Critical role of the heme axial ligand, Met95, in locking catalysis of the phosphodiesterase from Escherichia coli (Ec DOS) toward Cyclic diGMP

Heme-regulated phosphodiesterase from Escherichia coli (Ec DOS) is a gas-sensor enzyme that hydrolyzes cyclic dinucleotide-GMP, and it is activated by O(2) or CO binding to the Fe(II) heme. In contrast to other well known heme-regulated gas-sensor enzymes or proteins, Ec DOS is not specific for a single gas ligand. Because Arg(97) in the heme distal side in Ec DOS interacts with the O(2) molecule and Met(95) serves as the axial ligand on the distal side of the Fe(II) heme-bound PAS domain of Ec DOS, we explored the effect of mutating these residues on the activity and gas specificity of Ec DOS. We found that R97A, R97I, and R97E mutations do not significantly affect regulation of the phosphodiesterase activities of the Fe(II)-CO and Fe(II)-NO complexes. The phosphodiesterase activities of the Fe(II)-O(2) complexes of the mutants could not be detected due to rapid autoxidation and/or low affinity for O(2). In contrast, the activities even of the gas-free M95A and M95L mutants were similar to that of the gas-activated wild-type enzyme. Interestingly, the activity of the M95H mutant was partially activated by O(2), CO, and NO. Spectroscopic analysis indicated that the Fe(II) heme is in the 5-coordinated high-spin state in the M95A and M95L mutants but that in the M95H mutant, like wild-type Ec DOS, it is in the 6-coordinated low-spin state. These results suggest that Met(95) coordination to the Fe(II) heme is critical for locking the system and that global structural changes around Met(95) caused by the binding of the external ligands or mutations at Met(95) releases the catalytic lock and activates catalysis.     

Fibrillin I gene polymorphism is associated with tall stature of normal individuals

In order to test the hypothesis that polymorphisms of the Marfan syndrome gene (FBN1) might affect the stature (height) of normal individuals, we genotyped three exonic SNPs on 428 males, 219 with tall stature (>2 SD) and 209 with normal stature (within ±1 SD). One of the SNPs, rs8033037, in exon 15 showed a significant correlation (P = 0.0061) with the adult height, suggesting that FBN1 is one of the ‘stature genes’ of normal individuals.     

Evaluation of major membrane protein-II as a tool for serodiagnosis of leprosy

As serodiagnosis is the easiest way of diagnosing a disease, the utility of Mycobacterium leprae-derived major membrane protein-II (MMP-II), one of the immuno-dominant antigens, in the serodiagnosis of leprosy was examined. The percent positivity by an enzyme-linked immunosorbent assay for anti-MMP-II antibody was 82.4% for multi-bacillary leprosy, and the specificity of the test was 90.1%. For pauci-bacillary leprosy where cell-mediated immunity predominates, 39.0% showed positive results. These percentage values were significantly higher than these values obtained for existing phenolic glycolipid-I based methods, suggesting that MMP-II antibody detection would facilitate the diagnosis of leprosy.     

Effect of dilution rate on the microbial structure of a mesophilic butyrate-degrading methanogenic community during continuous cultivation

We constructed two mesophilic anaerobic chemostats that were continuously fed with synthetic wastewater containing butyrate as the sole source of carbon and energy. Steady-state conditions were achieved at dilution rates between 0.025 and 0.7 day⁻¹. Butyrate, fed into the chemostat, was almost completely mineralized to CH₄ and CO₂ at dilution rates below 0.5 day⁻¹. The butyrate-degrading methanogenic communities in the chemostats at dilution rates between 0.025 and 0.7 day⁻¹ were monitored based on the 16S rRNA gene, using molecular biological techniques including clone library analysis, denaturing gradient gel electrophoresis, and quantitative real-time polymerase chain reaction. The aceticlastic methanogen Methanosaeta and the hydrogenotrophic methanogen Methanoculleus dominated in methanogens at low dilution rates, whereas the aceticlastic methanogen Methanosaeta, Methanosarcina, the hydrogenotrophic methanogen Methanoculleus, and Methanospirillum dominated at high dilution rates. Bacteria affiliated with the family Syntrophaceae in the phylum Proteobacteria predominated at the low dilution rate of 0.025 day⁻¹, whereas bacteria affiliated with the phylum Firmicutes and Candidate division OP3 predominated at high dilution rates. A significant quantity of bacteria closely related to the genus Syntrophomonas was detected at high dilution rates. Dilution rate showed an apparent effect on archaeal and bacterial communities in the butyrate-fed chemostats.     

p18Ink4c, but not p27Kip1, collaborates with Men1 to suppress neuroendocrine organ tumors

Mutant mice lacking both cyclin-dependent kinase (CDK) inhibitors p18(Ink4c) and p27(Kip1) develop a tumor spectrum reminiscent of human multiple endocrine neoplasia (MEN) syndromes. To determine how p18 and p27 genetically interact with Men1, the tumor suppressor gene mutated in familial MEN1, we characterized p18-Men1 and p27-Men1 double mutant mice. Compared with their corresponding single mutant littermates, the p18(-/-); Men1(+/-) mice develop tumors at an accelerated rate and with an increased incidence in the pituitary, thyroid, parathyroid, and pancreas. In the pituitary and pancreatic islets, phosphorylation of the retinoblastoma (Rb) protein at both CDK2 and CDK4/6 sites was increased in p18(-/-) and Men1(+/-) cells and was further increased in p18(-/-); Men1(+/-) cells. The remaining wild-type Men1 allele was lost in most tumors from Men1(+/-) mice but was retained in most tumors from p18(-/-); Men1(+/-) mice. Combined mutations of p27(-/-) and Men1(+/-), in contrast, did not exhibit noticeable synergistic stimulation of Rb kinase activity, cell proliferation, and tumor growth. These results demonstrate that functional collaboration exists between p18 and Men1 and suggest that Men1 may regulate additional factor(s) that interact with p18 and p27 differently.     

