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Chromogranin A (CGA) is a member of a family of highly acidic proteins co-stored and co-released with catecholamines in the adrenal medullary cells as well as in other neurons and paraneurons. The nucleotide sequence encoding equine CGA was determined using RT-PCR and rapid amplification of complementary DNA (cDNA) ends (RACE) techniques. A total 1,828 bp of the nucleotide sequence reveals that equine CGA is a 448-residue protein preceded by an 18-residue signal peptide. Comparison of the amino acid sequence of equine CGA with those of human, porcine, bovine, mouse, rat and frog CGA showed high conservation at the NH2-terminal 1-77 amino acids regions (94.8%, 93.5%, 92.2%, 81.8%, 83.1% and 66.2%, respectively) and COOH-terminal 314-430 amino acids regions (90.6%, 81.4%, 90.6%, 80.5%, 83.3% and 39.0%, respectively), as well as a potential dibasic cleavage site, whereas the middle portion showed marked sequence variation (52.5%, 49.1%, 38.9%, 26.6%, 27.9% and 6.2%, respectively). Northern blot analysis and RT-PCR elucidated the tissue distribution of equine CGA mRNA. Its expression was confirmed not only in the adrenal medullary cells but also in other organs (cerebrum, cerebellum, pituitary gland, spinal cord, liver, thyroid gland, striated muscle, lung, spleen, kidney, parotid gland and sublingual gland). Further, in adrenal chromaffin cells and pituitary cells of the anterior-intermediate lobe, the expression was confirmed by in situ hybridization with anti-sense CGA cRNA probe.

Sensory innervation of the entire hard palate was investigated in the rat using serial sections immunostained for protein gene product 9.5 (PGP 9.5), a neuronal marker. PGP 9.5-immunoreactive nerve endings were widely distributed in the hard palate, but the innervation pattern and density differed among portions. They were numerous at papillary protrusions including the incisal papilla, antemolar/intermolar rugae, and postrugal filiform papillae. Immunoreactive free nerve endings gathered at the summits of the connective tissue papillae, some of them entering deeply into the epithelium. Electron microscopy demonstrated that nerves in the postrugal filiform papillae reached the stratum corneum. The atrial region, possibly the most sensitive in the hard palate, showed unique innervation: its anterior part, adjacent to incisors, developed intraepithelial networks of fine and beaded nerves, whereas its posterior part revealed cone-shaped nerve terminals formed on the connective tissue papillae of the atrial folds which comprised two lines of longitudinal flaps. Taste bud-like corpuscles gathered in the medial walls of the incisal canals and in the "Geschmacksstreifen" (taste stripes) present at the most anterior part of the soft palate. The hard palate of the rat is thus richly innervated, and is characterized by region-specific nerve endings which may be involved in mechano- and chemoreception in the oral cavity.

Background: Germinated barley foodstuffs (GBF), which are derived from brewer’s spent grain and are a highly safe food substance, increased butyrate production in the lower intestine and prevented mucosal damage and bloody diarrhoea in an acute experimental colitis model. As human histocompatibility leucocyte antigen (HLA)-B27 transgenic rats develop spontaneous and chronic intestinal inflammation resembling ulcerative colitis, we investigated the mechanisms underlying the preventive effects of GBF against a spontaneous and chronic colitis model. Specifically, the production of bacterial butyrate and the regulation of proinflammatory cytokine production were examined. Methods: A GBF diet and a cellulose (CE) diet were fed to HLA-B27 transgenic rats for 13 weeks. The presence of faecal occult blood, colonic mucosal protein, DNA and RNA content, colonic myeloperoxidase activity, nuclear factor kappa B (NFκB) DNA binding activity, the depth of the crypts and serum inflammatory parameters were then evaluated. Butyrate production in the caecal contents was also determined. Results: Feeding GBF significantly increased bacterial butyrate production and simultaneously attenuated the presence of faecal occult blood and colonic mucosal hyperplasia. Colonic mucosal NFκB-DNA binding activity and the production of interleukin-8 were also suppressed by the butyrate produced from GBF. Conclusions: Germinated barley foodstuffs feeding promotes bacterial butyrate production and attenuated inflammation in both spontaneous and chronic colitis in HLA-B27 transgenic rats. © 1999 Blackwell Science Asia Pty Ltd

Protein gene product 9.5 (PGP9.5), originally isolated as a neuron-specific protein, belongs to a family of ubiquitin carboxyl-terminal hydrolases that play important roles in the nonlysosomal proteolytic pathway. Antibodies against PGP9.5 have been used for immunohistochemical detection of neural elements, although some non-neuronal cells are also immunoreactive for PGP9.5. In the present study, developing testes of the mouse were immunostained after autoclave pretreatment of sections. In the testes of days 8 and 16, PGP9.5 was only localized on the spermatogonia, whereas on day 30 and in adults it appeared not only on spermatogonia, but also on Sertoli cells. In the testis of the male sterile W/Wv mutant, very little, but strong, immunoreactivity was detected at some Sertoli cells, which were phagocytizing Sertoli cell aggregations that had fallen from the basal membrane. Additionally, it was confirmed that the nucleotide sequence of PGP9.5 in mice was highly conserved, like that in other mammals. These results suggest that PGP9.5 is a useful marker for activated Sertoli cells, playing an important role in degradation of abnormal proteins. Mol. Reprod. Dev. 54:333–341, 1999. © 1999 Wiley-Liss, Inc.

