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71.
Wolins NE Quaynor BK Skinner JR Schoenfish MJ Tzekov A Bickel PE 《The Journal of biological chemistry》2005,280(19):19146-19155
Animals have evolved mechanisms to maintain circulating nutrient levels when energy demands exceed feeding opportunities. Mammals store most of their energy as triacylglycerol in the perilipin-coated lipid droplets of adipocytes. How newly synthesized triacylglycerol is delivered to perilipin-coated lipid droplets is poorly understood. Perilipin is a member of the evolutionarily related family of PAT proteins (Perilipin, Adipophilin, TIP47), which is defined by sequence similarity and association with lipid droplets. We previously showed that S3-12, which is also a member of this family, associates with a separate pool of lipid droplets that emerge when triacylglycerol storage is driven by adding oleate to the culture medium of adipocytes. Our current data extend these findings to demonstrate that nascent lipid droplets emerge with a coat composed of S3-12, TIP47, and adipophilin. After 100 min of oleate treatment, the nascent lipid droplets are more heterogeneous: S3-12 and TIP47 coat smaller, peripheral droplets and adipophilin coats a more medial population of droplets. Fractionation of untreated and oleate-treated adipocytes shows oleate-dependent redistribution of TIP47 and adipophilin from cytosolic fractions to the lipid droplet fraction. Inhibition of protein synthesis with cycloheximide does not block the oleate-induced formation of the nascent lipid droplets, nor does it prevent TAG accumulation. We suggest that the non-lipid droplet pools of S3-12, adipophilin, and TIP47 constitute a ready reservoir of coat proteins to permit rapid packaging of newly synthesized triacylglycerol and to maximize energy storage during nutrient excess. 相似文献
72.
The thermodynamic properties and DNA binding ability of the N-terminal DNA binding domains of interferon regulatory factors IRF-1 (DBD1) and IRF-3 (DBD3) were studied using microcalorimetric and optical methods. DBD3 is significantly more stable than DBD1: at 20 degrees C the Gibbs energy of unfolding of DBD3 is -28.6 kJ/mol, which is 2 times larger than that of DBD1, -14.9 kJ/mol. Fluorescence anisotropy titration experiments showed that at this temperature the association constants with the PRDI binding site are 1.1 x 10(6) M(-)(1) for DBD1 and 3.6 x 10(6) M(-)(1) for DBD3, corresponding to Gibbs energies of association of -34 and -37 kJ/mol, respectively. However, the larger binding energy of DBD3 is due to its larger electrostatic component, while its nonelectrostatic component is smaller than that of DBD1. Therefore, DBD1 appears to have more sequence specificity than DBD3. Binding of DBD1 to target DNA is characterized by a substantially larger negative enthalpy than binding of DBD3, implying that the more flexible structure of DBD1 forms tighter contacts with DNA than the more rigid structure of DBD3. Thus, the strength of the DBDs' specific association with DNA is inversely related to the stability of the free DBDs. 相似文献
73.
Shulzhenko N Yambartsev A Goncalves-Primo A Gerbase-DeLima M Morgun A 《Biochemical and biophysical research communications》2005,337(1):306-312
Use of internal reference gene(s) is necessary for adequate quantification of target gene expression by RT-PCR. Herein, we elaborated a strategy of control gene selection based on microarray data and illustrated it by analyzing endomyocardial biopsies with acute cardiac rejection and infection. Using order statistics and binomial distribution we evaluated the probability of finding low-varying genes by chance. For analysis, the microarray data were divided into two sample subsets. Among the first 10% of genes with the lowest standard deviations, we found 14 genes common to both subsets. After normalization using two selected genes, high correlation was observed between expression of target genes evaluated by microarray and RT-PCR, and in independent dataset by RT-PCR (r = 0.9, p < 0.001). In conclusion, we showed a simple and reliable strategy of selection and validation of control genes for RT-PCR from microarray data that can be easily applied for different experimental designs and tissues. 相似文献
74.
