Biomedical subjects

N Harada

Publications and source records attributed to N Harada.

At least 379 records · Page 21Linked to original sources

PubMed1994

Case of 46,XX/47,XY, +21 chimerism in a newborn infant with ambiguous genitalia.

We describe whole-body chimerism in a newborn infant with small phallus, pseudo-vaginal perineal hypospadias, and a bifid scrotum containing gonads. The human testis determining factor gene (SRY) was detected by PCR amplification. GTG-banding chromosome analysis in peripheral blood lymphocytes and cultured fibroblasts derived from right cubital skin showed a 46,XX/47,XY, +21 karyotype. Their ratios in each cell line were 294:5 and 178:7, respectively. QFQ-banding chromosome analysis documented 3 heteromorphic satellites on trisomic chromosomes 21 in the 47,XY, +21 cell line and a homozygous satellite pattern in the 46,XX cell line. Heteromorphic patterns of chromosomes 4, 13, 14, and 22 were also different between the two cell lines. To our knowledge, such disomy/trisomy chimeras have not been described previously.

Cells, Cultured↗

PubMed1994

Novel exon 1 of the aromatase gene specific for aromatase transcripts in human brain.

Aromatase in the brain is supposed to participate in sexual differentiation of the brain. The presence of a brain-specific exon 1 and promoter in the human aromatase gene was examined in the 5' regions of aromatase cDNAs isolated from the amygdala and hypothalamus areas. The DNA sequences of all the isolated clones were different in the 5' regions encoded by exon 1 from those encoded by any alternative exons 1 of human aromatase gene. This unique sequence was also found in the clones isolated from a human genomic library and indicated to be that of a new exon 1. It involved putative TATA and CAAT boxes, Ad4 sequence, and the androgen responsive element in the promoter region. Reverse transcriptase-polymerase chain reaction analysis of mRNAs in various tissues revealed that expression of this unique species of aromatase mRNA is specific for the brain, in particular in the fetal brain. These show that the aromatase gene in the human brain is expressed by means of the brain-specific exon 1 and promoter, and may be responsible for sexual differentiation of the brain in the fetal period.

Amygdala↗

PubMed1994

Distribution and steroid dependence of aromatase enzyme immunoreactivity in limbic nuclei of the female musk shrew brain.

In the female musk shrew (Suncus murinus) neural aromatization of testosterone to estradiol is critical for the expression of sexual behavior. To localize the brain regions capable of aromatization, we used immunocytochemistry to map the distribution of aromatase enzyme. Aromatase immunoreactivity (AROM-ir) has a discrete distribution primarily limited to the lateral septum (LS), central nuclei of the amygdala (Ce) and the bed nucleus of the stria terminalis (BST). In these nuclei the intensity of immunoreactivity varies with hormonal status. Ovariectomy (OVX) significantly reduces the optical density of AROM-ir neurons in all nuclei as compared with brains of normal females. Combined OVX and adrenalectomy (ADX) further reduces optical density readings in AROM-ir cells in the LS and BST, as compared with readings from the brains of OVX animals. Normal and ovariectomized females implanted with testosterone had qualitatively equivalent AROM-ir. High levels of aromatase activity have been measured in the preoptic area and hypothalamus in a number of mammals, including the musk shrew. However, in this experiment AROM-ir was absent in these areas. We present several hypotheses to account for this discrepancy between previously reported biochemical data and these histological data. In summary, these data suggest that limbic nuclei may play a role in the expression of sexual behavior in female musk shrews.

Adrenal Glands↗

PubMed1994

Morphometric studies demonstrate that aromatase-immunoreactive cells are the main target of androgens and estrogens in the quail medial preoptic nucleus.

