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Biomedical subjects

M Ihara

Publications and source records attributed to M Ihara.

At least 91 records · Page 5Linked to original sources
PubMed1994

Random mutagenesis of pullulanase from Klebsiella aerogenes for studies of the structure and function of the enzyme.

To study the structure and function of pullulanase from Klebsiella aerogenes, a method involving random mutagenesis of the entire gene for pullulanase was used. Out of 50,000 clones screened at high temperature, seven genes for mutant proteins were identified by DNA sequencing. The amino acid substitutions in the seven mutant proteins were clustered on the NH2-terminal side of the four conserved regions found in alpha-amylases. These mutant pullulanases were classified into two types: those whose catalytic activity was altered and those whose thermal stability was increased. The results presented here and in previous reports suggest that pullulanase from K. aerogenes has similar active sites to those of alpha-amylases with the four conserved regions, as well as another substrate-binding site closer to the NH2-terminus. The plate assay method used for isolation of thermostable variants may be applicable to the generation of useful variants of other enzymes.

Bacterial Proteins↗
PubMed1994

Synergistic inhibition by BQ-123 and BQ-788 of endothelin-1-induced contractions of the rabbit pulmonary artery.

In the rabbit isolated pulmonary artery, neither the ETA receptor antagonist, BQ-123 (10 microM), nor the ETB receptor antagonist, BQ-788 (10 microM), inhibited the contractions induced by 1 nM endothelin-1 (ET-1). However, the combination of BQ-123 and BQ-788 completely inhibited the ET-1-induced contraction. In contrast, the ETB-selective agonist, sarafotoxin S6c (1 nM)-induced contraction was completely inhibited by BQ-788 but not by BQ-123. In receptor binding assays, [125I]-ET-1 specific binding to pulmonary arterial membranes was inhibited by BQ-123 (1 microM) by approximately 20% and additive treatment with BQ-788 (1 microM) completely inhibited the BQ-123-resistant component of [125I]-ET-1 specific binding. The present study demonstrates synergistic inhibition by BQ-123 and BQ-788 of ET-1-induced contraction of the rabbit pulmonary artery and the coexistence of ETA and ETB receptors, suggesting that the activation of either only ETA or only ETB receptors may be sufficient to cause complete vasoconstriction. Therefore, blockade of both receptor subtypes would be necessary for the inhibition of some ETA/ETB composite types of responses.

Amino Acid Sequence↗
PubMed1994

Morphology of spontaneous Harderian gland tumors in aged B6C3F1 mice.

Spontaneous Harderian gland tumors in B6C3F1 mice were found in 18 (3.1%) (one male had bilateral tumors) of 589 males and 18 (3.0%) of 609 females. The tumor-bearing mice ranged in age from 73 to 109 weeks. These tumors comprised 32 adenomas (86.5%), four adenocarcinomas (10.8%) and one pleomorphic tumor (2.7%); the adenomas were classified, based on the growth patterns, into 17 papillary, nine cystic papillary, five acinar and one cystic types. Two adenocarcinomas grew papillary and invaded surrounding tissues, and the other two grew acinously. The pleomorphic tumor consisted of pleomorphic cells some of which had a keratin-positive epithelial feature and acini lined by atypical epithelial cells.

Adenocarcinoma↗
PubMed1994

Endothelin ETB receptors couple to two distinct signaling pathways in porcine kidney epithelial LLC-PK1 cells.

We characterized the endothelin (ET) receptor subtypes responsible for signal transduction in cultured porcine kidney epithelial LLC-PK1 cells. Both ET-1 (IC50, 43 pM) and ET-3 (IC50, 46 pM) inhibited the binding of [125I]ET-1 to LLC-PK1 cells to a similar extent. The binding affinity of LLC-PK1 cells was about 10,000 times higher for the ETB antagonist BQ-788 [N-cis-2,6-dimethyl-piperidinocarbonyl-L-tau-metylleucyl-D-+ ++Nin- methoxycarbonyltryptophanyl-D-norleucine] (IC50, 1.3 nM) than for the ETA antagonist BQ-123 [cyclo-(D-Trp-D-Asp-Pro-D-Val-Leu)] (IC50, 14 microM). ET-1 enhanced cyclic GMP (cGMP) production, but reduced vasopressin- and forskolin-stimulated cyclic AMP (cAMP) production. Both effects of ET-1 were antagonized by BQ-788, but not by BQ-123. The cAMP decrease, but not the cGMP increase, in response to ET-1 was inhibited by pertussis toxin, suggesting that the former response is mediated by pertussis toxin-sensitive Gi, whereas the latter is mediated by a pertussis toxin-insensitive G-protein. Therefore, the ETB receptors in LLC-PK1 cells couple to the two types of signal transduction cascades to reduce cAMP production and stimulate cGMP production via distinct G-proteins. ET-1 and probably also ET-3 may play a role in the regulation of renal epithelial transport by decreasing cAMP and increasing cGMP.

