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Biomedical subjects

M Fujino

Publications and source records attributed to M Fujino.

At least 127 records · Page 7Linked to original sources
PubMed1995

[A benign clear cell tumor of the lung that grew gradually over five years].

A 38-year-old man was found to have an abnormal shadow in his chest X-ray film during a general medical examination. A nodule with a homogeneous density and a clear margin was seen in the upper lobe of the right lung. Since this shadow gradually enlarged during a five-year follow-up period, he was admitted to our hospital. Chest contrast and dynamic CT scans and aortography showed characteristics of a benign tumor with somewhat rich vascularity. The doubling time of this tumor was about 430 days. Transbronchial brushing for cytologic study and a transbronchial biopsy showed no malignancy. However, the tumor was surgically resected because of its gradual growth over the five years. The resected tumor was histologically diagnosed as a benign clear cell tumor of the lung. This disease was first described by Leibow and Castleman in 1963, as a benign pulmonary tumor similar to metastatic renal cell carcinoma. This disease is extremely rare and only 61 cases have been reported: twenty cases, including the present case, in Japan and 41 in other countries.

Adenocarcinoma, Clear Cell↗
PubMed1995

Role of endogenous endothelin in the extension of myocardial infarct size studied with the endothelin receptor antagonist, TAK-044.

The effects of TAK-044 on the extension of ischemia/reperfusion-induced myocardial infarction were studied in rats. Acute myocardial infarction was induced by occlusion of the left coronary artery for 1 h followed by reperfusion for 24 h. Infarct size and the area at risk were determined histochemically. Infarct size as a percentage of the area at risk (IS) was significantly reduced in a dose-dependent manner by TAK-044 administered 30 min before reperfusion (0.3-3.0 mg/kg, i.v.). Size-limiting effects similar to those in rats were also obtained in rabbits and dogs. In addition, TAK-044 administered 3 h after reperfusion also reduced the IS significantly. Preconditioning (5 min of LAD occlusion preceding 1 h of coronary occlusion) inhibited the extension of the IS. These effects of preconditioning and TAK-044 were inhibited by glibenclamide (0.1 mg/kg, i.v.; an inhibitor of the ATP-sensitive K channel) but not by DPCPX (1 mg/kg, i.v.; an adenosine A1 receptor antagonist). These results indicate that endogenous endothelin plays an important role in the extension of myocardial infarction and that the cardioprotective effects of TAK-044 can be partially explained by a mechanism similar to that of preconditioning in rats.

Animals↗
PubMed1994

Cloning and expression of a complementary DNA encoding the bovine receptor for pituitary adenylate cyclase-activating polypeptide (PACAP).

A cDNA encoding a pituitary adenylate cyclase-activating polypeptide (PACAP) receptor was cloned from a bovine brain cDNA library using a synthetic oligonucleotide probe corresponding to the partial N-terminal amino acid sequence of the PACAP receptor purified from the bovine brain. The cloned cDNA encoded a polypeptide of 513 amino acid residues with seven putative transmembrane domains. The deduced amino acid sequence exactly matched the N-terminal amino acid sequence of the purified PACAP receptor. It also shared an apparent similarity with the vasoactive intestinal peptide (VIP), secretin, growth hormone releasing hormone, calcitonin, and glucagon receptors, suggesting that the PACAP receptor is a member of the secretin receptor subfamily of the guanine nucleotide-binding regulatory protein-coupled receptor family. Northern blot analysis showed that the size of the major mRNA band which hybridized with the cDNA was about 7 kb in the bovine cerebral-cortex and hippocampus. An expression vector containing the cloned cDNA for the PACAP receptor was introduced into Chinese hamster ovary (CHO) cells. The affinity of PACAP receptors expressed on the transfected CHO cells was quite similar to that of natural PACAP receptors on the bovine brain membranes. Competitive binding experiments showed that PACAP38 displaced the binding of 125I-labeled PACAP27 to the receptors on the CHO cells more efficiently than PACAP27, while VIP was less effective. In addition, both of PACAP27 and PACAP38 elevated the levels of cAMP and inositol phosphates in the transformed CHO cells. These results indicate that the PACAP receptors encoded by the cloned cDNA are identical to the purified PACAP receptors, and that they can stimulate dual signaling cascades.

