Biomedical subjects

H Shinohara

Publications and source records attributed to H Shinohara.

At least 19 recordsLinked to original sources

PubMed1992

Inhibition of in vitro ovarian cancer cell invasion by modulation of urokinase-type plasminogen activator and cathepsin B.

HOC-I ovarian cancer cells express the single-chain form of the urokinase-type plasminogen activator (uPA) and cathepsin B (cath B) on their cell surface. The significance of the expression of cell surface uPA/cath B activity to the invasive potential was examined by preincubating with uPA/cath B-modulating agents in in vitro invasion assay. The anti-uPA monoclonal antibody 394 effectively inhibited invasion in a dose-dependent manner. On the contrary, anti-cath B antibody did not affect the invasive potential of the cells. E-64, a specific inhibitor for cysteine proteases, blocked invasion as effectively as monoclonal antibody 394. The data reveal that the uPA and cysteine proteases contribute significantly to the invasive capacity of the cells. We suggest that the cysteine proteases facilitate the action of uPA, possibly by activating proenzyme uPA produced by cancer cells. Evidence for the role of a cathepsin-uPA activation cascade in HOC-I cell invasion is provided.

Cathepsin B↗

PubMed1992

Group II phospholipase A2 induced by interleukin-1 beta in cultured rat gingival fibroblasts.

Previously, we reported the presence of group II-like phospholipase A2 activity in the soluble fraction of rat gingiva. In the present study, we found that treatment of rat gingival cells with human recombinant interleukin-1 beta resulted in dose-dependent stimulation of intracellular and extracellular phospholipase A2 activity. Antisera against group II phospholipase A2 totally blocked the interleukin-1 beta-induced phospholipase A2 activity, but antisera against group I phospholipase A2 did not. Moreover, immunoblot analysis showed that the induced phospholipase A2 was group II phospholipase A2. These findings suggest that the induced enzyme belongs to the group II phospholipase A2 family of proinflammatory enzymes.

Animals↗

PubMed1992

Copurification of small heat shock protein with alpha B crystallin from human skeletal muscle.

Immunoreactive alpha B crystallin and a 28-kDa protein in an extract of human pectoral muscle were precipitated by (NH4)2SO4 at 40% saturation, and coeluted during column chromatography on DEAE-Sepharose and on Bio-Gel A-5m. The two proteins were separated on a column of S-Sepharose HP in the presence of 7 M urea. Further chromatography of each of the two resultant fractions on a column of Superdex 75 pg and on a TSK-SP 5PW column in the presence of urea yielded preparations of alpha B crystallin and the 28-kDa protein each of which gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The final preparation of 28-kDa protein contained at least two subtypes, which were separable on the TSK-SP column. However, fragmentation patterns of the two major 28-kDa proteins after digestion with endoproteinase Asp-N were identical. Amino acid sequences of peptides formed by cleavage of the purified 28-kDa protein and alpha B crystallin were identical to those of particular regions of the deduced amino acid sequences of human small heat shock protein (HSP28) and lens alpha B crystallin, respectively. Using an immunoassay method, with antibodies raised in rabbits, we found that HSP28 was present in all human tissues tested and at high levels (greater than 1 micrograms/mg protein) in the heart and other tissues composed of striated and smooth muscles. HSP28, found with alpha B crystallin, in extracts of several human and bovine tissues was trapped on and coeluted with alpha B crystallin from an affinity column prepared with antibodies against alpha B crystallin. This result suggests that the two proteins are associated in cells.

Amino Acid Sequence↗

PubMed1992

Translocation and induction of alpha B crystallin by heat shock in rat glioma (GA-1) cells.

Response to heat shock of alpha B crystallin expressed in rat astrocytoma GA-1 cells was analysed quantitatively using an immunoassay method. GA-1 cells contained a considerable amount of alpha B crystallin at growing phase. When the growing cells were heated at 45 degrees C, concentrations of alpha B crystallin in cell extracts were decreased to less than one-fifth of the original level within 15 min, with an increase in the insoluble fraction which was detected by immunoblotting. The low level of alpha B crystallin in the cytoplasm, that was observed for a few hours after heat shock, gradually recovered to the control level within several hours. At 10 h following heat shock (45 degrees C for 15 min), the concentration of alpha B crystallin in the soluble extract was about twice that of the control level, with little detectable amounts in the insoluble fraction. These results are additional evidence that suggest that alpha B crystallin is a small heat shock protein.

