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Although accumulating evidence indicates that alternative splicing is aberrantly altered in many cancers, the functional mechanism remains to be elucidated. Here, we show that epithelial and mesenchymal isoform switches of leucine-rich repeat Fli-I-interacting protein 2 (LRRFIP2) regulated by epithelial splicing regulatory protein 1 (ESRP1) correlate with metastatic potential of gastric cancer cells. We found that expression of the splicing variants of LRRFIP2 was closely correlated with that of ESRP1. Surprisingly, ectopic expression of the mesenchymal isoform of LRRFIP2 (variant 3) dramatically increased liver metastasis of gastric cancer cells, whereas deletion of exon 7 of LRRFIP2 by the CRISPR/Cas9 system caused an isoform switch, leading to marked suppression of liver metastasis. Mechanistically, the epithelial LRRFIP2 isoform (variant 2) inhibited the oncogenic function of coactivator-associated arginine methyltransferase 1 (CARM1) through interaction. Taken together, our data reveals a mechanism of LRRFIP2 isoform switches in gastric cancer with important implication for cancer metastasis. ESRP1 is reported to regulate the alternative splicing of some genes involved in epithelial–mesenchymal transition. Here the authors show that LRRFIP2 is a target of ESRP1 and that two spliced variants of LRRFIP2 have opposing functions in gastric cancer metastasis [ABSTRACT FROM AUTHOR]

The development of triple-negative breast cancer (TNBC) negatively impacts both quality of life and survival in a high percentage of patients. Here, we show that RING finger protein 208 (RNF208) decreases the stability of soluble Vimentin protein through a polyubiquitin-mediated proteasomal degradation pathway, thereby suppressing metastasis of TNBC cells. RNF208 was significantly lower in TNBC than the luminal type, and low expression of RNF208 was strongly associated with poor clinical outcomes. Furthermore, RNF208 was induced by 17β-estradiol (E2) treatment in an estrogen receptor alpha (ΕRα)-dependent manner. Overexpression of RNF208 suppresses tumor formation and lung metastasis of TNBC cells. Mechanistically, RNF208 specifically polyubiquitinated the Lys97 residue within the head domain of Vimentin through interaction with the Ser39 residue of phosphorylated Vimentin, which exists as a soluble form, eventually facilitating proteasomal degradation of Vimentin. Collectively, our findings define RNF208 as a negative regulator of soluble Vimentin and a prognostic biomarker for TNBC cells. Triple-negative breast cancer (TNBC) is an aggressive subtype lacking effective targeted therapies. Here, the authors show that RNF208, an estrogen-induced ubiquitin ligase, promotes the degradation of Vimentin, thereby suppressing lung metastasis of TNBC, and may serve as a biomarker for the disease. [ABSTRACT FROM AUTHOR]

Infective endocarditis (IE), a life-threatening condition predominantly occurring in patients with underlying heart disease, is mainly caused by bacteremia induced by invasive dental treatment. However, the amount of related information shared between cardiologists and dentists appears to be inadequate. In the present study, a survey regarding prevention of IE, composed of 13 major questions, 2 of which also allowed free comments, was sent to approximately 3000 dentists belonging to a prefectural dental association in Japan. Of the 13.6% who returned the forms, more than 80% were general dentists with more than 20 years of experience. Approximately, 55% of the responders reported that they had opportunities to prescribe antibiotics prior to performing treatments with risk of IE, though noted difficulties with designation of which patients with heart disease were at risk. Most of the dentists considered that oral surgery procedures have a high risk for IE, whereas less invasive procedures were considered to be not associated with the disease. Approximately, 35% selected oral amoxicillin, with a dose of 2.0 g (20%) or 500 mg (27%) prescribed for adults, and 50 mg (10%) or 30 mg (12%) per kg of body weight for children. However, the timing of the antibiotics administration varied. The present results reveal current knowledge regarding prevention of IE among general dentists in Japan, and should be valuable for construction of a protocol to establish consensus between dentists and cardiologists. [ABSTRACT FROM AUTHOR]

Rationale: High-fat diet (HFD) has been recently reported to induce sensorimotor gating deficits, but the underlying mechanisms are not well understood. Objective: The purpose of this study is to determine whether HFD induces long-lasting deficits in sensorimotor gating and to examine the involvement of altered dopamine (DA) function. Methods: C57BL/6J mice were fed HFD for 10 weeks and then normal diet (ND) for 4 weeks. DA D receptor (DR) knockout (KO) mice were also fed HFD for 10 weeks. The mice were evaluated for prepulse inhibition (PPI) of acoustic startle after HFD and the subsequent 4-week ND. We evaluated the effect of SCH23390, a D receptor (DR) antagonist, on PPI and measured protein expression levels of DR and DR in the prefrontal cortex (PFC) in HFD mice. The concentrations of monoamines and their metabolites in the cortices of 10-week HFD or ND mice were measured using high performance liquid chromatography. Results: Long-term HFD-induced PPI disruption in WT and DR KO mice. Even after 4 weeks of subsequent ND, PPI remained to be disrupted. SCH23390 mitigated the PPI disruption. In HFD animals, DR protein expression in the PFC was significantly decreased, while DA, homovanillic acid, and 3,4-dihydroxyphenylacetic acid levels in the cortex were increased. Conclusion: This is the first evidence that HFD can induce long-lasting deficits in sensorimotor gating through alteration of cortical levels of DA and its metabolites. Our data suggest that HFD-induced PPI deficits are related to altered DR signaling and that DR antagonists may have therapeutic effects on the deficits. [ABSTRACT FROM AUTHOR]

The relative proportion of kynurenine aminotransferase (KAT) I-IV activities in the brain is similar between humans and rats. Moreover, KAT II is considered to be the main enzyme for kynurenic acid production in the brain. Taken together, human KAT II knock-in (hKAT II KI) rats will become a valuable tool for the evaluation of KAT II targeted drugs as a human mimetic model. Although we initially tried the approach by conventional gene-targeting via embryonic stem cells (ESCs) to generate them, we had to give up the production because of no recombinant ESCs. Accordingly, we developed a method to improve the efficiency of homologous recombination (HR) in ESCs by the combination with the CRISPR/Cas system. Co-electroporation of Cas9 plasmid, single guide RNA plasmid and hKAT II KI vector increased the number of drug-resistant colonies and greatly enhanced the HR efficiency from 0 to 36 %. All the clones which we obtained showed the same sequence as designed. These recombinant clones resulted in chimeras that transmitted the hKAT II KI allele to their offspring. hKAT II KI rats showed no reduction of KATs mRNA expression and the amount of kynurenic acid was similar between the hKAT II KI rats and the wild type in their brains. These results indicate that the methodology presented in this report can overcome the problem encountered in conventional gene-targeting that prevented production of humanized rats. [ABSTRACT FROM AUTHOR]

