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The high-mobility group A protein 1a (HMGA1a) protein is known as an oncogene whose expression level in cancer tissue correlates with the malignant potential, and known as a component of senescence-related structures connecting it to tumor suppressor networks in fibroblasts. HMGA1 protein binds to DNA, but recent studies have shown it exerts novel functions through RNA-binding. Our previous studies have shown that sequence-specific RNA-binding of HMGA1a induces exon-skipping of Presenilin-2 exon 5 in sporadic Alzheimer disease. Here we show that HMGA1a induced exon-skipping of the estrogen receptor alpha (ERα) gene and increased ERα46 mRNA expression in MCF-7 breast cancer cells. An RNA-decoy of HMGA1a efficiently blocked this event and reduced ERα46 protein expression. Blockage of HMGA1a RNA-binding property consequently induced cell growth through reduced ERα46 expression in MCF-7 cells and increased sensitivity to tamoxifen in the tamoxifen-resistant cell line, MCF-7/TAMR1. Stable expression of an HMGA1a RNA-decoy in MCF-7 cells exhibited decreased ERα46 protein expression and increased estrogen-dependent tumor growth when these cells were implanted in nude mice. These results show HMGA1a is involved in alternative splicing of the ERα gene and related to estrogen-related growth as well as tamoxifen sensitivity in MCF-7 breast cancer cells.

Obesity commonly occurs in postmenopausal women, increasing the risk of various diseases. Estrogen can prevent obesity by activating lipid metabolism and suppressing depressive behavior. However, the reasons for obesity in postmenopausal women are not clearly elucidated. To mimic the effect of estrogen decline in postmenopausal women, we analyzed the behavior and the lipid metabolism-related genes, PPARγ and CD36 in ovariectomized (OVX) mice. The OVX mice showed increased visceral fat mass and PPARγ and CD36 expression in the visceral fat. In contrast, they were not significantly affected in terms of physical activity and food intake. Further, subcutaneous supplementation of estrogen effectively suppressed the increase in subcutaneous and visceral fat mass in OVX mice. We conclude that obesity in postmenopausal women is unlikely to be caused by overeating and reduction in physical activity, and subcutaneous supplementation of estrogen is an effective strategy to prevent obesity in postmenopausal women., Highlights • We studied the effects of estrogen deficiency/supplementation on obesity in mice. • After ovariectomy, the study animals received transdermal injections of estrogen. • Exogenous 17β-estradiol appears to mediate the effects of menopause on obesity. • Obesity in the mice was caused solely by estrogen deficiency, not overeating. • Estrogen deficiency altered the mechanics of fat deposition in the mice.

Purpose To evaluate the efficiency of automated function imaging (AFI) on transthoracic echocardiogram (TTE) for detecting left ventricular (LV) wall motion (LVWM) abnormalities, we compared longitudinal peak systolic strain (LPSS) measurements using AFI with naked eye TTE evaluations by experienced cardiologists and non-experienced residents. Materials and methods A total of 352 segments of LV myocardium from 22 consecutive subjects with LVWM abnormalities based on American Heart Association classifications (11 male, mean age 58±14years) on previous TTE (Vivid-7, GE) were evaluated. LPSS was measured using stored AFI data. Naked eye evaluation of LVWM was performed by 2 experienced cardiologists and 2 non-experienced residents. Results AFI successfully tracked 342 (97%) of all segments (mean LPSS −14.8±8.1%). A significant strong negative correlation was observed between LV ejection fraction using method of disks and global LPSS (R=−0.8974). Temporary AFI criteria of LPSS were normal 2. Of 342 segments, 239, 87, and 16 segments were diagnosed as normal, hypokinesis, and akinesis, respectively. Level of agreement and kappa coefficients between qualitative evaluation of LVWM by AFI temporary criteria and qualitative evaluation of LVWM by experienced cardiologist 2 (0.784 and 0.479, respectively) were inferior to those comparing experienced cardiologists (0.845 and 0.595) but superior comparing experienced cardiologist with non-experienced resident (0.696 and 0.323), and between the 2 non-experienced-residents (0.682 and 0.347). Conclusion Qualitative evaluation of LVWM using temporary AFI criteria had a 97% success rate and agreed well with findings of an experienced cardiologist. AFI can be a useful tool for training residents.

This paper presents the investigation of ablation plasma characteristics together with the flyer velocity for flyer acceleration when irradiating the 50-μm-Al target with a multi-pulsed ion beam. A one-dimensional hydrodynamic model is used in the calculations. The CIP method is used to describe the ablation plasma production and the acceleration mechanism by the repetition of ion beam irradiation. In the calculations, two shots of ion beam, which has a total energy density of 120 J/cm 2 , are used. The time intervals between the first pulse and the second pulse of 0 ns, 20 ns, 50 ns and 100 ns were used for the investigation of flyer (target) velocity. Results indicate that irradiating the second pulse for 20 ns, 50 ns and 100 ns after the first pulse increases the flyer velocity by approximately 16%, 32% and 40%, respectively.

In this study, the possible application of atmospheric pressure dielectric barrier discharge plasma for the annealing of metallic wire is examined and presented. The main purpose of the current study is to examine the surface cleaning effect for a cylindrical object by atmospheric pressure plasma. The experimental setup consists of a gas tank, plasma reactor, and power supply with control panel. The gas assists in the generation of plasma. Copper wire was used as an experimental cylindrical object. This copper wire was irradiated with the plasma, and the cleaning effect was confirmed. The result showed that it is possible to remove the tarnish which exists on the copper wire surface. The experiment reveals that atmospheric pressure plasma is usable for the surface cleaning of metal wire. However, it is necessary to examine the method for preventing oxidization of the copper wire.