Amino Acid Sequence↗
PubMed1993

A novel ligand, [125I]BQ-3020, reveals the localization of endothelin ETB receptors.

The precise localization of an endothelin (ET) receptor subtype, the ETB receptor, in porcine lung was elucidated by in vitro microautoradiography using a novel ETB-selective radioligand, [125I]BQ-3020 ([125I-Tyr]-N-acetyl-Leu-Met-Asp-Lys-Glu-Ala-Val-Tyr-Phe-Ala-His-Leu-Asp -Ile-Ile-Trp). Of the labeled native ET isopeptides, [125I]ET-3 is selective for ETB receptors. However, [125I]ET-3 was not suitable for autoradiography due to its high degree of non-specific binding. On the other hand, [125I]BQ-3020 showed extremely low non-specific binding on autoradiography. The distribution of [125I]BQ-3020 binding in porcine lung was clearly different from that of [125I]ET-1, which showed more widespread binding than [125I]BQ-3020 due to a high affinity to both ETA and ETB receptors. [125I]BQ-3020 was found to bind to parenchyma, parasympathetic ganglia, pulmonary and submucosal plexuses, but bound only slightly to circular smooth muscle layers and the epithelium of airway tracts. Although [125I]ET-1 bound to the smooth muscle layer of all blood vessels, the binding of [125I]BQ-3020 differed among blood vessels. [125I]BQ-3020 binding in blood vessels paralleled acetylcholinesterase activity, suggesting that ETB receptors in blood vessels are located on parasympathetic nerves. Thus, the radioligand [125I]BQ-3020 is very useful for studying the precise localization of ETB receptors.

Acetylcholinesterase↗
PubMed1993

Role of endothelin ETB receptors in bronchoconstrictor and vasoconstrictor responses in guinea-pigs.

In anesthetized and ventilated guinea-pigs, intravenous injections of endothelin (ET)-1 (0.5 nmol/kg), ET-3 (0.5 nmol/kg), and [Ala1,3,11,15]ET-1 (20 nmol/kg), an ETB-selective receptor agonist, induced bronchoconstrictor and transient vasoconstrictor responses. Only the ET-1-induced transient vasoconstriction was followed by a secondary sustained pressor response. The ETA-selective receptor antagonist, BQ-123 (1 mg/kg i.v.), attenuated only the sustained pressor response. These results indicate that the bronchoconstrictor and transient vasoconstrictor responses to endothelins in guinea-pigs are mediated by ETB receptors, whilst the sustained pressor response is mediated by ETA receptors. The thromboxane A2 receptor antagonist, L-670,596 (0.5 mg/kg, i.v.) and a high dose of BQ-123 (30 mg/kg i.v.) abolished the bronchoconstriction only without affecting the transient pressor response to endothelin isopeptides. These results suggest that the ETB-mediated bronchoconstriction depends on thromboxane A2 formation. The different sensitivity of these ETB-mediated transient responses to BQ-123 suggests the possible existence of distinct ETB receptor subtypes.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1993

A new photoreactivating enzyme that specifically repairs ultraviolet light-induced (6-4)photoproducts.

Cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts ((6-4)photoproducts) are the two major classes of cytotoxic, mutagenic and carcinogenic DNA photoproducts produced by ultraviolet light irradiation of cells. The phenomenon of photoreactivation, the reduction of the lethal and mutagenic effects of ultraviolet radiation by simultaneous or subsequent irradiation with near ultraviolet or visible light, has been identified in several organisms and in some cases the enzymes that catalyse this process have been characterized in sufficient detail. CPDs are the only known substrate for the photoreactivating enzymes so far analysed and enzymatic photoreactivation of (6-4)photoproducts has not yet been reported. We report here that an enzyme that catalyses the light-dependent repair of (6-4)photoproduct exists in Drosophila melanogaster. This is, to our knowledge, the first report of such photoreactivating activity specific for (6-4)photoproducts in any organism.

