Autoimmune myocarditis induced in mice by cardiac C-protein. Cloning of complementary DNA encoding murine cardiac C-protein and partial characterization of the antigenic peptides.

Autoimmune myocarditis is considered to play a major role in the pathogenesis of dilated cardiomyopathy. A new autoimmune myocarditis model was attained by repeated immunization using murine cardiac C-protein with the immunological adjuvant, Klebsiella pneumoniae O3 lipopolysaccharide. For further analysis of a pathological epitope, the cDNA encoding C-protein was isolated; a fusion protein encoded by part of this cDNA induced myocarditis in SMA mice as well as in three other strains: DBA/1J (H-2q), O20/A (H-2pz1), and SJL (H-2s). The nucleotide sequence and its deduced amino acid analysis revealed that this protein had immunoglobulin-like and fibronectin-like repeats. This study provides a new animal model of autoimmune myocarditis which may shed light on the pathogenesis of dilated cardiomyopathy.

Bacterial polysaccharide synthesis and gene nomenclature.

Gene nomenclature for bacterial surface polysaccharides is complicated by the large number of structures and genes. We propose a scheme applicable to all species that distinguishes different classes of genes, provides a single name for all genes of a given function and greatly facilitates comparative studies.

Pulse fluorimetry of N-(1-pyrenesulfonyl)dipalmitoyl-L-alpha-phosphatidylethanolamine in concanavalin A-stimulated human lymphocytes.

Human peripheral lymphocytes were cultured with a fluorescent probe, N-(1-pyrenesulfonyl)dipalmitoyl-L-alpha-phosphatidylethanolamine, and with concanavalin A. Fluorescence microscopic observations revealed that in lymphoblasts, pyrenesulfonyl dye was distributed mainly in vacuoles whereas in normal cells cultured without concanavalin A the dye was distributed exclusively in plasma membranes. The fluorescence spectra of the pyrenesulfonyl group incorporated into the cells exhibited two emission maxima, band A (monomer fluorescence of the pyrenesulfonyl group at about 400 nm) and band B (dimer fluorescence of the dye at about 500 nm). The values of the fluorescence lifetime measured at bands A and B indicated that in the absence of concanavalin A, the environment surrounding the pyrenesulfonyl group at the lipid/water interface became more hydrophilic with cultivation time. Concanavalin A made the environment of the interface more hydrophobic than that of lymphocytes cultured without concanavalin A. Fluorescence polarization measured at band A revealed that the mobility of pyrenesulfonyl monomers at the aqueous interface of the membranes was reduced upon concanavalin A stimulation.

Evolutionary relationship among rfb gene clusters synthesizing mannose homopolymer as O-specific polysaccharides in Escherichia coli and Klebsiella.

In order to clarify the evolutionary relationship among rfb gene clusters synthesizing mannose homopolymer as O-specific polysaccharides in Escherichia coli and Klebsiella, we studied the DNA sequence of the boundary region between the rfb and his genes in a series of strains possessing mannose homopolymer as O-specific polysaccharide. All had a characteristic gene organization carrying no gene between the rfb and his genes. Further, the recombination event was suggested to occur at the same site of the hisI gene in those strains. It was suggested that there was a close evolutionary relationship among rfb gene clusters synthesizing mannose homopolymer as O-specific polysaccharide in E. coli and Klebsiella.

Close evolutionary relationship between the chromosomally encoded beta-lactamase gene of Klebsiella pneumoniae and the TEM beta-lactamase gene mediated by R plasmids.

Sixty-three percent homology of nucleotide sequence and 67% homology of deduced amino acid sequence were found between the chromosomally encoded beta-lactamase gene of Klebsiella pneumoniae and the TEM beta-lactamase of transposon Tn3. Moreover, 22 out of 24 amino acid residues are identical around the predicted active site. It is therefore suggested that these two kinds of beta-lactamases share a common evolutionary origin. The 0.5 kb DNA fragment of the cloned gene hybridized specifically with the chromosomal DNA of all the K. pneumoniae strains tested which had been isolated in Japan, USA and Europe.

