Skp2 controls adipocyte proliferation during the development of obesity

The increase in the mass of adipose tissue during the development of obesity can arise through an increase in cell size, an increase in cell number, or both. Here we show that long term maintenance of C57BL/6 mice on a high fat diet (for approximately 25 weeks) induces an initial increase in adipocyte size followed by an increase in adipocyte number in white adipose tissue. The latter effect was found to be accompanied by up-regulation of expression of the gene for the F-box protein Skp2 as well as by downregulation of the cyclin-dependent kinase inhibitor p27(Kip1), a principal target of the SCF(Skp2) ubiquitin ligase, in white adipose tissue. Ablation of Skp2 protected mice from the development of obesity induced either by a high fat diet or by the lethal yellow agouti (A(y)) mutation, and this protective action was due to inhibition of the increase in adipocyte number without an effect on adipocyte hypertrophy. The reduction in the number of adipocyte caused by Skp2 ablation also inhibited the development of obesity-related insulin resistance in the A(y) mutant mice, although the reduced number of beta cells and reduced level of insulin secretion in Skp2-deficient mice resulted in glucose intolerance. Our observations thus indicate that Skp2 controls adipocyte proliferation during the development of obesity.     

Biochemical characterization of photosystem I complexes having different subunit compositions of fucoxanthin chlorophyll a/c-binding proteins in the diatom Chaetoceros gracilis

Photosynthesis Research - Diatoms are dominant phytoplankton in aquatic environments and have unique light-harvesting apparatus, fucoxanthin chlorophyll a/c-binding protein (FCP). Diatom...     

Rap1 is activated by erythropoietin or interleukin-3 and is involved in regulation of beta1 integrin-mediated hematopoietic cell adhesion

The CrkL adaptor protein is involved in signaling from the receptor for erythropoietin (Epo) as well as interleukin (IL)-3 and activates beta(1) integrin-mediated hematopoietic cell adhesion through its interaction with C3G, a guanine nucleotide exchange factor for Rap1. We demonstrate here that Epo as well as IL-3 activates Rap1 in an IL-3-dependent hematopoietic cell line, 32D, expressing the Epo receptor. The cytokine-induced activation of Rap1 was augmented in cells that inducibly overexpress CrkL or C3G. The CrkL-mediated enhancement of cell adhesion was inhibited by expression of a dominant negative mutant of Rap1, Rap1A-17N, whereas an activated mutant of Rap1, Rap1A-63E, activated beta(1) integrin-dependent adhesion of hematopoietic cells. In 32D cells, Rap1 was also activated by phorbol 12-myristate 13-acetate and ionomycin, which also enhanced cell adhesion to fibronectin, whereas, an inhibitor of phospholipase C, inhibited both cytokine-induced activation of Rap1 and cell adhesion. It was also demonstrated that Rap1 as well as CrkL is involved in signaling from the EpoR endogenously expressed in a human leukemic cell line, UT-7. These results suggest that Epo and IL-3 activate Rap1 at least partly through the CrkL-C3G complex as well as through additional pathways most likely involving phospholipase Cgamma and strongly implicate Rap1 in regulation of beta(1) integrin-mediated hematopoietic cell adhesion.     

Molecular mechanisms of the resistance to hydrogen peroxide of enzymes involved in the calvin cycle from halotolerant Chlamydomonas sp. W80

cDNA clones encoding NADP(+)-glyceraldehyde-3-phosphate dehydrogenase (NADP(+)-GAPDH) and sedoheptulose-1,7-bisphosphatase (SBPase) were isolated and characterized from halotolerant Chlamydomonas sp. W80 (C. W80) cells. The cDNA clone for NADP(+)-GAPDH encoded 369 amino acid residues, preceded by the chloroplast transit peptide (37 amino acid residues). The cDNA clone for SBPase encoded 351 amino acids with the chloroplast transit peptide. The activities of NADP(+)-GAPDH and SBPase from C. W80 cells were resistant to H(2)O(2) up to 1 mM, as distinct from spinach chloroplastic thiol-modulated enzymes. The illumination to the dark-adapted cells and dithiothreitol treatment to the crude homogenate had little effect on the activities of NADP(+)-GAPDH and SBPase in C. W80. Modeling of the tertiary structures of NADP(+)-GAPDH and SBPase suggests that resistance of the enzymes to H(2)O(2) in C. W80 is due to the different conformational structures in the vicinity of the Cys residues of the chloroplastic enzymes between higher plant and C. W80 cells.     