Acarbose attenuates experimental non-alcoholic steatohepatitis 总被引:7,自引:0,他引:7
Lieber CS Leo MA Mak KM Xu Y Cao Q Ren C Ponomarenko A DeCarli LM 《Biochemical and biophysical research communications》2004,315(3):699-703
The alpha-glucosidase inhibitor acarbose is beneficial in the prevention of type 2 diabetes. To determine whether it attenuates the commonly associated non-alcoholic steatohepatitis (NASH), we used an experimental NASH model. Rats were fed ad libitum a nutritionally adequate high fat diet (71% of calories as fat) with or without acarbose (200 mg/1000 calories) for 3 weeks. All rats given the high fat diet only developed typical NASH whereas acarbose attenuated several of the characteristic hepatic alterations of NASH: there was less steatosis and inflammation, with a significant reduction in the mRNA of the hepatic inflammatory cytokine TNF-alpha and of its protein. There was also a decrease in the CYP2E1 mRNA and in collagen, with similar trends for CYP2E1 protein and procollagen mRNA. Because acarbose attenuates many of the hepatic alterations associated with experimental NASH, it is now indicated to determine whether it exerts similar beneficial effects in patients afflicted by this disease. 相似文献
75.
Human nucleotide excision repair efficiently removes chromium-DNA phosphate adducts and protects cells against chromate toxicity 总被引:5,自引:0,他引:5
Reynolds M Peterson E Quievryn G Zhitkovich A 《The Journal of biological chemistry》2004,279(29):30419-30424
Intracellular reduction of carcinogenic Cr(VI) leads to the extensive formation of Cr(III)-DNA phosphate adducts. Repair mechanisms for chromium and other DNA phosphate-based adducts are currently unknown in human cells. We found that nucleotide excision repair (NER)-proficient human cells rapidly removed chromium-DNA adducts, with an average t((1/2)) of 7.1 h, whereas NER-deficient XP-A, XP-C, and XP-F cells were severely compromised in their ability to repair chromium-DNA lesions. Activation of NER in Cr(VI)-treated human fibroblasts or lung epithelial H460 cells was manifested by XPC-dependent binding of the XPA protein to the nuclear matrix, which was also observed in UV light-treated (but not oxidant-stressed) cells. Intracellular replication of chromium-modified plasmids demonstrated increased mutagenicity of binary Cr(III)-DNA and ternary cysteine-Cr(III)-DNA adducts in cells with inactive NER. NER deficiency created by the loss of XPA in fibroblasts or by knockdown of this protein by stable expression of small interfering RNA in H460 cells increased apoptosis and clonogenic death by Cr(VI), providing genetic evidence for the role of monofunctional chromium-DNA adducts in the toxic effects of this metal. The rate of NER of chromium-DNA adducts under saturating conditions was calculated to be approximately 50,000 lesions/min/cell. Because chromium-DNA adducts cause only small changes in the DNA helix, rapid repair of these modifications in human cells indicates that the presence of major structural distortions in DNA is not required for the efficient detection of the damaged sites by NER proteins in vivo. 相似文献
76.
Pain DJ Green RE Giebetaing B Kozulin A Poluda A Ottosson U Flade M Hilton GM 《Oecologia》2004,138(2):168-174
Understanding the links between breeding and wintering areas of migratory species has important ecological and conservation implications. Recently, stable isotope technology has been used to further our understanding. Stable isotope ratios vary geographically with a range of biogeochemical factors and isotope profiles in organisms reflect those in their food and environment. For inert tissues like feathers, isotope profiles reflect the environment in which they were formed. Following large-scale habitat destruction, the globally threatened aquatic warbler Acrocephalus paludicola has a fragmented breeding population across central Europe, largely in Belarus, Poland and Ukraine. The species sub-Saharan African wintering grounds have not yet been discovered, and this significantly hampers conservation efforts. Aquatic warblers grow their flight feathers on their wintering grounds, and we analysed stable isotope ratios (15N, 13C, D) in rectrices of adults from six main breeding sites (subpopulations) across Europe to determine whether different breeding subpopulations formed a single mixed population on the wintering grounds. 15N varies considerably with dietary trophic level and environmental factors, and D with the D in rainfall; neither varied between aquatic warbler subpopulations. Uniform feather 15N signatures suggest no major variation in dietary trophic level during feather formation. High variance and inter-annual differences in mean D values hinder interpretation of these data. Significant differences in mean 13C ratios existed between subpopulations. We discuss possible interpretations of this result, and consider differences in moulting latitude of different subpopulations to be the most parsimonious. 13C in plants and animals decreases with latitude, along a steep gradient in sub-Saharan Africa. Birds from the most north-westerly breeding subpopulation (Karsibor, Poland) had significantly lower variance in 13C and 15N than birds from all other sites, suggesting either that birds from Karsibor are less geographically dispersed during moult, or moult in an area with less isotopic heterogeneity. Mean 13C signatures from winter-grown feathers of different subpopulations were positively correlated with the latitude and longitude of breeding sites, suggesting a strong relationship between European breeding and African winter moulting latitudes. The use of stable isotopes provides novel insights into migratory connectivity and migration patterns in this little-known threatened species. 相似文献
77.