The volume and cytoarchitectonic organization of the sexually dimorphic medial preoptic nucleus (POM) of the quail are sensitive to plasma levels of testosterone (T). We previously showed that, in castrated quail, T or its estrogenic metabolite, estradiol (E2), increases the size of the large neurons located in the lateral part of POM. Embryonic treatments with estrogens are also known to affect permanently the size of these large neurons. Since the lateral POM also contains a dense population of aromatase-immunoreactive (ARO-ir) cells, and these are known to be a target for steroids, we hypothesized that the effects of steroids identified in previous experiments were primarily directed to these ARO-ir cells. This idea was tested in two experiments in which the size of these cells was measured in male quail under various endocrine conditions. In experiment 1, a detailed analysis of ARO-ir and of non-immunoreactive cells in the POM of adult, sexually mature males revealed that the immunoreactive perikarya are larger than the non-immunoreactive cells and that they constitute the vast majority of the large cells (area > 50 microns 2) in the POM. In experiment 2, it was shown that T and E2 actually increase the size of ARO-ir cells in the POM while the androgenic metabolite of T, dihydrotestosterone has no effect at this level. Taken together, these data suggest that the sex differences and the steroid-induced changes in cell size previously described in the study of POM sections stained for Nissl material largely concern aromatase-containing cells. Since aromatization of T plays a limiting role in the activation of male copulatory behavior, these changes may represent the morphological signature of the mechanisms causally involved in the control of this behavior.

Androgens↗

PubMed1994

Analysis of acute myeloid leukemia cells by flow cytometry, introducing a new light-scattering classification.

A combined flow-cytometric evaluation of light scattering and the immunophenotype of acute myeloid leukemia (AML) cells from 71 newly diagnosed consecutive patients was conducted. Light-scattering characteristic of AML cells examined by flow cytometry and multiple surface markers were also analyzed using the same samples, to enable a comparison with the French-American-British (FAB) classification. Our AML cases could be classified into three light-scattering classification (LSC) types according to their physical properties on flow cytometry. These were type A, where forward light scattering (FSC) of the leukemic cell population was larger than that of lymphocytes, while side light scattering (SSC) was the same or larger than that of lymphocytes but smaller than that of monocytes; type B, where FSC of the leukemic cell population was larger than that of lymphocytes and SSC spread toward that of monocytes; and type C, where both FSC and SSC of the leukemic cell population spread beyond those of monocytes. Although a clear relationship between the FAB classification and LSC classification by the light-scattering profile of AML was not established, we observed the following findings. The majority of cases were classified as type A (58%), while type B comprised 25% and type C comprised 17%. While CD7 expression on AML cells is considered to be an immature characteristic, CD7 was expressed more frequently among LSC type A cases. Furthermore, all but one of the FAB M1 cases were classified as type A. On the other hand, CD7 was not expressed on type C leukemic cells. The percentage of cases in which more than 60% of leukemic cells possessed another immature surface antigen, CD 34ö, was 13/18 (72%) among FAB M1 cases, much higher than among FAB M2 (35%) or FAB M4 (27%) cases. A negative correlation was observed between mature antigen CD33 and CD34 among the FAB M2 cases. The frequency of CD7 expression was 25% among the total cases, and CD7-positive cases were frequent among FAB M1 and M2, but not among FAB M3 cases. These findings concerning LSC and immunophenotyping indicate that the scattergram pattern analysis may contribute towards more precise immunophenotyping, in that it reflects the maturation stage of each AML case.

Acute Disease↗

PubMed1994

A point mutation at ATP-binding region of the ALD gene in a family with X-linked adrenoleukodystrophy.

A prenatal diagnosis was performed in a family with X-linked adrenoleukodystrophy (ALD). A fetus was at high risk of suffering the disease by segregation analysis and by very long chain fatty acid-CoA synthetase activity assay. A transition (G to A) at codon 617 of the candidate ALD gene was detected by reverse transcription PCR (RT-PCR) based sequencing of the fetal liver RNA. The mutation was located in highly conserved ATP-binding site in this gene and deduced amino acid transversion R617H was thought to be the cause of ALD in this family.

Adenosine Triphosphate↗

PubMed1994

Molecular analysis of a patient with Beckwith-Wiedemann syndrome, rhabdomyosarcoma and renal cell carcinoma.

