Biomedical subjects

H Murakami

Publications and source records attributed to H Murakami.

At least 163 records · Page 9Linked to original sources

PubMed1999

Enhanced phosphatidylinositol 3-kinase activity and high phosphorylation state of its downstream signalling molecules mediated by ret with the MEN 2B mutation.

We compared the intracellular signalling pathways through Ret tyrosine kinase activated by glial cell line-derived neurotrophic factor (GDNF), multiple endocrine neoplasia (MEN) 2A, or MEN 2B mutation. Tyrosine phosphorylation of Grb2-associated binder-1 (Gab1) and activation of phosphatidylinositol 3-kinase (PI 3-kinase) were induced at higher levels by GDNF stimulation or the MEN 2B mutation than by the MEN 2A mutation. Tyrosine-phosphorylated Gab1 was a major component that interacted with the active PI 3-kinase in vivo. In addition, we found that p62Dok and PKB/Akt were phosphorylated in a PI 3-kinase-dependent manner and the levels of their phosphorylation were significantly higher in the MEN 2B transfectant than in the MEN 2A transfectant. Tyrosine phosphorylation of p62Dok resulted in its complex formation with the Ras GTPase-activating protein (RasGAP) and the Nck adaptor protein. These findings thus suggested that high levels of activation of PI 3-kinase and of phosphorylation of its downstream signalling molecules may be associated with the clinical phenotype of MEN 2B.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Characterization of the promoter region of the human RFP gene.

The RFP gene encodes a Ring finger protein that has a tripartite motif consisting of a Ring finger, a B-box finger and a coiled-coil domain. In the present study, we cloned and characterized the promoter region of the human RFP gene. The nucleotide sequence of the promoter was GC-rich and had no typical TATA and CAAT boxes. Instead, it contained one AP2 and two Sp1 binding sites within 100 base pairs upstream of the transcription initiation site. Analysis by the luciferase assay revealed that the activity of this promoter region is very strong in both human and mouse cell lines, although the activity in human cells was approximately 10-15 fold higher than that in mouse cells. In addition, the AP2 and Sp1 binding sites appeared to synergistically function for the promoter activity. Thus, the promoter of the RFP gene could be useful for high levels of expression of various genes in culture cells.

3T3 Cells↗

PubMed1999

Biological and biochemical properties of Ret with kinase domain mutations identified in multiple endocrine neoplasia type 2B and familial medullary thyroid carcinoma.

Several mutations were identified in the kinase domain of the RET proto-oncogene in patients with multiple endocrine neoplasia (MEN) 2B, familial medullary thyroid carcinoma (FMTC) or sporadic medullary thyroid carcinoma. We introduced seven mutations (glutamic acid 768-->aspartic acid (E768D), valine 804-->leucine (V804L), alanine 883-->phenylalanine (A883F), serine 891-->alanine (S891A), methionine 918-->threonine (M918T), alanine 919-->proline (A919P) and E768D/A919P) into the short and long isoforms of RET cDNA and transfected the mutant cDNAs into NIH3T3 cells. The transforming activity of the long isoform of Ret with each mutation was much higher that that of its short isoform. Based on the levels of the transforming activity, these mutant RET genes were classified into two groups; a group with high transforming activity (A883F, M918T and E768D/A919P) and a group with low transforming activity (E768D, V804L, S891A and A919P) (designated high group and low group). Interestingly, the level of transforming activity correlated with clinical phenotypes; high group Ret with the A883F or M918T mutation and low group Ret with the E768D, V804L or S891A mutation were associated with the development of MEN 2B and FMTC, respectively. In addition, we found that substitution of phenylalanine for tyrosine 905 present in the kinase domain abolished both transforming and autophosphorylation activities of low group Ret whereas it did not affect the activities of high group Ret.

Amino Acid Substitution↗

PubMed1999

Recapping T-saw laminoplasty for spinal cord tumors.

