Biomedical subjects

K Ishihara

Publications and source records attributed to K Ishihara.

At least 541 records · Page 30Linked to original sources

PubMed1989

Amino acid sequence of exogastrula-inducing peptide C from the sea urchin, Anthocidaris crassispina.

The complete amino acid sequence of exogastrula-inducing peptide C from embryos of the sea urchin, Anthocidaris crassispina has been determined by analysis of the amino acid sequences in the S-pyridylethylated peptide C and the peptides generated after digestion of the peptide C with arginyl endopeptidase. Exogastrula-inducing peptide C was composed of 58 amino acid residues and its molecular weight was calculated to be 6464. The sequence was DTKGGCERATNNCNGHGDCVQGRWGQYYCKCTLPYRVGGSESSCYMPKDKEEDVEIET.

Amino Acid Sequence↗

PubMed1989

Effect of cooling rate on insulin release from frozen-thawed dispersed rat islet cells.

Rat islet cells, dissociated with EDTA-Dispase, were immersed in 10% dimethyl sulfoxide at 20 degrees C for 15 min and frozen to -40 degrees C at a cooling rate of 0.5 or 1.0 degree C/min and subsequently further to -80 degrees C at 3 degrees C/min by a programmable freezer. After being maintained at -80 degrees C for 10 min, they were rapidly thawed in a water bath at 37 degrees C. They were cultured for 12 h and preincubated in 3.3 mM glucose-containing Krebs-Henseleit bicarbonate buffer (KHBB) for 1 h. Groups of 10(4) cells were then incubated in 3.3 or 16.7 mM glucose-containing KHBB for another hour. As a control, non-frozen-thawed cultured islet cells were incubated similarly. The non-frozen rat islet cells released 1.29 pg insulin/cell.60 min in the presence of 3.3 mM glucose and this release level was significantly elevated to 1.64 pg insulin/cell.60 min in the presence of 16.7 mM glucose. The cells frozen at 0.5 degree C/min releasing 1.55 pg insulin/cell.60 min in the presence of 3.3 mM glucose also responded to 16.7 mM glucose and released the significantly high level of 1.87 pg insulin/cell.60 min. However, the islet cells frozen at a cooling rate of 1 degree C/min secreted 1.74 and 1.92 pg insulin/cell.60 min in the presence of 3.3 mM and 16.7 mM glucose respectively. There was no significant difference between these levels. These results indicate that cryopreservation at a cooling rate of 0.5 degree C/min may be adequate for the preservation of dispersed pancreatic endocrine cells.

Animals↗

PubMed1989

Effect of dimethylthiourea on syngeneic rodent islet transplantation.

We studied the effects of a hydroxyl radical scavenger, dimethylthiourea (DMTU), on syngeneic islet grafts. Six hundred fresh islets taken from two Wistar rats were transplanted intraportally into other Wistar rats made diabetic with streptozotocin. DMTU was given to five recipients intraperitoneally for a month while nine controls received only intraperitoneal saline. The DMTU-treated group had significantly lower fasting plasma glucose levels at 1.5, 4.5, 5, 5.5, 6, 7, 9 and 11 weeks and had higher mean fasting body weights between the 2.5th week and their eventual sacrifice. Their islets also survived significantly longer than did those of the controls (73.6 +/- 3.4 vs. 21.6 +/- 9.4 days). This suggests that oxygen free radical production endangers graft survival and that the hydroxyl radical scavenger DMTU protects syngeneic islet grafts.

Animals↗

PubMed1989

Sophisticated mesh filtration technique of a large-scale isolation of islets and their function.

A large-scale isolation of islets is required for islet transplantation. We improved our conventional method, and could obtain about three times more islets than by the conventional methods. Pancreata of adult Wistar rats were inflated by injection of buffer with (A) or without 1.3 mg/ml collagenase (B). The rats were bled from the inferior vena cava and the aorta in (A) simultaneously with the inflation. They were further digested with collagenase and filtered through two different meshes (pore size: 1190 and 590 microns) (A1) or three different meshes (pore size: 1190, 590 120 microns) (A2) in order. Insulin released from islets isolated in this manner was determined by 1-h incubation with 3.3 and 16.7 mM glucose. Besides, 600 islets each were transplanted into the liver of streptozotocin-induced diabetic Wistar rats and their fasting plasma glucose was measured at weekly intervals. (1) With these methods more numerous islets were harvested by A1 (mean: 554) and A2 (mean: 746) than B (mean: 224). (2) Insulin released at both glucose concentrations was similar among islets obtained by A1, A2 and B. (3) The plasma glucose-lowering effect was similar among the islets obtained by these methods. (4) A more selected range of islet sizes was obtained by A2 than A1. These observations indicate that the present techniques (A1 and A2) are less time-consuming and simpler for a large-scale isolation of islets.

