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Biomedical subjects

S Ohashi

Publications and source records attributed to S Ohashi.

At least 145 records · Page 8Linked to original sources
PubMed1992

Pituitary adenylate cyclase activating polypeptide stimulates insulin release from the isolated perfused rat pancreas.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a novel hypothalamic peptide structurally related to vasoactive intestinal peptide (VIP) and glucagon like peptide-1(7-36) amide (tGLP-1) in its N-terminal portion. Therefore, their levels of insulinotropic potency were compared using an isolated rat pancreas perfusion. It was found that 0.1 nM PACAP (1-27) amide (PACAP27) significantly stimulated insulin release under a perfusate glucose concentration of 5.5 mM, whereas 1 nM PACAP27 did not under a perfusate glucose concentration of 2.8 mM. The potency was evaluated as tGLP-1 greater than PACAP27 greater than VIP. These results indicate that PACAP is a glucagon superfamily peptide which stimulates insulin release in a glucose dependent manner.

Amino Acid Sequence↗
PubMed1992

Interaction of glucagon-like peptide-1(7-36) amide and gastric inhibitory polypeptide or cholecystokinin on insulin and glucagon secretion from the isolated perfused rat pancreas.

The interaction of three incretin candidates, glucagon-like peptide-1(7-36)amide (t-GLP-1), gastric inhibitory polypeptide (GIP), and sulfated COOH-terminal octapeptide of cholecystokinin (CCK-8-S), on insulin and glucagon release from the isolated perfused rat pancreas was studied. Under the perfusate condition of 8.3 mmol/L glucose, coinfusion of 0.1 nmol/L t-GLP-1 and 0.1 nmol/L GIP resulted in an augmented insulin release greater than that obtained by the same dose of each peptide alone. The degree of stimulation elicited by t-GLP-1 and GIP reached a plateau at 0.3 nmol/L for both infusates, and no cooperative effect was observed by coinfusion at 0.3 nmol/L. Coinfusion of 0.1 nmol/L t-GLP-1 and and 0.1 nmol/L CCK-8-S also resulted in an augmented insulin release greater than that obtained by the same dose of each peptide alone. A similar cooperative effect was observed by coinfusion at 0.3 nmol/L, 1 nmol/L, and 3 nmol/L. With the same perfusion experiments, glucagon release was not significantly affected by any peptide at concentrations of 0.1, 0.3, 1, or 3 nmol/L. The coinfusion of 1 nmol/L t-GLP-1 and GIP elicited a transient, but significant, increase in glucagon release. A similar result was obtained by the coinfusion of 0.3 nmol/L and 3 nmol/L t-GLP-1 and GIP, respectively. The coinfusion of t-GLP-1 and CCK-8-S did not affect the glucagon release.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Comparison of the insulinotropic activity of glucagon-superfamily peptides in rat pancreas perfusion.

Previous studies have demonstrated that glucagon-superfamily peptides stimulate insulin release from the pancreatic islets in a glucose dependent manner. In this study we have carried out a structure-activity study of their insulinotropic activity using a rat pancreas perfusion with 5.5 mM glucose concentration. The following peptides were examined: glucagon-like peptide-1(7-36)amide (tGLP-1), glucagon, gastric inhibitory peptide (GIP), peptide having an amino-terminal histidine and carboxy-terminal isoleucine amide (PHI), vasoactive intestinal polypeptide (VIP), growth hormone releasing factor(1-29)amide (GRF), GRF(1-27)amide and synthetic hybrid-peptides of PHI-GRF, PHI(1-11)-GRF(12-27) and PHI(1-20)-GRF(21-27). Their potencies were evaluated as: tGLP-1 = GIP > glucagon > PHI = VIP > PHI(1-20)-GRF(21-27) > PHI(1-11)-GRF(12-27) >> GRF(1-29) = GRF(1-27). It is clear that 0.1 nM tGLP-1 stimulated insulin release, whereas 1 microM GRF(1-29) did not. These results indicate that 1) in addition to N-terminal amino acid (histidine or tyrosine), position 4 (glycine), position 9 (aspartic acid) and position 11 (serine) in the amino acid sequence are important for their insulinotropic activity, 2) not only the N-terminal portion but also the C-terminal portion of these peptides contribute to their insulinotropic activity.

Amino Acid Sequence↗
PubMed1992

In-vitro activity of new carbapenem antibiotics: comparative studies with meropenem, L-627 and imipenem against pathogenic Nocardia spp.