SummarySex hormones are essential for neural circuit development and sex-specific behaviors. Male behaviors require both testosterone and estrogen, but it is unclear how the two hormonal pathways intersect. Circulating testosterone activates the androgen receptor (AR) and is also converted into estrogen in the brain via aromatase. We demonstrate extensive sexual dimorphism in the number and projections of aromatase-expressing neurons. The masculinization of these cells is independent of AR but can be induced in females by either testosterone or estrogen, indicating a role for aromatase in sexual differentiation of these neurons. We provide evidence suggesting that aromatase is also important in activating male-specific aggression and urine marking because these behaviors can be elicited by testosterone in males mutant for AR and in females subjected to neonatal estrogen exposure. Our results suggest that aromatization of testosterone into estrogen is important for the development and activation of neural circuits that control male territorial behaviors.

PHYSIOL BEH Exposure to estrogens during critical developmental periods and in adulthood affects sex differences in the brain. We examined the roles of estradiol (E2) and phytoestrogens, and their interactions, on potential sex differences in brain. We used aromatase knockout (ArKO) mice, which cannot produce endogenous estrogens, along with wild type (WT) littermates. Mice were gestated, raised and maintained on a diet either rich in phytoestrogens or a diet virtually void of soy-derived phytoestrogens. Adult males and females were gonadectomized and received implants filled with 17-β-estradiol to induce progestin receptors (PR), while controls received empty implants. Mice were sacrificed five days later and brain sections containing the posterodorsal medial amygdala (MePD) were processed for PR immunoreactivity. Activation of sex differences in PR required adult E2 treatment. A diet high in phytoestrogens was required for expression of sex differences in PR after E2 treatment. Our data underscore the important contribution of dietary phytoestrogens for the development of sex differences in PR-ir in the adult mouse medial amygdala. We hypothesize that both aromatization of androgens to estrogens and dietary sources of additional estrogens are part of the normal requirement for sex differences in the rodent brain.

A magnetohydrodynamic (MHD) accelerator is proposed as a next generation propulsion system. The fundamental acceleration performance has been, so far, evaluated using one-dimensional (1-D) numerical analysis. However, the actual phenomena in the accelerator channel are highly coupled with boundary layers and with electrode phenomena and are three-dimensional. In this paper, research for investigating the three-dimensional (3-D) phenomena of an MHD accelerator is described. The validity of the 3-D numerical analysis is assessed through comparisons with the results of 1-D analysis. An existence of a local Hall current not predicted through 1-D analysis enhances the Joule dissipation and induces a secondary flow. It was found that a significant local Hall current influences the growth of the velocity overshoot, and this phenomenon is promoted particularly in a high Hall parameter environment. This study of 3-D analysis offers a more realistic prediction of the accelerator performance of the MHD accelerator than that of the 1-D predictions.

Investigation of application of magnetohydrodynamic (MHD) acceleration process for advanced propulsion system has significantly increased. The diagonal MHD accelerator is possible for this acceleration technique because of its minimum weight, size and complexity. In this paper, the fundamental performance of a diagonal type MHD accelerator under chemical and thermal equilibrium conditions is described. A one dimensional numerical simulation is performed to characterize the acceleration along the specified channel designed and developed at the NASA Marshall Space Flight Center. In order to solve the set of differential equations with MHD approximations, the MacCormack scheme is employed. In the present calculation, a working gas of air-plasma composed of diatomic molecules of nitrogen and oxygen seeded with potassium is considered in order to actualize application of the MHD accelerator propulsion system. Numerical results show the gasdynamic characteristics (flow velocity, gas pressure and gas temperature), electrical properties (electrical conductivity, Hall parameter and Faraday current density) and electrical efficiency of the MHD accelerator.

The fundamental performance of a linear Faraday type magnetohydrodynamic (MHD) accelerator under chemical and thermal equilibrium conditions is studied in this paper. Quasi-one dimensional numerical simulation is performed to characterize acceleration along the specified channel. In order to solve the set of differential equations of magnetohydrodynamics, the MacCormack scheme is employed. In this present calculation, a working gas of air-plasma, consisting of diatomic molecules of nitrogen, oxygen and hydrogen, is considered in order to actualize application of the MHD accelerator propulsion system. Numerical results show the characteristics of flow velocity, gas temperature and electrical conductivity of the MHD accelerator with the working gas of air-plasma. The computations are compared with numerical simulation results using the inert gas of argon evaluated in past studies.

During aging, the decline of neuroendocrine, endocrine, and behavioral components of reproduction ultimately leads to reproductive failure. These studies considered both neuroendocrine and behavioral aspects of reproductive aging in Japanese quail, using chronological age and reproductive status to separate animals into experimental groups. In Study I, age-related changes in the gonadotropin releasing hormone (GnRH-I) system were investigated and a sharp decrease was observed in GnRH-I concentration in the median eminence of aging animals of both sexes, whereas preoptic-lateral septal region GnRH-I concentrations declined only in aging males. Immunohistochemistry confirmed these findings since aging females retained, whereas males lost GnRH-I cells. Functional changes were assessed by in vitro incubation of parasaggittal hypothalamic slices collected from young and old inactive males and females. Results showed reduced baseline GnRH-I release and diminished response to norepinephrine (NE). Deteriorating fertility also correlated with decreased male sexual behavior and loss of aromatase immunoreactive (AROM-ir) neurons in the medial, but not lateral preoptic nucleus (POA). Sexual behavior and AROM-ir were restored with exogenous testosterone, which was associated with increased cell size in the medial POA. Comparison of cell size and number of AROM-ir cells showed that aged sexually active males had fewer, larger AROM-ir cells when compared to young males, suggesting neuroplasticity of specific neural systems and a critical role of estradiol in maintaining reproductive function.

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.

