Biomedical subjects

H Morimoto

Publications and source records attributed to H Morimoto.

At least 109 records · Page 6Linked to original sources

PubMed1992

Studies on neurokinin antagonists. 2. Design and structure-activity relationships of novel tripeptide substance P antagonists, N alpha-[N alpha-(N alpha-acetyl-L-threonyl)-N1-formyl-D-tryptophyl]-N- methyl-N-(phenylmethyl)-L-phenylalaninamide and its related compounds.

Continuing studies on the chemical modification of the previously reported novel tripeptide SP antagonist, N alpha-[N alpha-[N alpha- (tert-butyloxycarbonyl)glutaminyl]-N1-formyl-D-tryptophyl]phenylalanine benzyl ester [Boc-Gln-D-Trp-(CHO)-Phe-OBzl (1)], are described herein. We initially investigated the stability of 1 in guinea pig plasma and liver homogenate to elucidate the most labile part in the structure. It was consequently revealed that the benzyl ester part was easily hydrolyzed to produce the inactive acid analog. Thus we searched for a benzyl ester surrogate that would be more resistant to hydrolytic enzymes. This approach found an isosteric amide structure, N-methyl-N-(phenylmethyl)amide, suitable in terms of potency and stability. Subsequent modification of the amino terminal into N alpha-acetyl-L-threonine led to the most potent compound, N alpha-[N alpha-(N alpha-acetyl-L-threonyl)-N1-formyl-D-tryptophyl]-N- methyl-N-(phenylmethyl)-L-phenylalaninamide [Ac-Thr-D-Trp(CHO)-Phe-NMeBzl (5a, FR113680)]. This compound 5a potently blocked 3H-SP binding to guinea pig lung membranes with IC50 of (5.8 +/- 0.78) x 10(-9) M. In vitro, 5a inhibited SP-induced contraction of isolated guinea pig trachea strips with IC50 of 2.3 x 10(-6) M and caused no contraction when used alone in this preparation up to 3.2 x 10(-5) M. In addition 5a exhibited no effect on the contraction induced by histamine or acetylcholine. Intriguingly, it was demonstrated in vivo that 5a suppressed the SP-induced bronchoconstriction and airway edema in guinea pigs with ED50 of 0.42 mg/kg and 0.66 mg/kg, respectively, when administered intravenously.

Acetylcholine↗

PubMed1992

Level of ETB receptor mRNA is down-regulated by endothelins through decreasing the intracellular stability of mRNA molecules.

Using ROS17/2 rat osteosarcoma cells as a model system, we examined the possibility that endothelin (ET)-induced down-regulation of ETB receptor was accompanied by a decrease in levels of ETB receptor mRNA. Northern blot analysis showed that low doses of ET-1 and ET-3 caused a transient decrease in ETB receptor mRNA in the cells. The maximum decrease in the levels of ETB receptor mRNA (80%) occurred after 2-4 h of exposure of the cells to ETs and was followed by a gradual recovery to control levels by 24 h. The effects were dose-dependent (EC50-1 nM), and ET-1 and ET-3 were almost equipotent in eliciting the response. The addition of either ionomycin, a Ca2+ ionophore, or phorbol dibutyrate, a protein kinase C activator, mimicked the effect of ETs. These results suggested that ETs-induced down-regulation of ETB receptor mRNA was mediated by the activation of ETB receptor and that it may have involved ETB receptor coupled second messenger pathways. We also showed that ETB receptor mRNA had a long intracellular life span which suggested that ETs-induced down-regulation of ETB receptor mRNA may have been due to a decrease in the stability of mRNA, rather than inactivation of the transcription of mRNA.

Animals↗

PubMed1992

Cloning and expression of rat preproendothelin-3 cDNA.

We report here the cloning and expression of a rat full-length cDNA encoding preproendothelin-3 (preproET-3). The predicted rat preproET-3 consisted of 167 amino acid residues. As in other ET-family peptides, the mature rat ET-3 was predicted to be produced through unusual processing from a 41-residue intermediate, the big ET-3 in rat. Transient transfection of COS-7 cells with the cloned preproET-3 cDNA resulted in the production of mature ET-3 and this production was inhibited by phosphoramidon, a metaloprotease inhibitor. This suggested that a phosphoramidon sensitive mechanism was involved in the production of ET-3 in the transfected COS-7 cells. Northern blot analysis showed that an approximately 3.0-kb rat preproET-3 mRNA was expressed in rat tissues, including the eye ball, submandibular gland, brain, kidney, jejunum, stomach and spleen. A 2.0-kb and a 3.3-kb mRNA were also detected in the eye ball and small intestine, respectively. The distinct distribution of rat preproET-3 mRNA from that of preproET-1 mRNA suggested that ET-1 and ET-3 played different roles.

