) in the lung and the bronchoalveolar lining fluid. Severe mitochondrial damage was also found in skeletal muscle. Treatment with ascorbate (1-2 mmol per kg of body weight per day), which led to greatly increased (approximately 2-fold) levels of lung and muscle mitochondrial glutathione, prevented damage to lamellar bodies and mitochondria as well as the decline of phosphatidylcholine levels in lung and alveolar lining fluid.

High resistance to cisplatin in human ovarian cancer cell lines is associated with marked increase of glutathione synthesis.

Exposure of human ovarian tumor cell lines to cisplatin led to development of cell lines that exhibited increasing degrees of drug resistance, which were closely correlated with increase of the levels of cellular glutathione. Cell lines were obtained that showed 30- to 1000-fold increases in resistance; these cells also had strikingly increased (13- to 50-fold) levels of glutathione as compared with the drug-sensitive cells of origin. These levels of resistance to cisplatin and the cellular glutathione levels are substantially greater than previously reported.

Inhibition of glutathione synthesis in the newborn rat: a model for endogenously produced oxidative stress.

A model for oxidative stress is described in which glutathione (GSH) synthesis is selectively blocked in newborn rats by administration of L-buthionine-(S,R)-sulfoximine (BSO). In this model, the normal endogenous physiological formation of reactive oxygen species is largely unopposed, and therefore oxidative tissue damage occurs; because GSH is used for reduction of dehydroascorbate, tissue ascorbate levels decrease. In lung there are decreased numbers of lamellar bodies and decrease of intraalveolar surfactant.

Exocrine pancreas transcription factor 1 binds to a bipartite enhancer element and activates transcription of acinar genes.

Exocrine pancreas (XP) enhancers, which contain a conserved core sequence, are active only in XP cells. A core enhancer-binding activity also appears to be restricted to XP nuclei. Here we describe the properties of a factor, purified approximately 100,000-fold from pancreas nuclei, which displays core enhancer-binding activity.