Hsc70 regulates the nuclear export but not the import of influenza viral RNP: A possible target for the development of anti-influenza virus drugs.

In our previous report, we demonstrated that the matrix 1 (M1) protein of influenza virus directly binds to heat shock cognate protein 70 (Hsc70). The down-regulation of Hsc70 resulted in the reduction of influenza virus production, thus suggesting that Hsc70 plays a crucial role for viral replication. However, the detailed role of Hsc70 in viral replication remains to be elucidated. Hsc70 has been suggested to play a significant role in both the nuclear import and export processes. In this report, using leptomycin B (LMB), a CRM1-mediated nuclear export inhibitor, we demonstrated that Hsc70 forms a complex with vRNP through M1 in infected cells and in the virion, thus playing a significant role in the export of vRNP from the nucleus but not in the import of vRNP into the nucleus. The regulation of Hsc70 may therefore lead to the development of new anti-influenza virus drugs without raising mutant viruses.

A novel G-protein-coupled receptor gene expressed in striatum.

Differential display screening for region-specific transcripts in rat brain revealed a novel striatum-specific transcript encoding an orphan G-protein-coupled receptor (GPCR) designated Strg/Gpr88 for striatum-specific GPCR. We isolated its homologues from human (HGMW-approved symbol GPR88) and mouse and mapped them to chromosomes 1p21.3 and 3G1, respectively. These loci are syntenic to each other, thereby suggesting their orthology. The predicted primary sequences of Strg/Gpr88 proteins are highly conserved between human and rodents and show the highest level of homology to receptors for biogenic amines. However, Strg/Gpr88 lacks some residues conserved in all known biogenic amine receptors and hence may represent a novel subtype of GPCR. Northern blot and in situ hybridization analyses revealed that Strg/Gpr88 transcripts are expressed almost exclusively in striatum in both human and rodents. Remarkable conservation in primary structure and a unique expression pattern may indicate a role for Strg/Gpr88 in the fundamental functions of striatum such as the control of motor behavior.

Molecular and functional characterization of HSC54, a novel variant of human heat-shock cognate protein 70.

A novel variant of human heat-shock cognate protein 70 (HSC70) transcript, named heat-shock cognate protein 54 (HSC54), was identified and characterized. The transcript encodes the protein lacking 153 amino acid residues of HSC70 in a part of the protein-binding and variable domains, resulting in a calculated molecular mass of 53.5 kDa. HSC54 mRNA was detected in all human cells and tissues examined. The protein was also detected in peripheral mononuclear cells and U937 human histiocytic lymphoma cells. Heat treatment of U937 cells up-regulated the expression of HSC54. The chaperoning activity of HSC54 was examined by luciferase renaturation assay. HSC70 recovered the luciferase activity in the presence of reticulocyte lysate as a source of cochaperones. However, HSC54 did not facilitate the recovery of denatured luciferase; besides, HSC54 significantly inhibited the HSC70-mediated chaperoning activity. In pull-down experiments, HSC54 interacted with cochaperones, p60, HSP40, and p48, as HSC70 did. The resonant mirror detection analysis showed that p60 binds to HSC54 with a higher association rate constant than HSC70 with a similar affinity constant. These results suggest that HSC54 is constitutively expressed and also inducible by stress and may function as an endogenous inhibitory regulator of HSC70 by competing the cochaperones.

[An application of fusion fluorescent proteins in the pharmacological study of intracellular protein trafficking].

The fluorescent proteins have expanded various aspects of biological research. Proteins fused to the fluorescent proteins such as green fluorescent protein (GFP) provided researchers an opportunity to visualize the intracellular trafficking of proteins in living cells. The trafficking of proteins including that of steroid receptors involves dynamic interactions with cellular proteins such as heat shock proteins, immunophilins. Such interactions, which can be monitored by GFP fusion proteins, may be altered by pathophysiological conditions and by drugs. Thus, GFP fusion proteins may be applied to the investigations for the pharmacological manipulation of protein trafficking.