Sexually relevant pheromonal cues are detected by the vomeronasal system which includes the posterodorsal part of the medial amygdala, the posteromedial part of the bed nucleus of the stria terminalis and the medial preoptic area. Copulatory behavior is impaired in mice lacking functional aromatase, the enzyme converting testosterone into estradiol. In this study, we used male aromatase knockout (ArKO) mice to investigate the role of aromatase in the differentiation and activation of preference for male- or female-related odorants. Moreover, using Fos immunoreactivity as a marker of neuronal activation we investigated the ability of sex-related pheromonal cues to activate the vomeronasal system. Both gonadally intact wild-type and ArKO mice preferred to investigate urine from females. The lack of estrogens did not reverse odor preferences, i.e. male ArKO mice did not show a preference for male odors. Exposure to soiled bedding from females induced Fos-protein in the posterodorsal part of the medial amygdala, in the posteromedial part of the bed nucleus of the stria terminalis, and in the periventricular part of the medial preoptic area of both the genotypes. Exposure to soiled bedding from intact males induced Fos in the posterodorsal part of the medial amygdala in wild-type mice and in the periventricular medial preoptic area in wild-type and ArKO mice. These results suggest that preference for female-related odors and the Fos-mediated activation of the vomeronasal system do not rely on estradiol. Furthermore, sensitivity to female chemosensory cues and copulatory behavior are uncoupled in this knockout model.

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.

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.

The functional differences between male and female brains commit to the existence of androgen that the testis secretes during the perinatal period. Androgen exerts its action on the brain after conversion to estrogen by brain aromatase. The aromatase appears in some neural nuclei such as in the hypothalamus and amygdala, and has been indicated to be involved in the expression of sexuality by the results of neurobehavioral analyses involving aromatase-knockout mice. We analyzed the brain-specific promoter in order to clarify the control mechanism for the expression of brain aromatase, which is deeply concerned in the sexual differentiation of the brain. The 202bp upstream region of brain-specific exon 1 contains at least three kinds of cis-acting elements, Arom-Aalpha, -Abeta and -B. In particular, the binding activities as to the Abeta sequence show a tissue-specific pattern. Gel shift analysis revealed that the Abeta binding factor recognizes the TTGGCCCCT sequence. Abeta binding activity is detectable at the perinatal stage, but is undetectable at the adult stage in the brain. Furthermore, a protein which binds to the Abeta sequence was purified from the fetal mouse brain. The molecular mass of the Abeta binding protein was estimated to be 49kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Environmental chemicals with estrogenic activities have been suggested to be able to interact with the endocrine system. Endogenous estrogen is synthesized in the ovarian theca cells of premenopausal women or in the stromal adipose cells of the breast of postmenopausal women and minor quantities in peripheral tissue. These cells, as well as breast tissue, express all the necessary enzymes for this synthesis, CYP17, CYP11a, CYP19, 17-β-hydroxysteroid hydrogenase, steroid sulfatase as well as enzymes further hydroxylating estradiol, such as CYP1A1, CYP3A4, CYP1B1, catechol- o -methyltransferase (COMT). Polymorphisms in these enzymes may have a possible role in the link between environmental estrogens and hormone-like substances and the interindividual risk of breast cancer.

The change in hepatic antioxidant defense system with the development of α-naphthylisothiocyanate (ANIT)-induced liver injury was examined in rats injected once with the toxicant (75 mg/kg body weight). Liver injury with cholestasis did not occur 12 h after ANIT injection, but appeared at 24 h, progressed at 48 h, and recovered at 72 h, judging from the serum levels of marker enzymes and components. Liver lipid peroxide content increased 12 h after ANIT injection and further increased 24 and 48 h, but this increase was attenuated at 72 h. Liver superoxide dismutase and catalase activities decreased 24 and 48 h, respectively, after ANIT injection, although the catalase activity increased at 12 h, but these decreases were attenuated at 72 h. Liver Se–glutathione peroxidase activity remained unchanged 24, 48, and 72 h after ANIT injection, although the activity increased at 12 h. Liver reduced glutathione content increased 24 h after ANIT injection, but the increase was reduced time dependently thereafter. Liver ascorbic acid content increased 12 h after ANIT injection and further increased at 24 h, but the increase was reduced time dependently thereafter. These results indicate that the change in hepatic antioxidant defense system occurs before and with the development of ANIT-induced liver injury in rats, and suggest that the reduction of hepatic antioxidant defense system mediated by SOD and catalase could contribute to the liver injury development through an enhancement of hepatic lipid peroxidation.

An association between lipid peroxidation and alpha-naphthylisothiocyanate (ANIT)-induced liver injury was examined in rats injected once with the toxicant (75 mg/kg body weight). The severity of liver injury was estimated 12, 24, 48, and 72 h after ANIT injection. Liver injury appeared 24 h after ANIT injection, progressed at 48 h, and recovered at 72 h, judging from the serum levels of marker enzymes and components. Serum lipid peroxide (LPO) concentration increased 24 h after ANIT injection and further increased at 48 h, but this increase was attenuated at 72 h. In contrast, liver LPO content increased 12 h after ANIT injection and further increased 24 and 48 h, but this increase was attenuated at 72 h. Similarly, myeloperoxidase (MPO) activity, an index of neutrophil infiltration, in the liver tissue increased 12 h after ANIT injection and further increased at 24 and 48 h, but this increase was attenuated at 72 h. Either serum LPO concentration or liver LPO content was significantly correlated with liver MPO activity (r = 0.661 for serum LPO concentration; r = 0.585 for liver LPO content). These results suggest that lipid peroxidation might be associated with ANIT-induced liver injury in rats and that this lipid peroxidation might occur via oxygen radicals derived from neutrophils infiltrated into the liver tissue of ANIT-intoxicated rats.

Aromatase is known to be involved in the control of morphological, neuroendocrine, and behavioral sex differences in the brain. To study the detailed functions of aromatase in mouse brain, we generated aromatase-deficient mice that lack exons 1 and 2 and the proximal promoter region of the cyp19 gene by homologous recombination. The genotypes of offspring from heterozygous matings conformed to the Mendelian rule. The aromatase knockout (ArKO) male and female mice displayed no significant external morphological abnormalities, although the uteri of adult ArKO female mice were remarkably underdeveloped. Male-typical sexual behavior directed toward females was, however, strongly modified in the ArKO male mice. The ArKO male mice showed significantly prolonged latencies to mount and decreased numbers of mounts in response to receptive stimulus females. These results suggest an important participation of the aromatase gene in the reproductive behaviors.

