Biomedical subjects

Nobuhiro Harada

Publications and source records attributed to Nobuhiro Harada.

At least 37 records · Page 2Linked to original sources

PubMed2004

Extrapleural thoracoscopic excision of a mediastinal bronchogenic cyst in a patient with dense pleural adhesions.

We report the case of a 70-year-old man with dense pleural adhesions who underwent an excision of a mediastinal bronchogenic cyst by video-assisted thoracoscopic surgery (VATS) through an extrapleural approach. The VATS approach for the diagnostic and therapeutic excision of a mediastinal tumor is easier and safer than an intrapleural approach if a patient has dense pleural adhesions.

Aged↗

PubMed2004

[Aromatase deficiency].

Aromatase, a key enzyme in estrogen synthesis, is tissue-specifically regulated in various tissues and plays an important role through endocrine and intracrine estrogen production in various physiological functions. Therefore, aromatase deficiency caused crucial impairments of physiological functions in the gonadal tissues as well as extra-gonadal tissues. Because aromatase is protective for androgenic exposure by catabolizing, virilization of a pregnant mother and pseudohermaphroditism of a baby girl consequently result from the deficiency. Similarly, because aromatase is productive for a multifunctional physiological factor, estrogens, impaired metabolisms of bone, carbohydrate, and fat etc. result from the deficiency. We discuss the etiology, clinical symptoms, and therapeusis by classifying it into two types of complete and incomplete aromatase deficiencies.

Androgens↗

PubMed2003

Adult male rat hippocampus synthesizes estradiol from pregnenolone by cytochromes P45017alpha and P450 aromatase localized in neurons.

In adult mammalian brain, occurrence of the synthesis of estradiol from endogenous cholesterol has been doubted because of the inability to detect dehydroepiandrosterone synthase, P45017alpha. In adult male rat hippocampal formation, significant localization was demonstrated for both cytochromes P45017alpha and P450 aromatase, in pyramidal neurons in the CA1-CA3 regions, as well as in the granule cells in the dentate gyrus, by means of immunohistochemical staining of slices. Only a weak immunoreaction of these P450s was observed in astrocytes and oligodendrocytes. ImmunoGold electron microscopy revealed that P45017alpha and P450 aromatase were localized in pre- and postsynaptic compartments as well as in the endoplasmic reticulum in principal neurons. The expression of these cytochromes was further verified by using Western blot analysis and RT-PCR. Stimulation of hippocampal neurons with N-methyl-d-aspartate induced a significant net production of estradiol. Analysis of radioactive metabolites demonstrated the conversion from [(3)H]pregnenolone to [(3)H]estradiol through dehydroepiandrosterone and testosterone. This activity was abolished by the application of specific inhibitors of cytochrome P450s. Interestingly, estradiol was not significantly converted to other steroid metabolites. Taken together with our previous finding of a P450scc-containing neuronal system for pregnenolone synthesis, these results imply that 17beta-estradiol is synthesized by P45017alpha and P450 aromatase localized in hippocampal neurons from endogenous cholesterol. This synthesis may be regulated by a glutamate-mediated synaptic communication that evokes Ca(2+) signals.

Animals↗

PubMed2003

A benzimidazole fungicide, benomyl, and its metabolite, carbendazim, induce aromatase activity in a human ovarian granulose-like tumor cell line (KGN).

Endocrine disruptor chemicals are known to cause a range of abnormalities in sexual differentiation and reproduction. One mechanism underlying such effects may be via alteration of aromatase activity, which is responsible for estrogen production. A good screening system for identifying endocrine disruptors has long been desired. We have recently established a human ovarian granulosa-like tumor cell line, KGN, which possesses a relatively high level of aromatase expression and is considered a useful mammalian model for investigating the in vitro effects of various chemicals on aromatase activity. In this study we screened 55 different candidate chemicals for endocrine disruptors by assaying aromatase activity. Only benomyl, known as both a benzimidazole fungicide and a microtubule-interfering agent, was found to induce aromatase activity in association with increased levels of aromatase mRNA in KGN cells. The effect of benomyl was presumed to be mediated by its metabolite carbendazim, because it produced an effect equivalent to that of benomyl. The mechanism underlying the benomyl-induced increase in aromatase activity appears independent of the cAMP-protein kinase A pathway. Treatment with taxol, another class of microtubule-interfering agents, also caused induction of aromatase in KGN cells. Both benomyl and taxol changed KGN cell morphology, including the development of cell roundness and a disorganized network of microtubules. These results indicate that benomyl is a potential endocrine disruptor that provides a novel estrogenicity and operates through a microtubule-interfering mechanism.