Chromogranin A (CgA) is a member of a family of highly acidic proteins, chromogranins, which are co-stored in the adrenergic neurons and paraneurons and co-released with adrenaline and noradrenaline (NAd) in response to adequate stimulation. The present study provides novel evidence that CgA-like immunoreactivity (IR) is stored in the exocrine cells in the granular convoluted tubule, and is secreted into saliva by stimulation with NAd and acetylcholine (ACh) in the isolated and perfused rat submandibular gland. NAd at 1 microM produced maximum secretion of CgA-like IR (<< 0.9 mM) and a marked increase in salivary flow. Further increases in NAd concentration (10 or 100 microM) yielded concentration-dependent decreases in both responses. ACh at 1 microM produced maximum salivary flow and a slight elevation of CgA-like IR secretion (6 microM); 100 microM ACh decreased the salivary flow but increased the CgA-like IR secretion (0.6 mM). Electron microscopic examination showed vigorous compound exocytosis of secretory granules in the cells of the granular convoluted tubule when the submandibular gland was stimulated with 1 microM NAd. These results provide an experimental basis for the view that the salivary CgA-like IR secretion may be a sensitive and quantitative index of the activity of the sympathetic nervous system innervating the gland.

Background and Aims: The consumption of germinated barley foodstuff (GBF) prevents inflammation and diarrhoea in a colitis model. In this study we investigated the mechanism of the preventative effect of GBF on experimental colitis in rats, in view of production of bacterial butyrate and preservation of intestinal barrier function. Methods: Sprague–Dawley rats administered with diets supplemented with 3.5% dextran sodium sulphate were used as an experimental colitis model. Butyrate was given to rats orally or intracaecally. Intestinal barrier function was estimated by light microscopic observation of the mucosa, intestinal permeability and bacterial translocation. Results: Mucosal damage was reduced by intracaecal administration of butyrate, but not by oral administration. Bacterial butyrate production and reduction of mucosal damage depended on the dose of GBF in diets. The action of endogenous bacterial butyrate, including the reduction of intestinal permeability and bacterial translocation, was inhibited by administration of an inhibitor of β-oxidation of short-chain fatty acids. Conclusions: The feeding of GBF promotes bacterial butyrate production and improves intestinal barrier function in rats, resulting in mitigation of experimental colitis.

BCNT, named after Bucentaur, is a protein that contains a 324-amino-acid region derived from part of a long interspersed DNA sequence element (LINE) in Ruminantia. However, the unique portion is completely missing in human and mouse BCNTs. Since no significant information on their function has been obtained by homology search, we at first examined cellular localization and biochemical characteristics of bovine BCNT to get a hint on its function. Subcellular fractionation and immunohistochemical analyses using a normal bovine epithelial cell line and bovine brain revealed that a significant amount of bovine BCNT is localized in the nuclei, while the major portion is present in the cytosol. Furthermore, it was shown that bovine BCNT is a phosphoprotein and that both bovine and human BCNTs are phosphorylated by casein kinase II in vitro. These results show that BCNTs consist of a unique family, probably a substrate of casein kinase II, which may contribute further to the understanding of gene evolution.

Fibroblast-like (Type B) synoviocytes are cells in the synovial membrane that are responsible for production of both synovial fluid and the extracellular matrix in the synovial intima. Immunostaining of the horse synovial membrane for protein gene product (PGP) 9.5, which is a neuron-specific ubiquitin C-terminal hydrolase, demonstrated selective localization of the immunoreactivity in a synoviocyte population different from acid phosphatase-positive Type A synoviocytes. The immunoreactive cells were lined up in the synovial intima and extended dendritic processes towards the joint cavity to form a dense plexus on the surface. Electron microscopic examination clearly identified the PGP 9.5-immunoreactive cells as Type B synoviocytes characterized by developed rough endoplasmic reticulum and free ribosomes. Immunoreactivity for PGP 9.5 was diffusely distributed throughout the cytoplasm, including the tips of fine processes. Western and Northern blot analyses could not distinguish the corresponding protein and mRNA obtained from the brain and synovial membrane. The existence of the neuron-specific PGP 9.5 in Type B synoviocytes suggests a common mechanism regulating the protein metabolism between neurons and synoviocytes, and also provides a new cytochemical marker for identification of the cells.

1. The aim of our study is to clarify the relationship between expression pattern of P2X receptors and the cell type of male adult rat (Wistar) dorsal root ganglion (DRG) neurons. We identified the nociceptive cells of acutely dissociated DRG neurons from adult rats type using capsaicin sensitivity. 2. Two types of ATP-activated currents, one with fast, the other with slow desensitization, were found under voltage-clamp conditions. In addition, cells with fast but not slow desensitization responded to capsaicin, indicating that there was a relationship between current kinetics and capsaicin-sensitivity. 3. Both types of neurons were responsive to ATP and alpha, beta methylene-ATP (alpha,betameATP). The concentration of alpha,(beta)meATP producing half-maximal activation (EC50) of neurons with fast desensitization was less (11 microM) than that of neurons with slow desensitization (63 microM), while the Hill coefficients were similar. Suramin and pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid tetrasodium (PPADS) antagonized alpha,betameATP-induced currents in both types of neurons. 4. In situ hybridization revealed that small cells of the DRG predominantly expressed mRNAs of P2X3 and medium-sized cells expressed mRNAs of P2X2 and P2X3. In contrast, both of mRNAs were not detected in large cells of the DRG. 5. These results suggest that capsaicin-sensitive, small-sized DRG neurons expressed mainly the homomeric P2X3 subunit and that capsaicin-insensitive, medium-sized DRG neurons expressed the heteromultimeric receptor with P2X2 and P2X3.

