Conjugated linoleic acid inhibits differentiation of pre- and post- confluent 3T3-L1 preadipocytes but inhibits cell proliferation only in preconfluent cells.
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Conjugated linoleic acid (CLA; 18:2) is a group of isomers (mainly 9-cis, 11-trans and 10-trans, 12-cis) of linoleic acid. CLA is the product of rumen fermentation and can be found in the milk and muscle of ruminants. Animals fed CLA have a lower body fat content. The objective of this study was to establish the possible mechanisms by which CLA affects adipogenesis. 3T3-L1 is a well-established cell line that is used extensively in studying adipocyte biology. These cells typically grow in a culture medium until they reach confluence, at which time they are induced to differentiate by hormonal treatment (d 0). Treatment of 3T3-L1 cells with 25 to 100 micromol/L CLA inhibited differentiation in a dose-dependent manner, while linoleic acid treatment did not differ from DMSO-treated controls. Continuous treatment from d -2, -1, 0 or 2 to d 8 and treatment from d -2 to d 0 and from d 0 to d 2 inhibited differentiation. Differentiation was monitored morphologically (oil Red-O staining), enzymatically (reduction of activity of glycerol-3-phosphate dehydrogenase), and by northern analysis of peroxisome proliferator-activated receptor gamma2, CCAAT/enhancer binding protein alpha and adipocyte specific protein 2 mRNA. CLA inhibited cell proliferation of nonconfluent cells but did not affect cell division of confluent cells, as indicated by 5-bromo-2'-deoxyuridine incorporation and mitochondria metabolism. Therefore, CLA inhibited differentiation before confluence and during induction. However, cellular proliferation was only inhibited prior to induction. These results imply that fat reduction caused by CLA treatment may be attributed to its inhibition of both proliferation and differentiation of preadipocytes in animals. (+info)
Two putative MAP kinase genes, ZrHOG1 and ZrHOG2, cloned from the salt-tolerant yeast Zygosaccharomyces rouxii are functionally homologous to the Saccharomyces cerevisiae HOG1 gene.
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The salt-tolerant yeast Zygosaccharomyces rouxii can adjust its osmotic balance when responding to osmotic shock by accumulating glycerol as the compatible osmolyte. However, the mechanism of glycerol production in Z. rouxii cells and its genetic regulation remain to be elucidated. Two putative mitogen-activated protein (MAP) kinase genes, ZrHOG1 and ZrHOG2, were cloned from Z. rouxii by their homology with HOG1 from Saccharomyces cerevisiae. The deduced amino acid sequences of ZrHog1p and ZrHog2p indicated close homology to that of Hog1p and contained a TGY motif for phosphorylation by MAP kinase kinase. When ZrHOG1 or ZrHOG2 was expressed in an S. cerevisiae hog1delta null mutant, the salt tolerance and osmotic tolerance characteristics of wild-type S. cerevisiae were restored. In addition, the aberrant cell morphology and low glycerol content of the hog1delta null mutant were corrected, indicating that ZrHog1p and ZrHog2p have functions similar to Hog1p. While the transcription of the glycerol-3-phosphate dehydrogenase gene (GPD1) of the ZrHOG1-harbouring S. cerevisiae mutant was similar to that of wild-type S. cerevisiae, the ZrHOG2-harbouring strain showed prolonged GPD1 transcription. Both Zrhog1delta and Zrhog2delta Z. rouxii null mutants showed a decrease in salt tolerance compared to the wild-type strain. The present study suggested the presence of a high-osmolarity glycerol response (HOG) pathway in Z. rouxii similar to that elucidated in S. cerevisiae. Two putative MAP kinase genes in Z. rouxii appeared to be significant in either osmotic regulation or ion homeostasis. (+info)
Different signalling pathways contribute to the control of GPD1 gene expression by osmotic stress in Saccharomyces cerevisiae.
