Electron microscopic observations of the anterior pituitary gland. Part I. The neurons in the "transitional zone" of the anterior pituitary gland.

Since [Westlud, K.N., Chils, G.V., 1982. Localization of serotonin fibers in the rat adenohypophysis. Endocrinology 111, 1761-1763] initially identified the serotonin nerve fibers in the anterior pituitary gland, attention has been paid to the rostral zone of the anterior lobe into which nerve fibers enter and subsequently spread to deeper regions of the lobe. The rostral zone is the trifurcated junction of the partes tuberalis, intermedia and distalis, and has the important role(s) for hormone secretion via the "transitional zone" [Sato, G, Shirasawa, N, Sakuma, E, Sato, Y, Asai, Y, Wada, I, Horiuchi, O, Sakamoto, A, Herbert, DC, Soji, T, 2005a. Intercellular communications within the rat anterior pituitary. XI: An immunohistochemical study of distributions of S-100 positive cells in the anterior pituitary of the rat. Tissue and Cell 37, 269-280.]. The objective of this study was to focus on the ultrastructure of this "zone." All of the animals studied were fixed by perfusion with glutaraldehyde via the left ventricle of the heart and examined by electron microscopy. In the "transitional zone," a cluster of neuronal elements was observed between the folliculo-stellate cell-rich area and the anterior lobe. This cluster consisted of myelinated fibers, unmyelinated fibers, neuroendocrine fibers, large cells, and supporting cells. The large cells were perikarya of neurons which made a "ganglion-like" structure with associated satellite cells. Agranular, folliculo-stellate cells were intermingled among the elements. This is the first report that neuronal elements form clusters in the "transitional zone." A relationship of the unmyelinated and neuroendocrine fibers in the basal layer and in the "transitional zone" is discussed.

Bilaterally existing sternoclavicularis anticus muscles.

A 92-year-old Japanese woman was found to have the sternoclavicularis anticus muscles existing bilaterally during the course of educational dissection at Nagoya City University Medical School. It was confirmed that these muscles received the nerve supply from small branches of the lateral pectoral nerves from the brachial plexus. Herein is documented the precise gross anatomical findings with some morphometric measurements. Moreover, the morphology of this muscle is discussed in relation to previously described variations of the pectoral sheet of muscles.

Distinctive localization of N- and E-cadherins in rat anterior pituitary gland.

In the rat anterior pituitary gland, folliculo-stellate cells aggregate preferably to form pseudofollicles, and each type of hormone-producing cell shows adhesive affinity with particular types of heterologous hormone-producing cells. Distribution of cadherin types in the rat anterior pituitary was examined immunohistochemically to clarify the unique cell arrangements caused by homologous and heterologous affinities among cells. N- and E-cadherins were detected continuously along cell membranes, while P-cadherin was not. N- and E-cadherins showed distinct isolation in localization, with N-cadherins localized in hormone-producing cells of distal and intermediate lobes in various amounts, and E-cadherins limited to folliculo-stellate cells and marginal layer cells facing the residual lumen of Rathke's pouch. A similar distribution of cadherins was observed in cell clusters of primary cultured anterior pituitary cells. These findings suggest that differential expression of cell adhesion molecules may be partially responsible for localization of hormone-producing cells and folliculo-stellate cells.

Intercellular communications within the rat anterior pituitary XI: an immunohistochemical study of distributions of S-100 positive cells in the anterior pituitary of the rat.

The distribution of the S-100 protein cell (folliculo-stellate cell) is very important to our understanding of the regulation of the anterior pituitary. In this study, 10 intact 60-day-old male Wistar-Imamichi rats, were separated equally into two groups. One was used for immunohistochemical study, and the other for electron microscopic analysis. Immunostained pituitary sections with S-100 protein antibody were photographed using a CCD camera equipped with a computer. The S-100 protein cells were then measured using NIH image software, and the three-dimensional distribution of the cells was analyzed. The distribution of the cells observed in each serial section showed that S-100 protein cells were dense at the basal zone of the gland and at the "transitional zone" where the pars tuberalis adjoined the anterior and intermediate lobes, where they represented over 50% of the total cell population. They then decreased in number with distance from this region to mid-way towards the sagittal axis before increasing again in the tail of the gland. The population of cells also decreased with increasing distance from the "transitional zone" to the wing and with distance from the basal zone. Portal vessels entered the anterior lobe through the "transitional zone" as thick capillaries, ran through the basal surface and penetrated into the central area of the anterior lobe. In all planes, S-100 protein cells encircled the capillaries. Ultrastructural observations confirmed the light microscopic findings indicating that clusters of agranular cells were densely located at the "transitional zone" and in the pars tuberalis. The distribution pattern of the folliculo-stellate cells and the capillaries showed good agreement and the spatial relationship between these two is detailed so as to better understand hypophyseal histophysiology.

