Association of a missense mutation in the bovine leptin gene with carcass fat content and leptin mRNA levels

Previously, we have shown that alleles of the BM1500 microsatellite, located 3.6 kb downstream of the leptin gene in cattle, were associated with carcass fat measures in a population of 154 unrelated beef bulls. Subsequently, a cytosine (C) to thymine (T) transition that encoded an amino acid change of an arginine to a cysteine was identified in exon 2 of the leptin gene. A PCR-RFLP was designed and allele frequencies in four beef breeds were correlated with levels of carcass fat. The T allele was associated with fatter carcasses and the C allele with leaner carcasses. The frequencies of the SNP alleles among breeds indicated that British breeds have a higher frequency of the T allele whereas the continental breeds have a higher occurrence of the C allele. A ribonuclease protection assay was developed to quantify leptin mRNA in a separate group of animals selected by genotype. Animals homozygous for thymine expressed higher levels of leptin mRNA. This may suggest that the T allele, which adds an extra cysteine to the protein, imparts a partial loss of biological function and hence could be the causative mutation.     

Ubiquinone redox cycle as a cellular antioxidant defense system

Ubiquinone (UQ) reductase responsible for reduction of non-mitochondrial UQ was investigated in rats toward demonstrating an antioxidant role of UQ. In the liver, most of cellular UQ-10 reductase activity was attributable to a novel NADPH-UQ reductase in cytosol. The enzyme was not inhibited by dicumarol and rotenone, and had a Km of 19 microM for NADPH and 307 microM for NADH at the optimum pH 7.4. The enzyme was purified 300-fold to apparent homogeneity from the liver cytosol by an affinity chromatographic method. The purified enzyme reduced UQ-10 in lecithin liposomes, and protected the liposomes from lipid peroxidation. Furthermore, supplementation of rats with UQ-10 was observed to increase the enzyme level in their livers without affecting levels of other antioxidant factors. The observations suggested that cytosolic NADPH-UQ reductase is responsible for cellular UQ redox cycle as an endogenous antioxidant.     

State of antioxidant defense and L-arginin-nitrogen oxide system in blood of patients with urogenital chlamydiosis

The system L-arginine-nitrogen oxide plays a significant role in maintenance of the anti-infectious protection of an organism. A condition of the given system and activity of a enzymatic part of antiradical protection in the blood of patients with chlamydiosis has been studied. Obtained data specify an intensification of processes of an oxidizing way of recycling of arginine in an organism of patients. Substantial increase of NO-synthase activity and insignificant activity of arginase in the blood is revealed. The level of nitrite-anion in blood cells of patients authentically increases: 1.7 times in erythrocytes, and 1.4 times in lymphocytes. It is shown, that in patients with chlamydiosis glutathione system is intensified, that is evidenced by an increase glutathione-peroxidase activity and authentic increase of glutathione level. It is assummed that the established features of nitrogen oxide exchange play a significant role in formation of a pathological condition at urogenital chlamydia infections.     

Oxidative burden and antioxidant defense system in polymorphonuclear leukocytes of human lung diseases

Superoxide anion (O2.-) and hydrogen peroxide (H2O2) production was significantly higher in blood neutrophils (PMNs) of patients with lung cancer and non-malignant lung diseases when compared to the controls (p<0.001). Superoxide dismutase (SOD) activity was significantly decreased in PMNs of patients with lung cancer (p<0.001). Similarly, catalase and glutathione peroxidase (GPx) activities were lower in PMNs of lung cancer patients as compared to non-malignant lung diseases and controls. There was an increase in HMP shunt activity as measured by rate of formation of 14CO2 from [1-14C]-glucose in PMNs of lung cancer patients. Modifications in the antioxidant defense system in PMNs of malignant and nonmalignant lung diseases, the changes being more in the malignancy are indicated.     