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Yeast cells respond to a shift to higher osmolarity by increasing the cellular content of the osmolyte glycerol. This response is accompanied by a stimulation of the expression of genes encoding enzymes in the glycerol production pathway. In this study the osmotic induction of one of those genes, GPD1, which encodes glycerol-3-phosphate dehydrogenase, was monitored in time course experiments. The response is independent of the osmolyte and consists of four apparent phases: a lag phase, an initial induction phase, a feedback phase and a sustained long-term induction. Osmotic shock with progressively higher osmolyte concentrations caused a prolonged lag phase. Deletion of HOG1, which encodes the terminal protein kinase of the high osmolarity glycerol (HOG) response pathway, led to an even longer lag phase and drastically lower basal and induced GPD1 mRNA levels. However, the induction was only moderately diminished. Overstimulation of Hog1p by deletion of the genes for the protein phosphatases PTP2 and PTP3 led to higher basal and induced mRNA levels and a shorter lag phase. The protein phosphatase calcineurin, which mediates salt-induced expression of some genes, does not appear to contribute to the control of GPD1 expression. Although GPD1 expression has so far not been reported to be controlled by a general stress response mechanism, heat-shock induction of the GPD1 mRNA level was observed. However, unregulated protein kinase A activity, which strongly affects the general stress response, only marginally altered the mRNA level of GPD1. The osmotic stimulation of GPD1 expression does not seem to be mediated by derepression, since deletion of the SSN6 gene, which encodes a general repressor, did not significantly alter the induction profile. A hypoosmotic shock led to a transient 10-fold drop of the GPD1 mRNA level. Neither the HOG nor the protein kinase C pathway, which is stimulated by a decrease in external osmolarity, is involved in this effect. It was concluded that osmotic regulation of GPD1 expression is the result of an interplay between different signalling pathways, some of which remain to be identified. (+info)
What controls glycolysis in bloodstream form Trypanosoma brucei?
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On the basis of the experimentally determined kinetic properties of the trypanosomal enzymes, the question is addressed of which step limits the glycolytic flux in bloodstream form Trypanosoma brucei. There appeared to be no single answer; in the physiological range, control shifted between the glucose transporter on the one hand and aldolase (ALD), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), and glycerol-3-phosphate dehydrogenase (GDH) on the other hand. The other kinases, which are often thought to control glycolysis, exerted little control; so did the utilization of ATP. We identified potential targets for anti-trypanosomal drugs by calculating which steps need the least inhibition to achieve a certain inhibition of the glycolytic flux in these parasites. The glucose transporter appeared to be the most promising target, followed by ALD, GDH, GAPDH, and PGK. By contrast, in erythrocytes more than 95% deficiencies of PGK, GAPDH, or ALD did not cause any clinical symptoms (Schuster, R. and Holzhutter, H.-G. (1995) Eur. J. Biochem. 229, 403-418). Therefore, the selectivity of drugs inhibiting these enzymes may be much higher than expected from their molecular effects alone. Quite unexpectedly, trypanosomes seem to possess a substantial overcapacity of hexokinase, phosphofructokinase, and pyruvate kinase, making these "irreversible" enzymes mediocre drug targets. (+info)
Mechanism of metabolite transfer in coupled two-enzyme reactions involving aldolase.
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Transient-state kinetic experiments and analyses have been performed to examine the validity of hitherto unchallenged evidence proposed to be indicative of a channelled transfer of triose phosphates from aldolase to glyceraldehyde-3-phosphate dehydrogenase and glycerol-3-phosphate dehydrogenase. The results lend no support to such proposals, but show that the kinetic behaviour of the examined aldolase-dehydrogenase reactions is fully consistent with a free-diffusion mechanism of metabolite transfer. (+info)
Proliferation and differentiation of unilocular fat cells in the bone marrow.