Intercellular communications within the rat anterior pituitary XII: immunohistochemical and physiological evidences for the gap junctional coupling of the folliculo-stellate cells in the rat anterior pituitary.

Since Farquhar [1957. "Corticotrophs" of the rat adenohypophysis as revealed by electron microscopy. Anat. Rec. 127, 291] was the first to report the presence of agranular folliculo-stellate cells as corticotrophs in the anterior pituitary gland, there were no reports about electro-physiological characteristics of the folliculo-stellate cells because of its no hormonal activity and the chaotic distribution of the parenchyma cells. Male Wistar rats, aged 7 weeks with weighing 250--300 g, were separated into two groups. One group was used for immunohistochemical and light microscopical studies to detect S-100 protein and connexin 43. The other group was used for the electro-physiological study and then for the electron microscopical study to know the fine structural character of folliculo-stellate cells after the electro-physiological experiment. Clusters of S-100 protein cells (agranulated folliculo-stellate cells) and numerous connexin 43 positive sites on S-100 protein cells were clear in the "transitional zone" at which the pituitary tissue made the transition from the pars tuberalis to the proximal part of the anterior lobe. Penetration of electrodes to the cells distributed in the transitional zone showed stable membrane potential ranged between--27 and--67mV with no spontaneous activity. Random penetration of electrode showed that larger populations of cell ( approximately 80%) had membrane potentials with -55.6+/-5.1 mV, and less than 20% of cells had the resting membrane potential with -36.0+/-4.4 mV. There were two types of cell couplings; one major group for the recordings from cells with similar deep resting membrane potentials and the other for the recordings from cells with different resting membrane potentials. The former indicated that two cells were electrically coupled while the latter no electrical couples were observed. Carbenoxolone depolarized the membrane by 12.3+/-5.5 mV and reduced the amplitude of electrotonic potentials, and the response recovered by removal of carbenoxolone by the superfusate. The transitional zones of the pituitary glands examined the electrical coupling were observed by an electron microscopy. Almost cytological profiles were observed as intact. The results clearly indicated that the folliculo-stellate cell system deeply participated in the regulation of the anterior pituitary parallel with the portal vessel system, which was the main regulatory system for pituitary hormone secretion.

[Anatomy education in medical and dental schools in Japan].

We studied the anatomy education and the view of anatomy professors on it in medical and dental schools in Japan. In most schools anatomy is taught in the second year. In medical schools, the systematic education separating macroscopic and microscopic anatomy is prevalent. Although the tutorial system has been introduced in 80% of medical schools, its introduction into anatomy education has remained at 30%. The tutorial system is regarded to be more effective by engaged professors than non-engaged. Some kinds of clinical anatomy education have been introduced in half of the medical schools surveyed. In dental schools, on the other hand, macroscopic and microscopic anatomy tend to be taught in combination. One third of the dental schools have introduced clinical anatomy but few schools have a tutorial system. The overwhelming majority of professors are evaluated by students and have regarded the evaluation useful for improving their teaching. They also have thought that the questionnaire and the timing of the evaluation must be considered carefully, and that the evaluation should not be directly used for purposes other than the improvement of education. We have made the proposals for further improvement in anatomy education based upon this study.

A rare anomaly of the extensor digitorum longus.

