Expression of synaptotagmin 1 in the taste buds of rat gustatory papillae

Synapses between taste receptor cells and primary sensory afferent fibers transmit the output signal from taste buds to the central nervous system. The synaptic vesicle cycle at the synapses involves vesicle docking, priming, fusion, endocytosis, and recycling. Many kinds of synaptic vesicle proteins participate in synaptic vesicle cycles. One of these, synaptotagmin 1, binds Ca(2+) phospholipids with high affinity and plays a role in Ca(2+) regulated neurotransmitter release in the central and peripheral nervous systems. However, the expression patterns of synaptotagmin 1 in rat taste tissues have not been determined. We therefore examined the expression patterns of synaptotagmin 1 and several cell specific markers of type II and III cells in rat taste buds. RT-PCR assay showed that synaptotagmin 1 mRNA was expressed in circumvallate papillae. In fungiform, foliate, and circumvallate papillae, the antibody against synaptotagmin 1 yielded the labeling of a subset of taste bud cells and intra- and subgemmal nerve processes. Double labeled experiments showed that synaptotagmin 1 positive cells co-expressed type III cell markers, PGP 9.5, and NCAM. Intragemmal nerve processes positive for synaptotagmin 1 co-expressed PGP 9.5. Conversely, all synaptotagmin 1 expressing cells did not co-expressed type II cell markers, PLCbeta2, or gustducin. These results show that synaptotagmin 1 may play some regulatory roles in vesicle membrane fusion events with the plasma membrane at the synapses of type III cells in rat taste buds.     

An electron microscopic study of apoptosis in the granulosa layer of ovarian follicles in rats treated with continuous illumination

Polycystic ovarian syndrome was induced in adult rats by treatment with continuous illumination to study follicular atresia. Granulosa cells of regressing antral follicles showed characteristic features of apoptosis ultrastructurally: condensed nuclei, apoptotic bodies, and phagocytosis of the apoptotic cells and their fragments by neighboring cells. In this study, moreover, it was observed that an intact granulosa cell was able to ingest another granulosa cell of normal appearance, presumably indicating an early stage of follicular atresia. The phagocytosing granulosa cell surrounded another granulosa cell to be phagocytosed by elongated cell processes. Gap junctions developed between the phagocytic cell and the phagocytosed cell. The phagocytic cell possessed abundant free ribosomes, well-developed rough endoplasmic reticulum and mitochondria, but few lysosomes. The ingested cell was similar in appearance to the phagocytic cell. In addition, granulosa cells were also observed ingesting karyopyknotic cells and degenerative organelles, both displayed in large vacuoles filled with fragments of nuclei and organelles. These findings suggest a process of apoptosis in the granulosa cells during follicular atresia. This study was presented at the 20th Annual Meeting of the Clinical Electron Microscopy Society of Japan, Fukuoka, September 1–3, 1988.     

Therapeutic effect of topical administration of SN50, an inhibitor of nuclear factor-kappaB, in treatment of corneal alkali burns in mice

We evaluated the therapeutic efficacy of topical administration of SN50, an inhibitor of nuclear factor-kappaB, in a corneal alkali burn model in mice. An alkali burn was produced with 1 N NaOH in the cornea of C57BL/6 mice under general anesthesia. SN50 (10 microg/microl) or vehicle was topically administered daily for up to 12 days. The eyes were processed for histological or immunohistochemical examination after bromodeoxyuridine labeling or for semi-quantification of cytokine mRNA. Topical SN50 suppressed nuclear factor-kappaB activation in local cells and reduced the incidence of epithelial defects/ulceration in healing corneas. Myofibroblast generation, macrophage invasion, activity of matrix metalloproteinases, basement membrane destruction, and expression of cytokines were all decreased in treated corneas compared with controls. To elucidate the role of tumor necrosis factor (TNF)-alpha in epithelial cell proliferation, we performed organ culture of mouse eyes with TNF-alpha, SN50, or an inhibitor of c-Jun N-terminal kinase (JNK) and examined cell proliferation in healing corneal epithelium in TNF-alpha-/- mice treated with SN50. An acceleration of epithelial cell proliferation by SN50 treatment was found to depend on TNF-alpha/JNK signaling. In conclusion, topical application of SN50 is effective in treating corneal alkali burns in mice.     