Anti-NO action of carvedilol in cell-free system and in vascular endothelial cells.

1. Carvedilol, an adrenoceptor blocker with antioxidant activity, was studied for its ability to interact with NO in a cell-free condition and in an endothelial cell line (ECV304). 2. In a cell-free system, carvedilol attenuated NO-dependent reduction of carboxy-2-phenyl-4,4, 5,5-tetramethyl-imidazoline-1-oxyl-3-oxide induced by a NO donor, 1-hydroxy-2-oxo-3-(aminopropyl)-3-isopropyl-1-triazene (NOC5), which was determined by electron paramagnetic resonance (EPR) spectrometry. The EPR study also showed that nitrosylhaemoglobin formation in rat red blood cells by the addition of NO-saturated solution was attenuated by prior incubation with 0.1 - 10 microM carvedilol. 3. NO-induced fluorescence in 4,5-diaminofluorescein-2 diacethyl (DAF-2DA)-loaded ECV304 cells was attenuated by carvedilol but not by labetalol. The IC(50) of carvedilol for NOC5 or sodium nitroprusside-induced fluorescence of DAF-2DA in ECV304 cells was 1. 0x10(-7) M, which was similar to the reported IC(50) of carvedilol for the antioxidant effect. 4. Cell toxicity induced by a NO donor determined by the number of viable cells after 24 h treatment with 2-2'(hydroxynitrosohydrazino)bis-ethanamine was significantly attenuated by pretreatment with 1 microM carvedilol. 5. Both free and cell-associated carvedilol quenched NO. Because NO mediates both physiological and pathophysiological processes, NO quenching by the drug may have diverse clinical implications depending upon specific functions of local NO in tissues where carvedilol is distributed.

Developmentally-regulated expression of mNapor encoding an apoptosis-induced ELAV-type RNA binding protein.

Proteins with RNA recognition motifs (RRMs) participate in many aspects of RNA metabolism, and some of them are required for the accomplishment of normal development. The neuroblastoma apoptosis-related RNA binding protein (NAPOR) is an ELAV-type RNA-binding protein with three characteristic RNP2/RNP1-type RRMs, which we identified as a gene induced during apoptosis of neuroblastoma cells. Here we isolated and characterized the cDNA for mNapor, the mouse homolog of NAPOR. The mNapor encodes mRNA sharing striking homology with that of NAPOR, not only in its open reading frame (98.5%) but also in the 3'-untranslated region (80.1%), and is mapped to chromosome 2 A2-A3, a region syntenic to the human NAPOR locus. In situ hybridization analysis revealed that the expression pattern of mNapor is spatially and temporally coincident with the occurrence of programmed cell death, suggesting its involvement in the development of the central nervous system in which apoptosis plays a crucial role.

The effect of acute cold exposure and norepinephrine on uncoupling protein gene expression in brown adipose tissue of monosodium glutamate-obese mice.

Abnormal regulation of mitochondrial uncoupling protein (UCP) gene expression was studied in brown adipose tissue (BAT) of monosodium glutamate (MSG)-induced obese mice. UCP mRNA levels in control mice increased markedly after acute cold exposure; however, MSG-obese mice showed an impaired response. In contrast, an injection of norepinephrine (NE) induced a comparable increase in UCP mRNA levels in control and MSG-obese mice. These results suggest that the impairment in the cold-induced increase in UCP mRNA is due to a deficient sympathetic input to BAT and/or to a diminished response of BAT to endogenous NE, which constitutes the mechanism of impaired thermoregulation in obese mice in a cold environment.

Molecular characterization of the mouse mtprd gene, a homologue of human TPRD: unique gene expression suggesting its critical role in the pathophysiology of Down syndrome.

