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Biomedical subjects

J Inoue

Publications and source records attributed to J Inoue.

At least 55 records · Page 3Linked to original sources
PubMed1999

Immunomodulatory effect of a plasmid expressing CD40 ligand on DNA vaccination against human immunodeficiency virus type-1.

CD40 ligand is a costimulatory molecule which acts a potent immunomodulator. We found the mice inoculated with human CD40 ligand expression plasmid (pMEhCD40L) combined with human immunodeficiency virus type-1 (HIV-1) DNA vaccine exhibited both humoral and cellular antigen-specific immunological enhancement. The expression of hCD40L induced predominantly antigen-specific immunoglobulin G (IgG) antibody response while it failed to induce mucosal IgA response. Delayed-type hypersensitivity (DTH) and cytotoxic T lymphocyte (CTL) activity were induced in a dose-dependent manner. Examination of the relative levels of the two IgG subclasses showed that co-injection of pMEhCD40L enhanced IgG2a response without suppressing IgG1 response. Similarly, the expression of pMEhCD40L enhanced not only T helper 1 (Th1)- but also Th2-type cytokine production. In conclusion, co-inoculation of pMEhCD40L with DNA vaccine was shown to be a useful way to enhance CTL responses without suppressing the humoral immune response in acquired immune deficiency syndrome (AIDS) patients.

AIDS Vaccines↗
PubMed1999

IKK-i, a novel lipopolysaccharide-inducible kinase that is related to IkappaB kinases.

Using the suppression subtractive hybridization technique, we isolated a novel kinase, IKK-i, whose message is drastically induced by lipopolysaccharide (LPS) in the mouse macrophage cell line RAW264. 7. The predicted protein contains the kinase domain in its N-terminus, which shares 30% identity to that of IKK-alpha or IKK-beta. The C-terminal portion contains a leucine zipper and a potential helix-loop-helix domain, as in the case of IKK-alpha and IKK-beta. IKK-i is expressed mainly in immune cells, and is induced in response to proinflammatory cytokines such as tumor necrosis factor-alpha, IL-1 and IL-6, in addition to LPS. Overexpression of wild-type IKK-i phosphorylated serine residues Ser32 and Ser36 of IkappaB-alpha (preferentially Ser36), and significantly stimulated NF-kappaB activation. These results suggest that IKK-i is an inducible IkappaB kinase which may play a special role in the immune response.

Amino Acid Sequence↗
PubMed1999

A novel splicing isoform of mouse sterol regulatory element-binding protein-1 (SREBP-1).

We cloned a cDNA encoding the NH2-terminal portion of mouse SREBP-1. The deduced amino acid sequence was 76% and 90% identical to human and hamster SREBP-1, respectively. We found out a novel splicing isoform of mouse SREBP-1 that lacks 42 amino acid residues composing a PEST sequence observed in unstable proteins. It has been reported that SREBP-1 is rapidly turned over in the nucleus. Although this isoform was not a dominant isoform, it might be possible that the produced protein functions differently from other isoforms including a complete PEST sequence.

Alternative Splicing↗
PubMed1999

Syntheses and SH-enzyme inhibitory activities of new epoxysuccinic acid piperazine derivatives against mu-calpain and cathepsin B.

New chiral epoxysuccinic acid derivatives 5 approximately 23 bearing various amino acids and N-substituted piperazines were synthesized to evaluate their inhibitory activities against mu-calpain and cathepsin B. After screening these compounds, 1-[(2S,3S)-epoxysuccinyl-L-leucyl]-4-(2-chlorophenyl)piperazine 9 proved to exhibit fairly strong inhibitory activity against both cysteine proteases. L-Valyl derivative 19 exhibited selective inhibitory activity against cathepsin B in comparison with that against mu-calpain.

Calpain↗
PubMed1998

Proton transfer in benzyl alcohol dehydrogenase during catalysis: alternate proton-relay routes.