The effects of nucleotides and potassium channel openers on the SUR2A/Kir6.2 complex K+ channel expressed in a mammalian cell line, HEK293T cells

 The effects of potassium channel opening drugs and intracellular nucleotides on the ATP-sensitive K+ (K_ATP) channel composed of SUR2A and Kir6.2 in HEK293T cells were examined using the patch-clamp technique. The SUR2A/Kir6.2 channel was activated effectively by pinacidil, marginally by nicorandil but not by diazoxide. The pinacidil-activated channel currents were inhibited by glibenclamide with a K _i value of 160 nM. Upon formation of inside-out (I-O) patches, spontaneous openings of the channels appeared, which were inhibited by intracellular ATP (ATP_i) equipotently in the presence and in the absence of intracellular Mg²⁺ (Mg²⁺ _i). The channel activity ran-down gradually in I-O patches. The run-down channels could be reactivated by ATP_i only in the presence of Mg²⁺ _i. Uridine 5’-diphosphate (UDP) antagonized the ATP_i-mediated inhibition of the channel activity before run-down. After run-down, UDP activated the channel without antagonizing ATP_i-mediated channel inhibition. Thus, the SUR2A/Kir6.2 reproduced the major properties of the native cardiac K_ATP channel well in terms of nucleotide regulation and pharmacology, and therefore can be a useful tool with which to elucidate the molecular mechanisms characterizing the K_ATP channel. Received: 24 October 1997 / Received after revision and accepted: 4 December 1997     

FGF signaling segregates biliary cell-lineage from chick hepatoblasts cooperatively with BMP4 and ECM components in vitro.

Intrahepatic bile ducts (IHBDs) are indispensable for transporting bile secreted from hepatocytes to the hepatic duct. The biliary epithelial cells (BECs) of the IHBD arise from bipotent hepatoblasts around the portal vein, suggesting the portal mesenchyme is essential for their development. However, except for Notch or Activin/TGF-beta signaling molecules, it is not known which molecules regulate IHBD development. Here, we found that FGF receptors and BMP4 are specifically expressed in the developing IHBD and the hepatic mesenchyme, respectively. Using a mesenchyme-free culture of liver bud, we showed that bFGF and FGF7 induce the hepatoblasts to differentiate into BECs, and that BMP4 enhances bFGF-induced BEC differentiation. The extracellular matrix (ECM) components in the hepatic mesenchyme induced BEC differentiation. Forced expression of a constitutively active form of the FGF receptor partially induced BEC differentiation markers in vivo. These data strongly suggest that bFGF and FGF7 promote BEC differentiation cooperatively with BMP4 and ECMs in vivo.     

Immunohistological analysis in the distribution of cells in the fetal porcine adenohypophysis

Adenohypophyses of porcine fetuses from 25 to 110 days of gestation were studied by immunohistochemical staining to ascertain the ontogeny of specific cell types and their spatial distribution in the pars distalis. No hormonecontaining cells were found before 30 days of gestation. ACTH cells were observed first at 40 days, while GH and LH cells appeared first at 60 days. PRL cells were initially detected at 105 days. ACTH immunoreactive cells were also observed in the pars intermedia at 40 days. Blood capillaries were interposed between cell cords of the pars distalis after 40 days of gestation. ACTH cells were evenly distribution in all areas of the pars distalis except the rostal area (sex zone). GH cells were densely distributed in lateral wings of the pars distalis and immediately anterior to Rathke's lumen. PRL cells resembled GH cells in their distribution pattern, but PRL cells were fewer in number. LH cells were scattered in the sex zone of the pars distalis from 60 to 80 days of gestation. After 90 days, they became scattered throughout the pars distalis but were more numerous in the sex zone than in other areas. The inductive elements of adenohypophysial cells from Rathke's pouch epithelia are discussed. We hypothesize that cell cords of specific areas facing Rathke's lumen may differentiate into specific cell types of the pars distalis during fetal life. © 1992 Wiley-Liss, Inc.     