We and others recently isolated a human TPRD gene, possessing a motif of the tetratricopeptide repeat (TPR), from the Down syndrome-critical region (DCR) of chromosome 21q22.2. In this study, we isolated a mouse homologue of TPRD cDNA, mtprd, and examined its expression profile in mouse embryos. The gene was mapped to mouse chromosome 16C3.3-4, consistent with the location of DCR, and encodes 1,979 amino acid residues with 76% identity to TPRD. The mtprd protein has three units of the TPR motif with 91% homology to TPRD. The protein also has two regions homologous to several matrix proteins with 86 and 70% identities to those of TPRD. Several splicing variants of the 5' portion of the open reading frame of mtprd were identified by RT-PCR and sequencing of mRNAs. In situ hybridization showed that mtprd is ubiquitously expressed in mouse embryos but predominantly in the central nervous system, including the telencephalon, mesencephalon, and metencephalon. These results suggest that the TPRD gene is one of the genes responsible for not only the morphological anomalies but also the neurological abnormalities observed in Down syndrome. The presence of splicing variants indicates that the protein may also have several isoforms in mice.

Defective stimulation of thyroxine 5'-deiodinase activity by cold exposure and norepinephrine in brown adipose tissue of monosodium glutamate-obese mice.

In order to examine the possible change in the thyroid hormone metabolism in the monosodium glutamate (MSG)-obese mice, we determined the iodothyronine deiodinase activity of brown adipose tissue (BAT), liver and kidney of male and female mice. There was no significant difference in the type II thyroxine 5'-deiodinase (T4 5'DII) activity in BAT between MSG-obese and control mice when they were kept at the ambient temperature of 22 degrees C. T4 5'DII activity in BAT of control mice increased markedly after exposure to cold (4 degrees C) for 4 h; however, the extent of cold-induced increase in T4 5'DII activity in BAT of MSG-obese mice was greatly reduced. Injection of norepinephrine (NE) (0.8 mg/kg, s.c.) 4 h previously increased T4 5'DII activity in BAT of control mice, but NE-induced increase in T4 5'DII activity was also markedly reduced in BAT of MSG-obese mice. Both cold- and NE-induced increase in T4 5'DII activity was greater in female, although similar tendency was obtained in male mice. Type I 3,3',5'-triiodothyronine deiodinase (rT3 5'DI) activity of liver and kidney, and serum thyroxine (T4) and 3,5,3'-triiodothyronine (T3) levels in MSG-obese mice were essentially the same as those of the control male and female mice irrespective of cold exposure. These results suggest that defective stimulation of T4 5'DII activity of BAT by cold in the MSG-obese mice is due to deficient sympathetic input to BAT and/or to diminished response of BAT to NE, and may contribute to a possible cause of inability of MSG-obese mice to maintain body temperature under cold exposure.

Nitric oxide mediates down regulation of lipoprotein lipase activity induced by tumor necrosis factor-alpha in brown adipocytes.

We previously reported that tumor necrosis factor-alpha (TNF-alpha)/cachectin suppresses lipoprotein lipase activity and its gene expression in brown adipocytes differentiated in culture. Recent evidence suggests that the effect of TNF-alpha over various cells is related to the enhanced production of nitric oxide (NO). The present study examined whether the suppressive effect of TNF-alpha on lipoprotein lipase activity is mediated by production of NO in the brown adipocytes. A reverse transcription-polymerase chain reaction (RT-PCR) assay revealed that TNF-alpha caused a concentration- and time-dependent expression of inducible NO synthase in brown adipocytes. Increasing concentrations of TNF-alpha (0.5-50 ng/ml) for 24 h resulted in a concentration-dependent decrease in lipoprotein lipase activity with reciprocal increase in nitrite production in the medium. The suppressive effect of TNF-alpha on lipoprotein lipase activity was significantly prevented by NO synthase inhibitors, NG-nitro-L-arginine methyl ester (L-NAME) and aminoguanidine, but not by D-NAME, an inactive isomer. Furthermore, 8-bromoguanosine 3',5'-cyclic monophosphate, cell permeant cGMP, suppressed lipoprotein lipase activity and 1 H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1-one, a selective inhibitor for soluble guanylate cyclase, restored the TNF-alpha-suppressed lipoprotein lipase activity. These results suggest that TNF-alpha stimulates brown adipocytes to express inducible NO synthase, followed by production of NO, which in turn mediates the suppressive effect of TNF-alpha on lipoprotein lipase activity. The effect of NO is mediated, at least partly, through production of cGMP.