A number of studies have been devoted to the analysis of the anatomical distribution, control by steroids and functional significance of aromatase (the enzyme metabolizing testosterone into 17beta-estradiol) in the quail brain. In particular, the sexually dimorphic nucleus preopticus medialis has been the main focus of investigation because testosterone aromatization in this structure mediates the activation of male sexual behavior and aromatase activity is itself testosterone-dependent in this nucleus. No information on the anatomical distribution of aromatase gene expression is, however, available so far in this avian species. In the present study we applied a non-radioactive in situ hybridization technique to describe the distribution of aromatase mRNA containing neurons in the quail prosencephalon. We also analyzed, at a neuronal level of resolution, the induction by testosterone of this mRNA in the medial preoptic nucleus. Dense clusters of aromatase gene expressing neurons were observed within the medial preoptic nucleus, the nucleus of the stria terminalis, the ventro-medial hypothalamus and the tuberal region. Scattered neurons expressing lower levels of aromatase mRNA were also found in the dorsal thalamic area and central gray. The specificity of the staining was confirmed by demonstrating the absence of signal in sections that had been hybridized with a sense probe. Moreover, the distribution of the aromatase mRNA containing cells completely overlapped with the distribution of the aromatase-immunoreactive cells. Aromatase-mRNA expression was controlled by testosterone (or its metabolites) in the entire medial preoptic nucleus. Castration resulted in a decrease in the number of aromatase mRNA-containing cells and this effect was totally reversed by testosterone treatment. These data further support the idea that testosterone regulates the rate of its own aromatization by modulating the expression of aromatase rather than by acting at a post transcriptional level.

A transient increase in aromatase activity is known to occur in the hypothalamus of rodents in pre- and postnatal periods. The mechanisms regulating such a developmental increase of brain aromatase was studied in fetal mouse diencephalic cells, by measuring aromatase mRNA levels by a quantitative reverse transcription-polymerase chain reaction (RT-PCR) method. When slices of diencephalon were cultured on embryonic day (E) 12, E13 and E15, the level of aromatase mRNA continued to increase for the first 2 to 3 days. A time-dependent increase of mRNA was also shown for 3 days in E13 neuronal cells dissociated with papain and cultured in chemically defined medium. However, no significant increase was observed in E10 or E11 brain cells cultured by either method. Aromatase mRNA was detected in neither cerebral cortex neurons nor astrocytes. An alpha1-selective adrenergic agonist, phenylephrine, increased aromatase in the E13 diencephalic neurons in culture, whereas prazosin, an alpha1-antagonist, suppressed the mRNA level. Ligands for alpha2- or beta-adrenergic receptors did not alter the mRNA level. Substance P, cholecystokinin, neurotensin, and brain natriuretic peptide as well as phorbol 12-myristate 13-acetate and dibutyryl-cyclic GMP all increased the mRNA level. We concluded that: (a) the developmental increase of aromatase mRNA in diencephalic neurons is an autonomous event and is perhaps genetically regulated after E12; (b) aromatase mRNA is expressed in a cell type- and region-specific manner; and (c) protein kinases C and G activated via receptors of the specific neurotransmitters may be involved in modulation of the developmental expression of aromatase mRNA.

Aromatase cDNA clones were isolated from cDNA libraries of mouse hypothalamus, amygdala and ovary. Analysis of the nucleotide sequences of the 5′ regions of the obtained cDNAs suggested that the mouse aromatase gene is tissue-specifically regulated by alternative exons 1. There were obvious differences between the 5′ regions of the brain and ovary aromatase cDNAs, but no difference was found between the sequences of the hypothalamus and amygdala ones. We further isolated a mouse genomic DNA clone containing brain- and ovary-specific exons 1. The brain specific exons 1 and their promoters were highly homologous in the human and mouse aromatase genes. In contrast there were several differences in the sequences among the promoter regions of the ovary-specific exons 1 of the mouse, human and rat aromatase genes, significant homology between their sequences was also observed. The present results demonstrate that expression of the mouse aromatase gene is also tissue-specifically regulated through the use of alternative exons 1 and promoters, as reported for man.

Treatment of castrated quail with testosterone (T) reliably activates male copulatory behavior and, at the same time, increases the aromatase activity (AA), the number of aromatase-immunoreactive (ARO-ir) cells and the concentration of aromatase mRNA as measured by RT-PCR in the brain. All these effects can be mimicked by estrogens. The behavioral effects of T can be blocked by a variety of aromatase inhibitors and, in parallel, the AA is strongly inhibited in the preoptic area (POA). We showed recently that the steroidal inhibitor, 4-OH-androstenedione (OHA) markedly decreases the immunostaining density of brain ARO-ir cells while the non-steroidal inhibitor, R76713 (racemic Vorozole; VOR) unexpectedly increased the density of this staining, despite the fact that the enzyme activity was completely inhibited. To generalize these findings and try to identify the underlying mechanism, we compared here the effects of two steroidal (OHA and androstatrienedione [ATD]) and two non-steroidal (VOR and Fadrozole [FAD]) aromatase inhibitors on the aromatase immunostaining and aromatase mRNA concentration in the brain of castrated quail concurrently treated with T. The 4 inhibitors significantly blocked the activation by T of male copulation. The two steroidal inhibitors decreased the immunostaining of brain ARO-ir cells but both VOR and FAD markedly enhanced the density of this staining. In parallel, OHA and ATD completely blocked the T-induced increase in aromatase mRNA concentration, while VOR and FAD had no effect on these RNA concentrations in the POA-anterior hypothalamus and they decreased them only slightly in the posterior hypothalamus. Taken together these results suggest that the inhibition of AA by ATD or OHA and the subsequent removal of locally produced estrogens blocks the synthesis of aromatase presumably at the transcriptional level. By contrast, the two non-steroidal inhibitors tested here block AA but in parallel increase the aromatase immunostaining. This effect does not result from an enhanced transcription and it is therefore speculated that these compounds increase either the translation of the aromatase mRNA or the half-life of the protein itself.

