Naoki Nishino

cDNA cloning of IL-1α and IL-1β from mRNA of U937 cell line

Clones of cDNAs encoding growth inhibitory factors for human melanoma cell line A375 were isolated from cDNA library prepared by using mRNA derived from human histiocytic lymphoma cell line U937 induced with PMA and further stimulated with LPS. Cloning was achieved using Okayama-Berg cDNA expression vector system that permits expression of the inserted cDNA segments in mammalian cells. By assaying the transfected COS-1 cells supernatants and cell extracts, we isolated two distinct cDNA clones encoding growth inhibitory factors. It was determined by the nucleotide sequences of the inserts, the cDNAs corresponded to IL-1 alpha and -1 beta. Our results indicate U937 cells can be induced to produce both interleukin-1s.

Cloning and chromosomal mapping of a novel ABC transporter gene (hABC7), a candidate for X-linked sideroblastic anemia with spinocerebellar ataxia

AbstractWe isolated a novel human ATP-binding cassette (ABC) transporter cDNA, determined its nucleotide sequence, and designated it human ABC7 (hABC7). The nucleotide sequence was highly homologous to the ATM1 gene in yeast, which encodes an ABC transporter (yAtm1p) located in the mitochondrial inner membrane. The deduced human product, a putative half-type transporter, consists of 752 amino acids that are 48.9% identical to those of yAtm1p. A computer-assisted protein structural and localization analysis revealed that the mitochondrial targeting signal of yAtm1p is conserved in the N-terminal region of the primary sequence of the hABC7 protein, and therefore this product is also likely to be located in the mitochondrial inner membrane. The evidence strongly suggests that the hABC7 gene is a counterpart of ATM1 and that its product is probably involved in heme transport. We mapped the hABC7 gene to chromosome Xq13.1–q13.3 by fluorescence in-situ hybridization. As band Xq13 has been implicated in X-linked sideroblastic anemia with spinocerebellar ataxia, hABC7 becomes a candidate gene for this heritable disorder.

Dexamethasone regulation of the expression of cytokine mRNAs induced by interleukin-1 in the astrocytoma cell line U373MG

BSF‐2/IL‐6, GM‐CSF and IL‐1β mRNAs were induced by recombinant IL‐1 in human astrocytoma cell line U373MG. The induction of BSF‐2/IL‐6 and IL‐1β mRNAs did not require de novo protein synthesis while that of GM‐CSF mRNA required a newly synthesized protein. Dexamethasone inhibited the induction of these cytokine mRNAs by IL‐1. This process seems to require continued protein synthesis. These results suggest that the production of these cytokines are positively and negatively controlled by IL‐1 and glucocorticoids, respectively, in astrocytes.

Promyelocytic leukemia cell line, HL-60, produces human hepatocyte growth factor

The human promyelocytic leukemia cell line, HL-60, stimulated with PMA, produced human HGF-like immunoreactivity (HL-60 HGF), which was detected with human HGF-specific ELISA. The purified HL-60 HGF was indistinguishable from human HGF in the plasma of patients with fulminant hepatic failure by studies of subunit constitution and amino acid composition. The HL-60 HGF mRNA corresponded to 6 kb, which was consistent with previous reported data in rat and human HGF mRNA, was detected in stimulated HL-60, by northern hybridization analysis using human HGF cDNA probe. These findings indicated that HL-60 HGF was identical to, or closely resembled, human plasma HGF. The HL-60 cell is an attractive model for studies of HGF-producing mechanisms, the manner of secretion and the nature of induction signals.

Cloning and sequence analysis of the arginine deiminase gene from Mycoplasma arginini

SummaryArginine deiminase from Mycoplasma arginini was purified. The purified enzyme has a molecular weight of 46000 daltons as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Its specific activity (20 units/mg protein) and amino acid composition showed a strong similarity to that of the Mycoplasma arthritidis arginine deiminase. The amino acid sequences of the N-terminal region and three internal peptides generated by enzymatic cleavage of the purified protein were determined. Using a synthetic oligonucleotide mixture complementary to part of the determined N-terminal amino acid sequence, the gene coding for arginine deiminase was isolated from a phage library. A nucleotide sequence of 2189 by encoding the gene was determined. An open reading frame (ORF) contained the amino acid sequences corresponding to the determined N-terminal region and the three internal peptides of arginine deiminase. Thus it was concluded that this ORF encoded the arginine deiminase, a 385 amino acid polypeptide (mol.wt. 43900 daltons). The three tryptophan residues in the sequenced peptides align with UGA codons in the nucleotide sequence, indicating that the nonsense codon UGA is used as a tryptophan codon in M. arginini.