Amino Acid Sequence↗

PubMed1992

Effects of the tripeptide substance P antagonist, FR113680, on airway constriction and airway edema induced by neurokinins in guinea-pigs.

FR113680 is a newly developed tripeptide substance P (SP) receptor antagonist. The effects of FR113680 on airway constriction and airway edema induced by neurokinins were investigated in guinea-pigs. In in vitro experiments, FR113680 inhibited the contraction of isolated guinea-pig trachea induced by SP and neurokinin A (NKA) in a dose-dependent manner with IC50 values of 2.3 x 10(-6) and 1.5 x 10(-5) M, respectively. The tracheal contraction induced by histamine and acetylcholine was not affected by FR113680. FR113680 (5 x 10(-5) M) also significantly inhibited the atropine-resistant contraction of isolated guinea-pig bronchi induced by electrical field stimulation. In in vivo experiments, FR113680 given i.v. inhibited SP-induced airway constriction in guinea-pigs at doses of 1 and 10 mg kg-1. However, FR113680 only inhibited NKA- and capsaicin-induced airway constriction by 40-50% even at a dose of 10 mg kg-1. FR113680 also inhibited SP-induced airway edema in guinea-pigs with the same potency as it inhibited SP-induced airway constriction. Histamine-induced airway constriction and airway edema were not affected at a dose of 10 mg kg-1. These results suggest that FR113680 preferentially inhibits responses induced by NK1 receptor activation (SP-induced airway constriction and airway edema), but is less effective on a NK2 receptor-induced response (airway constriction by NKA and neurogenic stimulation).

Amino Acid Sequence↗

PubMed1992

Studies on neurokinin antagonists. 1. The design of novel tripeptides possessing the glutaminyl-D-tryptophylphenylalanine sequence as substance P antagonists.

To discover a novel and low molecular weight substance P (SP) antagonist we postulated that the essential binding domain of peptide ligands was only a small portion in the whole structure. On the basis of this assumption, we selected the known octapeptide SP antagonist D-Pro-Gln-Gln-D-Trp-Phe-D-Trp-D-Trp-Phe-NH2 (1) as a lead and synthesized its fragment tripeptides which were evaluated for their activity to block 3H-SP binding on guinea pig lung membranes. The protected tripeptide N alpha-[N alpha-[N alpha-(tert-butyloxycarbonyl)-L-glutaminyl]-N1-formyl-D-tryptophyl]- L-phenylalanine benzyl ester [Boc-Gln-D-Trp(CHO)-Phe-OBzl (4a)], corresponding to the Gln-D-Trp-Phe part of 1, exhibited 7-fold potent inhibitory activity in comparison with 1. Studies on structure-activity relationships revealed that the D-tryptophan, L-phenylalanine, and benzyl ester were quite important to maintain the high binding affinity. It was also indicated that 4a antagonized the SP-induced contraction of isolated guinea pig trachea strips (IC50 = 4.7 x 10(-6) M).

Amino Acid Sequence↗

PubMed1992

Site-directed mutagenesis in hemoglobin: functional and structural role of inter- and intrasubunit hydrogen bonds as studied with 37 beta and 145 beta mutations.

In order to clarify the functional and structural role of intra- and intersubunit hydrogen bonds in human hemoglobin (Hb A), we prepared two artificial beta chain mutant hemoglobins by site-directed mutagenesis. The mutant Hb Phe-37 beta, in which Trp-37 beta is replaced by Phe to remove the intersubunit hydrogen bond between Asp-94 alpha and Trp-37 beta at the alpha 1-beta 2 interface in deoxy Hb A, showed a markedly increased oxygen affinity and almost completely diminished Bohr effect and cooperativity. However, 1H-NMR data indicated that the structure of deoxy Hb Phe-37 beta is rather similar to that of deoxy Hb A. The enhanced tetramer-to-dimer dissociation previously observed in Hb Hirose (Trp-37 beta----Ser) together with our observation of the effects of organic phosphate on the structure and function of Hb Phe-37 beta suggested that a large part of the abnormal properties of Hb Phe-37 beta observed for dilute solutions appears to result from partial dissociation into alpha beta dimers rather than direct destabilization of the T-quaternary structure in the deoxygenated state. Thus, the primary and direct role of the hydrogen bond between Asp-94 alpha and Trp-37 beta is to stabilize the tetrameric assembly, and thereby this hydrogen bond indirectly contributes to stabilization of the T-quaternary structure. The other mutant Hb Phe-145 beta has a Phe residue at the 145 beta site and lacks the intrasubunit hydrogen bond formed between Tyr-145 beta and the carbonyl group of Val-98 beta in deoxy Hb A.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1992

Use of fibrinogen to enhance the antitumor effect of OK-432. A new approach to immunotherapy for colorectal carcinoma.