His51 in horse liver alcohol dehydrogenase (ADHE) has been proposed to act as a proton donor/acceptor in the NAD+/NADH-dependent oxidation/reduction of alcohol/aldehyde. The residue corresponding to His51 of ADHE is Val51 (Val45 in the protein sequence) in benzyl alcohol dehydrogenase (BADH) encoded by TOL plasmid pWW0. The 3-D structure of BADH modeled from the crystal structure of ADHE suggests that His47 (His41 in the protein sequence, corresponding to Arg47 in ADHE) of BADH would play the role of His51 in ADHE. To test this hypothesis, mutants of BADH, in which His47 was replaced by Gln(His47Gln) and/or Val51 was replaced by His (Val51His), were constructed. The kcat/K(m) value of the His47Gln mutant for benzyl alcohol was 125-fold lower than that of wild-type BADH, while the kcat/K(m) value of the His47Gln/Val51His double mutant was 12-fold higher than that of the His47Gln mutant. The kcat/K(m) value of the His47Gln mutant increased with increasing concentration of exogenous amines. These results suggest that His47 in wild-type BADH, exogenous amines in the His47Gln mutant, and His51 in the double mutant act as a general base catalyst during alcohol oxidation.

Alcohol Oxidoreductases↗
PubMed1998

Cloning and characterization of a cDNA encoding the human homolog of tumor necrosis factor receptor-associated factor 5 (TRAF5).

A cDNA encoding the human homolog of the tumor necrosis factor receptor-associated factor 5 (TRAF5) protein has been molecularly cloned from a cDNA library of Human Daudi B cell line. The sequence analysis revealed that the cDNA encoded a protein of 557 aa residues with a calculated molecular weight of 64,236. The encoded protein has typical structural characteristics shown in the TRAF family of proteins and binds to the cytoplasmic region of lymphotoxin-beta receptor more efficiently than to that of CD40 and CD30. The TRAF5 gene was mapped to the human chromosome 1q32.3-q41.1. Overexpression of human TRAF5 activates NF kappa B transcription factor in human 293T kidney cells. These results suggest that the human TRAF5 protein could be involved in the signal triggered by various members of the tumor necrosis factor receptor (TNFR) superfamily including CD40, CD30 and lymphotoxin-beta receptor.

Amino Acid Sequence↗
PubMed1998

Endothelin-1 inhibits endothelin-converting enzyme-1 expression in cultured rat pulmonary endothelial cells.

BACKGROUND: The lung expresses large amounts of endothelin-converting enzyme-1 (ECE-1), which catalyzes a step in the biosynthesis of potent vasoactive endothelin-1 (ET-1) from the inactive intermediate big ET-1. Because there has been no report concerning a possible relationship between ET-1 and ECE-1, we investigated the effects of ET-1 on ECE-1 expression in cultured rat pulmonary endothelial cells. METHODS AND RESULTS: ECE-1 messenger RNA (mRNA) and protein expression in cultured endothelial cells were assayed by Northern and Western blotting, respectively. Incubation with ET-1 for 6 hours caused a significant decrease in ECE-1 mRNA expression. The action of ET-1 on ECE-1 mRNA expression was antagonized by pretreatment with BQ788, a specific ETB receptor antagonist, but not by pretreatment with BQ123, a specific ETA receptor antagonist. The expression of ECE-1 protein was also inhibited at 6 hours after incubation with ET-1. The effects of ET-1 on ECE-1 mRNA and protein expression were shown to be mimicked by ionomycin, a calcium ionophore, but not by 12-O-tetradecanoylphorbol 13-acetate, a protein kinase C activator. CONCLUSIONS: The present results demonstrate that ET-1 suppressed ECE-1 protein levels by inhibiting ECE-1 mRNA expression through the ETB receptor, suggesting the existence of a feedback action of ET-1 on ECE-1 in pulmonary endothelial cells.