Background Germinated barley foodstuff (GBF) has been shown to attenuate intestinal injury in animal models, largely by increasing luminal short-chain fatty acid production. Aim To investigate the safety and efficacy of GBF in the treatment of ulcerative colitis (UC). Methods Ten patients with active UC received 30 g of GBF daily for 4 weeks in an open-label treatment protocol while the baseline anti-inflammatory therapy was continued. The response to treatment was evaluated clinically and endoscopically. Pre- and post-treatment stool concentrations of short-chain fatty acids were measured by gas-liquid chromatography. Results Patients showed improvement in their clinical activity index scores, with a significant decrease in the score from 6.9 ± 1.4 to 2.8 ± 1.5 (mean ± S.E.M., P

Ratiometric images of cytoplasmic Ca2+ concentration ([Ca2+]c) in individual cells were recorded simultaneously with a confocal ultraviolet-laser microscope in the Indo-1-loaded islets isolated from mice. After changes in [Ca2+]c in response to glucose or amino acids were recorded, the islet was fixed, permeabilized, and stained by the indirect immunofluorescence method against insulin or glucagon in situ; the individual cells were then identified in the focal plain identical to that used for the [Ca2+]c imaging. Almost all cells identified as insulin-positive (beta-cells) by their distinct immunofluorescence responded to the increase in glucose concentration from 3 to 11 mmol/l with an increase in [Ca2+]c. Major populations of cells (approximately 65%) identified as glucagon-positive (alpha-cells) responded to the addition of arginine (5-10 mmol/l) to 3 mmol/l glucose solution with an increase in [Ca2+]c. About half of the alpha-cells (47.6%) responded to the addition of alanine (5-10 mmol/l) to 3 mmol/l glucose solution with an increase in [Ca2+]c. In contrast

Cryptopatches (CPs) are part of the murine intestinal immune compartment. Cells isolated from CPs of the small intestine that were c-kit positive (c-kit+) but lineage markers negative (Lin−) gave rise to T cell receptor (TCR) αβ and TCR γδ intestinal intraepithelial T cells after in vivo transfer or tissue engraftment into severe combined immunodeficient mice. In contrast, cells from Peyer's patches and mesenteric lymph nodes, which belong in the same intestinal immune compartment but lack c-kit+Lin−cells, failed to do so. These findings and results of electron microscopic analysis provide evidence of a local intestinal T cell precursor that develops in the CPs.

There have been no reports on an abundance of CD4−8−B220+ αβ T cells, seen in autoimmune mice carrying the lpr gene (abnormal Fas gene), in any immune organs of normal mice. We herein report, however, that such αβ T cells were abundant at intraepithelial sites of the appendix in normal mice. They lacked the expression of NK1.1 Ags (C57BL/6 mice), but had the morphology of granular lymphocytes and contained forbidden T cell clones in the minor lymphocyte-stimulating antigen (Mls) system (C3H/He mice with Mls-1b2a). In other words, many properties of intraepithelial T cells in the appendix resembled those ascribed to abnormal αβ T cells, which expand in the lymph nodes and spleen of lpr mice. In the case of lpr mice, CD4−8−B220+ αβ T cells first expanded in the appendix and then extended to other organs. CD4−8−B220+ αβ T cells seemed to originate in situ from c-kit+ stem cells in the appendix. These results suggest that the appendix is one of the primary sites in which CD4−8−B220+ αβ T cells exist, and that these cells carry many primordial properties as prototype T cells.

The distribution and entire shape of olfactory receptor cells were investigated by means of whole-mount preparations of the nasal mucosa. Whole mucosa isolated from the nasal septum of rats was processed, as "a free-floating section", and examined by the avidin-biotin complex (ABC) method using antisera against protein gene product 9.5 (PGP 9.5) and calbindin. Essentially all receptor cells were immunolabeled with the PGP 9.5 antiserum, but only half of PGP 9.5-immunoreactive cells were calbindin-immunoreactive. In the immunostaining of whole-mount preparations, pretreatment of tissues by freeze-thawing and dipping in ethanol and xylene greatly improved the permeability of antibodies. Overview of the nasal septum showed that the dorsal and ventral portions of the rostral olfactory area extended deeply into the respiratory area, making a "semi-lunar" shape. The boundary between the two areas was clearly demarcated, although several receptor cells were scattered in the respiratory area near the boundary. Observation at higher magnification clearly demonstrated that several axons derived from perikarya gathered to form nerve bundles showing a dendritic pattern. Proximal axons close to perikarya displayed beaded structures with intense immunoreactivity. They were electron-microscopically identified as swollen portions of axons which might be formed in association with the axonal flow. The present study showed that whole-mount preparation of the nasal mucosa for immunohistochemistry is a useful tool to analyze the morphology of olfactory receptor cells and axons.

ATP-sensitive K + (K ATP ) channels are known to play important roles in various cellular functions, but the direct consequences of disruption of K ATP channel function are largely unknown. We have generated transgenic mice expressing a dominant-negative form of the K ATP channel subunit Kir6.2 (Kir6.2G132S, substitution of glycine with serine at position 132) in pancreatic beta cells. Kir6.2G132S transgenic mice develop hypoglycemia with hyperinsulinemia in neonates and hyperglycemia with hypoinsulinemia and decreased beta cell population in adults. K ATP channel function is found to be impaired in the beta cells of transgenic mice with hyperglycemia. In addition, both resting membrane potential and basal calcium concentrations are shown to be significantly elevated in the beta cells of transgenic mice. We also found a high frequency of apoptotic beta cells before the appearance of hyperglycemia in the transgenic mice, suggesting that the K ATP channel might play a significant role in beta cell survival in addition to its role in the regulation of insulin secretion.

The putative membrane fatty acid transporter (FAT) protein and its mRNA, originally expressed in adipose tissue, were found in the tongue of rats. Northern blot analysis showed a significant expression of FAT mRNA in the epithelial layer of circumvallate papillae. Immunohistochemical staining revealed that immunoreactivity for FAT is specifically localized in the apical part of taste bud cells, possibly gustatory cells, in the circumvallate papillae.

Postnatal development of B lymphocytes and plasma cells containing different classes of immunoglobulins (IgG, IgA, and IgM) was immunohistochemically studied in the oviduct of the Dekalb strain of the White Leghorn chicken. B lymphocytes first appeared in the lamina propria of the chicken oviduct at 5 weeks of age. Their frequency of occurrence peaked at 15 weeks from the infundibulum to the uterus (glandular part), while in the vagina (aglandular part) it did so at 21 weeks. Intraepithelial B lymphocytes were very rare and exclusively located in the vagina at 19 and 21 weeks. Plasma cells first appeared in the lamina propria of the oviduct at 11 weeks of age, and this frequency peaked at 32 weeks. IgG-containing plasma cells were most numerous in the glandular part, whereas in the aglandular part IgA and IgM cells were more numerous than IgG cells. When 7-day-old-chickens were treated with sex hormones, B lymphocytes and plasma cells appeared 12 h and 5 days after the hormone injection, respectively. Their frequency of occurrence was statistically higher in diethylstilbestrol (DES)-treated chickens than in DES plus progesterone-treated chickens. This suggests that the postnatal development of B lymphocytes and plasma cells in the oviduct of the chicken is correlated to estrogen secretion.

