Biomedical subjects

M Kawata

Publications and source records attributed to M Kawata.

At least 145 records · Page 8Linked to original sources

PubMed1992

[Morphometric analysis in the rat spinal cord following peripheral nerve transection with the use of MAP2 immunohistochemistry].

The physiological function of the spinal cord is regulated by two different kinds of information, namely, afferent inputs from the primary sensory fibers and descending inputs from the brain. The former conveys the sensory information from the peripheral region via the dorsal root ganglia to spinal interneurons or directly to motor neurons and brings about the voluntary and/or involuntary muscle movement. In the present study, the time-related changes in the dendrocytoarchitecture of the rat spinal cord following the transection of afferent and efferent connections from/to the peripheral organs were observed by using immunohistochemistry for microtubule-associated protein 2 (MAP2) and Nissl staining. To investigate the changes quantitatively, morphometric analyses were performed on the section of gray matter of tissue samples stained with MAP2-antisera. One to 24 weeks after hemitransection of the dorsal and ventral roots, a remarkable reduction in the area and progressive changes in the shape of the gray matter were seen in both the anterior and posterior horns on the lesioned side. These changes were found to be due to the reduction of the width of the anterior and posterior horns on the lesioned side rather than of the height. At the later postoperative stage, atrophy of the Fasciculus gracilis and disappearance of motoneurons on the lesioned side were recognized in Nissl stained tissue samples.

Animals↗

PubMed1992

[Small GTP-binding protein and platelet function].

In platelets, nearly ten small GTP-binding proteins (G proteins) are identified. Among these small G proteins, smg p21 is the most abundant. The C terminus of smg p21 is processed by three post-translational modifications: geranylgeranylation, removal of three amino acids and carboxyl methylation. There are two regulatory proteins for smg p21, smg p21 GTPase activating protein (GAP) and smg GDP dissociation stimulator (GDS). Moreover, smg p21 is phosphorylated by A-Kinase and this phosphorylation stimulates the action of smg GDS. C-Kinase and Ca2+ induce platelet activation, while A-Kinase antagonizes this activation. Since smg p21 is the substrate of A-Kinase, it is possible that smg p21 mediates the A-Kinase action and inhibits the platelet function, such as aggregation and secretion. The mode of action of and the possible function of smg p21 in platelets are discussed.

Alkyl and Aryl Transferases↗

PubMed1991

[An attempt to detect genetic information by means of molecular cytochemistry].

In situ hybridization is a histochemical technique that attracts many cell biologists and others interested in developmental biology, virology, genetics and neuroendocrinology. This method gives us the precise localization and identification of individual cells which contain specific nucleic acid sequences, in a similar manner to the immunohistochemistry of cells which have a particular protein. There has been a wide range of applications for this technique. One of the most important and significant applications of in situ hybridization is the demonstration of specific mRNA in particular cells. This is quite valuable in heterogeneous tissue such as that of the hypothalamus with a various types of different cells. The combination with the immunohistochemistry enables us to study the dynamics of peptides or proteins in a certain tissue or cell. In this mini-review the logic and methodology of molecular cytochemistry, particularly, in situ hybridization, with its application in the endocrinological field was presented.

DNA Probes↗

PubMed1991

A novel prenyltransferase for a small GTP-binding protein having a C-terminal Cys-Ala-Cys structure.

smg p25A/rab3A p25 is a member of the small GTP-binding protein superfamily which is implicated in intracellular vesicle transport. smg p25A has a cDNA-predicted C-terminal structure of Cys-Ala-Cys. The protein purified from bovine brain membranes is geranylgeranylated at both the two cysteine residues and carboxyl-methylated at the C-terminal cysteine residue. Two types of prenyltransferase for small GTP-binding proteins have thus far been reported: ras p21 farnesyltransferase (ras p21 FT) and rhoA p21 geranylgeranyltransferase (rhoA p21 GGT). Neither of them geranylgeranylated smg p25A having a C-terminal Cys-Ala-Cys structure. In this paper, a smg p25A GGT was partially purified from bovine brain cytosol and separated from the ras p21 FT and rhoA p21 GGT by column chromatographies. smg p25A GGT transferred the geranylgeranyl moiety from geranylgeranyl pyrophosphate to both the two cysteine residues in the C-terminal Cys-Ala-Cys structure of smg p25A. smg p25A GGT did not use farnesyl pyrophosphate as a substrate and was also inactive on c-Ha-ras p21 and rhoA p21 with either farnesyl pyrophosphate or geranylgeranyl pyrophosphate as a substrate. These results indicate that there are at least three types of prenyltransferase for small GTP-binding proteins in mammalian tissues.