We described a patient with Beckwith-Wiedemann syndrome associated with rhabdomyosarcoma (RMS), and renal cell carcinoma (RCC). Karyotypes of peripheral lymphocytes and RMS cells were normal. DNA analyses showed maternal loss of heterozygosity (LOH) at 11p15 region in RMS but not in RCC. The insulin-like growth factor II gene (IGF2) was found to be expressed at a moderate level in RMS but not in RCC by in situ hybridization. Each of parental allele-derived IGF2 transcript was detected in RCC, while maternal allele-derived transcript was weak in RMS because of maternal LOH. These results suggest that (1) loss of imprinting (LOI) of IGF2 might be responsible for BWS, (2) on the other hand, LOI itself might not induce tumor occurrence in tissues where the control of tissue-specific expression of IGF2 is maintained, (3) increased expression of IGF2 due to maternal loss of a putative controller gene for IGF2 at 11p15 might predispose to sustaining tumorigenic mutations and tumor progression, (4) loss of a putative onco-suppressor gene at 11p15 might induce RMS occurrence. The cause of RCC was thought to be different from that of RMS.

Abdominal Neoplasms↗

PubMed1994

Direct inhibitory effect of atrial natriuretic peptide on isolated caecal circular smooth muscle cells via soluble guanylate cyclase.

Atrial natriuretic peptide (ANP) relaxes the vascular smooth muscle via particulate guanylate cyclase. Smooth muscle cells isolated from the caecal circular muscle layer of the guinea pig were used to examine the direct inhibitory effect of ANP on those cells. The role of adenylate cyclase, particulate guanylate cyclase, and soluble guanylate cyclase in the direct inhibitory effect of ANP on contraction of this muscle cell induced by carbachol was investigated. ANP inhibited the contractile response produced by 10(6)M carbachol in a concentration-dependent manner, with an IC50 value of 8nM. An inhibitor of adenylate cyclase (2',5'-dideoxyadenosine) and two inhibitors of particulate guanylate cyclase (HS-142-1, and PMA) had no significant effect on the relaxation produced by ANP. In contrast, an inhibitor of soluble guanylate cyclase (LY83583) significantly and completely inhibited the relaxation produced by ANP. This is the first report demonstrating the direct inhibitory action of ANP on the isolated caecal smooth muscle cells via soluble guanylate cyclase, which differs from the intracellular mechanism responsible for the relaxation of vascular smooth muscle produced by ANP.

Aminoquinolines↗

PubMed1994

Aromatase-immunoreactive cells in the quail brain: effects of testosterone and sex dimorphism.

We previously demonstrated that testosterone (T) increases aromatase activity (AA) and that AA is sexually dimorphic (males > females) in the quail preoptic area (POA). The precise anatomical localization of these effects is, however, impossible to obtain by biochemical assays even when samples are dissected by the Palkovits punch technique. We were recently able to set up an immunocytochemical (ICC) procedure that permits visualization of aromatase-immunoreactive (ARO-ir) cells in the quail brain. This showed that the ARO-ir cells of the quail POA actually outline the sexually dimorphic medial preoptic nucleus (POM). This ICC technique was used here to analyze the sex dimorphism of the quail preoptic aromatase and the localization of T effects on ARO-ir cells. In Experiment 1, the number of ARO-ir cells was counted in one section every 100 microns throughout the rostral to caudal extent of the POM of castrated birds that had been treated with increasing doses of T (5, 10, or 20 mm long Silastic implants). These T-treatments produced a dose-related increase in the sexual behavior of the birds and they increased the number of ARO-ir cells in POM, in the septal regions, and in the bed nucleus of the stria terminalis (BNST). The effect had a particularly large amplitude in the part of the POM located under the anterior commissure (AC). In Experiment 2, the same procedure was used to reanalyze the sex difference of the preoptic aromatase system. This showed that the POM of adult males contains more stained cells than the POM of females but only in a restricted region located just under and rostral to the AC. No significant sex difference was observed in the septum or in the BNST. In Experiment 3, the number of ARO-ir cells was determined in the POM of males and females that had been gonadectomized and treated with a same dose of T (40 mm implants). No sex difference in the number of ARO-ir cells could be detected in these conditions. This suggests that the sex difference in AA that had been previously observed in T-treated birds results either from a difference in aromatase concentration or activity in a similar number of positive cells or from a difference in the number of ARO-ir cells that is very discrete from the anatomical point of view.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1994

Immunolocalization of aromatase and other steroidogenic enzymes in human breast disorders.

