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The exact formula for the average hitting time (HT, as an abbreviation) of simple random walks from one vertex to any other vertex on the square $C^2_N$ of an $N$-vertex cycle graph $C_N$ was given by N. Chair [\textit{Journal of Statistical Physics}, \textbf{154} (2014) 1177-1190]. In that paper, the author gives the expression for the even $N$ case and the expression for the odd $N$ case separately. In this paper, by using an elementary method different from Chair (2014), we give a much simpler single formula for the HT's of simple random walks on $C^2_N$. Our proof is considerably short and fully combinatorial, in particular, has no-need of any spectral graph theoretical arguments. Not only the formula itself but also intermediate results through the process of our proof describe clear relations between the HT's of simple random walks on $C^2_N$ and the Fibonacci numbers.

Continuous exposure to peritoneal dialysis fluids (PDFs) is associated with pathological responses such as persistent micro-inflammation, which leads to ultrafiltration failure. Pentraxin-3 (PTX3), a multifunctional soluble pattern recognition receptor, is produced at sites of inflammation by a wide range of cell types. This study investigates the in vivo expression of PTX3 in the peritoneal membrane of a rat continuous peritoneal dialysis (PD) model, as well as the effect of high glucose on the in vitro expression of PTX3.The expression of PTX3 was analyzed using RT-PCR, real-time PCR, immunohistochemistry and western blotting in a PD rat model receiving saline or conventional PDF containing 3.86% glucose for 8 weeks. The effects of high glucose on the expression of PTX3 were examined in cultured rat peritoneal mesothelial cells (RPMCs), mouse macrophage-like cells, and mouse fibroblasts.In a rat model of PD, eight-week instillation of the conventional PDF produced increased submesothelial thickening, followed by substantially enhanced PTX3 protein levels in the submesothelial layer of peritoneal membrane. PTX3 was detected in peritoneal mesothelial cells, macrophages and fibroblasts in the thickened submesothelial area. Glucose was found to induce PTX3 protein expression in RPMCs as well as macrophage-like cells and fibroblasts.Continuous exposure to conventional PDF induces PTX3 expression in the peritoneal membrane of rats. High glucose may be involved in the mechanism of PDF-induced local micro-inflammation in the peritoneum.

The xenobiotic metabolizing system is considered to play important roles in the olfaction by the chemical homeostasis. Several phase I and phase II xenobiotic metabolizing enzymes are expressed in the olfactory epithelium in vertebrates. Multidrug resistance-related proteins (MRPs) are the phase III xenobiotic metabolizing pumps that eliminate some conjugated ligands from cells. However, the MRP-expressions in the olfactory epithelium have not been confirmed in the mammals. We investigated gene and protein expressions of MRP type 1 (MRP1) and type 2 (MRP2) isoforms in the adult rat olfactory epithelium in order to clarify the existence of phase III xenobiotic metabolizing pumps in the olfactory organs. Expressions of MRP1 mRNA were detected in the nasal cavity by reverse transcriptase polymerase chain reaction (RT-PCR). The nucleoside sequence of the RT-PCR products were completely identical to that found in other organs of rat. On the contrary, the analysis did not detect expressions of MRP2 mRNA in the nasal cavity. By in situ hybridization using a digoxigenin-labeled MRP1 cRNA probe, signals for MRP1 mRNA were observed preferentially in the perinuclear regions of supporting cells. However, the respiratory epithelial cells did not show the signals for MRP1 mRNA. By immunohistochemistry using a specific antibody to MRP1, MRP1-immunoreactivities were seen mainly on the supporting cells. These findings suggest that MRP1 is involved in olfaction as a part of the "olfactory signal termination" by the chemical homeostasis in the "perireceptor events" of the olfactory epithelium.

BACKGROUND: Bioincompatible peritoneal dialysis fluids (PDFs) cause pathological changes in the peritoneal membrane, related to membrane dysfunction and progressive peritoneal fibrosis. We investigated the effects of Pro-His-Ser-Arg-Asn (PHSRN) peptide, one of the fibronectin cell-binding domains that activates integrins and reinforces wound healing, on peritoneal remodelling in a rat peritoneal injury model undergoing peritoneal dialysis. METHODS: The peritoneal mesothelial monolayer was removed by a stripping procedure in rats receiving conventional high glucose-containing PDF supplemented with or without PHSRN or control His-Ser-Pro-Asn-Hrg (HSPNR) peptides. Effects of PHSRN on cell motility and signalling molecules were examined in cultured rat peritoneal mesothelial cells (RPMCs) and normal rat kidney fibroblasts (NRKs). RESULTS: The cytokeratin- and HBME-1-positive mesothelial cell monolayer was selectively removed by the procedure. By day 6, HBME-1-positive cells had regenerated to 53.3 +/- 6.5% of the peritoneal surface in the control group. Regeneration of the mesothelial layer was delayed in the PDF group (35.2 +/- 10.2%, P < 0.05), but PHSRN reversed the effects of PDF (51.7 +/- 9.6%, P < 0.05). PDF treatment increased thickening of granulomatous submesothelial tissue and numbers of ED1-, CD31- and alpha-smooth muscle actin-positive cells, but PHSRN ameliorated these effects. HSPNR had no effects on mesothelial regeneration or peritoneal wound healing. PHSRN, but not HSPNR, recovered glucose-induced inhibition of cell motility and phosphorylation of focal adhesion kinase and its downstream p130(Cas) in RPMCs and NRKs. CONCLUSIONS: These results suggest that PHSRN has beneficial effects on peritoneal regeneration by reducing the inhibitory effects of conventional PDF on integrin-mediated wound healing.

Despite the importance of olfactory receptor neurons (ORNs) for homing migration, the expression of olfactory marker protein (OMP) is not well understood in ORNs of Pacific salmon (genus Oncorhynchus ). In this study, salmon OMP was characterized in the olfactory epithelia of lacustrine sockeye salmon ( O. nerka ) by molecular biological and histochemical techniques. Two cDNAs encoding salmon OMP were isolated and sequenced. These cDNAs both contained a coding region encoding 173 amino acid residues, and the molecular mass of the two proteins was calculated to be 19,581.17 and 19,387.11 Da, respectively. Both amino acid sequences showed marked homology (90%). The protein and nucleotide sequencing demonstrates the existence of high-level homology between salmon OMPs and those of other teleosts. By in situ hybridization using a digoxigenin-labeled salmon OMP cRNA probe, signals for salmon OMP mRNA were observed preferentially in the perinuclear regions of the ORNs. By immunohistochemistry using a specific antibody to salmon OMP, OMP-immunoreactivities were noted in the cytosol of those neurons. The present study is the first to describe cDNA cloning of OMP in salmon olfactory epithelium, and indicate that OMP is a useful molecular marker for the detection of the ORNs in Pacific salmon.