Low birth weight due to intrauterine growth retardation (IUGR) is suggested to be a risk factor for various psychiatric disorders such as schizophrenia. It has been reported that developmental cortical dysfunction and neurocognitive deficits are observed in individuals with IUGR, however, the underlying molecular mechanisms have yet to be elucidated. Brain-derived neurotrophic factor (BDNF) and its receptor TrkB are associated with schizophrenia and play a role in cortical development. We previously demonstrated that BDNF induced glutamate release through activation of the TrkB/phospholipase C-γ (PLC-γ) pathway in developing cultured cortical neurons, and that, using a rat model for IUGR caused by maternal administration of thromboxane A2, cortical levels of TrkB were significantly reduced in IUGR rats at birth. These studies prompted us to hypothesize that TrkB reduction in IUGR cortex led to impairment of BDNF-dependent glutamatergic neurotransmission. In the present study, we found that BDNF-induced glutamate release was strongly impaired in cultured IUGR cortical neurons where TrkB reduction was maintained. Impairment of BDNF-induced glutamate release in IUGR neurons was ameliorated by transfection of human TrkB (hTrkB). Although BDNF-stimulated phosphorylation of TrkB and of PLC-γ was decreased in IUGR neurons, the hTrkB transfection recovered the deficits in their phosphorylation. These results suggest that TrkB reduction causes impairment of BDNF-stimulated glutamatergic function via suppression of TrkB/PLC-γ activation in IUGR cortical neurons. Our findings provide molecular insights into how IUGR links to downregulation of BDNF function in the cortex, which might be involved in the development of IUGR-related diseases such as schizophrenia. [ABSTRACT FROM AUTHOR]

Renal fibrosis is a common consequence of unilateral ureteral obstruction, which provides a useful model to investigate the pathogenesis of obstructive nephropathy and progressive renal fibrosis. Transforming growth factor (TGF-β1) has been recognized as a key mediator in renal fibrosis by stimulating matrix-producing fibrogenic cells and promoting extracellular matrix deposition. Therefore, considerable efforts have been made to regulate TGF-β signaling for antifibrotic therapy. Here, we investigated the mode of action of glucosamine hydrochloride (GS-HCl) on TGF-β1-induced renal fibrosis. In the obstructed kidneys and TGF-β1-treated renal cells, GS-HCl significantly decreased renal expression of α-smooth muscle actin, collagen I, and fibronectin. By investigating the inhibitory mechanism of GS-HCl on renal fibrosis, we found that GS-HCl suppressed TGF-β signaling by inhibiting N-linked glycosylation of the type II TGF-β receptor (TβRII), leading to an inefficient trafficking of TβRII to the membrane surface. Defective N-glycosylation of TβRII further suppressed the TGF-β1-binding to TβRII, thereby decreasing TGF-β signaling. Notably, GS-HCl treatment significantly reduced TGF-β1-induced up-regulation of Smad2/3 phosphorylation and transcriptional activity in vivo and in vitro. Taken together, GS-HCl-mediated regulation of TGF-β signaling exerted an antifibrotic effect, thereby ameliorating renal fibrosis. Our study suggests that GS-HCl would be a promising agent for therapeutic intervention for preventing TGF-β1-induced renal fibrosis in kidney diseases. Key message: [ABSTRACT FROM AUTHOR]

Gene-targeting technology using mouse embryonic stem (ES) cells has become the 'gold standard' for analyzing gene functions and producing disease models. Recently, genetically modified mice with multiple mutations have increasingly been produced to study the interaction between proteins and polygenic diseases. However, introduction of an additional mutation into mice already harboring several mutations by conventional natural crossbreeding is an extremely time- and labor-intensive process. Moreover, to do so in mice with a complex genetic background, several years may be required if the genetic background is to be retained. Establishing ES cells from multiple-mutant mice, or disease-model mice with a complex genetic background, would offer a possible solution. Here, we report the establishment and characterization of novel ES cell lines from a mouse model of Alzheimer's disease (3xTg-AD mouse, Oddo et al. in Neuron 39:409-421, ) harboring 3 mutated genes (APP, Tau, and PS1) and a complex genetic background. Thirty blastocysts were cultured and 15 stable ES cell lines (male: 11; female: 4) obtained. By injecting these ES cells into diploid or tetraploid blastocysts, we generated germline-competent chimeras. Subsequently, we confirmed that F mice derived from these animals showed similar biochemical and behavioral characteristics to the original 3xTg-AD mice. Furthermore, we introduced a gene-targeting vector into the ES cells and successfully obtained gene-targeted ES cells, which were then used to generate knockout mice for the targeted gene. These results suggest that the present methodology is effective for introducing an additional mutation into mice already harboring multiple mutated genes and/or a complex genetic background. [ABSTRACT FROM AUTHOR]

One of the remarkable achievements in knockout (KO) rat production reported during the period 2008-2010 is the derivation of authentic embryonic stem (ES) cells from rat blastocysts using a novel culture medium containing glycogen synthase kinase 3 and mitogen-activated protein kinase kinase inhibitors (2i medium). Here, we report gene-targeting technology via homologous recombination in rat ES cells, demonstrating its use through production of a protease-activated receptor-2 gene ( Par- 2) KO rat. We began by generating germline-competent ES cells from Dark Agouti rats using 2i medium. These ES cells, which differentiate into cardiomyocytes in vitro, can produce chimeras with high ES cell contribution when injected into blastocysts. We then introduced a targeting vector with a neomycin-resistant gene driven by the CAG promoter to disrupt Par- 2. After a 7-day drug selection, 489 neomycin-resistant colonies were obtained. Following screening by polymerase chain reaction (PCR) genotyping and quantitative PCR analysis, we confirmed three homologous recombinant clones, resulting in chimeras that transmitted the Par- 2 targeted allele to offspring. Par- 2 KO rats showed a loss of Par- 2 messenger RNA expression in their stomach cells and a lack of PAR-2 mediated smooth muscle relaxation in the aorta as indicated by pharmacological testing. Compared with mice, rats offer many advantages in biomedical research, including a larger body size; consequently, they are widely used in scientific investigation. Thus, the establishment of a gene-targeting technology using rat ES cells will be a valuable tool in human disease model production and drug discovery. [ABSTRACT FROM AUTHOR]