Animals↗

PubMed1992

Immunoreactive parvalbumin concentrations in parahippocampal gyrus decrease in patients with Alzheimer's disease.

By using a sensitive enzyme immunoassay system for rat parvalbumin, we determined parvalbumin contents in the 4 cerebrocortical regions (superior frontal gyrus of frontal lobe, parahippocampal gyrus of temporal lobe, superior parietal lobule of parietal lobe, and calcarine area of occipital lobe) of patients with Alzheimer's disease and age-matched controls. Among the 4 regions, concentrations of parvalbumin were the highest in calcarine area (68.6 +/- 6.7 ng/mg protein, rat parvalbumin equivalents, mean +/- SE) and the lowest in the parahippocampal gyrus (11.0 +/- 1.7 ng/mg protein) in the controls. A similar regional difference of the concentration was observed also in the patients with Alzheimer's disease. When compared with the controls, however, concentrations of parvalbumin in parahippocampal gyrus of patients with Alzheimer's disease (4.0 +/- 0.9 ng/mg protein) were significantly low (P less than 0.01), showing less than a half of the control values. In contrast, the concentrations in the 3 other regions showed little difference between Alzheimer's disease and the controls.

Alzheimer Disease↗

PubMed1992

Characterization and clinical evaluation of tumor-associated antigen CA54/61 identified by monoclonal antibodies MA54 and MA61 in epithelial ovarian cancer.

Monoclonal antibodies (moABs) MA54 and MA61, directed toward the O-linked mucin-type glycoprotein, have been established and showed highly specific reactivity with human ovarian cancer. Fetal intestinal and colonic mucosal cells expressed this antigen and meconium staining was also frequently positive. To investigate the characteristic of an epitopic carbohydrate recognized by these moABs, the reactivity of each moAB with meconium extract was monitored by solid-phase enzyme-linked immunosorbent assay with mono-, di-, and oligosaccharides. MA54 and MA61 react with meconium extract and the reactivities of these moABs are neuraminidase sensitive. Ovine submaxillary mucin had a strong inhibitory activity toward the reaction between meconium extract and MA54 as well as MA61, suggesting that these moABs recognize NeuAc 2-6GalNAc epitope in meconium. The second aim of this study is to investigate the possible application of moABs to diagnose ovarian cancer and to compare these levels with those of the CA125 antigen. While serum CA54/61 antigen levels were elevated in 44.4% of ovarian cancer cases and serum CA125 antigen levels were elevated in 86.7% of the same population, the use of both assays indicated a sensitivity of detection of 97.8% (44 of 45 patients) in the population studied.

Antibodies, Monoclonal↗

PubMed1992

Depressed CD4/CD8 ratio in TPHA-negative patients with syphilis.

The Treponema pallidum hemagglutination assay (TPHA) is a widely used method for screening syphilis. We report our experience with six elderly (age 72.4 +/- 8.3 years) patients with syphilis, whose TPHA was negative. Their cardiolipin (RPR) and absorbed fluorescence treponemal tests (FTA-ABS) were positive. TPHA-negative patients with syphilis were compared with TPHA-positive syphilitics by immunological analysis. We found that both the numbers and the percentage of CD4 cells in TPHA-negative syphilitics were significantly lower than those in TPHA-positive syphilitics (722 +/- 142 vs. 1,064 +/- 141/mm3, P less than 0.01: 35.1 +/- 6.9 vs. 48.4 +/- 6.4%, P less than 0.01) and that the ratio of CD4 to CD8 was also lower in TPHA-negative syphilitics compared with TPHA-positive syphilitics (1.08 +/- 0.46 vs. 2.24 +/- 1.07, P less than 0.05). These data suggest that the TPHA is insufficient for excluding elderly syphilitics because of immunological impairment seen in aged patients.

Adult↗

PubMed1992

Developmental and age-dependent changes of 28-kDa calbindin-D in the central nervous tissue determined with a sensitive immunoassay method.