GABA is known to produce membrane depolarization and secretion in adrenal medullary (AM) cells in various species. However, whether the GABAergic system is intrinsic or extrinsic or both in the adrenal medulla and the role that GABA plays are controversial. Therefore, these issues were addressed by combining a biochemical and functional analysis. Glutamic acid decarboxylase (GAD), a GABA synthesizing enzyme, and vesicular GABA transporter (VGAT) were expressed in rat AM cells at the mRNA and protein levels, and the adrenal medulla had no nerve fibre-like structures immunoreactive to an anti-GAD Ab. The double staining for VGAT and chromogranin A indicates that GABA was stored in chromaffin granules. The α1, α3, β2/3, γ2 and δ subunits of GABAA receptors were identified in AM cells at the mRNA and protein levels. Pharmacological properties of GABA-induced Cl− currents, immunoprecipitation experiments and immunocytochemistry indicated the expression of not only γ2-, but also δ-containing GABAA receptors, which have higher affinities for GABA and neurosteroids. Expression of GATs, which are involved in the clearance of GABA at GABAergic synapses, were conspicuously suppressed in the adrenal medulla, compared with expression levels of GABAA receptors. Increases in Ca2+ signal in AM cells evoked trans-synaptically by nerve stimulation were suppressed during the response to GABA, and this suppression was attributed to the shunt effect of the GABA-induced increase in conductance. Overall Ca2+ responses to electrical stimulation and GABA in AM cells were larger or smaller than those to electrical stimulation alone, depending on the frequency of stimulation. The results indicate that GABA functions as a paracrine in rat AM cells and this function may be supported by the suppression of GAT expression and the expression of not only γ2-, but also δ-GABAA receptors.

Our study was designed to clarify the role of antioxidant enzymes in the rat von Ebner gland during acute nickel toxicity. After treatment with nickel acetate, we monitored ultrastructural alterations in acinar and ductal cells, immunohistochemical staining for glutathione peroxidase (GPx) and glutathione S-transferases (GST mu and GST pi), and immunoreactivity for malondialdehyde (MDA). Immunoreactivity for MDA was present only in the acinar cells, and it was enhanced at 3 h after Ni treatment. In contrast, immunoreactivities for GPx and GSTs did not change in acinar cells but significantly increased in ductal cells after Ni treatment. Cytoplasmic vacuoles increased in acinar cells at 3 h after Ni treatment, but they almost completely disappeared at 24 h. No morphological changes were observed in taste bud cells from Ni-treated rats. Because lipid peroxidation, as monitored by immunoreactivity for MDA, was only transiently increased in the acinar cells, the enhanced antioxidant enzyme immunoreactivity in ductal cells of the von Ebner gland plays a crucial role in the self-defense system against nickel toxicity in the rat oral cavity.

The progression of rat liver fibrosis induced by intraperitoneal administration of thioacetamide (TAA) was evaluated by immunocytochemistry using anti-alpha-smooth muscle actin (alpha-SMA), antiendothelin-converting enzyme (ECE)-1, and anti-monocyte chemotactic protein (MCP)-1 antibodies. The fibrous septal spaces gradually increased after administration of TAA, and pseudolobules were established in the 7-week TAA-treated groups. Immunoreactivities against alpha-SMA were not detected in hepatic stellate cells (HSCs) of the control group without TAA treatment, although they were observed in the HSCs around the fibrous septal spaces in all TAA-treated groups, indicating that activation of HSCs occurs during the establishment of pseudolobules. Immunoreactivities against ECE-1 and MCP-1 were seen in such HSCs of the TAA-treated groups, but few or no immunoreactivities were detected in the HSCs of the control group. The most significant increase in the ECE-1 immunoreactivities was detected in the 1-week TAA-treated group, whereas that in MCP-1 was observed in the 7-week TAA-treated group. The present immunocytochemistry indicated a difference in the accelerated expression period between immunoreactivities against ECE-1 and MCP-1 in the HSCs during the progression of TAA-induced liver fibrosis, suggesting that ECE-1 is involved in the early phase of liver fibrosis and that MCP-1 plays a role during the later phase.

Wistar rats were deeply anesthetized and perfused by Hanks' solution bubbled with either 95% air and 5% CO2 (normoxic group) or 95% N2 and 5% CO2 (hypoxic group) from the thoracic aorta for 30 minutes. The isolated abdominal aortas were used for electron microscopy, immunocytochemistry of endothelin-1 (ET-1) and endothelin-converting enzyme-1 (ECE-1), and in situ hybridization of preproendothelin-1 mRNA. A remarkable increase in the number of Weibel-Palade bodies, storage sites of ET-1 and ECE-1, occurred in the hypoxic group when compared with the normoxic group. Immunoreactivities for ET-1 and ECE-1, and signals for preproendothelin-1 mRNA were seen along the endothelia of both groups, but the intensities were significantly elevated in the hypoxic group. The increase in the number of ECE-1 immunoreactive gold particles was noticed in Weibel-Palade bodies in the hypoxic group. These findings indicate the enhancement of preproendothelin-1 synthesis in the rat aortic endothelial cells and the acceleration of ET-1 processing in Weibel-Palade bodies of such cells in an acute hypoxic condition.

We previously reported that 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB) increased the 8-hydroxyguanine (8-OH-Gua) content in nuclear DNA and the base excision repair activity in mouse liver. However, to understand the mechanism of 3'-MeDAB carcinogenesis, a further investigation of the 8-OH-Gua repair systems was necessary. In this report, we examined the expression of the repair enzyme, 8-oxoguanine DNA glycosylase 1 (OGG1), in 3'-MeDAB-treated mouse liver. We prepared four kinds of anti-peptide polyclonal antibodies raised against mouse OGG1 (mOGG1). The sequences used as epitopes were designed from positions located close to the N-terminus, the nuclear localization signal (NLS), and the regions containing Lys(249) and Asp(267), which are involved in the catalytic mechanisms of mOGG1 (glycosylase and lyase, respectively). Immunoblotting, using all four antibodies, revealed a 32-kDa protein (mOGG1-32) in addition to the 38-kDa mOGG1 in the 3'-MeDAB-treated mouse liver. Moreover, immunostaining with mOGG1 antibody yielded strong, positive signals in the 3'-MeDAB-treated mouse liver nuclei. However, we could not detect any difference in the Ogg1 mRNA expression pattern. Although the function of mOGG1-32 remains unclear, these findings suggest that 3'-MeDAB may alter the function of the DNA repair protein, and this action may be related to 3'-MeDAB carcinogenesis.

Vasculogenesis is defined as a neovascularization manner by which endothelial progenitor cells are successively incorporated into the growing capillaries, whereas angiogenesis is another neovascularization manner which includes mitotic proliferations of endothelial cells of the preexisting capillaries and their migration to the vascular tips forming so called "vascular sprouts" or "endothelial buds". Although angiogenesis had been considered to be more prevalent during organogenesis as well as during a wound healing process of adult mammals, recent findings that endothelial progenitor cells were isolated from human peripheral blood and incorporated into sites of active neovascularization have led many researchers to recognize the significance of vasculogenesis in a phenomenon of neovascularization. This paper mainly deals with the history of morphological approaches to clarify the crucial roles of vasculogenesis during organogenesis and a wound healing process.