Mutation of the human TRPS1 gene leads to trichorhinophalangeal syndrome (TRPS), which is characterized by an abnormal development of various organs including the craniofacial skeleton. Trps1 has recently been shown to be expressed in the jaw joints of zebrafish; however, whether Trps1 is expressed in the mammalian temporomandibular joint (TMJ), or whether it is necessary for TMJ development is unknown. We have analyzed (1) the expression pattern of Trps1 during TMJ development in mice and (2) TMJ development in Trps1 knockout animals. Trps1 is expressed in the maxillo-mandibular junction at embryonic day (E) 11.5. At E15.5, expression is restricted to the developing condylar cartilage and to the surrounding joint disc progenitor cells. In Trps1 knockout mice, the glenoid fossa of the temporal bone forms relatively normally but the condylar process is extremely small and the joint disc and cavities do not develop. The initiation of condyle formation is slightly delayed in the mutants at E14.5; however, at E18.5, the flattened chondrocyte layer is narrowed and most of the condylar chondrocytes exhibit precocious chondrocyte maturation. Expression of Runx2 and its target genes is expanded toward the condylar apex in the mutants. These observations underscore the indispensable role played by Trps1 in normal TMJ development in supporting the differentiation of disc and synoviocyte progenitor cells and in coordinating condylar chondrocyte differentiation. [ABSTRACT FROM AUTHOR]

The transforming growth factor beta (TGF-β) pathway can play either a tumor-suppressing or a tumor-promoting role in human breast carcinogenesis. In order to determine whether expression of TGF-β signaling factors varies by age at onset and breast tumor characteristics that have prognostic significance, we undertook a study of 623 women with invasive breast carcinoma enrolled in a population-based case–control study conducted in Poland from 2000 to 2003. TGF-β signaling factors were analyzed by immunohistochemistry in tumor tissue microarrays. We found that most tumors expressed extracellular-TGF-β1 (78%), TGF-β2 (91%), TGF-β3 (93%), TGF-βR2 (72%), and phospho-SMAD2 (61%), whereas intracellular-TGF-β1 was expressed in 32% of tumors. Expression of TGF-β ligands (β1, β2, and β3) was associated with prognostically favorable pathological features including small size, and low grade, and these associations were similar for ER-positive and negative tumors. On the contrary, expression of the receptor TGF-βR2 was primarily associated with small tumor size among ER-negative tumors, while expression of the transcription factor phospho-SMAD2 was associated with positive nodal status among ER-negative tumors. The greater frequency of expression of phospho-SMAD2 in cancers associated with lymph node metastases is consistent with a pro-progression role for TGF-β. In addition, expression of extracellular-TGF-β1 ( P = 0.005), TGF-βR2 ( P = 8.2E-11), and phospho-SMAD2 ( P = 1.3E-8) was strongly associated with earlier age at onset, independent of ER status. Our data provide evidence that TGF-β signaling patterns vary by age and pathologic features of prognostic significance including ER expression. These results warrant analysis in studies of clinical outcomes accounting for age, ER status and treatment. [ABSTRACT FROM AUTHOR]

Stroke-prone spontaneously hypertensive rats (SHRSP) used as a model of essential hypertension cause a high incidence of brain stroke on the course of hypertension. Incidences and sizes of brain lesions are known to relate to the astrocyte activities. Therefore, relation between brain damage and the expression profile of the astrocytes was investigated with morphometric and immunohistochemical analyses using astrocyte marker antibodies of S100B and glial fibrillary acidic protein (GFAP) with or without arundic acid administration, a suppressor on the activation of astrocytes. Arundic acid extended the average life span of SHRSP. An increase in brain tissue weight was inhibited concomitant with a lower rate of gliosis/hemosiderin deposit/scarring in brain lesions. S100B- or GFAP-positive dot and filamentous structures were decreased in arundic acid-treated SHRSP, and this effect was most pronounced in the cerebral cortex, white matter, and pons, and less so in the hippocampus, diencephalon, midbrain, and cerebellum. Blood pressure decreased after administration of arundic acid in the high-dose group (100 mg/kg/day arundic acid), but not in the low-dose group (30 mg/kg/day). These data indicate that arundic acid can prevent hypertension-induced stroke, and may inhibit the enlargement of the stroke lesion by preventing the inflammatory changes caused by overproduction of the S100B protein in the astrocytes. [ABSTRACT FROM AUTHOR]

The ectomycorrhizal (ECM) fungi associated with Pinus thunbergii seedlings grown on sand dune were identified by molecular method, and the diversity of bacteria associated with ECM and Extraradical mycelium were examined by Denaturing Gradient Gel Electrophoresis (DGGE) of PCR-amplified 16S rDNA. The mycorrhizal formation rate of 1-year old P. thunbergii seedlings was more than 95%. Cenococcum geophilum was the most dominant ECM fungus, followed by T01, RFLP-8, Russula spp., and Suillus sp. Bacterial community was most diverse with C. geophilum- and RFLP-8-mycorrhiza. Sequencing analysis showed that Burkholderia spp. and Bradyrhizobium spp. were on the surface of ECM short root of seven ECM. The fungi detected as extraradical mycelium using DGGE of 18S rDNA were Suillus bovinus and RFLP-8-mycorrhiza. Bacterial community on the extraradical mycelium was more diverse than those on ECM root tip. Burkholderia spp. and Bradyrhizobium spp. were found also on extraradical mycelium. [ABSTRACT FROM AUTHOR]