Alteration of methamphetamine-induced striatal dopamine release in mint-1 knockout mice

Mint-1, which is also called as X11 or mammalian Lin10, protein has been implicated in the synaptic vesicle exocytosis and the targeting and localization of synaptic membrane proteins. Here, we established mint-1 gene knockout (mint-1 KO) mice and investigated vesicular and transporter-mediated dopamine (DA) release evoked by high K(+) and methamphetamine (METH), respectively. Compared with wild-type control, high K(+)-evoked striatal DA release was attenuated, but not significantly, in the KO mice as measured by microdialysis method. The METH-induced DA release was significantly attenuated in the KO mice. In addition, METH-induced stereotypy was also significantly attenuated in the KO mice. Mint-1 KO mice showed more sensitive and prominent behavioral response to an approaching object as compared with wild-type mice. These results suggest that mint-1 protein is involved in transporter-mediated DA release induced by METH.

Enhanced long-term potentiation in vivo in dentate gyrus of NELL2-deficient mice.

NELL2 is a neuron-specific thrombospondin-1-like extracellular protein containing six epidermal growth factor-like domains and is highly expressed in the hippocampus. We have previously shown that NELL2 promotes survival of neurons through mitogen-activated protein kinases. To clarify the function of NELL2 in vivo, we have generated a novel strain of mice with a targeted mutation in the NELL2 gene and assessed long-term potentiation (LTP) in vivo in the dentate gyrus of NELL2-deficient mice using extracellular recording techniques. Production of LTP at perforant path–granule cell synapses was significantly larger in NELL2-deficient mice than in wild-type controls. Thus, we propose that NELL2 plays an important role as a novel suppressor in LTP in vivo in the mouse dentate gyrus.

Expression of TMEFF1 mRNA in the mouse central nervous system : precise examination and comparative studies of TMEFF1 and TMEFF2

TMEFF1 and TMEFF2 are putative transmembrane proteins comprised of one epidermal growth factor (EGF)-like domain and two follistatin-like domains. Both TMEFF1 and TMEFF2 are predominantly expressed in the brain. We previously demonstrated that recombinant TMEFF2 protein can promote survival of neurons in primary culture and determined expression sites of TMEFF2 mRNA in the mouse central nervous system. To extend our understanding of TMEFF protein functions, we compared precise sites of expression of TMEFF1 and TMEFF2 mRNA using in situ hybridization analysis. Although both TMEFF genes are widely expressed in the brain, they exhibit different patterns of expression. TMEFF1 showed comparatively higher signals in the pyramidal cells of fifth layer of the cerebral neocortex, CA3, CA1 and subiculum regions of the hippocampus, locus coeruleus, and dentate cerebellar nucleus. In contrast, TMEFF2 is highly expressed in the medial habenular, CA2, CA3 and dentate gyrus region of the hippocampus, corpus callosum, cerebellar cortex and cranial nerve nuclei (III, IV, VII, X, XII). The results presented here indicate that expression of TMEFF1 and TMEFF2 are regulated differently and that they play region-specific roles in the central nervous system.

The transcription of the interleukin 1β gene is induced with PMA and inhibited with dexamethasone in U937 cells

Interleukin 1 beta mRNA was induced with phorbol ester, not LPS, in the human histiocytic lymphoma cell line U937, but interleukin 1 alpha mRNA was not induced. Nuclear run-on analysis showed that phorbol ester induced the transcription of interleukin 1 beta but did not induce it in the presence of cycloheximide. This indicates that the induction of the transcription with PMA requires de novo protein synthesis. Dexamethasone, an immunosuppressive and anti-inflammatory agent, decreased the level of interleukin 1 beta mRNA induced with phorbol ester. The transcriptional analysis showed that dexamethasone inhibits the transcription of the interleukin 1 beta gene.

Identification of EGF as an angiogenic factor present in conditioned medium from human salivary gland adenocarcinoma cell clones with varying degrees of metastatic potential

We have previously shown that conditioned medium (CM) from metastasizing human salivary gland adenocarcinoma cell clones contains factor(s) that stimulate the proliferation and migration of bovine aortic endothelial (BAE) cells, and inhibit the production of collagenases by BAE cells (Azuma M. et al. (1993) Cancer Lett., 73, 85-93). To further characterize this, we evaluated the expression level of epidermal growth factor (EGF) secreted by a non-metastasizing cell clone (HSGc) and its metastasizing cell clones, and analysed the effect of EGF on the biologic behaviors of BAE cells. When the secretion of EGF by cell clones was estimated by enzyme-linked immunosorbent assay, metastasizing cell clones released a large amount of EGF as compared with HSGc. However, the number of EGF receptor was detected consistently at a level that was similar in all cell clones. With regard to the effect of EGF on the malignant potential of cell clones such as proteolytic aggressiveness, EGF did not affect the secretion of both collagenases and their inhibitor from cell clones. Alternatively, exogenous EGF stimulated the proliferation and migration of BAE cells, and inhibited the secretion of collagenases from BAE cells. Neutralization with a neutralizing antibody of EGF released into CM abolished the inhibitory effect of CM on the secretion of collagenases from BAE cells. Thus, the CM-contained factor, which is responsible for the induction of biologic behaviors of BAE cells, can be attributed to EGF.