Staurosporine, a potentially important gift from a microorganism.
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Alkaloids↗
Biomedical subjects
H Takeshima
Publications and source records attributed to H Takeshima.
[Approach to the role of ryanodine receptors using mice with a targeted mutation].
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Animals↗
Expression of osteopontin in human glioma. Its correlation with the malignancy.
BACKGROUND: Osteopontin (OPN) is a phosphorylated glycoprotein that contains a functional Gly-Arg-Gly-Asp-Ser (GRGDS) cell-binding sequence. It is reported that the expression of OPN is increased in a number of transformed cell lines. This study demonstrates the expression of OPN in human glioma representative of brain tumors. EXPERIMENTAL DESIGN: The expression of OPN in human gliomas was determined with: (a) Northern blot analysis; (b) Southern blot analysis; (c) Immunofluorescent cytochemistry; (d) Western blot analysis; (e) Cloning and sequencing of complementary DNAs (cDNAS); (f) Immunohistochemistry. RESULTS: OPN mRNA was detected in all glioma cell lines examined, and Southern blot analysis showed that OPN DNA most likely exists as a single copy gene in these cell lines. Immunofluorescent cytochemistry showed strong staining mainly in the cytoplasm. These cell lines produced two major forms of OPN protein migrating at 53 and 47 kilodaltons, and two minor forms at 38 and 31 kilodaltons on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. We also isolated and sequenced three types of splice variants in OPN from human glioma cell lines through polymerase chain reaction. In addition, Northern blot analysis was carried out on RNA from human astrocytoma tissues of various malignancy grades. Expression of OPN mRNA was high in malignant astrocytomas, but low in benign astrocytomas and non-neoplastic tissue. Immunohistochemistry also showed strong staining in malignant astrocytomas and slight staining in benign astrocytomas. CONCLUSIONS: These findings indicate that OPN mRNA and proteins are expressed in human glioma cells, and that the extent of OPN expression may correlate with the malignancy grade of gliomas.
Alternative Splicing↗
Renal oncocytoma mimicking hemorrhagic renal cyst.
A case of oncocytoma, the radiological findings of which resembled a hemorrhagic renal cyst, is reported. Cyst fluid analysis demonstrated extremely high levels of carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9). Preoperatively the lesion was regarded a malignant cyst, and radical nephrectomy was performed. Pathological diagnosis was renal oncocytoma with cystic degeneration. Although cystic change occasionally has been mentioned in oncocytoma, a high level of CEA and CA 19-9 in the cyst fluid of the cystic oncocytoma have not been reported. We discuss the variant forms of oncocytoma and tumor markers in the cyst fluid.
Adenoma, Oxyphilic↗
Structure and chromosomal mapping of genes for the mouse kappa-opioid receptor and an opioid receptor homologue (MOR-C).
Recent cDNA cloning studies have defined four members of the opioid receptor family, i.e., delta-, mu- and kappa-subtypes, and an opioid receptor homologue for unknown ligands. In this report, we isolated and analyzed mouse genomic DNA segments containing the kappa-opioid receptor gene and a gene for the opioid receptor homologue (designated as MOR-C). The genes are closely related each other in exon-intron organization, suggesting their evolutional relationship. Using in situ hybridization, we show that the kappa-opioid receptor gene and the MOR-C gene map to mouse chromosome 1A2-3 and 2H2-4, respectively.
Amino Acid Sequence↗
Cerulenin-resistant mutants of Saccharomyces cerevisiae with an altered fatty acid synthase gene.
