Key amino acids for differential coupling of alpha1-adrenergic receptor subtypes to Gs

We have established that differing effects of alpha1-adrenergic receptor (AR) subtypes on cell proliferation are due to differential coupling to the Gs/cAMP pathway; thus, both alpha1A- and alpha1B-ARs couple to Gs, while alpha1D-AR does not. To identify the region responsible for this difference in subtype-specific Gs coupling, we constructed a series of chimeric and a set of point-mutated human alpha1A- and alpha1D-ARs, and examined their signaling ability. Here, we show that the amino acid residues Thr 136 and Val138 in the intracellular loop II of the human alpha1A-AR are intimately involved with Gs coupling.     

Inhibition of gastric inhibitory polypeptide signaling prevents obesity

Secretion of gastric inhibitory polypeptide (GIP), a duodenal hormone, is primarily induced by absorption of ingested fat. Here we describe a novel pathway of obesity promotion via GIP. Wild-type mice fed a high-fat diet exhibited both hypersecretion of GIP and extreme visceral and subcutaneous fat deposition with insulin resistance. In contrast, mice lacking the GIP receptor (Gipr(-/-)) fed a high-fat diet were clearly protected from both the obesity and the insulin resistance. Moreover, double-homozygous mice (Gipr(-/-), Lep(ob)/Lep(ob)) generated by crossbreeding Gipr(-/-) and obese ob/ob (Lep(ob)/Lep(ob)) mice gained less weight and had lower adiposity than Lep(ob)/Lep(ob) mice. The Gipr(-/-) mice had a lower respiratory quotient and used fat as the preferred energy substrate, and were thus resistant to obesity. Therefore, GIP directly links overnutrition to obesity and it is a potential target for anti-obesity drugs.     

Canine mitochondrial uncoupling proteins: structure and mRNA expression of three isoforms in adult beagles

Uncoupling proteins (UCPs) are members of the mitochondrial transporter family that dissipate the proton gradient as heat more than via ATP synthesis. In the present study, nucleotide and amino acid sequences of UCPs 1, 2 and 3 of a dog were determined, and their mRNA expression in various peripheral tissues was examined. The sequences were highly (76-97%) homologous to those of other species. Although lower homologies (60-74%) were found when compared among the three canine UCPs, their deduced amino acid sequences had some common domains, such as three mitochondrial carrier protein motifs, six transmembrane alpha-helix domains, and putative purine nucleotide binding domains. By Northern blot analyses, UCP1 mRNA was not detected in any tissues examined. UCP2 mRNA was expressed in most tissues, particularly abundantly in adipose tissue, spleen and lung. Two sizes of UCP3 mRNA were found exclusively in heart and skeletal muscle. These results suggest that canine UCPs have uncoupling activity, and are involved in the regulation of metabolic heat production and/or energy expenditure, as do those of other species.     

MagSNiPer: a new single nucleotide polymorphism typing method based on single base extension, magnetic separation, and chemiluminescence

We have developed a new method for typing single nucleotide polymorphisms (SNPs), MagSNiPer, based on single base extension, magnetic separation, and chemiluminescence. Single base nucleotide extension reaction is performed with a biotinylated primer whose 3' terminus is contiguous to the SNP site with a tag-labeled ddNTP. Then the primers are captured by magnetic-coated beads with streptavidin, and unincorporated labeled ddNTP is removed by magnetic separation. The magnetic beads are incubated with anti-tag antibody conjugated with alkaline phosphatase. After the removal of excess conjugates by magnetic separation, SNP typing is performed by measuring chemiluminescence. The incorporation of labeled ddNTP is monitored by chemiluminescence induced by alkaline phosphatase. MagSNiPer is a simple and robust SNP typing method with a wide dynamic range and high sensitivity. Using MagSNiPer, we could perform SNP typing with as little as 10(-17) mol of template DNA.     

Extended linkage disequilibrium surrounding the hemoglobin E variant due to malarial selection

The hemoglobin E variant (HbE; ( beta )26Glu-->Lys) is concentrated in parts of Southeast Asia where malaria is endemic, and HbE carrier status has been shown to confer some protection against Plasmodium falciparum malaria. To examine the effect of natural selection on the pattern of linkage disequilibrium (LD) and to infer the evolutionary history of the HbE variant, we analyzed biallelic markers surrounding the HbE variant in a Thai population. Pairwise LD analysis of HbE and 43 surrounding biallelic markers revealed LD of HbE extending beyond 100 kb, whereas no LD was observed between non-HbE variants and the same markers. The inferred haplotype network suggests a single origin of the HbE variant in the Thai population. Forward-in-time computer simulations under a variety of selection models indicate that the HbE variant arose 1,240-4,440 years ago. These results support the conjecture that the HbE mutation occurred recently, and the allele frequency has increased rapidly. Our study provides another clear demonstration that a high-resolution LD map across the human genome can detect recent variants that have been subjected to positive selection.     

