Metabolic adaptation of renal carbohydrate metabolism. V.In vivo response of rat renal-tubule gluconeogenesis to different diuretics

We have studied the effects of the diuretics mersalyl, furosemide and ethacrynic acid on renal gluconeogenesis in isolated rat-kidney tubules and on the activities of the most important gluconeogenic and glycolytic enzymes in both fed and fasted rats. Mersalyl (15 mg.kg^–1 animal weight) significantly decreased the rate of gluconeogenesis in well-fed rats (68%) as well as in 24 and 48-h fasted ones (33 and 37% respectively). This inhibition occurred when lactate, pyruvate, glycerol or fructose were used as substrates. Ethacrynic acid at a dose of 50 mg.kg^–1 animal weight provoked a transient inhibition of renal glucose production by almost 20% but only in fed rats with lactate as substrate, whereas the same dose of furosemide did not affect this metabolic pathway.Parallel to these changes, mersalyl caused a significant inhibition in the maximum activity of the most important gluconeogenic enzymes, phosphoenolpyruvate carboxykinase, fructose 1,6-bisphosphatase and glucose 6-phosphatase, in both fed and fasted rats. Neither ethacrynic acid nor furosemide produced any variations in the activities of these enzymes. The activity of the glycolytic enzymes phosphofructokinase and pyruvate kinase was not modified by these diuretics. Nevertheless, the activity of the thiol-enzyme glyceraldehyde 3-phosphate dehydrogenase was severely inhibited by mersalyl and to a lesser extent by the other diuretics. This inhibition was higher in fasted than fed rats. Hence, we conclude that the inhibitory effect of mersalyl on renal gluconeogenesis is due, at least partly, to a decrease in the flux through the gluconeogenic enzymes. Blood glucose was not modified after diuretic treatment in fed animals whereas mersalyl decreased the levels of blood glucose in 24-h fasted rats. Thein vivo effects of diuretics on gluconeogenesis correlate well with the previously observedin vitro effects, although ethacrynic acid was less potent as an inhibitorin vivo, probably because of its rapid clearance.Abbreviations EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycolbis (-aminoethylether) N,N,N,N-tetraacetic acid - DTT dithiothreitol - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - TRIS 2-amino-2-hydroxymethyl-1,3-propanediol Publication No. 166 from Drogas, Tóxicos Ambientales y Metabolismo Celular Research Group, Department of Biochemistry and Molecular Biology, University of Granada, Granada, Spain     

Condensation of supercoiled DNA induced by MnCl2.

Multivalent cations condense DNA in vitro, but it had been thought that a valence of at least + 3 was required in aqueous solution. We have found that Mn2+ can produce toroidal condensates of supercoiled plasmid DNA, but not of linearized plasmid. Mg2+ does not cause condensation, and neither MgCl2 nor NaCl can negate the effect of MnCl2, indicating that the condensation mechanism with Mn is not primarily electrostatic. Supercoiled MnDNA is more extensively digested than the linear form by S1 nuclease. Supercoiling appears to cooperate with Mn2+ in stabilizing helix distortions and also provides a "pressure" that enhances lateral association.     

Uptake of biotin by human hepatoma cell line,Hep G2: A carrier-mediated process similar to that of normal liver

