Effect of ammonium acetate-induced hyperammonemia on metabolism of guanidino compounds.

Guanidino compounds are synthesized from arginine in various tissues such as liver, kidney, brain, and skeletal muscle. Guanidino compounds such as arginine and creatine play an important role in nitrogen metabolism, whereas other guanidino compounds such as guanidinosuccinic acid and alpha-N-acetylarginine are known toxins. In order to understand the changes in the metabolism of guanidino compounds during ammonia toxicity, we investigated the effect of hyperammonemia induced by an ammonium acetate injection on the levels of guanidino compounds in plasma, liver, kidney, and brain of rats. Control animals were injected with an equal volume of saline. Blood and tissues were removed 1 h following ammonium acetate or saline injection and guanidino compounds were analyzed by high-performance liquid chromatography. Plasma and kidney levels of guanidinosuccinic acid were significantly elevated in rats challenged with ammonium acetate. Brain alpha-N-acetylarginine levels were also significantly higher in rats injected with ammonium acetate as compared to those in controls. Our results suggest that guanidinosuccinic acid and alpha-N-acetylarginine may play an important role in hyperammonemia.     

Experimental evaluation of the biological risks of introduction of the genetically modified microorganism (GMM) B. subtilis VKPM B-7092 into the environment

Experimental evaluation of the biological risks of introducing the genetically modified microorganism (GMM) B. subtilis VKPM B-7092, an active ingredient of the probiotic VETOM 1.1, into an open system was performed. The following features of the GMM were studied: the survival rate of the GMM in bovine gastroenteric tract; its influence on the microbiocenosis; the species composition of microflora of the gastroenteric tract of the animal species; the possibility of transfer of the DNA fragment cloned in the B. subtilis bacterium and containing the gene of human leukocyte alpha2 interferon to the representatives of intestinal microflora of animals fed on the probiotic VETOM 1.1, as well as the GMM transfer to other microorganism species spread in the areas of potential getting of the GMM into the environment (soil). The study revealed no negative effects of the GMM on the animal organism and the environment, including remote aftereffects.     

Dietary iron restriction increases plaque stability in apolipoprotein-E-deficient mice

Accumulative evidence has supported the role of iron in the development of atherosclerosis. To test whether iron-mediated oxidative stress influences plaque stability, apoliporotein-E (ApoE)-deficient mice (3 months old) were placed on a chow diet or a low-iron diet for 3 months, and the abundance of interstitial collagen and the expression of the matrix degradation-associated enzyme, matrix metalloproteinase-9 (MMP-9), in vascular lesions were assessed. A low-iron diet appeared to reduce iron deposition while substantially increasing collagen content of lesions in mice. Immunostaining demonstrated lower expression of MMP-9 in lesions of iron-restricted animals. Likewise, SDS-PAGE zymography revealed lower gelatinolytic activities in aortic tissues and sera of the same group of animals. When older ApoE-deficient mice (5 months old) received a low-iron diet for 2 months, development of the lesion area was not significantly affected. However, the lesional collagen content was much higher in the iron-restricted group of animals, and MMP-9 expression in aortic tissues from the same group of mice was significantly lower. Treatment of murine J774 macrophages with increasing concentrations of ferric ammonium citrate significantly enhanced the amount of MMP-9 secreted. Together, these data indicate that decreased vascular iron content following dietary iron restriction in ApoE-deficient mice leads to lower matrix degradation capacity and increased plaque stability.     

Morin a flavonoid exerts antioxidant potential in chronic hyperammonemic rats: a biochemical and histopathological study

Plant flavonoids are emerging as potent therapeutic drugs effective against a wide range of free radical-mediated diseases. Morin (3,5,7,2′,4′-pentahydroxyflavone), a member of flavonols, is an important bioactive compound by interacting with nucleic acids, enzymes and protein. In this study, we found that morin (30 mg/kg body weight) by oral administration offers protection against hyperammonemia by means of reducing blood ammonia, oxidative stress and enhancing antioxidant status in ammonium chloride-induced (100 mg/kg body weight; i.p) hyperammonemic rats. Enhanced blood ammonia, plasma urea, lipid peroxidation in circulation and tissues (liver and brain) of ammonium chloride-treated rats was accompanied by a significant decrease in the tissues levels of superoxide dismutase (SOD), catalase, reduced glutathione (GSH) and glutathione peroxidase (GPx). Morin administered rats showed a significant reduction in ammonia, urea, lipid peroxidation with a simultaneous elevation in antioxidant levels. Cotreatment with morin prevented the elevation of liver marker enzymes induced by ammonium chloride. The body weight of the animals decreased significantly on ammonium chloride administration when compared with control group. However, cotreatment with morin significantly prevented the decrease of the body weight caused by ammonium chloride. Hyperammonemic rats show liver fibrosis, steatosis, sinusoidal dilatation, etc., along with necrosis, microcystic degeneration in brain. All these changes were reduced in hyperammonemic rats treated with Morin, which too correlated with the biochemical observations. In conclusion, these findings indicate that morin exert antioxidant potential and offer protection against ammonium chloride-induced hyperammonemia. But the exact underlying mechanism needs to be elucidated.     