Amino Acid Sequence↗
PubMed1994

Enhanced production of pituitary adenylate-cyclase-activating polypeptide by 1, N6-dibutyryladenosine 3',5'-monophosphate, phorbol 12-myristate 13-acetate and by the polypeptide itself in human neuroblastoma cells, IMR-32.

We investigated a regulatory mechanism for the production of pituitary adenylate-cyclase-activating polypeptide (PACAP) using the human neuroblastoma cell line, IMR-32. Although immunoreactive (ir-) PACAP38 was produced by these cells, it was not accumulated in the culture medium. To overcome this problem, we cultured the cells in the presence of a biotinylated monoclonal antibody to PACAP38 in order to trap ir-PACAP38 in the medium. Then, the trapped ir-PACAP38 was measured with a two-site enzyme immunoassay based on the biotin-avidin interaction. These studies showed that confluent IMR-32 cells constitutively secreted at least 130 pg/10(6) cells per day of ir-PACAP38. Treatment of the cells with 1 mM 1, N6-dibutyryladenosine 3',5'-monophosphate (Bt2cAMP) or 0.1 microM phorbol 12-myristate 13-acetate stimulated ir-PACAP38 production at almost twice the basal level in IMR-32 cells, and these two reagents additively stimulated ir-PACAP38 production. Northern blot analysis indicated that treatment of the cells with both Bt2cAMP and the phorbol ester increased the intensity of several mRNA bands that hybridized with a cDNA probe for human PACAP precursor. We also found that IMR-32 cells possessed high-affinity type-I PACAP receptors (Kd = 230 pM, binding sites: 8.6 x 10(4) sites/cell), which had a 1000-fold higher affinity for PACAP38 and PACAP27 than for vasoactive intestinal polypeptide. The treatment of IMR-32 cells with PACAP and vasoactive intestinal polypeptide increased intracellular cAMP levels, and also increased ir-PACAP production. Northern blot analysis revealed that PACAP at 10 nM markedly stimulated the expression of a 3.7-kb mRNA band that hybridized with PACAP precursor cDNA. These results indicate that IMR-32 cells produce ir-PACAP38, and that PACAP stimulates the synthesis of PACAP mRNA and peptides by increasing intracellular cAMP. Although it remains to be investigated whether PACAP secreted by IMR-32 cells really acts in an autocrine fashion, the present study provides a useful model for further studies of the biological role of PACAP in neuronal cells that produce PACAP and also respond to PACAP.

Bucladesine↗
PubMed1994

Ontogeny of pituitary adenylate cyclase-activating polypeptide (PACAP) and its binding sites in the rat brain.

Endogenous levels of pituitary adenylate cyclase-activating polypeptide (PACAP) and its binding site densities were measured in eight brain regions in rats of different ages (2-240 days) by sandwich-enzyme immunoassay and autoradiography. PACAP levels were quite low at day 2, peaked in 30-60 days, and then remained constant in most regions. Such ontogenetic changes are similar to those of vasoactive intestinal polypeptide (VIP) and classical neurotransmitters. PACAP-binding sites were already dense at day 2 and varied only slightly up to day 240. These results suggest that PACAP may have modulatory effects on brain development.

Animals↗
PubMed1994

Simultaneous racemization and isomerization at specific aspartic acid residues in alpha B-crystallin from the aged human lens.

We provide evidence that the racemization and isomerization of aspartyl(Asp) residues occur simultaneously in the alpha B-crystallin in the lens of aged (mean age: 80 years) and young (age: 11 months) humans. We purified alpha B-crystallin and subjected it to tryptic digestion. The resulting peptides were separated by reverse-phase high-performance chromatography (RP-HPLC) and were characterized by amino-acid composition, sequence analysis and mass spectrometry. Two specific sites, Asp-36 (D/L of Asp: 0.92) and Asp-62(D/L of Asp: 0.57), among 13 Asp/asparginyl (Asn) residues in aged alpha B-crystallin, were found to be highly racemized and isomerized to form beta-Asp residues. The beta-Asp-containing peptides were clearly distinguished from normal Asp-containing (alpha-Asp) peptides by RP-HPLC. The racemization and isomerization of Asp residues in aged alpha B-crystallin may occur via a succinimide intermediate. In young alpha B-crystallin, we observed neither racemization nor isomerization. We also found that Met-68 was oxidized to form Met sulfoxide to a greater extent in aged alpha B-crystallin than in young alpha B-crystallin. We concluded that racemization, isomerization, and oxidation of alpha B-crystallin occur spontaneously in the aging process.