Little is known about the cellular and molecular regulation of the uptake process of the water-soluble vitamin biotin into liver cells, the major site of biotin utilization and metabolism. Such studies are best done using a highly viable and homogeneous cellular system that allows examination of prolonged exposure to an agent(s) or a particular condition(s) on the uptake process. Isolated hepatocytes when maintained in primary culture lose their ability to transport biotin by the specialized carrier system. The aim of the present study was, therefore, to examine the mechanism(s) of biotin uptake by the cultured human-derived liver cells, Hep G₂. Uptake to biotin by Hep G₂ cells was appreciable and linear for up to 10 min of incubation. The uptake process was Na⁺ gradient-dependent as indicated by studies of Na⁺ replacement and pretreatment of cells with gramicidin and ouabain. Biotin uptake was also dependent on both incubation temperature and intracellular energy. Unlabeled biotin and the structural analogs with free carboxyl groups (thioctic acid, desthiobiotin) but not those with blocked carboxyl group (biocytin, biotin methyl ester, and thioctic amide) caused significant inhibition of ³H-biotin uptake at 37°C but not 4°C. Initial rate of biotin uptake was saturable as a function of concentration at 37°C but was lower and linear at 4°C. Pretreatment of Hep G₂ cells with sulfhydryl group inhibitors (e.g., p-chloromer-curibenzene sulfonate) led to a significant inhibition in biotin uptake; this inhibition was effectively reversed by reducing agents (e.g., dithiothreitol). Biotin uptake was also inhibited by the membrane transport inhibitors probenecid (noncompetitively), DIDS and furosemide but not by amiloride. Pretreatment of Hep G₂ cells with valinomycin did not alter biotin uptake. The stoichiometric ratio of biotin to Na⁺ uptake in Hep G₂ cells was also determined and found to be 1:1. These findings demonstrate that biotin uptake by these cultured liver cells is mediated through a specialized carrier system that is dependent on Na⁺-gradient, temperature, and energy and transports the vitamin by an electroneutral process. These findings are similar to those seen with native liver tissue preparations and demonstrate the suitability of Hep G₂ cells for in-depth investigations of the cellular and molecular regulation of biotin uptake by the liver. © 1994 Wiley-Liss, Inc.

1 This article is a US Government work, and as such, is in the public domain in the United State of America

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Phylogenetic relationships among eutherian orders estimated from inferred sequences of mitochondrial proteins: Instability of a tree based on a single gene

The phylogenetic relationships among Primates (human), Artiodactyla (cow), Cetacea (whale), Carnivora (seal), and Rodentia (mouse and rat) were estimated from the inferred amino acid sequences of the mitochondrial genomes using Marsupialia (opossum), Aves (chicken), and Amphibia (Xenopus) as an outgroup. The overall evidence of the maximum likelihood analysis suggests that Rodentia is an outgroup to the other four eutherian orders and that Cetacea and Artiodactyla form a clade with Carnivora as a sister taxon irrespective of the assumed model for amino acid substitutions. Although there remains an uncertainty concerning the relation among Artiodactyla, Cetacea, and Carnivora, the existence of a clade formed by these three orders and the outgroup status of Rodentia to the other eutherian orders seems to be firmly established. However, analyses of individual genes do not necessarily conform to this conclusion, and some of the genes reject the putatively correct tree with nearly 5% significance. Although this discrepancy can be due to convergent or parallel evolution in the specific genes, it was pointed out that, even without a particular reason, such a discrepancy can occur in 5% of the cases if the branching among the orders in question occurred within a short period. Due to uncertainty about the assumed model underlying the phylogenetic inference, this can occur even more frequently. This demonstrates the importance of analyzing enough sequences to avoid the danger of concluding an erroneous tree.     

Capsid assembly and involved function analysis of twelve core protein mutants of duck hepatitis B virus.

The roles of different regions of the duck hepatitis B virus (DHBV) core protein on viral capsid assembly and related functions were examined. Twelve deletion and insertion mutations which covered 80% of the DHBV C open reading frame were constructed and expressed in Escherichia coli. The N-terminal region (amino acids 3 to 66) of DHBV core protein was important for its tertiary structure and function in E. coli. The expressed core mutants without this region apparently inhibited E. coli growth. The results of transmission electron microscopy of E. coli thin sections, capsid agarose gel, and sucrose gradient sedimentation demonstrated that a few DHBV core mutants with insertion in the N terminus and deletion in the C terminus retained the ability to form core-like particles in E. coli. However, other mutations in most of N-terminal and central regions strongly inhibited the self-assembly ability of DHBV core protein in E. coli. In addition, the mutant with a C-terminal region deletion (amino acids 181 to 228) lost most of the nucleic acid-binding activity of the DHBV core protein.     

