Mutations in Glucose Transporter 9 Gene SLC2A9 Cause Renal Hypouricemia

Renal hypouricemia is an inherited disorder characterized by impaired renal urate (uric acid) reabsorption and subsequent low serum urate levels, with severe complications such as exercise-induced acute renal failure and nephrolithiasis. We previously identified SLC22A12, also known as URAT1, as a causative gene of renal hypouricemia. However, hypouricemic patients without URAT1 mutations, as well as genome-wide association studies between urate and SLC2A9 (also called GLUT9), imply that GLUT9 could be another causative gene of renal hypouricemia. With a large human database, we identified two loss-of-function heterozygous mutations in GLUT9, which occur in the highly conserved “sugar transport proteins signatures 1/2.” Both mutations result in loss of positive charges, one of which is reported to be an important membrane topology determinant. The oocyte expression study revealed that both GLUT9 isoforms showed high urate transport activities, whereas the mutated GLUT9 isoforms markedly reduced them. Our findings, together with previous reports on GLUT9 localization, suggest that these GLUT9 mutations cause renal hypouricemia by their decreased urate reabsorption on both sides of the renal proximal tubules. These findings also enable us to propose a physiological model of the renal urate reabsorption in which GLUT9 regulates serum urate levels in humans and can be a promising therapeutic target for gout and related cardiovascular diseases.     

Retrieval of entire genes from environmental DNA by inverse PCR with pre-amplification of target genes using primers containing locked nucleic acids

We had been unsuccessful to amplify desired nucleotide sequences from various environmental DNA samples by using the inverse polymerase chain reaction (IPCR) technique, most probably because the copy numbers of target DNA sequences had been quite low. To enrich the target DNA sequences prior to IPCR, a rolling-circle amplification was used with a site-specific primer containing locked nucleic acids (LNAs). This pre-amplified IPCR (PAI-PCR) method increased the sensitivity of PCR almost 10 000 times compared with the standard IPCR in model experiments using Escherichia coli . We then applied the PAI-PCR method to isolate glycosyl hydrolase genes from DNAs extracted from vermiform appendixes of horses and termite guts. The flanking sequences of the target genes were amplified and cloned successfully using PAI-PCR, whereas standard IPCR resulted in no amplification.     

Switching of chromatin-remodelling complexes for oestrogen receptor-alpha

The female sex steroid hormone oestrogen stimulates both cell proliferation and cell differentiation in target tissues. These biological actions are mediated primarily through nuclear oestrogen receptors (ERs). The ligand-dependent transactivation of ERs requires several nuclear co-regulator complexes; however, the cell-cycle-dependent associations of these complexes are poorly understood. By using a synchronization system, we found that the transactivation function of ERalpha at G2/M was lowered. Biochemical approaches showed that ERalpha associated with two discrete classes of ATP-dependent chromatin-remodelling complex in a cell-cycle-dependent manner. The components of the NuRD-type complex were identified as G2/M-phase-specific ERalpha co-repressors. Thus, our results indicate that the transactivation function of ERalpha is cell-cycle dependent and is coupled with a cell-cycle-dependent association of chromatin-remodelling complexes.     

In vivo function of VDR in gene expression-VDR knock-out mice

Vitamin D exerts many biological actions through nuclear vitamin D receptor (VDR)-mediated gene expression. The transactivation function of VDR is activated by binding 1,25-dihydroxyvitamin D₃[1,25(OH)₂D₃], an active form of vitamin D. Conversion from 25(OH)D₃ is finely regulated in kidney by 25(OH)D₃ 1-hydroxylase[25(OH)D 1-hydroxylase], keeping serum levels of 1,25(OH)₂D₃ constant. Deficiency of vitamin D and mutations in the genes like VDR (type II genetic rickets) are known to cause rickets like lowered serum calcium, alopecia and impaired bone formation. However, the molecular basis of vitamin D–VDR system in the vitamin D action in intact animals remained to be established. In addition, the 1-hydroxylase gene from any species had not yet been cloned, irrespective of its biological significance and putative link to the type I genetic rickets. We generated VDR-deficient mice (VDR KO mice). VDR KO mice grew up normally until weaning, but after weaning they developed abnormality like the type II rickets patients. These results demonstrated indispensability of vitamin D–VDR system in mineral and bone metabolism only in post-weaning life. Using a newly developed cloning system, we cloned the cDNA encoding a novel P450 enzyme, mouse and human 1-hydroxylase. The study in VDR KO mice demonstrated the function of liganded VDR in the negative feed-back regulation of 1,25(OH)₂D₃ production. Finally, from the analysis of type I rickets patients, we found missense genetic mutations in 1-hydroxylase, leading to the conclusion that this gene is responsible for the type I rickets.     