A novel method for making nested deletions and its application for sequencing of a 300 kb region of human APP locus.

We developed a novel in vitro method for making nested deletions and applied it to a large-scale DNA sequencing. A DNA fragment to be sequenced (up to 15 kb long) was cloned with a new vector possessing two unique Sfi I sites, digested by Sfi I and ligated to generate a large head-to-tail concatemer. The large concatemer was randomly fragmented by sonication and then redigested by Sfi I to separate insert and vector DNAs. The fragments of various length were then cloned into the other vector(s) specifically designed for selective cloning of insert-derived DNA fragments to generate a library of nested deletions. This method allowed a single person to generate >20 nested deletion libraries sufficient to cover 100 kb in a few days. We applied the method for sequencing of P1 clones and successfully determined the complete sequence of approximately 300 kb of the human amyloid precursor protein (APP) locus on chromosome 21 with a redundancy of 3.8, reasonably low cost and very few gaps remaining to be closed. Development of some new instruments and software is also described which makes this method more applicable for large-scale sequencing.

Iodothyronine deiodinases in a mammalian hibernator, the chipmunk (Tamias asiaticus).

We examined the activities of iodothyronine deiodinase, a key enzyme for thyroid hormone metabolism, in selected tissues of the chipmunk (Tamias asiaticus), a mammalian hibernator, of both sexes in the summer season. Reverse T3 5'-deiodinase (5'-D) activity was the highest in the liver followed by the kidney; T4 5'-D activity was the highest in brown adipose tissue (BAT) and T3 5'-deiodinase (5-D) activity was the highest in the testes followed by the brain. Distributions of three types of deiodinase activities in liver kidney BAT, and brain were comparable to other mammals reported, except that the type III deiodinase was unique in testes. The 5'-D activity of liver and kidney of chipmunks was 52% and 24%, respectively, of male rats and the 5-D activity of brain and testes of chipmunks was 227% and 567%, respectively of male rats. In addition, the cold exposure increased BAT 5'-D activity in chipmunks as reported in the ground squirrels. Our results indicated that tissue distribution of deiodinases and response to cold exposure in BAT in hibernators are similar to nonhibernators. However, there was a quantitative difference of rT3 5'-D and T3 5-D activities in some tissues between chipmunks and rats, indicating different local thyroid hormone metabolisms in hibernators and nonhibernators.

Cationic amino acid transporter-2 mRNA induction by tumor necrosis factor-alpha in vascular endothelial cells.

Nitric oxide (NO) synthesis may be coupled to the activity of the cellular L-arginine transporter, namely the cationic amino acid transporter. The present study examined tumor necrosis factor (TNF)-alpha-induced alterations in the gene expression of the cationic amino acid transporter (CAT) and NO production in human umbilical vein endothelial cells. In quiescent endothelial cells, CAT-1 mRNA expression, determined by reverse transcription-polymerase chain reaction, was dominant to that of CAT-2. TNF-alpha (10 ng/ml for 1-24 h) induced a time-dependent increase in CAT-2 but not CAT-1 expression. Moreover, TNF-alpha (1-30 ng/ml) treatment for 6 h induced a concentration-dependent increase in CAT-2 mRNA expression. The upregulation of CAT-2 expression by TNF-alpha was associated with enhanced nitrite accumulation in the culture medium (70% increase compared with vehicle-treated cells at 24 h). Thus, induction of the cationic amino acid transporter may constitute one mechanism for the TNF-alpha-induced NO production in human umbilical vein endothelial cells.

Tolerance to lipopolysaccharide-induced increase in vascular permeability in mouse skin.