Keloids are tumor-like lesions that result from excessive scar formation during healing of wounds. Histologically, keloids show an increased blood vessel density compared with normal dermis or normal scars. However, the angiogenic activity of keloid fibroblasts remains unknown. In this study, we investigated angiogenic activity of keloid fibroblasts. Transforming growth factor-β1 (TGF-β1) and vascular endothelial growth factor (VEGF) were investigated as elements of the angiogenic factors. Expressions of TGF-β1 and VEGF in conditioned medium were measured with enzyme-linked immunosorbent assay (EIA) and Northern blot analysis. Participation of TGF-β1 in the production of VEGF was also investigated with addition of TGF-β1 and a neutralizing anti-TGF-β1 antibody. A modified Boyden chamber assay was performed to assess the chemotactic activity of vascular endothelial cells. Angiogenic activity in vivo was evaluated by neovascularization of nodules formed by implantation of fibroblasts into severe combined immunodeficiency (SCID) mice. EIA showed that the concentrations of TGF-β1 and VEGF in conditioned medium were increased 2.5- and 6-fold, respectively, after the culture of keloid fibroblasts compared with normal fibroblasts. Northern blot analysis revealed that the expression of TGF-β1 and VEGF mRNA was upregulated 3.6- and 6-fold, respectively, in keloid fibroblasts compared with normal fibroblasts. Addition of TGF-β1 to keloid fibroblast cultures increased VEGF production by 3.5-fold, while there was a 6-fold in culture of normal fibroblasts. A neutralizing anti-TGF-β1 antibody reduced VEGF secretion to control levels, suggesting that TGF-β1 mediated the upregulation of VEGF expression. A modified Boyden chamber assay demonstrated that the chemotactic activity of vascular endothelial cells was more strongly (sevenfold) induced by keloid fibroblast-conditioned medium than by normal fibroblast-conditioned medium. Anti-VEGF antibody inhibited chemotaxis to basal levels. When SCID mice underwent implantation of fibroblasts into the back, the nodules formed by keloid fibroblasts were three times larger than those formed by normal fibroblasts. Although abundant neovascularization was observed in keloid fibroblast nodules, neovascularization was scarce in normal fibroblast nodules. Both in vitro and in vivo studies confirmed the significantly higher angiogenic activity of keloid fibroblasts compared with normal fibroblasts, and TGF-β1 and VEGF were clearly shown to be involved. These results suggest that angiogenesis in keloids is promoted by endogenous TGF-β1 and VEGF. [ABSTRACT FROM AUTHOR]

PurposeTo examine the alteration of protein expression pattern of collagen type XV in cornea during embryonic development and adult tissue repair. Collagen type XV is a basement membrane collagen of a subfamily of multiplexins (multiple triple helix domains and interruptions). Its COOH-terminal peptide has an anti-angiogenic effect and its distribution in avascular tissue of cornea is of interest.MethodsEyes of mouse embryos [day (E) 10.5?18.5] and healing adult mouse corneas following either débridement injury or incision were embedded in paraffin. Deparaffinized sections were processed for immunofluorescent staining with anti-collagen XV antibody.ResultsAt E14.5 embryonic corneal epithelium, as well as fibroblasts in eyelids, began to express this collagen type very faintly, and at E18.5, besides corneal epithelial expression, epidermis, palpebral conjunctiva, and keratocytes started to express collagen type XV. In adult mouse cornea, collagen type XV was observed in basal and suprabasal epithelial cells and stroma, but not in the subepithelial basement membrane. Healing epithelial cells following débridement or incision injury down-regulated its protein expression.ConclusionsMouse embryonic corneal epithelium and keratocytes begin to express collagen type XV before birth. Healing murine corneal epithelium down-regulates collagen XV expression. The presence of collagen XV in corneal stroma may play a part in avascularity. [ABSTRACT FROM AUTHOR]

Abstract Background. We examined the effects of interferon-γ (IFN-γ) on protein production of extracellular matrix (ECM) components in cultured human subconjunctival fibroblasts or those stimulated by exogenous transforming growth factor β1 (TGFβ1). IFN-γ reportedly up-regulates Smad7, an inhibitory mediator of TGFβ-Smad signaling, and blocks TGFβ effects. Methods. Proliferation and migration as well as the ultrastructure of these cells were examined in the presence and absence of IFN-γ. Cell migration was examined using an in vitro wound healing model in monolayer fibroblast cultures. Results. The results showed that IFN-γ reduced ECM production in normal subconjunctival fibroblasts, as well as in those treated with TGFβ1, below the control levels. IFN-γ had no effect on cell proliferation and fibroblast ultrastructure. On the other hand, IFN-γ delayed defect closure in monolayer cell sheets in a dose-dependent manner. Immunohistochemistry also revealed that the addition of IFN-γ attenuated the translocation of Smads2/4 into the nuclei of TGFβ1-treated subconjunctival fibroblasts. Conclusion. These findings suggest that IFN-γ may be clinically effective in attenuating excessive ECM accumulation in conjunctiva after ocular surgery and in the presence of inflammatory ocular surface disorder. IFN-γ modulates the Smads2/4 pathway of TGFβ1 signal transduction toward the up-regulation of ECM components. [ABSTRACT FROM AUTHOR]

Purpose: To examine immunohistochemically whether extracellular matrix (ECM) of the filtering bleb and of cultured human subconjunctival fibroblasts contains latent TGFβ binding protein-1 (LTBP-1) and TGFβ. Methods: An enucleated human eye that had undergone trabeculectomy and cultured human subconjunctival fibroblasts were processed for light microscopic immunohistochemistry. Antibodies against LTBP-1, collagen types, fibrillin-1 and TGFβs were used. TGFβ1 was located by detecting β1-latency associated peptide (LAP). Results: LTBP-1, β1-LAP and fibrillin-1 were all located in the subepithelial ECM as well as in the basal epithelial cells of the conjunctiva over the filtering bleb. TGFβ2 and β3 were immunolocated to epithelium and/or fibroblasts/keratocytes. ECM deposited in confluent fibroblast cultures was positive for β1-LAP, LTBP-1 and fibrillin-1, whereas sparse cells were negative. Conclusions: LTBP-1, β1-LAP and fibrillin-1 are co-localized to the ECM of the filtering bleb and of cultured conjunctival fibroblasts. Both conjunctival epithelium and fibroblasts are considered to be the source of TGFβ in healing bleb. ECM secreted by in vivo and in vitro subconjunctival fibroblasts may works as a scavenger or repository of TGFβ. [ABSTRACT FROM AUTHOR]