Amino Acid Sequence↗

PubMed1991

Collateral sprouting of serotonergic fibers in the cingulate cortex and the septum following cortical-hippocampal lesions.

The parietal cortex and dorsal hippocampus of adult rats were unilaterally ablated. One and 3 months after this operation, changes in serotonergic fiber distribution in the forebrain were studied immunohistochemically. At 1 month, increased numbers of serotonergic fibers were seen in the cingulate cortex and the medial and lateral septal nuclei of the lesioned side. This increase continued to 3 months in the ipsilateral cingulate cortex. In the present study, two different processes of serotonergic fiber collateral sprouting were noted. Plastic changes in serotonergic fibers in the ipsilateral septal nuclei and cingulate cortex may be attributable to the collateral sprouting underlying reactive synaptogenesis and the pruning effect, respectively.

Animals↗

PubMed1991

C terminus of the small GTP-binding protein smg p25A contains two geranylgeranylated cysteine residues and a methyl ester.

smg p25A, also known as the rab3A protein, is a small GTP-binding protein that has been implicated in intracellular vesicle transport and the secretion of neurotransmitters. It has been shown to bind reversibly to membranes, though its cDNA-predicted sequence contains no obvious membrane-binding domains. However, smg p25A does contain a cDNA-predicted C-terminal Cys-Ala-Cys sequence at positions 218 through 220, which suggests that it may be posttranslationally modified. In the present study we used two different approaches to investigate this possibility. First, we incubated pheochromocytoma cells with [3H]mevalonolactone, examined the proteins that became labeled by two-dimensional gel electrophoresis, and demonstrated that two of these proteins exactly corresponded to smg p25A. Second, we purified smg p25A from bovine brain membranes and analyzed both the full-length protein and a proteolytically derived C-terminal peptide by a combination of high performance liquid chromatography and mass spectrometry. This approach revealed that the protein's C-terminal region is methyl-esterified and contains two geranylgeranyl groups linked via thioether bonds to Cys-218 and Cys-220. Since smg p25A is one of several small GTP-binding proteins that share a C-terminal Cys-Xaa-Cys consensus sequence (where Xaa is an unspecified amino acid), our results suggest that these proteins may be similarly geranylgeranylated and methyl-esterified.

Adrenal Gland Neoplasms↗

PubMed1991

The posttranslationally modified C-terminal structure of bovine aortic smooth muscle rhoA p21.

rhoA p21, a ras p21-like small GTP-binding protein, has the same C-terminal consensus motif of Cys-A-A-X (A is an aliphatic amino acid and X is any amino acid) as ras p21s, which is posttranslationally processed. We here determine the posttranslationally processed C-terminal structure of the rhoA p21 purified from bovine aortic smooth muscle. Incubation of rhoA p21-expressing insect cells with exogenous [3H]mevalonolactone caused the labeling of rhoA p21, suggesting that rhoA p21 is prenylated. Consistently, Raney nickel treatment of rhoA p21 released a geranylgeranyl moiety as estimated by gas chromatography/mass spectrometry. No lipid moiety was released by KOH or NH2OH treatment. Extensive digestion of rhoA p21 with Achromobacter protease I yielded a C-terminal peptide, Ser-Gly-Cys190, that lacked the three C-terminal amino acids predicted from the cDNA but was geranylgeranylated and carboxyl methylated at the cysteine residue. Bovine brain cytosol geranylgeranylated the bacterial rhoA p21 having the three C-terminal amino acids predicted from the cDNA but not the protein lacking the three C-terminal amino acids. Bovine brain membranes methylated the synthetic C-terminal peptide with 10 amino acids of rhoA p21 which was geranylgeranylated at its C-terminal cysteine residue but not the peptide which was not geranylgeranylated. These results suggest that rhoA p21 is first geranylgeranylated followed by removal of the three C-terminal amino acids and the subsequent carboxyl methylation of the exposed cysteine residue.

Alcaligenes↗

PubMed1991

Neural rosette formation within in vitro spheroids of a clonal human teratocarcinoma cell line, PA-1/NR: role of extracellular matrix components in the morphogenesis.