We investigated whether tolerance develops to the lipopolysaccharide-induced increase in vascular permeability of mouse skin on pretreatment with Salmonella typhimurium lipopolysaccharide. Lipopolysaccharide-induced plasma extravasation was assessed by determining Pontamine sky blue dye accumulation in the skin where lipopolysaccharide was injected s.c. 2 h previously. When mice were pretreated with lipopolysaccharide (0.15 mg/kg i.p.), the dye leakage induced by s.c. challenge with lipopolysaccharide (400 micrograms/site) was significantly, inhibited for 2-24 h after pretreatment, indicating the development of lipopolysaccharide tolerance. At 4 h after lipopolysaccharide (0.15 mg/kg i.p.), the dose-response curve of dye leakage against the challenge dose of lipopolysaccharide shifted about 2-fold to the higher dose. The dye leakage induced by lipopolysaccharide was inhibited by pretreatment with lipopolysaccharide in a dose-dependent manner (0.05-0.15 mg/kg i.p.). Lipopolysaccharide tolerance was not seen in adrenalectomized mice. When mice were pretreated with lipopolysaccharide and NG-nitro-L-arginine methyl ester (L-NAME), a nitric oxide (NO) synthase inhibitor, at the same time, the hyporesponsiveness to lipopolysaccharide challenge disappeared. However, L-NAME was ineffective to inhibit the development of lipopolysaccharide tolerance when administered 24 h after lipopolysaccharide pretreatment or just before the lipopolysaccharide challenge. Tumor necrosis factor-alpha and interleukin-1 alpha but not interleukin-6 induced a similar hyporesponsiveness to lipopolysaccharide. These results suggest that tolerance develops to the lipopolysaccharide-induced increase in vascular permeability in mouse skin after a single lipopolysaccharide administration and that endogenous glucocorticoids and NO are necessary for induction of lipopolysaccharide tolerance. Hyporesponsiveness induced by lipopolysaccharide pretreatment may be mediated by production of some cytokines such as tumor necrosis factor-alpha or interleukin-1 alpha.

Identification and cloning of a novel cDNA belonging to tetratricopeptide repeat gene family from Down syndrome-critical region 21q22.2.

We identified and cloned a novel 9,078-bp cDNA, designated TPRDI, from the Down syndrome-critical region by exon trapping. The cDNA encodes a putative protein (TPRDI) of 2,025 amino acid residues. Two isoforms, TPRDII (8,992 bp) and TPRDIII (7,416 bp), were also isolated. TPRDII, which is probably an alternative splicing product from the TPRD gene transcript, encodes two large open reading frames (ORFs) of 200 amino acid residues and 1,792 amino acid residues, respectively. TPRDIII, which is probably generated by transcription from an alternative start site of the TPRD gene, encodes a putative protein of 1,715 amino acid residues (TPRDIII). Northern blot analysis revealed that TPRDI and its isoforms are present in 7-17 day mouse embryo and in all the human adult and fetal tissues examined. TPRDI has three units of a 34-amino-acid repeat similar to the tetratricopeptide repeat (TPR) motif, which may mediate interaction with various proteins. A larger ORF encoded by TPRDII also has three units of TPR motif, but TPRDIII has only two-thirds of this motif unit. Thus, the TPRD gene may belong to the TPR gene family. Near-central and C terminal regions of TPRDs showed some homology to several matrix proteins such as trichohyalin and bullous pemphigoid antigen. It is possible that the TPRD gene is one of the genes whose overexpression causes several morphological anomalies observed in Down syndrome.

Endothelin-1, but not endothelin-3, suppresses lipoprotein lipase gene expression in brown adipocytes differentiated in culture.