For the quantitative analysis of vitamin D-dependent 28-kDa calcium-binding protein (calbindin-D) in the CNS, we have established a highly sensitive immunoassay method. The antisera were raised in rabbits with purified calbindin-D from rat kidneys, and the antibodies were purified with a calbindin-D-coupled Sepharose column. The purified antibodies were specific for calbindin-D, showing a single band on the immunoblot with the extract of rat kidney or cerebellum. The sandwich-type immunoassay system was prepared by the use of purified monospecific antibodies, and the minimum detection limit of the assay was 0.1 pg or 3.6 amol of calbindin-D, which was sufficiently sensitive for the measurement of calbindin-D content in isolated Purkinje cell bodies at the level of single cells. The average content of calbindin-D in a single Purkinje cell was 0.05 pg. Calbindin-D was detected in most of the rat tissues examined, but it was present predominantly in the kidney and CNS, especially in the cerebellum. Calbindin-D was detected at a similarly low level in the cerebral cortex, cerebellum, and brainstem of rat embryos of 15 gestational days, and it increased gradually but differently in these regions, reaching the respective adult levels by 4-5 weeks of postnatal age. In contrast, kidney calbindin-D increased sharply between 15 gestational days and 3 postnatal days, reaching the adult level by 6 days of age. Calbindin-D levels in the adult rat CNS were affected little by age, whereas the concentrations in human cerebral cortices were significantly low in the aged brain as compared with those in the young brain.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗

PubMed1992

Phospholipase A2 in rat gingival tissue.

Phospholipase A2 (PLA2) is a proinflammatory enzyme in the synovial fluids of all--and sera of some--patients with rheumatoid arthritis. Due to the similarities in pathogenesis between rheumatoid arthritis and periodontitis, we sought to study the enzymatic properties of PLA2 in periodontal tissue. In this study, we demonstrated PLA2 activity in rat gingival tissue, about 80% of which was present in the cytosolic fraction. We characterized the cytosolic PLA2 enzyme with respect to substrate specificity, sensitivity to detergent, Ca2+ ion dependency and optimum pH. We found that phosphatidylethanolamine, rather than phosphatidylcholine, was the preferred substrate, the Ca2+ ion was essential for the expression of PLA2 activity, the enzyme was active over a broad pH range, with the optimum at pH 9.0, and sodium-deoxycholate inhibited the enzyme activity strongly in a concentration-dependent manner. These results are consistent with those which have been obtained with synovial fluid PLA2 and suggest that gingival PLA2 may be involved in the pathogenic processes of gingivitis and periodontitis.

Analysis of Variance↗

PubMed1992

Differential localization of G proteins, Gi and Go, in the olfactory epithelium and the main olfactory bulb of the rat.

The immunohistochemical localization of proteins Gi1 (plus Gi3). Gi2 and Go was studied in the olfactory epithelium and the main olfactory bulb of rats, using purified antibodies to the respective alpha subunits and beta gamma subunits of these G proteins. In the olfactory epithelium, only a restricted population of olfactory cells was immunopositive for Gi2 alpha, but others were not. The immunoreactivity for Gi1 alpha/Gi3 alpha was not observed. The olfactory epithelium was immunopositive for both Go alpha and beta gamma, but its apical surface was immunopositive only for beta gamma. In the main olfactory bulb, all layers were intensely immunopositive for Go alpha and beta gamma but weakly for Gi2 alpha. In contrast to the negative or weak immunostainings in the olfactory nerve fiber layer and glomeruli, the molecular and the internal granular layers were intensely immunopositive for Gi1 alpha/Gi3 alpha. These findings suggest the functional difference among Gi1/Gi3, Gi2 and Go in the signal transduction in the olfactory system.

Animals↗

PubMed1992

Immunohistochemical study of carbonic anhydrase III in the extraocular muscles of human embryos.

The differentiation of extraocular muscles was studied immunohistochemically in externally normal human embryos (Carnegie stages 13-23), using antibodies to carbonic anhydrase (CA) III and beta-enolase as the markers of type 1 and type 2 muscle fibers, respectively. At stage 18, some myoblasts were immunoreactive to beta-enolase antibodies, however, CA-III immunoreactivity was not observed around the optic vesicle. At stage 20, CA-III immunoreactivity appeared in some muscle fibers of extraocular muscles. From stage 21 to stage 23, CA-III-immunoreactive fibers increased and almost equalled the number of beta-enolase-immunoreactive fibers. These findings suggest that CA-III-immunoreactive type 1 fibers appear in the late stage of myogenesis compared with beta-enolase-immunoreactive type 2 fibers.