From the human teratocarcinoma-derived cell line PA-1, we established a clonal line, PA-1/NR, that stably produced a distinct cellular arrangement of neural rosettes when cultured as in vitro multicellular spheroids for 3 weeks. On immunofluorescence staining and fluorescence-activated cell sorter analyses, PA-1/NR cells in monolayer expressed the neuroectoderm-associated antigens HNK-1, NC-1, and A2B5 and the neuroblastoma-associated antigens KP-NAC8 and KP-NAC10 but lacked human embryonal carcinoma antigens, SSEA-3 or K21 antigen. Here, we investigated the developmental process of rosette formation with respect to morphological features, distribution of mitotic cells, and expression of multiple lineage-related markers and extracellular matrix (ECM) components. Ultrastructural examination of these rosettes disclosed a well-defined cavity radially surrounded by wedge-shaped or pseudostratified cells, apical microvilli and junctional complexes, and basal laminae and collagen fibrils at their basal surface. In these rosettes, many proliferating cells were detected by the immunohistochemical staining of cells incorporating bromodeoxyuridine. PA-1/NR spheroids consistently displayed neuron-specific enolase, S-100 protein, and vimentin but not glial fibrillary acidic protein, neurofilament proteins, or myelin basic protein. The rosette formation accompanied a strikingly polarized and overlapped deposition of ECM components including tenascin-carrying HNK-1 epitopes, laminin, type IV collagen, heparan, and chondroitin sulfate proteoglycans. Immunoblotting analyses showed that laminin B1 and B2 chains were constitutively expressed, whereas a fully assembled form of laminin and type IV collagen appeared only after spheroid development, suggesting that these ECM components play a morphogenetically important role in rosette formation. Close similarities between these rosettes and the neural tube of humans and experimental animals in the morphogenetic process and ECM formation lead us to propose that the PA-1/NR spheroids provide an in vitro model for the study of the earliest stage of human neurogenesis.

Antibodies↗

PubMed1991

The effect of estrogen on the estrogen receptor-immunoreactive cells in the rat medial preoptic nucleus.

The localization of estrogen receptor-immunoreactive (ER-IR) cells in the medial preoptic nucleus (MPN) and the effect of estrogen upon these cells were quantitatively analyzed by immunohistochemistry using a monoclonal antibody to estrogen receptor (ER) protein in rat. ER was distributed in the nucleus of MPN neurons and was not detected in either cytoplasm or glial cells. There were more ER-IR cells with higher immunoreactivities in ovariectomized (OVX) rats than in estradiol (E2)-treated rats. The number of ER-IR cells in E2-treated compared with OVX rats was reduced by 43%. In particular, the number of ER-IR cells in the central part of the MPN was largely decreased. Our data indicate that the central part of the MPN is most sensitive to estrogen.

Animals↗

PubMed1991

A geranylgeranyltransferase for rhoA p21 distinct from the farnesyltransferase for ras p21S.

We have clarified that rhoA p21 purified from bovine aortic smooth muscle is geranylgeranylated at the cysteine residue in the C-terminal CAAX motif (A is an aliphatic amino acid and X is any amino acid). In this paper, a geranylgeranyltransferase for rhoA p21 (rhoA p21 GGT) was partially purified from bovine brain cytosol. This enzyme transferred a geranylgeranyl moiety from geranylgeranyl pyrophosphate to rhoA p21 having the CAAX motif (rhoA p21-CAAX) but not to rhoA p21 lacking the AAX portion. rhoA p21 GGT was separated from the previously reported farnesyltransferase for ras p21s (ras p21 FT) by column chromatographies and did not geranylgeranylate or farnesylate c-Ha-ras p21-CAAX. ras p21 FT did not geranylgeranylate or farnesylate rhoA p21-CAAX. These results indicate that rhoA p21 GGT distinct from ras p21 FT is present in bovine brain cytosol.

Alkyl and Aryl Transferases↗

PubMed1991

Preparation and evaluation of Eudragit gels. I: Eudragit organogels containing drugs as rectal sustained-release preparations.

New organogels containing salicylic acid, sodium salicylate, procaine, and ketoprofen were prepared by using Eudragit L and S, and propylene glycol. The physical properties (e.g., penetration and extensibility) of the organogels were affected considerably by the incorporation of a basic drug, such as procaine, rather than an acidic drug, such as salicylic acid. In in vivo evaluation using rabbits, it was found that 30% Eudragit L organogels containing 1.25% (w/w) salicylic acid or 2.5% (w/w) ketoprofen showed sustained plasma salicylic acid or ketoprofen levels and almost the same AUC as compared with Witepsol H-15 suppositories containing these drugs. Furthermore, the relative bioavailability for salicylic acid and ketoprofen released from 30% Eudragit L organogels, with 10% (w/w) linolic acid or oleic acid as absorption enhancer, increased to 1.55-1.75- and 1.46-1.85-fold, respectively, compared with 30% Eudragit L organogels without these unsaturated fatty acids.