Expression of ciliary neurotrophic factor activated by retinal Müller cells in eyes with NMDA- and kainic acid-induced neuronal death.

PURPOSE: To elucidate the role of retinal Muller cells in N-methyl-D-aspartate (NMDA)- or kainic acid (KA)induced retinal damage. METHODS: In experimental eyes, NMDA or KA was injected into the vitreous of rat eyes. Immunohistochemistry and western blot analysis were conducted to elucidate expression and localization of glial fibrillary acidic protein (GFAP) and ciliary neurotrophic factor (CNTF). In addition, the neuroprotective effects of CNTF were calculated by counting cells in the ganglion cell layer (GCL) and by measuring the thickness of the various retinal layers. RESULTS: Morphometric analysis of retinal damage in NMDA- and KA-injected eyes showed significant cell loss in the GCL and thinning of the inner plexiform layer (IPL) of the retina, but not of other retinal layers. Immunohistochemistry demonstrated disappearance and/or decrease in immunoreactivities of calbindin- and calretinin- positive cells and their neurites and upregulated expression of both GFAP and CNTF in experimental eyes. Western blot analysis showed an increase in protein expression for CNTF in retinas of experimental eyes. Confocal images and sequential localization demonstrated colocalization of CNTF and GFAP in the inner retinal layer and possibly in Muller cells. In addition, pretreatment with CNTF (1 microg) before the intravitreal injection of NMDA (or KA) demonstrated that CNTF has neuroprotective effects against NMDA- or KA-induced neuronal death in the retina. CONCLUSIONS: These studies revealed the upregulated expression of CNTF and GFAP in Muller cells in response to NMDA- and KA-induced neuronal death, suggesting that production of CNTF in Muller cells may be a part of the endogenous neuroprotective system in the retina.

Neuroglycan C, a neural tissue-specific transmembrane chondroitin sulfate proteoglycan, in retinal neural network formation.

PURPOSE: Neuroglycan C (NGC) is a transmembrane chondroitin sulfate proteoglycan present exclusively in central nervous system tissues. In the current study the expression pattern and characterization of NGC during the development of the retina were investigated. METHODS: Expressional changes of NGC mRNAs during rat retinal development were examined by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). The localization and characterization of NGC core proteins were investigated by immunoblot analysis and immunohistochemistry using an anti-NGC antibody. RESULTS: Immunohistochemical analysis revealed that NGC was highly expressed in the nerve fiber layer (NFL) and inner plexiform layer (IPL) in rat postnatal developing retina. At embryonal stages, NGC immunoreactivities were faint. In contrast, at postnatal developmental stages (approximately postnatal day [P]7), intense immunoreactivity was observed in the NFL and IPL, where active dendrite branching was observed, and conventional synapses began to be formed. As retinal layer differentiation proceeded (from P14 to P42), immunoreactivities in the inner retinal layers gradually became fainter. Immunoblot and semiquantitative RT-PCR analyses showed that the peak level of NGC expression occurred on approximately P7 and P14. Glycosylation of the NGC core protein changed as the retinal layers matured. In immunoelectron microscopic analysis, NGC immunoreactivity was located on the axonal membranes of neuronal cells in the postnatal retina, whereas immunoreactivity was reduced on membranes at the adult stage. In retinal ganglion cells in vitro, NGC was highly localized in their spiny budding neurites. CONCLUSIONS: The results show spatiotemporal expression patterns of NGC, and suggest that it plays a role in the formation of neural networks in retinal development.

Spatiotemporal expression patterns of 6B4 proteoglycan/phosphacan in the developing rat retina.

