Reaction on D-glucal by an inverting phosphorylase to synthesize derivatives of 2-deoxy-beta-D-arabino-hexopyranosyl-(1-->4)-D-glucose (2II-deoxycellobiose)

Four derivatives of 2(II)-deoxycellobiose were synthesized from d-glucal and acceptor sugars (d-glucose, d-xylose, d-mannose, and 2-deoxy-d-arabino-hexose) using a cellobiose phosphorylase from Cellvibrio gilvus. The enzyme was found to be an effective catalyst to synthesize the beta-(1-->4) linkage of 2-deoxy-d-arabino-hexopyranoside. The acceptor specificity for the d-glucal reaction was identical to that for the alpha-d-glucose 1-phosphate reaction, but the activity of d-glucal was approximately 500 times less than that of alpha-d-glucose 1-phosphate, using 10mM substrates.     

Phosphoinositide synthesis and degradation in isolated rat liver peroxisomes

Analyzing peroxisomal phosphoinositide (PId(#)) synthesis in highly purified rat liver peroxisomes we found synthesis of phosphatidylinositol 4-phosphate (PtdIns4P), PtdIns(4,5)P(2) and PtdIns(3,5)P(2). PtdIns3P was hardly detected in vitro, however, was observed in vivo after [(32)P]-phosphate labeling of primary rat hepatocytes. In comparison with other subcellular organelles peroxisomes revealed a unique PId pattern suggesting peroxisomal specificity of the observed synthesis. Use of phosphatase inhibitors enhanced the amount of PtdIns4P. The results obtained provide evidence that isolated rat liver peroxisomes synthesize PIds and suggest the association of PId 4-kinase and PId 5-kinase and PId 4-phosphatase activities with the peroxisomal membrane.     

Geranylgeranyl-pyrophosphate (GGPP) synthase is down-regulated during differentiation of osteoblastic cell line MC3T3-E1

Isoprenylation of geranylgeranyl-pyrophosphate (GGPP) is critical for activation of small GTPases. We examined the roles of GGPP synthase (GGPPS) during the differentiation induced by the cell-to-cell contact in osteoblastic cell line MC3T3-E1 cells. We found that (1) both mRNA and protein expression of GGPPS was reduced with decrement of its activity during the differentiation, (2) GGOH, which is converted to GGPP in the cells, inhibited differentiation. These results suggest that the decrement of GGPP is critical for the cell-to-cell contact-induced differentiation, in which the down-regulation of GGPPS might be involved.     

Detection of new anti-neutral glycosphingolipids antibodies and their effects on Trk neurotrophin receptors

We screened sera from patients with various neurological disorders for the presence of anti-neutral glycosphingolipids antibodies and only found them in sera from relapsing polychondritis with limbic encephalitis patients. Neutral glycosphingolipids are resident in membrane lipid rafts where high affinity nerve growth factor (NGF) receptor, Trk is co-localized. Therefore, we examined whether these antibodies influence the action of NGF in NGF-responsive cells. The results strongly suggest that these antibodies enhance NGF-induced Trk autophosphorylation and neurite outgrowth as well as neurofilament M expression. These data strongly indicate that these anti-neutral glycosphingolipids antibodies have a functional impact on NGF-Trk-mediated intracellular signal transduction pathway.     

gammaepsilon Sub-complex of thermophilic ATP synthase has the ability to bind ATP

The isolated epsilon subunit of F(1)-ATPase from thermophilic Bacillus PS3 (TF(1)) binds ATP [Y. Kato-Yamada, M. Yoshida, J. Biol. Chem. 278 (2003) 36013]. The obvious question is whether the ATP binding concern with the regulation of ATP synthase activity or not. If so, the epsilon subunit even in the ATP synthase complex should have the ability to bind ATP. To check if the ATP binding to the epsilon subunit within the ATP synthase complex may occur, the gammaepsilon sub-complex of TF(1) was prepared and ATP binding was examined. The results clearly showed that the gammaepsilon sub-complex can bind ATP.     

Naked gene therapy of hepatocyte growth factor for dextran sulfate sodium-induced colitis in mice

Ulcerative colitis (UC) is progressive and relapsing disease. To explore the therapeutic effects of naked gene therapy of hepatocyte growth factor (HGF) on UC, the SRalpha promoter driving HGF gene was intrarectally administered to the mice in which colitis was induced by dextran sulfate sodium (DSS). Expression of the transgene was seen in surface epithelium, lamina propria, and muscularis mucosae. The HGF-treated mice showed reduced colonic mucosal damage and increased body weights, compared with control mice (P < 0.01 and P < 0.05, respectively). The HGF-treated mice displayed increased number of PCNA-positive cells and decreased number of apoptotic cells than in control mice (P < 0.01, each). Phosphorylated AKT was dramatically increased after HGF gene administration, however, phosphorylated ERK1/2 was not altered. Microarray analysis revealed that HGF induced expression of proliferation- and apoptosis-associated genes. These data suggest that naked HGF gene delivery causes therapeutic effects through regulation of many downstream genes.     