Animals↗
PubMed1998

Identification and expression of the Xenopus homolog of mammalian p100-NFkappaB2.

We have molecularly cloned a cDNA encoding a new Rel-related protein in Xenopus laevis. The product is most homologous to mammalian p100-NFkappaB2. Furthermore, the putative protein kinase A-phosphorylation site (RRPS), which is found in most of the Rel family proteins and is replaced by KRKR in mammalian p100, is also replaced by KRKK in our clone, indicating that our cDNA most likely encodes the Xenopus p100 (Xp100). Like mammalian p52, a processed product of p100, Xp52 alone binds to the kappaB site but does not activate transcription, while the XRelB/Xp52 heterodimer activates transcription, which is inhibited by the carboxyl-terminal half of Xp100 (XIkappaBdelta). Xp100 transcripts are present at all stages of oocyte maturation and in all adult tissues examined. Xp100 transcripts decrease at the gastrula stage and resume their expression at the neurula stage, which is different from other Xenopus rel family. Xp100 is highly expressed in somitogenic mesoderm at the neurula stage, while in the gastrula and tailbud stages, Xp100 transcripts are not localized to restricted regions. These results suggest that Xp100 could be involved in the late-stage development of Xenopus laevis, especially in the maturation of somites.

Amino Acid Sequence↗
PubMed1998

Molecular analysis of constitutive IL-1alpha gene expression in human melanoma cells: autocrine stimulation through NF-kappaB activation by endogenous IL-1alpha.

Constitutive production of/and acquired resistance to anti-proliferative cytokines are implicated in pathogenesis and progression of human melanoma cells. Human melanoma cells A375-C6 are sensitive to interleukin 1 (IL-1) anti-proliferative effect and do not produce IL-1. After long period of culture we have obtained cells which acquired resistance to IL-1. The resistant cells exhibited constitutive production of IL-1α. To analyse the mechanisms that lead to the expression of IL-1α in the cells, we transfected of the resistant clone A375-R8 with CAT (chloramphenicol acetyltransferase) expression plasmids linked to a 5'-flanking deletion mutants of the human IL-1α gene. Two nucleotide regions (--103 to --70 bp) and (--70 to --47 bp) from the start of the first exon appeared to contain a positive regulatory element(s) while the one --421 to --103 bp contained a negative regulatory element(s).The --103 to --70 bp region contained the consensus NF(-k)B (nuclear factor-kB) binding motif.(Immunofluorescence analysis revealed that NF-kB is activated in A375-R8 cells. IL-1 receptor antagonist (IL-1Ra) decreased the level of IL-1α mRNA and production of IL-1α. IL-1Ra also inhibited the localization of p65 in the nuclei and CAT activity in transfectants with the plasmids containing NF-kB binding motif. These results indicate that endogenous IL-1α stimulates the gene expression and production of IL-1α in an autocrine manner through activation of NF-kB.

Autocrine Communication↗
PubMed1998

Effect of sorbitol dehydrogenase inhibition on sugar cataract formation in galactose-fed and diabetic rats.