Cerulenin, an antifungal antibiotic produced by Cephalosporium caerulens, is a potent inhibitor of fatty acid synthase in various organisms, including Saccharomyces cerevisiae. The antibiotic inhibits the enzyme by binding covalently to the active center cysteine of the condensing enzyme domain. We isolated 12 cerulenin-resistant mutants of S. cerevisiae following treatment with ethyl methanesulfonate. The mechanism of cerulenin resistance in one of the mutants, KNCR-1, was studied. Growth of the mutant was over 20 times more resistant to cerulenin than that of the wild-type strain. Tetrad analysis suggested that all mutants mapped at the same locus, FAS2, the gene encoding the alpha subunit of the fatty acid synthase. The isolated fatty acid synthase, purified from the mutant KNCR-1, was highly resistant to cerulenin. The cerulenin concentration causing 50% inhibition (IC50) of the enzyme activity was measured to be 400 microM, whereas the IC50 value was 15 microM for the enzyme isolated from the wild-type strain, indicating a 30-fold increase in resistance to cerulenin. The FAS2 gene was cloned from the mutant. Sequence replacement experiments suggested that an 0.8 kb EcoRV-HindIII fragment closely correlated with cerulenin resistance. Sequence analysis of this region revealed that the GGT codon encoding Gly-1257 of the FAS2 gene was altered to AGT in the mutant, resulting in the codon for Ser. Furthermore, a recombinant FAS2 gene, in which the 0.8 Kb EcoRV-HindIII fragment of the wild-type FAS2 gene was replaced with the same region from the mutant, when introduced into FAS2-defective S. cerevisiae complemented the FAS2 phenotype and showed cerulenin resistance.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Excitation-contraction uncoupling and muscular degeneration in mice lacking functional skeletal muscle ryanodine-receptor gene.
Contraction of skeletal muscle is triggered by the release of Ca2+ from the sarcoplasmic reticulum (SR) after depolarization of transverse tubules. The ryanodine receptor exists as a 'foot' protein in the junctional gap between the sarcoplasmic reticulum and the transverse tubule in skeletal muscle, and is proposed to function as a calcium-release channel during excitation-contraction (E-C) coupling. Previous complementary DNA-cloning studies have defined three distinct subtypes of the ryanodine receptor in mammalian tissues, namely skeletal muscle, cardiac and brain types. We report here mice with a targeted mutation in the skeletal muscle ryanodine receptor gene. Mice homozygous for the mutation die perinatally with gross abnormalities of the skeletal muscle. The contractile response to electrical stimulation under physiological conditions is totally abolished in the mutant muscle, although ryanodine receptors other than the skeletal-muscle type seem to exist because the response to caffeine is retained. Our results show that the skeletal muscle ryanodine receptor is essential for both muscular maturation and E-C coupling, and also imply that the function of the skeletal muscle ryanodine receptor during E-C coupling cannot be substituted by other subtypes of the receptor.
Animals↗
Gene expression of GLUT3 glucose transporter regulated by glucose in vivo in mouse brain and in vitro in neuronal cell cultures from rat embryos.
This study was designed to determine whether glucose regulates the gene expression of glucose transporter GLUT3 in neurons. We examined the regulation of GLUT3 mRNA by glucose in vivo in mouse brain and in vitro by using neuronal cultures from rat embryos. Hypoglycaemia (< 30 mg/dl), produced by 72 h of starvation, increased GLUT3 mRNA in mouse brain by 2-fold. Hybridization studies in situ demonstrated that hypoglycaemia-induced increases in GLUT3 mRNA expression were observed selectively in brain regions including the hippocampus, dentate gyrus, cerebral cortex and piriform cortex, but not the cerebellum. Primary neuronal cultures from rat embryos deprived of glucose for 48 h also showed an increase (4-fold over control) in GLUT3 mRNA, indicating that glucose can directly regulate expression of GLUT3 mRNA. In contrast with hypoglycaemia, hyperglycaemia produced by streptozotocin did not alter the expression of GLUT3 mRNA. We also confirmed previous findings that hypoglycaemia increases GLUT1 mRNA expression in brain. The increase in GLUT1 expression was probably limited to the blood-brain barrier in vivo, since GLUT1 mRNA could not be detected in neurons of the mouse cerebrum. Thus we conclude that up-regulation of neuronal GLUT3 in response to glucose starvation represents a protective mechanism against energy depletion in neurons.
Animals↗
cDNA cloning and regional distribution of a novel member of the opioid receptor family.
We have cloned a cDNA for a novel member of the opioid receptor family, designated as ROR-C, from the rat cerebrum cDNA library using the probe derived from the delta-opioid receptor subtype cDNA. The deduced amino acid sequence of ROR-C shows high homology with those of ROR-A (rat delta-opioid receptor subtype), ROR-B (rat mu-subtype) and ROR-D (rat kappa-subtype). RNA blot hybridization and in situ hybridization analysis revealed that ROR-C mRNA is expressed in discrete regions of the rat central nervous system.
Amino Acid Sequence↗
Isolation and characterization of a gene for a ryanodine receptor/calcium release channel in Drosophila melanogaster.