Acrylic Resins↗

PubMed1991

Preparation and evaluation of Eudragit gels. II: In vitro release of salicylic acid, sodium salicylate, and ketoprofen from Eudragit L and S organogels.

The in vitro dissolution characteristic of salicylic acid, sodium salicylate, and ketoprofen from Eudragit L and S organogels was investigated by the rotation disk method. The dissolution pattern of salicylic acid and erosion of Eudragit L polymer from the organogels followed apparent zero-order kinetics, providing strong evidence for a surface erosion mechanism and negligible diffusional release of salicylic acid. On the other hand, the dissolution of salicylic acid from Eudragit S organogels was a linear function of the square root of time. The apparent dissolution rate of salicylic acid from Eudragit S organogels increased with increasing temperature from 32 to 42 degrees C and agitation rate from 50 to 200 rpm. A linear relationship was obtained between the logarithm of apparent dissolution rate constants and the reciprocal of absolute temperatures. The activation energy for release of salicylic acid from Eudragit S organogels was in the range of 2.99 to 5.57 kcal/mol. From various experimental results, it was concluded that the release process of salicylic acid from Eudragit S organogels was diffusion controlled through the organogels matrix.

Acrylic Resins↗

PubMed1991

Target-specific innervation by autonomic and sensory nerve fibers in hairy fetal skin transplanted into the anterior eye chamber of adult rat.

Pieces of hairy skin tissue of fetal rat were transplanted into the anterior eye chamber of adult rats. The ability of autonomic and sensory nerve fibers from the host iris to innervate the grafted skin tissue was immunohistochemically and enzyme-histochemically examined using antisera against tyrosine hydroxylase (TH), substance P (SP), calcitonin gene-related peptide (CGRP) and vasoactive intestinal peptide (VIP), and a reaction medium for acetylcholinesterase (AchE). The grafted tissue was successfully implanted and connected with the host iris. Epidermis, dermis, subcutaneous tissue, hairs, hair follicles, sebaceous glands, and piloerector muscles developed in the graft. Two weeks after transplantation, TH-, SP-, and CGRP-immunoreactive fibers were observed in association with the blood vessels in the graft. Four weeks after transplantation, TH-immunoreactive fibers were distributed in the piloerector muscles, whereas SP- and CGRP-immunoreactive fibers were present around the hair follicles. VIP-immunoreactive and AchE-positive fibers were restricted to the host iris at all survival times. These results suggest that the outgrowth of autonomic and sensory nerve fibers from the host iris show target specificity for the grafted skin tissue.

Animals↗

PubMed1991

Cellular subsets of the milky spots in the human greater omentum.

The cellular composition of the human milky spots was investigated on surgically removed specimens of the greater omentum of three 8-month-old infants operated on for neuroblastoma. Monoclonal antibodies and immunohistochemical methods for recognition of macrophages. B-lymphocytes and T-lymphocytes and toluidine-blue staining for mast cells were used. The mean number of cells in one milky spot amounted to 570 +/- 33. This cell population was composed of 47.5% macrophages, 29.1% B-lymphocytes, 11.7% T-lymphocytes and 6.1% mast cells. Since inflammation was absent in the material investigated, the numerical data found in the present paper could be regarded as representative cell levels of normal milky spots.

B-Lymphocytes↗

PubMed1991

Genetic identification of pregnancies responsible for choriocarcinomas after multiple pregnancies by restriction fragment length polymorphism analysis.

The pregnancies responsible for two cases of choriocarcinomas were determined by the restriction fragment length polymorphism of human leukocyte antigen-DQ genes in the tumors and leukocytes from the patients, their husbands, and their children. The first case was preceded by three normal pregnancies, and the second case was preceded by two normal pregnancies and a complete hydatidiform mole. Comparison of the restriction fragment length polymorphism patterns revealed that the first case of choriocarcinoma carried the parental genome and originated from the third normal pregnancy, whereas the second case was of androgenetic origin from the complete mole. By choosing an appropriate probe (or probes), restriction fragment length polymorphism analysis is shown to be a useful method for identification of the pregnancy responsible for choriocarcinoma.