Several recent studies with the sorbitol dehydrogenase inhibitors 4-[4-(N,N-dimethylsulfamoyl)-piperazino]-2-methylpyrimidine, SDH-1, and its active metabolite 4-[4-(N, N-dimethylsulfamoyl)piperazino]-2-hydroxymethylpyrimidine , SDH-2, suggest that inhibition of sorbitol dehydrogenase may be beneficial in delaying the onset of diabetic complications due to their ability to ameliorate redox changes associated with polyol metabolism. To compare the relative importance of sorbitol dehydrogenase versus aldose reductase inhibition on sugar cataract formation, cataract formation was monitored in 50% galactose-fed and diabetic rats treated with/without the sorbitol dehydrogenase inhibitors SDH-1 or SDH-2 or the aldose reductase inhibitors AL 1576 or Ponalrestat. For these studies, diabetes was induced in young 50 g rats with streptozotocin while galactosemia was produced by feeding a diet containing 50% galactose. Inhibitors were administered in the diet with the diet containing 0.06% (w/w) of the sorbitol dehydrogenase inhibitors or Ponalrestat, and 0.0125% (w/w) of AL 1576. Cataract formation was monitored by hand-held slit lamp and polyol levels were measured by gas chromatography. Sugar cataract formation was accelerated in diabetic rats treated with sorbitol dehydrogenase inhibitors while no difference in cataract formation was observed in galactose-fed rats treated with/without SDH inhibitors. Cataract formation was inhibited in both diabetic and galactosemic rats by either Ponalrestat or AL 1576. These results support the concept that sugar cataract formation is initiated by the aldose reductase catalysed intracellular accumulation of polyols and that these sugar cataracts can be prevented through inhibition of aldose reductase.

Aldehyde Reductase↗
PubMed1998

Interactions between NFkappaB and its inhibitor ikappaB: biophysical characterization of a NFkappaB/ikappaB-alpha complex.

The N-terminal domain (1-318 amino acids) of mouse NFkappaB (p65) has been purified to homogeneity from the soluble fraction of Escherichia coli cells expressing this protein. Its complex with a full-length ikappaB-alpha (MAD3, 1-317 amino acids) molecule was generated by binding the E. coli-derived ikappaB-alpha to the purified NFkappaB and purifying the complex by sequential chromatography. The stoichiometry of NFkappaB to ikappaB in the complex was determined to be 2 to 1 by light scattering and SDS-polyacrylamide gel electrophoresis. The secondary structure of the NFkappaB (p65) determined by Fourier-transform infrared (FTIR) spectroscopy is in good agreement with that of the p50 in the crystal structure of the p50/DNA complex, indicating that no significant structural change in NFkappaB occurs upon binding of DNA. The FTIR spectrum of the NFkappaB/ikappaB complex indicates that its secondary structure is composed of 17% alpha-helix, 39% beta-strand, 18% irregular structures, and 26% beta-turns and loops. By comparing these data to the FTIR data for NFkappaB alone, it is concluded that the ikappaB (MAD3) in the complex contains 35% alpha-helix, 27% beta-strand, 22% irregular structures, and 16% beta-turns and loops. Circular dichroism (CD) analysis of a shorter form of ikappaB (pp40) indicates that it contains at least 20% alpha-helix and that the ikappaB subunit accounts for nearly all of the alpha-helix present in the NFkappaB/ikappaB complex, consistent with the FTIR results. The stabilities of NFkappaB, ikappaB, and their complex against heat-induced denaturation were investigated by following changes in CD signal. The results indicate that the thermal stability of ikappaB is enhanced upon the formation of the NFkappaB/ikappaB complex.

Animals↗
PubMed1998

A novel domain in the CD30 cytoplasmic tail mediates NFkappaB activation.

About 100 amino acid residues in the C-terminal region are conserved among human, rat and murine CD30, a member of the tumor necrosis factor receptor (TNFR) superfamily, and can be separated into three subdomains with relatively higher conservation (D1, D2 and D3). Activation of NFkappaB by CD30 was shown to be mediated through interaction of TNFR-associated factor (TRAF) 1, 2 and 5 with the D2 and D3 subdomains. However, the function of the other conserved subdomain, D1, remained to be determined. Deletion of the D2 and D3 subdomains abolished interactions with TRAF2 and 5 but it did not affect NFkappaB activation. Reporter gene assays using deletion and mutant constructs of CD30 revealed that the D1 subdomain is sufficient for NFkappaB activation, without interaction with TRAF2 or 5, and that each subdomain alone can activate NFkappaB. Electrophoretic mobility shift assays revealed constitutive and CD30-induced NFkappaB activation in stable transformants of 293 cells expressing CD30 or a deletion mutant lacking D2 and D3 subdomains. Deletion of C-terminal 19 amino acid residues of the D1 subdomain abolished activation of NFkappaB. Substitution of alanine for one of the two threonine residues (amino acid position 524 and 529), one of which is a potential phosphorylation site in the D1 subdomain, also abolished the NFkappaB activation. Overexpression of the TRAF domain of TRAF2 or 5 had a dominant negative effect on the NFkappaB activation mediated by the D1 subdomain, thereby suggesting involvement of TRAF proteins in the signaling. Thus, the C-terminal 100 amino acid region of CD30 is composed of three independent functional subdomains, two of which contain binding sites for TRAF proteins. A novel domain in the cytoplasmic tail mediates NFkappaB activation, without direct interaction of TRAF2 or 5. Our observations suggest involvement of an unknown TRAF protein(s) in the signal transduction pathway of CD30.