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Fat cells contribute not only to systemic lipid metabolism, but also to osteogenesis and hemopoiesis in the bone marrow. The present study represents the first attempt to culture mature unilocular fat cells of the bone marrow. Two methods devised in our laboratory were employed: one is the "ceiling culture method" that utilizes the floating property of the cells; the other, a three-dimensional collagen gel matrix culture that captures unilocular fat cells in the gel matrix. Using these methods, the proliferation of unilocular fat cells from the bovine metacarpal bone marrow was demonstrated. First, we confirmed the proliferative ability of unilocular fat cells derived from the bone marrow, using autoradiography to study 3H-thymidine incorporation into the nuclei. The unilocular fat cells de-differentiated into fibroblast-like fat cells and then proceeded to proliferate. When they underwent a contact inhibition of growth, re-differentiation from fibroblast-like fat cells into unilocular fat cells occurred at a high rate. A specific enzymatic marker of the fat cell, alpha-glycerophosphate dehydrogenase activity related to lipogenesis, was then demonstrated in the cultured fat cells. We examined the functional reactivity of the fat cells by treatment with insulin and cyclic-AMP, and both lipogenesis and lipolysis were also confirmed in them. We concluded that unilocular fat cells from the bone marrow de-differentiated, proliferated and re-differentiated in culture. The present results may help to clarify the various functions of fat cells in the bone marrow. (+info)
Dynamics of correlated genetic systems. IV. Multilocus effects of ethanol stress environments.
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Four replicate populations of Drosophila melanogaster, two reared on medium supplemented with ethanol and two reared on standard medium, were electrophoretically monitored for 28 generations. During the first 12 generations, allelic, genotypic and gametic frequencies were determined for eight polymorphic enzymes: GOT, alpha-GPDH, MDH, ADH, TO, E6, Ec and ODH. Samples from generation 18 and 28 were electrophoretically typed for ADH and alpha-GPDH. In addition, samples from generation 27 were analyzed for the presence of inversion heterozygotes. The experimental results showed rapid gene-frequency divergence between control and treatment populations at the Adh locus in a direction consistent with the activity hierarchy of Adh genotypes. Gene-frequency divergence between control and treatment populations also occurred at the alpha-Gpdh locus, although the agreement among replicates appeared to have broken down by generation 28. No differential gene-frequency change occurred at any of the six remaining marker loci. Furthermore, values of linkage disequilibria among all linked pairs of genes were initially small and remained small throughout the course of the experiment. Taking these facts into account, it is argued that the gene-frequency response observed at ADH is most probably caused by selection at the Adh locus. The gene frequency response at alpha-Gpdh can also be be accounted for in terms of the effect of ethanol on energy metabolism, although other explanations cannot be excluded. (+info)
Population structure and genetic divergence in Anopheles nuneztovari (Diptera: Culicidae) from Brazil and Colombia.
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Anopheles nuneztovari is considered an important vector of human malaria in several localities in Venezuela and Colombia. Its status as a vector of human malaria is still unresolved in areas of the Brazilian Amazon, in spite of have been found infected with Plasmodium sp.. For a better understanding of the genetic differentiation of populations of A. nuneztovari, electrophoretic analysis using 11 enzymes was performed on four populations from Brazil and two from Colombia. The results showed a strong differentiation for two loci: alpha-glycerophosphate dehydrogenase (alpha-Gpd) and malate dehydrogenase (Mdh) from 16 loci analyzed. Diagnostic loci were not detected. The populations of A. nuneztovari from the Brazilian Amazon showed little genetic structure and low geographic differentiation, based on the F(IS) (0.029), F(ST) (0.070), and genetic distance (0.001-0.032) values. The results of the isozyme analysis do not coincide with the indication of two lineages in the Amazon Basin by analysis of mitochondrial DNA, suggesting that this evolutionary event is recent. The mean F(ST) value (0.324) suggests that there is considerable genetic divergence among populations from the Brazilian Amazon and Colombia. The genetic distance among populations from the Brazilian Amazon and Colombia is ranges from 0.047 to 0.148, with the highest values between the Brazilian Amazon and Sitronela (SIT) (0.125-0.148). These results are consistent with those observed among members of anopheline species complexes. It is suggested that geographic isolation has reduced the gene flow, resulting in the genetic divergence of the SIT population. Dendrogram analysis showed three large groups: one Amazonian and two Colombia, indicating some genetic structuring. The present study is important because it attempted to clarify the taxonomic status of A. nuneztovari and provide a better understanding of the role of this mosquito in transmission of human malaria in northern South America. (+info)