Involvement of a Calcium-Dependent Protein Kinase in Hypoosmotic Turgor Regulation in a Brackish Water Characeae Lamprothamnium succinctum

Calcium ion is a key messenger in turgor regulation of internodalcells of Lamprothamnium succinctum in response to hypoosmotictreatment. An increase in the concentration of cytosolic freecalcium ion ([Ca²⁺]_c) is prerequisite for the turgor regulation[Okazaki and Tazawa (1990) J. Membr. Biol. 114: 189], We examinedwhether or not a calcium-dependent protein kinase (CDPK) isinvolved in the Ca²⁺-mediated turgor regulation of Lamprothamniumcells. A 53-kDa CDPK which phosphorylated preferentially histoneH1 but poorly myelin basic protein or casein, was detected inthe cell extract of Lamprothamnium by an in-gel protein kinaseassay. This protein kinase was detected by Western blottingand was immunoprecipitated using an anti-Dunaliella tertiolectaCDPK antibody which can neutralize the Dunaliella CDPK activity[Yuasa et al. (1995) Plant Cell Physiol. 36: 699]. The 53-kDaCDPK was partially purified from Lamprothamnium and its activitywas shown to be inhibited by the antibody and K-252a, a proteinkinase inhibitor. Microinjection of the antibody into the cytosblof Lamprothamnium cells inhibited the decrease in turgor pressurein response to hypoosmotic treatment. However, a transient increasein [Ca²⁺]_c, which was suggested by a transient reduction ofthe velocity of cytoplasmic streaming, was induced in antibody-injectedcells after hypoosmotic treatment. Turgor regulation upon hypoosmotictreatment was inhibited when the cells were treated with K-252a.These results imply that CDPK of Lamprothamnium functions ata down-stream position of Ca²⁺-mobilization in processing turgorregulation in response to hypoosmotic treatment. ² These authors contributed equally to the work.     

Two novel susceptibility loci for type 2 diabetes mellitus identified by longitudinal exome-wide association studies in a Japanese population

Recent genome-wide association studies identified genetic variants that confer susceptibility to type 2 diabetes mellitus (T2DM). However, few longitudinal genome-wide association studies of this metabolic disorder have been reported to date. Therefore, we performed a longitudinal exome-wide association study of T2DM, using 24,579 single nucleotide polymorphisms (SNPs) and repeated measurements from 6022 Japanese individuals. The generalized estimating equation model was applied to test relations of SNPs to three T2DM-related parameters: prevalence of T2DM, fasting plasma glucose level, and blood glycosylated hemoglobin content. Three SNPs that passed quality control were significantly (P < 2.26 × 10^? 7) associated with two of the three T2DM-related parameters in additive and recessive models. Of the three SNPs, rs6414624 in EVC and rs78338345 in GGA3 were novel susceptibility loci for T2DM. In the present study, the SNP of GGA3 was predicted to be a genetic variant whose minor allele frequency has recently increased in East Asia.     

Identification of six novel susceptibility loci for dyslipidemia using longitudinal exome-wide association studies in a Japanese population

Recent genome-wide association studies have identified various dyslipidemia-related genetic variants. However, most studies were conducted in a cross-sectional manner. We thus performed longitudinal exome-wide association studies of dyslipidemia in a Japanese population. We used ~244,000 genetic variants and clinical data of 6022 Japanese individuals who had undergone annual health checkups for several years. After quality control, the association of dyslipidemia-related phenotypes with 24,691 single nucleotide polymorphisms (SNPs) was tested using the generalized estimating equation model. In total, 82 SNPs were significantly (P < 2.03 × 10^?6) associated with dyslipidemia phenotypes. Of these SNPs, four (rs74416240 of TCHP, rs925368 of GIT2, rs7969300 of ATXN2, and rs12231744 of NAA25) and two (rs34902660 of SLC17A3 and rs1042127 of CDSN) were identified as novel genetic determinants of hypo-HDL- and hyper-LDL-cholesterolemia, respectively. A replication study using the cross-sectional data of 8310 Japanese individuals showed the association of the six identified SNPs with dyslipidemia-related traits.     

Megakaryoblastic leukemia factor-1 gene in the susceptibility to coronary artery disease

Coronary artery disease (CAD) is based on the atherosclerosis of coronary artery and may manifest with myocardial infarction or angina pectoris. Although it is widely accepted that genetic factors are linked to CAD and several disease-related genes have been reported, only a few could be replicated suggesting that there might be some other CAD-related genes. To identify novel susceptibility loci for CAD, we used microsatellite markers in the screening and found six different candidate CAD loci. Subsequent single nucleotide polymorphism (SNP) association studies revealed an association between CAD and megakaryoblastic leukemia factor-1 gene (MKL1). The association with a promoter SNP of MKL1, ?184C > T, was found in a Japanese population and the association was replicated in another Japanese population and a Korean population. Functional analysis of the MKL1 promoter SNP suggested that the higher MKL1 expression was associated with CAD. These findings suggest that MKL1 is involved in the pathogenesis of CAD.     

