Biomedical subjects

H Esumi

Publications and source records attributed to H Esumi.

At least 73 records · Page 4Linked to original sources

PubMed1997

Evidence for monoclonal expansion of epithelial cells in ovarian endometrial cysts.

Ovarian endometrial cysts, one of the typical manifestations of endometriosis, are generated by the retention of cyclic hemorrhages and are classified as tumor-like lesions rather than neoplasms. Clonality analysis provides important information about the histogenesis and progression of neoplastic diseases. As it is generally accepted that most neoplasms are monoclonal in origin, however, the clonality of endometrial cysts remains uncertain. Using the human androgen receptor gene (HUMARA) as an X-linked polymorphic marker, we examined the clonal status of epithelial cells in endometrial cysts. We separated 21 fresh epithelial cell samples from 11 endometrial cysts and found that all were monoclonal in the methylation pattern of the HUMARA alleles. Moreover, in each of the five cysts from which epithelial cells were sampled from multiple and distant areas, the methylation patterns of all samples from a single cyst were identical. These data indicate that endometrial cysts are monoclonal in origin and suggest their neoplastic potentiality.

Adult↗

PubMed1997

Combined analysis of microsatellite instability and K-ras mutation increases detection incidence of normal samples from colorectal cancer patients.

Microsatellite instability (MI) and K-ras oncogene mutation have been widely used as biomarkers of genetic changes in colorectal cancer (CRC). Each of these biomarkers was independently found in normal-appearing colonic mucosa at stages preceding the development of CRC, albeit at a relatively low incidence. To assess the potential value of combined MI and K-ras mutation analysis in the detection of normal-appearing colonic mucosa samples taken from patients with CRC, we have chosen to analyze multiple (3-7) normal colonic mucosa samples and the respective colorectal tumor tissues from 20 patients with CRC. As a control, we have used 54 normal mucosa samples obtained from 9 autopsies of patients without CRC. In at least 1 of 5 loci analyzed, MI was found in 8 of 20 patients via analysis of multiple normal-appearing colonic mucosa samples from each patient. Combined analysis of MI and mutant ras alleles in normal-appearing colonic mucosa samples enabled the identification of 11 of 20 patients with CRC. None of the 54 normal colonic mucosa samples obtained from 9 patients without CRC were found to carry mutant ras or MI. The ability to detect 55% of patients with CRC via the analysis of normal mucosa samples provides an important advance in our approach toward early detection of individuals who may be at risk to develop this tumor type.

Aged↗

PubMed1996

Clonal analysis of isolated single fundic and pyloric gland of stomach using X-linked polymorphism.

In order to understand the histogenesis of precancerous and cancerous lesions, we investigated the clonality of normal single fundic gland and single pyloric gland using X-chromosome inactivation. Sixty percent of single fundic glands were cell populations methylated at the same alleles of the X-chromosome (homotypic) and 40% consisted of mixed cells methylated at different alleles of the X-chromosome (heterotypic). Among 28 fundic glands in which the clonality of the upper parts and that of the lower parts were analyzed separately, 11 glands were heterotypic in either part and 4 glands were homotypic with different allelic methylation in the upper part and in the lower part. Ninety-six percent of the single pyloric glands were homotypic and 4% were heterotypic. The results show the differences in the development of the fundic glands and the pyloric glands.

Female↗

PubMed1996

Regulation of the mouse inducible-type nitric oxide synthase gene promoter by interferon-gamma, bacterial lipopolysaccharide and NG-monomethyl-L-arginine.