MICs of two new carbapenems, meropenem and L-627, and imipenem were determined against 98 strains of the Nocardia asteroides group (i.e. N. asteroides sensu stricto, Nocardia farcinica and Nocardia nova), 46 strains of Nocardia brasiliensis and 17 strains of Nocardia otitidiscaviarum. Meropenem and L-627 were less active against the N. asteroides group than imipenem. Among the three species of the N. asteroides group, N. nova was the most sensitive to all the carbapenems. Meropenem was more active than imipenem against both N. brasiliensis and N. otitidiscaviarum with MIC50 values of 28.3-53.3 mg/L. L-627 was less active than meropenem.

Carbapenems↗
PubMed1992

Dihydrofolate reductase as a new "affinity handle".

Dihydrofolate reductase (DHFR) has been demonstrated to be a versatile "affinity handle" for expression of recombinant proteins. The DHFR "handle" has advantages not only in terms of efficiency of expressing the fusion protein as a soluble form but also in stabilizing unstable polypeptides and facilitating purification of the expressed protein by means of methotrexate-bound affinity chromatography and by making use of the enzyme activity. Fifteen genes encoding different lengths of polypeptides of 5 to 44 amino acids were chemically synthesized and introduced into expression vectors, pTP70-1 or its derivatives. All the polypeptide genes were efficiently expressed in Escherichia coli cells as fusion proteins which show DHFR activity. The respective fusion proteins were highly purified from cell-free extracts by monitoring the DHFR activity at each purification step. The use of methotrexate-bound affinity chromatography was very effective. In order to cut out the polypeptides, the purified fusion proteins were treated with either BrCN or site-specific protease according to the spacer sequence. The objective polypeptide was purified by means of a reversed-phase high-pressure liquid chromatography (HPLC) system. Specific cleavage of the purified fusion protein actually yielded very few peptide fragments, so the assignment and isolation of the objective polypeptide were carried out without difficulty.

Amino Acid Sequence↗
PubMed1992

Expression and purification of growth hormone-releasing factor with the aid of dihydrofolate reductase handle.

Expression of a fusion protein composed of dihydrofolate reductase and a derivative of growth hormone-releasing factor resulted in the formation of inclusion bodies in Escherichia coli at 37 degrees C. Among various chemicals, such as detergents, protein denaturants, and acetic acid, tested for the ability to dissolve the inclusion bodies, acetic acid, Brij-35, deoxycholic acid sodium salts, guanidine-HCl, and urea showed a strong solubilizing effect without damaging the DHFR activity. Acetic acid was useful in terms of preparing GRF derivatives, since it could be easily removed by lyophilization, and this made it easy to perform the succeeding BrCN treatment for cutting out the GRF derivative from the fusion protein. The GRF derivative was purified by reversed phase HPLC from the BrCN digest of the acetic acid extract, and its growth hormone-releasing activity was demonstrated. However, for obtaining a highly purified fusion protein itself, solubilization of inclusion bodies by urea was preferred because urea was the only agent which did not cause serious precipitation of the regenerated fusion protein after 10-fold dilution of the extracted inclusion bodies with buffer. The fusion protein was highly purified by means of a methotrexate affinity chromatography.

Acetates↗
PubMed1992

Efficient production of a small peptide by expression as a multimeric form fused with the dihydrofolate reductase affinity handle.

A pentapeptide which potently inhibits primary IgE antibody formation, Asp-Ser-Asp-Gly-Lys (DSDGK), has been efficiently produced with the aid of the dihydrofolate reductase (DHFR) handle [M. Iwakura, et al. (1992) J. Biochem. 111, 37-45]. The genes coding fused proteins comprising DHFR and multimeric forms of DSDGK, namely, DHFR-(DSDGK)3, DHFR-(DSDGK)14, and DHFR-(DSDGK)28, were constructed and expressed in Escherichia coli. The C-terminal peptides attached to DHFR did not affect the expression or the function of the DHFR handle, even when the length of the C-terminal peptide was as long as 160 amino acid residues. The fused proteins were easily purified by methotrexate affinity chromatography, one of the major advantages of the DHFR handle. The fused proteins were digested with trypsin and the monomeric peptide, DSDGK, was purified by HPLC. The yields of the peptide were estimated to be 11, 43, and 99 mg per 1 gram of the total cell proteins from E. coli cells producing DHFR-(DSDGK)3, DHFR-(DSDGK)14, and DHFR-(DSDGK)28, respectively.

Amino Acid Sequence↗
PubMed1991

Fluctuation and rotation of human growth hormone-releasing factor in the presence and the absence of phospholipid bilayer analyzed by time-resolved fluorescence depolarization.