Binding Sites↗
PubMed1998

Multiple DNA elements for sterol regulatory element-binding protein and NF-Y are responsible for sterol-regulated transcription of the genes for human 3-hydroxy-3-methylglutaryl coenzyme A synthase and squalene synthase.

The expression of the human SREBP-2 gene is transcriptionally regulated in a cooperative manner by sterol regulatory element-binding proteins (SREBPs) and the general transcription factor NF-Y [Sato, R., Inoue, J., Kawabe, Y., Kodama, T., Takano, T., and Maeda, M. (1996) J. Biol. Chem. 271, 26461-26464]. To understand the sterol-dependent transcriptional regulation by these factors in detail, we have examined the regulation of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) synthase and squalene synthase genes, whose promoters have multiple potential sterol regulatory elements (SRE, SREBP binding site) and NF-Y binding sites. The promoter of the human HMG CoA synthase gene was cloned, sequenced, and functionally characterized by means of reporter gene assays. The results indicate that an inverted CCAAT box, two SRE motifs and two Sp1 sites localized in a 90-bp region coordinately regulate the transcription. In the case of the human squalene synthase promoter, two SRE motifs and an inverted CCAAT box between the motifs localized in a 51-bp region are responsible for the sterol-regulated transcription of the gene. Gel mobility shift assay reveals that these two inverted CCAAT boxes are recognized by NF-Y. The involvement of multiple responsive elements in the transcription of HMG CoA synthase and squalene synthase seems to induce a higher level of sterol-dependent regulation (3.5 to 5. 8-fold) compared with that of the SREBP-2 promoter, which contains a single pair of SRE motif and CCAAT box (1.8 to 2.6-fold). Reporter gene assays using constructs containing various nucleotide spacing lengths between the SRE motif and the CCAAT box demonstrate that the 16 to 20-bp spacing range is required for maximal transcriptional regulation. These results agree with the findings that the distances between the two motifs in the known sterol responsive elements in several genes, including the human HMG CoA synthase and squalene synthase genes, are in this range.

Base Sequence↗
PubMed1998

Genetic analysis of the epithelial sodium channel in Liddle's syndrome.