Association of a polymorphism of the apolipoprotein E gene with chronic kidney disease in Japanese individuals with metabolic syndrome

The purpose of the present study was to identify genetic variants that confer susceptibility to chronic kidney disease (CKD) in Japanese individuals with metabolic syndrome. The study population comprised 2150 Japanese individuals with metabolic syndrome, including 411 subjects with CKD [estimated glomerular filtration rate (eGFR) < 50 mL/min/1.73m²] and 1739 controls (eGFR ≥ 60 mL/min/1.73m²). The genotypes for 100 polymorphisms of 80 candidate genes were determined. The chi-square test, multivariable logistic regression analysis with adjustment for covariates, as well as a stepwise forward selection procedure revealed that nine polymorphisms of APOE, ABCA1, PTGS1, TNF, CPB2, AGTR1, OR13G1, and GNB3 were associated (P < 0.05) with the prevalence of CKD. Among these polymorphisms, the ? 219G → T polymorphism of APOE (rs405509) was most significantly associated with CKD in Japanese individuals with metabolic syndrome.     

Mitochondrial ATPase subunit 6 and cytochrome B gene polymorphisms in young obese adults.

We hypothesized that the mutational strand asymmetry is more strongly exerted upon the mitochondrial cytochrome b (Cytb) gene, which is distant from the origin of the light-strand replication (Ori(L)), than upon the ATPase subunit 6 (ATP6) gene, which is close to the Ori(L). To test this hypothesis, we determined the sequences of these two genes in 96 Japanese young obese adults. The frequency of G-->A transitions was significantly higher than that of C-->T transitions in the Cytb gene, whereas the frequencies of G-->A and C-->T transitions were not significantly different in the ATP6 gene. The marked mutational strand asymmetry in the Cytb gene can be explained by the deamination of C to uracil in the long single-stranded state of the heavy strand during replication. The ratio of the nonsynonymous substitutions at the second codon positions to those at the first codon positions was significantly lower in the Cytb gene than in the ATP6 gene. The physicochemical differences between the standard and the replaced amino acid residues were significantly smaller in the Cytb gene than in ATP6 one. The present study indicates that amino acid sequences are less variable for Cytb than for ATP6 in spite of the strong mutational strand asymmetry for the Cytb gene.     

Association of polymorphisms in CYP17A1, MTP, and VLDLR with bone mineral density in community-dwelling Japanese women and men

We examined whether a -34T --> C polymorphism of the gene for cytochrome P450, family 17, subfamily A, polypeptide 1 (CYP17A1), a -493G --> T polymorphism of the microsomal triglyceride transfer protein gene (MTP), and a CGG repeat polymorphism of the very low density lipoprotein receptor gene (VLDLR) were associated with bone mineral density (BMD) in community-dwelling Japanese women and men. The -34T --> C polymorphism of CYP17A1 was associated with BMD in postmenopausal women, with the CC genotype being related to increased BMD. The -493G --> T polymorphism of MTP was associated with BMD in premenopausal women, with the TT genotype being related to increased BMD. The CGG repeat polymorphism of VLDLR was associated with BMD in men, with two (CGG)(n > or= 8) alleles being related to increased BMD. These results suggest that CYP17A1 and MTP are susceptibility loci for increased BMD in postmenopausal and premenopausal Japanese women, respectively, and that VLDLR constitutes such a locus in Japanese men.     

ADRB2 and LEPR gene polymorphisms: synergistic effects on the risk of obesity in Japanese

The objective of the present study was to validate a recently reported synergistic effect between variants located in the leptin receptor (LEPR) gene and in the β-2 adrenergic receptor (ADRB2) gene on the risk of overweight/obesity. We studied a middle-aged/elderly sample of 4,193 nondiabetic Japanese subjects stratified according gender (1,911 women and 2,282 men). The LEPR Gln223Arg (rs1137101) variant as well as both ADRB2 Arg16Gly (rs1042713) and Gln27Glu (rs1042714) polymorphisms were analyzed. The primary outcome was the risk of overweight/obesity defined as BMI ≥25 kg/m(2), whereas secondary outcomes included the risk of a BMI ≥27 kg/m(2) and BMI as a continuous variable. None of the studied polymorphisms showed statistically significant individual effects, regardless of the group or phenotype studied. Haplotype analysis also did not disclose any associations of ADRB2 polymorphisms with BMI. However, dimensionality reduction-based models confirmed significant interactions among the investigated variants for BMI as a continuous variable as well as for the risk of obesity defined as BMI ≥27 kg/m(2). All disclosed interactions were found in men only. Our results provide external validation for a male specific ADRB2-LEPR interaction effect on the risk of overweight/obesity, but indicate that effect sizes associated with these interactions may be smaller in the population studied.     

Purification and Substrate Specificity of Yeast Isoamylase

Yeast isoamylase was highly purified by means of salting-out with ammonium sulfate, chromatography on DEAE-cellulose, and gel filtration on Sephadex G-100. More than 200-fold purification was achieved through these procedures from crude yeast extract. While the purified enzyme did not attack α-1, 6-glucosidic linkages in panose, isopanose (6-malto-sylglucose), branched triose (4,6-diglucosylglucose), and isomaltosylmaltose (6³-α-glucosylmaltotriose), it acted on α,β-limit dextrin to liberate glucose as well as maltose and higher oligosaccharides. Substrate specificity of the yeast isoamylase was discussed in comparison with that of plant and bacterial isoamylases (R-enzvme and pullulanase), and the mechanism of debranching of glycogen by yeast enzymes was also discussed.