Numerous studies have shown that human breast cancer tissue has the potential to synthesize estrogen through aromatization, which may act as a local growth factor of hormone-dependent cancer cells. This study was performed to localize the site of aromatization in human breast disorders by immunohistochemistry and correlate the findings with steroid receptors, clinicopathological findings, and other steroidogenic enzymes. Specimens from 60 cases of breast disorders, including 33 cases of breast cancer and 27 cases of benign proliferative disorders, were studied immunohistochemically for aromatase. In the carcinoma cases estrogen receptor (ER) and progesterone receptor (PgR) status was determined by enzyme immunoassay and immunohistochemistry, and other steroidogenic enzymes, including P450scc (side-chain cleavage), 3 beta HSD (hydroxysteroid dehydrogenase), and P450c17, were immunolocalized. Aromatase was immunolocalized in interstitial cells and adipocytes as well as other cells in both benign and malignant breast tissues. However, strong immunoreactivity was observed in adipocytes adjacent to carcinoma in all carcinoma cases and in interstitial or stromal cells around carcinomatous glands in 20 carcinoma cases. Intratumoral staining for aromatase did not correlate significantly with age, clinical stage, histopathological type, lymph nodes metastasis, or ER and PgR status. P450scc and 3 beta HSD were focally observed in 18 and 12 cases of carcinoma, respectively, but P450c17 was never observed. Aromatase expression in stromal or interstitial cells, including adipocytes, in breast cancer may be induced by carcinoma cells and locally synthesized estrogens could function as paracrine hormones. Intratumoral aromatase in human breast neoplasms correlated with malignant phenotypes but not with ER status or prognostic parameters, suggesting that other synthetic systems probably generate any biologically significant locally synthesized estrogens in hormone-dependent breast malignancy.

3-Hydroxysteroid Dehydrogenases↗

PubMed1994

Detection and localization of interleukin-8 mRNA and protein in human placenta and decidual tissues.

It has been reported that interleukin-8 (IL-8) is secreted from the placental and decidual tissues and that IL-8 levels in the amniotic fluids are significantly elevated by chorioamnionitis or labor pain. The present study was aimed at defining the localization of IL-8 mRNA as well as IL-8 protein at the feto-maternal interface using in situ hybridization and immunohistochemical staining. Both IL-8 mRNA and protein were localized in cytotrophoblast, syncytiotrophoblast and Hofbauer cells of the placenta, decidual stromal cells, decidual lymphocytes and endometrial gland cells. IL-8 secretion from glandular cells has not previously been reported. In addition, we confirmed IL-8 mRNA expression and secretion of IL-8 by an endometrial cancer cell line (Ishikawa) using the reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) methods, respectively.

Base Sequence↗

PubMed1994

Cell type- and region-specific expression of aromatase mRNA in cultured brain cells.

The expression of aromatase mRNA in cultured mouse brain cells was measured by a quantitative reverse transcription-PCR method using an internal standard. Aromatase mRNA was expressed in the cultured neurons prepared from diencephalon at 0.037 +/- 0.005 attomol/microgram total RNA. However, the mRNA was detected in neither the neurons from cerebral cortex nor astrocytes. These results demonstrate that expression of aromatase mRNA is regulated in cell type- and region-specific manners in cultured brain cells. The aromatase mRNA levels in neurons obtained from diencephalon were not affected by administration of testosterone, estradiol, dexamethasone, forskolin, or 12-O-tetradecanoyl 13-acetate. The results are in apparent disagreement with previous reports concerning regulation by androgens of brain aromatase activity in vivo and may suggest that aromatase expression in brain neurons is not directly induced by androgens. Androgen induction of brain aromatase may be mediated by several steps including cell-cell (neuron-neuron and/or neuron-glia) interaction.