STUDY DESIGN: A prospective study of patients whose spinal cord tumors were managed surgically with a unique posterior method of removing and replacing the posterior spinal elements using T-saw ("recapping T-saw laminoplasty"). OBJECTIVES: To examine the safety and efficacy of the recapping T-saw laminoplasty technique for spinal canal surgery. SUMMARY OF BACKGROUND DATA: Laminectomy, laminoplasty, and/or laminotomy typically are used to approach intraspinal lesions. When removal and replacement of the posterior elements have been attempted, the effectiveness of the technique has been limited by the amount of bone sacrificed when using burrs or osteotomes. The authors thought to adapt a unique "threadwire saw" (T-saw) in these cases, because its use results in minimal bone loss. METHODS: Patients underwent recapping T-saw laminoplasty in the thoracic or lumbar spine for extirpation of spinal cord tumors. The T-saw was used for division of the posterior elements. After resection of the lesion, the excised laminae were replaced exactly in situ to their original anatomic position. The mean follow-up period was 47 months (range, 31-71 months). Patients were observed neurologically and radiologically. RESULTS: One to eight laminae were excised and replaced in 24 patients. Findings on computed tomography scans confirmed primary bony union in 23 patients by 6 months after surgery, and in one patient by 12 months after surgery. No complications such as postoperative spinal canal stenosis, facet arthrosis, or kyphosis were observed. CONCLUSIONS: Recapping laminoplasty afforded anatomic reconstruction of the vertebral arch after excision of spinal cord tumors. This procedure appears to warrant further evaluation as an alternative to wide laminectomies for exposure of intraspinal tumors.

Adult↗

PubMed1999

Localized expression of aromatase in human vascular tissues.

The atheroprotective effects of estrogen are well established and the presence of an estrogen receptor in vascular tissues has recently been reported. Therefore, we investigated the localization of the estrogen-producing enzyme aromatase in vascular tissues to assess the possible contribution of endocrine, paracrine, and autocrine modes of action. Aromatase was found in human vascular smooth muscle cells (SMCs) but not in endothelial cells on in situ hybridization. These observations were further supported by quantitative analysis of aromatase mRNA and the activity in 15 human vascular specimens. Only trace levels of expression were detected in the 3 infants examined, whereas 0.0088 to 0.0806 amol/ microg RNA of aromatase mRNA and 12.9 to 122.3 fmol. h-1. mg-1 protein of the activity were detected in 12 of the adult individuals. The switching of tissue-specific exon 1 of the human aromatase gene was also observed in some cases. Aromatase was found to be expressed only in cultured SMCs and not in cultured endothelial cells of human aorta and pulmonary artery and to be regulated through dexamethasone and the signaling pathways of protein kinase A and C. Study results revealed the localized expression of aromatase in vascular SMCs, which indicated a possible direct action of locally produced estrogen in an autocrine or paracrine manner, with possible cross talk between smooth muscle and endothelial cells.

Adolescent↗

PubMed1999

Human histamine H2 receptor gene: multiple transcription initiation and tissue-specific expression.

We have characterized the genomic structure of 12.3 kb of the 5'-flanking region of the human histamine H2 receptor gene. The multiple transcription initiation sites of the human histamine H2 receptor gene were mapped utilizing the 5'-end cap structure of mRNA. We found that a 85 bp segment (-610-(-)525 bp) immediately upstream of the initiation site exhibits a strong promoter activity in the gastric adenocarcinoma, MNK45, expressing human histamine H2 receptor. A 4.8 kb transcript of the human histamine H2 receptor gene was found in the placenta, spinal cord, lymph node and bone marrow in addition to the previously reported tissues including the heart, brain and stomach, whereas a 1.8 kb transcript was observed in almost all tissues examined. 3'-rapid amplification of cDNA ends revealed the corresponding length of the 3'-untranslated region. These results suggest that the 3'-untranslated region may be involved in the differential expression.