We encountered the absence of the tendon to the fourth toe of the extensor digitorum longus muscle of the right leg in the body of a 73-year-old Japanese woman during the course of educational dissection at Nagoya City University Medical School. The tendon to the fourth toe of the extensor digitorum longus muscle was solely absent. To our knowledge, this anatomical variation has never been cited in the medical literature. We document the precise gross anatomical findings with some morphometric measurements. Moreover, we discuss the morphology of this anomaly in relation to previously described variations and anomalies of the extensor digitorum longus muscle.

Intercellular communication within the rat anterior pituitary gland: X. Immunohistocytochemistry of S-100 and connexin 43 of folliculo-stellate cells in the rat anterior pituitary gland.

Since Rinehart and Farquhar reported the presence of agranulated cells in the anterior pituitary gland in 1953, the functions of the folliculo-stellate cell remain to be clarified. Intercellular junctions have been described in the monkey, rat, and teleost anterior pituitary glands, indicating the existence of cell-to-cell communication within the organ. We pointed to their possible role in the rapid dissemination of information through a complex interconnecting system of follicles involving gap junctions. The gap junctional/folliculo-stellate cellular network was essential in the maturation and regulation of the pituitary gland system such as the hypothalamic-pituitary-gonadal axis. It has been was shown that a network participated in the conduction of electrophysiological information over a long distance using the ion Ca(++), which propagates to other folliculo-stellate cells by signaling through gap junctions. Sixty-day-old male rats were used in this study for light microscopic immunohistochemistry of S-100 protein, type I collagen, and connexin 43, and for electron microscopy to observe the morphological relationships between the cellular networks of folliculo-stellate cells and granulated pituitary cells. Clusters of anti-S-100 protein-positive cells were clearly observed in a region of the hypophysis tentatively named the transition zone. Anti-S-100 protein-positive cells and their cytoplasmic processes were also present in the anterior lobe and assembled together to form follicular lumina. Type I collagen was clearly shown outlining the incomplete lobular or ductule-like structure making cell cords in the anterior pituitary gland. Numerous microvilli were present within the follicular lumen while around the lumina, junctional specializations including gap junctions were positive for the connexin 43 protein. A nonuniform distribution of the connexin 43-positive sites were observed. Small or dot-shaped positive sites were noted where two clusters of cells were connected; the cells were identified as S-100 cells. Double immunohistochemical staining of the connexin 43 and growth hormone (GH) or connexin 43 and luteinizing hormone (LH) was also performed, demonstrating no direct relationship between the connexin 43 and either the GH or LH cells. These findings indicate that there are two kinds of messages necessary for the hormone release in the pituitary gland. One is via the portal vein system, the other is through the gap junction-mediated networks of folliculo-stellate cells. The granulated cells directly associate with cell membrane of folliculo-stellate cells are able to discharge secretory granules through communication via gap junctions, while those granulated cells that are more distant from the folliculo-stellate cells are only able to discharge hormones via the pituitary hormone-releasing hormone from the portal vein system.

The changes of gap junctions between pituitary folliculo-stellate cells during the postnatal development of Zucker fatty and lean rats.

We investigated the effect of leptin on the postnatal development of gap junctions between folliculo-stellate cells by using Zucker fatty (fa/fa) rats that have defects of the functional leptin receptor. Male Zucker fatty rats (fa/fa) and male Zucker lean rats (+/+) were used at each of the following postnatal ages: 20, 30, 40, 50, 60, 70, 80, 90 days, and 1 year. On one of the aforementioned dates, the anterior pituitary glands were prepared for observation by transmission electron microscopy. We quantified the number of follicles and gap junctions, and calculated the rate of occurrence as the ratio of the number of gap junctions existing between folliculo-stellate cells per intersected follicular profile. In Zucker lean male rats, the number of gap junctions remained relatively constant from days 50 to 90 (0.44 ± 0.02 to 0.49 ± 0.03), and was similar in 1 year old rats (0.47 ± 0.03). These data were statistically higher compared to Zucker fatty male rats. In Zucker fatty male rats, very few gap junctions were observed in 30-day-old rats (0.04 ± 0.01: mean ± SE). This disruption of gap junction formation persisted, and the number of gap junctions remained constant and showed a low level from days 40 to 90 (0.11 ± 0.02 to 0.17 ± 0.02); this finding was similar in 1-year-old rats (0.17 ± 0.02). These observations indicate that the effect of leptin over the gap junction formation within the anterior pituitary glands was directly mediated by interaction with the functional leptin receptor present on the folliculo-stellate cells.