Aromatase↗

PubMed2003

Intratumoural mRNA expression of genes from the oestradiol metabolic pathway and clinical and histopathological parameters of breast cancer.

INTRODUCTION: The expression of the oestrogen receptor (ER) is one of the more important clinical parameters of breast cancer. However, the relationship between the ER and its ligand, oestradiol, and the enzymes that synthesise it are not well understood. The expression of mRNA transcripts of members of the oestradiol metabolic and signalling pathways including the ER was studied in detail. METHOD: mRNA transcripts for aromatase (CYP19), 17-beta-hydroxysteroid dehydrogenase I, 17-beta-hydroxysteroid dehydrogenase II, ERalpha, ERbeta, steroid sulfatase (STS), oestradiol sulfotransferase (EST), cyclin D1 (CYCLD1) and ERBB2 were fluorometrically quantified by competitive RT-PCR using an internal standard in 155 breast carcinomas. In addition, the transcripts of CYP19 were analysed for alternative splicing/usage of exon 1 and an alternative poly A tail. RESULTS: A great variability of expression was observed, ranging from 0 to 2376 amol/mg RNA. The highest levels were observed for STS and EST, and the lowest levels (close to zero) were observed for the 17-beta-hydroxysteroid dehydrogenase isoenzymes. The levels of mRNA expression were analysed with respect to clinical and histopathological parameters as well as for disease-free survival. High correlation of the mRNA expression of STS, EST and 17-beta-hydroxysteroid dehydrogenase in the tumours suggested a common regulation, possibly by their common metabolite (oestradiol). Hierarchical clustering analysis in the 155 patients resulted in two main clusters, representing the ERalpha-negative and ERalpha-positive breast cancer cases. The mRNA expression of the oestradiol metabolising enzymes did not follow the expression of the ERalpha in all cases, leading to the formation of several subclasses of tumours. Patients with no expression of CYP19 and patients with high levels of expression of STS had significantly shorter disease-free survival time (P > 0.0005 and P < 0.03, respectively). Expression of ERbeta mRNA was a better prognostic factor than that of ERalpha in this material. CONCLUSION: Our results indicate the importance of CYP19 and the enzymes regulating the oestrone sulfate metabolism as factors of disease-free survival in breast cancer, in addition to the well-known factors ER and ERBB2.

Adult↗

PubMed2003

Specific innervation of aromatase neurons by substance P fibers in the dorsal horn of the spinal cord in quail.

The enzyme aromatase catalyzes the production of estrogens in the dorsal horn of the spinal cord where most of the nociceptive primary afferent fibers terminate. Numerous estrogen receptors are present in this area and the control of spinal aromatase activity is thought to play an important role in the estrogenic control of nociception. The coexistence of aromatase and nociceptive terminals suggests a role for aromatase cells in pain-related processes, but whether terminals releasing nociceptive neuropeptides (e.g., substance P) actually contact aromatase neurons is unknown and the factors that control spinal aromatase activity have not yet been identified. In the present study we analyzed by double-label immunocytochemistry the distribution in the Japanese quail spinal cord, of aromatase and of substance P or its receptor (neurokinin 1 receptor). All antigens were mainly localized in laminae I and II as observed in mammals. Most aromatase neurons were colocalized with neurokinin 1 receptors and were in close apposition with substance P-immunoreactive fibers. These results suggest that aromatase neurons are responsive to noxious stimulation and may participate in the control of nociception. Furthermore, spinal aromatase activity could be controlled by substance P through a regulation of the aromatase gene transcription as reported for the mouse diencephalon and/or through neurokinin 1 receptor-dependent phosphorylation of the aromatase protein.

Androgens↗

PubMed2003

Age-related neurodegenerative changes in the central nervous system of estrogen-deficient follitropin receptor knockout mice.

