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R Semba

Publications and source records attributed to R Semba.

At least 73 records · Page 4Linked to original sources
PubMed1987

Highly sensitive immunoassay for the alpha subunit of the GTP-binding protein go and its regional distribution in bovine brain.

Antisera were raised in rabbits against the alpha subunit of a GTP-binding protein, Go. Because the antisera cross-reacted weakly with the alpha subunit of inhibitory GTP-binding protein of adenylate cyclase (Gi), they were purified with a Go alpha-coupled Sepharose column. Purified antibodies reacted only with Go alpha and did not cross-react with the Gi alpha subunit or beta gamma subunits in an immunoblot assay. Using these purified antibodies, a highly sensitive enzyme immunoassay method for the quantification of bovine brain Go alpha was developed. The assay system consisted of polystyrene balls with immobilized antibody F(ab')2 fragments and the same antibody Fab' fragments labeled with beta-D-galactosidase from Escherichia coli. The minimal detection limit of the assay was 0.1 fmol, or 4 pg. The assay was specific for Go alpha, and it did not cross-react with Gi alpha or beta gamma. Samples from various regions of bovine brain were solubilized with 2% sodium cholate and 1 M NaCl, and the concentrations of Go alpha were determined. Go alpha was detected in all the regions, and the highest concentration was observed in the cerebral cortex. The immunohistochemical study showed that the neuropil was rich in Go alpha.

Animals↗
PubMed1987

Different behaviors among lysosomal enzymes in the cerebellum of jaundiced Gunn rats with cerebellar hypoplasia.

Activities of six lysosomal enzymes in the cerebellum of jaundiced homozygous (jj) Gunn rats were examined from 5 to 20 days of life and compared with those in heterozygotes (j+). Significantly higher enzyme activities were first detected at 8 days. The jj/j+ activity ratios of all enzymes peaked at 15 days. The ratios of beta-glycerophosphatase, beta-mannosidase, and acid lipase were only 1.3-1.7, whereas those of arylsulfatase and cathepsin were 2.0 and 3.1, respectively. The most striking increase in activity was observed with beta-glucuronidase, the ratio of which was 8.4. These results indicate a selective increase in activities of certain lysosomal enzymes in the hypoplastic cerebellum of jj rats.

Aging↗
PubMed1986

Immunohistochemical localization of gamma-enolase in early human embryos.

gamma-Enolase has been believed to be distributed only in the neurons and it was frequently labelled as neuron-specific enolase. However, recent precise studies have suggested a wider distribution of the protein. It can also be found in neuroendocrine cells, some mesodermal tissues, and some malignant tumors originating from tissues without the antigen in a normal condition. In early rat embryos, before the formation of neural tissues, a sensitive immunoassay system revealed a substantial amount of gamma-enolase, though it is not yet clear where the antigen is located. In the present study, tissue distribution of gamma-enolase in early human embryos was studied using an immunohistochemical method and it was suggested that the protein is present not only in neural tissue primordium but also in most tissues in the youngest embryo of 6.3 mm crown-rump length. Many of the immunoreactive non-neural tissues, however, lost immunoreactivity with the advancement of the embryonic stage while neural tissues became more intensely stained. In the embryo of 24 mm length, the staining pattern was almost the same as that of reported adult men. In embryonic tissues such as notochord and mesonephros, which disappear in the due course of growth, the antigen was also found. These findings will suggest that the antigen is rather common in undifferentiated tissues but then localizes in neural elements with advancing age. Our findings may be useful in explaining why early rat embryos, before the formation of neural tissues, showed a considerable amount of gamma-enolase and why many undifferentiated tumors, originating from tissues which did not have the antigen in normal condition, revealed the antigen.

Embryo, Mammalian↗
PubMed1986

New candidates for GABAergic neurons in the rat cerebellum: an immunocytochemical study with anti-GABA antibody.

Cerebellar cortical neurons immunoreactive to anti-gamma-aminobutyric acid (GABA) antibody were examined in the rat. In addition to the Purkinje, Golgi, basket and stellate cells, spindle-shaped cells lying just below the Purkinje cell layer were found to be strongly immunoreactive to the antibody. By the combination of immunofluorescence and hematoxylin stainings, these GABA-positive cells were shown to be the Lugaro cells. Unlike the immunopositive small Golgi cell, the pale cell was not immunoreactive to the antibody.

Animals↗
PubMed1986

Highly sensitive immunoassay for rat brain-type creatine kinase: determination in isolated Purkinje cells.