Base Sequence↗

PubMed1999

Rho-dependent and -independent tyrosine phosphorylation of focal adhesion kinase, paxillin and p130Cas mediated by Ret kinase.

Glial cell line-derived neurotrophic factor (GDNF) signals through a unique receptor system that includes Ret receptor tyrosine kinase and a glycosyl-phosphatidylinositol-linked cell surface protein. In the present study, we have identified several proteins in neuroblastoma cells that are phosphorylated on tyrosine in response to GDNF. The phosphorylated proteins include focal adhesion kinase (FAK), paxillin and Crk-associated substrate, p130Cas, all of which are known to be associated with focal adhesions. Of these, paxillin and p130Cas interacted with Crk proteins in GDNF-treated neuroblastoma cells. GDNF also induced reorganization of the actin cytoskelton. Tyrosine phosphorylation of FAK, paxillin and p130Cas was inhibited by cytochalasin D or two specific inhibitors of phosphatidylinositol-3' kinase (PI-3' kinase), wortmannin and LY294002, indicating that their tyrosine phosphorylation depends on the formation of actin stress fiber and activation of PI-3' kinase. In addition, phosphorylation of FAK but not of paxillin and p130Cas was markedly impaired by the Clostridium botulinum C3 exoenzyme that specifically ADP-ribosylates and inactivates Rho. These results suggested the presence of Rho-dependent and -independent signaling pathways downstream of PI-3' kinase that mediate tyrosine phosphorylation of FAK, paxillin and p130Cas through Ret kinase.

ADP Ribose Transferases↗

PubMed1999

Mechanism of Ret activation by a mutation at aspartic acid 631 identified in sporadic pheochromocytoma.

Mutations at aspartic acid 631 in Ret were reported in sporadic pheochromocytoma and medullary thyroid carcinoma. We replaced this aspartic acid with four other amino acids including tyrosine, glycine, asparagine, and alanine and investigated the transforming activity of these mutant cDNAs. Among them, RET cDNA with a mutation of aspartic acid to tyrosine (D631Y) that was reported in sporadic pheochromocytoma showed high transforming activity. The D631Y mutation activated Ret by inducing its disulfide-linked dimerization in the transfectant as observed for multiple endocrine neoplasia (MEN) 2A mutations at cysteine 609, 611, 618, 620, 630, or 634. Further mutation analysis suggested that cysteine 630 or 634 could be involved in the disulfide-linked Ret dimerization induced by the D631Y mutation.

3T3 Cells↗

PubMed1999

Effects of 7-nitroindazole on renal sympathetic nerve activity during acute cardiac tamponade in conscious rabbits.

To investigate whether nitric oxide (NO) in the central nervous system is involved in the decrease in renal sympathetic nerve activity (RSNA) during acute cardiac tamponade in conscious rabbits, we examined the effect of 7-nitroindazole (7-NI), a selective inhibitor of neuronal nitric oxide synthase in vivo, on RSNA during acute cardiac tamponade in chronically installed conscious rabbits. Cardiac tamponade was produced by intrapericardial infusion of physiological saline at 2 ml/30 s. Mean arterial pressure (MAP) remained constant initially but RSNA increased to 218+/-24% when we started injection of physiological saline into the pericardial space. Concomitantly after MAP fell to 51+/-1 mm Hg by subsequent injection of the saline into the pericardial space, RSNA decreased to 45+/-6%. If 7-NI (50 mg/kg) was administered intraperitoneally 35 min before the beginning of cardiac tamponade, the decline in RSNA caused by cardiac tamponade was markedly counteracted. Brain nitric oxide synthase (NOS) activity in the cerebral cortex and medulla oblongata, assessed by the conversion of labelled arginine to citrulline, was inhibited by 48% and 44% after the intraperitoneal administration of 7-NI. These results indicate that acute cardiac tamponade elicits a biphasic effect on RSNA, which rises during non-hypotensive period and then falls during hypotension in conscious rabbits. The decrease in RSNA was abolished by treatment with 7-NI, suggesting that the abrupt decrease in RSNA during hypotension induced by acute cardiac tamponade is mediated by NO in the central nervous system.