In order to clarify the role of antioxidant enzymes in the male rat submandibular gland against short-term normobaric oxygenation, we performed immunocytochemical staining of manganese-containing superoxide dismutase (Mn-SOD), copper- and zinc-containing SOD (Cu/Zn-SOD), catalase (CAT), glutathione peroxidase, and glutathione S-transferases (GST alpha, GST mu, and GST pi) between days 1 and 7 after normobaric oxygenation. Ultrastructural alterations and immunoreactivities for malondialdehyde (MDA), a lipid peroxidation-related molecule, of the acinar and ductal cells after the oxygenation were also investigated. Immunoreactivity for MDA was exhibited in the acinar cells throughout the experiment. On the other hand, immunoreactivity for the SODs, CAT, and GSTs was not altered, when compared to that of controls, but was significantly elevated in the granular, striated, and excretory ductal cells. Since an increase of lipid peroxidation as indicated by enhanced immunoreactivity for MDA was detected in the acinar and intercalated ductal cells, the results indicate that the enhanced antioxidant enzymes in the granular, striated, and excretory ductal cells play a crucial role in the self-defense system of the male rat submandibular gland against normobaric oxygenation.

Male rats received daily intraperitoneal injections of cadmium sulfate (2.0 mg/kg) for 3, 6, and 8 days (cadmium-treated groups) or physiological saline for 8 days (control group). The thoracic aortae from both groups were used for electron microscopy and immunocytochemistry for big endothelin (ET)-1, ET-1 and ET-converting enzyme (ECE)-1, and the blood plasma and homogenized thoracic aortae were prepared for assays of big ET-1 and ET-1 concentrations. A remarkable increase in the number of Weibel-Palade (WP) bodies, enhanced immunoreactivities for ET-1 and ECE-1 along the endothelium, and elevated concentrations of ET-1 in the blood plasma as well as in homogenized thoracic aortae were observed in the cadmium-treated groups. However, immunoreactivity for big ET-1 and the plasma and aortic tissue concentrations of big ET-1 did not show any significant changes between the control and cadmium-treated groups. By immunoelectron microscopy, immunoreactivities for ET-1 and ECE-1 were much more pronounced in the increased WP bodies. Since WP bodies are involved in the extracellular release of ET-1 in the manner of a regulated pathway, these findings indicate that cadmium administration induces the enhanced release of ET-1, which is actively processed by ECE-1 in the WP bodies.

Background: Desmoplastic trichoepithelioma is a benign neoplasm considered to have follicular differentiation. Its sweat gland- or sebaceous-lines of differentiation have been also reported. There have been, however, only a few reports regarding extensive immunohistochemical and ultrastructural investigations of this neoplasm. Methods: Histopathological and immunohistochemical studies were performed on three cases of desmoplastic trichoepithelioma, comparing it with normal skin. One of these cases was ultrastructurally investigated. Results: The cord-like basaloid nests were reacted with the anti-cytokeratin (CK)1/5/10/14, -CK5/8, -CK14 and -CK15 antibodies, but not with the anti-CK6 antibody. Similar findings were observed in the outer layers of the normal follicular outer root sheath. Basaloid cell nests in one case, which showed ductal structures in the nests, also expressed CK7, CK8/18 and CK19. These keratins were also detected in the normal sweat glands. In addition, CK8/18 and CK19 were expressed in the basal cells of the outer root sheath. Keratinous cysts had inner reactions with the anti-CK10/11 and -CK6 antibodies, and outer reactions with anti-CK5/8 and -CK14 antibodies. Ultrastructurally, the cells in the cord-like nests were basically immature and basaloid in appearance. A few cells contained Odland bodies, which were also observed in the normal apocrine acrosyringeum. The ductal structure was lined by the cells which bore numerous microvilli in the luminal surface. Conclusion: The cells in desmoplastic trichoepithelioma are suggested to be in close association with the basal cells in the outer root sheath, which can differentiate into various parts of the folliculosebaceous apocrine unit.

Acute toxicity of bis (tributyltin) oxide in the sweat glands in the rat footpad was investigated by electron microscopy and an energy-dispersive X-ray microanalyzer. Male Wistar rats received an intramuscular injection of 0.5 ml/kg bis (tributyltin) oxide. After 6–8 h, swelling of mitochondria appeared in the secretory cells of the sweat glands. After 12 h, the secretory cells began to show intracytoplasmic edema. After 16–20 h, secretory cells in some sweat glands showed marked hydropic degeneration with swollen cytoplasm. Using X-ray microanalysis, tin peaks were preferentially obtained from the swollen mitochondria of the affected secretory cells. Mitochondria dysfunction due to the toxic effects of bis (tributyltin) oxide induced changes in the secretory cells of rat sweat glands. After 24–48 h, the secretory portion of the sweat glands contained three types of cells: degenerating dark cells, regenerating cells carrying injured mitochondria, and light cells which were morphologically very similar to the cells in the transitional portion of the sweat gland. These light cells appeared to differentiate into active secretory cells after settling down in the secretory portion. Based on these observations, we concluded that the cells in the transitional portion could play an important role at least as reserve cells against secretory cell toxicity. In association with the regenerating process of the damaged secretory portions, increased mitotic activities were seen in different areas of all the dermal sweat ducts. The above-mentioned morphological observations for cell damage and subsequent regeneration and renewal of secretory cells in sweat gland intoxication have not been reported so far.

We first detected alpha-calcitonin gene-related peptide (alpha-CGRP) precursor mRNA in the enteric nervous system (ENS) of rat small intestine by reverse transcriptase polymerase chain reaction (RT-PCR). The nucleotide sequence of the RT-PCR product was completely identical to that found in other organs. By in situ hybridization using digoxygenin-labeled alpha-CGRP precursor cRNA probe, we found that antisense probes detected a signal on nerve cell bodies of both submucosal and myenteric plexuses. Our findings indicate that the rat ENS participates in synthesis of alpha-CGRP precursor.

Profilin binds to actin monomer to regulate actin poly- merization, and to phosphatidylinositol 4,5-bisphosphate to inhibit hydrolysis by phospholipase Cg1. This study investigated the expression of profilin in rat anti-Thy-1.1 mesangial proliferative glomerulonephritis (GN) and examined the effect of growth fac- tors on its expression in cultured rat mesangial cells. Profilin mRNA was constitutively expressed in isolated glomeruli of un- treated rats. However, in glomeruli of anti-Thy-1.1 GN rats, its expression was upregulated beginning on day 1, reaching a peak level on day 4 (3.9-fold versus control glomeruli), and decreased on day 14, as determined by competitive reverse transcription- PCR. Increased expression of profilin protein was confirmed using immunoblotting and immunohistochemistry. Immunoelec- tron microscopy revealed the presence of profilin in plasma mem- brane and the rough endoplasmic reticulum of mesangial cells, indicating that profilin was produced in mesangial cells. In cul- tured rat mesangial cells, expression of profilin mRNA and pro- tein was upregulated by basic fibroblast growth factor but not by platelet-derived growth factor or transforming growth factor- b. Suppression of profilin expression using an antisense oligonucle- otide against profilin inhibited ( 3 H)thymidine uptake. These find- ings indicated the involvement of profilin in anti-Thy-1.1 GN and suggest that the upregulation of profilin might be involved in the progression of anti-Thy-1.1 GN possibly by affecting cell growth.