Arpita Kalla Vyas Joseph C. Koster Anatoly Tzekov Paul W. Hruz 《The Journal of biological chemistry》2010,285(47):36395-36400
HIV protease inhibitors acutely block glucose transporters (GLUTs) in vitro, and this may contribute to altered glucose homeostasis in vivo. However, several GLUT-independent mechanisms have been postulated. To determine the contribution of GLUT blockade to protease inhibitor-mediated glucose dysregulation, the effects of ritonavir were investigated in mice lacking the insulin-sensitive glucose transporter GLUT4 (G4KO). G4KO and control C57BL/6J mice were administered ritonavir or vehicle at the start of an intraperitoneal glucose tolerance test and during hyperinsulinemic-euglycemic clamps. G4KO mice exhibited elevated fasting blood glucose compared with C57BL/6J mice. Ritonavir impaired glucose tolerance in control mice but did not exacerbate glucose intolerance in G4KO mice. Similarly, ritonavir reduced peripheral insulin sensitivity in control mice but not in G4KO mice. Serum insulin levels were reduced in vivo in ritonavir-treated mice. Ritonavir reduced serum leptin levels in C57BL/6J mice but had no effect on serum adiponectin. No change in these adipokines was observed following ritonavir treatment of G4KO mice. These data confirm that a primary effect of ritonavir on peripheral glucose disposal is mediated through direct inhibition of GLUT4 activity in vivo. The ability of GLUT4 blockade to contribute to derangements in the other molecular pathways that influence insulin sensitivity remains to be determined. 相似文献
78.
Correct timing and spatial location of growth factor expression is critical for undisturbed brain development and functioning. In terminally differentiated cells distinct biological responses to growth factors may depend on cell type specific activation of signalling cascades. We show that the hematopoietic growth factors thrombopoietin (TPO) and granulocyte colony-stimulating factor (GCSF) exert cell type specific effects on survival, proliferation and the degree of phosphorylation of Akt1, ERK1/2 and STAT3 in rat hippocampal neurons and cortical astrocytes. In neurons, TPO induced cell death and selectively activated ERK1/2. GCSF protected neurons from TPO- and hypoxia-induced cell death via selective activation of Akt1. In astrocytes, neither TPO nor GCSF had any effect on cell viability but inhibited proliferation. This effect was accompanied by activation of ERK1/2 and inhibition of STAT3 activity. A balance between growth factors, their receptors and signalling proteins may play an important role in regulation of neural cell survival. 相似文献
79.
80.
pH is one of the key parameters that affect the stability and function of proteins. We have studied the effect of pH on the pyridoxal-5'-phosphate-dependent enzyme phosphoserine aminotransferase produced by the facultative alkaliphile Bacillus circulans ssp. alkalophilus using thermodynamic and crystallographic analysis. Enzymatic activity assay showed that the enzyme has maximum activity at pH 9.0 and relative activity less than 10% at pH 7.0. Differential scanning calorimetry and circular dichroism experiments revealed variations in the stability and denaturation profiles of the enzyme at different pHs. Most importantly, release of pyridoxal-5'-phosphate and protein thermal denaturation were found to occur simultaneously at pH 6.0 in contrast to pH 8.5 where denaturation preceded cofactor's release by approximately 3 degrees C. To correlate the observed differences in thermal denaturation with structural features, the crystal structure of phosphoserine aminotransferase was determined at 1.2 and 1.5 A resolution at two different pHs (8.5 and 4.6, respectively). Analysis of the two structures revealed changes in the vicinity of the active site and in surface residues. A conformational change in a loop involved in substrate binding at the entrance of the active site has been identified upon pH change. Moreover, the number of intramolecular ion pairs was found reduced in the pH 4.6 structure. Taken together, the presented kinetics, thermal denaturation, and crystallographic data demonstrate a potential role of the active site in unfolding and suggest that subtle but structurally significant conformational rearrangements are involved in the stability and integrity of phosphoserine aminotransferase in response to pH changes. 相似文献