Cloning of a rabbit brain glucose transporter cDNA and alteration of glucose transporter mRNA during tissue development

A full-length cDNA clone that codes for glucose transporter protein was isolated from a rabbit brain cDNA library by using synthetic oligonucleotide probe derived from the sequence of human glucose transporter cDNA. The coding region shared 93.2% nucleotide and 97.0% amino-acid similarities with those of human glucose transporter and 89.4% nucleotide and 97.4% amino-acid similarities with those of rat transporter. Northern blot analysis revealed that glucose transporter mRNA is most abundant in the placenta and that it is also abundant in the brain. The fat tissue, heart, liver, and skeletal muscle of adult rats contained a very small amount of mRNA, while heart, liver, skeletal muscle and kidney of fetal rats contained a very high amount of glucose transporter mRNA. These results suggest that this type of glucose transporter might be closely related with cell proliferation and tissue development.     

Improved identification of agonist-mediated Gα(i)-specific human G-protein-coupled receptor signaling in yeast cells by flow cytometry

Flow cytometry enables comparative quantification, population analysis, and high-throughput screening of agonist-mediated G-protein-coupled receptor (GPCR) signaling in genetically engineered yeasts. By using flow cytometry, we found that transformation of yeast cells with a low plasmid number is critical both for the construction of large screening libraries and for stable signal transmission in cell ensembles. Based on these findings, we constructed an engineered yeast strain for the improved identification of signal promotion by Gα(i)-specific human GPCRs using flow cytometry.     

Dentists’ Dietary Perception and Practice Patterns in a Dental Practice-Based Research Network

Background

Dental caries are largely preventable, and epidemiological evidence for a relationship between diet and oral health is abundant. To date, however, dentists’ perceptions about the role of diet and dentists’ practice patterns regarding diet counseling have not been clarified.

Objective

The purposes of this study were to: (1) examine discordance between dentists’ perception of the importance of diet in caries treatment planning and their actual provision of diet counseling to patients, and (2) identify dentists’ characteristics associated with their provision of diet counseling.

Design

The study used a cross-sectional study design consisting of a questionnaire survey in Japan.

Participants

The study queried dentists working in outpatient dental practices who were affiliated with the Dental Practice-Based Research Network Japan (JDPBRN), which aims to allow dentists to investigate research questions and share experiences and expertise (n = 282).

Measurement

Dentists were asked about their perceptions on the importance of diet and their practice patterns regarding diet counseling, as well as patient, practice, and dentist background data.

Results

The majority of participants (n = 116, 63%) recognized that diet is “more important” to oral health. However, among participants who think diet is “more important” (n = 116), only 48% (n = 56) provide diet counseling to more than 20% of their patients. Multiple logistic regression analysis suggested that several variables were associated with providing diet counseling; dentist gender, practice busyness, percentage of patients interested in caries prevention, caries risk assessment, and percentage of patients who receive blood pressure screening.

Conclusions

Some discordance exists between dentists’ perception of the importance of diet in caries treatment planning and their actual practice pattern regarding diet counseling to patients. Reducing this discordance may require additional dentist education, including nutritional and systemic disease concepts; patient education to increase perception of the importance of caries prevention; or removing barriers to practices’ implementation of counseling.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01680848","term_id":"NCT01680848"}}NCT01680848     

Characterization of the carboxyl terminal-truncated endothelin B receptor coexpressed with G protein-coupled receptor kinase 2.

The role of phosphorylation of the C-terminal tail of endothelin B receptor (ETBR) in agonist-induced desensitization was investigated, using a mutant lacking C-terminal 40 amino acids (delta 40 ETBR). In cells expressing the wild type or delta 40 ETBR, ET-1 caused rapid desensitization of calcium responses. The wild type ETBR was phosphorylated by biotinylated ET-1, and the phosphorylation was markedly enhanced by coexpression with G protein-coupled receptor kinase 2 (GRK2). However, delta 40 ETBR was not phosphorylated regardless of coexpression with GRK2. On the other hand, ET-1-induced IP3 formation in these cells was decreased by coexpression with GRK2 or catalytically inactive Lys220Arg GRK2 to the similar extent. The present study demonstrates the presence of phosphorylation-independent desensitization mechanism in delta 40 ETBR and suggests that GRK2 might play a role other than that as a kinase.     