Surfactant-induced lipid peroxidation in a tropical euryhaline teleost Oreochromis mossambicus (Tilapia) adapted to fresh water

Exposure to anionic (sodium dodecyl sulfate, SDS), cationic (cetyl trimethyl ammonium bromide CTAB) and non ionic (Triton X-100) surfactants at a sub lethal concentration of 1 ppm resulted in severe oxidative stress in the hepatic, renal and cardiac tissues of fresh water adapted Oreochromis mossambicus. Hepatic catalase showed significant increase (P<0.001) in all the surfactant exposed fish, but the renal enzyme was significantly increased only in CTAB dosed fish (P<0.001) and the cardiac enzyme showed significant increase in Triton (P<0.05) and CTAB dosed fish (P<0.001). SOD levels were significantly increased (P<0.001) in hepatic, renal and cardiac tissues of all the surfactant-treated fish. Glutathione reductase also was significantly increased (P<0.001) in the hepatic and renal tissues of surfactant dosed fish except cardiac tissues of CTAB exposed animals. Glutathione levels in the tissues studied were significantly higher in the surfactant treated animals (P<0.001) whereas malondialdehyde levels were significantly elevated only in the hepatic tissues of animals exposed to Triton (P<0.001). The surfactants based on their charge, antioxidant profile and in vivo metabolism may be arranged in the order of decreasing toxicity as CTAB > Triton > SDS. Thus it may be inferred from the present study that the antioxidant defenses and the in vivo metabolism of the surfactants are key factors in deciding the surfactant toxicity.     

In vivo study of the appearance and fluctuations of insulin binding sites in different tissues during rat development

The appearance and fluctuations of specific insulin binding sites in several tissues in vivo during rat development, have been determined. After intravenous administration of 125I-insulin to fetal, suckling and adult rats, changes on specific hormone uptake were observed depending on the tissues tested and on the age of animals. Thus, in liver, specific insulin uptake was much greater in 19 day-old fetuses and 10 day-old suckling animals than in adult rats. By contrast, brown fat and spleen insulin uptake was undetected in fetal animals but present in suckling rats, while lung insulin uptake was absent in the adults but present in fetal and suckling animals. Of interest were the specific insulin uptakes by three different muscle tissues. In fact, heart insulin uptake was much higher in younger animals than in adult rats, while in the diaphragm it was significantly smaller in all groups and in skeletal muscles hormone uptake was much smaller than in the other two muscle tissues and was even absent in the fetuses. In those tissues that had previously been shown to exhibit a specific insulin uptake, the iodinated hormone uptake decreased proportionally with simultaneous injection of increasing amounts of unlabelled insulin. These results indicate that insulin binding sites appear at different times and fluctuate in a different manner according to the tissues tested during rat development; this might be important in the stimulation of the functional activities of those tissues during perinatal age.     

Mode of action of iron on arylsulphatases under acute lead acetate treated conditions in rats

Arylsulphatases A, B and C were found to be inhibited in liver and kidney tissues under lead acetate-treated conditions (both in vivo and in vitro) in rats. When lead acetate-treated animals (in vivo) were supplemented with ferric ammonium citrate (in vivo), a remarkable recovery was found in the activities of all arylsulphatases A, B and C whereas ferric ammonium citrate itself had no effect on the activities of arylsulphatases. When both the in vivo and in vitro lead acetate-treated arylsulphatases were supplemented with the purified ferritins (in vitro) it was observed that lead-induced inhibition of the activities of arylsulphatases was successfully reversed. It was also found that ferritins were able to bind a large quantity of lead. These results indicated that ferritins were directly involved for reactivation of arylsulphatases which were inhibited by lead. It was well established that a response to iron administration in rats was an immediate de novo stimulation of ferritin biosynthesis. Iron might therefore protect the enzymatic activities of arylsulphatases by enhancing the level of ferritin in liver and kidney tissues which is known to bind a large quantity of lead thereby ameliorating their toxic effects in the living system.     

Superoxide Production and Antioxidant Enzymes in Ammonia Intoxication in Rats

Injection of large doses of ammonium salts lead to the rapid death of animals. However, the molecular mechanisms involved in ammonia toxicity remain to be clarified. We have tested the effect of injecting 7 mmol/kg of ammonium acetate on the production of superoxide and on the activities of some antioxidant enzymes in rat liver, brain, erythrocytes and plasma. Glutathione peroxidase, superoxide dismutase and catalase activities were decreased in liver and brain (both in cytoso-lic and mitochondrial fractions) and also in blood red cells, while glutathione reductase activity remained unchanged. Superoxide production in submitochon-drial particles from liver and brain was increased by more than 100% in both tissues. Both diminished activity of antioxidant enzymes and increased superoxide radical production could lead to oxidative stress and cell damage, which could be involved in the mechanism of acute ammonia toxicity.     