When verotoxin (VT)-producing attaching and effacing Escherichia coli (AEEC, serotype O5: H-) were inoculated perorally into 10-day-old rabbits, attaching of the E. coli to enterocytes and effecting of their microvillous portion were observed extensively from the ileum to the colon. Subsequent apoptotic changes of the infected enterocytes were demonstrated by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) reaction and electron microscopy. Apoptosis was also induced in cultured Vero cells by inoculation of VT extracted from the AEEC. This study clarified that VT-producing AEEC induce apoptosis of enterocytes, causing mucosal damage.

A novel protein harboring a 280-amino acid region from an Alu-linked repetitive sequence (bovine Alu-like dimer-driven family) was isolated from a bovine brain S-100 fraction using monoclonal antibodies against a rat GTPase-activating protein that shares the same epitope. The protein has an apparent molecular mass of 97 kDa (p97). Western blot analysis using extracts prepared from various tissues showed p97 to be predominantly detected in brain and moderately in liver and lung. From sequence analysis of the cDNA encoding p97, it was found that the 840-base pair sequence homologous to a part of the bovine Alu-like dimer-driven family, which has never been shown to be expressed, occurs in the middle of the protein coding region. The protein also contains a pair of intramolecular repeats composed of 40 highly hydrophilic amino acids at the C terminus. Human cDNA homologous to p97 was cloned, and its nucleotide sequence demonstrates that the 840-base pair repetitive sequence and one of the intramolecular repeats are missing. We named p97 bovine BCNT after Bucentaur. These results show that bovine BCNT is a unique molecule and suggest that an analysis of the relationship between bovine bcnt and its human homologue may help further the understanding of gene organization and evolution.

Brown adipose tissue (BAT) is responsible for cold- and diet-induced thermogenesis, and thereby contributes to the control of whole-body energy expenditure (EE) and body fat content. BAT activity can be assessed by fluoro-2-deoxyglucose (FDG)-positron emission tomography (PET) in human subjects. Grains of paradise (GP, Aframomum melegueta), a species of the ginger family, contain pungent, aromatic ketones such as 6-paradol, 6-gingerol and 6-shogaol. An alcohol extract of GP seeds and 6-paradol are known to activate BAT thermogenesis in small rodents. The present study aimed to examine the effects of the GP extract on whole-body EE and to analyse its relation to BAT activity in men. A total of nineteen healthy male volunteers aged 20–32 years underwent FDG-PET after 2 h of exposure to cold at 19°C with light clothing. A total of twelve subjects showed marked FDG uptake into the adipose tissue of the supraclavicular and paraspinal regions (BAT positive). The remaining seven showed no detectable uptake (BAT negative). Within 4 weeks after the FDG-PET examination, whole-body EE was measured at 27°C before and after oral ingestion of GP extract (40 mg) in a single-blind, randomised, placebo-controlled, crossover design. The resting EE of the BAT-positive group did not differ from that of the BAT-negative group. After GP extract ingestion, the EE of the BAT-positive group increased within 2 h to a significantly greater (P

RNA blot analysis has shown that an ionotropic ATP receptor P2X4 is widely distributed, as compared with other P2X subtypes. Cellular distribution of the P2X4 ATP receptor mRNA was investigated by in situ hybridization technique in the brain and non-neuronal organs. In the brain, transcripts of the P2X4 were widely distributed in various regions, with the highest signals in the cerebellar Purkinje cells and granule cells. In other organs, intense and selective expression was detected in seminiferous tubules of the testis and intestinal crypts of the colon, suggesting an involvement of ATP in spermatogenesis and intestinal secretion. A low but significant level of the P2X4 mRNA was detected in the adenohypophysis and the cortex of the adrenal gland. The tissue- or cell-specific expression of the P2X4 mRNA is of importance to understand the functional roles of the purinergic transmission system.

Extrathymic T cells exist in the liver and are often seen in close contact with Kupffer cells in the hepatic sinusoids. Since selective depletion of Kupffer cells has become possible by using liposome-encapsulated clodronate, it was investigated whether elimination of Kupffer cells influences the level of extrathymic T cells in the liver. Extrathymic T cells were identified as interleukin-2 receptor beta-chain (IL-2R beta) intermediate TCR (TCRint) cells by two-color staining for CD3 or T cell receptor (TCR) and IL-2R beta. The elimination of Kupffer cells did not significantly affect levels of TCRint cells up to 7 days after treatment. We then examined monocyte colony stimulating factor (M-CSF)-deficient op/op mice (low levels of Kupffer cells). Extrathymic T cells both in the liver and spleen of these mice were detected at a level comparable to that of control mice. Since extrathymic T cells in the liver are sometimes located in the parenchymal space, the relationship between extrathymic T cells and hepatocytes was then examined. Electron microscopy revealed that some hepatic T cells adhered directly to hepatocytes. When hepatocytes were damaged by a single injection of CCl4, hepatocyte death and subsequent hepatic fibrosis were induced. Beginning 3 days after injection, CD3int cells, but not other type of cells, decreased prominently. Purified CD3int cells, as well as whole lymphocytes in the liver, were cytotoxic against syngeneic hepatoma. In parallel with the above-mentioned hepatic damage, the cytotoxic activity of lymphocytes against such targets was impaired in the liver. These results suggest that extra-thymic generation of TCRint cells and their acquisition of cytotoxic function are relatively independent of Kupffer cells, but are dependent on hepatocytes.

