Genome-wide association study of idiopathic hypersomnia in a Japanese population

Sleep and Biological Rhythms - Idiopathic hypersomnia (IH) is a rare sleep disorder characterized by excessive daytime sleepiness, great difficulty upon awakening, and prolonged sleep time. In...     

Determination of the epinephrine-refractory hypoglycaemic activity of whole-cell pertussis vaccine in mice

A new assay method has been developed for the quantitative estimation of the inhibitory effect of pertussis vaccine on epinephrine-induced hyperglycaemia in mice. The statistical analysis of the assay was based on logarithm-transformed estimates of the blood glucose levels. The method was sufficiently sensitive to detect the activity of 0.004 millilitre of commercial combined diphtheria-tetanus-whole cell pertussis vaccine. The estimated common variance was as small as 0.0034 and the assay was highly reproducible. Among commercial vaccines there was a significant difference in activity. The activity of a stock pertussis vaccine was inactivated by 5 mM glutaraldehyde at 37 degrees C for 30 min, but resisted treatment with 40 mM formaldehyde at 37 degrees C for 5 days. The extent of inactivation with the chemicals was calculated by a parallel line assay as the activity relative to that of untreated control pertussis vaccine.     

ARPP-16/ARPP-19: a highly conserved family of cAMP-regulated phosphoproteins

ARPP-16 and ARPP-19 are closely related cAMP-regulated phosphoproteins that were initially discovered in mammalian brain as in vitro substrates for protein kinase A (PKA). ARPP-16 is enriched in dopamine-responsive medium spiny neurons in the striatum, while ARPP-19 is ubiquitously expressed. ARPP-19 is highly homologous to alpha-endosulfine and database searches allowed the identification of novel related proteins in D. melanogaster, C. elegans, S. mansoni and yeast genomes. Using isoform-specific antibodies, we now show that ARPP-19 is composed of at least two differentially expressed isoforms (termed ARPP-19 and ARPP-19e/endosulfine). All ARPP-16/19 family members contain a conserved consensus site for phosphorylation by PKA (RKPSLVA in mammalian ARPP-16 and ARPP-19), and this site was shown to be efficiently phosphorylated in vitro by PKA. An antibody that specifically recognized the phosphorylated form of ARPP-16/19/19e was used to examine the phosphorylation of ARPP-16/19 family members in intact cells. In striatal slices, the phosphorylation of ARPP-16 was increased in response to activation of D(1)-type dopamine receptors, and decreased in response to activation of D(2)-type dopamine receptors. In non-neuronal cells, ARPP-19 was highly phosphorylated in response to activation of PKA. These results establish that ARPP-16/19 proteins constitute a family of PKA-dependent intracellular messengers that function in all cells. The high levels of ARPP-16 in striatal neurons and its bi-directional regulation by dopamine suggest a specific role in dopamine-dependent signal transduction. The conservation of this protein family through evolution suggests that it subserves an important cellular function that is regulated by PKA.     

5'-upstream structure of the gene coding for chicken riboflavin-binding protein and its relation to estrogen induction

To elucidate the mechanism of the estrogen-dependent induction of chicken riboflavin-binding protein (RfBP), we analyzed the 5'-upstream structure of its gene. A noncoding exon exists there, and around this sequence, 9 widely spaced half-palindromic estrogen-response element (ERE) motifs (5'-GGTCA or 5'-TGACC) were found. Furthermore, an imperfect ERE-like palindromic sequence (5'-ATGTCANNNTGACAT-3') was also found at the 2.25 kb upstream region. No consensus palindromic ERE was observed. By luciferase reporter assay, the regions containing the half ERE motifs and the imperfect ERE showed estrogen-dependent enhancer activities, suggesting that these two characteristic sequences might confer estrogen-inducibility upon the chicken RfBP gene. However the activities were lower than that of a consensus ERE. It remains uncertain whether these sequences act cooperatively.     