PURPOSE: To investigate expression of 6B4 proteoglycan/phosphacan, the major constituent of chondroitin sulfate proteoglycan and a possible modulator of neural network formation in the developing central nervous system, in developing rat retina. METHODS: Changes in expression and localization of 6B4 proteoglycan in developing rat retina were investigated by reverse transcription-initiated polymerase chain reaction (RT-PCR), immunohistochemistry, and immunoblot analysis. RESULTS: Semiquantitative RT-PCR revealed that mRNA expression of 6B4 proteoglycan in retinas peaked at postnatal day 14 (P14) and then decreased at P42. Immunohistochemical analyses using MAb 6B4, a monoclonal antibody against 6B4 proteoglycan, revealed faint immunoreactivity in the inner aspects of the retina at embryonal day 16 (E16). At birth, weak immunoreactivity was present in the nerve fiber layer (NFL) and inner plexiform layer (IPL). At P7 and P14, the NFL, IPL, and outer plexiform layer (OPL) stained intensely, but the ganglion cell layer (GCL) remained unstained. Between P21 and P42, immunoreactivity in the NFL and IPL weakened slightly. Immunoblot analyses showed a MAb 6B4 immunopositive band in the retinal soluble fraction treated with chondroitinase ABC. The amount of the immunopositive band increased rapidly as retinal development proceeded. Surprisingly, a significant amount of the immunopositive band was present in the retina even before digestion with chondroitinase ABC, indicating that at least part of 6B4 proteoglycan in rat retina exists in a non-proteoglycan form. CONCLUSIONS: The existence of 6B4 proteoglycan/phosphacan was thus demonstrated in rat retina, although some biochemical parameters were different from those of the 6B4 proteoglycan seen in brain.

Inhibitory effects of neurocan and phosphacan on neurite outgrowth from retinal ganglion cells in culture.

PURPOSE: Neurocan and phosphacan are nervous tissue-specific chondroitin sulfate proteoglycans (CSPGs) that are highly expressed in postnatal rat retina. To elucidate potential roles of neurocan and phosphacan on neurite outgrowth from retinal ganglion cells (RGCs), in vitro experiments were conducted with purified RGCs. METHODS: Neurocan and phosphacan were purified from postnatal rat brain by DEAE-column chromatography and subsequent gel chromatography. RGCs were obtained from postnatal rat retinas by a two-step immunopanning procedure using an anti-Thy 1,1 antibody and an anti-macrophage antibody. Neurite outgrowth from RGCs was examined on poly-L-lysine (PLL)-conditioned plates, and PLL-conditioned plates treated with neurocan or phosphacan. RESULTS: Compared with PLL-conditioned plates, neurocan and phosphacan inhibited neurite outgrowth from RGCs at 48 and 72 hours after seeding. When chondroitin sulfate side chains linked to the core proteins were digested by chondroitinase ABC, the inhibitory effect remained, indicating that the core proteins are related to the effect. Furthermore, the digestion of chondroitin sulfate side chains linked to phosphacan core protein significantly promoted the inhibitory effect of phosphacan on neurite outgrowth from RGCs. CONCLUSIONS: Neurocan and phosphacan, which are highly expressed in postnatal rat retina, inhibit neurite outgrowth from postnatal rat RGCs, indicating that these proteoglycans may be inhibitory factors against neurite outgrowth from RGCs during retinal development.

Upregulated expression of neurocan, a nervous tissue specific proteoglycan, in transient retinal ischemia.

PURPOSE: Neurocan, a nervous tissue-specific chondroitin sulfate proteoglycan synthesized primarily by neurons, is expressed abundantly in developing rat retina, whereas it is rarely expressed in adult rat retinas. This study investigated the reexpression of neurocan in a pathologic condition of adult rat retina. METHODS: Transient retinal ischemia was produced by occlusion of the retinal artery for 60 minutes. After transient retinal ischemia, neurocan expression was investigated by reverse transcription-initiated polymerase chain reaction (RT-PCR), immunohistochemistry, and immunoblot analysis. RESULTS: Semiquantitative analysis using RT-PCR revealed that mRNA expression for neurocan increased at 24 hours after reperfusion. Furthermore, on immunoblot analysis using an anti-neurocan antibody, MAb 1G2, the intensity of the 220-kDa band as well as the 150-kDa band increased markedly at 24 and 72 hours after reperfusion. The 220-kDa band was predominant at 24 hours after reperfusion, whereas the intensity of the 150-kDa band became almost the same as that of the 220-kDa band at 72 hours after reperfusion. Immunohistochemical analysis revealed that upregulated neurocan immunoreactivity was associated with glial Müller cells. CONCLUSIONS: Thus, upregulated expression of neurocan in transient retinal ischemia was demonstrated. Furthermore, the immunohistochemical analysis revealed that the upregulated expression of neurocan is derived from Müller cells, although it has been thought that neurocan is synthesized by neurons so far. The neurocan expression by Müller cells suggests that this proteoglycan plays a role in the damage and repair processes in diseased retina.