The nucleotide sequence of a 25.7 kilobase Drosophila melanogaster genomic DNA segment containing a gene for a ryanodine receptor/calcium release channel homologue has been determined. Computer analysis and partial cDNA cloning revealed 26 exons comprising the protein-coding sequence in this gene. The predicted protein is homologous in amino acid sequence and shares characteristic structural features with the mammalian ryanodine receptors. In blot hybridization analysis, a approximately 16 kilobase RNA species was identified abundantly in a 6-12 h embryo as the transcript from this gene. In situ hybridization to polytene chromosomes indicated that this gene locates at band position 44F on the second chromosome.
Amino Acid Sequence↗
Mu opioid receptor: expression and vagotomy-induced depletion of the mRNA in medullary preganglionic neurons.
By in situ hybridization with digoxigenin-labeled RNA probes, distinct expression of mu opioid receptor mRNA was found in rat medullary preganglionic neurons for vagal parasympathetic outflow. Two days or more following unilateral vagotomy, the expression was almost completely abolished on the operated side. The results, taken together with previous findings indicating proximodistal axonal transport of radioligand-labeled opioid receptors, suggest that the medullary preganglionic neurons thus identified are the source for mu opioid receptor acting in cardiorespiratory and gastrointestinal tissues.
Animals↗
Expression and localization of messenger RNA and protein for monocyte chemoattractant protein-1 in human malignant glioma.
Expression of monocyte chemoattractant protein-1 (MCP-1) in human glioma cell lines and surgical specimens was studied by Northern blot analysis, reverse-transcription polymerase chain reaction, in situ hybridization, and immunohistochemistry. The samples tested consisted of 11 human glioma cell lines and eight specimens of human malignant glioma (seven from glioblastomas and one from a malignant ependymoma). Messenger ribonucleic acid (mRNA) of MCP-1 was detected by either Northern blot or reverse-transcription polymerase chain reaction analysis in all cell lines and tumor specimens examined. In vivo expression of MCP-1 mRNA and protein was found predominantly in glioma cells with large and pleomorphic nuclei rather than in areas of small nucleated glioma cells. Adjacent brain tissue did not produce a significant level of MCP-1 mRNA or protein. Tumor vessels with endothelial proliferation expressed a moderate level of MCP-1 protein. Macrophages were found among the glioma cells, and the degree of macrophage infiltration was grossly correlated with the level of MCP-1 expression. The study results suggest that MCP-1 produced by the glioma cells may mediate macrophage infiltration into the glioma tissue.
Aged↗
Detection of cellular proteins that interact with the NF2 tumor suppressor gene product.
The neurofibromatosis type 2 (NF2) gene was recently cloned, and the protein it encodes (merlin) was revealed to belong to a family of proteins that link cytoskeletal components with proteins in the cell membrane. To elucidate the biological function of merlin, we produced a bacterial fusion protein consisting of glutathione S-transferase and merlin and used it to detect five merlin-binding cellular proteins, designated p165, p145, p125, p85 and p70, by a protein-binding assay. p165 and merlin were phosphorylated on serine/threonine residues, and immunoprecipitation showed that p85 bound the native form of merlin. Although the entire merlin-ezrin-radixin-moesin (MERM) homology domain of merlin was found to be essential for binding to all five proteins, the MERM homology domains of ezrin and moesin did not bind to any of the five proteins. Since most reported NF2 mutations are in the region we determined was necessary for binding, the mutations probably impair binding. Therefore, the formation of the protein complex is probably crucial for tumor suppression.
Base Sequence↗
Multiple types of ryanodine receptor/Ca2+ release channels are differentially expressed in rabbit brain.
