Close correlation of streptococcal DNase B (sdaB) alleles with emm genotypes in Streptococcus pyogenes

DNase B is a major nuclease and a possible virulence factor in Streptococcus pyogenes. The allelic diversity of streptococcal DNase B (sdaB) gene was investigated in 83 strains with 14 emm genotypes. Of the 15 alleles identified, 11 alleles carried only synonymous nucleotide substitutions. On the other hand, 4 alleles had a non-synonymous substitution other than synonymous substitutions, resulting in the substitution of a single amino acid. The distribution of each allele was generally emm genotype-specific. Only sdaB7 was found in both emm2 and emm4. The promoter region was highly conserved and DNase B protein was similarly expressed in all alleles.     

Effects of melatonin feeding on smoltification in masu salmon (Oncorhynchus masou)

Influences of photoperiod on plasma melatonin profiles and effects of melatonin administration on long-day-induced smoltification in masu salmon (Oncorhynchus masou) were investigated in order to reveal the roles of melatonin in the regulation of smoltification in salmonids. Under light-dark (LD) cycles, plasma melatonin levels exhibited daily variation, with higher values during the dark phase than during the light phase. The duration of nocturnal elevation under short photoperiod (LD 8:16) was longer than that under long photoperiod (LD 16:8). Melatonin feeding (0.01, 0.1 and 1 mg/kg body weight) elevated plasma levels of melatonin in a dose-dependent manner for at least 7 h but not for 24 h. When masu salmon reared under short photoperiod were exposed to long photoperiod (LD 16:8) and fed melatonin (1 mg/kg body weight) 7 hours before the onset of darkness, a significantly smaller proportion of smolts appeared in the melatonin-fed group after 32 days than in the control group. However, after 59 days of the treatment, there was no difference in the proportion of smolts between the control and melatonin-treated groups. Thus, melatonin feeding mimicked the effects of short photoperiod, which delays but does not completely suppress smoltification. These results indicate that the day length is transduced into changes in the duration of nocturnal elevation in plasma melatonin levels, and that artificial modification of the plasma melatonin pattern possibly delays the physiological processes of smoltification induced by long-day photoperiodic treatment.     

Formation of embryogenic cell clumps from carrot epidermal cells is suppressed by 5-azacytidine, a DNA methylation inhibitor

Using a direct somatic embryogenesis system in carrot, we examined the role of DNA methylation in the change of cellular differentiation state, from somatic to embryogenic. 5-Azacytidine (aza-C), an inhibitor of DNA methylation suppressed the formation of embryogenic cell clumps from epidermal carrot cells. Aza-C also downregulated the expression of DcLEC1c, a LEC1-like embryonic gene in carrot, during morphogenesis of embryos. A carrot DNA methyltransferase gene, Met1-5 was expressed transiently after the induction of somatic embryogenesis by 2,4-dichlorophenoxyacetic acid (2,4-D), before the formation of embryogenic cell clumps. These findings suggested the significance of DNA methylation in acquiring the embryogenic competence in somatic cells in carrot.     

Selective Assembly of V-ATPase Subunit Isoforms in Mouse Kidney

The kidney plays vital roles in acid–base homeostasis, and the reabsorption of water, ions, and proteins. These processes are achieved through acidification of urine and endosomes of proximal tubule epithelial cells. Multisubunit vacuolar-type proton ATPase (V-ATPase) is one of the major acidification-machinery proteins that localizes to the apical or basolateral plasma membranes of intercalated cells in collecting ducts and the endosomal region at the base of brush border microvilli in proximal tubules. Multiple subunit isoforms of V-ATPase, which are expressed in kidney, have been identified. One obvious question is whether the pumps at different locations in the kidney have their own unique subunit identities. We have used a combination of methods to study this enzyme in kidney including immunocytochemical staining and immunoprecipitation analyses. The subunit isoforms of V-ATPase exhibited selective association/assembly in kidney: kidney-specific isoforms predominantly formed the intercalated cell proton pump, whereas the pump located in the brush border comprised ubiquitously expressed counterparts.     

Crystal structures of novel vascular endothelial growth factors (VEGF) from snake venoms: insight into selective VEGF binding to kinase insert domain-containing receptor but not to fms-like tyrosine kinase-1

Vascular endothelial growth factor-A (VEGF-A(165)) exerts multiple effects upon binding to the fms-like tyrosine kinase-1 (Flt-1) and the kinase insert domain-containing receptor (KDR). We recently identified two novel snake venom VEGFs (vammin and VR-1) having unique properties. These VEGFs, designated VEGF-Fs, are highly specific ligands for the kinase insert domain-containing receptor and exhibit potent biological activity both in vitro and in vivo when compared with VEGF-A(165). Here, we solved the crystal structures of vammin and VR-1 at 1.9 and 2.0 A resolutions, respectively. Both structures are very similar to each other, and these structures exhibit similar but significantly different features from the known structures of other VEGFs. These differences include a conformational difference in receptor-binding loop 3 caused by an amino acid residue insertion and a difference in surface potential on the possible binding surface for domain 3 of the receptor. These structural differences may be related to the highly selective ligand properties of VEGF-F.     