The aromatase (cytochrome P-450AROM) gene contains multiple untranslated exons I that are differentially transcribed in a tissue-specific manner. DNA sequences within the initial −301 upstream of placenta-specific exon I (exon Ia) are sufficient for placenta-specific expression of aromatase. In gel mobility shift assay, three separate domains in this region form specific binding complexes with proteins extracted from choriocarcinoma JEG-3 nuclei. A fragment containing these domains activates transcription driven by a heterologous promoter in a cell type-specific manner. Two of the binding domains that form major complexes in gel shift assay compete with each other and with a DNA fragment containing the trophoblast-specific element (TSE), which is derived from the enhancer region of the human chorionic gonadotropin α-subunit gene and is believed to confer placenta-specific expression of the gene. The core sequence RNCCTNNRG is sufficient for recognition of the TSE-binding protein, which is detected only in nuclear extracts prepared from placenta and choriocarcinoma. A mutation introduced in the distal TSE core in aromatase promoter resulted in marked reduction of transcriptional activity, although TSE region by itself did not show enhancer activity as that in human chorionic gonadotropin α-subunit gene.

Mouse and quail aromatase cDNAs were isolated from libraries of mouse ovary and quail brain by using a human aromatase cDNA fragment (hA-24) as a probe. These three cDNAs were inserted into plasmid vectors and expressed in Escherichia coli. Antisera against these purified recombinant proteins were raised in rabbit and purified by ammonium sulfate fractionation and affinity chromatography. The three antibodies directed against recombinant human, mouse and quail proteins were used to visualize aromatase-immunoreactive cells in the quail brain. They were compared with the antibody raised against human placental aromatase used in previous experiments and with another antibody recently developed by similar methods. The signal obtained with all antibodies was completely abolished by preadsorption with the homologous recombinant antigens and the signal produced by the two antibodies raised against placental aromatase was similarly abolished by a preadsorption with recombinant quail aromatase. The antibodies raised against recombinant proteins identified the major groups of aromatase cells previously described in the quail brain. The antibodies directed against the mouse and quail antigen identified more positive cells and stained them more densely than the antibodies raised against human recombinant antigen or purified placental aromatase. The new cell groups identified by the antibody raised against quail recombinant aromatase were located in an area ventral to the fasciculus prosencephali lateralis, the nucleus accumbens, the paleostriatum ventrale, the nucleus taeniae, the area around the nucleus ovoidalis, the caudal tuber and the mesencephalic central gray. A critical re-examination of the distribution and nomenclature of the aromatase-positive cells is proposed based on these new findings.

In the hamster liver, 2,5,2',5'-tetrachlorobiphenyl (TCB) is metabolized to 3-hydroxy- and 4-hydroxy-2,5,2',5'-TCB to a similar extent, and formation of the former metabolite is stimulated by phenobarbital pretreatment of the animals, while that of the latter metabolite is stimulated by 3-methylcholanthrene pretreatment. In the present study, we identified a new isoform (designated P450HPB-1) of cytochrome P450 which proved to be phenobarbital-inducible and responsible for 3-hydroxylation of this TCB isomer. This isoform was purified from liver microsomes of phenobarbital-treated hamsters and characterized. P450HPB-1 has a molecular mass of 50 kDa, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the absorption maxima of the oxidized form at 417 nm and of the reduced CO-complex form at 450 nm. The sequence of 28 amino acids of P450HPB-1 at the amino-terminal has a 68% similarity with those of rat P450 2B1 and mouse P450 2b10, 57% similarity with that of guinea pig P450GP-1, and 54% similarity with that of guinea pig P450GP-1, and 54% similarity with those of rabbit P450 2B4 and dog P450 2B11. P450HPB-1 in the reconstituted system catalyzed the 3- but not 4-hydroxylation of 2,5,2',5'-TCB, at a rate of 19.0 pmol/min/nmol P450. The isoform also has high catalytic activity for 17-oxidation of testosterone but low activity for the N-demethylation of benzphetamine and 16 alpha- and 16 beta-hydroxylations of testosterone. In microsomal metabolism of 2,5,2',5'-TCB, rabbit antiserum against P450HPB-1 almost completely inhibited 3- but not 4-hydroxylation. Immunoblot analysis of hamster liver microsomes revealed that P450HPB-1 was constitutive and phenobarbital-inducible but was decreased by pretreatment with 3-methylcholanthrene or 3,4,5,3',4'-pentachlorobiphenyl. These results suggest that P450HPB-1 belongs in the P450 2B subfamily and apparently plays a major role in the 3-hydroxylation of 2,5,2',5'-TCB, in hamster liver.

The aromatase enzyme (estrogen synthetase) catalyzes the conversion of testosterone to estrogen in peripheral and central nervous tissue. Light and electron microscopic immunocytochemistry was used to study the localization of this enzyme in the septal area of adult male and female albino rats. Aromatase-immunoreactivity was found restricted to neuronal somata and dendritic arbors, and no sex differences were detected in its distribution or intensity. Most aromatase-immunoreactive neurons formed two oblique bands in the lateral and the medial zones of the lateral septum; in addition, labeled cells were present in the septohippocampal nucleus and the laterodorsal portion of the bed nucleus of the stria terminalis. Electron microscopy revealed that the majority of aromatase-positive neurons in the lateral septum exhibit somatic spines, a characteristic marker of a neuron population that is known to contribute to local and extraseptal projections. The presence of aromatase in lateral septal somatospiny neurons suggests that estrogen formed by these neurons may be critically involved in the septal control of steroid-dependent behaviors.