BACKGROUND: Liddle's syndrome is an autosomal inheritable disorder that causes hypertension due to excess function of sodium channel. OBJECTIVE: To analyze the DNA sequence of the amiloride-sensitive epithelial sodium channel (ENaC) in three patients who had low-renin hypertension with hypokalemia. The patients included a 24-year-old woman and her 20-year-old brother whose mother was hypertensive. The third patient was a 15-year-old girl with no family history of hypertension. METHODS: The DNA sequence of the ENaC was analyzed as follows. Venous blood samples were collected from the patients and total genomic DNA was prepared by standard methods. Specific primers were used for direct polymerase chain reaction; one set of primers for amplifying the C terminus (codon 523-638) of the , subunit of ENaC, and two sets of primers for amplifying the C terminus (codons 525-587 and 568-650) of the y subunit of ENaC. Polymerase chain reaction products were purified and subjected to direct DNA sequence analysis. RESULTS: Direct sequence analysis demonstrated the presence of a single-base substitution in one segment of the 0 subunit of ENaC, a C-T transition that changed the encoded Pro (CCC) at codon 616 to Ser (TCC) in the siblings (cases 1 and 2). In case 3, we found a missense mutation of Pro (CCC) to Leu (CTC) at codon 616. Case 3 is considered to be sporadic, since DNA sequencing of the PY motif of her parents gave normal results. CONCLUSIONS: The DNA sequences of the ENaC in three patients with Liddle's syndrome were analyzed. In one family case, we found a new missense mutation of Pro (CCC) to Ser (TCC) at codon 616 in the 0 subunit of ENaC. A genetic analysis of the amiloride-sensitive epithelial sodium channel is recommended in assessing patients with low-renin, salt-sensitive hypertension whose blood pressure is not responsive to spironolactone treatment.

Adolescent↗
PubMed1998

New diagnostic peritoneal lavage criteria for diagnosis of intestinal injury.

BACKGROUND: Although diagnostic peritoneal lavage (DPL) is a well-established, reliably objective method of diagnosis of intraperitoneal injury, it is too sensitive to be used as an indicator for emergency celiotomy. Therefore, since the development of ultrasonography and advanced computed tomographic scanners, the role of DPL has been markedly reduced. Despite such remarkable advances, however, radiologic diagnosis of intestinal injury cannot always provide definitive results, and DPL may still be valuable in such instances. We have developed a new DPL criteria specifically designed to aid in the diagnosis of intestinal injury and have evaluated its effectiveness. METHODS: From August 1988 to December 1995, we performed DPL in 250 patients with blunt abdominal trauma and analyzed the diagnostic accuracy of our new criteria. We used the standard quantitative white blood cell (WBC) criterion for detection of intestinal injury supplemented by a positive-negative borderline adjusted to WBC > or = red blood cell (RBC)/150, where RBC > or = 10 x 10(4)/mm3. RESULTS: Our criteria had a diagnostic sensitivity of 96.6% and a specificity of 99.4% for intestinal injury after exclusion of 57 patients in whom DPL was performed within 3 hours or after 18 hours from the time of injury. In 133 patients with hemoperitoneum, emergency celiotomy was performed in only 48; the remaining 85 patients with negative DPL based on the WBC criterion avoided surgery, and conservative management resulted in no complications. CONCLUSION: With the proposed criteria, DPL can be used to diagnose or exclude intestinal injury even in the presence of hemoperitoneum.

Abdominal Injuries↗
PubMed1998

Induction of apoptosis in human pancreatic carcinoma cells by a synthetic bleomycin-like ligand.

Histidine-pyridine-histidine-3 (HPH-3) is an oxygen-activating ligand based on the structure of bleomycin. HPH-3 induced the death of human pancreatic adenocarcinoma AsPC-1 cells in 24 h, causing apoptotic morphology and internucleosomal degradation of DNA. HPH-3-induced cell death was not inhibited by antioxidants such as reduced glutathione and N-acetylcysteine, whereas hydrogen peroxide-induced cell death was inhibited by them, indicating that hydrogen peroxide is not involved in the induction of apoptosis by HPH-3. Induction of apoptosis by HPH-3 was inhibited by zinc and copper ions, indicating that chelation with ferrous ion is responsible for induction of apoptosis, as is the case in chelation by bleomycin to cleave DNA. Bleomycin A2 and its fragment having no DNA-binding region, glycopeptide-3, did not induce apoptosis in AsPC-1 cells. Bleomycin A2 induced G2/M block in flow-cytometric analysis, but HPH-3 did not and instead induced an apoptotic pre-G1 peak. Thus, HPH-3 induced apoptosis in human pancreatic carcinoma cells, which is a unique characteristic among bleomycin-related compounds.

Adenocarcinoma↗