Aged↗
PubMed1994

Effects of a new endothelin antagonist, TAK-044, on post-ischemic acute renal failure in rats.

Protective effects of a new endothelin (ET) receptor antagonist, TAK-044, were studied in a model of acute renal failure (ARF) in rats. ARF was induced by clamping the left renal pedicle for 45 minutes with contralateral nephrectomy and subsequent reperfusion of the left kidney. Plasma creatinine concentration (Pcr) increased to 2.28 mg/dl 24 hours after reperfusion of the ischemic kidney. Intravenous administration of TAK-044 (1-10mg/kg) prior to renal occlusion dose-dependently but partially attenuated the increase in Pcr and the morphological damages of the kidney. ET-1 and ET-3 increased perfusion pressure in isolated kidney preparations with similar potency, indicating that the renal vasoconstriction evoked by these ET isomers is mainly via ETB receptors, and TAK-044 (10nM) shifted the ET-1 dose-response curve to the right by a factor about 10. In a rat renal membrane fraction, ET-1 showed competitive inhibition of specific [125I]ET-1 binding with an IC50 value of 0.34nM and a Hill slope of 1.10. ET-3 did so with a higher IC50 value (3.3nM) and a lower Hill slope (0.56), suggesting that rat kidney contains both ETA and another receptor subtype, probably ETB. TAK-044 inhibited ET-1 binding with an IC50 value of 6.6nM and a Hill slope of 0.41. Plasma concentrations of immunoreactive TAK-044 were maintained over 7nM for 8 hours following i.v. injection of 10mg/kg TAK-044. These results suggest that endogenous ET is involved in the pathogenesis of post-ischemic ARF, at least, in part and that TAK-044 provided protective effects against ARF by blocking ET receptors, possibly both ETA and ETB receptors in renal vasculature and parenchymal cells.

Acute Kidney Injury↗
PubMed1994

Endothelin family peptides in human plasma and urine: their molecular forms and concentrations.

Molecular forms and concentrations of endothelin (ET) family peptides in normal human plasma and urine were investigated using five different sandwich-type enzyme immunoassays (sandwich-EIAs) and reverse-phase high performance liquid chromatography (RP-HPLC). We found that immunoreactive (IR-) big ET-3 and IR-big ET-2 were the major species in both plasma and urine from normal humans. Big ET-2(1-38) was found to be the major form of IR-big ET-2 in plasma. Urinary IR-big ET-2 and plasma and urinary IR-big ET-3 were heterogeneous, and the IR-big ET-3s were larger (M(r) 6,000-12,000) than authentic big ET-3. IR-big ET-1, IR-ET-1, and IR-ET-3 were found at relatively low concentrations in human plasma and urine, and their major forms were identical to authentic big ET-1, ET-1, and ET-3, respectively. Although ET-1 and other ET family peptides have often been measured using RIAs with board-spectrum antibodies, the presence of high concentrations of IR-big ET-2 and IR-big ET-3, but not IR-ET-1, in plasma and urine indicates that the clinical significance of ET family peptides should be investigated with assay methods specific to each ET and big ET.

Adult↗
PubMed1994

Abnormal p53 expression in human lung cancer is associated with histologic subtypes and patient smoking history.

Among the most common mutations in human lung cancer are those affecting the p53 gene. The expression of p53 in the nucleus is considered an immunohistochemical reflection of the nuclear accumulation of mutant p53 protein, which is coded by the p53 gene with missense mutation and has a prolonged half-life. In the present study, p53 expression detected by means of immunohistochemistry occurred frequently in human lung cancer and was associated with histologic subtypes. The alteration in the p53 gene was found to be a relatively early genetic event in the development and progression of lung cancer and to be maintained in the process of metastasis: abnormal p53 expression was found in both the early and late clinical stages, and identical p53 expression was detected consistently among primary and metastatic lesions from the same patients. Furthermore, an observed association between abnormal p53 expression and the patients' smoking history suggests that the p53 gene could be a common target of tobacco-associated carcinogenesis in lung cancer.

Carcinoma, Non-Small-Cell Lung↗
PubMed1994

Biosynthesis of human endothelins in transformants expressing cDNAs for human prepro-endothelins.