Ultrasensitive enzyme immunoassay method for the measurement of rat brain-type creatine kinase BB (CK-BB) was developed by use of purified antibodies specific to the B subunit of creatine kinase. The antibody immunoglobulin G was purified with immunoaffinity chromatography of the antiserum raised in rabbits by injecting the purified rat CK-BB. The assay system consisted of polystyrene balls with immobilized antibody F(ab')2 fragments and the same antibody Fab' fragments labeled with beta-D-galactosidase from Escherichia coli. The assay was specific to the B subunit of CK (CK-B), showing about 10% cross-reactivity with CK-MB, but it did not cross-react with CK-MM and neuron-specific gamma gamma enolase. The minimum detection limit of the assay was 0.1 pg or 1 amol CK-BB, being sufficiently sensitive for the measurement of CK-B contents in the isolated Purkinje cell bodies at the level of single cells. The average content of CK-B in a single Purkinje cell was 1.64 pg. The CK-B concentration in rat cerebellum (about 22 micrograms/mg protein) was about twofold higher than that (about 13 micrograms/mg protein) in the cerebrum. High levels (greater than 5 micrograms/mg protein) of CK-B were also found in the peripheral tissues such as gastrointestinal tract and urinary bladder, all of which are composed of smooth muscle. Immunohistochemical localization of CK-B antigens in the CNS revealed that the antigens is distributed not only in the neurons but also in the glial cells.

Animals↗
PubMed1985

Improved separation at low temperature of glycoproteins by Con A-Sepharose affinity chromatography in the presence of sodium dodecyl sulfate (SDS).

The Con A-Sepharose affinity chromatography of glycoproteins was even more effective at 4 degrees C than that at room temperature (26-28 degrees C) in the presence of sodium dodecyl sulfate (SDS). Application of this methodology to the separation of several glycoproteins from SDS-solubilized membrane proteins in rat cerebellum, including a glycoprotein characteristic of the Purkinje cells, was successful.

Animals↗
PubMed1985

Tissue distribution, developmental profiles and effect of denervation of enolase isozymes in rat muscles.

The tissue distribution of muscle-type alpha beta and beta beta enolases in rats were determined with the sandwich-type enzyme immunoassay method which utilized the purified antibodies specific to the alpha and to the beta subunit of enolase, and beta-D-galactosidase from Escherichia coli as label. All the tissues examined contained detectable levels of both alpha beta and beta beta enolases. The beta beta enolase was found at high levels in the skeletal muscle tissues (tongue, esophagus, diaphragm and leg muscles) and in the cartilages (xipoid process and auricular cartilage). The alpha beta enolase was distributed at a relatively high concentration in the heart and in the above-mentioned tissues. The beta beta enolase in the leg muscles, diaphragm and tongue was present on the day of birth at a concentration higher than that of the alpha alpha and alpha beta enolases, and its concentration further increased in a manner apparently related to the functional state of each tissue. Denervation of the leg muscles by cutting the sciatic nerve in adult rats resulted in a drastic change in the isozymes profile. The concentration of beta beta enolase in the tibialis anterior gastrocnemius lateralis and extensor digitorum longus (about 800 pmol/mg protein) decreased to about a half in a few weeks after denervation. In contrast, the concentrations of alpha alpha (2 pmol/mg) and alpha beta (80 pmol/mg) usually showed a slight increase by the treatment (alpha alpha, 7 pmol/mg; alpha beta, 100 pmol/mg after 2 weeks). As compared with these three muscles, the soleus had normally a low enolase level and the effect of denervation was less drastic. These results seem to suggest that the concentration of beta beta enolase is closely correlated with the functional state of the muscle tissue.

Animals↗
PubMed1985

Bovine mitochondrial DNA polymorphism in restriction endonuclease cleavage patterns and the location of the polymorphic sites.

Cleavage patterns of mitochondrial DNA (mtDNA) by restriction endonuclease analysis were examined in four Japanese Black cows, three Japanese Shorthorn cows, and six Holstein cows. Seventeen restriction enzymes which recognize six base pairs and two restriction enzymes which recognize four base pairs were used in this study. Polymorphism was observed with three restriction enzymes, HindIII, TaqI, and MspI, and was detected within the breeds. Nucleotide substitution was determined in the HindIII polymorphic site by DNA cloning and sequencing; this is C----T at position 10126 of the URF-3 region. Furthermore, the MspI and TaqI polymorphic sites were located on the physical map.

Animals↗
PubMed1985

Studies on a cerebellar 50,000-dalton protein associated with cerebellar hypoplasia in jaundiced Gunn rats: its identity with glial fibrillary acidic protein as evidenced by the improved immunoblotting method.