Intercellular communications within the rat anterior pituitary. XVI: postnatal changes of distribution of S-100 protein positive cells, connexin 43 and LH-RH positive sites in the pars tuberalis of the rat pituitary gland. An immunohistochemical and electron microscopic study.

The architecture of luteinizing hormone-releasing hormone (LH-RH) nerve ends and the S-100 protein containing folliculo-stellate cells forming gap junctions in the pars tuberalis is basically important in understanding the regulation of the hormone producing mechanism of anterior pituitary glands. In this study, intact male rats 5-60 days old were prepared for immunohistochemistry and electron microscopy. From immunostained sections, the S-100 containing cells in pars tuberalis were first detected on day 30 and increased in number to day 60; this was parallel to the immunohistochemical staining of gap junction protein, connexin 43. LH-RH positive sites were clearly observed on just behind the optic chiasm and on the root of pituitary stalk on day 30. On day 60, the width of layer increased, while follicles and gap junctions were frequently observed between agranular cells in 10 or more layers of pars tuberalis. In the present study, we investigated the sexual maturation of the anterior pituitary glands through the postnatal development of S-100 positive cells, connexin 43 and LH-RH nerves. It is suggested that the folliculo-stellate cell system including the LH-RH neurons in the pars tuberalis participates in the control of LH secretion along with the portal vein system.

Intercellular communication within the rat anterior pituitary gland. XV. Properties of spontaneous and LHRH-induced Ca2+ transients in the transitional zone of the rat anterior pituitary in situ.

In the transitional zone of the rat anterior pituitary, spontaneous and LHRH-induced Ca(2+) dynamics were visualized using fluo-4 fluorescence Ca(2+) imaging. A majority of cells exhibited spontaneous Ca(2+) transients, while small populations of cells remained quiescent. Approximately 70% of spontaneously active cells generated fast, oscillatory Ca(2+) transients that were inhibited by cyclopiazonic acid (10 µm) but not nicardipine (1 µm), suggesting that Ca(2+) handling by endoplasmic reticulum, but not Ca(2+) influx through voltage-dependent L-type Ca(2+) channels, plays a fundamental role in their generation. In the adult rat anterior pituitary, LHRH (100 µg/ml) caused a transient increase in the Ca(2+) level in a majority of preparations taken from the morning group rats killed between 0930 h and 1030 h. However, the second application of LHRH invariably failed to elevate Ca(2+) levels, suggesting that the long-lasting refractoriness to LHRH stimulation was developed upon the first challenge of LHRH. In contrast, LHRH had no effect in most preparations taken from the afternoon group rats euthanized between 1200 h and 1400 h. In the neonatal rat anterior pituitary, LHRH caused a suppression of spontaneous Ca(2+) transients. Strikingly, the second application of LHRH was capable of reproducing the suppression of Ca(2+) signals, indicating that the refractoriness to LHRH had not been established in neonatal rats. These results suggest that responsiveness to LHRH has a long-term refractoriness in adult rats, and that the physiological LHRH surge may be clocked in the morning. Moreover, LHRH-induced excitation and associated refractoriness appear to be incomplete in neonatal rats and may be acquired during development.

Involvement of the adrenal glands and testis in gap junction formation via testosterone within the male rat anterior pituitary gland.

We investigated the influence of testicular and adrenal androgens on the presence of gap junctions between folliculo-stellate cells in the anterior pituitary glands of 60-day-old Wistar-Imamichi strain male rats. The animals were separated into six groups: Group A served as the controls and had free access to a normal diet and water, Group B was given a normal diet and 0.9% NaCl for their drinking water as the controls of adrenalectomized groups, Group C was castrated, Group D was adrenalectomized, Group E was both castrated and adrenalectomized, and Group F was also both castrated and adrenalectomized. In addition, the animals of Group F were administered a dose of testosterone that is known to produce high physiological levels of the hormones in plasma. Five rats from each group were sacrificed 1, 2, 3, 4, 5, 6, and 7 days after their respective operation, and the anterior pituitary glands were removed and prepared for observation by transmission electron microscopy. We quantified the number of follicles and gap junctions and calculated the rate of occurrence as the ratio of the number of gap junctions existing between folliculo-stellate cells per intersected follicle profile. Simultaneous removal of adrenal glands with castration resulted in a significantly decrease in the number of gap junctions, whereas the administration of testosterone to these rats compensated for this change. These observations indicate that the preservation of gap junctions between folliculo-stellate cells is mainly dependent on androgens from both the testes and adrenal glands in adult male rats.