Cytokines and bacterial lipopolysaccharides (LPSs) stimulate nitric oxide production in macrophages by inducing transcription of the gene coding for the inducible isoform of nitric oxide synthase (iNOS). We have cloned the mouse iNOS gene promoter and analysed its structural features and its response to interferon-gamma (IFN-gamma) and Escherichia coli LPS in RAW 264.7 mouse macrophage-like cells. Transcription of a recombinant reporter gene including the promoter and 4 kb of its 5'-flanking DNA, linked to the bacterial chloramphenicol acetyltransferase (CAT) reporter gene, is stimulated by IFN-gamma and, more efficiently, by LPS upon transient transfection in RAW 264.7 cells. Two upstream DNA regions are required for maximal promoter activation of LPS: the first maps between positions -1541 and -775 and the other between -420 and -47, with respect to the major transcriptional start site of the iNOS gene. The upstream-most region also mediates promoter trans-activation by IFN-gamma. As reported earlier for transcription of the endogenous iNOS gene, combined stimulation of RAW 264.7 cells with IFN-gamma and LPS results in lower activation of the transfected promoter, when compared with LPS alone. NG-Monomethyl-L-arginine, a competitive inhibitor of nitric oxide synthase activity, enhances iNOS gene mRNA induction and promoter activation by IFN-gamma and LPS, indicating that nitric oxide can influence negatively the reponsiveness of this gene to inducers. These results suggest the possibility of a negative regulatory feedback exerted by iNOS on the transcriptional activation of its own gene.

Animals↗

PubMed1996

Nitric oxide synthase expression in human neuroblastoma cell line induced by cytokines.

The human neuroblastoma cell line NB-39-nu expressed mRNA coding for inducible nitric oxide synthase (iNOS) following treatment with a combination of interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha). The level of iNOS mRNA peaked 24 h after stimulation and had declined by about 25% after 48 h. Trace levels of iNOS mRNA were detected after treatment with IFN-gamma alone, and its mRNA level was synergistically enhanced by simultaneous treatment with TNF-alpha. Neither bacterial lipopolysaccharide nor interleukin-1 beta (IL-1 beta) showed synergistic effects as great as that of TNF-alpha on iNOS gene expression. Dexamethasone inhibited the induction of iNOS mRNA by IFN-gamma and TNF-alpha. Induction of iNOS was confirmed by NADPH-diaphorase staining and by immunostaining with human iNOS-specific antibody.

Drug Synergism↗

PubMed1996

Neuronal nitric oxide synthase expression in neuronal cell differentiation.

Expression of neuronal nitric oxide synthase (n-NOS) was investigated during neuronal cell differentiation. Trace amounts of n-NOS mRNA were detected in the primary culture of neural precursor cell (NPC) at embryonic day 10, and a high level of n-NOS mRNA was observed after a further 7 days cultivation of NPC. Both n-NOS activity and its mRNA level were also increased in the human neuroblastoma cell line, TGW, following trans-retinoic acid (RA)-induced neuronal differentiation. These findings indicate that there is positive regulation of n-NOS mRNA during neuronal cell differentiation. However TGW cell differentiation with trans-RA could not be reversed by treatment with specific inhibitors of NOS. Thus, the signal transduction mechanism which is involved in the induction of neuronal cell differentiation by trans-RA appears to be distinct from the NO-mediated pathway.

Animals↗

PubMed1996

Possible involvement of tyrosine kinase activation in lipopolysaccharide-induced expression of Ca(2+)-insensitive but calmodulin-coupling nitric oxide synthase in rat glial cells.

To clarify the properties of an inducible type of nitric oxide synthase (i-NOS) in the brain, we examined whether lipopolysaccharide (LPS) induces NOS in glial cells cultured from neonatal rats. NOS activities (NO2- accumulation and L-[14C]citrulline formation) were detected by treatment with LPS at 10 micrograms/ml for 6-72 hr. L-[14C]citrulline formation by LPS-induced i-NOS was inhibited by NG-monomethyl-L-arginine (a NOS inhibitor) and diphenyleneiodonium (a flavo-protein inhibitor). The activity was not markedly changed in the presence or absence of Ca2+. The induction of i-NOS by LPS was abolished by cycloheximide, actinomycin D, or dexamethasone. In addition, the induction was inhibited by herbimycin A (a tyrosine kinase inhibitor), but was not by staurosporine, W-7, or FK-506. After LPS stimulation, 130 kDa proteins were reacted with anti-rat liver i-NOS antibody 5-72 hr. i-NOS induced from glial cells coupled tightly with endogenous calmodulin (CaM) even in the absence of Ca2+. These results suggest that LPS induces expression of 130-kDa i-NOS through an activation of tyrosine kinase, after which i-NOS couples with CaM, and that NO is formed for 6-72 hr in glial cells.