Effects of rho-associated protein kinase inhibitor Y-27632 on intraocular pressure and outflow facility.

PURPOSE: To elucidate the roles of Rho-associated protein kinase (ROCK) in regulating intraocular pressure (IOP) and outflow facility in the rabbit eye. METHODS: A specific ROCK inhibitor Y-27632 was used. The IOP, the outflow facility, and the pupil diameter were determined before and after the topical, intracameral, or intravitreal administration of Y-27632 in rabbits. Western blot analysis was used to identify specific ROCK isoform in human trabecular meshwork (TM) cells and bovine ciliary muscle (CM) tissues. The cell morphology and distribution of actin filaments and vinculin in TM cells were studied by cell biology techniques. Carbachol (Cch)-induced contraction of isolated bovine CM strips after administration of Y-27632 was measured in a perfusion chamber. RESULTS: In rabbit eyes, administration of Y-27632 resulted in a significant decrease in IOP in a dose-dependent manner. An increase of the outflow facility and pupil size dilation was also observed in Y-27632-treated eyes. Western blot analysis revealed the presence of p160ROCK in human TM cells and bovine CM tissues. In cultured human TM cells, exposure to Y-27632 caused retraction and rounding of cell bodies as well as disruption of actin bundles and impairment of focal adhesion formation. Y-27632 in addition inhibited Cch-induced contraction of isolated bovine CM strips. CONCLUSIONS: Administration of Y-27632 caused a reduction in IOP and an increase in the outflow facility. The in vitro experiments suggest that the IOP-lowering effects of Y-27632 may be related to the altered cellular behavior of TM cells and relaxation of CM contraction. These studies suggest that ROCK inhibitors may have great potential to be developed for treatment of glaucoma and other ocular diseases.

Intact and subcellular form of thymocytes of rats are exceptionally immunogenic in mice for inducing anti-Thy-1 T cell-independent class 2 antibody responses.

Results of the present study show that the primary anti-Thy-1.1 antibody response to rat antigen in Thy-1.2 mice is induced exclusively by thymocyte antigen. Thy-1 antigens of brain and bone marrow, which expressed much Thy-1 antigen, were poorly immunogenic if at all. Brain Thy-1 antigen considerably inhibited the immunogenicity of thymocyte Thy-1. Moreover, we found that subcellular form of thymocytes induce as high antibody responses as intact thymocytes do. The subcellular thymocyte Thy-1 antigen behaved as TI-2 antigen, inducing a good response in athymic nude mice but not in CBA/N mice with a B cell defect. The significance of these findings is discussed in relation to the possible physiological activity of Thy-1 or Thy-1-linked molecules on thymocytes specifically mediating lymphocyte differentiation.

Experimental murine model for autoimmune myocarditis using Klebsiella pneumoniae O3 lipopolysaccharide as a potent immunological adjuvant.

Experimental autoimmune myocarditis could be produced in mice by repeated injection of syngeneic heart extract together with Klebsiella pneumoniae O3 lipopolysaccharide (KO3 LPS) as a powerful adjuvant. Histological changes in the cardiac lesions were characterized by infiltration with mononuclear cells in the myocardium, degeneration and loss of myocardial fibers, and replacement of granulation tissues. No such cardiac lesions were produced in mice receiving injections of heart extract alone or KO3 LPS alone. Development of the autoantibody and the delayed type-hypersensitivity (DTH) against syngeneic heart extract was found in mice immunized repeatedly with the mixture of heart extract and KO3 LPS. Moreover, definite cardiac lesions were produced in normal recipient mice by transfer of sensitized spleen cells from hyperimmunized mice. Therefore, it was suggested that those cardiac lesions were caused by the autoimmune mechanism. Our methodology provided a new experimental murine model for autoimmune myocarditis.