On the basis of our previous findings that a 50,000-dalton protein (GR-50) shows a marked increase in the hypoplastic cerebellum of jaundiced homozygous Gunn rats and its electrophoretic behavior is similar to that of glial fibrillary acidic protein (GFAP), we tried to identify GR-50 as GFAP by two-dimensional electrophoresis of rat cerebellar membrane proteins using an improved immunoblotting method. In this method a blot immunostained for a specific antigen was also visualized for other proteins, thereby enabling us to determine the location of the antigen on the blot more precisely. With the methodology it was found that GFAP antigen occupied exactly the same position as GR-50 on the blot, suggesting strongly the identity of both proteins. Immunohistochemical studies revealed that in the cerebellum of homozygotes compared with that of heterozygotes GFAP antigen was greatly increased and that it was especially rich in the homozygous rat cerebellar lobules with a high degree of hypoplasia. Further, it was shown that not only the fibers of the Bergmann glial cells but also their somata were intensely immunostained in the affected lobules. A 47,000-dalton protein (SG-47), which has been reported to be increased in staggerer mutant mice with cerebellar hypoplasia, also immunoreacted with the antiserum to GFAP.

Animals↗
PubMed1983

Two proteins associated with cerebellar hypoplasia in jaundiced Gunn rats.

Two cerebellar proteins with apparent molecular weights of 250,000 (GR-250) and 50,000 (GR-50) are closely associated with cerebellar hypoplasia in jaundiced homozygous Gunn rats. These proteins, found in Gunn rat cerebellum (4-60 days of age) and cerebrum as well as staggerer mouse cerebellum, were studied with electrophoretic techniques. After 8 days of life, GR-250 decreased and GR-50 increased in the homozygous Gunn rat cerebellum. The pI's of GR-250 and GR-50 were 4.7-5.8 and 4.6-4.9, respectively, and the former protein was shown to bind to Concanavalin A. A comparative study between cerebella of Gunn rats and staggerer mice revealed that GR-250 and P400, a protein generally thought to be characteristic of the Purkinje cells, were identical. Evidence was also obtained showing that GR-250 was present in the Gunn rat cerebrum. GR-50 was not detectable in the staggerer mouse cerebellum but instead, a protein (MW 47,000) was found to be increased in the mutant mouse cerebellum.

Aging↗
PubMed1983

Increase in S-100 protein levels in blood plasma by epinephrine.

The level of S-100 protein in rat blood plasma (about 1.4 ng/ml), which was detectable by the present immunoassay method, was markedly increased by a single injection of epinephrine (5 h after injection, 19.7 +/- 5.2 ng/ml). The S-100 protein thus increased was eluted in the same fractions as that of rat brain on butyl-Sepharose 4B column chromatography. The S-100 protein levels were also enhanced moderately by norepinephrine and slightly by isoproterenol, but not by insulin. It is strongly suggested that the increased S-100 protein in blood plasma is derived mainly from the adipose tissue through the effect of catecholamines.

Animals↗
PubMed1982

Distribution of nervous system-specific forms of enolase in peripheral tissues.

The distribution of 3 forms of rat enolase (alpha alpha, alpha gamma and gamma gamma forms), including nervous system-specific forms (alpha gamma and gamma gamma), was determined in various tissues with a sensitive enzyme immunoassay system. The brain and spinal cord contained more than 100 pmol/mg protein of the alpha alpha, alpha gamma and gamma gamma enolases. Organs such as the lungs, heart, spleen, liver, and kidney contained similarly high levels of alpha alpha enolase, but these tissues contained alpha gamma and gamma gamma enolases at levels less than 1% of the central nervous tissues. High levels of the alpha gamma (greater than 10 pmol/mg) and the gamma gamma (greater than 1.5 pmol/mg) forms were found in rectum, bladder, and uterus. In gut, major portions of the nervous system-specific forms were localized in the muscle layers. Skeletal muscle and diaphragm, which are composed of striated muscle, contained low levels of 3 forms of enolase. Megakaryocytes separated from the suspension of bone marrow contained 11.3, and 0.53 amol/cell of the alpha alpha and gamma gamma enolases, respectively, with little, if any, of the alpha gamma form.

Animals↗
PubMed1982

Increased nervous system-specific enolases in rat plasma and cerebrospinal fluid in bilirubin encephalopathy detected by an enzyme immunoassay.