Acute Disease↗

PubMed1999

Expression of functional Na+/H+ antiporters of Helicobacter pylori in antiporter-deficient Escherichia coli mutants.

An open reading frame with a sequence homologous to Escherichia coli Na+/H+ antiporter A (ENhaA) was found in the total genomic sequence of Helicobacter pylori, a pathogenic bacterium of gastric inflammation, and was named HNhaA. The primary sequences and the hydropathy profiles of ENhaA and HNhaA were very homologous except for one additional region found in HNhaA. This sequence has about 40 hydrophilic amino acid residues inserted at the position next to residue 235 of ENhaA which corresponds to residue 245 of HNhaA. HNhaA was expressed in E. coli mutants deficient in Na+/H+ antiporters and complemented the salt-sensitive phenotype of the mutants. Membrane vesicles prepared from these transformants of HNhaA using mutants deficient in the antiporters had the antiporter activities. Surprisingly, the antiporter activity in the transformant membranes was high at acidic and neutral pH, while ENhaA did not function at these pHs. A hydrophilic region around residue 235 in ENhaA and the additional hydrophilic region of about 40 residues in the same region found in HNhaA might be responsible for this difference in activity by acting as putative pH sensors.

Amino Acid Sequence↗

PubMed1999

Characteristics of genomic breakpoints in TLS-CHOP translocations in liposarcomas suggest the involvement of Translin and topoisomerase II in the process of translocation.

Fusion of TLS/FUS and CHOP gene by reciprocal translocation t(12;16)(q32;q16) is a common genetic event found in myxoid and round-cell liposarcomas. Characterization of this genetic event was performed by three methods, Southern blot, RT-PCR, and genomic long-distance PCR in nine myxoid and three round-cell liposarcomas. All but one tumors showed genetic alternations indicating the fusion of TLS/FUS and CHOP gene. Two novel types of fusion transcripts were found, of which one lacked exon 2 sequence of CHOP gene, and the other lacked 3' half of exon 5 of TLS gene. The latter case was caused by a cryptic splicing site which was created by the genomic fusion. Detailed analyses genomic fusion points revealed several sequence characteristics surrounding the fusion points. Homology analyses of breakpoint sequences with known sequence motifs possibly involve in the process of translocation uncovered Translin binding sequences at both of TLS/ FUS and CHOP breakpoints in two cases. Translocations were always associated with other genetic alterations, such as deletions, duplications, or insertions. Short direct repeats were almost always found at both ends of deleted or duplicated fragments some of which had apparently been created by joining of sequences that flank the rearrangement. Finally, consensus topoisomerase II cleavage sites were found at breakpoints in all cases analysed, suggesting a role of this enzyme in creating staggered ends at the breakpoint. These data suggested that sequence characteristics may play an important role to recruit several factors such as Translin and topoisomerase II in the process of chromosomal translation in liposarcomas.

Artificial Gene Fusion↗

PubMed1999

Myelodysplastic syndromes with nephrotic syndrome.