Filamentous phage replication initiator protein gpII forms a covalent complex with the 5' end of the nick it introduced

Rolling circle type DNA replication is initiated by introduction of a nick in the leading strand of the origin by the initiator protein, which in most cases binds covalently to the 5' end of the nick. In filamentous phage, however, such a covalent complex has not been detected. Using a suitable substrate and short reaction time, we show that filamentous phage initiator gpII forms a covalent complex with nicked DNA, which rapidly dissociates unless gpII is inactivated. A peptide-DNA complex was isolated from trypsin digest of the complex by ion-exchange column chromatography and gel filtration, and its peptide sequence was determined. The result indicated that gpII was linked to DNA by the tyrosine residue at position 197 from the N-terminus. The mutant protein in which this tyrosine was replaced by phenylalanine did not show any detectable activity to complement gene II amber mutant phage in vivo. In vitro, the mutant protein recognized the origin and bent DNA as well as the wild-type does, but failed to introduce a nick and to relax the superhelicity of cognate DNA.     

Essential role for ADAM19 in cardiovascular morphogenesis

Congenital heart disease is the most common form of human birth defects, yet much remains to be learned about its underlying causes. Here we report that mice lacking functional ADAM19 (mnemonic for a disintegrin and metalloprotease 19) exhibit severe defects in cardiac morphogenesis, including a ventricular septal defect (VSD), abnormal formation of the aortic and pulmonic valves, leading to valvular stenosis, and abnormalities of the cardiac vasculature. During mouse development, ADAM19 is highly expressed in the conotruncus and the endocardial cushion, structures that give rise to the affected heart valves and the membranous ventricular septum. ADAM19 is also highly expressed in osteoblast-like cells in the bone, yet it does not appear to be essential for bone growth and skeletal development. Most adam19(-/-) animals die perinatally, likely as a result of their cardiac defects. These findings raise the possibility that mutations in ADAM19 may contribute to human congenital heart valve and septal defects.     

Induction of osteoblast differentiation indices by statins in MC3T3-E1 cells

Statins inhibit 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, which catalyzes conversion of HMG-CoA to mevalonate, a rate-limiting step in cholesterol synthesis. The present study was undertaken to understand the events of osteoblast differentiation induced by statins. Simvastatin at 10(-7) M markedly increased mRNA expression for bone morphogenetic protein-2 (BMP-2), vascular endothelial growth factor (VEGF), alkaline phosphatase, type I collagen, bone sialoprotein, and osteocalcin (OCN) in nontransformed osteoblastic cells (MC3T3-E1), while suppressing gene expression for collagenase-1, and collagenase-3. Extracellular accumulation of proteins such as VEGF, OCN, collagenase-digestive proteins, and noncollagenous proteins was increased in the cells treated with 10(-7) M simvastatin, or 10(-8) M cerivastatin. In the culture of MC3T3-E1 cells, statins stimulated mineralization; pretreating MC3T3-E1 cells with mevalonate, or geranylgeranyl pyrophosphate (a mevalonate metabolite) abolished statin-induced mineralization. Statins stimulate osteoblast differentiation in vitro, and may hold promise drugs for the treatment of osteoporosis in the future.     

Production of <Emphasis Type="SmallCaps">l</Emphasis>-lactic acid from a mixture of xylose and glucose by co-cultivation of lactic acid bacteria

The production of optically pure lactic acid in a high yield from xylose or a mixture of xylose and glucose, which is a model hydrolysate of lignocellulose, is described. In a single cultivation, Enterococcus casseliflavus produced 38 g/l of lactic acid with an optical purity of 96% enantiomeric excess (ee) and 6.4 g/l of acetic acid from 50 g/l of xylose when MRS medium was used. When a mixture of 50 g/l of xylose and 100 g/l of glucose was used as the carbon source in a cultivation of E. casseliflavus alone, glucose was converted to lactic acid in the early phase of the cultivation but xylose was hardly consumed. In a co-cultivation where E. casseliflavus and Lactobacillus casei specific for glucose were simultaneously inoculated, little or no lactic acid was produced after the glucose was almost consumed. A co-cultivation with two-stage inoculation (in which E. casseliflavus was added at a cultivation time of 40 h after L. casei cells were inoculated) resulted in complete consumption of 50 g/l of xylose and 100 g/l of glucose. In the co-cultivation, 95 g/l of lactic acid with a high optical purity of 96% ee was obtained at 192 h. Such a co-cultivation using two microorganisms specific for each sugar is considered to be one promising cultivation technique for the efficient production of lactic acid from a sugar mixture derived from lignocellulose.