WE investigated the cardiovascular effects of bilateral microinjection of antisense oligodeoxynucleotides (oligos) into the nucleus tractus solitarii (NTS) to neuronal nitric oxide synthase (nNOS) to suppress the expression of nNOS molecular biologically. In urethane-anesthetized, paralyzed Wistar-Kyoto rats, bilateral microinjection of nNOS antisense oligos (20 pmol in a 50 nl volume) into the NTS produced a significant increase in mean arterial blood pressure at 30-60 min after injection, compared with rats injected with nNOS sense or scrambled oligos. Immunohistochemical study demonstrated that nNOS immunoreactivity in the rat NTS was suppressed by nNOS antisense oligos. These results indicate that suppression of the nNOS gene using antisense in the NTS increases blood pressure.

Vasoactivity after treatment with endothelin (ET)-1 and immunoreactivity for ET-1 and its receptors were investigated in the rat superior mesenteric vasculature (SMV). By measurements of corrosion cast images of the SMVs, it was seen that ET-1 induces remarkable vasocontraction of the distal arterial branches, consisting of small arteries and arterioles, and localized vasoconstriction throughout the venous branches which possess localized medial thickenings. Immunoreactivity for ET-1 was preferentially seen along the endothelia of the proximal arterial branches. Cisterns of the rough endoplasmic reticulum and Weibel-Palade (WP) bodies of the endothelial cells were immunoreacted. Immunoreactivity for the ETa receptor was preferentially seen on the media of the distal arterial branches. These findings indicate that endothelial cells of the proximal arterial branches synthesize ET-1 and store it in the WP bodies. Because WP bodies are involved in the release of ET-1, this suggests that this endogenous ET-1, which is released from the proximal arterial branches, may be involved in the regulation of blood flow through the distal arterial branches by mediation of the ETa receptor. In addition, it seems likely that ET-1-induced vasoconstriction of the venous branches may act to impel the portal blood flow.

Immunoreactivities of endothelin-1, endothelin-3, endothelin receptor type A, and Na,K-ATPase were investigated in the stria vascularis of adult male WBB6F1 +/+ mice and in that of W/Wv mutants lacking strial intermediate cells. In the +/+ mice, electron microscopic immunoreactivity for the endothelins was seen on the rough endoplasmic reticulum, Golgi apparatus, cytoplasmic vesicles and lysosomes exclusively in the strial intermediate cells by the postembedment method. Immunoreactive endothelin receptor A was localized along the plasma membrane of strial marginal cells of both wild and mutant types although the immunoreactivity of the latter was much less than that of the former by the preembedment method. These findings suggest that the endothelins, which are produced in the strial intermediate cells, may play a role in the maintenance of the stria vascularis function in the +/+ mice. Since the plasma membrane of the marginal cells of the W/Wv mice, which do not generate a high positive endocochlear potential, also showed immunoreactivity for Na,K-ATPase, it seems likely that the endothelins are involved in the activation of sodium pump of the strial marginal cells by mediation of endothelin receptor A. In addition, the role of lysosomes in the crinophagy of the endothelins in the strial intermediate cells is proposed in the +/+ mice.

In the rat hind limb bud aged between prenatal days 14 and 16, immunoreactions of fibronectin in the apical ectodermal ridge were localized on the plasma membranes of epidermal cells and cytoplasmic projections of the underlying mesenchymal cells, which are in contact with the basal lamina. Those of integrin alpha 3 and alpha 5 subunits also appeared on such areas. Definite immunoreactions of fibronectin and both integrin subunits were seen in cell to cell contact areas of mesenchymal cells which are associated with the marginal vein, or with each other forming solid cell cords, and appeared on the basal plasma membrane of endothelial cells of the growing capillaries arising from the marginal vein. These findings suggest that fibronectin may work as a ligand for alpha 3 beta 1 and/or alpha 5 beta 1 integrins expressed by the mesenchymal and vasoformative cells in developing limb bud.

BACKGROUND Apical tubules (ATs) in marginal cells (MCs) of the stria vascularis appear in limited stages of differentiation of the MCs, but their origin and roles remain uncertain. The present study was designed to solve the problem of whether the ATs are intracellular compartments derived from the Golgi apparatus (GA). METHODS The cochleae of Wistar rats at ages of postnatal days 1, 3, and 5 were prepared for electron microscopy and cytochemistry using thiamine pyrophosphatase (TPPase) and coenzyme A phosphatase (CoA-Pase) as marker enzymes of trans Golgi cisterns and fluorescent labelled lectin, griffonia simplicifolia agglutinin-I (GS-1). RESULTS The ATs appeared in the apical cytoplasm of the MCs between postnatal days 1 and 5. Reaction products of TPPase and CoA-Pase activities were localized in the trans-Golgi cisterns and the ATs, which were occasionally in a close apposition to the GA. The reaction was found along the apical plasma membrane of the MCs only in case of TPPase. Heavy reactions to GS-1 were seen in the supranuclear region as well as along the apical plasma membrane of the MCs. CONCLUSIONS The present ultrastructural and cytochemical studies indicate that the ATs, which appear in the MCs at limited perinatal stages, originate from the trans-Golgi cisterns. These ATs may be involved in the apical plasma membrane supply for the differentiation of the MCs prior to the generation of EP.

Effects of bFGF, TGFalpha and TGFbeta on cultured mesenchymal cells of rabbit phalli were investigated. A remarkable cell proliferation reaching confluency was only obtained from the media containing bFGF by the first week of culture. By subsequent 4 weeks culture using such confluent cells in the media containing bFGF, TGFalpha or TGFbeta, respectively, cells cultured in the media containing bFGF showed an active transformation to endothelial precursor cells determined by positive immunoreactions for factor VIII-related antigen at 2 weeks. By electron microscopy, such vasoformative cells appeared in contact with each other, occasionally forming a solid cell cord. The use of Matrigel's basement membrane matrix (Matrigel) in each culture bed promoted the capillary ingrowth, implying that certain extracellular matrix (ECM) components included in Matrigel may accelerate the formation of neocapillaries. Enhancement in the production of ECM components and transformation to smooth muscle cells determined by positive immunoreactions for actin were also seen in cells cultured in the media containing TGFalpha and TGFbeta, respectively. The present results indicate that the above growth factors play crucial roles in the differentiation of the rabbit cavernous body.

