Interleukin-1alpha inhibits insulin signaling with phosphorylating insulin receptor substrate-1 on serine residues in 3T3-L1 adipocytes

Proinflammatory cytokines are recently reported to inhibit insulin signaling causing insulin resistance. IL-1alpha is also one of the proinflammatory cytokines; however, it has not been clarified whether IL-1alpha may also cause insulin resistance. Here, we investigated the effects of IL-1alpha treatment on insulin signaling in 3T3-L1 adipocytes. IL-1alpha treatment up to 4 h did not alter insulin-stimulated insulin receptor tyrosine phosphorylation, whereas tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and the association with phosphatidylinositol 3-kinase were partially inhibited with the maximal inhibition in around 15 min. IRS-1 was transiently phosphorylated on some serine residues around 15 min after IL-1alpha stimulation, when several serine kinases, IkappaB kinase, c-Jun-N-terminal kinase, ERK, and p70S6K were activated. Chemical inhibitors for these kinases inhibited IL-1alpha-induced serine phosphorylation of IRS-1. Tyrosine phosphorylation of IRS-1 was recovered only by the IKK inhibitor or JNK inhibitor, suggesting specific involvement of these two kinases. Insulin-stimulated Akt phosphorylation and 2-deoxyglucose uptake were not inhibited only by IL-1alpha. Interestingly, Akt phosphorylation was synergistically inhibited by IL-1alpha in the presence of IL-6. Taken together, short-term IL-1alpha treatment transiently causes insulin resistance at IRS-1 level with its serine phosphorylation. IL-1alpha may suppress insulin signaling downstream of IRS-1 in the presence of other cytokines, such as IL-6.     

Vitamin E deficiency accelerates nitrate tolerance via a decrease in cardiac P450 expression and increased oxidative stress

Organic nitrates, such as nitroglycerin (NTG), have been used to relieve the symptoms of angina pectoris. However, their biochemical mechanisms of action, particularly in relation to the development of tolerance, are incompletely defined. It has been reported that supplemental antioxidants such as vitamin E attenuate the development of nitrate tolerance. Therefore, we examined the role of vitamin E in the regulation of nitrate tolerance. Continuous NTG infusion induced nitrate tolerance in rats after 48 h, and vitamin E concentrations decreased in a time-dependent manner in tissues and plasma. Vitamin E supplementation (0.5 g/kg diet) maintained higher concentrations of vitamin E during NTG infusion. The onset and extent of the tolerance, estimated by the decrease in blood pressure following NTG bolus injection during the infusion of NTG, were accentuated in the vitamin E-deficient group. Vitamin E supplementation inhibited nitrate tolerance 48 h after NTG infusion. Cardiac P450 expression (CYP1A2) assessed by immunoblotting, markedly decreased 48 h after NTG administration in control rats. The supplementation of vitamin E significantly attenuated the decrease in P450. Treatment of NTG enhanced vascular superoxide production (L-012 chemiluminescence, DHE fluorescence). The peak of lipid peroxidation and free radical generation in the heart was reached before tolerance developed. In contrast, vitamin E-deficient hearts had lower P450 expression and higher free radical generation than control hearts. To evaluate other vitamin E-inhibitable mechanisms of nitrate tolerance, we studied the NO-cGMP pathway. NTG markedly reduced the vasodilator-stimulated phosphoprotein (VASP) serine 239 phosphorylation (specific substrate of cGMP-activated protein kinase I; cGK-I) in tolerant hearts. Vitamin E inhibited the depletion of pVASP. In conclusion, because continuous NTG infusion causes vitamin E depletion as well as nitrate tolerance, vitamin E deficiency may further accelerate nitrate tolerance via an increase in oxidative stress, the reduced bioconversion because of decreased P450 expression, and impairment of the NO/cGMP pathway in tolerant heart tissues.     

Characteristics of the β-Globin Gene Cluster Haplotypes of Three Han Chinese Populations at Beijing, Xi’an, and Kunming as Compared with Those of Other Asian Populations

Haplotype frequencies of the beta-globin gene cluster of Han Chinese at Beijing, Xi'an, and Kunming were estimated, and their mutual genetic relationships were examined and compared to those of Buryats, Khalkhs, Evenkis, Oroqens, Koreans, and Colombian Amerindians. A major 5' subhaplotype (5' to the delta-globin gene), a major 3' subhaplotype (in and 3' to the beta-globin gene), and a major haplotype (combination of 5' and 3' subhaplotypes) are represented as + - - - -, - +, and + - - - - - +, respectively, and found in all three Han Chinese. A rare 5' subhaplotype, - - - - -, which is one of the possible ancestral types, was found only in Han Chinese at Kunming at low frequency (0.013), and a rare 3' subhaplotype, - -, was also observed in all three Han Chinese at low frequencies (0.009-0.014). The present haplotype frequency study suggested that the highest genetic affinity was found between Han Chinese at Beijing and those at Xi'an; the next highest was between Han Chinese at Beijing and Koreans, followed by that between Han Chinese at Beijing and Khalkhs, then that between Han Chinese at Xi'an and those at Kunming or Khalkhs, and finally that between Han Chinese at Beijing and those at Kunming. A genetic boundary between northern and southern Han Chinese was not evident in the present study.     