At least eight inherited neurodegenerative diseases are caused by expanded CAG repeats encoding polyglutamine (polyQ) stretches. Although cytotoxicities of expanded polyQ stretches are implicated, the molecular mechanisms of neurodegeneration remain unclear. We found that expanded polyQ stretches preferentially bind to TAFII130, a coactivator involved in cAMP-responsive element binding protein (CREB)-dependent transcriptional activation, and strongly suppress CREB-dependent transcriptional activation. The suppression of CREB-dependent transcription and the cell death induced by polyQ stretches were restored by the co-expression of TAFII130. Our results indicate that interference of transcription by the binding of TAFII130 with expanded polyQ stretches is involved in the pathogenetic mechanisms underlying neurodegeneration. [ABSTRACT FROM AUTHOR]

Purpose: To establish the alteration in expression pattern of transforming growth factor (TGF)-βs and their receptors during repair of lens capsules after cataract surgery, we immunohistochemically located TGF-β isoforms and their receptors in human lens capsules before and after cataract surgery. Methods: Ten post-cataract surgery capsular specimens were obtained during vitrectomy. Three sections of the anterior capsules were obtained during cataract surgery. A whole lens capsular bag immediately after lens extraction was obtained during vitrectomy. Cryosections of these specimens were processed for immunohistochemical analysis for TGF-β1, TGF-β2, TGF-β3, TGF-β receptor type I (TβR-I), type II (TβR-II) and type III (TβR-III), and were observed under light micros-copy. Results: Lens epithelial cells (LECs) lining the inner surface of the anterior capsules exhibited immunoreactivity for TGF-β2 and TβR-II. Immunoreactivity for TGF-β1, -β3, TβR-I and TβR-III was negative. In the whole capsular bag specimen, equatorial LECs were positive for TGF-β1 and -β2, but not for -β3. In post-cataract surgery specimens, antibodies for each TGF-β isoform labelled either the LECs or ECM accumulated on the capsules. Post-surgical LECs expressed TβR-I and TβR-II, and had also TβR-III in seven of the nine specimens examined. Conclusion: Expression pattern of TGF-β s in quiescent LECs showed regional heterogeneity. Anterior LECs exhibited TGF-β2 immunoreactivity, while equatorial LECs were positive for TGF-β1 and -β2. Quiescent LECs expressed TβR-II. LECs proliferating around IOLs expressed proteins of each TGF-β isoform and each TβR. TGF-β s were also localized in the ECM on capsules undergoing repair. TGF-β3, TβR-I and TβR-III are up-regulated in LECs after cataract surgery. [ABSTRACT FROM AUTHOR]

Radiation therapy to the head and neck area frequently causes severe salivary gland dysfunction and xerostomia. Morphological studies of irradiated salivary glands have suggested that the submandibular/sublingual gland may be less radiosensitive than the parotid gland. The purpose of this study was to evaluate the effect of radiation on major salivary gland functions in rats with radiation-induced xerostomia. The effect of salivary gland irradiation on salivary function was examined in specific pathogen-free Sprague-Dawley rats. The animals were irradiated with a single exposure of either 22 Gy or 32 Gy. Stimulated saliva excretion time was measured for the parotid and submandibular/ sublingual glands, and the total protein in saliva was analysed. Our results showed that the saliva flow rate and protein concentration of parotid saliva were significantly reduced in the 32 Gy-irradiated rats. [ABSTRACT FROM AUTHOR]

Preduodenal portal vein (PDPV) is a rare developmental anomaly. We recently encountered this anomaly in a 73-year-old woman who had gastric cancer and colonic cancer with liver metastasis. The PDPV was diagnosed preoperatively by computed tomography and angiography. To the best of our knowledge, there are only 19 adult cases reported in the world literature, with this additional case being reported here. In infants the anomaly has often been associated with duodenal obstruction, but in adults it is often symptomless. We discuss the disease process, embryology, diagnosis, association with other anomalies, and surgical treatment. [ABSTRACT FROM AUTHOR]

The present study was undertaken to examine the localization of calbindin D28k (CB)-like immunoreactivity (-LI) during the root formation of the rat molar. In the adult rat, CB-LI was detected in some of the cells of the epithelial rest of Malassez at the bifurcational region and in certain cells between the root dentin and cementum at the apical region. These cells had indented nuclei and many tonofilaments, and cementocytes lacked CB-LI. Moreover, CB-LI was observed in the periodontal fibroblasts in the alveolar half of the apical region. During root formation, the cells in the Hertwig’s epithelial root sheath (HERS) lacked CB-LI, but most fragmented cells along the root surface began to express CB-LI when HERS was disrupted. Preodontoblasts and odontoblasts at the apical portion of the root also showed CB-LI. After the formation of cellular cementum, the CB-immunoreactive (-IR) cells were entrapped between the root dentin and cementum in the apical portion of the root. The number of CB-IR cells at the root surface decreased gradually, while that between the root dentin and cementum increased. The fibroblasts in the periodontal ligament began to express CB-LI after commencement of the occlusion, and the number and the staining intensity of CB-IR fibroblasts increased gradually with the passage of time. The present results suggest that CB may play an important role in the survival of the epithelial cells, in the cellular responses of periodontal fibroblasts against mechanical forces caused by the occlusion, and in the initial mineralization by the odontoblasts through the regulation of intracellular Ca2+ concentration. [ABSTRACT FROM AUTHOR]

Unlabelled: A 32-month-old Japanese boy exhibited haemophagocytic syndrome (HPS) during the recurrent course of Kawasaki disease. Despite repeated gamma-globulin therapy, he developed cytopenia with marked hepatomegaly and evidence of histiocytic haemophagocytosis in the bone marrow. Serum levels of interferon-gamma and tumour necrosis factor, but not of interleukin-1 beta, increased in parallel with his symptoms. No confirmation was obtained of the association of toxic reactions to the used drugs. No coronary lesions remained as sequelae.Conclusion: Cytopenia in Kawasaki disease could herald HPS, and the hypercytokinaemia involved in the two febrile syndromes might be of distinct nature. [ABSTRACT FROM AUTHOR]

