Disruption of adiponectin causes insulin resistance and neointimal formation

The adipocyte-derived hormone adiponectin has been proposed to play important roles in the regulation of energy homeostasis and insulin sensitivity, and it has been reported to exhibit putative antiatherogenic properties in vitro. In this study we generated adiponectin-deficient mice to directly investigate whether adiponectin has a physiological protective role against diabetes and atherosclerosis in vivo. Heterozygous adiponectin-deficient (adipo(+/-)) mice showed mild insulin resistance, while homozygous adiponectin-deficient (adipo(-/-)) mice showed moderate insulin resistance with glucose intolerance despite body weight gain similar to that of wild-type mice. Moreover, adipo(-/-) mice showed 2-fold more neointimal formation in response to external vascular cuff injury than wild-type mice (p = 0.01). This study provides the first direct evidence that adiponectin plays a protective role against insulin resistance and atherosclerosis in vivo.     

Cleavage of a DNA-RNA-DNA/DNA chimeric substrate containing a single ribonucleotide at the DNA-RNA junction with prokaryotic RNases HII

We have analyzed the cleavage specificities of various prokaryotic Type 2 ribonucleases H (RNases H) on chimeric DNA-RNA-DNA/DNA substrates containing one to four ribonucleotides. RNases HII from Bacillus subtilis and Thermococcus kodakaraensis cleaved all of these substrates to produce a DNA segment with a 5'-monoribonucleotide. Consequently, these enzymes cleaved even the chimeric substrate containing a single ribonucleotide at the DNA-RNA junction (5'-side of the single ribonucleotide). In contrast, Escherichia coli RNase HI and B. subtilis RNase HIII did not cleave the chimeric substrate containing a single ribonucleotide. These results suggest that bacterial and archaeal RNases HII are involved in excision of a single ribonucleotide misincorporated into DNA.     

Homologous pairing and ring and filament structure formation activities of the human Xrcc2*Rad51D complex

The Xrcc2 and Rad51D/Rad51L3 proteins, which belong to the Rad51 paralogs, are required for homologous recombinational repair (HRR) in vertebrates. The Xrcc2 and Rad51D/Rad51L3 genes, whose products interact with each other, have essential roles in ensuring normal embryonic development. In the present study, we coexpressed the human Xrcc2 and Rad51D/Rad51L3 proteins (Xrcc2 and Rad51D, respectively) in Escherichia coli, and purified the Xrcc2*Rad51D complex to homogeneity. The Xrcc2 small middle dotRad51D complex catalyzed homologous pairing between single-stranded and double-stranded DNA, similar to the function of the Xrcc3*Rad51C complex, which is another complex of the Rad51 paralogs. An electron microscopic analysis showed that Xrcc2*Rad51D formed a multimeric ring structure in the absence of DNA. In the presence of ssDNA, Xrcc2*Rad51D formed a filamentous structure, which is commonly observed among the human homologous pairing proteins, Rad51, Rad52, and Xrcc3*Rad51C.     

Hic-5 interacts with GIT1 with a different binding mode from paxillin

Hic-5, a member of the paxillin family of adaptor molecules, is localized at focal adhesion and implicated in integrin-mediated signaling. Hic-5 and paxillin exhibit structural homology and share interacting factors, however, diverse functions are suggested for them. In this study, we carried out yeast two-hybrid screening to identify Hic-5 interacting factors using its LD3-4 region, which includes the Hic-5-specific amino acid sequence, as a bait. Through the screening, we identified GIT1, an Arf GTPase-activating protein, as a Hic-5 binding protein. The interaction of these two proteins was mediated by the LD3 motif of Hic-5 and the C-terminal region, which includes a paxillin-binding subdomain, of GIT1. Although GIT1 is known as a paxillin-binding protein, we only observed weak association of paxillin with GIT1 in the overexpression system. In contrast, Hic-5 firmly bound to GIT1 under the same conditions. In addition, the paxillin/GIT1 complex contained PIX, a guanine nucleotide exchange factor, whereas the Hic-5/GIT1 complex contained a smaller amount of PIX. These results suggested that paxillin and Hic-5 associate with GIT1 with different binding modes, and that the Hic-5 complex possesses static features compared with the paxillin complex, which contains both positive and negative regulators of GTPases involved in actin dynamics. Moreover, Hic-5-mediated inhibition of cell spreading was restored by co-expression of the C-terminal fragment of GIT1, which perturbs the interaction of Hic-5 with endogenous GIT1. Thus, it was demonstrated that Hic-5 and GIT1 interact functionally in addition to showing a physical association.     

Use of 13C-labeled glucose for measuring exogenous glucose oxidation in mice

The author modified a respiratory gas analyzer to analyze the respiratory 13CO2 of 12 small laboratory animals all at once. To investigate the practical use of this system, mice were orally (OR) or intravenously (i.v.) given glucose solutions containing three different amounts of 13C-labeled glucose. Expired 13CO2 derived from exogenous glucose was detected within 10 minutes after administration in OR mice, but about 30 minutes in i.v. mice. The height of the peak of 13CO2 expiration was correlated with the administered 13C-glucose mass.     