Age-related neurodegenerative conditions are characterized by neuronal death and degeneration that lead to a progressive functional decline. Among the factors influencing degenerative processes during aging are altered levels of neurotrophic ovarian steroid 17beta-estradiol (E2). The follitropin receptor knockout (FORKO) female mouse displays hormonal imbalance characterized by very low levels of circulating E2 and high levels of testosterone. FORKO mice (24 days and 20 months) were used to investigate structural and functional changes in the central nervous system. We now show that the lifelong depletion of the sex hormone E2 in female FORKO mice correlates with abnormal behavior associated with defined alterations in brain morphology early in life, especially in aged animals. Immunohistochemical studies showed significant increases in the size and number of immunoreactive glial fibrillary acidic protein glial cells found in several brain regions (cortex and hippocampus) and a dramatic decline in estrogen receptors alpha and beta in the amygdala of FORKO females. These changes were associated with increased signs of anxiety in these animals. In the present study, we provide evidence that the chronic depletion of sex hormone E2 from early development leads to neural impairments in adult and aged FORKO mice that are associated with hypertrophy of glial cells, cell loss in distinct brain regions, and abnormal behavior. We suggest that the hormonal imbalance found in the female FORKO mouse provides an experimental paradigm for the study of morphological correlates of the behavioral changes that often accompany menopause in women.

Age Factors↗

PubMed2003

Neurological effects of aromatase deficiency in the mouse.

In the brain, the conversion from androgen into estrogen is an important process for the differentiation of the brain function in male rodents. The aromatase is expressed in some nucleus of the brain. To assess the functional significance of the aromatase gene in development and activation of sex-specific behavior, we analyzed behavioral phenotypes of the aromatase knockout (ArKO) male mice. ArKO males obviously decreased their fertility and showed deficits in male sexual behavior including mount, intromission and ejaculation. Noncontact penile erection was not significantly affected by defect of the aromatase gene. A reduction of aggressive behavior against male intruders was also observed in ArKO males, while they tend to exhibit aggression toward estrous females during male copulatory tests. Moreover, the infanticide toward the pups was observed in the ArKO males, whereas characteristic parental behavior, but not infanticide was observed in wild-type males. These results indicate that aromatase gene expression is a critical step not only for motivational and consummatory aspects of male sexual behavior, but also for aggressive and parental behaviors in male mice.

Aggression↗

PubMed2003

Unique regulation of expression of human aromatase in the placenta.

The expression of human placental aromatase is transcriptionally regulated through the promoter region of exon 1a (I.1) of the gene. We examined the transcriptional regulation by using human choriocarcinoma-derived JEG-3 cells which also express aromatase mRNA transcribed under the control of the placenta-specific promoter of the exon 1a. Aromatase in the cells was induced by forskolin (cAMP) and phorbol ester (TPA) in both levels of the activity and the mRNA. However, any elements responsible for the cAMP-responsiveness have not yet identified. To identify and characterize the specific elements, CAT assay of the placenta-specific promoter was performed. We reconstructed an 11.5 kb gene structure consisting of exons 1a (I.1), 1b (I.4), 1c (I.3), and 1d (PII) and their proximal promoter regions to mimic the native structure of human aromatase gene and performed a promoter assay by the transient expression of a CAT reporter carrying the mini-gene structure. The construct was transcribed from exon 1a in JEG-3 cells and exon 1b in HepG2 cells to produce tissue-specific mRNAs from the exons 1-CAT hybrid gene, indicating that the mini-gene structure contained promoter regions essential for the tissue-specific expression. However, unexpectedly exons 1-CAT hybrid mRNA in JEG-3 cells was not induced by forskolin. Then, we prepared JEG-3 cells transformed by incorporation of the exons 1-CAT hybrid gene into the chromosomal DNA. The cells stably expressed the hybrid reporter gene which was transcribed from exon 1a and induced by forskolin and TPA. These results suggest that enhancers on the promoter regions of exons 1b, 1c, and 1d might interact with a transcriptional machinery of exon 1a in the induction by forskolin and TPA. Finally, a beta-galactosidase gene connected with the 11.5 kb gene structure was introduced into mouse eggs to produce transgenic mice. The hybrid gene was transcribed from exon 1c in the gonadal tissues of all lines of the transgenic mice in accordance with the tissue-specificity of human aromatase gene, whereas it was not transcribed from exon 1a, but from exons 1b and 1c in the all placentae. The results suggest that the mouse placenta might lack in the transcriptional elements or factors essential for the placenta-specific expression of human aromatase gene.

