Double-stranded RNA-dependent protein kinase regulates insulin-stimulated chondrogenesis in mouse clonal chondrogenic cells, ATDC-5.

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.

Preclinical in vitro evaluation of immune suppression induced by GYM329, Fc-engineered sweeping antibody.

Fc-engineering is commonly used to improve the therapeutic potency of antibody (Ab) treatments. Because FcγRIIb is the only inhibitory FcγR that contains an immunoreceptor tyrosine-based inhibition motif (ITIM), Fc-engineered Abs with enhanced binding affinity to FcγRIIb might provide immune suppression in clinical contexts. GYM329 is an anti-latent myostatin Fc-engineered Ab with increased affinity to FcγRIIb which is expected to improve muscle strength in patients with muscular disorders. Cross-linking of FcγRIIb by immune complex (IC) results in phosphorylation of ITIM to inhibit immune activation and apoptosis in B cells. We examined whether the IC of Fc-engineered Abs with enhanced binding affinity to FcγRIIb causes phosphorylation of ITIM or B cell apoptosis using GYM329 and its Fc variant Abs in human and cynomolgus-monkey (cyno) immune cells in vitro. IC of GYM329 with enhanced binding affinity to human FcγRIIb (×5) induced neither ITIM phosphorylation nor B cell apoptosis. As for GYM329, FcγRIIb should work as an endocytic receptor of small IC to sweep latent myostatin, so it is preferable that GYM329 induces neither ITIM phosphorylation nor B cell apoptosis to prevent immune suppression. In contrast, IC of myo-HuCy2b, the Ab with enhanced binding affinity to human FcγRIIb (×4), induced ITIM phosphorylation and B cell apoptosis. The result of the present study demonstrated that Fc-engineered Abs with similar binding affinity to FcγRIIb had different effects. Thus, it is important to also investigate FcγR-mediated immune functions other than binding to fully understand the biological effects of Fc-engineered Abs.

Enhanced expression of nuclear factor I/B in oxaliplatin-resistant human cancer cell lines.

Oxaliplatin is a third-generation platinum drug that has favorable activity in cisplatin-resistant cells. However, the molecular mechanisms underlying oxaliplatin resistance are not well understood. To investigate the molecular mechanisms involved, resistant cell lines were independently derived from colon cancer (DLD1) and bladder cancer (T24) cells. Oxaliplatin-resistant DLD1 OX1 and DLD1 OX2 cell lines were approximately 16.3-fold and 17.8-fold more resistant to oxaliplatin than the parent cell lines, respectively, and had 1.7- and 2.2-fold higher cross-resistance to cisplatin, respectively. Oxaliplatin-resistant T24 OX2 and T24 OX3 cell lines were approximately 5.0-fold more resistant to oxaliplatin than the parent cell line and had 1.9-fold higher cross-resistance to cisplatin. One hundred and fifty-eight genes commonly upregulated in both DLD1 OX1 and DLD1 OX2 were identified by microarray analysis. These genes were mainly involved in the function of transcriptional regulators (14.6%), metabolic molecules (14.6%), and transporters (9.5%). Of these, nuclear factor I/B (NFIB) was upregulated in all oxaliplatin-resistant cells. Downregulation of NFIB rendered cells sensitive to oxaliplatin, but not to cisplatin. Forced expression of NFIB induced resistance to oxaliplatin, but not to cisplatin. Taken together, these results suggest that NFIB is a novel and specific biomarker for oxaliplatin resistance in human cancers.

Double stranded RNA-dependent protein kinase is involved in osteoclast differentiation of RAW264.7 cells in vitro.

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.

Functional expression of double-stranded RNA-dependent protein kinase in rat intestinal epithelial cells.

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.

An integrin-activating peptide, PHSRN, ameliorates inhibitory effects of conventional peritoneal dialysis fluids on peritoneal wound healing.

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.

Molecular mechanisms supporting a paracrine role of GABA in rat adrenal medullary cells.

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.

Corrigendum to "High glucose concentration-induced expression of pentraxin-3 in a rat model of continuous peritoneal dialysis".

This correct the article DOI: 10.14670/HH-11-756.

High glucose concentration-induced expression of pentraxin-3 in a rat model of continuous peritoneal dialysis.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Pathological alterations of strial capillaries in dominant white spotting W/Wv mice.

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.

Enhancement of immunoreactivity for endothelin-1 and endothelin-converting enzyme-1 in the cadmium-treated rat thoracic aorta.

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.

The enhancement of preproendothelin-1 synthesis and the acceleration of endothelin-1 processing in the acute hypoxic rat aorta.

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.

[Vasculogenesis and angiogenesis].

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.

Possible participation of acidic pH in bone resorption in middle ear cholesteatoma.

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.

Expressions of the multidrug resistance-related proteins in the rat olfactory epithelium: a possible role in the phase III xenobiotic metabolizing function.

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.

Bisphenol A induces endoplasmic reticulum stress-associated apoptosis in mouse non-parenchymal hepatocytes.

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.

Antibody recycling by engineered pH-dependent antigen binding improves the duration of antigen neutralization.

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.

Antioxidant enzyme immunoreactivity in rat von Ebner gland after nickel treatment.

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.

Immunoreactivities for glutathione S-transferases and glutathione peroxidase in the lateral wall of pigmented and albino guinea pig cochlea.

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.

Increased immunoreactivities against endothelin-converting enzyme-1 and monocyte chemotactic protein-1 in hepatic stellate cells of rat fibrous liver induced by thioacetamide.

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.