Three forms of enolase isozymes (alpha alpha, alpha gamma, gamma gamma), including nervous system-specific forms, were measured in the cerebrospinal fluid and the blood plasma of jaundiced or nonjaundiced infant rats by means of enzyme immunoassay systems capable of detecting each form of enolase at the 1 amol (10(-18) mol) level. Average enolase levels in cerebrospinal fluid in normal rat were 2.0, 0.2 and 0.1 pmol/ml for alpha alpha, alpha gamma, gamma gamma forms, respectively. Levels of alpha gamma and gamma gamma forms (nervous system-specific enolases; NSE) in jaundiced rats, which suffer Purkinje cell degeneration due to the inborn hyperbilirubinemia, were three to four times as high as the normal values. When kernicterus was induced in jaundiced rats by an injection of bucolome, the NSE level in cerebrospinal fluid was elevated up to more than 30-fold the control, together with a significantly higher level of alpha gamma form in blood plasma are helpful in detecting neuronal damage in the central nervous system.

Animals↗
PubMed1981

Determination of brain enolase isozymes with an enzyme immunoassay at the level of single neurons.

Ultrasensitive enzyme immunoassay systems for the assay of rat brain enolase isozymes (alpha alpha, alpha gamma, and gamma gamma forms) were prepared by use of beta-D-galactosidase from Escherichia coli as label and the purified rabbit antibodies to alpha alpha and gamma gamma enolases. The antibodies were purified from the immunoglobulin G (IgG) fractions of antisera by immunoaffinity chromatography with a column of the corresponding antigen-coupled Sepharose. Sandwich-type immunoassay systems with the galactosidase-labeled antibody Fab' fragments and the antibody F(ab')2-immobilized polystyrene beads could determine amounts as small as 1 amol (10(-18) mol) of each isozyme. Purkinje cell bodies picked up from the bulk-separated fraction by means of a nylon loop were subjected to the assay at the level of single cells. In contrast to previous report, this neuron contained not only the gamma gamma but also the alpha gamma and alpha alpha enolases at a level of amol per cell body, although the concentration of gamma gamma was the highest. Immunohistochemical experiments on te cerebellum with the peroxidase-labeled antirabbit IgG antibody and the unlabeled antibody method confirmed the above results, and indicated that both alpha and gamma subunits of hte enolase were stained intensely in axons.

Animals↗
PubMed1979

Contributions to semithin sectioning on a conventional rotary microtome.

Procedures for obtaining sections 1 micrometer thick on a conventional rotary microtome are described. Hydrophilic resin blocks with adequate hardness and elasticity for semithin sectioning are made by addition of divinylbenzene and methylmethacrylate to a commercial embedding kit. The blocks are pinched between two simple adapters and mounted in the specimen holder of a microtome. A glass knife of the Ralph type with an effective blade length of 25 mm is made from a glass slide and attached to a metal bar with paraffin. The low cost assembly is set in the steel knife holder of a conventional rotary microtome. Sections 1 micron in thickness can be cut from the resin embedded blocks. Staining with the usual staining solutions may be weak due to the thinness of the sections, but the fine resolution and low distortion achieved are compensating gains.

Animals↗
PubMed1978

Relationship between plasma unbound-bilirubin concentration and cerebellar bilirubin content in homozygous Gunn rat sucklings.

Suckling increases and starvation (16 h) decreases plasma unbound-bilirubin concentration in 15-day-old homozygous Gunn rats. The effect of suckling-induced increase of plasma unbound-bilirubin concentration on cerebellar bilirubin content was examined. Cerebellar bilirubin content of the suckled rats did not increase, but rather decreased, in spite of the elevated concentration of plasma unbound-bilirubin . There were significant negative correlations between cerebellar bilirubin content and b. wt both in the suckled and starved groups.

Animals↗
PubMed1978

Danger of bucolome in infants with hyperbilirubinaemia. Experimental evidence.

Effects of bucolome on congenitally jaundiced Gunn rats were examined. Plasma total bilirubin level fell immediately after a single injection of bucolome and the lowered level persisted for more than 6 hours. Plasma unbound-bilirubin level and cerebellar bilirubin content increased simultaneously with the drop in total plasma bilirubin level. Kernicterus was observed in the brains of the treated rats 6 hours after the treatment. LD50 of the drug in jaundiced rats was about 37 mg/kg, about one-tenth of the value in nonjaundiced rats. It is suggested that bucolome displaces bilirubin from albumin, transferring bilirubin from blood into tissues including the brain, and resulting in kernicterus. The use of bucolome in infants with hyperbilirubinaemia is inadvisable.

Animals↗