Castrated quail were treated with Silastic implants filled with testosterone (T) in association with injections of the aromatase inhibitors, R76713 (racemic vorozole; 1 mg/kg twice a day) or 4-hydroxyandrostenedione (OHA; 5 mg/bird twice a day). Control birds received no treatment (CX group) or were implanted with T capsules only (CX + T group). Both R76713 and OHA strongly inhibited the T-activated male copulatory behavior. This inhibition had the same magnitude in both groups. The growth of the cloacal gland, a strictly androgen-dependent process was not affected by these compounds. The treatments significantly affected the number of aromatase-immunoreactive (ARO-ir) cells in each of the six brain areas that were studied: the anterior and posterior parts of the sexually dimorphic medial preoptic nucleus (POM), the septal region, the bed nucleus of the stria terminalis (BNST) and the anterior and posterior parts of the tuber. This number was significantly increased in all areas by T. In agreement with our previous study, R76713 significantly inhibited this effect of T in the tuberal hypothalamus but not in the anterior POM nor in the BNST. By contrast the effect of T on the number of ARO-ir cells was completely blocked by OHA in all brain nuclei. The two inhibitors had statistically different effects in all brain regions. Like in a previous study, R76713 increased the intensity of the staining of all ARO-ir cells. This effect took several days to develop suggesting a progressive build-up of the enzyme concentration. This was also suggested by the fact that a rebound in aromatase activity was observed 16 to 24 h after a single injection of R76713. The increased immunoreactivity was not observed in OHA-treated birds. The denser immunoreactivity in R76713-treated birds and the better tissue preservation due to the aldehyde fixative that had been used provided here a clearer picture of the cellular and subcellular localization of ARO-ir material. This allowed to identify new groups of immunoreactive cells, namely in the nucleus accumbens, in the area of the paleostriatum ventrale, in the nucleus taeniae, in the medial and caudal hypothalamus and in the medial part of the mesencephalon and of the pons. Most of the immunoreactive material was located in perikarya but some of these cells were also surrounded by dense networks of ARO-ir fibers often associated with immunopositive punctate structures. These fibers and punctate structures were seen also in areas that were quite distant from the closest ARO-ir cells. They were detected in periventricular position throughout the preoptic area and hypothalamus.

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), 3βHSD (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 3βHSD 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.

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 μm 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. These experiments also indicate that ARO-ir cells in the region of POM located just under and rostral to AC are sexually dimorphic and T-sensitive, which suggests their involvement in the control of male sexual behavior. This conclusion is also supported but our recent studies based on electrolytic lesion and stereotaxic implantation of T in POM.

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.

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.

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.

It was found that cytochrome P-450(arom) purified from human placenta microsomes is glycosylated, and the sugar chain was cleaved with endoglycosidase H (Endo H). The core glycosylation of P-450(arom) was examined with two heterologous expression systems, cultured insect cells and in vitro translation system. The P-450(arom) protein expressed in the insect cells was glycosylated, and the sugar chain was sensitive to Endo H. It was also glycosylated when translated with the wheat germ cell-free system in the presence of rough microsomal membrane, and the sugar chain could be removed by Endo H treatment. Since the P-450(arom) molecule has two potential glycosylation sites (Asn-12 and Asn-180), we replaced each of the 2 asparagine residues with alanine by site-directed mutagenesis and examined the glycosylation of the two mutant proteins in the cell-free system. The core glycosylation did not occur when the Asn-12 residue was mutated, whereas the mutant protein with modified Asn-180 residue was glycosylated. These results demonstrated that the potential glycosylation site (Asn-12) in the N-terminal portion of P-450(arom) is the site of glycosylation. We conclude that the N terminus of P-450(arom) is translocated across the endoplasmic reticulum membrane to be glycosylated at the luminal side.

Experimental results of the FUJI-1 disk generator have been discussed from the calculated results based on the quasi-one-dimensional model. The calculations have been performed for generators with different channel shape to investigate the effects of the area ratio on power generation performance. The increase of output power at the high load resistance regime with increase of area ratio has been qualitatively shown by calculations, however absolute values of the plasma properties in an MHD channel obtained from the calculations have not been in agreement with experiment values. To clarify the pressure loss mechanism, the influences of the plasma properties on the changes of stagnation pressure have been investigated analytically. It has been found that the strong Lorentz force takes a significant role in the pressure loss processes as compared with wall friction under a strong MHD interaction conditions. Furthermore, it has been suggested that a 100 MW class thermal input generator is required to reduce the stagnation pressure loss by wall friction with high gas velocity and high output power.

An analytical method for quantitation of the RNA transcripts of murine bcl-2 gene was developed. The PCR products from bcl-2 alpha and bcl-2 beta mRNA were fluorometrically analyzed and their specific contents were calculated by the internal standard method. Both bcl-2 mRNAs in adult mice were transcribed at the highest level in the thymus and at a comparable level in the spleen. Aside from the immune system, the brain gave the most abundant levels of the bcl-2 mRNAs. The ratios of bcl-2 beta mRNA to bcl-2 alpha mRNA in the thymus and spleen were significantly higher than those in other tissues. During development of the brain, the bcl-2 alpha and bcl-2 beta mRNA levels were highest on embryonic day 15, and about two and three times higher than those of adult, respectively. The results suggest that the bcl-2 gene functions to regulate development and survival of neurons in the central nervous system.