It is sometimes reported that the immunological abnormalities in myelodysplastic syndromes (MDS) induce autoimmune disease (i.e., acute systemic vasculitic syndrome, chronic cutaneous vasculitis, polyneuropathy, relapsing polychondritis, and steroid-responsive pulmonary disorders). We investigated the clinical features of patients with MDS accompanied by nephrotic syndrome. We enrolled 125 patients with MDS who were admitted between January 1979 and May 1996 in this study. The renal function was assessed based on the laboratory data and the findings at the physical examination. The diagnoses of nephrotic syndrome and glomerular disease were established when 24-hr urinary excretion was more than 3.5 g and serum total protein was less than 6.0 g/dl, and when the 24-hr protein excretion was more than 1.5 g. Five patients (4%) had glomerular disease, and three (2.4%) had nephrotic syndrome. Of the five patients with glomerular disease, two had refractory anemia (RA), and three had chronic myelomonocytic leukemia (CMMOL). Three of the total 11 patients with CMMOL were diagnosed as having nephrotic syndrome. Among the CMMOL patients, those with nephrotic syndrome showed higher absolute monocyte numbers than did those without nephrotic syndrome (8830 +/- 4677/microl vs. 3061 +/- 2887/microl, P = 0.03). One CMMOL patient was treated with VP-16 and hydroxyurea. As the white blood cell count in this patient decreased, the 24-hr urine protein excretion and the serum tumor necrosis factor alpha level decreased. The relationship between nephrotic syndrome and CMMOL was not clear. High monocyte count and the serum cytokines in MDS patients may play a partial role in the evolution of glomerulonephritis, and CMMOL may be closely related to nephrotic syndrome.

Aged↗

PubMed1999

A successful surgical repair of the hepatic hydrothorax using pneumoperitoneum: report of a case.

A successful surgical repair of a right hepatic hydrothorax in the absence of ascites is reported. A technetium-99m scintigram that was injected intraperitoneally provided evidence of a one-way flow of fluid from the peritoneal to pleural cavity. To identify any possible minute defects in the diaphragm, carbon dioxide was insufflated into the peritoneal cavity during the operation. We performed a direct suture of the defect observed on the diaphragm. The pleural effusion subsequently vanished after the operation.

Humans↗

PubMed1999

Prognostic value of morphological classifications and clinical variables in elderly and young patients with multiple myeloma.

The prognostic value of morphological classifications and clinical variables was compared between 31 elderly (> or = 65 years) and 43 young (< 65 years) patients with myeloma. Prognostic factors were divided into three groups: factors useful in elderly patients, e.g., calcium, albumin; factors useful in young patients, e.g., platelet, creatinine, light-chain type; and factors useful in both patients, e.g., clinical stage, hemoglobin, LDH, CRP, bone marrow plasma cell and plasmablast percentages, light- and electron-microscopic classifications. The 5-year survival rates of elderly patients with calcium < 12 and > or = 12 mg/dl were 66.2 and < 11.1%, respectively (p<0.01). Those of the young patients were 64.1 and 33.3%, respectively. The 5-year survival rates of elderly patients with platelets > or = 200 x 10(9)/l and < 100 x 10(9)/l were 59.7 and 50.0%, respectively. Those of the young patients were 68.9 and 33.3%, respectively (p<0.05). The 5-year survival rates of elderly patients with few and numerous electron-microscopic abnormalities were 90 and 0%, respectively (p<0.01), those of young patients were 92.9 and < 14.3%, respectively (p<0.01). These findings suggest that individual clinical variables may differ in prognostic importance in elderly and young patients.

Age Factors↗

PubMed1999

Primary nonsecretory plasma cell leukemia: a rare variant of multiple myeloma.

We report a patient with primary nonsecretory plasma cell leukemia. These cells resembled prolymphocytes and expressed only CD38 and CD56. Due to the atypical morphology and lack of monoclonal immunoglobulins in both serum and urine, it was difficult to make a correct diagnosis. Electron-microscopic and immunocytochemical cytoplasmic studies were useful.

Aged↗

PubMed1999

Determination of the prognostic significance of cyclin B1 overexpression in patients with esophageal squamous cell carcinoma.