The phototropic response of Spirogyra sp. filaments and its relation with the different phases of their diurnal movements were studied. The filaments rapidly responded to unilateral irradiation; curvature towards the light source began in their tip region but shifted down to more basal regions. However, this typical and steady phototropic curvature could be observed only in the GnSt phase of the diurnal movement. In the other phases competitive states between the phototropic movement and other kinds of movement were evident. Thus, from the results of our previous and present studies it is proposed that the diurnal movement of Spirogyra filaments is composed of various individual movements, including a phototropic one; among these movements there exists a certain balance determined by the culture conditions and the time of day, and the phototropic movement is relatively inferior to the other movements. [ABSTRACT FROM AUTHOR]

Lysyl oxidase (LOX) initiates the crosslinking of the lysine-derived aldehyde and plays an essential role in maturation of collagen, for example in wound healing. Although the activity of this enzyme has been examined in various disorders, and a further intriguing aspect of the relationship between LOX and tumorigenesis has recently emerged, its gene expression pattern in tissues is still unknown. We examined LOX gene expression during wound healing in rat skin. In addition, type III collagen gene expression was studied to determine the formation of fibrils. The LOX mRNA level reached a peak by day 3 after injury, which was earlier than that of type III collagen, and continued at a high level until day 22. The type III collagen mRNA level began to rise from day 3 and had increased intensely by day 22. In situ hybridization revealed grains corresponding to LOX mRNA in the fibroblasts of the granulomatous tissue. These results suggest that LOX is produced before collagen synthesis in preparation for crosslinking in the early phase of wound healing. [ABSTRACT FROM AUTHOR]

Tissue distribution of type V collagen in normal human skin was studied using an indirect immunofluorescent technique to determine whether type V collagen is present in the interstitium or in the basement membrane. Type V collagen was isolated from the human placenta by pepsin digestion and was purified with fractioning salt precipitations. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that type V collagen contained α1(V) and α2(V) chains, but not the α3(V) chain. Specificity of the rabbit antibodies to type V collagen was assessed using enzyme-linked immunosorbent assay (ELISA) and an immunoblotting method. Antibodies showed no cross-reactivity to other collagens, laminin, and fibronectin. With an indirect immunofluorescent technique, type V collagen was found to be widely distributed throughout the dermis. Intense fluorescent staining was noted in the papillary dermis and adnexal dermis surrounding hair follicles and eccrine glands. The basement membrane of the dermoepidermal junction, skin appendages, and capillaries was not stained. By indirect immunoperoxidase double staining, type V collagen was not found to be deposited on type IV collagen present in the basement membrane. Immunoelectron microscopic studies showed that type V collagen was not located in the basal lamina. These results suggest that type V collagen is distributed in the interstitium, but not in the basement membrane of normal human skin. [ABSTRACT FROM AUTHOR]

Type IV collagen and laminin are major components of basement membrane (BM), whereas prolyl hydroxylase (PH) is a key enzyme in the hydroxylation of proline to hydroxyproline in collagen synthesis. In order to elucidate the exact mechanism of the formation of BM, immune electron microscopic observation of type IV collagen, laminin and PH was made in rat liver with marked proliferation of bile ducts following ligation of the common bile duct. Extracellular localization of type IV collagen was found in the BM of bile ducts and blood vessels and in the space of Disse in both normal rat liver and the liver of rats undergoing operation. Type IV collagen was localized in lamina rara and lamina densa. Laminin was codistributed with type IV collagen in BM but rarely in the space of Disse even in the liver of rats undergoing operation. Immunostaining of laminin was diffusely spread in lamina densa, but sparsely in lamina rara. Though no reaction products of type IV collagen and laminin were detected in the cytoplasm of normal biliary epithelial cells, they were found in rough endoplasmic reticulum (rER) and the vesicles close to the basal surfaces of the plasma membrane of the proliferating biliary epithelial cells. No evident localization of these components in Golgi apparatus was found. PH was found in rER of the biliary epithelial cells, hepatocytes, endothelial cells of vessels, fibroblasts and perisinusoidal cells except for Kupffer cells in normal rat liver. More intense and diffuse staining of PH was observed in rER in the proliferating biliary epithelial cells of the liver of rats undergoing operation in concomitance with the evident localization of type IV collagen in this organelle. These findings suggest that the major components of BM, such as type IV collagen and laminin in the proliferating biliary epithelial cells, are produced in rER and secreted by vesicles to the basal extracellular spaces, thus forming new BM in these circumstances. [ABSTRACT FROM AUTHOR]

The effects of X-ray irradiation to the salivary glands on the induction of dental caries were examined in specific pathogen-free Sprague-Dawley rats fed either a high (containing 56% sucrose) or a low-sucrose (containing 28% sucrose and 28% wheat flour) diet and inoculated with a strain of Streptococcus mutans. Hyposalivation was induced by irradiation (35 Gy) and dental caries was significantly enhanced in irradiated rats fed a high-sucrose diet. However, the recovery of the strain of S. mutans from rats fed high-sucrose was not significantly higher than that of rats fed a low-sucrose diet. [ABSTRACT FROM AUTHOR]

The effects of X-ray irradiation to the salivary glands on the induction of dental caries and on the oral microflora were examined in specific pathogen-free Sprague-Dawley rats fed a sucrose diet and inoculated with a strain of Streptococcus mutans. Hyposalivation was induced by irradiation of 35 Gy and dental caries was significantly enhanced in irradiated rats infected with S. mutans. Bacteriological examinations at the end of the experiment demonstrated that the recoveries of lactobacilli and staphylococci significantly increased in hyposalivated rats with 50 Gy of irradiation. However, the recovery of the strain of S. mutans from hyposalivated rats were not significantly higher than that of non-irradiated rats. These findings suggest that some acidogenic microorganisms such as lactobacilli and staphylococci may have played some role in promoting dental caries. [ABSTRACT FROM AUTHOR]