Adult↗

PubMed1991

Immunohistochemical study on gastrin-releasing peptide-containing peripheral nerve fibers in rat, macaque and human.

Distribution of gastrin-releasing peptide (GRP)-containing peripheral nerve fibers in the respiratory organs, digestive tracts and hearts of rat, monkey and human was studied with immunohistochemical method. In the trachea, GRP-immunoreactive nerve fibers were observed in the lamina propria and surrounded ducts and acini of tracheal serous glands with a basket-like appearance. In the lung, immunoreactive nerve fibers were scattered in the lamina propria of bronchi and bronchioles, and also the adventitia of veins. In the digestive organs, such as esophagus, duodenum and jejunum, numerous GRP-immunoreactive nerve fibers were distributed in the lamina propria and muscle layer; especially in the former layer where they were seen running up to the apical part of villi. GRP-containing nerve bundles and branched fibers were also observed in the atrial muscle layer. These results gave a morphological basis to future studies of the functional significance of GRP on autonomic mechanisms.

Aged↗

PubMed1991

Rapid changes in the content of proenkephalin A and corticotrophin releasing hormone mRNAs in the paraventricular nucleus during morphine withdrawal in urethane-anaesthetized rats.

Quantitative in situ hybridization was used to measure corticotrophin-releasing hormone (CRH) and proenkephalin A mRNA in the medial parvocellular paraventricular nucleus (PVN) 4 h after test procedures. Urethane anaesthesia alone resulted in a significant increase in both CRH and proenkephalin transcripts. The additional stimulus of i.p. hypertonic saline, however, resulted in a further significant increase in both mRNA species. Female rats were given intracerebroventricular (i.c.v.) infusion for 5 days of either morphine sulphate to induce tolerance and dependence, or vehicle, via a subcutaneous osmotic minipump implanted under ether anaesthesia. The rats were then anaesthetized with urethane, fitted with an intravenous cannula for injections and 65 min later either naloxone (5 mg/kg) or vehicle was injected. Naloxone alone in the i.c.v. vehicle rats had no effect on CRH or proenkephalin A mRNA. In i.c.v. morphine-infused rats proenkephalin a mRNA in the PVN was significantly less than in controls. Naloxone given to i.c.v. morphine-infused rats resulted in a doubling of hybridization to proenkephalin mRNA in the PVN which was significantly greater than that seen in the i.c.v. vehicle group. CRH mRNA in the PVN was not altered either by naloxone in control rats, or by chronic i.c.v. morphine infusion. By contrast, naloxone did increase CRH mRNA by ca. 40% in morphine-infused rats. The results show that stress-induced increases in CRH and enkephalin mRNAs in the PVN do not require conscious appreciation of the stress.(ABSTRACT TRUNCATED AT 250 WORDS)

Anesthesia, General↗

PubMed1991

A transient decrease in N-myc expression and its biological role during differentiation of human embryonal carcinoma cells.

The human embryonal carcinoma (EC) cell line, NEC14 can be induced to morphologically differentiate by the addition of 10(-2) M N,N'-hexamethylene-bis-acetamide (HMBA) in vitro. The expression of several cellular oncogenes (c-onc) in NEC14 cells was examined after induction of differentiation by HMBA. The level of N-myc expression was the highest in undifferentiated cells but decreased transiently to less than 1/10 of the original level shortly after the induction of differentiation. To investigate the role of the transient decrease in N-myc level on NEC14 cell differentiation, a chimeric human N-myc gene in which transcription is initiated at the human beta-actin gene promoter was constructed and introduced into NEC14 cells. Several transformants expressing the exogenous N-myc gene constitutively were established. These transformants showed 10- to 70-fold increases in plating efficiency and shorter population doubling times as compared with the parental NEC14 cells. The transformants were hard to induce, spontaneously differentiated cells on the periphery of cell clusters in culture, unlike parental NEC14 cells, and took longer for HMBA-induced morphological differentiation. The populations of the cells expressing HLA and SSEA-1 antigens increased from 10%-20% to nearly 100% in NEC14 cells after the induction of differentiation, while the populations expressing these antigens increased only to 50%-60% in one of the transformants, S11. The transformants gained an increased tumorigenic potential in nude mice, and the tumors produced consisted exclusively of EC stem cells. These results suggest that the additional expression of the exogenous N-myc gene (increased about two-fold) confers the more transformed state on the cells.

Antigens, Surface↗