Animals↗

PubMed2003

Alteration in sex-specific behaviors in male mice lacking the aromatase gene.

Brain aromatase (P450arom) is a key enzyme in estrogen biosynthesis from testicular androgens. This local aromatization in neural tissues is thought to be an important process for sexual differentiation and activation of sexual behavior in male rodents. To determine the functional significance of the aromatase gene in development and activation of sex-specific behavior, we analyzed a series of behavioral profiles in gonadally intact male mice with targeted disruption of exons 1 and 2 of the aromatase gene (ArKO). In most cases, ArKO males were infertile and showed deficits in male sexual behavior including mount, intromission and ejaculation. Noncontact penile erection was not significantly affected by deletion of the aromatase gene. A great reduction of aggressive behavior against male intruders was also observed in ArKO males, while they tended to exhibit aggression toward estrous females during male copulatory tests. Furthermore, 73% of ArKO males showed infanticide toward pups, whereas characteristic parental behavior, but not infanticide, was observed in wild-type males. These results support the brain aromatization hypothesis and indicate that aromatase gene expression is a critical step not only for motivational and consummatory aspects of male sexual behavior, but also for aggressive and parental behaviors in male mice.

Aggression↗

PubMed2002

The aromatase knock-out mouse provides new evidence that estradiol is required during development in the female for the expression of sociosexual behaviors in adulthood.

We used estrogen-deficient aromatase knock-out (ArKO) mice to determine whether estrogens contribute to the development of the brain and behavior in females. Female mice of three different genotypes [i.e., wild type (WT), heterozygous (HET), and homozygous (ArKO)] were ovariectomized in adulthood and subsequently tested for odor preferences (choice: intact male vs estrous female) in a Y-maze. When treated with testosterone, ArKO females spent significantly less time sniffing odors (both volatile and nonvolatile) from either male or female stimuli compared with WT and HET females. When given direct access to anesthetized stimulus animals or when given a choice between odor and visual cues from both stimulus animals, ArKO females continued to spend less time investigating the stimuli compared with WT and HET females. These defects in olfactory investigation of ArKO females were partially corrected with estradiol treatment in adulthood. Estradiol-treated ArKO females no longer differed from WT and HET females in the time spent investigating either nonvolatile odors or the anogenital region of anesthetized animals. However, ArKO females still investigated volatile odors and/or visual cues less than WT and HET females. Sexual receptivity was severely impaired in ArKO females after treatments with estradiol and progesterone that successfully induced receptivity in WT and HET females. Furthermore, ArKO females showed diminished levels of male sexual behaviors, whereas WT and HET females readily mounted an estrous female. Together, these findings demonstrate that estrogen is required for normal female development. The concept that the female brain develops in the absence of any hormonal stimulation should therefore be reconsidered.

Animals↗

PubMed2002

Estrogen-dependent regulation of the expression of hepatic Cyp2b and 3a isoforms: assessment using aromatase-deficient mice.

The role of estrogen in the expression and induction of hepatic Cyp2b and Cyp3a isoforms was studied using mice [Ar (-/-) mice] lacking aromatase, a key enzyme for estrogen biosynthesis. The expression of P450s was determined by reverse transcription-polymerase chain reaction, immunoblotting, and measuring testosterone 6beta- and 16alpha-hydroxylase activity as markers. Basic expression of Cyp3a11 mRNA and protein was seen in both sexes of Ar (+/+) mice. Disruption of the aromatase gene caused an increase in the expression of Cyp3a11 protein, although the mRNA level remained unchanged. Female-specific Cyp3a41 disappeared in Ar (-/-) mice, and this could not be reversed by administration of exogenous beta-estradiol to adult knockout mice. The constitutive expression of female-specific Cyp2b9 also disappeared on disrupting the aromatase gene. However, in clear contrast to Cyp3a41, some individual Ar (-/-) mice exhibited expression of this form following treatment with exogenous beta-estradiol. Disruption of the aromatase gene had no effect on PB-mediated induction of Cyp2b10 or on the noninducible nature of Cyp2b9, Cyp3a11, and Cyp3a41. These results suggest that (1) Cyp3a11 is suppressed by estrogen; (2) the expression of female-specific Cyp3a41 is programmed by neonatal and/or infantile exposure to estrogen; (3) maintenance of the expression of female-specific Cyp2b9 requires estrogen in adults; and (4) endogenous estrogen plays little, if any, role in the mechanism by which PB induces Cyp2b10.