We investigated the distribution of extracellular matrix components such as fibronectin, laminin, type III, IV, V, and VI collagens and heparan sulfate proteoglycan (HSPG) in normal and diseased glomeruli using the indirect immunofluorescence method. This study included 96 renal biopsies: 7 controls, 3 minimal change nephrotic syndrome (MCNS), 47 mesangial proliferative glomerulonephritis (PGN), 25 membranous nephropathy (MN) and 14 membranoproliferative glomerulonephritis (MPGN) including 3 lupus nephritis. Fibronectin was detected predominantly in the mesangium and less prominently in the glomerular basement membrane (GBM) of normal glomeruli. Laminin and type IV collagen were present in the mesangium and GBM, type III collagen in the interstitium, and type V collagen in the mesangium, interstitium and a part of GBM. Type VI collagen was observed in the mesangium, interstitium and slightly in GBM. Anti-HSPG antibody reacted with the mesangium and GBM. MCNS showed a distribution of these antigens similar to that in normal controls. The finding that staining for HSPG was not decreased in the GBM and mesangium indicated that there was no change in the core protein of HSPG. Fibronectin, laminin, type IV collagen and HSPG were increased in the thickened GBM of MN and in the expanded mesangium of PGN. In MPGN, these matrix components were increased in the mesangium and GBM with remarkable increase of type V and VI collagens. While type III collagen was not found in normal glomeruli, it became detectable in the mesangium and a part of GBM in MPGN. No significant decrease in the intensity of fluorescence for HSPG was observed in the glomeruli from nephrotic patients. These findings suggest that proteinuria might be caused by the structural alteration in the glycosaminoglycan portion of HSPG, changes in any anionic material other than HSPG, or both, and also indicate that the glomerular mesangial sclerosis is closely related to the increase of type V and VI collagens. [ABSTRACT FROM AUTHOR]

An immunohistochemical study was performed on autopsy material from a patient with chronic neutrophilic leukaemia (CNL) using antibodies against various cell lineage-related antigens. Proliferation of immature neutrophils with occasional clusters of erythroblasts and megakaryocytes were noted in the retroperiotoneal lymph nodes, spleen, and kidneys as well as in the bone marrow. Predominance of immature neutrophils in the lymph nodes suggested the emergence of a blast crisis, although there was no increase of blasts in the peripheral blood. Since immature myeloid cells are difficult to distinguish from malignant lymphoid cells on tissue sections, we suggest that immunohistochemical identification of cell lineage-related molecules on these cells is necessary for the more accurate interpretation of lymph node lesions in myeloid neoplasms. [ABSTRACT FROM AUTHOR]

Immunolocalization of type III collagen and procollagen in cirrhotic human liver was studied using monoclonal antibody specific for the helical determinant of type III collagen extracted from human placenta. Deparaffinized, trypsin-treated cirrhotic liver sections from 8 autopsy cases were examined by the unlabeled peroxidase-antiperoxidase and immunofluorescence techniques. These techniques revealed the localization of this epitope shared by type III collagen and procollagen not only in the extracellular matrix of hepatocytes and sinusoidal cells but also in the cytoplasm. In hepatocellular carcinoma concurrent with cirrhosis, neoplastic cells were shown to react with this antibody as well. These results are consistent with data obtained using antiserum specific for bovine type III procollagen aminopeptide which appeared in our previous report. [ABSTRACT FROM AUTHOR]

· Purpose: We used immunohistochemistry to characterize cellular and proteinaceous deposits on the surfaces of explanted posterior chamber intraocular lenses (PC-IOLs). · Methods: A total of 30 PC-IOLs were immunostained for the α- or β-subunits of prolyl 4-hydroxylase, which is involved in collagen biosynthesis; cellular fibronectin; αB crystalline; and CD68, a macrophage marker, to characterize the cellular deposits that adhere to the IOL surfaces and to evaluate the distribution of cells involved in the deposition of extracellular matrix on IOLs. · Results: Cellular or proteinaceous deposits were observed on all 30 PC-IOLs. Cells that showed positive staining for αB crystallin were classified as lens epithelial cells; CD68-positive cells were considered to be of macrophagic origin. Positivity for cellular fibronectin, including the macrophages and related cells, appeared to be responsible for the accumulation of fibronectin on the surfaces of PC-IOLs. Prolyl 4-hydroxylase-positive cells were involved in the deposition of collagen on PC-IOLs. · Conclusion: Immunohistochemical study revealed that macrophages, foreign-body giant cells, and lens epithelial cells adhered to explanted PC-IOLs. Such adherent cells are responsible for the deposition of extracellular matrix on the surfaces of PC-IOLs and may regulate the assembly of the extracellular matrix, influencing the biocompatibility of PC-IOLs. [ABSTRACT FROM AUTHOR]

• Purpose: To examine the deposition of extracellullar matrix on silicone intraocular lenses (IOLs) implanted experimentally into rabbit eyes by electron microscopy and to determine the immunolocalization of extracellular matrix components, including collagen types and cellular fibronectin, on these IOLs. • Methods: We performed phacoemulsification and aspiration of the crystalline lens and implanted a foldable silicone IOL in the capsular bag of one eye of each of 26 adult albino rabbits under general anesthesia. After 8 weeks the animals were killed and the eyes were enucleated. The silicone IOLs were processed for electron microscopy and for immunohistochemical detection of collagen types I, III, and IV and cellular fibronectin. • Results: Electron microscopy revealed deposition of a presumed cell matrix complex on the optic portion of all silicone IOLs, as well as the adhesion of presumed macrophages and foreign-body giant cells. Cellular deposits showed immunoreactivity for cellular fibronectin. Fibrous or membranous deposits exhibited immunoreactivity for cellular fibronectin and collagen types I and III. A few type IV collagen-immunoreactive deposits were also seen. • Conclusion: Deposits of extracellular matrix components were observed on silicone IOLs. These deposits may form the scaffolding for the adhesion and proliferation of cells. These matrix components appeared to be the products of cells adhering to the surfaces of IOLs, including lens epithelial cells, macrophages and foreign-body giant cells, indicating that the process of granulation was incomplete. [ABSTRACT FROM AUTHOR]