Animals↗

PubMed1989

Islet function after storage at -2 degrees C and -196 degrees C.

The radical treatment of type 1 diabetes by transplantation requires the extracorporeal storage of islets, and this has frequently been studied. Damage from ice formation, however, has prevented the development of any satisfactory method for preservation. We compared islet function after frozen storage with that after non-frozen storage. Isolated rat islets immersed in 10% dimethyl sulfoxide were kept at -2 degrees C (group A) and -196 degrees C (group B) for 7 days. After one day of culture, some of the islets were incubated in 3.3 and 16.7 mM glucose-containing Krebs--Henseleit bicarbonate buffer for 60 min. The other islets were incubated with 3H-leucine for 2 h. The radioactivity of whole-islet homogenate and the insulin extracted from it were measured. We also counted the number of islets before and after the 7-day storage. The islets thus preserved were transplanted into streptozotocin-induced diabetic rats and the fasting plasma glucose was determined weekly. Non-cooled islets were used as controls (group C). Insulin release in the presence of 16.7 mM glucose did not significantly differ between groups C and A, whereas it was lower in group B than in groups A or C. The islet uptake of 3H-leucine was lower in A and B compared with C, but the insulin synthesis was similar in all three groups. More islets were recovered from A than B. Fasting plasma glucose was lowered similarly in the diabetic rats after transplantation of islets from A and B. The relative ease of preservation at -2 degrees C, and the positive results of this experiment, favor this method of preservation.

Animals↗

PubMed1989

Production of anti-insulin monoclonal antibody and its application to immunoassay of insulin and immunohistochemistry.

An unlimited supply of suitable antisera is wanted for immunoassays, analysis of antigenic determinants and precise localization of antigens in biological systems. Therefore, we produced a monoclonal antiporcine insulin antibody by the hybridoma technology and assessed it in comparison with polyclonal antibody. The spleen cells of BALB/c mice immunized against porcine insulin were hybridized with mouse myeloma cells (P3-X63-Ag8-U1). The monoclonal antibody thus generated was shown to have high binding capacity and specificity to porcine insulin in radioimmunoassay. It reacted with human insulin as well, but did not crossreact with other polypeptide hormones produced in the pancreatic islets such as glucagon, somatostatin and pancreatic polypeptide. In immunohistochemistry human and dog islets were stained by this monoclonal antibody. Rat islets were not stained, although they reacted with polyclonal anti-insulin antibody. The insulin of human serum samples measured using the monoclonal antibody was tightly correlated with that using the polyclonal antibody. These observations indicate that our hybridoma-derived monoclonal antibody is useful for immunoassay as well as localization of insulin.

Animals↗

PubMed1989

Disparate functions of I-A and I-E molecules on B cells as evidenced by the inhibition with anti-I-A and anti-I-E antibodies of polyclonal B cell activation.

Polyclonal differentiation of unprimed B cells into IgM-producing cells induced by lipopolysaccharide (LPS) or T cell-derived lymphokine B151-TRF2 has been shown to contain a process of I-A-restricted B-B cell interaction, so that the B cell responses are inhibited by monoclonal antibodies (mAb) specific for I-A molecules. On the other hand, the B cell responses are also inhibited by anti-I-E mAb, although I-E molecules are not involved in such B-B cell interaction. In this study, we examined the mechanism underlying the anti-I-E-mediated inhibition of the B cell responses. The B cell responses induced by LPS or B151-TRF2 were inhibited by either anti-I-A or anti-I-E mAb added on day 0 over a 5-day culture period, whereas when added on day 3 the responses were inhibited only by anti-I-E mAb and not by anti-I-A mAb. To gain insight into the mechanism underlying the anti-I-E-mediated inhibition, we prepared monovalent Fab and divalent F(ab')2 fragments of anti-I-A and anti-I-E mAb and examined their effects on the B cell responses. We found that the B cell responses were inhibited by the F(ab')2 but not Fab fragment of anti-I-E mAb, whereas the Fab fragment of anti-I-A mAb still gave effective inhibition. The F(ab')2 but not Fab fragment of anti-I-E mAb induced increases in cyclic AMP (cAMP) levels in B cells, whereas the undigested anti-I-A mAb did not induce such increases. Furthermore, adenylate cyclase inhibitors, which inhibit cellular cAMP accumulation, circumvented the B cell responses inhibited by anti-I-E but not anti-I-A mAb. Thus, these results indicate that the anti-I-E-mediated inhibition of the B cell responses requires increases in intracellular cAMP levels induced by cross-linking of I-E molecules. In contrast, anti-I-A mAb inhibits the B cell responses without cross-linking of I-A molecules and cAMP accumulation. These results reinforce a unique function of I-A molecules as restriction elements in the Ia-restricted B-B cell interaction.