The tunneling nanotube (TNT) is a structure used for intercellular communication, and is a thin membrane protrusion mediating transport of various signaling molecules and cellular components. M-Sec has potent membrane deformation ability and induces TNT formation in cooperation with the Ral/exocyst complex. Here, we show that the N-terminal polybasic region of M-Sec directly binds phosphatidylinositol (4,5)-bisphosphate for its localization to the plasma membrane during the initial stage of TNT formation. We further report a crystal structure of M-Sec, which consists of helix bundles arranged in a straight rod-like shape, similar to the membrane tethering complex subunits. A positively charged surface in the C-terminal domains is required for M-Sec interaction with active RalA to extend the plasma membrane protrusions. Our results suggest that the membrane-associated M-Sec recruits active RalA, which directs the exocyst complex to form TNTs.

Previous studies have demonstrated that the epithelium of the respiratory portion of rat nasal mucosa is amply supplied by nerve fibers with immunoreactivities for calcitonin gene-related peptide (CGRP) and substance P (SP), these fibers most likely acting as sensory mediators in the mucosa. The present study demonstrates that some intraepithelial fibers contain a VIP-immunoreactivity whose occurrence in these nerves has previously been neglected. The present study further aims to confirm the occurrence of NO-producing intraepithelial nerve fibers in the rat nasal mucosa and to examine its colocalization with CGRP and with VIP. Double staining methods were used to evaluate the colocalization of NADPH-diaphorase. The reactivity for NADPH-diaphorase and that for CGRP coexisted in only a small part, if any, of the nerve fibers distributed at the basal portion of the epithelium. In the perpendicularly and obliquely oriented transepithelial nerve fibers, both reactivities were clearly demonstrated to be separated in different fibers. VIP immunoreactivity was also present in a part of the intraepithelial nerve fibers of the nasal mucosa, and their entire population was shown to be positive for NADPH-diaphorase. The NADPH-diaphorase-positive reaction was displayed in only a small population of neurons in the trigeminal ganglion, whereas it was seen in numerous neurons in sphenopalatine ganglion, being colocalized with VIP.

The regional distribution of calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibers in the epithelium throughout the nasal cavity was determined using semiserial cryostat sections and whole mount preparations. Both respiratory and olfactory epithelia displayed, in their basal portion, an extensive nerve plexus projecting beaded nerve fibers toward the luminal surface. However, the density of the intraepithelial CGRP nerve fibers conspicuously varied according to regions. They were abundant on the septal mucosa, the ventromedial side of the nasoturbinates and the dorsal surface of the maxilloturbinates, while less numerous on the lateral side of the naso- and maxilloturbinates and on the lateral nasal wall. Another difference in the regional distribution of nerves was recognized between the anterior and posterior portions of the respiratory area: the anterior portion received a denser supply of intraepithelial CGRP fibers than the posterior portion. Characteristically, the transepithelial CGRP-immunoreactive nerve fibers were densest on the anteroventral aspect of the nasoturbinates and on the anterodorsal surface of the maxilloturbinates. Some of them appeared to penetrate through the epithelium to come into contact with the lumen of the nasal cavity. These results suggest that the CGRP fibers in the epithelium display a region-specific distribution, apparently disposed more densely over the areas which are more directly exposed to inhaled air, possibly that containing irritants and toxicants.

Epithelial cells of the gut are characterized by rapid, constant cell renewal. The death of epithelial cells at the villus tips occurs so regularly that it must be regarded as a well-controlled cell death, designated as apoptosis. However, only limited information has been available on the mechanism of this phenomenon, including the disposal of the effete cells. In the small intestine of the guinea pig and monkey, macrophages are densely aggregated at the lamina propria of the villus tips and vigorously engaged in the phagocytosis of effete epithelial cells. Intraepithelial lymphocytes possessing cytoplasmic granules, possibly intense in cytotoxicity, are topographically associated with the dying enterocytes, suggesting lymphocyte-mediated killing. After the engulfing of apoptotic enterocytes is left within the epithelium, maintaining the epithelial barrier until it is pinched off by the pushing of surrounding enterocytes. In the rat and mouse, on the other hand, effete enterocytes are exfoliated as a whole from the villus tips into the gut lumen. Macrophages, also numerous at the villus tips in these species, are less intense in phagocytotic activity. At the shoulder region of the villus, subepithelial macrophages extend thick processes deep into the epithelium; the processes appear to push out some enterocytes with typical apoptotic signs into the gut lumen. Lamina propria macrophages in the rat and mouse do not engulf enterocytes, but are believed to be involved in the induction of their apoptosis. The species difference in the mechanism of the apoptosis of enterocytes provides clues for understanding apoptosis.

Until recently, little has been known about the morphological features of dying enterocytes at the villus tips of the human small intestine. The present study aimed to show the exfoliating processes of effete enterocytes at the villus tips. Cellular elements of the duodenal lumen and jejunal tissue in humans were fixed and processed for DNA nick end labeling (TUNEL), and transmission and scanning electron microscopy (TEM and SEM). Most cellular elements in the duodenal lumen were enterocytes having TUNEL-positive nuclei. By SEM, protruding enterocytes were discerned at the villus tips. Using the SEM samples embedded in epoxy resin, protruding enterocytes were observed at the villus tips by TEM; they were shrunk by forming numerous clear and autophagic vacuoles, took dome-like profiles, and possessed nuclei with chromatin condensation. The intercellular spaces beneath these protruding or effete enterocytes were often occupied by large lymphocytes. By TUNEL reaction, positive stainings appeared in the epithelium not only at the tip of the villi but also around the site. The results suggest that effete enterocytes at the villus tips of human small intertine are first shrunk by forming clear and autophagic vacuoles, and showed that their nuclei exhibit chromatin condensation immediately before being exfoliated into the lumen.