Rat hind limb buds, aged between prenatal days 14 and 18, were used for electron microscopy and immunocytochemistry of fibronectin, laminin and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling staining indicative of DNA fragmentation. Fibronectin and laminin were actively synthesized in the rough endoplasmic reticulum of epidermal cells in the apical ectodermal ridge between prenatal days 14 and 15, but most cells underwent apoptosis after prenatal day 15. As the regression of the apical ectodermal ridge progressed, mesenchymal cells associated with the marginal veins were successively incorporated into the endothelium devoid of the basal lamina. No mitotic figures of endothelial cells were recognized either in the marginal vein or in the surrounding growing capillaries. Extracellular matrix components connected the adjacent mesenchymal cells, with the endothelium of such vessels immunoreacting to fibronectin and laminin. In addition, fibronectin-immunoreactive networks among the interstices of the mesenchymal cell cords developed in the avascular zone between the epidermis and the growing capillaries at prenatal day 15, but became inconspicuous at prenatal day 16. These results indicate that the apoptosis of the epidermal cells is the major reason for the regression of the apical ectodermal ridge, and that the capillary ingrowth from the marginal veins to the avascular zone is accelerated by transformation of mesenchymal cells to endothelial ones. Fibronectin and laminin seem to play crucial roles in capillary growth, especially in the adhesion between endothelial cells of the pre-existing vessels and mesenchymal cells.

Objectives/Hypothesis The etiopathology of bone resorption in cholesteatoma is unclear. We studied pH in middle ear cholesteatoma tissue and the permeability of the cholesteatoma epithelium in an attempt to elucidate the mechanism of bone resorption in this disease. Study Design Laboratorial study. Methods Cholesteatoma tissue was collected from patients with primary acquired middle ear cholesteatoma. The pH of the keratin debris of cholesteatoma was measured using a pH meter. The cholesteatoma epithelium was examined under a confocal laser scanning microscope, and under a transmission electron microscope. Expression of filaggrin in the cholesteatoma tissue was explored by fluorescence immunohistochemistry and by quantitative reverse transcription-polymerase chain reaction. Results The pH of the keratin debris of cholesteatoma was acidic. The pH of the basal layer of the cholesteatoma epithelium was significantly lower than that of the antrum mucosa. Transmission electron microscope showed distinct penetration of lanthanum in the intercellular space of the basal, spinous, and granular layers of the cholesteatoma epithelium, but only a small amount of lanthanum in the granular layer in the normal skin. The expression of filaggrin mRNA was significantly lower in the cholesteatoma tissue than in the normal skin. Conclusions These results indicate that acid leakage through the cholesteatoma epithelium probably participates in the resorption of the underlying bone structure. The increased permeability of the cholesteatoma epithelium may be explained by a decrease in filaggrin expression. Level of Evidence N/A. Laryngoscope, 124:245–250, 2014

Double-stranded RNA-dependent protein kinase (PKR) is an interferon-induced protein that has been identified and characterized as a translational inhibitor in an interferon-regulated antiviral pathway. PKR is also reported to play important roles in the regulation of cell growth and differentiation. We have previously demonstrated that PKR inactivation suppresses osteoblast calcification and osteoclast formation. However, reports concerning the roles of PKR in chondrogenesis are limited. In this study, we have demonstrated that PKR is required for the in vitro differentiation of the mouse clonal chondrogenic cell line ATDC-5. ATDC-5 cells treated with insulin differentiated into chondrocytes and produced an alcian-blue-positive cartilage matrix. The protein expression of signal transducers and activators of transcription (STAT) peaked at day 7 of differentiation, whereas the expression of SRY-box-containing gene 9 (Sox-9), which is a transcription factor for chondrocyte differentiation, increased gradually. When the cells were treated with a PKR inhibitor (2-aminopurine), the cartilage matrix formation decreased. The protein expression of STAT1 continued to increase up to day 21, whereas the expression of Sox-9 was low and did not increase. We also demonstrated that PKR was localized to a marginal region of the mandibular condyle cartilage in mouse embryos. Our findings suggest that PKR has important functions in the differentiation of chondrocytes through the modulation of STAT1 and Sox-9 expression.

Effects of phosphines on palladium catalyzedethoxycarbonylation of 4, 4'-dihalobiphenyl derivatives under high pressure of carbon monoxide were studied in the presence of amine and ethanol. In ethoxycarbonylation of 4, 4'-dibromobiphenyl (Ia), the presence of excess triphenylphosphine (Ph3P) ligand over palladium promoted selective formation of diethyl biphenyl-4, 4'-dicarboxylate (IIa), however, palladium complex with low Ph3P/Pd ratios gave ethyl 4'-bromobiphenyl-4-carboxylate (IIIa) selectively. Chelate diphosphines, 1, 3-bis(diphenylphosphino)propane (dppp) and 1, 4-bis(diphenylphosphino)-butane (dppb) produced palladium complexes having much higher activities than Ph3P in ethoxycarbonylation of several 4, 4'-dibromobiphenyl derivatives. Ia, 2, 7-dibromo-9, 10-dihydrophenanthrene (Ib), 2, 7-dibromofluorene(Ic), and 2, 7-dibromofluorenone (Id) quantitatively yielded their corresponding diesters by using Pd-dppp catalysts. Mechanisms of the palladium catalyzed reactions are discussed.

Immunoelectron microscopic localization of endothelin (ET) using the mouse monoclonal anti-ET sera was performed on the pre-and postnatal rabbit umbilical vein during stages when Weibel-Palade (WP) bodies increase in number in the endothelial cells. The immunoreactions are localized in the rough endoplasmic reticulum, Golgi cisterns, and WP bodies that are actively segregated from the Golgi apparatus. After degranulation and extracellular release of WP bodies was induced by compound 48/80, heavy immunoreactions were seen in both vascular lumen and cytoplasm near the degranulated WP bodies. These results indicate that ET is stored in WP bodies after segregation from the Golgi apparatus and released from the endothelial cells concomitantly with other components of these granules by exocytosis. Because the immunoreactions are also seen in pinocytotic vesicles that are occasionally in contact with WP bodies, it seems likely that WP bodies are also involved in the uptake and storage of ET from blood plasma. © 1993 Wiley-Liss, Inc.

The synthetic and secretory processes of human chorionic gonadotropin (hCG) and human placental lactogen (hPL) were investigated by immunoelectron microscopy which included the use of the double immunolabeling technique. The syncytiotrophoblast contains three kinds of granules: Small-sized granules (SG), middle-sized granules (MG), and large dense bodies (LB). Immunoreactions of hCG are localized in the MG and LB, while those of hPL are in the SG and LB. The double immunolabeling method proved without a doubt that hCG and hPL do not co-exist in the same SG or MG, but they are co-localized in the LB. This means that hCG and hPL are separately sorted into different secretory granules in the Golgi apparatus, but the LB are a kind of cytolysosome which are involved in the uptake and storage of both hPL and hCG.

Acetylcholinesterase (AChE) activities in the prenatal rat lower limb were investigated by both cytochemistry and immunocytochemistry. Results indicate that the epidermal cells show immunoreactions of AChE at a limited stage at prenatal day 15, and mesenchymal cells which are occasionally in contact with the basal lamina or with the adjacent myotubes begin to show AChE activities at prenatal day 17. Such AChE-positive mesenchymal cells, involved in the formation of the muscular tissues, have almost disappeared in the subepidermis by prenatal day 19. This suggests that AChE independent of the neuromuscular system may be involved in the mesenchymal cell differentiation especially in the inductive process during myogenesis.