旋毛虫肌幼虫ES抗原的基因克隆及高效表达

作者对编码旋毛虫肌幼虫ES抗原的部分结构基因进行了克隆、鉴定和表达。用RNA PCR技术直接从旋毛虫肌幼虫总RNA中反转录并扩增出0．7kh的靶DNA,酶切分析后将其克隆到融合表达载体pEx3lC中。SDS—PAGE电泳表明,含重组子的大肠杆菌能够表达出一分子量为37kDa的融合蛋白(P37),后者占菌体总蛋白的22%以上,并以包含体形式存在于菌体中。经对纯化后表达蛋白的ELlSA检测,证明它能被猪旋毛虫病阳性血清和抗旋毛虫单克隆抗体识别。研究结果揭示,重组蛋白P37对于研制旋毛虫病诊断抗原和免疫抗原具有潜在的应用价值。     

双价外壳蛋白基因植物表达载体的构建及马铃薯转基因植株的鉴定

在克隆了马铃薯Ｘ病毒（ＰＶＸ）、马铃薯Ｙ 病毒（ＰＶＹ）和马铃薯卷叶病毒（ＰＬＲＶ）的外壳蛋白基因的基础上,构建同时包含ＰＶＸ和ＰＶＹ 与ＰＶＹ 和ＰＬＲＶ 两个外壳蛋白基因植物表达框架的表达载体,通过农杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍ ｔｕｍｅｆａｃｉｅｎｓ）介导转化烟草（Ｎｉｃｏｔｉａｎａｔａｂａｃｕｍ ）和生产上常用的几个马铃薯（Ｓｏｌａｎｕｍ ｔｕｂｅｒｏｓｕｍ ）优良品种：“Ｆａｖｏｒｉｔａ”、“虎头”、“克４”。经ＰＣＲ检测证明外源基因已整合到植物的染色体上,得到批量转基因植株。在转ＰＶＸ＋ＰＶＹ 外壳蛋白基因的烟草上接种ＰＶＸ （５ μｇ／ｍ Ｌ）、ＰＶＹ（２０ μｇ／ｍ Ｌ）病毒,得到有一定抗性的植株     

Regeneration of haploid plants from isolated microspores of asparagus (Asparagus officinalis L.)

High percentages of micro-calli and micro-derived embryos were produced from isolated asparagus microspores at late uninucleate stage on MS liquid medium supplemented with 1.0 mg l^–1 2,4-D and 0.5 mg l^–1 BA. Two types of calli, namely compact callus (CC) and loose callus (LC), were found. Plantlets were regenerated via organogenesis, when these calli were transferred onto MS solid medium supplemented with 1.0 mg l^–1 BA and 0.2 mg l^–1 IBA 6 weeks. Embryos were produced from liquid cultured microspores, or from solid cultured micro-calli. The frequencies of haploid plant production from organogenesis and embryogenesis were compared. Effects of plant growth regulators on callus production, plantlet regeneration, and haploid plant production were tested. The combination of BA 1.0 mg l^–1 and IBA 0.2 mg l^–1 resulted the highest precentage of haploid plant production (7.7% from CC, 4.3% from LC).Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IBA 3-indolybutyric acid - BA 6-binzyladinine - NAA naphtalene acetic acid - MS Murashige and Skoog     

Enhanced expression of group II phospholipase A2 in human hepatocellular carcinoma

Enzyme activity, protein contents, and mRNA contents of group II phospholipase A₂ (PLA₂) in hepatocellular carcinoma (HCC) surgically obtained from 8 patients were compared with those in either its neighboring liver tissues or control liver tissues. The PLA₂ specific activity towards the mixed micelles of 1-palmitoyl-2-oleoyl-phosphatidylglycerol and cholate was significantly greater in the tumor tissues (6.62 ± 1.46 nmol/min/mg) than those in the surrounding liver tissues (1.33 ± 0.22 nmol/min/mg) and controls (0.43 ± 0.04 nmol/min/mg). The results of immunoblot analysis using a specific anti-human group II PLA₂ antibody and of Northern blot analysis using a human group II PLA₂ cDNA as a probe demonstrated that group II PLA₂ was responsible for the increased enzyme activity. The contents of immunoreactive group II PLA₂ in the tumor tissues (8.81 ± 1.24 ng/mg) were significantly higher than those in the surrounding liver tissues (1.77 ± 0.27 ng/mg); those in the control tissues were below the analytical range of the method used. The group II PLA₂ mRNA was also significantly increased in the tumor tissues, compared with that in the surrounding liver tissues, whereas it was not detectable in th controls. This indicates that group II PLA₂ in HCC is induced at the pretranslational level.