Animals↗

PubMed1989

Adhesive bone cement both to bone and metals: 4-META in MMA initiated with tri-n-butyl borane.

In order to develop a new bone cement which is expected to prevent loosening of a prosthesis by better adhesion, the cement composed of 4-methacryloyloxyethyl trimellitate anhydride (4-META) and methyl methacrylate (MMA) as monomers and tri-n-butyl borane (TBB) as an initiator was evaluated. The tensile bond strength between bone and metals adhered with 4-META/MMA-TBB cement was above 7 MPa. The values were higher than that with conventional bone cement (1 MPa) or MMA-TBB cement (2 MPa). Therefore, 4-META was effective for improving adhesion. When cohesive failure of the bone was observed with the scanning electron micrography after the tensile test, fracture occurred on the bone side below the interface between the bone and the 4-META/MMA-TBB cement. This result showed that the cement adhered tightly to both the bone and metals. Thus, it is concluded that the 4-META/MMA-TBB cement is useful as an adhesive bone cement.

Adhesiveness↗

PubMed1989

Limited atrial compensation to reduced early diastolic filling in hypertensive patients with advanced left ventricular hypertrophy: a Doppler echocardiographic study.

To assess atrial contribution to left ventricular (LV) filling in hypertension, we studied, using pulsed Doppler echocardiography, 22 hypertensive patients without apparent LV hypertrophy (LVH), 12 hypertensive patients with LVH, and 24 age-matched normal subjects. From mitral flow velocity waveform, we determined peak velocity of early diastolic filling flow (peak E), peak velocity of late diastolic filling flow (peak A), and the peak A/peak E ratio (peak A/peak E). Peak E decreased in hypertensives without apparent LVH and showed a further decrease in hypertensives with LVH compared with normal subjects (57 +/- 8 [mean +/- SD]; P less than 0.001, 46 +/- 7; P less than 0.0001, vs 65 +/- 10 cm/s). On the other hand, peak A/peak E increased in hypertensives without apparent LVH, and greatly increased in hypertensives with LVH (1.06 +/- 0.14; P less than 0.001, 1.40 +/- 0.29; P less than 0.0001, vs 0.79 +/- 0.21). However, increased peak A was not significantly different between the hypertensive groups (60 +/- 8 vs 64 +/- 8; NS, both; P less than 0.001 vs 50 +/- 10 cm/s for normal subjects). In hypertensives, we found no significant correlation between peak A and the wall thickness index (WTI, determined as mean LV wall thickness normalized by LV diastolic dimension), whereas peak E was significantly correlated with WTI (r = -0.65; P less than 0.001). Our findings indicate that atrial contraction can not fully compensate the decrease in early diastolic filling caused by advanced LVH. We conclude that atrial compensation for reduced early diastolic filling is limited in hypertensive patients with advanced left ventricular hypertrophy.

Adult↗

PubMed1989

Vacor inhibits insulin release from islets in vitro.

It has been reported that Vacor, a rodenticide containing N-3-pyridylmethyl-N'-p-nitrophenyl urea, causes insulin-dependent diabetes mellitus. The pathomechanism of Vacor-induced diabetes mellitus has not been clarified yet. The effect of Vacor, therefore, was studied in terms of insulin release from isolated rat pancreatic islets. Vacor suppressed glucose-stimulated insulin release, but did not affect the insulin release induced by theophylline or 12-o-tetra-decanoylphorbol 13-acetate. It is suspected that the suppression of insulin release from pancreatic islets by Vacor may contribute to the pathogenesis of Vacor-induced diabetes mellitus and that this suppression might not be related to cAMP and C-kinase.