Animals↗

PubMed1994

Intravital microreflectometry of individual pial vessels and capillary region of rat.

A microscopic reflectance spectrophotometer was constructed to obtain the spectra of single pial vessels and of a region containing only capillaries (capillary region). The difference in the oxygen saturation (SO2) of hemoglobin between the regional arteriole and venule [R(A - V)] and that between the regional arteriole or capillaries [R(A - C)] were calculated. The reduction of cytochrome aa3 was also estimated in the capillary region. This method was applied to the brain surface of spontaneously breathing rats subjected to hypoxic and anemic hypoxia. On decreasing the inhaled O2 from 100 to 15%, elevation of R(A - V) and R(A - C) with slight arteriolar dilatation (though statistically not significant) was observed. Below 10% O2 (especially at 4 and 3% O2), the R(A - V) and R(A - C) decreased in spite of significant arteriolar dilatation with progressive reduction of cytochrome aa3, indicating suppression of oxygen transport to mitochondria. In the case of hemodilution down to 37% hematocrit (Ht), elevation of R(A - V) and R(A - C) occurred with a slight tendency toward arteriolar dilatation. Below 32% Ht, the R(A - V) decreased but the R(A - C) remained steady, while reduction of cytochrome aa3 progressed. Altogether, the SO2 in the capillary region decreased and the reduction of cytochrome aa3 progressed with the decline of arteriolar O2 supply in both hypoxic and anemic hypoxia.

Anemia↗

PubMed1994

ESR spectral transition by arteriovenous cycle in nitric oxide hemoglobin of cytokine-treated rats.

Nitric oxide (NO) generation was induced in rats by Escherichia coli lipopolysaccharide (LPS) as detected by electron spin resonance (ESR) signals of NO hemoglobin (HbNO). However, there were inconsistencies in ESR spectral shape among them. We have therefore carried out a systematic study to clarify the in vivo spectral changes. First, the spectra of the alpha-NO heme species had the distinct three-line hyperfine structure in venous blood but not in arterial blood in all rats treated with tumor necrosis factor, interleukin-1, and/or LPS, and methemoglobin was not detected at the g = 6 (high-spin methemoglobin) region. Second, when the treated rats died, the three-line hyperfine structure was very distinct even in arterial blood. Third, even if HbNO was formed by injection of nitrite to rats, the three-line hyperfine structure of HbNO in venous blood was more marked than that in arterial blood, independent of the appearance of the methemoglobin signal. Fourth, an ex vivo study using whole blood demonstrated that the three-line hyperfine structure intensified lineally when O2 saturation of hemoglobin decreased but disappeared on reoxygenation of hemoglobin. These results directly demonstrate in vivo quaternary structural transition of the hemoglobin tetramer from the high-affinity state in the arterial cycle to the low-affinity state in the venous cycle. The transition makes the diverse ESR spectra of HbNO in vivo.

Animals↗

PubMed1994

Stromal spindle cells contain aromatase in human breast tumors.

In situ synthesis of estrogens by breast cancer tissue provides a potential explanation for the high concentrations of estradiol in mammary neoplasms in postmenopausal women. A major metabolic pathway for estrogen biosynthesis is the conversion of androstenedione to estrone via the enzyme aromatase. Biochemical studies have demonstrated aromatase in tumor tissue, but at relatively low and not clearly biologically significant levels. The present study tested the hypothesis that tumor levels of aromatase, albeit low, could be biologically important if present in high concentrations in focal clusters of specific cell types. A pilot study used an immunohistochemical method in frozen sections of fresh breast tumors as an optimal means to detect aromatase. Twelve of 18 tumors contained aromatase-positive cells, some with highly intense staining. A follow-up study then attempted to precisely define the types of cells containing aromatase and correlate the immunohistochemical findings with biochemical aromatase activity. A modified H-score (histological scoring system) was used to semiquantitate the amount of aromatase staining in tumor epithelial, stromal spindle, stromal inflammatory, and normal breast epithelial cells. We found that immunohistochemical staining for aromatase predominated in stromal spindle cells with a median H-score of 13, whereas tumor epithelial, stromal inflammatory, and normal breast elements contained lesser amounts (median H-scores of 4.8, 0.03, and 0.5, respectively). The H-score for stromal spindle cells, but not those for other cell types, correlated highly with the biochemical aromatase assay (P < 0.01). Using a cut-off parameter estimated by a sensitivity/specificity (receiver operating curve) analysis, 62% of tumors were classified as aromatase positive based on stromal spindle cell staining. A similar number were also positive by biochemical assay, with concordance between the two methods of 77%. These observations provide substantial evidence for the presence of aromatase in human breast tumors, particularly in stromal spindle cells, and support the biological importance of aromatase for in situ production of estradiol.