Animals↗

PubMed2002

Less-invasive thoracic aortic aneurysm repair.

To minimize surgical trauma, we performed graft replacement of a descending aortic aneurysm through a minithoracotomy (12 cm) with the use of thoracoscopy and special vascular clamps. Contrast magnetic resonance angiography can be useful for preventing postoperative paraplegia by revealing the Adamkiewicz artery. The patient was satisfied with the postoperative comfort and good cosmetic result. Further refinement of the technique and instrumentation would make this technique a valuable adjunct to conventional thoracic aortic surgery.

Aged↗

PubMed2002

Distribution of aromatase immunoreactivity in the forebrain of red-sided garter snakes at the beginning of the winter dormancy.

Until recently, it has been difficult to identify the exact location of aromatase containing cells in the brain. The development of new antibodies has provided a sensitive tool to analyze the distribution of aromatase immunoreactive (ARO-ir) material at a cellular level of resolution. In the present study we examined, for the first time, the distribution of ARO-ir cells in the brain of a reptile, the red-sided garter snake, at the beginning of the winter dormancy. ARO-ir cells were found at all rostro-caudal levels in the red-sided garter snake brain. Although weakly stained cells were distributed throughout the brain, more intensely immunoreactive cells were primarily concentrated in the preoptic area, anterior hypothalamus, septum and nucleus sphericus. Although androgens are elevated upon emergence from hibernation in the male red-sided garter snake, initiation of courtship behavior appears to be independent of direct androgen control. To date, the only known stimulus found to initiate courtship is a period of low temperature dormancy followed by exposure to warm temperatures. Circumstantial data, however, suggest an indirect role in the activation of male copulatory behavior for estrogenic metabolites of testosterone produced in the brain by aromatization during the winter dormancy. This study provides the first documentation of the distribution of ARO-ir cells in a reptilian species and demonstrates that while the aromatase enzyme occurs in most regions of the brain, the ARO-ir cells that appear to contain the highest concentration of enzyme are clustered in brain areas classically associated with the control of courtship behavior and mating in vertebrates. These data are consistent with the idea that estrogens locally produced in the brain may participate in some way to the activation of sexual behavior in this species also. This notion should now be experimentally tested by analyzing annual changes in aromatase activity and immunoreactivity and assessing the effects of pharmacological blockade of the enzyme activity at different times of the year.

Animals↗

PubMed2002

Semi-quantitative immunohistochemical analysis of aromatase expression in ductal carcinoma in situ of the breast.

Although estrogens whose production is catalyzed by aromatase are considered to play a role in human breast carcinogenesis, it remains unclear whether aromatase expression occurs in ductal carcinoma in situ (DCIS) of the breast. Aromatase expression in 61 cases of pure DCIS and 101 cases of invasive ductal carcinoma (IDC) was investigated by immunohistochemical analysis using a polyclonal anti-aromatase antibody. The level of aromatase expression was semiquantified by the H-score which was estimated by the percentage of positive-staining cells and the intensity of staining. The levels of aromatase expression were compared between the DCIS and IDC samples, and were also compared among the tumor cells and stromal cells in the DCIS and IDC samples. Positive cytoplasmic staining for aromatase expression was found not only in stromal cells but also in tumor cells. The levels of aromatase expression in the tumor cells and stromal cells from the DCIS samples were significantly higher than those in the respective cells from the IDC samples. Among the DCIS samples, those specimens from patients of ages 50 years or over showed higher levels of aromatase expression in stromal cells, than those from patients below 50 years. The finding that significantly higher aromatase expression levels were found in DCIS than in IDC indicates that it may be possible to treat DCIS patients with aromatase inhibitors, especially as an adjuvant hormonal therapy for postmenopausal patients.

Adult↗

PubMed2002

A calcium-deficient diet caused decreased bone mineral density and secondary elevation of estrogen in aged male rats-effect of menatetrenone and elcatonin.