Structural basis for the heterotropic and homotropic interactions of invertebrate giant hemoglobin

The oxygen binding properties of extracellular giant hemoglobins (Hbs) in some annelids exhibit features significantly different from those of vertebrate tetrameric Hbs. Annelid giant Hbs show cooperative oxygen binding properties in the presence of inorganic cations, while the cooperativities of vertebrate Hbs are enhanced by small organic anions or chloride ions. To elucidate the structural basis for the cation-mediated cooperative mechanisms of these giant Hbs, we determined the crystal structures of Ca2+- and Mg2+-bound Hbs from Oligobrachia mashikoi at 1.6 and 1.7 A resolution, respectively. Both of the metal-bound structures were determined in the oxygenated state. Four Ca2+-binding sites and one Mg2+-binding site were identified in each tetramer subassembly. These cations are considered to stabilize the oxygenated form and increase affinity and cooperativity for oxygen binding, as almost all of the Ca2+ and Mg2+ cations were bound at the interface regions, forming either direct or hydrogen bond-mediated interactions with the neighboring subunits. A comparison of the structures of the oxygenated form and the partially unliganded form provides structural insight into proton-coupled cooperativity (Bohr effect) and ligand-induced transitions. Two histidine residues are assumed to be primarily associated with the Bohr effect. With regard to the ligand-induced cooperativity, a novel quaternary rotation mechanism is proposed to exist at the interface region of the dimer subassembly. Interactions among conserved residues Arg E10, His F3, Gln F7, and Val E11, together with the bending motion of the heme molecules, appear to be essential for quaternary rearrangement.     

Biosynthetic origin of graphenone in cultured lichen mycobionts of Graphis handelii

The biosynthetic origins of the carbon skeleton in graphenone were verified by feeding the culture of spore-derived mycobionts of the lichen Graphis handelii with sodium [1-13C]-acetate, sodium [1,2-13C2]-acetate, sodium [2-13C]-pyruvate, [1,2,3-13C]-glycerol, [13CH3]-methionine and sodium [1,4-13C2]-succinate.     

Selection of differentiating cells by different levels of delta-like 1 among neural precursor cells in the developing mouse telencephalon

During the neurogenic phase of mammalian brain development, only a subpopulation of neural precursor cells (NPCs) differentiates into neurons. The mechanisms underlying this selection remain unclear. Here we provide evidence that the Notch-Delta pathway plays an important role in this selection in the developing mouse telencephalon. We found that the expression patterns of the Notch ligand delta-like 1 (Dll1) and of the active form of Notch1 were mutually exclusive and segregated into distinct NPC subpopulations in the ventricular zone of the telencephalon. When Dll1 was overexpressed in a small, but not a large, proportion of NPCs, these cells underwent neuronal differentiation in vitro and in vivo. This Dll1-induced neuronal differentiation did not occur when cells were plated at lower densities in an in vitro culture. Importantly, conditional deletion of the Dll1 gene in a small proportion of NPCs reduced neurogenesis in vivo, whereas deletion in a large proportion promoted premature neurogenesis. These results support the notion that different levels of Dll1 expression determine the fate of NPCs through cell-cell interactions, most likely through the Notch-Delta lateral inhibitory signaling pathway, thus contributing to the selection of differentiating cells.     

Recombinase-mediated cassette exchange reveals the selective use of Gq/G11-dependent and -independent endothelin 1/endothelin type A receptor signaling in pharyngeal arch development