OK-432 (5 KE), an immunomodulatory agent prepared from an attenuated strain of Streptococcus pyogenes, was dissolved in 1 ml of aprotinin (1000 KIE) and mixed with 80 mg of fibrinogen containing Factor XIII. A single intratumoral injection of the mixture was performed preoperatively under endoscopy in 20 patients with colorectal carcinoma. Postoperative histopathologic examinations revealed the formation of fibrin fibers at the site of injection and marked infiltration of inflammatory cells into the tumor stroma on the day after injection; the formation of granulomas containing many giant cells after 4 to 7 days; and extensive regression of tumor tissue after 14 days. This study suggests that the high concentration of exogenous fibrinogen gelatinized enough to trap OK-432 in tumor stroma and that OK-432 induced granulomatous hypersensitivity to degenerate tumor stroma, thereby causing regression of the tumors.

Adenocarcinoma↗

PubMed1992

Comparative effects of a recombinant and a mutein type of granulocyte colony stimulating factor on the growth of Meth-A fibrosarcoma with 5-fluorouracil chemotherapy.

The present study was designed to evaluate the effects of a recombinant human G-CSF (rhG-CSF) and a mutein G-CSF (KW-2228) on leucopenia and tumor growth in mice treated with 5-fluorouracil (5-FU). In normal mice, the number of leucocytes (white blood cell, WBC) reached the peak 12 hours after a single injection of either type of G-CSF and decreased to the normal level after 24 hours. Daily administration induced a continuous increase in the WBC count, however, administrations at intervals did not. Meth-A fibrosarcoma was subcutaneously inoculated into the backs of syngeneic BALB/c mice. The mice were treated with 5-FU alone or with G-CSFs. Chemotherapy with 5-FU alone resulted in leucopenia and an insignificant inhibition of tumor growth. The conjunctive administration of G-CSFs with 5-FU resulted in a significantly augmented inhibition of tumour growth, and leukopenia was not seen. This augmenting effect was more prominent with KW-2228. These results suggest that in 5-FU chemotherapy G-CSFs may be beneficial in restoring the number of leucocytes from leucopenic state and in augmenting the tumor inhibitory effect. Furthermore, KW-2228 may be more beneficial than the natural type rhG-CSF.

Amino Acid Sequence↗

PubMed1992

Immunomodulation by orally administered protein-bound polysaccharide PSK in patients with gastrointestinal cancer.

The present study was designed to assess the effects of the protein-bound polysaccharide PSK on the immunological status of patients with gastrointestinal cancer. Twenty-nine gastric and 18 colorectal cancer patients were randomly assigned to either the control or PSK group. Patients in the PSK group were given 3.0 g of PSK orally before surgery, either daily or every other day. Patients in the control group received no PSK. The data of peripheral blood lymphocytes (PBL) were compared before and after administration of PSK, and those of the regional node lymphocytes (RNL) were compared between the control and the PSK group. The results indicate that the effects of PSK were significantly influenced by the duration of administration, but not by the frequency of administration. In the patients belonging to the short term PSK group (administration less than 14 days), the response of the PBL to PSK and Con A become significantly stronger compared to before the administration of PSK, whereas the cytotoxicity against K562 and KATO-3, and the proportion of CD16+ cells increased significantly in those patients belonging to the long term PSK group (greater than or equal to 14 days). In addition, the proportion of CD9 + 11b + suppressor T cells decreased in the RNL of the short term PSK group, whereas the proportion of CD4 + Leu8 - helper T cells in the RNL increased in the long term PSK group. These results suggest that the oral administration of PSK leads to the suppression of suppressor cells in the RNL.(ABSTRACT TRUNCATED AT 250 WORDS)

Adjuvants, Immunologic↗

PubMed1992

Establishment of cytotoxic CD4+ T cell clones from cancer patients treated by local immunotherapy.