Animals↗

PubMed1996

Effects of ubiquinone enriched diet on deterioration of mitochondrial respiratory function caused by fried beef derived mutagenic factor in rats.

2-Amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP), a potent mutagenic agent produced during thermal processing of meats, shows detrimental effects on mitochondrial respiratory function. Effects of dietary ubiquinone-10 was examined on PhIP-induced mitochondrial respiratory dysfunction in rats. Rats fed with 0.2% ubiquinone-10 diet mitigated the PhIP-induced decrease in mitochondrial complex I activities observed in rat heart, diaphragm, and psoai major. Therefore, dietary anti-oxidants such as ubiquinone-10 could cancel out the unfavorable effects of PhIP and contribute to the preservation of mitochondrial function.

Animals↗

PubMed1996

Thiocyanate-independent nitrosation in humans with carcinogenic parasite infection.

Infection with the liver fluke, Opisthorchis viverrini, is a causative agent of cholangiocarcinoma. One possible contributing factor in this carcinogenesis is the chronic, local generation of nitric oxide by inflammatory cells expressing inducible nitric oxide synthase and the production of N-nitroso compounds via the reaction between amines and nitrosating agents derived from nitric oxide. Our previous studies provided evidence that nitric oxide synthesis is elevated during human liver fluke infection. Here we present data on the same sample of men which definitively demonstrates increased nitrosation of proline and thioproline (thiazolidine-4-carboxylic acid) among infected men compared to uninfected control subjects on a low nitrate diet. This difference was specifically abolished by co-administration of ascorbic acid with proline and by elimination of parasites by praziquantel treatment. Multivariate statistical models demonstrate the importance of salivary thiocyanate levels to variation in the nitrosation of proline among uninfected individuals, but not among those with current fluke infection. This suggests that considerable generation of nitrosating agents (N203/N204) in infected people may be occurring via oxidation of arginine by nitric oxide synthase in inflamed tissue which is thiocyanate insensitive. Analyses revealed positive associations between N-nitrosoproline excretion and nitrate/nitrite levels in urine, plasma and saliva and with usual alcohol intake; with variation in these trends between groups. In conclusion, we have confirmed the relationship between O.viverrini infection and enhanced endogenous nitrosation, showing evidence of its extragastric site. New information is also provided on the determinants of N-nitrosamino acid excretion in men on a controlled low nitrate diet without smoking, conditions which reduce exogenous sources of nitrosating agents.

Adult↗

PubMed1995

Emergence of adenomatous aberrant crypt foci (ACF) from hyperplastic ACF with concomitant increase in cell proliferation.

To investigate the relationship between aberrant crypt foci (ACF) and colon neoplasia in colorectal carcinogenesis, we evaluated 433 ACF, which were collected from the grossly normal mucosa of surgical specimens from 57 patients with colorectal cancer. The ACF ranged in size from 3 to 412 aberrant crypts/focus. Large ACF (> or = 50 crypts/focus) comprised 25% of the total ACF studied. Histopathologically, 65% (67 of 103) of large ACF were diagnosed as hyperplasia, 10% (10 of 103) as adenoma, and 1% (1 of 103) as within normal colorectal mucosa. The remaining 24% (25 of 103) were diagnosed as "stage I abnormality crypts," which were characterized by their extension of the proliferative compartment to the surface of crypts but with no changes in the major site of proliferation, as designated by E. E. Deschner [Pathol. Annu., 18 (Part 1): 205-219, 1983]. Of the 25 stage I abnormality ACF, 7 ACF coexisted with hyperplasia. Of 10 adenomatous ACF, two coexisted with stage I abnormality crypts. A K-ras codon 12 mutation was identified in 85% (93 of 109) of large ACF. The proliferative activity of stage I crypts was significantly higher than that of hyperplastic crypts in the same ACF. These observations suggest that some hyperplastic ACF may develop into adenomatous ACF by way of stage I abnormality ACF with concomitant acquisition of higher proliferative activity through some genetic and/or epigenetic changes.