For many antibodies, each antigen-binding site binds to only one antigen molecule during the antibody's lifetime in plasma. To increase the number of cycles of antigen binding and lysosomal degradation, we engineered tocilizumab (Actemra), an antibody against the IL-6 receptor (IL-6R), to rapidly dissociate from IL-6R within the acidic environment of the endosome (pH 6.0) while maintaining its binding affinity to IL-6R in plasma (pH 7.4). Studies using normal mice and mice expressing human IL-6R suggested that this pH-dependent IL-6R dissociation within the acidic environment of the endosome resulted in lysosomal degradation of the previously bound IL-6R while releasing the free antibody back to the plasma to bind another IL-6R molecule. In cynomolgus monkeys, an antibody with pH-dependent antigen binding, but not an affinity-matured variant, significantly improved the pharmacokinetics and duration of C-reactive protein inhibition. Engineering pH dependency into the interactions of therapeutic antibodies with their targets may enable them to be delivered less frequently or at lower doses.

Aims The effects of bisphenol A (BPA) on NCTC Clone 1469, non-parenchymal hepatocytes, were examined to clarify the molecular basis of BPA-induced liver injury. Main methods We analyzed the expression of C/EBP homologous protein ( CHOP ) mRNA, Bcl2 mRNA, caspase12, and glucose-regulated protein 78 kDa (GRP78)/Ig heavy chain-binding protein (BiP), to determine whether endoplasmic reticulum (ER) stress was involved in the 100 μM BPA-induced cell death. To examine the features of damaged hepatocytes, we analyzed the morphological changes in BPA-treated NCTC Clone 1469 by transmission electron microscopy. In addition, we analyzed the intracellular reactive oxygen species (ROS) level in BPA-treated NCTC Clone 1469 by the 2′, 7′-dichlorofluorescein diacetate (DCFDA) method. Key findings Increases in the expression of CHOP mRNA, caspase-12, and GRP78/BiP in NCTC Clone 1469-treated with 100 μM BPA were detected (CHOP, 1.42 fold; caspase-12, 1.33 fold; GRP78/Bip, 1.36 fold). These observations suggested that BPA induced ER stress-associated apoptosis. A morphological analysis revealed remarkable elongation of the rough ER, supporting the finding of ER stress. Intracellular ROS production was increased in NCTC Clone 1469-treated with BPA, and N -acetyl- l -cysteine (NAC) prevented the cell damage induced by BPA. However, these effects of BPA were not inhibited by estrogen receptor inhibitors. Significance We found that BPA induced ER stress-associated apoptosis in non-parenchymal hepatocytes. The ER stress is due to ROS production and is independent of estrogen receptors.

Double-stranded RNA-dependent protein kinase (PKR) plays a critical role in antiviral defence of the host cells. PKR is also involved in cell cycle progression, cell proliferation, cell differentiation, tumorigenesis, and apoptosis. We previously reported that PKR is required for differentiation and calcification of osteoblasts. However, it is unknown about the role of PKR in osteoclast differentiation. A dominant-negative PKR mutant cDNA, in which the amino acid lysine at 296 was replaced with arginine, was transfected into RAW264.7 cells. We have established the cell line that stably expresses the PKR mutant gene (PKR-K/R). Phosphorylation of PKR and α-subunit of eukaryotic initiation factor 2 was not stimulated by polyinosic-polycytidylic acid in the PKR-K/R cells. RANKL stimulated the formation of TRAP-positive multinuclear cells in RAW264.7 cells. However, TRAP-positive multinuclear cells were not formed in the PKR-K/R cells even when the cells were stimulated with higher doses of RANKL. A specific inhibitor of PKR, 2-aminopurine, also suppressed the RANKL-induced osteoclast differentiation in RAW264.7 cells. The expression of macrophage fusion receptor and dendritic cell-specific transmembrane protein significantly decreased in the PKR-K/R cells by real time PCR analysis. The results of RT-PCR revealed that the mRNA expression of osteoclast markers (cathepsin K and calcitonin receptor) was suppressed in the PKR-K/R cells and RAW264.7 cells treated with 2-aminopurine. Expression of NF-κB protein was suppressed in the PKR-K/R cells and 2-aminopurine-treated RAW264.7 cells. The level of STAT1 protein expression was elevated in the PKR-K/R cells compared with that of the wild-type cells. Immunohistochemical study showed that PKR was localized in osteoclasts of metatarsal bone of newborn mouse. The finding that the PKR-positive multinuclear cells should be osteoclasts was confirmed by TRAP-staining. Our present study indicates that PKR plays important roles in the differentiation of osteoclasts.

Intestinal epithelial cells (IECs) are exposed to external environment, microbial and viral products, and serve as essential barriers to antigens. Recent studies have shown that IECs express Toll-like receptors (TLRs) and respond to microbial components. The antimicrobial and antiviral barriers consist of many molecules including TLRs. To investigate the further component of this barrier in intestine, we examined the expression of double-stranded RNA-dependent protein kinase (PKR). PKR is a player in the cellular antiviral response and phosphorylates alpha-subunit of the eukaryotic translation initiation factor 2 (eIF-2alpha) to block protein synthesis and induces apoptosis. In this study, we showed that the expression of PKR was restricted to the cytoplasm of absorptive epithelial cells in the intestine of adult rat. We also demonstrated that PKR was expressed in the cultured rat intestinal epithelial cells (IEC-6). The level of PKR protein expression and the activity of alkaline phosphatase (ALP) increased in the cultured IEC-6 cells in a time-dependent manner. Inhibition of PKR by the 2-aminopurine treatment decreased ALP activity in the IEC-6 cells. Treatment of IEC-6 cells with synthetic double-stranded RNA (dsRNA) induced cell death in a dose-dependent manner. The addition of hydrocortisone also provoked suppression of PKR expression and ALP activity. This modulation might be mediated by signal transducers and activators of transcription-1 (STAT-1) protein. We concluded that PKR is expressed in IECs as potent barriers to antigens and is a possible modulator of the differentiation of rat IECs.

Simultaneous immunoelectron microscopic localization of histamine and factor VII-related antigen was examined on the same ultrathin section of the endothelium of the human umbilical vein from full-term deliveries by means of the double-immunolabeling technique. Small gold particles demonstrating antibody reaction with histamine are preferentially located in the cytoplasmic matrix and organelles, especially in mitochondria and on the luminal membrane surface of the endothelial cells. The gold particles representing histamine immunoreactivity also located on some of Weibel Palade (WP) bodies. In contrast, large gold particles demonstrating factor VII-related antigen are concentrated preferentially on most WP bodies. Single labeling of either histamine or factor VIII-related antigen shows similar results to those of the double labeling. The present study indicates that some WP bodies are involved in storage of both factor VIII-related antigen and histamine, but others store factor VIII-related antigen only. This difference in contents of WP bodies may be induced during the development and maturation process of this inclusion. At any rate, it is reasonable to consider that WP bodies have important roles in both vascular tonus and hemostasis during the vascular obliteration.