The characteristics of nitrogen metabolism in the tissues of rabbits with ammonium toxicosis]

Disturbances of nitrogen metabolism under acute ammonium toxicosis have been studied in tissues of rabbit. A sharp increase of the ammonium content in the blood and tissues of the liver and kidneys is accompanied by an increase in the glutamine and glutamate level in all tissues. The level of urea nitrogen in the blood of rabbits increases. The activity of phosphate-independent and phosphate-activated glutaminase also increases in tissues of the liver and kidneys, while arginase activity decreases as compared with the control, which is connected with fall of the ATP level under hyperammonemia. A nomograph method of representation of the redox state has been used.     

Development of uterine adenomyosis after treatment with dopamine antagonists in mice

Development of uterine adenomyosis was studied in SHN mice treated with psychotherapeutic drugs, sulpiride and perphenazine, and gastroenteric drug, metoclopramide, which act as dopamine antagonists to increase prolactin release from the pituitary gland. Administration of these drugs twice daily for 40-70 or 40-90 days of age induced an elevation in serum level of prolactin. Furthermore, the treated mice showed a prolongation of metestrous plus diestrous phase and a high incidence of uterine adenomyosis compared with vehicle-treated control mice. These results indicate that hyperprolactinemia produced by continuous treatment with psychotherapeutic and gastroenteric drugs is responsible for the occurrence of irregular estrous cycles and the genesis of uterine adenomyosis in mice.     

Electrophysiological abnormalities produced by ammonium in isolated perfused brook trout, Salvelinus fontinalis, hearts

Electrocardiographic (ECG) abnormalities and arrhythmias have been observed in hyperammonemic patients and in animals injected with ammonium salts. Ammonium is excreted directly into the environment by fish, and it is a potent fish toxin, but the effects of ammonium on the cardiovascular system of fish are unknown. This study investigated the cardiotoxic effects of ammonium on isolated perfused fish hearts. These were compared with ammonium-induced cardiac electrophysiological abnormalities observed in humans and other experimental animals. Isolated perfused fish hearts exhibit ammonium-induced cardiac abnormalities similar to the ammonium-induced abnormalities seen in the hearts of higher vertebrates. Therefore, we conclude that the myocardium of ammonotelic species does not possess a specialized ammonium detoxification mechanism, and that ammonium is cardiotoxic to salmonid fish.     

Effect of ammonia-stress on the total autolytic levels of proteins in tissues of an air-breathing fish,Channa punctatus (Bloch)

G season-specific effect of a sub-lethal ambient ammonium chloride concentration on the total autolysis of protein in different tissues of the Indian air-breathing murrel,Chauna punctatos (Bloch), has been demonstrated. While its effect on the autotytie levels of protein of different tissues (except white muscle) was marginal in the winter-adapted fish, the same in the summer-adapted fish was more pronounced. In general a reduction in the autolysic levels of the tissue protein of the ammonium chloride-stressed fish was observed. In certain tissues like white muscle of an ammonium chloride-stressed fish and gill of the ammonium hydroxide-stressed fish, augmented levels of autolysis were noticed. The present findings suggest that the accumulation of amino acids in different tissues of the ammonia-stressed fish, as observed earlier, can be explained best by mechanisms other than ammonia induced increased breakdown of proteins.     

Effect of bicarbonates and CO2 concentration increase in tissues on assimilation of ammonium nitrogen in cattle

The feeding of carboxyline and cobalt salts to cattle young fattener receiving the concentrate-silo rations with synthetic nitrogen-containing substances (diammonium phosphate and urea) is accompanied by an increase in the concentration of bicarbonates and CO2 in blood and citric acid in blood plasma with a decrease of the ketonic bodies content in it. The level of carbon dioxide in tissues being increased, the content of ammonium nitrogen in the rumen fluid lowers and the activity of transaminases in blood plasma, the content of glutamate in the liver and that of urea in the rumen fluid increase which evidences for an intensified transformation of the ration nitrogen in the organism. The performance of animals is increased.     

Structure-specific abnormalities associated with mutations in a DNA replication accessory factor in Drosophila

We have phenotypically and molecularly analyzed the cutlet locus in Drosophila. Homozygous cutlet flies exhibit abnormal development of a subset of adult tissues, including the eye, wing, and ovary. We show that abnormal development of these tissues is due to a defect in normal cell growth. Surprisingly, cell growth is affected in all developing precursor tissues in cutlet mutant animals, including those that give rise to phenotypically wild-type adult structures. The cutlet gene encodes a Drosophila homologue of yeast CHL12 and has similarity to mammalian replication factor C. In addition, cutlet genetically interacts with multiple subunits of Drosophila replication factor C. Our results suggest that the cutlet gene product acts as an accessory factor for DNA replication and has different requirements for the formation of various adult structures during Drosophila development.     