Comparative effects of fish oil given by gavage and fish oil-enriched diet on leukocytes

The comparative effects of fish oil given by gavage and fish oil enriched diet on metabolism and function of lymphocytes and macrophages were investigated. For this purpose, the following parameters were examined: 1) phagocytosis capacity, production of superoxide (O2*-) and hydrogen peroxide (H2O2) by macrophages, 2) lymphocytes proliferation capacity, 3) antioxidant enzyme activities in the mesenteric lymph nodes (MEN) and liver, 4) Thiobarbituric Acid Reactive Substances (TBARS) content in MLN, liver, and plasma, 5) total antioxidant capacity of the plasma, and 6) fatty acid composition of macrophages, MLN, liver and plasma. Both FO treatments did not affect phagocytosis capacity but increased hydrogen peroxide production by macrophages in the presence of PMA. FO given by gavage markedly increased lymphocytes proliferation both in the absence (5.8-fold) and in the presence (16.7-fold) of Con A, whereas FO-rich diet showed an increase in the presence of Con A only (53.3%). FO given by gavage raised the proliferation index by 2.9-fold and FO-rich diet increased by 29% only as compared to controls. Concomitantly, FO given by gavage was more effective to increase TBARS content in plasma. The proportion of some fatty acids in the tissues and cells was also differently changed depending on the way FO was administered to rats: in particular: myristic, arachidonic, and eicosapentaenoic acids. This fact may partially explain the differences between both FO treatments.     

Identification and characterization of ligands for L-selectin in the kidney. II. Expression of chondroitin sulfate and heparan sulfate proteoglycans reactive with L-selectin

Ligands for the leukocyte adhesion molecule L-selectin are expressed not only in lymph node high endothelial venules (HEV) but also in the renal distal tubuli. Here we report that L-selectin-reactive molecules in the kidney are chondroitin sulfate and heparan sulfate proteoglycans of 500-1000 kDa, unlike those in HEV bearing sialyl Lewis X-like carbohydrates. Binding of L-selectin to these molecules was mediated by the lectin domain of L-selectin and required divalent cations. Binding was inhibited by chondroitinase and/or heparitinase but not sialidase. Thus, L-selectin can recognize chondroitin sulfate and heparan sulfate glycosaminoglycans structurally distinct from sialyl Lewis X-like carbohydrates.     

Physical interaction between interleukin-12 receptor beta 2 subunit and Jak2 tyrosine kinase: Jak2 associates with cytoplasmic membrane-proximal region of interleukin-12 receptor beta 2 via amino-terminus

IL-12 is a heterodimeric cytokine, composed of p40 and p35 subunits, that exerts its biological effects by binding to specific cell surface receptors. Two human IL-12 receptor proteins, designated IL-12R beta 1 and IL-12R beta 2, have been previously identified. IL-12R beta 2 has box 1 motif, box 2 motif, and three tyrosine residues in its cytoplasmic domain. In response to IL-12, Jak2 and Tyk2, family members of Janus family protein tyrosine kinases, are phosphorylated in PHA-activated T lymphocytes. The present study demonstrates that Jak2 binds to the cytoplasmic membrane-proximal region of IL-12R beta 2, and box 2 motif and tyrosine residues in the cytoplasmic domain were not required for binding. The amino-terminus of Jak2 is necessary for association with IL-12R beta 2.     

Expression of equine interleukin-18 by baculovirus expression system and its biologic activity

The equine interleukin-18 (IL-18) cDNA that contains the coding sequence was cloned and a recombinant baculovirus, named AcEIL-18, was constructed. The recombinant protein of the equine IL-18 was expressed by AcEIL-18 and its expression was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Insect cells infected with AcEIL-18 secreted a precursor IL-18 with 24 kilo dalton (kDa) into the culture supernatant. Western blot analysis showed that mature equine IL-18 about 18 kDa was also confirmed without co-expression of caspase-1. Culture supernatant from AcEIL-18 infected cells showed a synergistic effect with recombinant human interleukin-12 for induction of interferon-gamma gene expression in equine peripheral mononuclear cells, indicating that the recombinant equine IL-18 expressed in this study also has biological activity without any treatment.