The endothelin (Edn) system comprises three ligands (Edn1, Edn2 and Edn3) and their G-protein-coupled type A (Ednra) and type B (Ednrb) receptors. During embryogenesis, the Edn1/Ednra signaling is thought to regulate the dorsoventral axis patterning of pharyngeal arches via Dlx5/Dlx6 upregulation. To further clarify the underlying mechanism, we have established mice in which gene cassettes can be efficiently knocked-in into the Ednra locus using recombinase-mediated cassette exchange (RMCE) based on the Cre-lox system. The first homologous recombination introducing mutant lox-flanked Neo resulted in homeotic transformation of the lower jaw to an upper jaw, as expected. Subsequent RMCE-mediated knock-in of lacZ targeted its expression to the cranial/cardiac neural crest derivatives as well as in mesoderm-derived head mesenchyme. Knock-in of Ednra cDNA resulted in a complete rescue of craniofacial defects of Ednra-null mutants. By contrast, Ednrb cDNA could not rescue them except for the most distal pharyngeal structures. At early stages, the expression of Dlx5, Dlx6 and their downstream genes was downregulated and apoptotic cells distributed distally in the mandible of Ednrb-knock-in embryos. These results, together with similarity in craniofacial defects between Ednrb-knock-in mice and neural-crest-specific Galpha(q)/Galpha(11)-deficient mice, indicate that the dorsoventral axis patterning of pharyngeal arches is regulated by the Ednra-selective, G(q)/G(11)-dependent signaling, while the formation of the distal pharyngeal region is under the control of a G(q)/G(11)-independent signaling, which can be substituted by Ednrb. This RMCE-mediated knock-in system can serve as a useful tool for studies on gene functions in craniofacial development.     

The major and minor wall teichoic acids prevent the sidewall localization of vegetative DL-endopeptidase LytF in Bacillus subtilis

Cell separation in Bacillus subtilis depends on specific activities of DL-endopeptidases CwlS, LytF and LytE. Immunofluorescence microscopy (IFM) indicated that the localization of LytF depended on its N-terminal LysM domain. In addition, we revealed that the LysM domain efficiently binds to peptidoglycan (PG) prepared by chemically removing wall teichoic acids (WTAs) from the B. subtilis cell wall. Moreover, increasing amounts of the LysM domain bound to TagB- or TagO-depleted cell walls. These results strongly suggested that the LysM domain specifically binds to PG, and that the binding may be prevented by WTAs. IFM with TagB-, TagF- or TagO-reduced cells indicated that LytF-6xFLAG was observed not only at cell separation site and poles but also as a helical pattern along the sidewall. Moreover, we found that LytF was localizable on the whole cell surface in TagB-, TagF- or TagO-depleted cells. These results strongly suggest that WTAs inhibit the sidewall localization of LytF. Furthermore, the helical LytF localization was observed on the lateral cell surface in MreB-depleted cells, suggesting that cell wall modification by WTAs along the sidewall might be governed by an actin-like cytoskeleton homologue, MreB.     

Localization of polypyrimidine-tract-binding protein is involved in the regulation of albumin synthesis by branched-chain amino acids in HepG2 cells

Long-term supplementation of branched-chain amino acids (BCAA) improves hypoalbuminemia in patients with cirrhosis. Our previous findings have suggested that the binding of polypyrimidine-tract-binding protein (PTB) to rat albumin mRNA attenuates its translation. The aim of the present study was to investigate the role of PTB in the regulation of albumin synthesis by BCAA in human hepatoma cells. HepG2 cells were cultured in a medium containing no amino acids (AA-free medium), a medium containing only 1 amino acid (a BCAA: valine, leucine or isoleucine) or a medium containing all 20 amino acids (AA-complete medium). HepG2 cells cultured in AA-complete medium secreted much more albumin than cells cultured in AA-free medium, with no difference in albumin mRNA levels. In cells cultured in AA-free medium, nuclear export of PTB was observed, and the level of the albumin mRNA-PTB complex was greater than in cells cultured in AA-complete medium. Addition of amino acids stimulated nuclear import of PTB. However, addition of amino acids with rapamycin inhibited the nuclear import of PTB. The addition of leucine, but not of valine or isoleucine, to AA-free medium increased albumin secretion and stimulated the nuclear import of PTB. These data indicate that the mammalian target of rapamycin is involved in the regulation of PTB localization and that leucine promotes albumin synthesis by inhibiting the formation of the albumin mRNA-PTB complex.     

A novel sugar-probe biosensor for the deadly plant proteinous toxin, ricin

Because of the illegal use of highly toxic ricin from the castor-oil plant, Ricinus communis, in bioterrorism and suspected white powder cases, anti-terrorism measures for the toxin are urgently required. Here we demonstrate a facile and sensitive detection method using synthetic analogues of beta-lactosyl- and beta-d-galactosyl ceramides as the ligands based on the fact that ricin binds cell-surface oligosaccharides. Sugar-probes having lipoic acids as anchor functions were synthesized via either a chemical or chemoenzymatic way and were immobilized on the sensor chips by a self-assembled monolayer technique. Surface plasmon resonance (SPR) analysis using these carbohydrate probes allowed us to detect the toxin in a highly sensitive and facile manner (10 pg/mL, 5 min), being the best benchmark as a method for detecting the toxin. In addition, a visual monitoring method was developed, in which sugar-coated Au nanoparticles were utilized for discriminating ricin from other proteins in a facile manner, taking 10-30 min for judgment.