We have previously reported that the antitumor effect of OK-432, a Streptococcal preparation, is markedly augmented when injected intratumorally together with fibrinogen (Cancer, 69: 636-642, 1992). In order to elucidate the mechanism of the antitumor effects, we established T cell clones from regional lymph nodes of colorectal cancer patients who received this local immunotherapy. By culture of lymph node lymphocytes, in the presence of IL-2 and OK-432, 4 clones of T cells were established from 4 patients treated by local immunotherapy. These clones had a helper T cell phenotype (CD3+, CD4+, CD8-, CD56-, WT31+) and were successfully maintained for several months. The cells strongly expressed CD25 when stimulated with OK-432 and exhibited a high level of cytotoxic activity in part explained by the increased expression of ICAM-1 and LFA-1, and the release of TNF beta. These results suggest that the CD4+ T cells play a role in the antitumor mechanism of local immunotherapy.

CD4-Positive T-Lymphocytes↗

PubMed1992

In vitro growth ability and chemosensitivity of gastric and colorectal cancer cells assessed with the human tumour clonogenic assay and the thymidine incorporation assay.

A human tumour cloning assay (HTCA) has been performed on 191 samples of gastric and 152 samples of colorectal cancers, and a thymidine incorporation assay (TIA) on 178 samples of gastric and 109 samples of colorectal cancers. The rate of evaluable assays was significantly higher in the TIA than in the HTCA (P less than 0.01). In terms of in vitro growth potential in the two assays, gastric cancer cells were less active than the colorectal cancer cells (P less than 0.05). In frequency of in vitro sensitivity to drugs, gastric cancer was more chemosensitive than colorectal cancer in both assays. The in vitro/in vivo correlations of high resistance-predictive ratios and low sensitivity-predictive ratios were similar in both assays. The results indicate that the TIA is more applicable than the HTCA to screening of active agents against fresh gastrointestinal cancers.

Colorectal Neoplasms↗

PubMed1992

A human monoclonal antibody derived from axillary lymph nodes of a breast cancer patient reactive to a sulfated glycolipid.

A human monoclonal antibody, BMMK-33G, was established by a fusion of human B-lymphoblastoid cells, HO-323, with lymphocytes of axillary lymph nodes obtained from a breast cancer patient. High-performance thin-layer chromatography (HPTLC)-immunostaining and enzyme-linked immunosorbent assay (ELISA) revealed that BMMK-33G was interestingly directed to enough sulfatide (Galactosylceramid-I2-sulfate), which is one of the sulfate ester containing glycolipids. By immunohistochemical staining, BMMK-33G intensely reacted to breast cancer, pancreatic cancer and gastric cancer. It also reacted to many normal human tissues including mammary glands, but these stainings were weaker than those for cancer. This report describes BMMK-33G, a human monoclonal antibody against sulfatide which may be very useful for studying not only tumor immunology but also autoimmune diseases.

Antibodies, Monoclonal↗

PubMed1992

Immunohistochemical study of p53 expression in microwave-fixed, paraffin-embedded sections of colorectal carcinoma and adenoma.

Expression of p53, a tumor-suppressor gene product, was studied immunohistochemically in microwave-fixed, paraffin-embedded sections of 84 colorectal carcinomas and 44 adenomas. Using a monoclonal antibody (PAb1801), nuclear p53 was detected successfully in 51 of 84 (60.7%) cases of carcinomas, and no stain for p53 was demonstrated in the adjacent normal mucosa. The results in the microwave-fixed, paraffin-embedded sections correlated with those in the frozen sections. The incidence of p53 expression in colorectal carcinomas was high in the cases with distant metastasis, but it was not affected by clinicopathologic features such as tumor size or depth of invasion. In colorectal adenomas, only 4 of 44 (9%) adenomas expressed p53. This expression of p53, however, was restricted to only a few glands within tubular adenomas with mild dysplasia. Thus, p53 protein was expressed preferably by malignant tumors of the colorectum. The present study demonstrated the usefulness of microwave fixation to preserve p53. The immunohistochemical detection of p53 in microwave-fixed, paraffin-embedded sections of colorectal carcinoma and adenoma can provide valuable information about the mechanism of carcinogenesis in colorectal epithelium.

Adenoma↗

PubMed1992

Use of a local immunotherapy as an adjunctive tool for the generation of human monoclonal antibodies from regional lymph nodes of colonic cancer patients.

Human hybridomas were generated through the fusion of the human B-lymphoblastoid cell line HO-323 with the regional lymph node lymphocytes of colonic cancer patients who had received a local immunotherapy. A total of 353 hybridomas were obtained from 4 patients and 116 of these were found to secrete greater than or equal to 100 ng/ml human immunoglobulin. The efficiency was remarkably high as compared with that from patients without the local immunotherapy. Further immunohistological examination showed that 5 hybridomas secreted IgM which selectively reacted with colonic cancers. The results indicate that local immunotherapy could be an adjunctive tool for the generation of highly potent human hybridomas through augmenting the host's immunity.