Altered distribution of interstitial cells in the guinea pig bladder following bladder outlet obstruction.

AIMS: We investigated the effects of bladder outlet obstruction (BOO) on the distribution of interstitial cells (ICs) in the guinea-pig bladder. METHODS: Bladder overactivity of BOO animals was validated with urodynamic studies. Immunohistochemical analyses for Kit and vimentin as markers for ICs were performed on both BOO and control bladders. Morphological and functional properties of detrusor smooth muscle (DSM) were examined with alpha-smooth muscle actin staining and intracellular recording, respectively. Electron microscopy was also carried out to characterize ultrastructural morphology of ICs. RESULTS: Two weeks after surgery, BOO animals showed an increased voiding frequency and a reduced voiding volume. Filling cystometry demonstrated a frequent incidence of non-voiding contractions, a reduced interval between voiding contractions and an increased voiding pressure in BOO bladders. In BOO bladders, the thickness of suburothelial and subserosal connective tissue layers was increased, whilst that of detrusor smooth muscle (DSM) layer was less affected. Population of Kit or vimentin immunoreactive ICs was increased in subserosal layers, and their distribution was altered in suburotherial layer in BOO bladders. Neither alpha-actin immunoreactivity nor spontaneous electrical activity of DSM was altered in BOO bladders. ICs were characterized by their numerous mitochondria and caveolae, and had a close contact with each other and with neighboring DSM or nerves. CONCLUSIONS: These results demonstrated the increased population of ICs in the BOO guinea-pig model for the first time, and suggest that the altered distribution of ICs may contribute to the pathophysiology of bladder overactivity.

Ultrastructural changes at the myotendinous junction induced by exercise.

BACKGROUND: Although exercise is believed to reduce the risk of rupture of the myotendinous junction, exercise-induced structural changes in this region have not been studied. We examined exercise-induced ultrastructural changes in the myotendinous junction of the lower legs in rats. METHODS: Ten adult male LETO rats were used. Five rats were randomly placed in the Exercise group; the remaining five were used as controls and placed in the non-Exercise group. Running exercise was performed every day for 4 weeks. The tibialis anterior and gastrocnemius muscles were then removed from both legs from each animal in the two groups. The specimens were subsequently examined by transmission electron microscopy (TEM). Numerous finger-like processes were observed at the myotendinous junction. The changes in frequency of branching of the finger-like process (the number of times one finger-like process branched) and the direction of the processes (the angle of the major axis of a finger-like process to the longitudinal direction of the muscle fiber) were studied. To evaluate the two indicators above, each 10 fingerlike process was randomly and separately selected from the tibialis anterior and gastrocnemius muscles of rats, providing 50 finger-like processes of both muscles for evaluation per group. RESULTS: In terms of the frequency of branching of the fingerlike processes, the mean values obtained in the non-Exercise group were 0.04 and 0.18 times, respectively, in the tibialis anterior and gastrocnemius muscles and were 0.38 and 1.16 times, respectively, in these two muscles in the Exercise group. Regarding the direction of the finger-like processes, the values were 4.1 degrees and 3.6 degrees, respectively in the non-Exercise group and 10.4 degrees and 14.5 degrees , respectively in the Exercise group. The differences between the two animal groups were significant. CONCLUSIONS: Morphological changes in the myotendinous junction occurred as an adaptation to tension increased by exercise.

Uptake behavior of embryonic chick liver cells.