Longitudinal analysis of deciduous tooth emergence: IV. Covariate effects in Japanese children

Emergence of the deciduous teeth is generally considered to be robust to moderate environmental insults, malnutrition, and disease. Consequently, deciduous tooth emergence has been used to assess growth and development and for age estimation in children. In this paper, we examine the way in which nutritional status and other covariates affect deciduous tooth emergence in a sample of 114 Japanese children born in Tokyo in 1914 and 1924. Parametric survival analysis was used to quantify the effects of nutritional status, breastfeeding behavior, and sex on the hazard of deciduous tooth emergence. Children of poor nutritional status exhibited significantly delayed emergence of all deciduous teeth, with effects that ranged from 14-29% increases in mean emergence times. Children of medium nutritional status exhibited increases in mean emergence times of 5-9% for the canines and lower molars, and 13-17% for the incisors. Partial breastfeeding had no effect on tooth emergence, but children who were not breastfed at all showed delayed emergence of the upper incisors. No significant sex differences in emergence were found. The findings contradict the idea that moderate malnutrition has little effect on deciduous tooth emergence. Furthermore, nutritional differences may account for some of the observed differences among populations in the timing of tooth emergence.     

Conformational changes in the alpha-subunit coupled to binding of the beta 2-subunit of tryptophan synthase from Escherichia coli: crystal structure of the tryptophan synthase alpha-subunit alone

When the tryptophan synthase alpha- and beta(2)-subunits combine to form the alpha(2)beta(2)-complex, the enzymatic activity of each subunit is stimulated by 1-2 orders of magnitude. To elucidate the structural basis of this mutual activation, it is necessary to determine the structures of the alpha- and beta-subunits alone and together with the alpha(2)beta(2)-complex. The crystal structures of the tryptophan synthase alpha(2)beta(2)-complex from Salmonella typhimurium (Stalpha(2)beta(2)-complex) have already been reported. However, the structures of the subunit alone from mesophiles have not yet been determined. The structure of the tryptophan synthase alpha-subunit alone from Escherichia coli (Ecalpha-subunit) was determined by an X-ray crystallographic analysis at 2.3 A, which is the first report on the subunits alone from the mesophiles. The biggest difference between the structures of the Ecalpha-subunit alone and the alpha-subunit in the Stalpha(2)beta(2)-complex (Stalpha-subunit) was as follows. Helix 2' in the Stalpha-subunit, including an active site residue (Asp60), was changed to a flexible loop in the Ecalpha-subunit alone. The conversion of the helix to a loop resulted in the collapse of the correct active site conformation. This region is also an important part for the mutual activation in the Stalpha(2)beta(2)-complex and interaction with the beta-subunit. These results suggest that the formation of helix 2'that is essential for the stimulation of the enzymatic activity of the alpha-subunit is constructed by the induced-fit mode involved in conformational changes upon interaction between the alpha- and beta-subunits. This also confirms the prediction of the conformational changes based on the thermodynamic analysis for the association between the alpha- and beta-subunits.     

Cloning of the hamster p16 gene 5' upstream region and its aberrant methylation patterns in pancreatic cancer

The hamster model of pancreatic carcinogenesis is useful for understanding the development of human pancreatic cancer. However, there is only a small amount of hamster genetic information available for analyzing the gene alterations in hamster pancreatic cancers. Here, we determined the nucleotide sequence of the 5' upstream region of the hamster p16 gene using a suppression polymerase chain reaction method combined with gene-specific primers. Based on this sequence, we analyzed the methylation status of the 5' region by bisulfite sequencing in three normal pancreatic tissues and five pancreatic duct adenocarcinomas (PDAs). All five PDAs were highly methylated in the 5' upstream region and showed reduced expressions of the p16 gene, while the three normal samples were demethylated. The method described in this study is a highly effective and rapid technique for determining the 5' upstream region, and is applicable to epigenetic studies of the methylation status of this region.     

Stabilization mechanism of the tryptophan synthase alpha-subunit from Thermus thermophilus HB8: X-ray crystallographic analysis and calorimetry

In order to elucidate the thermo-stabilization mechanism of the tryptophan synthase alpha-subunit from the extreme thermophile Thermus thermophilus HB8 (Tt-alpha-subunit), its crystal structure was determined and its stability was examined using DSC. The results were compared to those of other orthologs from mesophilic and hyperthermophilic organisms. The denaturation temperature of the Tt-alpha-subunit was higher than that of the alpha-subunit from S. typhimurium (St-alpha-subunit) but lower than that of the alpha-subunit from P. furiosus (Pf-alpha-subunit). Specific denaturation enthalpy and specific denaturation heat capacity values of the Tt-alpha-subunit were the lowest among the three proteins, suggesting that entropy effects are responsible for the stabilization of the Tt-alpha-subunit. Based on a structural comparison with the St-alpha-subunit, two deletions in loop regions, an increase in the number of ion pairs and a decrease in cavity volume seem to be responsible for the stabilization of the Tt-alpha-subunit. The results of structural comparison suggest that the native structure of the Tt-alpha-subunit is better adapted to an ideally stable structure than that of the St-alpha-subunit, but worse than that of the Pf-alpha-subunit. The results of calorimetry suggest that the residual structure of the Tt-alpha-subunit in the denatured state contributes to the stabilization.