Effects of protein kinase inhibitor, HA1077, on intraocular pressure and outflow facility in rabbit eyes.

OBJECTIVE: To elucidate the roles of protein kinase in regulating the intraocular pressure (IOP) and outflow facility in rabbit eyes. MATERIALS AND METHODS: A protein kinase inhibitor, 1-(5-isoquinolinesulfonyl)-homopiperazine (HA1077), was used. The IOP and the outflow facility were measured before and after topical, intracameral, or intravitreal administration of HA1077 in rabbits. Western blot analysis was performed to detect the 20-kd light chain of myosin in human trabecular meshwork (TM) cells and bovine ciliary muscle (CM) tissues. The cell morphologic condition and distribution of actin filaments and vinculin in TM cells were studied using cell biology techniques. Carbachol-induced contraction of isolated bovine CM strips following administration of HA1077 was examined in a perfusion chamber. RESULTS: In rabbit eyes, the administration of HA1077 resulted in a significant decrease in IOP in a dose-dependent manner. An increased outflow facility was also observed. Western blot analysis revealed the presence of 20-kd light chain of myosin in human TM cells and bovine CM tissues. In cultured human TM cells, exposure to HA1077 disrupted actin bundles and impaired focal adhesion formation. In addition HA1077 showed relaxation of bovine CM strips. CONCLUSIONS: Use of HA1077 caused a reduction in IOP and an increase in the outflow facility. The results of in vitro experiments suggest that the IOP-lowering effects of HA1077 may be related to the altered cellular behavior of TM cells and relaxation of CM contraction. The results of these studies suggested that protein kinase inhibitors have the potential to be developed into a treatment modality for glaucoma.

Identification of the hemidesmosomal 500 kDa protein (HD1) as plectin.

HD1 is a 500 kDa hemidesmosomal plaque protein recognized by monoclonal antibody mAb-121. Recent research on inherited skin disease has suggested that it might be identical to plectin or an isoform. To cast light on this question, we have prepared several monoclonal antibodies that recognize a 500 kDa protein in the hemidesmosome fraction. Unexpectedly, some staining pattern heterogeneity was observed on immunofluorescence microscopy. Attention was focused on two monoclonal antibodies which gave different localization in bovine skin and retinal pigment epithelial cells. Determination of the amino-terminal sequence of an antigenic 100 kDa polypeptide fragment derived from the 500 kDa component of an insoluble fraction of bovine hepatocytes revealed it was identical to that of plectin. Using the two antibodies, we screened a cDNA library derived from BMGE+H, a bovine mammary gland epithelial cell line. The isolated cDNA clones corresponded to the rod domain of bovine plectin, with two separate epitope regions for each of the antibodies. From these results we conclude that the hemidesmosomal 500 kDa component HD1 is identical to plectin. As judged on rough estimation of molar ratios on this basis, hemidesmosomes are composed of plectin, BP230, the integrin beta4 subunit, and alpha6 in a 1:1:1:1 ratio.

A unique DNA sequence of human enterotoxigenic Escherichia coli enterotoxin encoded by chromosomal DNA.

We detected Ent plasmids in 300 strains of human enterotoxigenic Escherichia coli, but one strain, E. coli 240-3, had neither a small nor a large plasmid and encoded the heat-labile enterotoxin (LTh(240-3)) gene on its chromosome. DNA sequences showed that LTh(240-3) differed by 12 and 14 base pairs from LT (LTh) and LT (LTp) from human H10407 and porcine EWD299 strains, respectively. In deduced precursor toxins, LTh(240-3), LTh and LTp differed from LTh, LTp and LTh(240-3) at nine, eight and eleven positions, respectively. These data suggest that although LTh(240-3) encoded in the chromosome is antigenically similar to LTh, it cannot be grouped with LTh due to differences in its DNA and amino acids sequences.

Somatostatin and GABA correlate with cervical autonomic nerve activity.