Viscoelastic properties of isolated rat colon smooth muscle cells

The measurement of the biomechanical properties of gastrointestinal smooth muscle cells is important for the basic understanding of digestive function and the interaction of muscle cells with the matrix. Externally applied forces will deform the cells depending upon their mechanical properties. Hence, the evoked response mediated through stretch-sensitive ion-channels in the smooth muscle cell membrane will depend upon membrane properties and the magnitude of the external force. The aim of this study was to test the hypothesis that gastrointestinal smooth muscle cells behave in a viscoelastic manner. Smooth muscle cells were dissociated from the muscle layers of the descending colon. The viscoelastic properties of the isolated cells were characterized by measuring the mechanical deflection response of the cell membrane to a negative pressure of 1cm H(2)O applied across the cell through a micropipette and fitting the response to a theoretical viscoelastic solid model. The viscoelastic mechanical constants of the isolated cells (N=9) were found to be as follows: k(1)=19.99+/-2.86 Pa, k(2)=7.19+/-1.21 Pa, mu=25.36+/-6.14 Pas and tau=4.84+/-0.95 s. This study represents, to the best of our knowledge, the first quantitative mechanical properties of isolated living smooth muscle cells from the gastrointestinal tract. The mechanical properties determined in this study will be of use in future analytical and numerical smooth muscle cell models to better predict the mechanism between the magnitude of mechanical stimuli, mechanosensitivity and the evoked afferent responses.     

A crucial role of mitochondrial Hsp40 in preventing dilated cardiomyopathy

Many heat-shock proteins (Hsp) are members of evolutionarily conserved families of chaperone proteins that inhibit the aggregation of unfolded polypeptides and refold denatured proteins, thereby remedying phenotypic effects that may result from protein aggregation or protein instability. Here we report that the mitochondrial chaperone Hsp40, also known as Dnaja3 or Tid1, is differentially expressed during cardiac development and pathological hypertrophy. Mice deficient in Dnaja3 developed dilated cardiomyopathy (DCM) and died before 10 weeks of age. Progressive respiratory chain deficiency and decreased copy number of mitochondrial DNA were evident in cardiomyocytes lacking Dnaja3. Profiling of Dnaja3-interacting proteins identified the alpha-subunit of DNA polymerase gamma (Polga) as a client protein. These findings suggest that Dnaja3 is crucial for mitochondrial biogenesis, at least in part, through its chaperone activity on Polga and provide genetic evidence of the necessity for mitochondrial Hsp40 in preventing DCM.     

Decreased serum concentrations of nitric oxide metabolites among Chinese in an endemic area of chronic arsenic poisoning in inner Mongolia

Prolonged exposure to arsenic results in peripheral and cardiovascular manifestations, as does impaired production of endothelial nitric oxide (NO). In vitro studies have indicated that endothelial cells undergo damage by arsenic. However, no information has been available on the relationship between NO synthesis and chronic arsenic poisoning in humans. The present study was designed to reveal this question. The subjects were 33 habitants who continued to drink well water containing high concentrations of inorganic arsenic (mean value = 0.41 microg/ml) for about 18 years in Inner Mongolia, China, and 10 other people who lived in this area but exposed to minimal concentrations of arsenic (mean value = 0.02 microg/ml) were employed as controls. Mean blood concentration of total arsenic was six times higher in exposed subjects than controls; 42.1 vs. 7.3 ng/ml, p <.001. Mean serum concentration of nitrite/nitrate, stable metabolites of endogenous NO, was lower in arsenic-exposed subjects than in controls: 24.7 vs. 51.6 microM, p<.001. In total samples, an inverse correlation with serum nitrite/nitrate levels was strong for blood inorganic arsenic (r = -0.52, p <.001) and less strong for its metabolites, monomethyl arsenic (r = -0.45, p<.005) and dimethyl arsenic (r = -0.37, p<.05). Furthermore, serum nitrite/nitrate concentration was significantly correlated with nonprotein sulfhydryl level in whole blood (r = 0.58, p<.001). In an in vitro study, we demonstrated that inorganic arsenite or arsenate suppresses the activity of endothelial NO synthase in human umbilical vein endothelial cells. These results suggest that long-term exposure to arsenic by drinking well water possibly reduces NO production in endothelial cells, resulting in a decrease in reduced nitrite/nitrate concentrations. Peripheral vascular disorders caused by arsenic may be attributable in part to impairment of NO production in vivo.