Leptin induces elongation of cardiac myocytes and causes eccentric left ventricular dilatation with compensation

One of the major manifestations of obesity is an increased production of the adipocyte-derived 16-kDa peptide leptin, which acts mainly on hypothalamic leptin receptors. Leptin receptors are widely distributed in various tissues, including the heart. Whereas increased plasma leptin levels have been reported in patients with congestive heart failure, systemic alterations induced by obesity can affect cardiac hypertrophy, and the direct effects of leptin on cardiac structure and function still remain to be determined. We first exposed primary cardiac myocytes from neonatal rats to leptin for 48 h. This resulted in a significant increase in myocyte long-axis length (P < 0.05 at 50 ng/ml) but not in the short-axis width. Leptin induced the rapid phosphorylation of STAT3 and its DNA binding in cardiac myocytes. Administration of a JAK2 inhibitor, AG-490, completely inhibited all of these effects by leptin. Furthermore, we examined the effect of continuous infusion of leptin for 4 wk following myocardial infarction in mice. Echocardiography demonstrated that left ventricular fractional shortening in the leptin-infused group (28.4 +/- 2.8%) was significantly higher than that in the PBS-infused group (18.4 +/- 2.2%) following myocardial infarction. Interestingly, left ventricular diastolic dimension in the leptin-infused group (4.56 +/- 0.12 mm) was also higher than that in the PBS-infused group (4.13 +/- 0.09 mm). These results demonstrate that leptin induces the elongation of cardiac myocytes via a JAK/STAT pathway and chronic leptin infusion causes eccentric dilatation with augmented systolic function after myocardial infarction.     

Construction of a yeast strain with regulatable phospholipid synthesis for analysis of the uptake and metabolism of phosphatidylethanolamine with short acyl chains

We constructed a yeast stain, TKY12Ga, in which phosphatidylethanolamine (PE) synthesis can be controlled by means of the carbon source in the medium. When PE synthesis was blocked, its growth was inhibited. However, in the presence of exogenous didecanoyl PE (diC10PE), TKY12Ga grew despite an inability to synthesize PE. Our system, which employs TKY12Ga strain and diC10PE, provides a valuable tool to study the transport and metabolism of PE in yeast.     

CPF and CPFL, two related gene families encoding cuticular proteins of Anopheles gambiae and other insects

Cuticular proteins (CPs) are structural proteins of insects as well as other arthropods. Several CP families have been described, among them a small family defined by a 51 amino acid motif [Andersen, S.O., Rafn, K., Roepstorff, P., 1997. Sequence studies of proteins from larval and pupal cuticle of the yellow meal worm, Tenebrio molitor. Insect Biochem. Mol. Biol. 27, 121-131]. We identified four proteins of this family in Anopheles gambiae that we have named CPF. We have also identified CPFs from other insects by searching databases. Alignment of these CPF proteins showed that the conserved region is only 44 aa long and revealed another conserved motif at the C-terminus. A dendrogram divided the CPF proteins into four groups, one basal and three specialized. We also identified several proteins of another CP family, CPFL, which has similarities to CPFs. CPFs and CPFLs share some protein motifs. Expression studies with real-time qRT-PCR of the A. gambiae CPFs and CPFLs showed that the four CPFs and one CPFL gene are expressed just before pupal or adult ecdysis, suggesting that they are components of the outer layer of pupal and adult cuticles. The other CPFLs appear to contribute to larval cuticle. Recombinant CPF proteins did not bind to chitin in the assay we used.     

Structural analysis of four and half LIM protein-2 in dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is a cardiac disease characterized by dilated ventricle and systolic dysfunction. Most of the DCM patients are sporadic cases, but a certain population of DCM patients can be familial cases caused by mutations in genes for sarcomere/Z-disc components including titin/connectin. However, disease-causing mutations could be identified only in a part of the familial DCM patients, suggesting that there should be other disease causing genes for DCM. To explore a novel disease gene for DCM, we searched for mutations in FHL2, encoding for four and half LIM protein 2 (FHL2) in DCM patients, because FHL2 is known to associate with titin/connectin. A missense mutation, Gly48Ser, was identified in a patient with familial DCM. Functional analysis demonstrated that the FHL2 mutation affected the binding to titin/connectin. Because FHL2 protein is known to tether metabolic enzymes to titin/connectin, these observations suggest that the Gly48Ser mutation may be involved in the pathogenesis of DCM via impaired recruitment of metabolic enzymes to the sarcomere.