Androgen receptor gene knockout male mice exhibit impaired cardiac growth and exacerbation of angiotensin II-induced cardiac fibrosis

Androgen has anabolic effects on cardiac myocytes and has been shown to enhance left ventricular enlargement and function. However, the physiological and patho-physiological roles of androgen in cardiac growth and cardiac stress-induced remodeling remains unclear. We aimed to clarify whether the androgen-nuclear androgen receptor (AR) system contributes to the cardiac growth and angiotensin II (Ang II)-stimulated cardiac remodeling by using systemic AR-null male mice. AR knock-out (ARKO) male mice, at 25 weeks of age, and age-matched wild-type (WT) male mice were treated with or without Ang II stimulation (2.0 mg/kg/day) for 2 weeks. ARKO mice with or without Ang II stimulation showed a significant reduction in the heart-to-body weight ratio compared with those of WT mice. In addition, echocardiographic analysis demonstrated impairments of both the concentric hypertrophic response and left ventricular function in Ang II-stimulated ARKO mice. Western blot analysis of the myocardium revealed that activation of extracellular signal-regulated kinases (ERK) 1/2 and ERK5 by Ang II stimulation were lower in ARKO mice than those of WT mice. Ang II stimulation caused more prominent cardiac fibrosis in ARKO mice than in WT mice with enhanced expression of types I and III collagen and transforming growth factor-beta1 genes and with increased Smad2 activation. These results suggest that, in male mice, the androgen-AR system participates in normal cardiac growth and modulates cardiac adaptive hypertrophy and fibrosis during the process of cardiac remodeling under hypertrophic stress.     

Two distinct pathways responsible for the loading of CENP-A to centromeres in the fission yeast cell cycle

CENP-A is a centromere-specific histone H3 variant that is- essential for faithful chromosome segregation in all eukaryotes thus far investigated. We genetically identified two factors, Ams2 and Mis6, each of which is required for the correct centromere localization of SpCENP-A (Cnp1), the fission yeast homologue of CENP-A. Ams2 is a cell-cycle-regulated GATA factor that localizes on the nuclear chromatin, including on centromeres, during the S phase. Ams2 may be responsible for the replication-coupled loading of SpCENP-A by facilitating nucleosomal formation during the S phase. Consistently, overproduction of histone H4, but not that of H3, suppressed the defect of SpCENP-A localization in Ams2-deficient cells. We demonstrated the existence of at least two distinct phases for SpCENP-A loading during the cell cycle: the S phase and the late-G2 phase. Ectopically induced SpCENP-A was efficiently loaded onto the centromeres in G2-arrested cells, indicating that SpCENP-A probably undergoes replication-uncoupled loading after the completion of S phase. This G2 loading pathway of SpCENP-A may require Mis6, a constitutive centromere-binding protein that is also implicated in the Mad2-dependent spindle attachment checkpoint response. Here, we discuss the functional relationship between the flexible loading mechanism of CENP-A and the plasticity of centromere chromatin formation in fission yeast.     

Cooperative effect of two surface amino acid mutations (Q252L and E170K) in glucose dehydrogenase from Bacillus megaterium IWG3 on stabilization of its oligomeric state

A thermostable glucose dehydrogenase (GlcDH) mutant of Bacillus megaterium IWG3 harboring the Q252L substitution (Y. Makino, S. Negoro, I. Urabe, and H. Okada, J. Biol. Chem. 264:6381-6385, 1989) is stable at pH values above 9, but only in the presence of 2 M NaCl. Another GlcDH mutant exhibiting increased stability at an alkaline pH in the absence of NaCl has been isolated previously (S.-H. Baik, T. Ide, H. Yoshida, O. Kagami, and S. Harayama, Appl. Microbiol. Biotechnol. 61:329-335, 2003). This mutant had two amino acid substitutions, Q252L and E170K. In the present study, we characterized three GlcDH mutants harboring the substitutions Q252L, E170K, and Q252L/E170K under low-salt conditions. The GlcDH mutant harboring two substitutions, Q252L/E170K, was stable, but mutants harboring a single substitution, either Q252L or E170K, were unstable at an alkaline pH. Gel filtration chromatography analyses demonstrated that the oligomeric state of the Q252/E170K enzyme was stable, while the tetramers of the enzymes harboring a single substitution (Q252L or E170K) dissociated into dimers at an alkaline pH. These results indicated that the Q252L and E170K substitutions synergistically strengthened the interaction at the dimer-dimer interface. The crystal structure of the E170K/Q252L mutant, determined at 2.0-angstroms resolution, showed that residues 170 and 252 are located in a hydrophobic cavity at the subunit-subunit interface. We concluded that these residues in the wild-type enzyme have thermodynamically unfavorable effects, while the Q252L and E170K substitutions increase the subunit-subunit interactions by stabilizing the hydrophobic cavity.