• Background: We examined by transmission electron microscopy the accumulation of extracellular matrix on intraocular lenses (IOLs) implanted experimentally into rabbit eyes, and evaluated the immunolocalization of such extracellular matrix components as collagen types I, III, and IV, and cellular fibronectin on these IOLs. • Methods: Phacoemulsification and aspiration of the crystalline lens were performed and an IOL was implanted into the capsular bag of each eye of each of 16 adult albino rabbits under general anesthesia. After up to 12 weeks, the animals were killed and the IOLs were removed. Specimens were processed for transmission electron microscopy or for immunohistochemical detection collagen types I, III, and IV, and cellular fibronectin. • Results: Transmission electron microscopy revealed an accumulation of extracellular matrix between the residual anterior lens capsule and the surface of an IOL explanted 4 weeks after surgery. Collagen types I and III and cellular fibronectin were detected immunohistochemically on each IOL in association with cellular deposits. Type IV collagen-immunoreactive matrix was not seen on the optic portion, but was detected on the haptic portion of one of six IOLs examined. • Conclusion: Each component of the extracellular matrix that is deposited on the IOL supplies scaffolding for the adhesion and proliferation of cells. These components are considered to be produced by cells such as lens epithelial cells and macrophages that adhere to the IOL surface. [ABSTRACT FROM AUTHOR]

• Background: We evaluated ultrastructurally the presence and distribution of lens epithelial cells and extracellular matrix on the posterior capsule after implantation of a silicone or a polymethylmethacrylate (PMMA) intraocular lens (IOL) in rabbits. The immunolocalization of prolyl 4-hydroxylase, an enzyme involved in procollagen synthesis, was also examined in such cells. • Methods: Anesthetized adult albino rabbits ( n=12) underwent phacoemulsification and aspiration (PEA) of the crystalline lens and implantation of a silicone or a PMMA IOL into the capsular bag of one eye. They were killed by an intravenous overdose of pentobarbital sodium. The lens capsules were processed for transmission electron microscopy and for immunohistochemical detection of the β-subunit of prolyl 4-hydroxylase. • Results: Ultrastructurally, lens epithelial cells on the posterior capsule evidenced an accumulation of collagenous extracellular matrix. Prolyl 4-hydroxylase immunoreactivity was detected in the cytoplasm of cells distributed on the capsules. • Conclusion: Lens epithelial cells are involved in the production of collagenous matrix on lens capsules after PEA and implantation of IOL, resulting in a fibrotic process of the lens capsule. [ABSTRACT FROM AUTHOR]

• Background: The excessive accumulation of extracellular matrix (ECM) with the repopulation of fibroblasts may lead to an unsuccessful outcome of glaucoma filtering surgery. We examined the immunolocalization of ECM components and prolyl 4-hydroxylase, an enzyme involved in collagen biosynthesis, in cultured Tenon's capsule fibroblasts (TCFs) of humans to evaluate the production of ECM in the cells. • Methods: We used light microscopy to evaluate the immunolocalization of prolyl 4-hydroxylase and ECM components, collagen types I, III, and IV, cellular fibronectin, and laminin in TCFs. Ultrastructural localization of the enzyme was also evaluated by electron microscopy. • Results: Immunoreactivity with monoclonal antibodies against the α and β subunits of the enzyme or with the polyclonal antibody against it was detected in the cytoplasm of the cells in a fine granular pattern, indicating its localization in the indoplasmic reticulum (ER). Immunoreactivity for the enzyme was detected in the cisternae of the ER on electron microscopy. Types I and III collagen reactivities were also observed in the cytoplasm in a fine granular pattern. T reactivity was present diffusely on the cell surface. The distribution of laminin reactivity in the cytoplasm resembled that of types I and III collagen. Cellular fibronectin reactivity was observed in the ECM in a reticular pattern. • Conclusion: Prolyl 4-hydroxylase was located in the cisternae of the ER. TCFs produced a variety of ECM components in vitro. The results provide insight into the fibrotic process during scar formation at the site of a bleb following filtering surgery. [ABSTRACT FROM AUTHOR]

• Background: Minoxidil is an inhibitor of lysyl hydroxylase, an enzyme involved in collagen production, and decreases collagen production in vitro. We investigated the in vitro effects of minoxidil on behavior such as proliferation and migration of rabbit subconjunctival fibroblasts (SCFs). The ultrastructural effect of the drug on SCFs was also examined. • Methods: Proliferation of SCFs and closure of the defect produced in monolayer cultures in the presence or absence of minoxidil was studied. The ultrastructure of SCFs treated with minoxidil was also examined. • Results: Minoxidil inhibited SCF proliferation and the closure of the defect produced in monolayer cell sheets. Ultrastructural observations revealed extensive areas of irregularly dilated endoplasmic reticulum in cells treated with minoxidil, indicating the accumulation of protein, probably underhydroxylated collagen precursors, in the cisternae of endoplasmic reticulum. • Conclusions: The results indicated that minoxidil attenuated cellular activities of SCFs such as proliferation and migration in vitro. The exact mechanism of the inhibitory effects of minoxidil on these cellular activities is unknown. The findings suggest that the drug might help to prevent bleb scarring after glaucoma filtering surgery. [ABSTRACT FROM AUTHOR]

⊎ Background: The spreading of epithelium is critical in the healing of corneal wounds. Such epithelial spreading requires the continuous production of protein and glycoprotein. To determine whether collagen production is required for the spreading of corneal epithelium, we studied the effects of inhibitors for collagen production on spreading of corneal epithelium in vitro. ⊎ Methods: We examined the effect of two proline analogs, l-azetidine 2-carboxylic acid and cis-hydroxyproline, a prolyl hydroxylase inhibitor, ethyl-3,4-dihydroxybenzoate, and a lysyl hydroxylase inhibitor, minoxidil, on the spreading of epithelium of organ-cultured rabbit cornea. ⊎ Results: Both analogs and inhibitors inhibited epithelial spreading in a dose-dependent manner. ⊎ Conclusion: These observations indicate that collagen production may be involved in the spreading of corneal epithelium. [ABSTRACT FROM AUTHOR]

Three primary teeth from familial and sporadic X-linked hypophosphatemic vitamin D-resistant rickets, one permanent tooth from regional odontodysplasia, and three sound permanent teeth were prepared for ground sections of 50 μm thickness. To estimate the crystallinity of globular dentin for each ground section, microbeam X-ray diffraction analysis and transmission electron microscopic examination were performed. The crystallinity of globular dentin in any of the teeth examined was higher than that of normal dentin, and the size of hydroxyapatite crystals within the globular dentin was larger than that at normal dentin. These results clearly indicate that the globular dentin generally develops larger crystals, probably due to the presence of unmineralized spaces around the crystal nuclei such as interglobular dentin, resulting in the high crystallinity of dentin. [ABSTRACT FROM AUTHOR]