Transforming growth factor-beta1 is responsible for maturation-dependent spontaneous apoptosis of cultured gastric pit cells

In this study, we established a system of high concentration serum-dependent spontaneous apoptosis of guinea pig gastric pit cells in primary culture, which seems to mimic the spontaneous apoptosis of matured gastric pit cells at gastric surface in vivo. In addition to induction of the spontaneous apoptosis, cell growth was inhibited in the presence of 10% serum compared with 0.5% serum. Transforming growth factor-beta1 (TGF-beta1), which is known to cause both apoptosis and growth inhibition in mammalian cells, was present in serum of both fetal calf and guinea pig. The addition of recombinant TGF-beta1 to the culture medium containing 0.5% fetal calf serum caused both induction of apoptosis and inhibition of cell growth. On the other hand, immunodepletion of TGF-beta1 from fetal calf serum caused inability to induce both the spontaneous apoptosis and inhibition of cell growth. These data suggest that TGF-beta1 is involved in the spontaneous apoptosis of guinea pig gastric pit cells in primary culture.     

Crystal structure of the RuvA-RuvB complex: a structural basis for the Holliday junction migrating motor machinery

We present the X-ray structure of the RuvA-RuvB complex, which plays a crucial role in ATP-dependent branch migration. Two RuvA tetramers form the symmetric and closed octameric shell, where four RuvA domain IIIs spring out in the two opposite directions to be individually caught by a single RuvB. The binding of domain III deforms the protruding beta hairpin in the N-terminal domain of RuvB and thereby appears to induce a functional and less symmetric RuvB hexameric ring. The model of the RuvA-RuvB junction DNA ternary complex, constructed by fitting the X-ray structure into the averaged electron microscopic images of the RuvA-RuvB junction, appears to be more compatible with the branch migration mode of a fixed RuvA-RuvB interaction than with a rotational interaction mode.     

Isolation and characterization of a halotolerant <Emphasis Type="Italic">Bacillus</Emphasis><Emphasis Type="Italic">subtilis</Emphasis> BBK-1 which produces three kinds of lipopeptides: bacillomycin L,plipastatin, and surfactin

Twenty-three halotolerant and biosurfactant producing strains were collected from salty conditions in central Thailand. One of the strains designated BBK-1 produced the biosurfactants with the highest activity. BBK-1 was isolated from fermented foods and was identified as B. subtilis based on its physiological characteristics and 16S rRNA gene sequence. We show that the strain grows in media containing NaCl up to 16% (w/v) and produces biosurfactants in NaCl up to 8%. We found that B. subtilis BBK-1 produces three kinds of surface-active lipopeptides simultaneously. By their respective molecular weights and amino acid compositions, it is indicated that these lipopeptides are bacillomycin L, plipastatin, and surfactin. In order to analyze the production mechanism of lipopeptides further in the strain, a generally important biosynthetic gene encoding 4'-phosphopantetheinyl transferase was cloned and sequenced. The gene existed in a single copy in the genome and the deduced amino acid sequence was almost identical to that of Lpa-14 from B. subtilis strain RB14, which co-produces iturin A and surfactin.     

Absolute stereostructure of potent alpha-glucosidase inhibitor, Salacinol, with unique thiosugar sulfonium sulfate inner salt structure from Salacia reticulata

A most potent alpha-glucosidase inhibitor named salacinol has been isolated from an antidiabetic Ayurvedic traditional medicine, Salacia reticulata WIGHT, through bioassay-guided separation. The absolute stereostructure of salacinol was determined on the basis of chemical and physicochemical evidence, which included the alkaline degradation of salacinol to 1-deoxy-4-thio-D-arabinofuranose and the X-ray crystallographic analysis, to be the unique spiro-like configuration of the inner salt comprised of 1-deoxy-4-thio-D-arabinofuranosyl sulfonium cation and 1'-deoxy-D-erythrosyl-3'-sulfate anion. Salacinol showed potent inhibitory activities on several alpha-glucosidases, such as maltase, sucrase, and isomaltase, and the inhibitory effects on serum glucose levels in maltose- and sucrose-loaded rats (in vivo) were found to be more potent than that of acarbose, a commercial alpha-glucosidase inhibitor.     

Hexameric ring structure of the ATPase domain of the membrane-integrated metalloprotease FtsH from Thermus thermophilus HB8

FtsH is a cytoplasmic membrane-integrated, ATP-dependent metalloprotease, which processively degrades both cytoplasmic and membrane proteins in concert with unfolding. The FtsH protein is divided into the N-terminal transmembrane region and the larger C-terminal cytoplasmic region, which consists of an ATPase domain and a protease domain. We have determined the crystal structures of the Thermus thermophilus FtsH ATPase domain in the nucleotide-free and AMP-PNP- and ADP-bound states, in addition to the domain with the extra preceding segment. Combined with the mapping of the putative substrate binding region, these structures suggest that FtsH internally forms a hexameric ring structure, in which ATP binding could cause a conformational change to facilitate transport of substrates into the protease domain through the central pore.