Adult↗

PubMed1995

Mutant K-ras in apparently normal mucosa of colorectal cancer patients. Its potential as a biomarker of colorectal tumorigenesis.

BACKGROUND: The best way to reduce the incidence of colorectal cancer mortality would be to prevent this cancer. However, none of the biomarkers proposed can accurately identify persons at increased risk of colorectal cancer or those at low risk. As a possible genetic biomarker, K-ras mutations, which are frequently found in colorectal cancers, were analyzed in apparently normal colorectal mucosa. METHODS: Nonneoplastic mucosa and tumor tissues were collected at surgery from 70 patients with colorectal cancer: one sample each from 50 patients (group A) and multiple samples from the other 20 patients (group B). Mutant K-ras codon 12 was analyzed by the enriched polymerase chain reaction (EPCR), by which one mutant can be detected among 10(3) to 10(4) normal alleles. RESULTS: Only with the aid of EPCR was mutant K-ras detected in nonneoplastic mucosa of nine patients (18%) in Group A and five patients (25%) in Group B. This increased incidence could be attributed to the multiple tissue sampling. The presence of mutant K-ras in nonneoplastic mucosae was not consistently correlated with that in the tumors. These findings suggest that the mutant K-ras identified in nonneoplastic mucosa actually represents de novo mutations, which may be initiated by different etiologic factors and at different times. CONCLUSION: Mutant K-ras detected in apparently normal mucosa should be a useful biomarker for identifying persons at higher risk of colorectal cancer. Our study also emphasizes the need for improving the method for sample collection to achieve true representation of the colorectal mucosa.

Adenocarcinoma↗

PubMed1995

Frequent and characteristic K-ras activation in aberrant crypt foci of colon. Is there preference among K-ras mutants for malignant progression?

BACKGROUND: To investigate very early lesion of colorectal cancer, K-ras activation and nuclear p53 accumulation were studied in aberrant crypt focus (ACF). METHODS: ACF were microscopically identified in grossly normal mucosa of patients with colorectal cancer who underwent surgery. Each ACF was microdissected from the surgical specimen and divided into two pieces, one for histologic and immunohistochemical examinations and the other for K-ras activation. K-ras mutations in codons 12 and 13 were sequenced after being screened by polymerase chain reaction amplification followed by restriction fragment length polymorphism analysis. Intranuclear accumulation of p53 protein was immunostained with the avidin-biotin complex method. RESULTS: ACF was predominantly distributed in the sigmoid colon and rectum, and its incidence was increased with age. Unexpectedly, ACF was very rare in colons of three patients with hereditary nonpolyposis colorectal carcinoma. K-ras mutations were detected in 58% (33 of 57) of ACF cases and in 44% (11 of 25) of adenocarcinoma cases. Although GTT mutation in codon 12 was predominantly observed in adenocarcinoma (10 of 11), GAT mutation (12 of 33) was as frequent as GTT mutation (11 of 33) in ACF together with mutation at codon 13 (7 of 33). No accumulation of p53 protein was found in any ACF. CONCLUSION: ACF were not diagnosed as neoplasms histologically, but they were considered to be neoplastic lesions, and K-ras activation is one of the key events to form ACF. The G-T substitution in K-ras codon 12 may undergo malignant growth easily compared with G-A substitution in colorectal carcinogenesis.