The neuronal Ca2+ signal is induced by a rise in the intracellular free Ca2+ concentration ([Ca2+]i), and is thought to be important for higher brain function. Dynamic changes in [Ca2+]i are affected by the spatial distributions of various Ca(2+)-increasing molecules (channels and receptors). The ryanodine receptor (RyR) is an intracellular channel through which Ca2+ is released from intracellular stores. To define the contribution of neuronal Ca2+ signaling via the RyR channel, we examined RyR type-specific gene expression in rabbit brain by in situ hybridization histochemistry. The neuronal RyR was composed of three distinct types, two types dominant in skeletal (sRyR) and cardiac (cRyR) muscle, respectively, and a novel brain type (bRyR). sRyR was distinguished by its high level of expression in cerebellar Purkinje cells. cRyR was predominantly expressed throughout nearly the entire brain, and was characterized by its markedly high level of expression in the olfactory nerve layer, layer VI of the cerebral cortex, the dentate gyrus, cerebellar granule cells, the motor trigeminal nucleus, and the facial nucleus. bRyR expression was the least widely distributed throughout the brain, and was high in the hippocampal CA1 pyramidal layer, caudate, putamen, and dorsal thalamus. This investigation demonstrates that the heterogeneous distribution of neuronal RyRs may be implicated in distinct Ca(2+)-associated brain functions. Moreover, it should be noted that cRyR, a typical CICR channel, is distributed widely throughout the brain, suggesting that in a variety of cell types, the amplification of neuronal Ca2+ signals is functionally accompanied by a rise in [Ca2+]i, such as Ca2+ influx stimulated by neuronal activity. This widespread distribution of the neuronal RyR family indicates that Ca2+ signals via the intracellular stores should be considered in studies of neuronal Ca2+ dynamics.
Animals↗
Primary structure and expression from cDNAs of the ryanodine receptor.
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Amino Acid Sequence↗
Cyclic AMP-dependent phosphorylation and regulation of the cardiac dihydropyridine-sensitive Ca channel.
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Animals↗
Quantitative study of monocyte chemoattractant protein-1 (MCP-1) in cerebrospinal fluid and cyst fluid from patients with malignant glioma.
BACKGROUND: Monocyte chemoattractant protein-1 (MCP-1) is a 76-amino acid protein that attracts monocytes. In vitro studies have reported high levels of MCP-1 messenger RNA expression, as well as the presence of MCP-1, in malignant glioma cells. PURPOSE: Our purpose was to determine whether an MCP-1 assay could be used in a clinical setting 1) to differentiate malignant from benign gliomas and from nontumor disorders of the central nervous system and 2) to detect subarachnoid dissemination of glioma cells. METHODS: MCP-1 levels in cerebrospinal fluid (CSF) and cyst fluid were measured with a sandwich enzyme-linked immunosorbent assay (ELISA) that we had previously developed. We measured MCP-1 levels in CSF samples from 19 patients with malignant glioma (glioblastoma, 10; anaplastic astrocytoma, six; anaplastic oligodendroglioma, two; and ependymoblastoma, one), nine patients with benign glioma, and seven patients with nontumor disorders of the central nervous system. Cyst fluids from four patients with malignant glioma (anaplastic astrocytoma) were also tested. The correlation between MCP-1 concentration in the CSF and subarachnoid dissemination of malignant glioma cells was also studied. RESULTS: The MCP-1 concentration (mean +/- SE) in CSF samples from patients with malignant glioma (2.3 +/- 0.4 ng/mL) was significantly higher than that from patients with benign glioma (0.6 +/- 0.1 ng/mL) (P < .01) or from patients with no tumor (0.5 +/- 0.1 ng/mL) (P < .01). Furthermore, CSF samples from patients with subarachnoid dissemination of malignant glioma contained significantly higher amounts of MCP-1 than those from patients without dissemination (P < .05). Cyst fluids from four of the patients with malignant glioma contained high concentrations of MCP-1. CONCLUSIONS: These results indicate that MCP-1 is produced by malignant glioma in vivo as well as in vitro and suggest that testing for MCP-1 in CSF may be useful in the clinic to differentiate malignant glioma from benign glioma and to detect subarachnoid dissemination of the tumor cells. IMPLICATIONS: The MCP-1 ELISA in CSF may lead to more accurate diagnosis of malignant glioma and detection of subarachnoid dissemination of tumor cells, facilitating selection of patients with these conditions for appropriate therapy.
Adolescent↗
cDNA cloning and pharmacological characterization of an opioid receptor with high affinities for kappa-subtype-selective ligands.
The amino acid sequence of a rat opioid receptor, designated as ROR-D, has been deduced by cloning and sequencing the cDNA. The ROR-D expressed from the cDNA exhibits high affinities for ligands selective for the opioid receptor kappa-subtype and low affinities for ligands selective for the delta- and mu-subtypes. RNA blot hybridization analysis indicated that ROR-D mRNA is distributed in cerebrum and brainstem but not in cerebellum.
Amino Acid Sequence↗