Animals↗
PubMed1993

Two receptor subtypes are involved in the contractile component of the guinea pig ileum responses to endothelins.

Endothelin-1 (ET-1) and endothelin-3 (ET-3) induced a biphasic response (relaxation and contraction) in the guinea pig ileum. Short-term activation of protein kinase C (PKC) with phorbol 12,13-dibutyrate (PDBu) inhibited the contractile but not the relaxing component of the responses, as did H-7. Long-term pretreatment with PDBu reversed the short-term inhibition but did not affect the tachyphylaxis induced by ET-1. These results suggest that PKC contributes to ET-1 contraction but not to tachyphylaxis. The ETA antagonist BQ-123, at 34 nM, competitively inhibited ET-1 contraction, but at 340 nM the inhibition was noncompetitive. Dose-response curves for ET-1 relaxation were shifted to the left. For ET-3, BQ-123 (340 nM) only decreased the maximal contractile response of the dose-response curve without affecting the relaxation. We suggest that in this tissue there is one receptor for ET-1-induced contraction, which is competitively antagonized by BQ-123, and another one for ET-3-induced contraction, which is noncompetitively antagonized by BQ-123.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1992

Suppressive effects of the endothelin receptor (ETA) antagonist BQ-123 on ET-1-induced reduction of lipoprotein lipase activity in 3T3-L1 adipocytes.

Endothelin (ET)-1 reduced heparin-releasable lipoprotein lipase (LPL) activity in 3T3-L1 adipocytes in a concentration-dependent manner. However, a selective ETB receptor agonist, [Ala1,3,11,15]ET-1, did not act like ET-1. The ET-1-induced decrease in LPL activity was suppressed by a selective ETA receptor antagonist, BQ-123: the concentration-response curve for the ET-1 reduction of LPL activity was shifted to the right in the presence of BQ-123 in a concentration-dependent manner. This antagonistic effect of BQ-123 clarifies that the ETA receptor is responsible for the ET-1-induced reduction of LPL activity in 3T3-L1 adipocytes, which suggests that there is therapeutic potential for ETA antagonists in LPL-related lipoprotein disorders.

3T3 Cells↗
PubMed1992

A novel ETA antagonist (BQ-123) inhibits endothelin-1-induced phosphoinositide breakdown and DNA synthesis in rat vascular smooth muscle cells.

The effects of a novel cyclic pentapeptide (BQ-123), an endothelin (ET) antagonist selective for the ETA receptor subtype, on phosphoinositide breakdown and DNA synthesis stimulated by ET-1 were studied in cultured rat vascular smooth muscle cells (VSMC). BQ-123 competitively inhibited the binding of [125I]ET-1 to VSMC with the apparent Ki of 4 x 10(-9) M. BQ-123 dose-dependently inhibited formation of inositol-1,4,5-trisphosphate and [3H]thymidine uptake stimulated by ET-1. These data suggest that the ET-1-induced DNA synthesis in VSMC is mainly mediated by ETA receptor subtype.

Animals↗
PubMed1992

Different distribution of endothelin receptor subtypes in pulmonary tissues revealed by the novel selective ligands BQ-123 and [Ala1,3,11,15]ET-1.

We have demonstrated the different distribution of two distinct endothelin (ET) receptor subtypes in porcine pulmonary tissues using a radioligand binding assay. The clear differentiation of the subtypes was made possible by the discovery of two compounds, BQ-123 and [Ala1,3,11,15]ET-1 (4AlaET-1), that are highly selective for ETA and ETB receptors, respectively. In the bronchus and lung parenchyma, BQ-123 inhibited 65% and 30% of [125I]ET-1 binding on the sensitive sites, while 4AlaET-1 displaced 25% and 60%, respectively. The combination of the two compounds completely inhibited ET-1 binding in both tissues. An autoradiographic study of [125I]ET-1 binding using BQ-123 and 4AlaET-1 also supported the different localization of two ET receptor subtypes in pulmonary tissues. In particular, the blood vessels and bronchi are rich in ETA, but the lung parenchyma is rich in ETB.