Adenocarcinoma↗

PubMed1995

Functional expression of three isoforms of human nitric oxide synthase in baculovirus-infected insect cells.

Complementary DNAs encoding three human isoforms (neuronal, inducible, and endothelial) of nitric oxide synthase were cloned into the baculovirus expression vector pVL1392/1393. Transfection of Sf-9 insect cells with the recombinant baculovirus resulted in the expression of high levels of nitric oxide synthases. The expressed proteins of neuronal and inducible nitric oxide synthase were predominantly soluble, whereas the endothelial enzyme was for the most part, particulate. Recombinant enzymes were purified with 2',5'-ADP Sepharose affinity chromatography. The effects of reference enzymatic inhibitors (NG-methyl-L-arginine, NG-nitro-L-arginine and N-iminoethyl-L-ornithine) on recombinant expressed proteins were not significantly different from native nitric oxide synthase enzyme preparations. L-aminoguanidine was found to be much less potent in inhibiting recombinant or native human inducible nitric oxide synthase compared to the murine isoform. These findings indicate previously unappreciated interspecies differences in the action of nitric oxide synthase enzymatic inhibitors. The functional expression of human nitric oxide synthase isoforms in a heterologous expression system allowed screening of novel inhibitors. Studies indicated that S-ethylisothiourea and 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine were potent novel inhibitors of human nitric oxide synthases.

Amino Acid Oxidoreductases↗

PubMed1995

Increases in pulmonary artery pressure and cardiac output due to the inhibition of nitric oxide synthesis during operative stress.

Pulmonary artery pressure (PAP), cardiac output (CO), and urinary nitrate, a stable endproduct of nitric oxide (NO), were measured pre- and postoperatively in eight patients who underwent esophagectomy for squamous cell carcinoma of the thoracic esophagus. A significant elevation of PAP and CO on the day of operation (POD 0) was accompanied by a low concentration of urinary nitrate. A reduction in PAP and CO, and an increase in nitrate to the preoperative levels, were found on PODs 2 and 3, respectively, but urinary nitrate decreased again after POD 3. Consequently, the changes in PAP and CO were closely correlated with the nitrate concentration. These results suggest that operative stress inhibited NO synthesis with a transitory induction of endogenous NO synthesis postoperatively.

Aged↗

PubMed1995

Frequent and characteristic K-ras activation and absence of p53 protein accumulation in aberrant crypt foci of the colon.

BACKGROUND/AIMS: The relationship of aberrant crypt foci (ACF) to colorectal carcinogenesis is still controversial. Histological examination and analyses of K-ras mutations and p53 gene expression were performed to characterize ACF. METHODS: ACF were identified microscopically in grossly normal colorectal mucosa. The ACF were separated into two pieces, one for histological and immunohistochemical examinations and the other for molecular analysis. K-ras mutations in codons 12 and 13 were analyzed by polymerase chain reaction amplification, followed by restriction fragment length polymorphism and sequencing analyses. Intranuclear p53 protein was immunostained by the avidin-biotin complex method. RESULTS: Histologically, elongation and apical branching of the crypts in ACF were striking. K-ras mutations were detected in 58% of ACF (33 of 57; 46% [26 of 57] in codon 12, and 12% [7 of 57] in codon 13) and in 44% of adenocarcinomas (11 of 25; all in codon 12). In ACF, GAT mutations (12 of 26) were as frequent as GTT mutations (11 of 26) in codon 12, although GTT mutations in codon 12 were predominant in adenocarcinomas (10 of 11). No accumulation of p53 protein was detected in any ACF, although it was detected in 52% (13 of 25) of the colorectal carcinomas. CONCLUSIONS: ACF do not seem histologically to be neoplasms, although genetically they are monoclonal lesions. K-ras mutation is critical in the formation of ACF, but p53 alteration could play a causal role in tumor progression.

Adult↗

PubMed1995

Expression of the inducible isoform of nitric oxide synthase in the central nervous system of mice correlates with the severity of actively induced experimental allergic encephalomyelitis.