Spectral characteristics of skin sympathetic nerve activity in heat-stressed humans

Skin sympathetic nerve activity (SSNA) exhibits low- and high-frequency spectral components in normothermic subjects. However, spectral characteristics of SSNA in heat-stressed subjects are unknown. Because the main components of the integrated SSNA during heat stress (sudomotor/vasodilator activities) are different from those during normothermia and cooling (vasoconstrictor activity), we hypothesize that spectral characteristics of SSNA in heat-stressed subjects will be different from those in subjects subjected to normothermia or cooling. In 17 healthy subjects, SSNA, electrocardiogram, arterial blood pressure (via Finapres), respiratory activity, and skin blood flow were recorded during normothermia and heat stress. In 7 of the 17 subjects, these variables were also recorded during cooling. Spectral characteristics of integrated SSNA, R-R interval, beat-by-beat mean blood pressure, skin blood flow variability, and respiratory excursions were assessed. Heat stress and cooling significantly increased total SSNA. SSNA spectral power in the low-frequency (0.03-0.15 Hz), high-frequency (0.15-0.45 Hz), and very-high-frequency (0.45-2.5 Hz) regions was significantly elevated by heat stress and cooling. Interestingly, heat stress caused a greater relative increase of SSNA spectral power within the 0.45- to 2.5-Hz region than in the other spectral ranges; cooling did not show this effect. Differences in the SSNA spectral distribution between normothermia/cooling and heat stress may reflect different characteristics of central modulation of vasoconstrictor and sudomotor/vasodilator activities.     

Histochemical studies of mucosubstances in metaplastic epithelium of the stomach,with special reference to the development of intestinal metaplasia

Summary Processes in the development of intestinal metaplasia of the stomach were investigated from the morphological and histochemical approaches using light and electron microscopic techniques. The specimens taken from 38 gastric carcinomas and 15 gastric and/or duodenal ulcers were subjected to this study. Morphological appearances of the intestinal metaplasia observed in routine examination with hematoxylin and eosin staining was able to be divided into complete and incomplete metaplasia by the light and electron microscopic histochemical stainings of the mucosubstances. The columnar cells at the area of the incomplete metaplasia had both the properties of the intestinal epithelia and the gastric foveolar epithelia. The incomplete as well as the complete metaplasia arose from the generative cells at the isthmus of the gland. The generative cells, however, sometimes gradually transformed to produce the complete metaplastic cells. The two processes of the development of the intestinal metaplasia were proposed and discussed.     

The Effects of Cyanate in Urea Solutions on the Gel Electrophoresis of the Subunits of Soybean 11S Globulin

For the screening of potent proteases of plant origin, gelatinolytic activities were measured for various vegetables and fruits. Green asparagus, kiwi fruit and miut were found to possess high proteolytic activities. Optimum temperatures for the activities of green asparagus and miut were 40 to 45°C and that of kiwi fruit was 60°C. Optimum pHs for the three activities were in neutral or slightly alkaline regions. The proteolytic enzymes of kiwi fruit and miut were stimulated by cysteine and EDTA but that of green asparagus was unaffected by them.     

Phosphorylation state and biological function of a mutant human insulin receptor Val996