The capacity of selective uptake by liver cells, focusing particularly on the parenchymal and perisinusoidal stellate cells during chick liver development (8-18 days of incubation), was ultrastructurally examined after injection of 240-nm-diameter lecithin (phosphatidylcholine) -coated or noncoated beads into the extraembryonic circulation. Cytoplasmic projections of both cells as well as extrasinusoidal macrophages reached into the sinusoid-like vascular spaces. The primitive perisinusoidal stellate cells were identified by immunocytochemistry as being rich in desmin-positive cytoplasmic intermediate filaments. The cells demonstrated selective uptake of noncoated beads by means of their cytoplasmic projections. These findings were significant in the early period of incubation, indicating that the phagocytic activity is a characteristic and transient phenomenon of developmental differentiation. Large numbers of coated and a few noncoated beads penetrated into the perivascular spaces. The parenchymal cells incorporated only the coated beads that passed through the endothelial lining, suggesting that these cells express selective but limited phagocytic capacity against large "foreign" substances even long before their maturation. The cell projections were not engaged in uptake function. Extrasinusoidal macrophages, Kupffer cells, and intraluminal primitive macrophages all took up both beads; however, lecithin coating of the beads clearly suppressed their uptake function. These data suggest that the uptake function of large "foreign" substances appears to be intrinsic to liver cells and lecithin coating would be useful for delivering large substances to parenchymal cells.

Intercellular communication within the rat anterior pituitary: XIV electron microscopic and immunohistochemical study on the relationship between the agranular cells and GnRH neurons in the dorsal pars tuberalis of the pituitary gland.

Although numerous investigators in 1970s to 1980s have reported the distribution of LH-RH nerve fibers in the median eminence, a few LH-RH fibers have been shown to be present in the pars tuberalis. The significance of the finding remains to be elucidated, and there are few studies on the distribution of LH-RH neurons in the pars tuberalis, especially in the dorsal pars tuberalis (DPT). Adult male Wistar-Imamichi rats were separated into two groups: one for electron microscopy and the other for immunohistochemistry to observe LH-RH and neurofilaments. Pituitary glands attached to the brain were fixed by perfusion, and the sections were prepared parallel to the sagittal plane. The typical glandular structure of the pars tuberalis was evident beneath the bottom floor of the third ventricle, and the thick glandular structure was present in the foremost region. Closer to the anterior lobe, the glandular structure changed to be a thin layer, and it was again observed at the posterior portion. Then the pituitary stalk was surrounded with the dorsal, lateral, and ventral pars tuberalis. LH-RH and neurofilaments fibers were noted in the bottom floor, and some of them vertically descended to the gland. Adjacent to the glandular folliculostellate cells in the pars tuberalis, Herring bodies with numerous dense granules invading into the gland were present between the pituitary stalk and DPT. It was postulated that the "message" carried by LH-RH might have been transmitted to the cells in the DPT to aid in the modulation of LH release.

A single-nucleotide synonymous mutation in the gag gene controlling human immunodeficiency virus type 1 virion production.

Viral factors as well as host ones play major roles in the disease progression of human immunodeficiency virus type 1 (HIV-1) infection. We have examined cytotoxic T-lymphocyte activity and HIV-1 DNA PCR results of 312 high-risk seronegative drug users in northern Thailand and identified four seronegative cases positive for both assays. Furthermore, we have identified a synonymous mutation in nucleotide position 75 of the gag p17 gene (A426G) of HIV-1 that belongs to the CRF01_AE virus circulating in Thailand. The replication-competent HIV-1 clone containing the A426G mutation demonstrated a dramatic reduction of virion production and perturbation of viral morphogenesis without affecting viral protein synthesis in cells.

Interaction between spontaneous and neurally mediated regulation of smooth muscle tone in the rabbit corpus cavernosum.