To understand the biosynthesis of the human ET family, three kinds of cloned cDNA for human prepro-endothelin-1 (prepro-ET-1), prepro-ET-2, and prepro-ET-3 were stably expressed in Chinese hamster ovary cells (CHO-K1). Immunoreactive (ir-) ET polypeptides in the culture media of the transformants were purified by reverse-phase high-performance liquid chromatography (HPLC) coupled with sandwich-type enzyme immunoassay (EIA). Amino acid sequencing and FAB mass spectrometry of the purified ir-ET polypeptides revealed the presence of human big ET-1 (1-38), big ET-2 (1-38), and big ET-3 (1-41)NH2 as intermediate forms. These results directly revealed the biosynthetic pathways of three human ETs at the peptide level.

Amino Acid Sequence↗
PubMed1994

[Serum pyridoxal concentration in children during theophylline therapy for bronchial asthma].

This study was designed to investigate the effect of sustained-release theophylline therapy on serum pyridoxal concentration in children with bronchial asthma. Forty-two children with bronchial asthma were divided into two groups according to duration of theophylline administration: The 22 children in group A had been treated with theophylline for less than 4 weeks, whereas the 20 children in group B had been treated for more than 5 weeks. The results obtained from this study were as follows: 1) The serum pyridoxal concentration in group B was significantly lower than that those in group A (p < 0.01). 2) The serum pyridoxal concentration was not significantly correlated with the serum theophylline concentration. These findings suggest that long-term theophylline therapy can depress vitamin B6 status in children with bronchial asthma. Theophylline-induced seizure may be caused by the possible decreased in gamma-aminobutyric acid concentration in the brain as a result of decrease in gamma-aminobutyric acid concentration in the brain as a result of decrease in vitamin B6 status, even if the serum theophylline concentrations are within the therapeutic range.

Asthma↗
PubMed1994

A new endothelin receptor antagonist, TAK-044, shows long-lasting inhibition of both ETA- and ETB-mediated blood pressure responses in rats.

The present study describes the pharmacological profile of an endothelin (ET) receptor antagonist, TAK-044, in anesthetized rats. TAK-044 given 10 min before administration of ET-1 (0.3 nmol/kg i.v.) partially inhibited the ET-1-induced pressor response at 0.1 and 1 mg/kg i.v. and almost completely inhibited the response at a dose of 10 mg/kg. The transient depressor response induced by ET-1 was also inhibited by 1 and 10 mg/kg TAK-044. BQ-123 partially inhibited the pressor response at 0.1-10 mg/kg i.v., whereas it did not inhibit the depressor response except at a dose of 10 mg/kg. These inhibitory effects of TAK-044 were longer lasting than those of BQ-123: 3 hr for TAK-044 and 1 hr for BQ-123 at 10 mg/kg. A selective ETB agonist, sarafotoxin S6c (0.3 nmol/kg i.v.), induced both depressor and pressor responses similar to ET-1. The initial depressor response was inhibited by TAK-044 in a dose-dependent manner (0.1-10 mg/kg i.v.) and by BQ-123 at the highest dose, whereas the sustained pressor response was inhibited only by TAK-044 at a dose of 10 mg/kg. Similar differences between TAK-044 and BQ-123 were observed for sarafotoxin S6c-induced renal vasoconstriction: TAK-044 but not BQ-123 inhibited the response at a dose of 3 mg/kg i.v. We conclude that TAK-044 inhibited both ETA- and ETB-mediated blood pressure responses and that these effects were longer lasting than those of BQ-123. In addition, the ETB-mediated vasoconstriction and pressor responses were inhibited by TAK-044 and not by BQ-123.

Animals↗
PubMed1994

Repair of osteochondral defects with grafts of cultured chondrocytes. Comparison of allografts and isografts.

Isogeneic and allogeneic chondrocytes cultured in collagen gel were transplanted into osteochondral defects in knee joints of inbred strains of rats. At 12 weeks, all eight isografted defects had healed successfully, compared with only four of eight allografted defects (p < 0.05). At 26 and 52 weeks, all defects except one had healed successfully and there was no significant difference in the success rate between the isografted and allografted groups. Control defects that had been implanted with only collagen gel did not heal successfully.