In view of the fact that a deficient calcium (Ca) intake results in osteoporosis in elderly males, we conducted an animal experiment on aged male Wistar rats given a Ca-deficient diet. The rats were divided into 2 groups according to diet: a Ca-deficient diet group (Ca content, 0.08% to 0.1%) and a regular diet group (Ca content, 0.8% to 1.2%). The Ca-deficient diet reduced bone mineral density (BMD) by approximately 12%. Administration of menatetrenone or elcatonin was able to reverse the reduction in BMD induced by Ca deficiency. The mean estradiol level in sera of rats fed the Ca-deficient diet was significantly increased to 4.3 times that in the regular diet group. However, the increased estradiol concentration was reduced after the administration of menatetrenone or elcatonin. The estrone concentrations in sera of menatetrenone- or elcatonin-treated rats fed the Ca-deficient diet decreased to a level lower than that of animals fed the regular diet. Testicular aromatase cytochrome P450 (P450(arom); estrogen synthetase) activity was significantly increased by 2.4-fold in the Ca-deficient diet group compared to that in the regular diet group, and the aromatase mRNA level was also significantly increased 1.45-fold. Testicular aromatase activity was strongly correlated with aromatase mRNA level and serum estradiol level. These data suggest that the change in testicular aromatase expression might be, in part, a compensatory mechanism for the bone mineral deficiency induced by the Ca-deficient diet in aged male rats.

Aging↗

PubMed2002

Immunohistochemical localization of steroidogenic enzymes and prolactin receptors in the corpus luteum and placenta of spotted seals (Phoca largha) during late pregnancy.

To study luteal function in the late gestational period of Phocidae (seals), we analyzed the localization of steroidogenic enzymes (P450scc, 3betaHSD and P450arom) and prolactin receptors in the corpora lutea of pregnant spotted seals (Larga seal; Phoca largha) immunohistochemically. P450scc, 3betaHSD and prolactin receptors were present in all luteal cells of each corpus luteum, and most luteal cells were immunostained for P450arom. Although we analyzed only two specimens, P450scc, 3betaHSD and prolactin receptors were negatively immunostained in the placentae. P450arom was present in the syncytiotrophoblast of placentae. These findings suggest that 1) the corpus luteum of the spotted seal synthesizes pregnenolone, progesterone and estrogen during late gestational period, 2) the placenta of this species do not possess the capacity to synthesize progesterone, and 3) like other terrestrial carnivores, this species requires prolactin to maintain the corpus luteum during pregnancy. These characteristics support the recent classification of family Phocidae in the order Carnivora, and suggest a relationship between prolactin and reproductive failure during the post-implantation period in pinnipeds.

3-Hydroxysteroid Dehydrogenases↗

PubMed1998

From Endocrinology to Intracrinology.

Recently, in situ formation of active sex steroids at the sites of their actions from biologically inactive precursors in the circulation have been demonstrated to play very important roles in sex steroid-dependent neoplasms. These tissues in which the conversion occurs are designated as intracrine tissues and their mechanisms of actions can be designated as intracrinology in contrast to endocrinology. Aromatase, which converts serum androgens to estrone, and 17B-hydroxysteroid dehydrogenase I, which is involved primarily in the conversion of estrone to estradiol, are two major enzymes which function in the in situ formation of biologically active estrogens from circulatory androgens. In human estrogen-dependent neoplasms, including breast, endometrioid endometrial, and common epithelial ovarian carcinoma, we recently demonstrated overexpression of aromatase, especially in stromal cells at sites of frank invasion possibly under a new promoter usage and that of 17B-hydroxysteroid dehydrogenase I in these carcinoma cells. These estrogen-dependent carcinomas are considered to have a common characteristic in estrogen metabolism (i.e., the expression of aromatase in the stromal cells and of 17B-hydroxysteroid dehydrogenase I in the epithelial cells). With these in situ mechanisms of generating biologically active estrogens from circulating androgens, that is, "intracrine manner," these estrogen-dependent neoplasms can exert estrogenic actions on carcinoma cells despite low circulating serum estrogen levels, as observed in postmenopausal women. Evaluation of intracrine mechanisms can provide new insights into various estrogen-related biological phenomena in humans.

Journal Article↗