A cytokine-mediated excessive increase in nitric oxide (NO) by macrophages or glial cells via an inducible isoform of NO synthase (iNOS) has been proposed to play an important role in demyelinating diseases. To further investigate the role of iNOS in demyelination, experimental allergic encephalomyelitis (EAE), a known animal model of multiple sclerosis (MS) in mice, was chosen in this study. A semiquantitative reverse transcriptase-polymerase chain reaction (RT/PCR) analysis revealed an increase in the mRNA levels of iNOS and cytokines known to induce iNOS or inflammatory cytokines (interleukin (IL)-1 alpha, IL-1 beta, IL-2, IL-6, interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha and TNF-beta) in the spinal cord corresponding to the severity of the disease without significant change in the mRNA levels of immunoregulatory cytokines (IL-4, IL-10 and transforming growth factor (TGF)-beta) during the course of EAE. An immunohistochemical examination of the spinal cord using an iNOS-specific antibody showed iNOS-positive cells to be mainly inflammatory cells with a higher frequency of iNOS-positive cells at the peak of EAE than in the early phase. These iNOS-positive cells at the peak appeared to be composed of infiltrating macrophages and most of them were located in the necrotic area. These results suggested that cytokine-induced excessive NO via iNOS by macrophages caused tissue damage in the central nervous system in EAE.

Animals↗

PubMed1995

Sympathetic preganglionic neurons contain nitric oxide synthase and project to the superior cervical ganglion: combined application of retrograde neuronal tracer and NADPH-diaphorase histochemistry.

Nitric Oxide (NO), which was initially identified as an endothelium-derived relaxing factor, has recently been demonstrated to be a neuronal messenger in central and peripheral nervous systems. In the present study, we examined the possibility of NO producing neurons in teh intermediolateral (IML) cell collum of the thoracic spinal cord (Th) project to the superior cervical ganglion (SCG). First, we observed the NADPH-diaphorase-positive/nitric oxide synthase (NOS)-immunoreactive neurons of the IML and the dorsal part of the central canal at the level of Th1-Th3, and numerous fiber-stainings in the superior cervical ganglion. Second, after injecting WGA-HRP (wheat germ agglutinin-horse radish peroxidase complex), a retrograde neuronal tracer, into the SCG, and developing WGA-immunohistochemistry and the NADPH-diaphorase histochemistry in the same sections, we detected double-labeled neurons in the IML. These findings provide evidence that sympathetic preganglionic NO producing neurons directly innervate to the SCG.

Amino Acid Oxidoreductases↗

PubMed1995

Inducible nitric oxide synthase in a human glioblastoma cell line.

Nitric oxide synthase (NOS) activity was induced in the cytosol of A-172 cells by treatment with lipopolysaccharide, tumor necrosis factor-alpha, and interferon-gamma. A 130-kDa human inducible NOS (iNOS) protein was detected with anti-rat iNOS antibody by western blot analysis. Northern blot analysis showed that the iNOS mRNA was approximately 4.5 kb, using a cDNA fragment for human iNOS, isolated from stimulated A-172 cells by reverse transcriptase-PCR, as a probe. The mRNA was induced by interferon-gamma at a trace level, and its expression was synergistically enhanced by simultaneous addition of lipopolysaccharide, tumor necrosis factor-alpha, and, to a larger extent, interleukin-1 beta. The mRNA expression was blocked by coincubation with actinomycin D or cycloheximide. Furthermore, by transfecting the mouse iNOS gene promoter into A-172 cells, transcriptional activation of the iNOS gene was detected in these cells upon stimulation with lipopolysaccharide and cytokines. The pattern of promoter activation correlated well with that of iNOS mRNA expression upon stimulation. These data indicate that expression of iNOS is transcriptionally regulated in A-172 cells. This process requires de novo protein synthesis with a mechanism similar to that in place in mouse macrophages.

Amino Acid Oxidoreductases↗