The localization of nitric oxide synthase (NOS) and its cofactor, nicotinamide-adenine dinucleotide hydrogen phosphate (NADPH)-diaphorase, was examined in the nasal mucosa of the rat by immunohistochemical and histochemical methods. In addition to cryostat sections, whole mount preparations were used to examine the distribution of nerves. Both in the nasal mucosa and in associated ganglia, the distribution of NOS-immunoreactive nervous structures essentially corresponded to that of NADPH-diaphorase-positive ones. The NOS-immunopositive nerve fibers in the respiratory area of the nasal mucosa were distributed around blood vessels and in submucosal glands. Part of the respiratory area was supplied with intraepithelial arborizations of the immunopositive fibers. The epithelial cells in the respiratory area were NADPH-diaphorase positive but NOS immunoreactivity negative. In the olfactory area, the NADPH-diaphorase- and NOS-positive nerve fibers were restricted to blood vessels located deep in the submucosa. Throughout the nasal mucosa, arterial endothelium was NADPH-diaphorase positive but NOS immunoreactivity negative. Both NOS immunoreactivity and NADPH-diaphorase activity were found in major populations of neuronal somata in the sphenopalatine ganglion. The present study provides the direct evidence supporting the notion that nitric oxide is richly produced in autonomic nerves of the nasal mucosa derived from the sphenopalatine ganglion.

Galectin-1 and galectin-3, β-galactoside-binding lectins, are specifically expressed in the regressing corpus luteum (CL) of mice; however, their function remains unclear. In this study, we examined the effects of prolactin (PRL) and prostaglandin F2 α (PGF2 α), two main regulatory molecules of mouse CL function, on galectin expression. In situ hybridization analysis clearly demonstrated an initial increase in galectin-1 in the newly formed CL (CLN) after postpartum ovulation 48 h after compulsory weaning. This was accompanied by a decline in 3β-hydroxysteroid dehydrogenase (3β-HSD) and LH receptor (LH-R) expression, suggesting a withdrawal of PRL stimulation. At 72 h after the weaning, the expression of both galectins in CLN was remarkably increased, being associated with an intense expression of progesterone degradation enzyme (20α-HSD). Compulsory weaning did not significantly alter both galectin expression in the remaining CL of pregnancy (CLP), while PGF2 α strongly upregulated both galectin expression only in the remaining CLP, which lacked LH-R in postpartum mice. Administration of bromocriptine, an antagonist for PRL secretion, to nonpregnant cyclic mice induced an accumulation of galectin-1 – but not galectin-3 – in all CL of various generations, and additional PRL treatment reduced its accumulation, suggesting a direct suppressive effect of PRL on galectin-1 expression. Although the function and regulatory mechanism of galectin in the CL is not fully understood, PGF2 α is an excellent candidate that regulates galectin expression, but its effect may be abolished by LH-R-mediated signal. PRL withdrawal seems to be necessary for an initiation of luteolysis and the following PGF2 α-induced galectin expression.

Brown adipose tissue (BAT) is involved in the regulation of whole-body energy expenditure and adiposity. The activity and prevalence of BAT decrease with age in humans. To examine the effects of single nucleotide polymorphisms of the genes for uncoupling protein 1 (UCP1) and β3-adrenergic receptor (β3AR), key molecules of BAT thermogenesis, on age-related decline of BAT activity and accumulation of body fat in humans. One hundred ninety-nine healthy volunteers (20–72 years old (y.o.)) underwent fluorodeoxyglucose-positron emission tomography (FDG-PET) and computed tomography (CT) after 2-h cold exposure to assess BAT activity. The visceral and subcutaneous fat areas at the abdominal level were estimated from the CT images. They were genotyped for −3826 A/G polymorphism of the UCP1 gene and 64 Trp/Arg mutation of the β3AR gene. BAT was detected in 88 subjects out of 199 (44%), more in younger (⩽30 y.o., 55%) than older subjects (>40 y.o., 15%). BAT prevalence of older subjects tended to be lower in the UCP1 G/G group than the A allele group (A/A and A/G), and also in the β3AR Arg allele group (Trp/Arg and Arg/Arg) than the Trp/Trp group. When compared subjects who had two or more base substitutions on the two genes (the 2–4 allele group) with those who had less than two base substitutions (the 0–1 allele group), BAT prevalence was comparable in younger subjects (62% vs 50%) but lower in older subjects (0% vs 24%, P

We examined cytotoxic effects of nicotine/tar-free cigarette smoke extract (CSE) on C6 glioma cells. The CSE induced plasma membrane damage (determined by lactate dehydrogenase leakage and propidium iodide uptake) and cell apoptosis {determined by MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] reduction activity and DNA fragmentation}. The cytotoxic activity decayed with a half-life of approximately 2 h at 37°C, and it was abolished by N-acetyl-l-cysteine and reduced glutathione. The membrane damage was prevented by catalase and edaravone (a scavenger of •OH) but not by superoxide dismutase, indicating involvement of •OH. In contrast, the CSE-induced cell apoptosis was resistant to edaravone and induced by authentic H2O2 or O2− generated by the xanthine/xanthine oxidase system, indicating involvement of H2O2 or O2− in cell apoptosis. Diphenyleneiodonium [NADPH oxidase (NOX) inhibitor] and bisindolylmaleimide I [BIS I, protein kinase C (PKC) inhibitor] abolished membrane damage, whereas they partially inhibited apoptosis. These results demonstrate that 1) a stable component(s) in the CSE activates PKC, which stimulates NOX to generate reactive oxygen species (ROS), causing membrane damage and apoptosis; 2) different ROS are responsible for membrane damage and apoptosis; and 3) part of the apoptosis is caused by oxidants independently of PKC and NOX.[Supplementary methods and Figure: available only at http://dx.doi.org/10.1254/jphs.11166FP] Keywords:: cigarette smoke extract (CSE), reactive oxygen species (ROS), NADPH oxidase (NOX), apoptosis, protein kinase C (PKC)