Animals↗
PubMed1993

Purification and characterization of the receptor for pituitary adenylate cyclase-activating polypeptide.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a 38-amino acid peptide (PACAP38) or a truncated peptide with the same 27 amino-terminal residues (PACAP27). The PACAP receptor was solubilized from bovine brain membranes with digitonin and purified 30-fold by the combination of DEAE-Toyopearl and hydroxylapatite chromatographic analyses. The partially purified PACAP receptors were mixed with biotinylated PACAP27 to form receptor-ligand complexes and then adsorbed onto avidin-agarose. The adsorbed PACAP receptors were eluted with an acidic buffer containing 1.0 M NaCl (pH 4.0). The eluted receptors were purified further by hydroxylapatite and gel filtration chromatography. A single protein band with a M(r) = 55,000-60,000 was found in the final preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. Affinity labeling of the purified receptors with 125I-PACAP27 labeled the M(r) 55,000-60,000 protein specifically. The dissociation constant and the specific activity of the purified receptors were 25.8 pM and 17.2 nmol of ligand binding per mg of protein, respectively. Inhibitory constants determined by competitive binding experiments were 30.0 pM for PACAP27, 4.6 pM for PACAP38, and 37.3 nM for vasoactive intestinal peptide. Therefore, the purified PACAP receptor retained high affinity and ligand specificity. The sequence of the amino-terminal 29 residues was derived from the purified receptor.

Amino Acid Sequence↗
PubMed1993

Endothelin-1 analogues substituted at both position 18 and 19: highly potent endothelin antagonists with no selectivity for either receptor subtype ETA or ETB.

Novel endothelin-1 (ET-1) analogues which are highly potent endothelin antagonists at both receptor subtype ETA and ETB are reported. The replacement of Asp18 with the Thr18 and of Ile19 with a hydrophobic amino acid whose side-chain branches on the gamma-carbon such as Leu, cyclohexylalanine, and gamma-methylleucine (gamma-MeLeu) resulted in loss of or significantly decreased the biological activity of ET-1, while high affinity for the ETA (IC50 = 0.42-0.70 nM) and ETB (IC50 = 0.17-0.43 nM) receptor was retained. These compounds were shown to have high antagonist activities in ET-1-induced vasoconstriction of porcine coronary artery (pA2 7.4-7.7) and in Sarafotoxin S6c-induced vasoconstriction of rabbit pulmonary artery ([Thr18, gamma-MeLeu19]ET-1: pA2 8.4). Among these compounds, [Thr18, gamma-MeLeu19]ET-1 has the desirable characteristic of possessing no agonist activity at either receptor subtype.

Amino Acid Sequence↗
PubMed1993

Molecular cloning and functional expression of a cDNA encoding a human pituitary adenylate cyclase activating polypeptide receptor.

A functional cDNA clone for a human pituitary adenylate cyclase activating polypeptide (PACAP) receptor was isolated from a human pituitary cDNA library. The cDNA encoded a polypeptide consisting of 525 amino acids with putative seven hydrophobic domains. Chinese hamster ovary (CHO)-K1 cells transfected with the cDNA specifically bound PACAP and mediated PACAP-triggered intracellular accumulation of cAMP, indicating that this cDNA encoded a functional human PACAP Type I receptor. This receptor was structurally related to the vasoactive intestinal peptide (VIP), secretin, calcitonin and parathyroid hormone receptors and is much more homologous to a rat PACAP receptor. Northern blot analysis revealed that PACAP receptor mRNAs were expressed mainly in the brain and widely distributed in the central nervous system.

Amino Acid Sequence↗
PubMed1993

Molecular cloning and functional expression of rat cDNAs encoding the receptor for pituitary adenylate cyclase activating polypeptide (PACAP).

Two types of cDNA encoding PACAP receptors were isolated from the rat brain cDNA library by homology screening with a cDNA probe for VIP receptor. Nucleotide sequence analysis indicated that these two types of receptor mRNA were generated by alternative splicing mechanisms. These two cloned cDNAs were introduced into CHO cells respectively. Resultant transformants showed specific binding to [125I]PACAP27 which was displaced by unlabeled PACAP27 but not by VIP. Thus, these receptors are two subtypes of Type I PACAP receptor (Type I-A and Type I-B). The amino acid sequences of rat PACAP receptors deduced by the cDNAs showed a remarkable similarity with rat receptors for VIP, secretin, glucagon, and GHRH. A 6.5 kb significant hybridizing signal of the PACAP receptor mRNA was detected in the rat brain, and slight signal was also detected in the lung and the liver.

Alternative Splicing↗