Changes in dietary sodium consumption modulate GLUT4 gene expression and early steps of insulin signaling

Previous studies have shown that chronic salt overload increases insulin sensitivity, while chronic salt restriction decreases it. In the present study we investigated the influence of dietary sodium on 1) GLUT4 gene expression, by No the n and Western blotting analysis; 2) in vivo GLUT4 protein translocation, by measuring the GLUT4 protein in plasma membrane and microsome, before and after insulin injection; and 3) insulin signaling, by analyzing basal and insulin-stimulated tyrosine phosphorylation of insulin receptor (IR)-beta, insulin receptor substrate (IRS)-1, and IRS-2. Wistar rats we e fed no mal-sodium (NS-0.5%), low-sodium (LS-0.06%), o high-sodium diets (HS-3.12%) fo 9 wk and were killed under pentobarbital anesthesia. Compared with NS ats, HS ats inc eased (P < 0.05) the GLUT4 protein in adipose tissue and skeletal muscle, whereas GLUT4 mRNA was increased only in adipose tissue. GLUT4 expression was unchanged in LS ats compared with NS ats. The GLUT4 translocation in HS ats was higher (P < 0.05) both in basal and insulin-stimulated conditions. On the other hand, LS ats did not increase the GLUT4 translocation after insulin stimulus. Compared with NS ats, LS ats showed reduced (P < 0.01) basal and insulin-stimulated tyrosine phosphorylation of IRS-1 in skeletal muscle and IRS-2 in live, whereas HS ats showed enhanced basal tyrosine phosphorylation of IRS-1 in skeletal muscle (P < 0.05) and of IRS-2 in live. In summary, increased insulin sensitivity in HS ats is elated to increased GLUT4 gene expression, enhanced insulin signaling, and GLUT4 translocation, whereas decreased insulin sensitivity of LS ats does not involve changes in GLUT4 gene expression but is elated to impaired insulin signaling.     

Localization of hydrogen-bonds within modules in barnase

Proteins in eukaryotes are composed of structural units, each encoded by discrete exons. The protein module is one such structural unit; it has been defined as the least extended or the most compact contiguous segment in a globular domain. To elucidate roles of modules in protein evolution and folding, we examined roles of hydrogen bonds and hydrophobic cores, as related to the stability of these modules. For this purpose we studied barnase, a bacterial Rnase from Bacillus amylolique-faciens. Barnase is decomposed into at least six modules, M1–M6; the module boundaries are identified at amino acid residues 24, 52, 73, 88, and 98. Hydrogen bonds are localized mainly within each of the modules, with only a few between them, thereby indicating that their locations are designed to primarily stabilize each individual module. To obtain support for this notion, an analysis was made of hypothetical modules defined as segments starting at a center of one module and ending at the center of the following one. We found that the hydrogen bonds did not localize in each hypothetical module and that many formed between the hypothetical modules. The native conformations of modules of barnase may be specified predominantly by interactions within the modules. © 1993 Wiley-Liss, Inc.     

Effect of the effluent released from the canine internal mammary artery after intraluminal and extraluminal perfusion of acetylcholine and adenosine diphosphate

Segments of the canine internal mammary artery (35 mm in length) were suspended in vitro in an organ chamber containing physiological salt solution (95% O₂/5% CO₂, pH = 7.4, 37°C). Segments were individually cannulated and perfused at 5 ml/minute using a roller pump. Vasorelaxant activity of the effluent from the perfused internal mammary arteries was bioassayed by measuring the decrease in tension induced by the effluent of the coronary artery endothelium-free ring which had been contracted with prostaglandin F_2α (2 × 10^-6 M). Intraluminal perfusion of adenosine diphosphate (10^-5 M) induced significant increase in relaxant activity in the effluent from the perfused blood vessel. However, when adenosine diphosphate (10^-5 M) was added extraluminally to the internal mammary artery, no change in relaxant activity in the effluent was noted. In contrast, acetylcholine produced significant increase in the relaxant activity on the effluent of the perfused internal mammary artery with both intraluminal and extraluminal perfusion. The intraluminal and extraluminal release of endothelium-derived relaxing factor (EDRF) by acetylcholine (10^-5 M) can be inhibited by site-specific administration of atropine (10^-5 M). These experiments indicate that certain agonists can induce the release of EDRF only by binding to intravascular receptors while other agonists can induce endothelium-dependent vasodilatation by acting on neural side receptors.     