Cryptosporidial infections in captive wild animals

Neonatal diarrhea was an important cause of morbidity and mortality in a hand-rearing facility for exotic ruminants at the San Diego Wild Animal Park. Studies undertaken to determine the causes of the problem revealed that oocysts of Cryptosporidium sp. were demonstrable in auramine O stained fecal smears from 52 of 183 (28.4%) animals examined. Cryptosporidial infection was identified in 21 of 40 species of exotic ruminants with diarrhea. In addition, cryptosporidia were associated with gastroenteric disease in two primates and two reptiles. It was observed also that auramine O stained coccidial oocysts of the genus Eimeria, which were present in five of 183 (2.7%) of the specimens examined.     

Rapid,simple and sensitive microassay for skeletal and cardiac muscle myoglobin and hemoglobin: use in various animals indicates functional role of myohemoproteins

A novel, simple, rapid, sensitive and reproducible microassay is described for determination of myoglobin and hemoglobin content of myocardial and skeletal muscle biopsy specimens from various mammals, birds and fish. As little as 50 mg of tissue is needed and myoglobin concentrations lower than 1 mg% can be detected. Myoglobin and hemoglobin are separated at alkaline pH by ammonium sulfate extraction followed by ultrafiltration. Heme content is determined by absorption of the Soret band when the hemoprotein extract is visibly colored or more sensitively by its peroxidase activity when the extract has low color. The heme reacts with tertiary-butyl hydroperoxide and orthotolidine to generate a blue color. Hemoglobin content is correlated with myoglobin content and is related to aerobic capacity and blood flow to the tissue. Myoglobin content varied over 5 orders of magnitude up to 7 per cent of the weight of tissue, whereas hemoglobin content varied over 2 orders of magnitude up to 6 per cent of tissue weight. Myoglobin content is increased in species with high basal metabolic rate, high physical activity, prolonged diving capacity, fatigue resistance, and red muscle, whereas it is decreased in white muscle, iron-deficient animals, animals with sedentary lifestyles, and in animals and tissues with small fiber diameters such as avian or fish hearts.     

The Use of SCID Mice in Biotechnology and as a Model for Human Disease

Abstract

The use of SCID (severe combined immunodeficient) mice in medical research and biotechnology has increased tremendously in recent years. This review outlines the major characteristics of these animals and the impediments that they pose to the engraftment of human cells and tissues. The development of our SCID mice pretreatment protocol (anti-asialo GM1 antisera and radiation) is described, and the results of xenotransplantation studies of human cells and tissues in these pretreated animals are outlined. Wherever possible, data from transplantation studies (of human tissues and cells) in pretreated and nonpretreated animals are compared. The potential of our pretreated SCID mice for medical research and biotechnology is discussed.     

Translational regulation of ferritin synthesis in rat spleen: effects of iron and inflammation

Translational control of ferritin synthesis was studied in rat spleen, and compared with that for liver, heart and brain, in response to iron and inflammation. Spleen concentrations of total RNA in the ribonucleoprotein (mRNP) fraction was comparable to that for liver, while polyribosomal RNA was less. Both fractions were ten-fold lower in heart and brain. In untreated animals, the mRNP fraction of all tissues had the largest portion of the ferritin mRNA, as determined by slot blot hybridization with 32P-labeled cDNA for the L subunit. Acute treatment with ferric ammonium citrate shifted the spleen ferritin mRNA to the polyribosome fraction. This was also so in liver but not in the heart and brain which took up much less iron. The findings were confirmed by hybridization studies of mRNPs and polyribosomes separated in sucrose gradients. Turpentine-induced inflammation also caused a shift in ferritin mRNA from the mRNP to the polyribosome fraction of spleen and liver, over 12 h. We conclude that as in liver, spleen ferritin synthesis is under translational control by iron, and that both tissues also respond to inflammation by shifting of ferritin mRNA to the polyribosomes.     

吡咯喹啉醌(PQQ)营养作用研究进展

吡咯喹啉醌(PQQ)是细菌脱氢酶氧化还原反应的辅助因子,广泛存在于微生物、植物、动物及人体中。迄今为止,PQQ催化氧化还原反应的能力远超过已知的生物活性分子。体内外研究表明,PQQ能够刺激微生物生长,增强其对极端环境的适应能力,并对植物和动物的生长、发育和繁殖十分重要。本文阐述了PQQ的理化性质、自然分布和营养作用的研究进展,以推动其在食品、医疗及农林渔业领域的发展应用。