Placental aromatase deficiency, which was characterized by maternal and fetal virilization and by a low level of estrogen excretion into urine during pregnancy, was studied by biochemical and molecular genetical techniques. Among enzymes participating in the electron transport system of the patient's placental microsomes, only aromatase activity was observed to be reduced (< 3% of normal levels). Northern and Western blotting analyses showed that the transcription of the aromatase gene and the translation of its mRNA seemed to proceed normally in the patient's tissue. However, the aromatase cDNA isolated from the patient was found to contain an extra DNA fragment of 87 base pairs (bp) which encoded 29 amino acids in frame but no termination codon. The insertion was located at the splicing point between exon 6 and intron 6 of the normal aromatase gene. The extra DNA fragment represented the first part of intron 6 except that its initial GT was altered to GC. These findings indicated that, in the patient's aromatase gene, the splicing between exon 6 and intron 6 did not occur at the normal position. This reflected the presence of one point mutation in its consensus sequence which caused the next cryptic consensus sequence 87 bp downstream, to be used according to the canonical GT/AG rule. The protein molecule thus translated contained an extra 29 amino acids. Furthermore, the patient's aromatase cDNA was observed to produce a protein molecule with a trace of activity in the transient expression system of COS-7 cells and in the high level expression system of baculovirus-insect cells. Direct DNA sequencing of aromatase genes from the patient and parents confirmed that this deficiency is a hereditary disease with an autosomal recessive inheritance pattern. The patient and parents are homozygote and heterozygotes, respectively, for this mutation.

The aromatase (estrogen synthetase) enzyme catalyzes the conversion of androgens to estrogens in peripheral tissues, as well as in the brain. Our study aimed at comparing the brain distribution of aromatase-immunoreactive neurons in male and female, normal and gonadectomized rats. Light microscopic immunostaining was employed using a purified polyclonal antiserum raised against human placental aromatase. Two anatomically separate aromatase-immunoreactive neuronal systems were detected in the rat brain: A “limbic telencephalic” aromatase system was composed by a large population of labeled neurons in the lateral septal area, and by a continuous “ring” of neurons of the laterodorsal division of the bed nucleus of stria terminalis, central amygdaloid nucleus, stria terminalis, and the substantia inominata-ventral pallidum-fundus striati region. The other, “hypothalamic” aromatase system consisted of neurons scattered in a dorsolateral hypothalamic area including the paraventricular, lateral and dorsomedial hypothalamic nuclei, the subincertal nucleus as well as the zona incerta. In addition, a few axon-like processes (unresponsive to gonadectomy) were present in the preoptic-anterior hypothalamic complex, the ventral striatum, and midline thalamic regions. No sexual dimorphism was observed in the distribution or intensity of aromatase-immunostaining. However, 3 days, 2, 3, 8, 16, or 32 weeks after gonadectomy, aromatase-immunoreactive neurons disappeared from the hypothalamus, whereas they were still present in the limbic areas of both sexes. The results indicate the existence of two distinct estrogen-producing neuron systems in the rat brain: (1) a “limbic ring” of aromatase-labeled neurons of the lateral septum-bed nucleus-amygdala complex unresponsive to gonadectomy; and (2) a sex hormone-sensitive “hypothalamic” aromatase neuron system.

Biochemical and molecular genetic studies were made on a case of placental aromatase (P-450AROM) deficiency. Of the enzymes participating in the electron transport system of placental microsomes, only aromatase activity was decreased specifically in the patient, being less than 0.3% of the normal activity. Northern and Western blotting analyses showed that the transcription of the aromatase gene and the translation of its mRNA proceeded normally in the placenta of this patient. However, aromatase cDNA isolated from a placental cDNA library of the patient was found to have an insert of 87 base pairs, encoding 29 amino acids in frame with no termination codon. The insert was located at the splicing point between exon 6 and intron 6 of the normal aromatase gene, and the extra DNA fragment was the first part of intron 6, except that its initial GT was altered to GC. These findings indicated that in this patient with aromatase deficiency, splicing between exon 6 and intron 6 did not occur at the normal position because of a point mutation in its consensus sequence and was forwarded to GT in the next cryptic consensus sequence 87 base pairs downstream according to the canonical GT/AG rule, resulting in translation of an abnormal protein molecule with 29 extra amino acids. During the transient expression in COS-7 cells, the aromatase cDNA of the patient was found to produce a protein with a trace of activity. This is the first report of a genetic defect for aromatase deficiency.

Testosterone (T) increases brain aromatase activity (AA) in quail and other avian and mammalian species. It was shown both in quail and in rat that this enzymatic induction results from a synergistic action of androgens and estrogens. These studies provide little information on possible anatomical or cellular specificity of the effect. Using a polyclonal antiserum against human placental aromatase, we have previously identified aromatase-immunoreactive (ARO-ir) neurons in the quail brain and demonstrated that T increases the number of ARO-ir cells in the quail preoptic area (POA) supporting previous evidence that T increases AA in the brain. However, which T metabolites are involved, the actual mechanism of regulation and the possibility of anatomical specificity for these effects are not yet clear. In the present study, we disassociated the effects of androgens and estrogens in aromatase induction by comparing ARO-ir neurons of quail treated with T alone or T in the presence of a potent aromatase inhibitor (R76713), which has been shown to depress AA levels and to suppress T-activated copulatory behavior. T increased the number of ARO-ir cells in POA, bed nucleus striae terminalis (BNST) and tuberal hypothalamus (Tu). The T effect was inhibited by concurrent treatment with aromatase inhibitor in Tu, but not in POA and BNST. This differential effect of the aromatase inhibitor fits in very well with our previous studies of the co-localization of aromatase and estrogen receptors. The T effect was blocked by R76713 in areas where ARO-ir and estrogen receptor-ir are generally co-localized (Tu) and was not affected in areas with mainly ARO-ir positive, estrogen receptor-ir negative cells (POA, BNST). This suggests anatomical differences in the expression or clearance of aromatase which may be differentially sensitive to androgens and estrogens and dependent upon the presence of sex steroid receptors.