Our previous ultrastructural study showed that, in the small intestine of the guinea pig, large granular lymphocytes (LGLs) are closely associated with effete epithelial cells at the villus tip, suggesting their involvement in the apoptosis of enterocytes. However, morphological signs of such lymphocyte-mediated apoptosis were not recognizable in the rat intestine. In the present study, perforin, a cytolytic substance of LGLs, was immunohistochemically examined in the small intestine of guinea pigs and rats. Perforin-immunoreactive lymphocytes predominated within the epithelium in guinea pigs

Extrathymic T-cell proliferation becomes active in the liver of autoimmune MRL-lpr/lpr mice at the onset of disease as well as in the liver of aged mice. Such T cells have several unique properties, including the expression of intermediate TCR (i.e., intermediate TCR cells), a consistent expression of IL-2 receptor β-chain, and a partial constitution of double-negative CD4 - 8 - cells. To analyze the site of extrathymic T-cell proliferation in the liver, MRL-lpr/lpr mice were used before and after the onset of disease. In general, hepatic lymphocytes, including extrathymic T cells and NK cells, existed in the lumen of the sinusoids. Cinematography showed that they moved against the blood stream, adhering to the sinusoidal walls

Luminal stimulation of enterochromaffin (EC) cells is known to cause their release of serotonin which in turn may induce a variety of reflexes via mucosal intrinsic neurons. To morphologically support this idea, the present study demonstrates a close topographical relationship between EC cells and nerves, using whole-mount preparations. Beaded nerve fibers containing vasoactive intestinal polypeptide (VIP) were observed in close proximity to serotonin-immunoreactive EC cells in the small and large intestines of the rat and guinea pig; in whole-mount preparations, the VIP nerve fibers appeared to underlie EC cells. This finding correlates with the physiological datum that the intra-arterial infusion of serotonin causes VIP release from the intestine. Canine urethral serotonin cells, a counterpart of the intestinal EC cells, were shown to contact intraepithelial nerves immunoreactive for calcitonin gene-related peptide. The neuroparaneuronal connection in the urethra may play an important role in the serotonin-evoked urethrogenital reflex. Intestinal and urethral serotonin-containing paraneurons, which are sensory in nature, may release serotonin in response to luminal stimuli, and directly activate adjacent peptidergic neurons to initiate the reflex arcs.

Our previous study reported the rich existence of multinucleated giant cells in an autoimmune myocarditis experimentally induced in rats. The present study investigated the histochemical and ultrastructural characteristics of these giant cells. Histochemistry for an acid phosphatase clearly demonstrated multinucleated giant cells dispersed at the inflammatory foci. Ultrastructurally, the giant cells were shown to be single cells, but not clustered cells. Their ultrastructural characteristics were very similar to the basic features of macrophages, except that the giant cells were poor in lysosomes and phagosomes. It was noticeable that some macrophages possessed three or more nuclei, displaying an intermediate form between mononuclear macrophages and multinucleated giant cells. These findings suggest that the giant cell in the experimental autoimmune myocarditis is a single multinucleated cell, and possibly derived from macrophages by cell-to-cell fusion.

Leptin reduces body fat by decreasing food intake and increasing energy expenditure. Uncoupling protein (UCP) 1, a key molecule for brown adipose tissue (BAT) thermogenesis, was reported to contribute to the stimulatory effect of leptin on energy expenditure. To clarify whether UCP1 is also involved in the anorexigenic effect of leptin, in this study we examined the effect of leptin on food intake using wild-type (WT) and UCP1-deficient (UCP1-KO) mice. Repeated injection of leptin decreased food intake more markedly in WT mice than in UCP1-KO mice, while a single injection of leptin showed similar effects in the two groups of mice. As chronic leptin stimulation induces UCP1 expression in BAT and ectopically in white adipose tissue (WAT), we mimicked the UCP1 induction by repeated injection of CL316,243 (CL), a highly specific β3-adrenoceptor agonist, and measured food intake in response to a single injection of leptin. Two-week treatment with CL enhanced the anorexigenic effect of leptin in WT mice, but not in UCP1-KO mice. Three-day treatment with CL in WT mice also enhanced the anorexigenic effect of leptin and leptin-induced phosphorylation of signal transducer and activator of transcription 3 (STAT3) in the arcuate nucleus of the hypothalamus, without any notable change in adiposity. These results indicate that UCP1 enhances leptin action at the hypothalamus level, suggesting UCP1 contributes to the control of energy balance not only through the regulation of energy expenditure but also through appetite control by modulating leptin action.