The toxic effects of bis (tributyltin) oxide (TBTO) on the rat liver were studied with an electron microscope and the accumulation sites of tin were determined with an X-ray microanalyzer. The activities of serum enzymes and the concentration of serum bilirubin were also analyzed. Male Wistar rats received an intramuscular injection of 0.5 ml/kg of TBTO. Marked swelling of the mitochondria appeared in the hepatocytes 4 h after injection of TBTO. Cytoplasmic vacuoles, which contained degenerated mitochondria, gradually increased in number in these hepatocytes. This in turn may have caused a decrease in the volume of hepatic cell cords and an enlargement of sinusoids in the entire hepatic lobule. However, fine structures of intrahepatic bile ducts were not altered. By X-ray microanalysis, tin peaks were preferentially obtained from swollen mitochondria of the hepatocytes. By polarographic analysis of the respiratory responses of mitochondria, it was demonstrated that rates of state 4 respiration and respiratory control ratio were significantly disturbed in TBTO-treated rats in comparison with those of controls. The activities of AST (aspartate aminotransferase) and ALT (alanine aminotransferase) were significantly increased after TBTO treatment, but those of ALP (alkaline phosphatase), LAP (leucine aminopeptidase) and total bilirubin were not changed. These results indicated that parenterally administered TBTO accumulated in the liver cell mitochondria and disturbed oxidative phosphorylation. Mitochondrial dysfunction might induce severe damage of the hepatocytes. Four days after injection of TBTO, hepatic structures and chemical indices were almost restored by the regeneration of hepatocytes.

The role of the submandibular glands in the excretion of parenterally administered bis (tributyltin) oxide (TBTO) was studied. Fine structural alterations of the submandibular glands were observed with an electron microscope. Accumulation sites of TBTO were determined with an X-ray microanalyzer and tin concentrations in saliva and blood were measured by atomic absorption spectrophotometry.

Dose-dependent contractions of the in vitro swine coronary artery were induced by application of histamine and acetylcholine, but not of angiotensin II, ergonovine, noradrenaline, prostaglandin F2α and serotonin. Ultra-structural changes especially of the tunica intima during the contractions were observed at 2,5 and 30 min after application of histamine and acetylcholine. The intimal gutter spirally running along the longitudinal axis of the vessel was obscured, and the intimal surface became extensively indented. Exclusively in the histamine-treated samples, the increase in number and size of the intracellular vacuoles and the dilation of the intercellular clefts to the extent of the intercellular vacuoles were observed in the endothelium. Moreover, the enhancement of the endothelial permeability was indicated by the marker experiments using horseradish peroxidase. Such endothelial cell damages and the enhancement of the endothelial permeability may amplify the coronary artery contraction.

The effects of dietary zinc deficiency on testicular protein secretion, mainly that by Sertoli cells, were examined by electron microscopy and two-dimensional polyacrylamide gel electrophoresis of [35S] methionine-labeled secretory proteins from mouse testes. Zinc deficiency caused a significant decrease in the gonadosomatic index and a distinct increase in deoxyribonucleic acid concentration. Sertoli cells maintained normal fine-structural features; junctional complexes among Sertoli cells continued to divide seminiferous tubules into basal and adluminal compartments in the zinc-deficient mouse testes. Severe atrophic changes were observed in spermatogenic cells after meiotic division in the adluminal compartment, but not in spermatogonia located in the basal compartment. Zinc replacement treatment caused spermatogenesis to recover normally. Although total protein secretion was not affected by zinc deficiency, one polypeptide spot appeared due mainly to the loss of its target spermatogenic cells. The present study indicates that zinc is indispensable for spermatogenic cells after meiosis and that testicular protein secretory functions can be preserved in the absence of zinc.

GABA is known to produce membrane depolarization and secretion in adrenal medullary (AM) cells in various species. However, whether the GABAergic system is intrinsic or extrinsic or both in the adrenal medulla and the role that GABA plays are controversial. Therefore, these issues were addressed by combining a biochemical and functional analysis. Glutamic acid decarboxylase (GAD), a GABA synthesizing enzyme, and vesicular GABA transporter (VGAT) were expressed in rat AM cells at the mRNA and protein levels, and the adrenal medulla had no nerve fibre-like structures immunoreactive to an anti-GAD Ab. The double staining for VGAT and chromogranin A indicates that GABA was stored in chromaffin granules. The alpha1, alpha3, beta2/3, gamma2 and delta subunits of GABA(A) receptors were identified in AM cells at the mRNA and protein levels. Pharmacological properties of GABA-induced Cl(-) currents, immunoprecipitation experiments and immunocytochemistry indicated the expression of not only gamma2-, but also delta-containing GABA(A) receptors, which have higher affinities for GABA and neurosteroids. Expression of GATs, which are involved in the clearance of GABA at GABAergic synapses, were conspicuously suppressed in the adrenal medulla, compared with expression levels of GABA(A) receptors. Increases in Ca(2+) signal in AM cells evoked trans-synaptically by nerve stimulation were suppressed during the response to GABA, and this suppression was attributed to the shunt effect of the GABA-induced increase in conductance. Overall Ca(2+) responses to electrical stimulation and GABA in AM cells were larger or smaller than those to electrical stimulation alone, depending on the frequency of stimulation. The results indicate that GABA functions as a paracrine in rat AM cells and this function may be supported by the suppression of GAT expression and the expression of not only gamma2-, but also delta-GABA(A) receptors.

Dark-skinned people are known to be more tolerant of ototraumatic noise than are light-skinned people, and pigmented animals are more tolerant of ototraumatic noise and aminoglycoside ototoxicity than are albino animals. Such tolerance may be dependent on the local ability of detoxification and antioxidant enzymes, including glutathione S-transferase (GST) and glutathione peroxidase (GSPx). In the present study, we examined the difference in GST/GSPx expression in the lateral wall of the cochlea between pigmented and albino guinea pigs. Eight-week-old male pigmented and albino guinea pigs were killed by transcardiac perfusion with 2% paraformaldehyde. The cochlear ducts were isolated, further fixed with 4% paraformaldehyde, decalcified, and then embedded in paraffin. Sections prepared at 5-microm thickness were incubated with anti-GST-alpha,-mu,-pi, or anti-GSPx antibody, reacted with Alexa Fluorconjugated secondary antibody, and examined under a Carl Zeiss Axioskop 2 plus fluorescence microscope. The cochlea ducts were also subjected to immunoelectron microscopy for GST-pi by the postembedment method. The stria vascularis of pigmented guinea pigs was strongly immunoreactive for GST-alpha,-mu,-pi, and GSPx, whereas no or only weak immunoreactivities were seen in the stria vascularis of albino guinea pigs. The spiral ligament showed positive but different immunoreactivities for these enzymes between the strains. Double-stained immunofluorescence micrographs for GST-pi and GSPx showed a close resemblance of localization between the two enzymes in both pigmented and albino guinea pigs. At the ultrastructural level, immunoreactivity for GST-pi was localized preferentially in the melanin cells of pigmented guinea pigs. These results suggest that correlation between pigmentation and inner ear susceptibility is, at least partially, attributed to the different distribution of GST/GSPx in the stria vascularis.