Chinese hamster ovary (CHO) cell transfectants that expressed human insulin receptors whose glycine 996 was substituted by valine were studied. Receptor processing and insulin binding were unaffected by this mutation; however, this mutant insulin receptor had little or no tyrosine kinase activity. Nevertheless, the Val996 mutant exhibited seryl and threonyl phosphorylation in both the basal and insulin-stimulated state in intact cells. This is in contrast to the Lys----Ala1018 tyrosine kinase deficient mutant (Russell, D. S., Gherzi, R., Johnson, E. L., Chou, C-K., and Rosen, O. M. (1987) J. Biol. Chem. 262, 11833-11840). Cells expressing the normal human receptor were 10-fold more sensitive to insulin than the untransfected CHO cells with respect to phosphorylation of a cellular substrate (pp 185) on tyrosyl residues, glucose incorporation into glycogen, thymidine incorporation into DNA, and phosphorylation of ribosomal protein S6. Cells expressing the mutant receptor exhibited the same insulin sensitivity as the untransfected CHO cells. Insulin was rapidly internalized in cells expressing the normal human receptor and the number of receptors expressed on the cell surface was decreased in response to exposure to insulin. However, little insulin was internalized in cells expressing the mutant receptor, and the number of receptors on the cell surface was not significantly diminished in response to exposure to insulin. It is concluded that despite the occurrence of seryl and threonyl phosphorylations, post-receptor effects of insulin described above are not mediated by the tyrosine kinase-deficient receptor, Val996.     

Effect of a continuously applied compressive pressure on mouse osteoblast-like cells (MC3T3-E1) in vitro

Bone metabolism is often affected by a variety of mechanical forces, but the cytological basis of their action is not known. In this study, we examined the effect of a continuously applied compressive pressure (CCP) on the growth and differentiation of clonal mouse osteoblast-like cells (MC3T3-E1) cultured in a specifically devised culture chamber. The gas phase of the chamber was maintained at a pressure of 2 atmospheres (atm) above ambient (3 atm total, 3.1 kg/cm2; 3.0 x 10(5) Pa) by continuously infusing a compressed mixed gas (O2: N2:CO2 = 7.0%:91.3%:1.7%). The pO2, pCO2, and pH in the culture medium at 37 degrees C under 3 atm were maintained at the same levels as those under 1 atm. MC3T3-E1 cells were cultured in alpha-minimal essential medium containing 10% fetal bovine serum under either 3 atm in the CCP culture chamber or 1 atm in an ordinary CO2 incubator. Alkaline phosphatase activity, a marker of osteoblasts, was greatly suppressed by the CCP treatment. The inhibition of alkaline phosphatase activity was rapidly restored when the cells were transferred to an ordinary CO2 incubator under 1 atm, indicating that the inhibition of alkaline phosphatase activity by CCP is reversible. Cell growth was not altered under CCP. The CCP treatment greatly increased the production and secretion of prostaglandin E2 (PGE2). Adding either conditioned medium from the CCP culture or exogenous PGE2 to the control culture under 1 atm suppressed alkaline phosphatase activity dose-dependently. The CCP treatment also suppressed collagen synthesis and calcification. These results suggest that CCP causes the cells to produce and secrete PGE2, which, in turn, inhibits differentiation of osteoblasts and the concomitant calcification.     

Sodium/Proton Exchange in Cultured Bovine Adrenal Medullary Cells

We investigated the presence of Na+/H+ exchange in cultured bovine adrenal medullary cells. The intracellular pH in control cells measured by 5,5-dimethyl[2-14C]oxazolidine-2,4-dione was 7.13 +/- 0.02 (n = 6). Removal of Na+ from the incubation medium shifted the intracellular pH down to 6.67 +/- 0.12 (n = 6). Reintroduction of Na+ to the medium caused a rapid recovery in intracellular pH to 7.20-7.30 that was associated with an increase in uptake of 22Na+ by the cells. Both increases in intracellular pH and uptake of 22Na+ were inhibited by amiloride, an inhibitor of Na+/H+ exchange. The recovery of intracellular pH by addition of Na+ was partially inhibited by quinidine, another inhibitor of Na+/H+ exchange, but not by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, an anion-exchange (Cl-/HCO3-) inhibitor. Li+ could substitute for Na+ in the recovery of intracellular pH. Carbachol caused an increase in intracellular pH from 7.12 +/- 0.01 to 7.21 +/- 0.02 (n = 10). This increase in intracellular pH caused by carbachol was inhibited by amiloride. These results suggest the existence of an amiloride-sensitive Na+/H+ exchange that regulates the intracellular pH in adrenal medullary cells.