A missense mutation in the bovine leptin receptor gene is associated with leptin concentrations during late pregnancy

The leptin receptor (LEPR) gene consists of 20 exons divided over 1.75 Mb. Parts of bovine LEPR exon 4 (79 bp), exon 11 (95 bp) and exon 20 (513 bp) of 20 cows (Holstein-Friesian) were sequenced (AJ580799; AJ580800; AJ580801) in an attempt to find polymorphisms. In exons 4 and 11 no SNPs were found. In exon 20, a T to C missense mutation was found at nucleotide 115, which causes an amino acid substitution at residue 945 (T945M). Frequencies for alleles C and T were 0.93 and 0.07 respectively, in a population of 323 Holstein-Friesian cows and TT animals were not detected. Using genotypes of these cows an association study was performed for leptin concentrations during late pregnancy and lactation. Leptin concentrations were determined by radioimmunoassay (RIA). The T945M mutation showed an association with circulating leptin concentrations only during late pregnancy (P < 0.05) but not during lactation (P > 0.05). The CC genotype had higher concentrations than the CT genotype during this period. A combined effect with previously described leptin polymorphisms on prepartum leptin concentrations was observed, with one genotype combination having significantly lower levels of leptin up to 50 days, but interaction effects were not significant. The T945M polymorphism may have induced a structural change in the intracellular domain of the LEPR, which may have influenced the signal transduction pathway. However, the effect was found only for the heterozygous genotype because the TT genotype was not detected in this population of 323 Holstein-Friesian cows.     

The human gene mutation database.

The Human Gene Mutation Database (HGMD) represents a comprehensive core collection of data on published germline mutations in nuclear genes underlying human inherited disease. By September 1997, the database contained nearly 12 000 different lesions in a total of 636 different genes, with new entries currently accumulating at a rate of over 2000 per annum. Although originally established for the scientific study of mutational mechanisms in human genes, HGMD has acquired a much broader utility to researchers, physicians and genetic counsellors so that it was made publicly available at http://uwcm.ac.uk/uwcm/mg/hgmd0.html in April 1996. Mutation data in HGMD are accessible on the basis of every gene being allocated one web page per mutation type, if data of that type are present. Meaningful integration with phenotypic, structural and mapping information has been accomplished through bi-directional links between HGMD and both the Genome Database (GDB) and Online Mendelian Inheritance in Man (OMIM), Baltimore, USA. Hypertext links have also been established to Medline abstracts through Entrez , and to a collection of 458 reference cDNA sequences also used for data checking. Being both comprehensive and fully integrated into the existing bioinformatics structures relevant to human genetics, HGMD has established itself as the central core database of inherited human gene mutations.     

The glutathione-dependent system of placenta antioxidant defense in miscarriage

The glutathione-dependent system of antioxidant defense was studied in the chorionic and placental tissues of women with miscarriage. In the case of spontaneous abortion, the level of glutathione peroxidase reached the maximum even in trimester I and remained more than 1.5-fold higher during the whole gestation period than in the placental tissue of women with physiological pregnancy and delivery. The activity of glutathione reductase in miscarriage was insignificantly different from that in the control group. The activity of glutathione S-transferase in miscarriage was similar to that in the control group during trimester I and remained low during the whole gestation, contributing to a decrease in nonspecific defense in the mother-placenta-fetus system, leading to pathology of the fetus and infant. It is concluded that oxidative stress in the placental tissues is an essential pathogenic factor of miscarriage.     

Effect of Terminalia arjuna on antioxidant defense system in cancer

Constant production of reactive oxygen species (ROS) during aerobic metabolism is balanced by antioxidant defense system of an organism. Although low level of ROS is important for various physiological functions, its accumulation has been implicated in the pathogenesis of age-related diseases such as cancer and coronary heart disease and neurodegenerative disorders such as Alzheimer’s disease. It is generally assumed that frequent consumption of phytochemicals derived from vegetables, fruits, tea and herbs may contribute to shift the balance towards an adequate antioxidant status. The present study is aimed to investigate the effect of aqueous extract of medicinal plant Terminalia arjuna on antioxidant defense system in lymphoma bearing AKR mice. Antioxidant action of T. arjuna is monitored by the activities of catalase, superoxide dismutase and glutathione S transferase which constitute major antioxidant defense system by scavenging ROS. These enzyme activities are low in lymphoma bearing mice indicating impaired antioxidant defense system. Oral administration of different doses of aqueous extract of T. arjuna causes significant elevation in the activities of catalase, superoxide dismutase and glutathione S transferase. T. arjuna is found to down regulate anaerobic metabolism by inhibiting the activity of lactate dehydrogenase in lymphoma bearing mice, which was elevated in untreated cancerous mice. The results indicate the antioxidant action of aqueous extract of T. arjuna, which may play a role in the anti carcinogenic activity by reducing the oxidative stress along with inhibition of anaerobic metabolism.     