Dominant white spotting W/W(v) and W(v)/W(v) mice are well-known mutants that lack strial intermediate cells in their cochlea and manifest hereditary sensorineural hearing loss. We recently reported marked thickening of and IgG deposition on the basement membrane of strial capillaries in W/W(v) mutant mice, similar to observations made in aged animals and in animals with autoimmune sensorineural hearing loss. The present study aimed to clarify the age-dependent changes in these pathological findings of strial capillaries in the W/W(v) mice. Male WBB6F1 +/+ and dominant white spotting W/W(v) mutant mice were sacrificed by transcardiac perfusion with paraformaldehyde solution. The cochlear ducts were isolated and subjected to light- and electron-microscopy, immunohistochemistry, immunoelectron microscopy. Alternatively, lanthanum chloride tracer examination in the isolated cochlear ducts was performed in order to compare the permeability of the strial capillaries between +/+ and W/W(v) mice. In the W/W(v) mice, thickening of and IgG deposition on the basement membrane of strial capillaries were observed as early as 1 week after birth and became more noticeable with age. Deposited IgG was preferentially localized to the thickened basement membrane and was also observed in partially the intercellular space between adjacent of endothelial cells. In addition, pinocytotic vesicles both in the apical and basal lesions of such cells also showed IgG deposition. Lanthanum chloride was retained along apical plasma membrane of the endothelial cells in the +/+ mice but penetrated through the endothelial layer in the W/W(v) mice. These results indicate that active transport via pinocytotic vesicles as well as increased permeability of strial capillaries in the W/W(v) mice occur in the early stage after birth, resulting in the morphological alterations in the strial capillaries of these mice.

In mammals, glutathione S-transferase (GST) in the olfactory epithelium is involved in assistance of the olfactory reception by the xenobiotic metabolism. We previously reported the protein and gene expressions of salmon olfactory GST class pi (soGST) in the olfactory receptor cells (ORCs) of the salmonid fish. However, the chronological appearances of soGST in ORCs during ontogeny and cell proliferation are still unknown in this species. In this study, we performed immunohistochemistry of soGST using an antibody specific to soGST in the olfactory system (olfactory placode, olfactory pit, olfactory epithelium, olfactory nerve and olfactory bulb) of lacustrine sockeye salmon ( Oncorhynchus nerka) embryos and 5-bromo-2'-deoxyuridine (BrdU) experimental fish. The projection of olfactory nerve bundles from the olfactory pit to the presumptive olfactory bulb was identified at embryonic day 28 after fertilization. The olfactory cilia were first detected on the apical surface of ORCs at day 43. soGST-immunoreactivity was first detected within the olfactory pit cells at day 55. At 58 day, the number of soGST-immunoreactive cells increased markedly in the olfactory epithelia, and soGST-immunoreactive fibers were observed in the olfactory nerves and olfactory bulbs. By in vivo uptake of BrdU in 1-year-old fish, we observed for the first time at day 7 after labeling that the olfactory epithelia showed ORCs in which both soGST-immunoreactivity and BrdU coexisted. These results indicate that soGST is synthesized in the mature ORCs of lacustrine sockeye salmon after cell formation and differentiation.

Desmoplastic trichoepithelioma is a benign neoplasm considered to have follicular differentiation. Its sweat gland- or sebaceous-lines of differentiation have been also reported. There have been, however, only a few reports regarding extensive immunohistochemical and ultrastructural investigations of this neoplasm.Histopathological and immunohistochemical studies were performed on three cases of desmoplastic trichoepithelioma, comparing it with normal skin. One of these cases was ultrastructurally investigated.The cord-like basaloid nests were reacted with the anti-cytokeratin (CK)1/5/10/14, -CK5/8, -CK14 and -CK15 antibodies, but not with the anti-CK6 antibody. Similar findings were observed in the outer layers of the normal follicular outer root sheath. Basaloid cell nests in one case, which showed ductal structures in the nests, also expressed CK7, CK8/18 and CK19. These keratins were also detected in the normal sweat glands. In addition, CK8/18 and CK19 were expressed in the basal cells of the outer root sheath. Keratinous cysts had inner reactions with the anti-CK10/11 and -CK6 antibodies, and outer reactions with anti-CK5/8 and -CK14 antibodies. Ultrastructurally, the cells in the cord-like nests were basically immature and basaloid in appearance. A few cells contained Odland bodies, which were also observed in the normal apocrine acrosyringeum. The ductal structure was lined by the cells which bore numerous microvilli in the luminal surface.The cells in desmoplastic trichoepithelioma are suggested to be in close association with the basal cells in the outer root sheath, which can differentiate into various parts of the folliculosebaceous apocrine unit.

Immunocytochemistry was used to investigate the distribution of cells reacting with specific antibodies against glutathione S-transferase (GST) mu and pi in rat circumvallate and foliate taste buds; the findings were confirmed by Western blotting. Double immunofluorescence staining for protein gene product (PGP) 9.5 and GST subunits allowed the classification of taste bud cells of both papillae into: (i) cells immunoreactive to either PGP 9.5 or GST subunit antibody; (ii) cells immunoreactive to both antibodies; and (iii) cells that did not react with either of these antibodies. Immunoelectron microscopy revealed that most GST subunit-immunoreactive cells seemed to be either type II or type III cells based on their ultrastructure. Since PGP 9.5 is now widely used as a marker for type III cells in mammalian taste buds, it seems reasonable to believe that most GST subunit-immunoreactive cells are type II cells. Whether cells immunoreactive for both PGP 9.5 and GST subunits constitute a small subpopulation of type III cells or whether they are intermediate forms between type II and III cells is under investigation. No type I cells reacted with antibodies against GST subunits in the present study. GST subunits in taste bud cells may participate in xenobiotic metabolism of certain substances exposed to taste pits, as already shown for olfactory epithelium.

We examined human umbilical vein endothelial cells (HUVECs) under prolonged culture by electron microscopy and by light and electron immunocytochemistry including double immunolabeling. Based on the cell area of HUVECs through multiple passages, we divided the cells into first, second, and third stages, which exhibited distinct morphological and immunocytochemical characteristics. During the first stage, HUVECs were polygonal in shape and had already formed the monolayer confluence. During the second stage, they were characterized by an increased number of Weibel-Palade (WP) bodies, which were actively segregated from Golgi cisterns. Endothelin (ET)-1 and von Willebrand factor, an endothelial cell marker, were occasionally colocalized in WP bodies. The increase in WP bodies correlated with high ET-1 concentration in the cultured medium, suggesting that these inclusions are involved in storage and release of ET-1 in a manner indicating a regulatory pathway. During the third stage, fibronectin and interleukin (IL)-1alpha were expressed in HUVECs as well as in multinucleated giant cells, which originated from HUVECs, but WP bodies decreased in number in association with a decrease in ET-1 immunoreactivity and concentration. The foregoing changes in immunoreactivities for ET-1, fibronectin, and IL-1alpha affecting HUVECs under prolonged culture may reflect a senescent process of these cells.