Glycans are expected to be one of the potential signal molecules for controlling drug targeting/delivery or long-term circulation of biopharmaceuticals. However, the effect of the carbohydrates of artificially glycosylated derivatives on in vivo dynamic distribution profiles after intravenous injection of model animals remains unclear due to the lack of standardized methodology and a suitable platform. We report herein an efficient and versatile method for the preparation of multifunctional quantum dots (QDs) displaying common synthetic glycosides with excellent solubility and long-term stability in aqueous solution without loss of quantum yields. Combined use of an aminooxy-terminated thiol derivative, 11,11'-dithio bis[undec-11-yl 12-(aminooxyacetyl)amino hexa(ethyleneglycol)], and a phosphorylcholine derivative, 11-mercaptoundecylphosphorylcholine, provided QDs with novel functions for the chemical ligation of ketone-functionalized compounds and the prevention of nonspecific protein adsorption concurrently. In vivo near-infrared (NIR) fluorescence imaging of phosphorylcholine self-assembled monolayer (SAM)-coated QDs displaying various simple sugars (glyco-PC-QDs) after administration into the tail vein of the mouse revealed that distinct long-term delocalization over 2 h can be achieved in cases of QDs modified with α-sialic acid residue (Neu5Ac-PC-QDs) and control PC-QDs, while QDs bearing other common sugars, such as α-glucose (Glc-PC-QDs), α-mannose (Man-PC-QDs), α-fucose (Fuc-PC-QDs), lactose (Lac-PC-QDs), β-glucuronic acid (GlcA-PC-QDs), N-acetyl-β-D-glucosamine (GlcNAc-PC-QDs), and N-acetyl-β-D-galactosamine (GalNAc-PC-QDs) residues, accumulated rapidly (5-10 min) in the liver. Sequential enzymatic modifications of GlcNAc-PC-QDs gave Galβ1,4GlcNAc-PC-QDs (LacNAc-PC-QDs), Galβ1,4(Fucα1,3)GlcNAc-PC-QDs (Le(x)-PC-QDs), Neu5Acα2,3Galβ1,4GlcNAc-PC-QDs (sialyl LacNAc-PC-QDs), and Neu5Acα2,3Galβ1,4(Fucα1,3)GlcNAc-PC-QDs (sialyl Le(x)-PC-QDs) in quantitative yield as monitored by direct matrix-assisted laser desorption ionization time-of-flight mass spectrometry analyses. Live animal imaging uncovered for the first time that Le(x)-PC-QDs also distributed rapidly in the liver after intravenous injection and almost quenched over 1 h in similar profiles to those of LacNAc-PC-QDs and Lac-PC-QDs. On the other hand, sialyl LacNAc-PC-QDs and sialyl Le(x)-PC-QDs were still retained stably in the whole body after 2 h, while they showed significantly different in vivo dynamics in the tissue distribution, suggesting that structure/sequence of the neighboring sugar residues in the individual sialyl oligosaccharides might influence the final organ-specific distribution. The present results clearly visualize the evidence of an essential role of the terminal sialic acid residue(s) for achieving prolonged in vivo lifetime and biodistribution of various glyco-PC-QDs as a novel class of functional platforms for nanomaterial-based drug targeting/delivery. A standardized protocol using multifunctional PC-QDs should facilitate live animal imaging of ligand-displayed QDs using versatile NIR fluorescence photometry without influence of size-dependent accumulation/excretion pathway for nanoparticles (e.g., viruses) >10 nm in hydrodynamic diameter by the liver.

The primary cilium is now considered to function as a fundamental, not rudimentary, structure for mechanical and chemical sensing by individual cells. Primary cilia in neurons express type III adenylyl cyclase (ACIII) and GPCRs for somatostatin (somatostatin receptor 3, SSTR3), serotonin, and melanin-concentrating hormone. The present immunohistochemical and electron microscopic study revealed an abundant occurrence of SSTR3-expressing solitary cilia in insulin- and growth hormone-secreting cells of the mouse. The SSTR3 immunoreactivity was restricted to the plasma membrane of cilia in both cell types, differing from previously reported immunohistochemical localization of SSTRs to cell bodies. The primary cilia in the islet cells were longer than those in the pituitary cells and extended for a long distance in the intercellular canalicules endowed with microvilli. No other endocrine organs were provided with the SSTR3-expressing primary cilia, while the primary cilia in these organs were frequently immunolabeled with ACIII antibody. Since the somatostatin inhibition of both insulin and GH release is regulated mainly by SSTR1 and SSTR5, the primary cilia expressing SSTR3 may be involved in a signaling which differs from that via other SSTR subtypes expressing in cell bodies.

Hexose transporters play a pivotal role in the absorption of food-derived monosaccharides in the gastrointestinal tract. Although a basic knowledge of the hexose transporters has already been gained, their detailed distribution and comparative intensities of expression throughout the gastrointestinal tract have not been fully elucidated. In this study, we quantitatively evaluated the expression of SGLT1, GLUT1, GLUT2, and GLUT5 by in situ hybridization and real-time PCR techniques using a total of 28 segments from the gastrointestinal tract of 9-week-old mice. GLUT2 and GLUT5 mRNA expressions were detected predominantly from the proximal to middle parts of the small intestine, showing identical expression profiles, while SGLT1 mRNA was expressed not only in the small intestine but also in the large intestine. Notably, GLUT1 mRNA was expressed at a considerable level in both the stomach and large intestine but was negligible in the small intestine. Immunohistochemistry demonstrated the polarized localization of hexose transporters in the large intestine: SGLT1 on the luminal surface and GLUT1 on the basal side of epithelial cells. The present study provided more elaborate information concerning the localization of hexose transporters in the small intestine. Furthermore, this study revealed the significant expression of glucose transporters in the large intestine, suggesting the existence of the physiological uptake of glucose in that location in mice.

Background: We previously showed that at the villus tips in the small intestine of guinea pigs effete enterocytes are not simply exfoliated into the lumen but phagocytosed by subepithelial macrophages, leaving only a thin apical cell portion in the epithelial lining. The aim of the present study is to investigate the fate of these apical pieces of enterocytes. Methods: The ileum of guinea pigs was perfusion-fixed and processed for transmission and scanning electron microscopic observation. Results: The apical cytoplasmic plates were found to be pushed by neighboring enterocytes and protruded from the epithelial surface, finally being pinched off into the lumen. In this process observed at the villus tips, the junctional complexes between the apical cytoplasmic plate and the adjacent enterocytes were preserved until the pinching-off of the plate. Luminal cell elements revealed a rich existence of cupshaped or spherical cell fragments covered with microvilli; nuclei were never observed in the luminal fragments. Conclusions: The findings in the small intestine of the guinea pig are the first to account for the mechanism of the epithelial barriers being preserved while apoptotic enterocytes drop out at the tips of the villi.

In order to make an immunohistochemical analysis of the development of the caudal neurosecretory system of the chum salmon, Oncorhynchus keta, we employed the peroxidase-anti-peroxidase technique using antisera specific for urotensins (U) I and II on artificially reared embryos, larvae, and juveniles of this species. Immunoreactivities for UI and UII were first demonstrated in the embryo immediately before hatching, showing labeled perikarya and fibers in the most caudal region of the spinal cord where the presumptive caudal neurosecretory system is located. However, distinct differentiation of the histological neurohemal organ had not yet begun in the embryo. Immunoreactive perikarya and fibers gradually increased in number, and an elaborate urophysis comparable to that of adults was demonstrated in the larvae about 5 months after hatching. At this stage, weak immunoreactivity against UI was detected in the neurohypophysis.