Ethanol-induced alterations of the antioxidant defense system in rat kidney

We report here the effects of chronic ethanol consumption on the antioxidant defense system in rat kidney. Thirty-two male Wistar rats were randomly divided in two identical groups and were treated as follows: control group (water for fluid) and the ethanol-fed group (2 g/kg body weight/24 h). The animals were sacrificed after 10 weeks, and respectively 30 weeks of ethanol consumption, and the renal tissue was isolated and analyzed. Results revealed that kidney alcohol dehydrogenase activities increased significantly after ethanol administration, but the electrophoretic pattern of alcohol dehydrogenase isoforms was unmodified. The SDS polyacrylamidegel electrophoretic study of kidney proteins has revealed the appearance of two new protein bands after long-term ethanol consumption. The kidney reduced glutathione/oxidized glutathione ratio decreased, indicating an oxidative stress response due to ethanol ingestion. The malondialdehyde contents and xanthine oxidase activities were unchanged. The antioxidant enzymatic defense system showed a different response during the two periods of ethanol administration. After 10 weeks, catalase, glutathione peroxidase, glutathione reductase, and glucose-6-phosphate dehydrogenase were activated, while superoxide dismutase, glutathione transferase, and gamma-glutamyltranspeptidase levels were stationary. After 30 weeks, superoxide dismutase and glutathione peroxidase activities were unmodified, but catalase, glutathione transferase, gamma-glutamyltranspeptidase, glutathione reductase, and glucose-6-phosphate dehydrogenase activities were significantly increased. Remarkable changes have been registered after 30 weeks of ethanol administration for glutathione reductase and glucose-6-phosphate dehydrogenase activities, including an increase by 106 and 216' of control values, respectively. These results showed specific changes in rat kidney antioxidant system and glutathione status as a consequence of long-term ethanol administration.     

Sulphadoxine-pyrimethamine alters the antioxidant defense system in blood of rabbit

Sulphadoxine-pyrimethamine (SP) despite reported resistance remains an important drug of choice for the treatment and control of malaria in most endemic areas. Exacerbation of intra-erythrocytic oxidative stress might contribute to the process of elimination of malaria parasites in the body. The effect of treatment with SP on the antioxidant defense system was investigated using rabbit as a model. Ten male rabbits were divided into two groups of five animals each. The first group was administered with normal saline and served as control. The second group received a single dose of SP (26.25mg/kg body weight). Blood samples were collected before and at 6, 12 and 24 h after drug administration. Activity of cellular enzymatic antioxidants, superoxide dismutase (SOD) and catalase (CAT), and level of reduced glutathione (GSH) were assayed using standard spectrophotometric methods. Serum lipid peroxidation was assessed by the formation of thiobarbituric acid reactive species (TBARS) while protein content was assayed by the method of Lowry et al., 1951. SOD activity was observed to increase progressively by 4.9, 63.4 and 120.8% at 6, 12 and 24 h respectively, after drug administration. Similarly, CAT activity increased by 44.5, 82.6 and 116.3% at 6, 12 and 24 h, respectively. TBARS level also increased significantly by 45.5, 118.2 and 186.4%, respectively. However, the level of GSH decreased by 41.9% at 6 h and remained so up till the 12 h, but by 24 h after drug administration, the level of the thiol substance has increased considerably up to 48.4% above the baseline level. SP treatment altered the antioxidant defense system in blood and may therefore induce oxidative stress by generating reactive oxygen species. This might play significant role in the therapeutic and adverse effects associated with the drug.     