Time-resolved fluorescence depolarization measurements were carried out for human growth hormone-releasing factor analog ([Trp10]-hGRF (1-29) NH2), where the Trp10 residue was incorporated as a fluorescent probe, in the presence and the absence of 1,2-dimyristoyl-sn-glycero-3-phospho-rac- glycerol(DMPG) liposome and in aqueous 2,2,2-trifluoroethanol (TFE) solution. The fluorescence lifetimes and the rotatory correlation times of the peptide in each medium were determined. The apparent volumes of the rotatory Brownian motion unit calculated from these fluorescent parameters indicate the different mode of the fluctuation and/or the rotation of the peptide in each medium, such as: (i) In the aqueous solution, several segments of the peptide fluctuate individually. (ii) In the DMPG bilayer, both the local fluctuation of Trp residue alone and the rotation of the whole molecule exist. (iii) In the aqueous TFE solution, the monomeric peptide rotates as a rigid ellipsoid.

Amino Acid Sequence↗
PubMed1991

Solution structure of human growth hormone-releasing factor fragment (1-29) by CD: characteristic conformational change on phospholipid membrane.

The conformations of synthetic human growth hormone-releasing factor fragment (1-29) in the presence and the absence of 1,2-dimyristoyl-sn-glycero-3-phosphorylglycerol liposome as well as in aqueous 2,2,2-trifluoroethanol solution were investigated by CD spectroscopy. The secondary structure of the peptide in each solution was analyzed by two methods. Both results show that the peptide has an unordered structure in the aqueous solution, whereas it folds into helical structure in the aqueous alcohol and in the phospholipid solution. In addition, although the peptide exists as almost complete helix in the 50 vol% aqueous alcohol (80-90% helicity), it does not reach full helicity even in the solution containing excess amount of phospholipid liposome (maximum 65-70% helicity). The conformational difference is explained by the characteristic amphipathy of the peptide, i.e., the necessity to twist the separated amphipathic helical parts in the interaction with the phospholipid membrane probably makes the helicity of the peptide decrease.

Amino Acid Sequence↗
PubMed1991

Nucleotide specificity of the enzymatic and motile activities of dynein, kinesin, and heavy meromyosin.

The substrate specificities of dynein, kinesin, and myosin substrate turnover activity and cytoskeletal filament-driven translocation were examined using 15 ATP analogues. The dyneins were more selective in their substrate utilization than bovine brain kinesin or muscle heavy meromyosin, and even different types of dyneins, such as 14S and 22S dynein from Tetrahymena cilia and the beta-heavy chain-containing particle from the outer-arm dynein of sea urchin flagella, could be distinguished by their substrate specificities. Although bovine brain kinesin and muscle heavy meromyosin both exhibited broad substrate specificities, kinesin-induced microtubule translocation varied over a 50-fold range in speed among the various substrates, whereas heavy meromyosin-induced actin translocation varied only by fourfold. With both kinesin and heavy meromyosin, the relative velocities of filament translocation did not correlate well with the relative filament-activated substrate turnover rates. Furthermore, some ATP analogues that did not support the filament translocation exhibited filament-activated substrate turnover rates. Filament-activated substrate turnover and power production, therefore, appear to become uncoupled with certain substrates. In conclusion, the substrate specificities and coupling to motility are distinct for different types of molecular motor proteins. Such nucleotide "fingerprints" of enzymatic activities of motor proteins may prove useful as a tool for identifying what type of motor is involved in powering a motility-related event that can be reconstituted in vitro.

Actins↗
PubMed1991

The structure-function relationship of GLP-1 related peptides in the endocrine function of the canine pancreas.

In order to clarify the relationship between the structure and function of glucagon-like peptide (GLP) 1 in the endocrine function of the pancreas, the response of insulin and glucagon to various synthetic GLP-1-related peptides was investigated in anesthetized dogs. GLP-1-related peptides were administered in a dosage of 400 pmol within 10 min into the pancreatic artery during glucose or arginine infusion and the changes in plasma insulin and glucagon in the pancreatic vein were studied. GLP-1 (7-36) and (7-37), as well as glucagon enhanced insulin release during glucose infusion, whereas neither GLP-1 (1-37), (7-20), (6-37) nor (8-37) stimulated insulin release. The administration of GLP-1 (1-37), (7-36) and (7-37) reduced glucagon release during glucose infusion. When arginine was infused, GLP-1 (7-20), (7-36), (7-37), and glucagon enhanced insulin release. In contrast, glucagon release was increased by the administration of GLP-1 (7-20), (8-37), and (7-37). The present study indicates that histidine at the 7th position of GLP-1 is important in eliciting biological action and that only truncated GLP-1 (7-36), (7-37), and (7-20) showed an insulinotropic action as strong as glucagon in dogs. Furthermore, it is suggested that the response of insulin and glucagon to GLP-1-related peptides is dependent on a background condition.

Animals↗
PubMed1991

A new method for evaluating gastric ulcer healing by endoscopic ultrasonography.