Carbonic Anhydrases↗

PubMed1992

Heat induced conformational changes generate mitogenicity to splenocytes by sclerogen from Sclerotinia sclerotiorum IFO 9395.

The fungal mitogen, sclerogen, obtained from sclerotia of Sclerotinia sclerotiorum IFO 9395 showed significant mitogenic activity to murine splenocytes after heat denaturation in relation to polymerization. To evaluate the conditions generating mitogenicity, we performed several chromatographic and spectral analyses. After heat denaturation of sclerogen, significant reduction of intrinsic fluorescence, significant changes on the ultraviolet absorption spectrum, significant changes on the circular dichroism spectrum, and an extreme change of the surface charge to anionic, were observed. These results strongly suggested that local as well as overall conformational changes of sclerogen associated with a high molecular mass and polyanionic charges are important in generating mitogenicity to murine splenocytes.

Animals↗

PubMed1992

Decrease of lactase activity in the small intestine of jejunum-bypassed rats.

The effect of jejunum-bypass operation on lactase in rat small intestine was examined. Three groups of four or five rats were designated as jejunum-bypassed, sham-operated and normal rats. All animals including normal rats received by pair-feeding 5% glucose/1% NaCl for 5 days following the operation; thereafter they were fed ad libitum the laboratory chow diet. Three weeks after the jejunal bypass operation, the proximal ileum exhibited a hyperplasia as evidenced by a concomitant increase in mucosal contents of both total proteins and DNA. The specific activity of lactase in this segment was significantly lower in the operated rats than sham-operated controls, whereas the specific activity of sucrase in this segment was significantly elevated. The reduction of lactase activity was also evident in the proximal jejunal segment as well as in the distal jejunum which was deprived of luminal nutrition, suggesting that some hormonal factor(s) might be involved in the decrease of lactase activity in jejunum-bypassed animals. Electroimmunoassay revealed that the amount of immunoreactive lactase also declined in the operated rats relative to the sham-operated controls. Our results thus suggest that lactase activity in residual ileum is not only unable to compensate for the loss of digestive-absorptive surface of jejunum, but lactase activity even decreases following jejunum-bypass operation.

Animals↗

PubMed1992

Differential localization of G-proteins Gi and Go in the accessory olfactory bulb of the rat.

To clarify the functional differences among G-proteins, we investigated the localization of Gi and Go in the olfactory bulb of rats by both immunohistochemical and immunochemical techniques, using purified antibodies specific to the alpha-subunits of Gi1 (Gi1 alpha), Gi2 (Gi2 alpha), and Go (Go alpha), respectively. We found that Gi2 alpha is localized exclusively in the accessory olfactory bulb, but it is present at only low levels in the main olfactory bulb. The unique pattern of immunoreactivity specific for Gi2 alpha and Go alpha within the glomeruli of the accessory olfactory bulb and the results of immunoassays indicate that the accessory olfactory bulb is divided into two parts: the anterior region is rich in Gi2, while the posterior region is rich in Go. These findings suggest that the accessory olfactory bulb has two different functions. In addition, we found that the concentration of Gi2 alpha in the accessory olfactory bulb increases during puberty and reaches the adult level at 12 weeks after birth, while that in the main olfactory bulb remains constant. By contrast, the concentrations of Go alpha in the accessory olfactory bulb and the main olfactory bulb increase with similar kinetics. These findings suggest that Gi2 is a key protein in signal transduction in the accessory olfactory bulb, and increases in its level seem to be related to sexual maturation.

Animals↗

PubMed1991

Immunoreactive alpha A crystallin in rat non-lenticular tissues detected with a sensitive immunoassay method.