Recent studies have identified a family of proteins referred to as cyclins, which control the cell cycle. Cyclin B1 activates cdc2, which regulates cell progression through the G2 and M phases. The main aim of this study was to examine the relationships between the cyclin B1 expression in human esophageal squamous cell carcinoma (SCC) and clinicopathological factors and prognosis of the patients. Eighty-seven cases of primary human SCC consecutively obtained at esophagectomy were immunohistochemically studied using an anti-human cyclin B1 protein antibody (2H1-H6). The relationship between cyclin B1 expression and clinicopathological factors, including prognosis, were also statistically assessed. Positive immunostaining of cancer cells, mainly in the cytoplasm, was detected in 72.4% (63/87): heterogeneous pattern in 37.9% (33/87) and homogeneous pattern in 34.5% (30/87). The prevalence of cyclin B1 expression was significantly higher in cases with invasion deeper than the muscularis propria (P<0.005) and with venous invasion (P<0.01) than in other cases. Patients whose SCCs expressed high levels of cyclin B1 protein had a significantly poorer prognosis than did the other patients (P<0.05). Multivariate analysis demonstrated that cyclin B1 status was an important factor affecting survival (P<0.05). These findings demonstrated that overexpression of cyclin B1 protein is associated with tumor behavior and prognosis for patients with human esophageal SCC.

Adult↗

PubMed1999

The visualization and evaluation of bone architecture in the rat using three-dimensional X-ray microcomputed tomography.

Microcomputed tomography allows the true three-dimensional structure of bone to be assessed by a nondestructive analysis. This article describes how this technique has for the first time been applied to rat bone to determine the effects of aging, ovariectomy, and antiresorptive drugs on bone structure and how these results compare with those determined by histological and histomorphometric techniques. During the procedure, a micro X-ray source is directed toward the bone sample. Modifications in the X-ray beam induced by bone crystals are determined for a range of acquisitions before three-dimensional reconstruction of bone architecture is performed. Morphometric parameters determined were trabecular bone volume/tissue volume, trabecular number, and trabecular thickness. The results show that ovariectomy has a dramatic effect on rat bone structure. Following treatment with the bone resorption inhibitor tiludronate, the morphometric parameters were significantly improved. The results obtained with three-dimensional microcomputed tomography were in agreement with observations made using classical techniques. Microcomputed tomography should prove useful for evaluating the antiresorptive effects of bisphosphonates on bone architecture and in allowing between-drug comparisons.

Animals↗

PubMed1999

Surgery for mitral valve disease in the pediatric age group.

OBJECTIVES: We reviewed a 20-year experience with the surgical treatment of mitral valve disease in the pediatric age group at our institution with 2 objectives: to clarify the long-term results over the last 2 decades and to evaluate the recent advances in mitral valve operation in children. METHODS: Since December 1978, 56 patients have undergone a total of 36 mitral valve repairs and 30 mitral valve replacements. Associated cardiac anomalies were present in 46 patients (82%), and concurrent repair of associated lesions was performed in 37 patients (66%). The age of the patients ranged from 3 months to 15 years (mean, 3.6 years) at mitral valve repair, and ranged from 2 months to 16 years (mean, 5.7 years) at mitral valve replacement. Mean follow-up period was 92.0 months (range, 1-235 months). RESULTS: There were 2 hospital deaths and 2 late deaths in patients who underwent mitral valve repair. Reoperation was performed in 4 patients. Three of these patients underwent mitral valve replacement because of residual mitral incompetence. No hospital deaths occurred in patients who underwent mitral valve replacement. Two late deaths occurred after mitral valve replacement. Six patients had a total of 10 episodes of prosthetic valve thrombosis. Thrombolytic therapy with urokinase was successful in all episodes without serious complications. Five patients required reoperations 49 to 141 months (mean, 78.4 months) after the initial valve replacement for relative prosthetic valve obstruction as the result of somatic growth. A valve 2 or 3 sizes larger than the original prostheses was inserted without death. Actuarial survival and freedom from cardiac events at 10 years after the operation were 87.2% and 72.7% in children who underwent mitral valve repair, and 90.3% and 67.3% for those children who underwent mitral valve replacement. CONCLUSIONS: The current risk of mitral valve operation in the pediatric age group is low, and the long-term results are satisfactory, irrespective of severe deformation of the mitral valve apparatus and associated complex cardiac anomalies.

Actuarial Analysis↗