We recently showed, using a new immunocytochemical technique, that aromatase-immunoreactive neurons are a specific marker for the sexually dimorphic medial preoptic nucleus (POM) in quail and that the number of these immunoreactive cells is markedly increased by a systemic treatment with testosterone (T). Since the POM is a key site for the activation of copulatory behavior by T and this androgen must be converted into estrogen by local aromatization within the POM before it can exert its behavioral effects, we used aromatase immunocytochemistry to map, at a cellular level of resolution, the areas that are destroyed by electrolytic lesions or that are stimulated by the stereotaxic implantation of T in the preoptic area (POA). These measures of the cellular action of T in the preoptic area were then correlated with the behavior of the animals to identify the parts of the POA that are critical in the activation of behavior. The electrolytic lesions of the POA disrupted the activation of male sexual behavior by T only if they destroyed a significant part of the POM. All lesions reduced the volume of the dimorphic nucleus and the absolute number of its aromatase-immunoreactive neurons, but the density of these cells in the remaining POM was not affected, suggesting that the volume change in the nucleus reflected a centripetal displacement of its boundaries rather than an overall shrinkage of the structure. Stereotaxic T implants in or close to POM activated male copulatory behavior and increased the volume of the POM and the number of its aromatase-immunoreactive cells. These neuroanatomical effects were more prominent on the side of the implant, but they were also detected on the contralateral side. Correlative analyses suggested that a part of the POM just rostral to the anterior commissure is critical for the activation of copulatory behavior. The best correlations between the behavioral deficits induced by electrolytic lesions and the size of the lesions were indeed observed in this area. In addition, high correlations were also observed between the behavior activated by T implants and the POM size or number of aromatase-immunoreactive cells that were induced by T in this area. Aromatase immunocytochemistry therefore appears as a useful tool to map the brain areas in which T action is presumably critical for the activation of male sexual behavior. It has allowed us to identify in the present studies a small part of the sexually dimorphic POM that is closely associated with behavior. Experimental studies involving the lesion, disconnection or specific stimulation by steroids of this area should now be undertaken to confirm the causal meaning of these correlations.

The expression of estrogen synthetase (aromatase), catalyzing a rate limiting reaction in estrogen formation, was examined in 3T3-L1 cells during adipose differentiation. The expression of another P-450 enzyme, cholesterol side-chain cleavage enzyme (P-450scc) by the cells was also studied for comparison. The level of specific mRNA for aromatase increased 17-fold during adipogenic conversion and the elevated level was maintained in fully differentiated adipocytes. The level of specific mRNA for P-450scc increased about 5-fold, mainly due to net increase of cellular RNA. Various reagents, such as dexamethasone, testosterone and 1-methyl-3-isobutylxanthine, affected the expression of specific mRNA for aromatase markedly in adipocytes but had scarcely any effect on its level in fibroblasts. In contrast, these reagents caused similar increases in the level of mRNA for P-450scc in the two types of cells. Thus the 3T3-L1 cell line during adipogenic differentiation may be a useful system for studies on the mechanism regulating aromatase gene expression.

The estrogen synthetase (aromatase, cytochrome P-450AROM) gene has been isolated from human genomic libraries and characterized. The restriction map of 43 positive clones obtained indicated that this enzyme is present as a single copy gene. The aromatase gene is unexpectedly large compared with other forms of the cytochrome P-450 superfamily, spanning at least 70 kilobases. The gene consists of 10 exons and its 5′-untranslated region is divided into 2 exons by an intron of more than 35 kilobases long. This organization of the first exon in the aromatase gene is unique in the cytochrome P-450 superfamily. All the exon-intron junctional sequences conform to the canonical GT AG rule. The sequences of a TATA box and a CAAT box are present 27 and 83 base pairs upstream from the transcriptional initiation site. Within 3 kilobases upstream from the initiation site, there are no typical consensus sequences of responsive elements for glucocorticoid and c-AMP, which regulate aromatase expression.

We isolated overlapping cDNA clones encoding human histidase (histidine ammonia-lyase) from a human λgt10 library. The cDNA predicted a 657 amino acid protein of 72 651 Da. The human histidase amino acid sequence was 93% conserved with both rat and mouse histidase sequences, including four N -glycosylation consensus sites.

Corrigendum to “The Japanese quail: a model for studying reproductive aging of hypothalamic systems” [Experimental Gerontology 39/11–12 (2004) 1679–1693] Mary Ann Ottinger*, Mahmoud Abdelnabi, Qichang Li, Kehong Chen, Nicola Thompson, Nobuhiro Harada, Carla Viglietti-Panzica, Gian Carlo Panzica Department of Animal & Avian Sciences, Animal Science Center, University of Maryland, Room 3113, College Park, MD 20742, USA Department of Biochemistry, School of Medicine, Fujita Health University, Toyoake, Aichi 470-1192, Japan Department of Anatomy, Pharmacology and Forensic Medicine, University of Turin, Torino, Italy

Females gender itself is a risk factor for drug-induced torsades de pointes (TdP) arrhythmia which is associated with QT prolongation caused by blockade of human ether-a-go-go related gene (hERG) currents. Some clinical evidence suggests that estrogen is a determinant of the gender-differences in drug-induced QT prolongation and baseline QTC intervals. Although the chronic effects of estrogen have been studied, it remains unclear whether the gender differences are due entirely to transcriptional regulations through estrogen receptors. We here found that the most bioactive estrogen, 17beta-estradiol (E2), acutely delayed cardiac repolarization within the physiological serum level (0.1-1 nM). E2 slightly but significantly suppressed hERG currents (Kd = 0.6 nM) by modifying channel gating kinetics. Mutagenesis study showed the interaction of E2 with F656, a common drug-binding site at the inner pore-cavity of hERG. E2 enhanced both hERG suppression and QTC prolongation by its blocker, E4031. The lack of effects of testosterone on hERG currents and E4031-sensitivity implicates the critical role of aromatic centroid present in E2 but not in testosterone, which is supported by data from aromatase-null mice that cannot produce estrogen. The aromatase-null mice showed lower sensitivity to E4031-induced QT prolongation compared with those of wild type mice, and i.v. application of exogenous E2 (0.1 μg/kg) subsequent to E4031 administration rapidly prolonged QT intervals, indicating that aromatized estrogen emphasize the effect of E4031 on cardiac repolarization in vivo. Our data indicate that E2 acutely affects the hERG channel gating and the E4031-induced QTC prolongation, and may provide a novel mechanism for the higher susceptibility to drug-induced arrhythmia in women.