Antibodies, Monoclonal↗

PubMed1992

FR 113680: a novel tripeptide substance P antagonist with NK1 receptor selectivity.

1. We have discovered a novel tripeptide substance P (SP) antagonist, FR 113680 [N alpha-[N alpha-(N alpha-acetyl-L-threonyl)-N'-formyl-D- tryptophyl]-N-methyl-N-phenylmethyl-L-phenylalaninamide]. In binding experiments, FR 113680 inhibited [3H]-SP binding to guinea-pig lung membranes (NK1) in a competitive manner but had not effect on [3H]-SP binding to rat cerebral cortical membranes (NK1), [3H]-neurokinin A ([3H]-NKA) binding to rat duodenum smooth muscle membranes (NK2) and [3H]-eledoisin (Ele) binding to rat cerebral cortical membranes (NK3). 2. In bioassay experiments, FR 113680 dose-dependently inhibited SP-induced guinea-pig ileum contraction (NK1), but did not inhibit either NKA-induced rat vas deferens contraction (NK2) or neurokinin B (NKB)-induced contraction of rat portal vein (NK3). According to Schild plot analysis, the inhibitory effect of FR 113680 on SP-induced guinea-pig ileum contraction is competitive and the pA2 value is 7.53. 3. The inactivity of FR 113680 on NK1 receptors in rat compared to guinea-pig may represent species-specific forms of the NK1 receptor. 4. These findings suggest that FR 113680 interacts selectively with the NK1 neurokinin receptor.

Animals↗

PubMed1992

Pharmacological profile of a high affinity dipeptide NK1 receptor antagonist, FK888.

1. In our search for compounds that inhibit the binding of [3H]-substance P (SP) to guinea-pig lung membranes, the dipeptide SP antagonist, FK888, was developed by chemical modification of the parent compound, (D-Pro4, D-Trp7,9,10, Phe11)SP4-11. 2. In a [3H]-SP binding assay using guinea-pig lung membranes and rat brain cortical synaptic membranes, FK888 displaced [3H]-SP binding with a Ki value of 0.69 +/- 0.13 nM and 0.45 +/- 0.17 microM, respectively, in a competitive manner. 3. FK888 inhibited the contraction of guinea-pig isolated ileum induced by SP in the presence of atropine and indomethacin (a NK1 receptor bioassay) with a pA2 value of 9.29 (8.60-9.98). 4. FK888 inhibited contractions of rat vas deferens by NKA (a NK2 receptor bioassay) and of rat portal vein by NKB (a NK3 receptor bioassay) at concentrations at least 10,000 times greater than that required to inhibit contractions of guinea-pig ileum. 5. FK888 also inhibited SP-induced airway oedema in guinea-pig after both intravenous and oral administration. 6. These data demonstrate that FK888 is a potent and selective NK1 antagonist which is active both in vitro and in vivo.

Administration, Oral↗

PubMed1992

Measurement of biventricular septal-to-free wall diameters using sonomicrocrystals.

An ultrasonic system for the measurement of biventricular septal-to-free wall internal diameters in the dog was described. The ventricular endocardial ultrasonic microcrystals were implanted via the left and right atrial appendages and arranged in a straight line, with minimal invasion of the myocardium and pericardium. Reliable signals were obtained during right ventricular pressure overload and septal myocardial ischemia.

Animals↗

PubMed1992

Expression of the ret proto-oncogene in human medullary thyroid carcinomas and pheochromocytomas of MEN 2A.

We studied the expression of the ret proto-oncogene (proto-ret) in human medullary thyroid carcinomas (MTCs) and pheochromocytomas of multiple endocrine neoplasia type 2A (MEN 2A) by Northern blot analysis. Expression of the normal-sized transcripts was detected in all 12 MTCs and in 6 of 8 pheochromocytomas. In situ localization of proto-ret mRNA revealed that the signal was confined to the cytoplasm of MTC cells. By Southern blot analysis neither amplification nor gross genetic changes of proto-ret were found in the tumors. Although no transcripts were detected in the normal portion of the thyroid from one MEN 2A patient, faint signals were detected in normal adrenal glands by Northern blot analysis, probably due to minor populations of C-cells and chromaffin cells in specimens from which MTC and pheochromocytoma might later develop. Proto-ret may play an important role in differentiation of a specific cell lineage from neuroectoderm, and it may be involved in development of MEN 2A tumors.

Adrenal Gland Neoplasms↗