For the quantitative analysis of the A subunit of alpha crystallin (alpha A) in the lens and for the survey of possible existence of alpha A in the non-lenticular tissues, we have established a highly sensitive and specific immunoassay method for alpha A. Antisera to alpha A were raised in rabbits with alpha A purified from bovine lens, or the C-terminal decapeptide (EEKPSSAPSS) of alpha A (alpha Apep). The antibodies to alpha A and alpha Apep were purified by the use of an alpha A-coupled Sepharose 4B column. The F(ab')2 fragments of purified anti-alpha A IgG were immobilized on polystyrene balls and the Fab' fragments of purified anti-alpha Apep IgG were labeled with beta-D-galactosidase from Escherichia coli. The minimum detection limit of the sandwich-type immunoassay using the two antibody preparations was less than 10 pg alpha A without any cross-reactivity with alpha B. By employing the present methods, it was found that a significant amount of immunoreactive alpha A was present in rat spleen and thymus. Very low levels of immunoreactive alpha A were detected in the rectum, caecum, liver, kidney, adrenal, cerebellum and brainstem. The immunoreactive alpha A in the spleen extract was purified partially (about 50% purity) by the use of anti-alpha Apep-coupled Sepharose. The concentration of alpha A in the spleen was less than 1 ng/mg protein before 3 weeks of age. After 5 weeks of age, however, it increased lineally reaching about 20 ng/mg protein by 18 weeks of age. Immunohistochemically, the alpha A was localized in the reticular cells in the spleen and thymus.

Animals↗

PubMed1991

Increment of alpha B-crystallin mRNA in the brain of patient with infantile type Alexander's disease.

To estimate the expression level of alpha B-crystallin in the brain of infantile type Alexander's disease, the amounts of protein and mRNA of alpha B-crystallin were measured by enzyme immunoassay (EIA) and Northern blot analysis, respectively, in the brain of patient and controls, and in the tissues from glioblastoma and astrocytoma. The alpha B-crystallin protein in the brain of patient was remarkably increased as compared with those of controls. The amount of alpha B-crystallin mRNA of patient was increased about 7-fold compared to the mean value of the control group and higher than that of glioblastoma tissue. These data suggest that increment of alpha B-crystallin mRNA in astrocytes leads to the overexpression of this protein and may be one of the main causes of infantile type Alexander's disease.

Adolescent↗

PubMed1991

Sensitive immunoassay for rat parvalbumin: tissue distribution and developmental changes.

A sensitive enzyme immunoassay for measurements of rat parvalbumin was established using antibodies raised in rabbits with parvalbumin purified from skeletal muscles. Antibodies in the antiserum were purified with a parvalbumin-coupled Sepharose column. The sandwich-type immunoassay system for parvalbumin was composed of polystyrene balls with immobilized purified antibodies and the same antibodies labeled with beta-D-galactosidase from Escherichia coli. The assay was highly sensitive and the minimum detection limit was 1 pg parvalbumin/tube. The assay did not cross-react with other calcium binding proteins, including human S-100a0 and S-100b proteins, rat 28-kDa calbindin-D, and bovine calmodulin. High concentrations of parvalbumin were observed in the skeletal muscles, especially in those composed of fast-twitch fibers, and in the diaphragm and tongue, but not in heart muscle. A relatively high concentration was estimated in the central nervous tissue. Parvalbumin was detected in the cerebral cortex and cerebellum of gestational 15-day fetuses. However, the levels of parvalbumin in the muscle tissues and central nervous tissue were very low in rats before 1 week of age. Thereafter, they increased sharply, reaching the adult levels by 5 weeks in most of the tissues. Parvalbumin concentrations in adult rat soleus muscle increased less than 20-fold within 10 days after transection of the ipsilateral sciatic nerve, while the concentrations in the extensor digitorum longus muscle did not change in the same period.

Animals↗

PubMed1991

Histochemically demonstrable phosphotyrosine protein phosphatase in the rat hippocampal formation.

Using o-phospho-L-tyrosine as substrate, a possible localization of phosphotyrosine protein phosphatase (PTPPase) activity was histochemically demonstrated in the rat hippocampal formation. The PTPPase activity was found in almost all layers of the hippocampal formation, with a high activity in the stratum moleculare. The activity was inhibited by vanadate and molybdate, but not by NaF and Zn2+. The activity was localized in the dendritic cytoplasm, particularly on the postsynaptic density, of hippocampal neurons.

Animals↗