RNAi induction and activation in mammalian muscle cells where Dicer and eIF2C translation initiation factors are barely expressed

Dicer plays an important role in the course of RNA interference (RNAi), i.e., it digests long double-stranded RNAs into 21-25 nucleotide small-interfering RNA (siRNA) duplexes functioning as sequence-specific RNAi mediators. In this study, we investigated the expression levels of Dicer and eIF2C1 approximately 4, which, like Dicer, appear to participate in mammalian RNAi, in various mouse tissues. Results indicate that the levels of eIF2C1 approximately 4 as well as Dicer are lower in skeletal muscle and heart than in other tissues. To see if RNAi could occur under such a condition with low levels of expression of Dicer and eIF2C1 approximately 4, we examined RNAi activity in mouse skeletal muscle fibers. The results indicate that RNAi can be induced by synthetic siRNA duplexes in muscle fibers. We further examined RNAi activity during myogenic differentiation of mouse C2C12 cells. The data indicate that although the expression levels of Dicer and eIF2C1 approximately 4 decrease during the differentiation, RNAi can be induced in the cells. Altogether, the data presented here suggest that muscle cells retain the ability to induce RNAi, although Dicer and eIF2C1 approximately 4 appear to be barely expressed in them.     

Haplotype-specific sequence encoding the protein kinase, interferon-inducible double-stranded RNA-dependent activator in the human leukocyte antigen class II region

The protein kinase, interferon-inducible double-stranded (ds)RNA-dependent activator (PRKRA) is a dsRNA-binding protein which activates a protein kinase participating in the antiviral activity of interferon. Our previous studies indicated that the nucleotide sequence encoding PRKRA, which appeared to be an intronless gene, was present in PAC HS265J14 containing the human leukocyte antigen (HLA) DR subregion. In this study, we further investigated and characterized the PRKRA gene on the human genome by means of Southern blotting and polymerase chain reaction with homozygous typing cell lines for HLA genes. Results indicated that the presence of PRKRA in the DR subregion was dependent on the DR53 group. Consistently, fluorescence in situ hybridization profiles with PRKRA as a probe showed that the hybridization signal on Chromosome (Chr) 6p21.3 was seen only in the samples carrying the DR haplotypes that belonged to the DR53 group. Interestingly, another hybridization signal, which was mapped on Chr 2q31.2-q32.1, was always detected in the samples examined, i.e., even in the samples negative for the DR53 group. The outcome of a sequence-database homology search further indicated that the PRKRA gene with introns appeared to be present in a recently opened draft-sequence, RP11-65L3 (GenBank accession number AC009948), which is located between D2S335 and D2S2257. Together, the data presented here indicate that the PRKRA gene in the DR subregion is a processed pseudogene (PRKRApsi), which could have been generated only on the DR53 common ancestor's genome, and that the master copy of PRKRApsi is most probably present on Chr 2q31.2-q32.1.     

Hypoxic conditions restore lost sensitivity to the growth inhibitory effect of transforming growth factor beta-1 (TGF beta-1) in proliferating rat hepatocytes in vitro

TGF beta-1 is known to be a growth inhibitor of regenerating liver, and an inducer of hepatocyte apoptosis in primary culture. However, hepatocytes can proliferate after partial hepatectomy even at high serum TGF beta-1 concentrations. In this study we used the primary cultures of rat hepatocytes for 10 days to investigate how TGF beta-1 affects proliferating hepatocytes. DNA synthesis peaked on day 8 of culture, and TGF beta-1-induced apoptosis was significantly suppressed on day 8 compared to days 2, 5, and 10. Flow-cytometric analysis revealed that hepatocytes that had incorporated BrdU were resistant to the apoptotic effect of TGF beta-1, and Northern blot analysis showed that TGF beta receptor mRNA was down-regulated on day 8. Hypoxic conditions restores TGF beta receptor mRNA expression and the lost sensitivity of proliferating hepatocyte to TGF beta-1.     

Geographic Variations in Dorsal and Anal Ray Counts of Juvenile Japanese Flounder, Paralichthys olivaceus, in the Japan Sea

Meristic counts of early and late wild settled juveniles of Japanese flounder, Paralichthys olivaceus (Paralichthyidae), in Wakasa Bay, Japan, were compared, with specimens collected from 26 sites spread along ca. 1700km of coastline facing the Japan Sea. Mean dorsal and anal ray counts of the early settled group at Kyoto were significantly larger than those of the late settled group. A discontinuous geographic boundary in mean dorsal and anal ray counts was found around Wakasa Bay and the Noto Peninsula with both means significantly larger in the southern group than in the northern group. Mean ray counts of the early and late settlers at Kyoto were similar to those of the southern and northern groups, respectively. The effect of water temperature on meristic characters was tested on two groups of laboratory-reared juveniles. Mean dorsal and anal ray counts were larger in juveniles reared at higher water temperatures. The early and late settler groups were estimated to hatch in late February and late April, respectively, representing negative response of meristic characters to water temperatures if these two groups hatched out in Wakasa Bay. Differences in the dorsal and anal ray counts between the early and late settled groups at Kyoto can, therefore be attributed to genotypic, not phenotypic variation. This suggests the possibility of transport of larvae of the early group from areas further south to Wakasa Bay by the warm Tsushima Current, a branch of the Kuroshio, which flows northeastward along the coast of the Japan Sea, particularly when the temperature gradation between the south and north areas during these seasons is considered.