The effect of endothelins on lipoprotein lipase activity and lipoprotein lipase mRNA levels was studied in brown adipocytes differentiated in culture. Lipoprotein lipase activity was determined in two fractions; lipoprotein lipase released by heparin (10 IU/ml, 1 h) into the medium (heparin-releasable fraction) and lipoprotein lipase activity remaining in cells (extractable fraction). Time-course studies showed that endothelin 1 (10(-7) M) progressively decreased both lipoprotein lipase fractions (heparin-releasable, extractable), until nadir at 24 h. Endothelin-1 reduced both lipoprotein lipase activities (heparin-releasable, extractable) in a concentration-dependent manner, whereas endothelin-3 did not produce any significant changes in either of them. Northern blot analysis revealed that endothelin-1 (10(-7)-10(-11) M) caused a concentration-dependent decrease in lipoprotein lipase mRNA obtained from cells on day 9. Furthermore, pretreatment of brown adipocytes with endothelin ETA receptor antagonist FR139317 antagonized the endothelin-1-induced reduction of lipoprotein lipase activity and lipoprotein lipase mRNA. These results suggest that endothelin-1 decreases lipoprotein lipase activity by inhibiting the lipoprotein lipase gene expression in brown adipocytes differentiated in culture, possibly through endothelin ETA receptors on cell membranes. Because of marked reduction of lipoprotein lipase activity and its mRNA as a marker of adipogenic differentiation, endothelin-1 may have an inhibitory role in the differentiation of brown adipocytes.

Possible role of nitric oxide in 5-hydroxytryptamine-induced increase in vascular permeability in mouse skin.

In order to test the hypothesis that a 5-hydroxytryptamine (5-HT)-induced increase in vascular permeability results from a cascade triggered by activation of the synthesis of nitric oxide (NO), the vascular permeability was investigated using the Pontamine sky blue leakage method in male mice. Subcutaneous injection of 5-HT induced a dose-related increase of vascular permeability at the injection site. The vascular permeability induced by 5-HT was inhibited by pretreatment with intraperitoneal injection of ketanserin (5-HT2A antagonist) and methysergide (5-HT1/2A antagonist), less efficiently by 1-(2-methoxyphenyl)-4-[4-(2-phthalimido)butyl] piperazine (NAN-190) (5-HT1A antagonist), but not by granisetron (5-HT3 antagonist). Increase in vascular permeability induced by 5-HT was inhibited by concurrent intravenous administration of NO synthase inhibitors NG-nitro-L-arginine methyl ester (L-NAME) and methylene blue but not by the inactive enantiomer NG-nitro-D-arginine methyl ester (D-NAME). These results suggest that 5-HT increases vascular permeability by activating the 5-HT receptors and that endogenous NO is involved in this effect of 5-HT.

Possible involvement of L-arginine-nitric oxide pathway in modulating regional blood flow to brown adipose tissue of rats.

To evaluate whether the L-arginine-nitric oxide (NO) pathway is involved in the regulation of regional blood flow to brown adipose tissue (BAT), the effects of two specific NO synthase inhibitors, NG-nitro-L-arginine methyl ester (L-NAME) and NG-monomethyl-L-arginine (L-NMMA), on the blood flow to interscapular brown adipose tissue (IBAT) were studied in urethane-anesthetized rats. Regional blood flow in IBAT was measured with laser-Doppler flowmetry. An intravenous injection of L-NAME and L-NMMA, but not of either D-enantiomer, caused a transient and dose-dependent increase in IBAT blood flow. Dose-response curves for these NO synthase inhibitors showed that L-NAME was more potent than L-NMMA in increasing IBAT blood flow. We also observed a concomitant pressor effect accompanied by a slight decrease in heart rate following intravenous injection of L-NAME and L-NMMA. An elevation of IBAT blood flow and blood pressure induced by both L-NAME and L-NMMA was reversed by L-arginine in an enantiomerically specific manner. The increase in IBAT blood flow induced by NO synthase inhibitors was of shorter duration and less sensitive to L-arginine than the increase in blood pressure. Our results show that the IBAT blood flow is increased by inhibition of NO synthase and that the response of IBAT vasculature to NO synthase inhibitors is different from that of the resistance vessels which regulate blood pressure. The involvement of L-arginine-NO pathways in modulating microcirculation in IBAT is suggested.