The mode of inhibitory action of centrally administered SRIF on the efferent activity of autonomic nerves was investigated in the rat by assessing the SRIF-induced change in the activity of the superior laryngeal nerve with or without pretreatment with various drugs. After picrotoxin or bicuculline treatment, the inhibition of nerve activity by SRIF was abolished while reserpine and atropine failed to abolish the SRIF effect. The centrally administered GABA inhibited the activity of the superior laryngeal nerve and the cervical sympathetic trunk. However, SRIF did not affect the sympathetic trunk. Arterial blood pressure was increased by SRIF while GABA produced hypotension. These data provide evidence for a GABAergic system as the mediator of SRIF action in the brain and for the selectivity of SRIF action on the particular intermediary GABAergic neurones.

Neuroprotective effects of brain-derived neurotrophic factor in eyes with NMDA-induced neuronal death.

PURPOSE: To determine if brain-derived neurotrophic factor (BDNF) has a neuroprotective effect against N-methyl-D-aspartate (NMDA)-induced cell death in retina. METHODS: NMDA was injected into the vitreous of rat eyes. NMDA-induced neuronal death was measured by morphometric analyses on cell counts of ganglion cell layer cells and thickness of retinal layers. Also, we conducted additional experiment using retrograde labeling with a fluorescent tracer (Fluoro-Gold) for exact counting of retinal ganglion cells (RGCs). In addition, intravitreal glutamate levels were measured with the use of a high-performance liquid chromatography (HPLC) system. RESULTS: Morphometric analysis of retinal damage in NMDA-injected eyes showed that BDNF could protect inner retinal cells from glutamate receptor-mediated neuronal death. Also, counts of RGCs labeled with a fluorescent tracer showed that BDNF could protect RGCs from glutamate receptor-mediated neuronal death. Furthermore, measurements of intravitreal glutamate levels indicated an increase in this excitatory amino acid in the vitreous after NMDA injection. CONCLUSIONS: Exogenous BDNF can protect inner retinal cells (possible RGCs and amacrine cells) from NMDA-induced neuronal death. However, increased intravitreal glutamate levels in response to NMDA-mediated neurotoxicity may augment retinal degeneration.

Dual effects of interleukin-1beta on N-methyl-D-aspartate-induced retinal neuronal death in rat eyes.

In this study we determine if interleukin-1beta (IL-1beta) modulates N-methyl-D-aspartate (NMDA)-induced retinal damage. Sprague-Dawley rats were anesthetized with inhalation of halothane, after which a single injection of 5 microl of IL-1beta (0.1 to 10 ng/eye) (and/or IL-1 receptor antagonist (IL-1ra)) for experimental eyes was administered. Two days later (or simultaneously), NMDA (20 nmol) was injected into the vitreous space. One week later, each eye was enucleated and transverse sections were subjected to morphometric analysis. Enzyme-linked immunosorbent assay (ELISA) was conducted for the determination of IL-1beta levels in retina. Immunohistochemical and immunoblot studies were also performed. In eyes that received an intravitreal injection of IL-1beta (0.1 to 10 ng/eye), significant thinning of the inner plexiform layer (IPL) was observed (P<0.05). Immunohistochemical and ELISA studies demonstrated upregulated expression of IL-1beta in retinas that had undergone NMDA injection. Treatment with 10 ng of IL-1ra induced a protective effect against NMDA-induced retinal damage. Pretreatment with IL-1beta induced a significant protective effect on NMDA-induced retinal damage. Our studies suggest that IL-1beta induces neuronal cell death directly, as shown by the protective effects of IL-1ra, but has a protective effect on NMDA-induced retinal damage indirectly after an incubation time of at least 2 days.

Upregulated expression of N-syndecan, a transmembrane heparan sulfate proteoglycan, in differentiated neural stem cells.

Adult rat hippocampus-derived neural stem cells are incorporated into neural tissues, and differentiate to neuronal and glial cells. However, the cell surface protein molecules are, to date, undefined. RT-PCR, immunoblotting and immunocytochemistry showed the increased expression of N-syndecan, a transmembrane heparan sulfate proteoglycan, in the neural stem cells after the differentiation induced by retinoic acid. Our data indicate that N-syndecan may be involved in the differentiation of neural stem cells.