Amino Acid Sequence↗
PubMed1992

Immunochemical detection of unrepaired cyclobutane-type pyrimidine dimers of DNAs extracted from human skin tumours.

Unrepaired cyclobutane-type pyrimidine dimers of DNA extracted from human skin tumours were examined by an immunoblotting method using polyclonal antibodies raised against UV-irradiated calf thymus DNA. A total of 40 DNA samples extracted from seven SCC lesions, two AK lesions, two lymphomas, one basal cell epithelioma, one eccrine poroma, one neurofibroma of Recklinghausen's disease, on verruca vulgaris, four femoral normal skins and white blood cells of 21 humans were studied by immunoblotting using this antibody. Two of the 40 DNAs examined, one from facial actinic keratosis (AK) and one from a squamous cell carcinoma (SCC) which developed form facial AK formed immunoprecipitates. It was found, using photoreactivation enzyme plus visible light, that both immunoprecipitates were cyclobutane-type pyrimidine dimers. In addition, immunofluorescent studies on AK tissue were positive in an immunoblotting assay and revealed that the unremoved photodamage in DNA remained in the nucleus of AK cells. These findings indicate that these tumour cells may be deficient in the enzyme function for repairing photoproduct damage. The unrepaired cyclobutane-type pyrimidine dimer in AK cells might reflect the genetic process in multistage carcinogenesis as well as in xeroderma pigmentosum.

Adult↗
PubMed1992

Biological profiles of highly potent novel endothelin antagonists selective for the ETA receptor.

We describe novel potent endothelin (ET) antagonists that are highly potent and selective for the ETA receptor (selective to ET-1). Of the synthetic analogs based on ETA antagonist BE-18257A isolated from Streptomyces misakiensis (IC50 value for ETA receptor on porcine aortic smooth muscle cells (VSMCs); 1.4 microM), the compounds BQ-123 and BQ-153 greatly improved the binding affinity of [125I]ET-1 for ETA receptors on VSMCs (IC50; 7.3 and 8.6 nM, respectively), whereas they barely inhibited [125I]ET-1 binding to ETB receptors (nonselective with respect to isopeptides of ET family) in the cerebellar membranes (IC50; 18 and 54 microM, respectively). Associated with the increased affinity for ETA receptors, these peptides antagonized ET-1-induced constriction of isolated porcine coronary artery. However, there was a small amount of ET-1-induced vasoconstriction resistant to these antagonists, which paralleled the incomplete inhibition of [125I]ET-1 binding in the membrane of the aortic smooth muscle layer. These data suggest that the artery has both ETA and ETB receptors responsible for ET-1-induced vasoconstriction. The antagonists shifted the concentration-response curve to the right for ET-1 in the coronary artery, and increased the apparent dissociation constant in the Scatchard analysis of [125I]ET-1 binding on the VSMCs without affecting the binding capacity, indicative of the competitive antagonism for ETA receptor. In conscious rats, pretreatment with the antagonists markedly antagonized ET-1-induced sustained pressor responses in dose-dependent fashion without affecting ET-1-induced transient depressor action, suggesting that the pressor action is mediated by ETA receptors, while the depressor action is mediated by ETB receptors. In addition, pretreatment with the potent antagonists prevented ET-1-induced sudden death in mice. Thus, these potent ETA antagonists should provide a powerful tool for exploring the therapeutic uses of ETA antagonists in putative ET-1-related disorders.

Amino Acid Sequence↗
PubMed1992

Analysis of responses to endothelins in isolated porcine blood vessels by using a novel endothelin antagonist, BQ-153.