We observed the quantitative estimation of the transmural changes associated with gastric ulcer healing by using endoscopic ultrasonography (EUS). It was possible to diagnose the depth of ulcer by EUS. Forty-eight patients were divided into three treatment groups. Group A (n = 16) was treated with 800 mg cimetidine daily, group B (n = 22) with 20 mg omeprazole daily, and group C (n = 10) with 400 mg cimetidine + 300 mg gefarnate daily. EUS was performed before and after 2, 4, and 8 weeks of treatment. The groups were compared from the viewpoints of endoscopic findings and contraction rate of the length and the cross-sectional area of the ulcer in EUS pictures. The best healing of both the endoscopic and EUS findings was seen in group B. By estimating the changes inside the ulcer, EUS may provide useful information for choice of anti-ulcer agents.

Anti-Ulcer Agents↗
PubMed1991

Evaluation of acetic acid-induced gastric ulcers in rats by scanning acoustic microscopy.

To clarify the mechanism of gastric ulcer recurrence, we studied acetic acid-induced gastric ulcers. The healing of these gastric ulcers was then studied for 4-21 weeks after the injection. Forty-eight ulcers were examined by macroscopic observation and scanning acoustic microscopy (SAM) and by histologic study of the ulcers. SAM has a higher radio frequency and provides more detailed information about the gastric wall than endoscopic ultrasonography (EUS). Thirteen healed and 35 non-healed gastric ulcers were observed by macroscopic observation over 4-21 weeks. SAM enabled us to observe the normal gastric wall of the rat in five different layers. The SAM echo patterns of the healed ulcers were all one pattern. On the other hand, we were able to classify the echo patterns of the non-healed ulcers into two different patterns; one pattern consisted of a high-echogenic region in the first and second layers with an underlying relatively high echogenic region in a horizontal pattern. The other pattern showed a poorly defined and spotted low-echogenic region in the first and second layers which fanned out toward the fifth layer. However, these two different ulcers could not be distinguished macroscopically. Histologic findings supported the results of the SAM study which showed one pattern in healed ulcers and two patterns in non-healed ulcers. In the non-healed ulcers the two different patterns may be ascribed to the proliferation of collagen fibers. SAM makes it possible to image the proliferation of collagen fibers and identify the two different types with an accuracy of 97%.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetates↗
PubMed1991

[Endoscopic ultrasonography in the assessment of invasive cardiac cancer].

The aim of this study was to assess the accuracy and limitation of Endoscopic ultrasonography (EUS) in the preoperative evaluation of the cardiac cancer (cancer in the area less than 2 cm from the esophago-gastric junction). EUS was preoperatively performed in 20 patients with cardiac cancer. These cases were visualized by the water immersion method and balloon contact method. The results were correlated with the histology of resected specimens. EUS was accurate in assessing the depth of tumor infiltration. Over all accuracy of EUS for cardiac cancer was 90%. Recognition of ultrasonographic patterns had enabled us to judge the mode of tumor infiltration whether expansive or infiltrative. Microscopical spread over 5 mm to the esophagus of the cardiac cancer could be detected by EUS. EUS was accurate in diagnosing thoracic lymph nodes (No. 110, 111, 112) metastasis of the cardiac cancer (accuracy 86%). EUS was valuable in the clinical diagnosis of the cardiac cancer.

Adult↗
PubMed1990

Captopril-stimulated renal vein renin in hypertensive patients with or without renal artery stenosis.

To examine the efficacy and usefulness of captopril-enhanced renal vein renin (RVR) measurements in detecting the functional significance of renal artery stenosis found in hypertensives, we compared these values in 22 patients with arteriographically documented renovascular hypertension due to unilateral (URVH: 14 patients) or bilateral renal artery stenosis (BRVH: 8 patients) and 12 patients with high renin essential hypertension (EHT). Before captopril administration, RVR ratio was less than 1.5 in 8 patients (36.4%) with renovascular hypertension and all patients (100%) with EHT. Captopril enhanced the lateralization of renal vein renin in renovascular hypertension; the postcaptopril RVR ratio was greater than 2.0 in 18 patients (81.8%) and greater than 1.5 in all the patients (100%). On the other hand, RVR ratio remained unchanged in most patients with EHT. There was no significant difference in the postcaptopril RVR ratios between URVH and BRVH. However, the postcaptopril RVR ratio was higher in atherosclerosis (10 patients) than in fibromuscular dysplasia (11 patients) (P less than .05). Captopril also elucidated contralateral renin suppression as expressed by a contralateral/peripheral renin ratio of less than 1.0, which was associated with a favorable outcome of unilateral surgical intervention. Captopril-stimulated RVR indices were valuable in detecting the functionally significant renal artery stenosis and predicting surgical curability in renovascular hypertension.

1-Sarcosine-8-Isoleucine Angiotensin II↗