Effect of N-acetylcysteine and deferoxamine on endogenous antioxidant defense system gene expression in a rat hepatocyte model of cocaine cytotoxicity

In the present study we investigated on cultures of hepatocytes from phenobarbital-pretreated rats, the effect of the antioxidants, 0.5 mM N-acetylcysteine (NAC) or 1.5 mM deferoxamine (DFO), previously incubated for 24 h and coincubated with cocaine (0-1000 microM) for another 24 h. Cocaine cytotoxicity was monitored by either the lysis of the cell membranes or apoptosis. Lysis of the cell membranes was evidenced by lactate dehydrogenase leakage, apoptosis was observed by detecting a hypodiploid peak (<2C) in DNA histograms obtained by flow cytometry, peroxide production was quantified with 2', 7'-dichlorodihydrofluorescein diacetate and gene expression of the antioxidant enzymes: Mn- and Cu,Zn-superoxide dismutases, catalase and glutathione peroxidase were measured by Northern blot analysis. NAC and DFO significantly decreased the extent of lysis of cell membranes and apoptosis, and the antiapoptotic effect was parallel to peroxide generation. By the effect of NAC and DFO, significant increases were detected in the levels of mRNA of catalase, manganese superoxide dismutase and glutathione peroxidase. From these results we conclude that NAC or DFO, when incubated in the presence of cocaine, exerted a protective effect against cocaine toxicity at the level of both lysis of the membranes and apoptosis. This protective effect, in the case of NAC, was directed towards an increase in antioxidant enzyme expression, and in the case of DFO against reactive oxygen species generation.     

Response of antioxidant defense system to chromium (VI)-induced cytotoxicity in human diploid cells

The aim of this study is to establish antioxidant indicators of chromium toxicity in fetal human lung fibroblasts (HLF). The results obtained corroborate and develop our earlier observation of low-dose and long-term action of Cr(VI) on human cells in culture. In the case of a nontoxic chromium dose, temporary oxidative stress is overcome by increased activity of the antioxidant system with correlation to cell cycle re-entry. The toxic concentrations misbalance the cell antioxidant defense systems and cause irreversible growth arrest and massive cell death by apoptosis. Sub-toxicity is defined as toxicity stretched in time. The activity of GPx (glutathione peroxidase) is proposed as a biomarker of oxidative stress caused by Cr(VI), and the GR (glutathione reductase) inhibition is considered as a marker of the toxicity developed under the complex Cr(VI) action. In HLF cells the glutathione dependent defense system is the first system destroyed in response to toxic chromium action. Only the balance between SOD (superoxide dismutase) and H₂O₂ degrading enzymes (catalase and GPx), should play an important role in the fate of a cell, not individual enzymes.     

Glutathione peroxidase-1 gene knockout on body antioxidant defense in mice

To determine the in vivo role of cellular glutathione peroxidase (E.C.1.11.1.9, GPX1), we challenged the GPX1 knockout [GPX1(-/-)], the GPX1 overexpressing [GPX1(+)], and their respective wild-type (WT) mice of different Se and vitamin E status with acute oxidative stress. After these mice were injected with pro-oxidants paraquat or diquat at 12 to 125 mg/kg of body weight, their survival rate and time were a function of their GPX1 activity levels. The GPX1 protection was associated with attenuation of NADPH and NADH oxidation, protein carbonyl and F(2)-isoprostanes formation, and alanine transaminase release in various tissues, and was irreplaceable by high levels of dietary vitamin E or other selenoproteins. The GPX1 expression was also protective against moderate oxidative stress induced by low levels of paraquat or diquat, particularly in the Se-deficient mice. Alteration of GPX1 expression showed no impact on the expression of other selenoproteins and antioxidant enzymes in unstressed mice. Total Se content in liver of the Se-adequate GPX1(-/-) mice was reduced by 60% the WT controls. In conclusion, normal expression of GPX1 is essential and overexpression of GPX1 is beneficial to protect mice against acute oxidative stress.