We examined the effects of a novel ETA-selective endothelin (ET) antagonist, BQ-153, on vascular responses to ET-1 and ET-3 in isolated porcine coronary and pulmonary blood vessels, to clarify the roles of ET receptor subtypes in the regulation of vascular smooth muscle tension. With endothelium-denuded vascular tissues, the concentration-contraction curve (CCC) for ET-1 appeared as a single sigmoidal shape for all types of tissue. The CCC for ET-1 was antagonized by BQ-153 (2 and 10 microM) in all tissues, but part of the contraction was resistant. The CCC for ET-3 usually consisted of two different phases with higher (first phase) and lower (second phase) sensitivities to the peptide. Only the second phase of CCC for ET-3 was completely inhibited by BQ-153 (2 microM) in all tissues, while the first phase was resistant. The BQ-153-resistant contractile phases of ET-1 and ET-3-induced vasoconstriction appeared to have similar sensitivity in all tissues, and the contractile activity varied with each type of tissue. With endothelium-intact materials, the potencies of ET-1 and ET-3 for endothelium-dependent vasorelaxation in pulmonary artery were almost equivalent. BQ-153 (10 microM) did not inhibit ET-induced vasorelaxation. These results indicate that ET-induced vasoconstriction is mediated not only through ETA but also through ETnonA (probably ETB), and that the relative proportions of the ET-receptor subtypes mediating contractions vary in different vascular areas. In addition, results showed that ET-induced endothelium-dependent vasorelaxation is mediated through ETB.

Animals↗
PubMed1992

A novel radioligand [125I]BQ-3020 selective for endothelin (ETB) receptors.

A linear endothelin (ET) analog, N-acetyl-LeuMetAspLysGluAlaValTyrPheAlaHisLeu-AspIleIleTrp (BQ-3020), is highly selective for ETB receptors. BQ-3020 displaces [125I]ET-1 binding to ETB receptors (nonselective to ET isopeptides) in porcine cerebellar membranes (IC50: 0.2nM) at a concentration 4,700 times lower than that to ETA receptors (selective to ET-1) on aortic vascular smooth muscle cells (VSMC) (IC50: 940nM). BQ-3020 as well as ET-1 and ET-3 elicits vasoconstriction in the rabbit pulmonary artery. The ETA antagonist BQ-123 failed to inhibit this BQ-3020-induced vasoconstriction. Furthermore, BQ-3020 elicits endothelium-dependent vasodilation. These data indicate that BQ-3020 has ETB agonistic activity. The radioligand [125I]BQ-3020 binds to cerebellar membranes at single high affinity sites (Kd = 34.4pM), whereas it scarcely binds to VSMC. [125I]BQ-3020 binding to the cerebellum was displaced by BQ-3020, ET-1 and ET-3 in a nonselective manner (IC50: 0.07-0.17nM). However, the binding of [125I]BQ-3020 was insensitive to the ETA antagonist BQ-123 and other bioactive peptides. Both [125I]ET-1 and [125I]BQ-3020 show slow onset and offset binding kinetics to ETB receptors. These data indicate that the radioligand [125I]BQ-3020 selectively labels ETB receptors and that the slow binding kinetics of ET-1 are dependent on the peptide sequence from Leu6 to Trp21, but not on the structure formed by its two disulfide bridges.

Amino Acid Sequence↗
PubMed1992

In vitro biological profile of a highly potent novel endothelin (ET) antagonist BQ-123 selective for the ETA receptor.

The novel endothelin (ET) receptor antagonists BE-18257A and BE-18257B were isolated from the fermentation products of Streptomyces misakiensis. The above-mentioned compounds inhibited [125I]ET-1 binding to ETA receptors (selective for ET-1) on porcine aortic vascular smooth muscle cells (VSMCs) with IC50 values of 1.4 and 0.47 microM, respectively. [125I]ET-1 binding to ETB receptors (nonselective to ET isopeptides) in cerebellar membranes was not inhibited by either of these compounds even at 100 microM. The synthesized analogue BQ-123 induced extremely potent inhibition of [125I]ET-1 binding to ETA receptors (IC50 of 7.3 nM), but it barely inhibited [125I]ET-1 binding to ETB receptors (IC50 of 18 microM) and binding of various other peptides to their receptors. BQ-123 shifted the concentration-response curve for ET-1 toward the right in porcine isolated coronary arteries, indicative of competitive antagonism for the ETA receptor. However, there was a small amount of BQ-123-insensitive vasoconstriction that paralleled the incomplete inhibition of [125I]ET-1 binding in the membrane of the vascular smooth muscle layer. These data suggest that the artery contracts via both ETA and ETB receptors and that BQ-123 selectively inhibits ETA-mediated contraction. Furthermore, BQ-123 revealed large tissue and species differences in the distribution of ETA receptors. Thus, the potent ETA antagonist BQ-123 should be useful in clarifying the (patho)physiological roles of ETA receptors.

Amino Acid Sequence↗