Analysis of wild-type and e antigen-defective hepatitis B viruses during the course of a short-term corticosteroid therapy in chronic hepatitis B

Hepatitis B virus (HBV), with a G-to-A point mutation at nucleotide 83 in the precore region (mutant HBV83), accounts for most cases of hepatitis B e antigen (HBeAg)-defective HBV. However, it is still not clear how mutant HBV83 is associated with HBe seroconversion. Twenty-six HBeAgpositive patients with chronic hepatitis B who received oral prednisolone (30 mg/day) for 3 weeks were studied to clarify the prevalence of mutant HBV83 during the treatment using polymerase chain reaction with a restriction fragment length polymorphism assay. Twelve (46%) patients seroconverted to anti-HBe 1 year after treatment, whereas 14 (54%) did not. The proportion of mutant HBV83 to whole HBV remained unchanged in both groups during an acute exacerbation induced by withdrawal of corticosteroids. Among 12 anti-HBe-0seroconverted patients, five (56%) of nine patients with only wild-type HBV at baseline developed detectable levels of mutant HBV83 while all three patients with a mixed viral population of wild-type HBV and mu tant HBV83 at baseline developed a higher pro portion of mutant HBV83 one year after treat ment. In contrast, these changes were observed in only one (14%) of seven who failed to seroconvert. The results indicate that a flare-up of hepa titis precedes emergence or selection of mutant HBV83, followed by HBe seroconversion in patients with chronic hepatitis B. © 1995 WiIey-Liss, Inc.     

The expression of vascular endothelial growth factor-A and -C, and receptors 1 and 3: correlation with lymph node metastasis and prognosis in tongue squamous cell carcinoma

We determined whether the expression of VEGF-A and -C and their receptors, Flt-1 and Flt-4, are associated with primary tumor size, regional lymph node metastasis, distant metastasis and prognosis in patients with tongue carcinoma. The expression of VEGF-A and -C, and their receptors, Flt-1 and Flt-4, in biopsy specimens taken from 73 patients with tongue carcinoma were examined by immunohistochemical staining. VEGF-A expression was associated with distant failure and VEGF-C expression correlated with locoregional recurrence and distant failure. Furthermore, VEGF-C expression was associated with lymph node recurrence in N0 cases. Multivariate analysis revealed that VEGF-C expression was an exclusively independent factor influencing lymph node metastasis. In terms of the overall 5-year survival rate, there was no significance correlation between the overall 5-year survival rate and expression of VEGF-A, Flt-1 and Flt-4 expression, whereas there was a significant difference between VEGF-C-positive and VEGF-C-negative cases (VEGF-C-positive, 51.7% vs VEGF-C-negative, 94.2%). Furthermore, there was a significant difference between positive and negative expression for both VEGF-A and VEGF-C. Multivariate analysis revealed that lymph node metastasis and VEGF-C expression were exclusive, independent factors influencing the overall survival rate. VEGF-C expression may be a predictive factor of regional lymph node recurrence and prognosis in patients with tongue carcinoma.     

CUSHING'S SYNDROME WITH BILATERAL MULTINODULAR ADRENAL HYPERPLASIA

An unusual case of Cushing's syndrome of a 59-year-old man with bilateral multinodular adrenal hyperplasia and microadenoma of the pituitary gland is presented. Failure to suppress plasma Cortisol with large doses of dexamethasone may suggest autonomous growth of hyperplastic nodules of the adrenals, which were at first induced by prolonged stimuli of ACTH from the microadenoma of the pituitary gland. ACTH could not be detected in the microadenoma cells on paraffin sections, while Crooke's cells were strongly positive for ACTH. The interrelation between bilateral multinodular adrenal hyperplasia and pituitary microadenoma is discussed.     

Direct in vivo protein transduction into a specific restricted brain area in rats

Attempts at protein transduction into specific restricted brain areas have remained unsuccessful. We attempted targeted, direct in vivo protein transduction by microinjecting beta-galactosidase (beta-gal) with hemagglutinating virus of Japan envelope (HVJ-E) vector into the rat nucleus tractus solitarius (NTS). The medulla oblongata including the NTS was removed 6h post-injection and cryostat sections were histochemically stained to detect beta-gal enzymatic activity. beta-gal-positive cells were present in these sections as was beta-gal activity determined by colorimetric analysis. beta-gal-positive cells were not present in the rats microinjected only beta-gal protein without HVJ-E vector. Our findings suggest that direct in vivo protein transduction into specific restricted brain areas is possible. The type of targeted delivery system we present may have wide applications in the administration of therapeutic proteins to the central nervous system.