Animals↗

PubMed1989

Idebenone and vinpocetine augment long-term potentiation in hippocampal slices in the guinea pig.

The effects of idebenone and vinpocetine which reportedly prevent impairment of learning and memory were studied in vitro, on the long-term potentiation of the population spike in the pyramidal layer of CA3 region of slices of hippocampus in the guinea pig. Idebenone (10(-9) M-10(-6) M) or vinpocetine (10(-7) M-10(-6) M) significantly augmented long-term potentiation in the mossy fibre-CA3 pyramidal cell system, without any significant changes in population spikes in the absence of tetanic stimulation. These results suggest that both drugs have direct actions on the hippocampal neurones to augment long-term potentiation at fairly small concentrations. Further, when the two drugs were applied together, the augmenting effects were additive.

2-Amino-5-phosphonovalerate↗

PubMed1989

Production of anti-insulin monoclonal antibody and its clinical application.

Hybridoma-produced monoclonal antibody (MoAb) against insulin is useful for insulin assays because of its specificity and plentiful supply. The spleen cells of male BALB/c mice immunized against monocomponent porcine insulin were hybridized with mouse myeloma cells (P3-X63-Ag8-U1). The resulting anti-insulin antibody (Ab) was purified and characterized by radioimmunoassay (RIA) using 125I-porcine insulin and sandwich-method enzyme immunoassay (EIA) using Ab-conjugated beads and beta-galactosidase. For reference, we used anti-insulin polyclonal antibody raised in guinea pigs (PoAb). Using the Ouchterlony technique, we identified the antibody as being of subclass IgG 1. We judged this antibody to be MoAb because it did not react at all with porcine insulin during EIA, in concentrations between 0 and 12.5 ng/ml; in contrast, PoAb reacted dose-dependently. During RIA, this Ab did not cross-react with glucagon, somatostatin or pancreatic polypeptide. It did cross-react with human and bovine insulins but not with rat insulin. The proper concentration of this MoAb for RIA proved to be 1:1,500,000 and the smallest detectable level of porcine insulin by RIA using this Ab was 0.5 ng/ml. These levels were similar to those obtained with PoAb. The binding activity of this MoAb to human insulin was quite similar to that of porcine insulin. RIA insulin determinations using our MoAb correlated well with those employing PoAb.

Animals↗

PubMed1989

The exogastrula-inducing peptides in embryos of the sea urchin, Anthocidaris crassispina--isolation and determination of the primary structure.

Four exogastrula-inducing peptides, A, B, C, and D have been isolated from the homogenates of embryos of the sea urchin, Anthocidaris crassispina, with successive chromatographic fractionations. The complete amino acid sequences of the peptides A and D were determined by analysis of the peptides generated by their digestion with lysyl endopeptidase. They were composed of 52 and 53 amino acid residues, and their molecular weights were calculated to be 5754 and 5737, respectively. The sequences of peptides A and D were DSVYQCNRDTNSCDGFGKCEKSTFGRTTGQYICNCDDGYRNNAYGGCSPRTE, and DTVARCERDTKNCDGHGTCQLSTFGRRTGQYICFCDAGYRKPNSYGGCSPSSA, respectively. The biological significance of the exogastrula-inducing peptides was discussed.

Amino Acid Sequence↗

PubMed1989

Cluster formation among small resting B lymphocytes leading to B cell activation.

Intercellular contacts are thought to be a crucial event in various aspects of immune responses. We have recently suggested that the self class II MHC-restricted B-B cell interaction is a prerequisite process in the polyclonal B cell differentiation induced by a novel T cell-derived lymphokine B151-TRF2 or LPS. The results have led us to the possibility that such a class II MHC-restricted B-B cell interaction promotes the formation of B cell clusters responsible for the development of IgM-producing cells upon subsequent stimulation with the polyclonal B cell activator. To examine directly this possibility, the present study has utilized a recently devised ACAS470 work station capable of sorting out desired cells in a tissue culture plate in situ. Clusters and non-clusters were observed after 2 day preculture of murine resting B cells in the absence of the polyclonal B cell activator. Interestingly, B cells in clusters purified by the ACAS470 gave rise to IgM-producing cells when stimulated with B151-TRF2 or LPS for an additional 2 days, whereas non-clustered B cells failed to differentiate. Moreover, such a B cell activation was not observed when the 2 day preculture of the resting B cells was conducted in the presence of anti-class II MHC mAb but not of anti-class I MHC mAb. Thus, the present results support the notion that cluster formation among small resting B cells accompanying the class II MHC-restricted B-B cell interaction makes the B cells responsive to differentiation signals of polyclonal B cell activator.