Revisiting gap locations in amino acid sequence alignments and a proposal for a method to improve them by introducing solvent accessibility

In comparative modeling, the quality of amino acid sequence alignment still constitutes a major bottleneck in the generation of high quality models of protein three-dimensional (3D) structures. Substantial efforts have been made to improve alignment quality by revising the substitution matrix, introducing multiple sequences, replacing dynamic programming with hidden Markov models, and incorporating 3D structure information. Improvements in the gap penalty have not been a major focus, however, following the development of the affine gap penalty and of the secondary structure dependent gap penalty. We revisited the correlation between protein 3D structure and gap location in a large protein 3D structure data set, and found that the frequency of gap locations approximated to an exponential function of the solvent accessibility of the inserted residues. The nonlinearity of the gap frequency as a function of accessibility corresponded well to the relationship between residue mutation pattern and residue accessibility. By introducing this relationship into the gap penalty calculation for pairwise alignment between template and target amino acid sequences, we were able to obtain a sequence alignment much closer to the structural alignment. The quality of the alignments was substantially improved on a pair of sequences with identity in the "twilight zone" between 20 and 40%. The relocation of gaps by our new method made a significant improvement in comparative modeling, exemplified here by the Bacillus subtilis yitF protein. The method was implemented in a computer program, ALAdeGAP (ALignment with Accessibility dependent GAp Penalty), which is available at http://cib.cf.ocha.ac.jp/target_protein/.     

Soybean and sunflower oil‐induced insulin resistance correlates with impaired GLUT4 protein expression and translocation specifically in white adipose tissue

Free fatty acids are known for playing a crucial role in the development of insulin resistance. High fat intake is known for impairing insulin sensitivity; however, the effect of vegetable‐oil injections have never been investigated. The present study investigated the effects of daily subcutaneous injections (100 µL) of soybean (SB) and sunflower (SF) oils, during 7 days. Both treated groups developed insulin resistance as assessed by insulin tolerance test. The mechanism underlying the SB‐ and SF‐induced insulin resistance was shown to involve GLUT4. In SB‐ and SF‐treated animals, the GLUT4 protein expression was reduced ～20% and 10 min after an acute in vivo stimulus with insulin, the plasma membrane GLUT4 content was ～60% lower in white adipose tissue (WAT). No effects were observed in skeletal muscle. Additionally, both oil treatments increased mainly the content of palmitic acid (～150%) in WAT, which can contribute to explain the GLUT4 regulations. Altogether, the present study collects evidence that those oil treatments might generate insulin resistance by targeting GLUT4 expression and translocation specifically in WAT. These alterations are likely to be caused due to the specific local increase in saturated fatty acids that occurred as a consequence of oil daily injections. Copyright © 2010 John Wiley & Sons, Ltd.     

Enlarged FAMSBASE: protein 3D structure models of genome sequences for 41 species

Enlarged FAMSBASE is a relational database of comparative protein structure models for the whole genome of 41 species, presented in the GTOP database. The models are calculated by Full Automatic Modeling System (FAMS). Enlarged FAMSBASE provides a wide range of query keys, such as name of ORF (open reading frame), ORF keywords, Protein Data Bank (PDB) ID, PDB heterogen atoms and sequence similarity. Heterogen atoms in PDB include cofactors, ligands and other factors that interact with proteins, and are a good starting point for analyzing interactions between proteins and other molecules. The data may also work as a template for drug design. The present number of ORFs with protein 3D models in FAMSBASE is 183 805, and the database includes an average of three models for each ORF. FAMSBASE is available at http://famsbase.bio.nagoya-u.ac.jp/famsbase/.     

Quantification of C4d deposition and hepatitis C virus RNA in tissue in cases of graft rejection and hepatitis C recurrence after liver transplantation

Histology is the gold standard for diagnosing acute rejection and hepatitis C recurrence after liver transplantation. However, differential diagnosis between the two can be difficult. We evaluated the role of C4d staining and quantification of hepatitis C virus (HCV) RNA levels in liver tissue. This was a retrospective study of 98 liver biopsy samples divided into four groups by histological diagnosis: acute rejection in patients undergoing liver transplant for hepatitis C (RejHCV+), HCV recurrence in patients undergoing liver transplant for hepatitis C (HCVTx+), acute rejection in patients undergoing liver transplant for reasons other than hepatitis C and chronic hepatitis C not transplanted (HCVTx-). All samples were submitted for immunohistochemical staining for C4d and HCV RNA quantification. Immunoexpression of C4d was observed in the portal vessels and was highest in the HCVTx- group. There was no difference in C4d expression between the RejHCV+ and HCVTx+ groups. However, tissue HCV RNA levels were higher in the HCVTx+ group samples than in the RejHCV+ group samples. Additionally, there was a significant correlation between tissue and serum levels of HCV RNA. The quantification of HCV RNA in liver tissue might prove to be an efficient diagnostic test for the recurrence of HCV infection.