Aromatase↗

PubMed1994

Intracellular calcium changes by hyposmotic activation of cochlear outer hair cells in the guinea pig.

During continued exposure to a hypotonic solution, isolated outer hair cells (OHCs) from the guinea pig cochlea showed a regulatory volume decrease (RVD) after initial cell swelling. In the absence of extracellular Ca2+, RVD was significantly inhibited. Using Ca(2+)-sensitive dye fura-2, accompanying changes of the intracellular Ca2+ concentrations ([Ca2+]i) of OHC were investigated. Hyposmotic activation resulted in a [Ca2+]i increase associated with cell shortening and swelling. In a Ca(2+)-free solution, [Ca2+]i was not significantly increased during hyposmotic activation although shortening and swelling of the OHC was observed. These results suggest that the increase in [Ca2+]i during hyposmotic activation is mainly based on an influx or extracellular Ca2+ which precedes the RVD.

Animals↗

PubMed1994

Glutamate-induced intracellular Ca2+ elevation in isolated spiral ganglion cells of the guinea pig cochlea.

Intracellular calcium concentrations ([Ca2+]i) in acutely isolated spiral ganglion cells (SGCs) of the guinea pig cochlea were measured using digital imaging microscopy and the Ca(2+)-sensitive fluorescence dye fura-2. L-glutamate increased [Ca2+]i in SGCs with neuritic processes but did not lead to an increase in [Ca2+]i in SGCs without neuritic processes. The depolarization induced by high K+ (150 mM) solution increased [Ca2+]i in both SGCs, with and without neuritic processes. The L-glutamate-induced [Ca2+]i elevation was abolished in the absence of extracellular Ca2+. We thus propose that the increase of [Ca2+]i during L-glutamate stimulation is mainly related to an influx of extracellular Ca2+. The excitatory amino acids, probably L-glutamate, may function as a neurotransmitter of the hair cell-afferent nerve synapse in the guinea pig cochlea.

Animals↗

PubMed1994

Correlations among examination findings, subjective symptoms and classification of stages in vibration syndrome.

From data collected in 1966, 1973, 1986 and 1989, we analyzed the correlations among examination findings, subjective symptoms, stages in the disorder of vibration syndrome and vibration exposure. As vibration syndrome progressed and vibration exposure accumulated, abnormality of examination findings, the prevalence of vibration induced white finger (VWF), numbness (N) and other subjective symptoms increased. Abnormality of examination findings and prevalence of subjective symptoms in the VWF(+) N(+) group were greatly different from those in the VWF(-) N(-) group. Both findings and symptoms showed closer correlations with VWF(+) than with N(+). Stages in the disorder traced the progression from VWF(-)N(-) to VWF(-)N(+), then to VWF(+)N(+/-), and finally to VWF(+/++)N(+/++). Pathophysiologically, it seems that VWF(+) and N(+) relayed many more cold and pain signals from the hand to the central nervous system. Such signals may activate autonomic nervous activity. In the recovery, subjective symptoms correlated more closely with N(+) than with VWF(+). This is because VWF involves the autonomic nervous system's hyperactivity and hypersensitivity to cold, both of which subside gradually in the recovery; N, however, involves pathological changes in nerve tissue which are irreversible.

Adult↗