Interaction between spontaneous and neurally mediated regulation of tone in the corpus cavernosum smooth muscle (CCSM) of the rabbit was investigated. Changes in isometric muscle tension, intracellular Ca2+ concentration ([Ca2+]i) and membrane potential were recorded. CCSM developed spontaneous contractions, transient increases in [Ca2+]i (Ca2+ transients) and depolarizations. This spontaneous activity was abolished by blocking L-type Ca2+ channels (nicardipine, 1 mum), sarcoplasmic reticulum Ca2+ pump activity (cyclopiazonic acid, 10 microm), Ca2(+)-activated Cl- channels (niflumic acid, 10 mum) or cyclooxygenase-2 (COX-2; NS-398, 1 microm). Transmural nerve stimulation initiated either alpha-adrenergic contractions or nitrergic relaxations of CCSM depending on the level of muscle tone. NS-398 suppressed nerve-evoked contractions by about 70% but caused only a 40% reduction in the corresponding Ca2+ transient. Blocking nitric oxide synthase with N(omega)-nitro-l-arginine (LNA, 100 microm) reinforced nerve-evoked Ca2+ transients by about 150%, whilst increasing the corresponding Ca2+ transients by only 20%. In CCSM preparations that had been pre-contracted with either noradrenaline (0.3 microm) or prostaglandin F(2alpha) (0.1 microm), nerve stimulation inhibited about 70% of the contraction and caused only a 20% decrease in [Ca2+]i. Fluorescent immunohistochemistry with COX-2 antibodies and the reverse transcriptase-polymerase chain reaction (RT-PCR) method showed that the enzyme and its mRNA were highly expressed in the CCSM. These results suggest that spontaneously produced prostaglandins (PGs) not only contribute to the generation of spontaneous contractions but also facilitate nerve-evoked contractions. Conversely, spontaneously released nitric oxide (NO) suppresses excitation. Thus, interaction between spontaneous and neurally mediated regulation of CCSM tone may be fundamental to maintaining the muscle contractility. In addition, both PGs and NO appear to alter CCSM tone with only small changes in [Ca2+]i.

Replication-defective HIV isolated from seronegative individuals at high risk for HIV infection.

We have identified a mutant human immunodeficiency virus type 1 (HIV-1) CRF01_AE that contains a single nucleotide mutation in gag gene from 4 HIV-1 seronegative drug users in Thailand. We found A to G mutation at the nucleotide position 75 of gag p17 gene (A75G) not changing the amino acid sequence. The mutant HIV-1 molecular clones were examined for their replication capability. Although the mutation dramatically reduced the level of virion production, it did not affect the amounts of viral protein synthesis within the transfected cells. In addition, this mutation did not affect the levels of Gag polyproteins. Furthermore, electron microscopic examinations have revealed a dramatic reduction of the virion production and perturbation of viral morphogenesis at the cytoplasmic membrane. These results indicate that the A75G mutation is attributable to the long-term sero-negativity of individuals at high risk of HIV-1 infection and suggest a novel mechanism that regulates HIV production.

The effects of sex steroids on the formation of gap junctions between folliculo-stellate cells; a study in castrated male rats and ovariectomized female rats.

We investigated the relationship between gap junction formation and the sex steroids testosterone, progesterone and 17beta-estradiol in the anterior pituitary glands of castrated male rats and ovariectomized female rats. Male and female 30-day-old Wistar-Imamichi strain rats were castrated or ovariectomized, and 30 days later they were subcutaneously injected with the above sex steroids. They were divided into six groups according to the injected materials: sesame oil (control), testosterone, progesterone, 17beta-estradiol, testosterone with 17beta-estradiol, and progesterone with 17beta-estradiol. Five rats from each group were sacrificed 1, 2, 3, 4 and 5 days after the injections, and the anterior pituitary glands were prepared for observation by transmission electron microscopy. We quantified the number of follicles and gap junctions and calculated the rate of occurrence of gap junctions as the ratio of the number of gap junctions existing between folliculo-stellate cells per intersected follicle profile in electron photomicrographs. The administration of testosterone to castrated male rats increased the rate of gap junctions between folliculo-stellate cells; however, progesterone and 17beta-estradiol did not affect the formation of gap junctions. The administration of progesterone to ovariectomized female rats increased the rate of gap junctions between folliculo-stellate cells; this progesterone effect was prevented by the simultaneous administration of 17beta-estradiol, which by itself did not affect the rate of gap junctions between folliculo-stellate cells. These observations indicate that the formation of gap junctions within the anterior pituitary gland is regulated differently by sex steroids in castrated male and ovariectomized female rats.