Animals↗

PubMed1989

Clinical features and distribution of malignant melanoma and pigmented nevi on the soles of the feet in Japan.

This study, based on 842 cases of malignant melanoma from 108 medical schools and medical centers in Japan (from 1983 to 1985), presents information regarding the incidence, sex and age distribution, anatomical location, and clinical features of malignant melanoma in Japan and compares it with data from the United States. In the white population, the epidemiologic evidence indicates that exposure to sunlight may be an important causal factor of the disease. On the other hand, in more heavily pigmented races, such as Negroids and Orientals, many of the malignant melanomas occur on the skin of the unexposed soles of the foot. In our survey, 241 out of 833 cases of melanoma (28.9%) in Japanese patients involved the skin of the soles. Whites tend to have a wide distribution of cutaneous melanoma over the whole body surface, showing the greatest aggregation of lesions on the head, neck, and trunk, with small number on the soles of the feet. This comparative study revealed that the anatomical distribution and clinical types of malignant melanoma in the United States and Japan were different. The racial difference--the highest frequency or proportion of melanoma on the skin of the soles in the pigmented races, and the lowest ratio of this anatomical area in the least-pigmented races--remains to be investigated.

Age Factors↗

PubMed1989

Loss of heterozygosity at polymorphic chromosomal loci in patients with malignant melanoma.

One type of aberration of DNA found in human cancers is loss of heterozygosity at chromosomal loci. This loss suggests that inactivation of particular genes is involved in the genesis of cancer cells. We have analyzed tumor DNA and normal DNA from eight patients with malignant melanomas using 24 polymorphic DNA probes and detected loss of heterozygosity in three patients at five loci on four different chromosomes. These results indicate that the frequency of loss of heterozygosity at chromosomal loci is relatively high in malignant melanomas, but that the regions lost are not restricted to specific chromosomes.

Adult↗

PubMed1989

Current status of melanoma treatment with interferon, cytokines and other biologic response modifiers in Japan.

This paper introduces the current status of melanoma treatment with various biologic response modifiers (BRMs) in Japan, with an emphasis on the clinical results of Interferon therapies. The authors also refer briefly to the current situation of interleukin-2 (IL-2) and tumor necrosis factor (TNF) in Japan. Many BRMs have been used in treatment of melanoma, e.g., IFN, IL-2, TNFs, BCG, MY-1 (DNA extracted from BCG), WPG (CWs of Bifidobacterium infantis, ATCC 15697), OK-432 (Picibanil, Streptococcus pyogenes preparation), bestatin, and forphenicinol. Some of these have completed clinical trials, while others are still undergoing clinical testing. Among IFN-alpha, beta, and gamma, intralesional administration of natural IFN-beta was found to be more effective than IFN-alpha for metastatic skin melanoma, the survival time of patients being prolonged by the administration of IFN-beta. IFN-gamma appeared to have lower efficacy than IFN-alpha and beta. The frequency of BRM application to melanoma treatment will increase. The authors foresee that combinations with radio- and/or other chemotherapy will be more common than the single use of a BRM, especially in the case of IFN.

Biological Factors↗

PubMed1989

Cisplatin combination chemotherapy in squamous cell carcinoma and adenoid cystic carcinoma of the skin.

Seven patients with skin cancers, six with squamous cell carcinoma (SCC) and one with adenoid cystic carcinoma, were treated with cisplatin in combination with vindesine or adriamycin. Partial response was observed in three patients with squamous cell carcinomas: two cases with metastatic lung lesions and one with a primary skin lesion and lymph node metastasis. Two of the responding SCC had been resistant to previous chemotherapy, including peplomycin and mitomycin C. Multiple metastatic lesions of adenoid cystic carcinoma of the skin completely regressed after two courses of the combination chemotherapy with cisplatin and adriamycin. This report showed that cisplatin combination chemotherapy may be useful for the treatment of cutaneous squamous cell carcinoma, which is resistant to peplomycin, and adenoid cystic carcinoma of the skin.

Adult↗