Nifedipine inhibits activation of transcription factor NF-kappaB.

This study was performed to examine the effects of the calcium channel blockers, nifedipine, amlodipine, diltiazem, and verapamil on the activation of the transcription factor NF-kappaB. A549 cells, a human epithelium-like lung carcinoma cell line, were transfected with the NF-kappaB reporter plasmid, which contains the luciferase gene driven by promoters containing a TATA element and 5 copies of the kappaB cis-acting element, and co-transfected with 0.2 microg of pSV2neo vector using LipofectAMINE. Nifedipine significantly decreased the expression of luciferase protein stimulated with IL-1beta (1 ng/mL) compared with controls: 80+/-4% at 3 micromol/L, 47+/-2% at 10 micromol/L and 30+/-2% at 30 micromol/L (each, n=3, p<0.0001). The inhibitory effect of nifedipine on promoter activity was concentration-dependent, with a maximal effect obtained at 30 micromol/L. In contrast, high concentrations (30 micromol/L) of amlodipine, diltiazem or verapamil decreased promoter activity to only 89+/-3%, 90+/-3% or 87+/-2% of control, respectively. A comparable inhibitory effect of nifedipine was observed when cells were stimulated with tumor necrosis factor (TNF)-alpha (50 ng/mL), or phorbol 12-myristate 13-acetate (PMA, 100 ng/mL). Electrophoretic mobility shift assay by lipopolysaccharide stimulation, using the RAW 264.7 macrophage cell line, also showed inhibition of NF-kappaB activation by nifedipine in concentrations of 30 and 50 micromol/L. Nifedipine possesses the unique property of inhibiting NF-kappaB, which may be independent of its calcium channel blocking activity, and may, in part, explain its immunosuppressive effect.

Pimobendan inhibits the activation of transcription factor NF-kappaB: a mechanism which explains its inhibition of cytokine production and inducible nitric oxide synthase.

Pimobendan, an inhibitor of phosphodiesterase III with calcium sensitizing properties, inhibits the production of cytokines and nitric oxide. In the present study, the effects of pimobendan and other inotropic agents on the activation of transcription factor NF-kappaB were examined. Pimobendan significantly decreased the expression of luciferase protein in A549 cells transfected with the NF-kappaB reporter plasmid, stimulated with interleukin (IL)-1beta, tumor necrosis factor-alpha, or phorbol 12-myristate 13 acetate. However, high concentrations of amrinone, vesnarinone, or NKH 477 decreased promoter activity only slightly. Electrophoretic mobility shift assay also showed inhibition of NF-kappaB activation by pimobendan. Pimobendan possesses the unique property of inhibiting NF-kappaB, which may be independent of phosphodiesterase inhibition.

The Fcgamma receptor genotype as a risk factor for generalized early-onset periodontitis in Japanese patients.

BACKGROUND: Genetic polymorphisms of immunoglobulin G (IgG) Fc receptors (FcgammaR) were recently shown to be associated with recurrence rates of adult periodontitis (AP). The purpose of this study was to evaluate whether FcgammaR polymorphisms are also associated with generalized early-onset periodontitis (G-EOP) in Japanese patients. METHODS: Thirty-eight Japanese patients with G-EOP and 83 Japanese patients with AP were identified according to established clinical criteria, including measurements of probing depth, clinical attachment level, and alveolar bone level. FcgammaR genotypes for 3 bi-allelic polymorphisms were determined in these G-EOP and AP patients and 104 race-matched healthy controls by means of allele-specific polymerase chain reactions. RESULTS: There was a significant difference in the distribution of FcgammaRIIIb genotypes between G-EOP patients and healthy controls (P = 0.02). Additionally, a significant over-representation of FcgammaRIIIb-NA2 allele was observed in G-EOP patients as compared to AP patients and controls (P= 0.02, P= 0.009, respectively). Moreover, we found a strong association between G-EOP and the composite genotype comprising FcgammaRIIIb-NA2 and FcgammaRIIIa-158F (G-EOP versus controls: odds ratio 2.4, 95% CI 1.0-6.0, chi2 = 4.13, P= 0.04). CONCLUSIONS: This study indicates that the FcgammaRIIIb-NA2 allele and possibly FcgammaRIIIa-158F could be associated with susceptibility to G-EOP in Japanese patients.

Expression of human inducible nitric oxide synthase gene in T-cell lines infected with human T-cell leukemia virus type-I and primary adult T-cell leukemia cells.

We examined the expression of messenger RNA (mRNA) of the human inducible nitric oxide synthase (hiNOS) gene in a panel of human T-cell lines. Reverse transcriptase-polymerase chain reaction showed that human T-cell leukemia virus type-I (HTLV-I)-infected T-cell lines (MT-1, SLB-1, and C5/MJ) expressed mRNA for the hiNOS, but TL-Om1 or uninfected Jurkat, H9, and CCRF-CEM did not. The MT-1, SLB-1, and C5/MJ cell lines are infected with HTLV-I and express the viral transactivator Tax, whereas TL-Om1 cells, although derived from adult T-cell leukemia (ATL) leukemic cells, do not express Tax. There was, thus, a correlation between Tax and hiNOS mRNA expression. The transcriptional regulatory region of the hiNOS gene was activated by Tax in Jurkat, in which endogenous hiNOS is induced by Tax. Deletion analysis showed that the region of hiNOS encompassing nucleotides -159 to -111 contained the minimum Tax-responsive elements. Mutations in the NF-kappaB element at position -115 and -106 bp in the hiNOS promoter were still activated by Tax, and a Tax mutant defective for activation of the NF-kappaB pathway retained the ability to activate the hiNOS promoter. In addition, overexpression of the dominant-negative mutants of IkappaBalpha and I kappaBbeta failed to reduce Tax-induced activation of hiNOS gene. Furthermore, hiNOS mRNA was detected in leukemic cells from ATL patients. Our results show that the hiNOS promoter contains a minimum Tax-responsive element located between nucleotides -159 and -111, and imply that the expression of the hiNOS gene is involved in the pathogenesis of HTLV-I-associated diseases.

Expression of human inducible nitric oxide synthase is regulated by both promoter and 3'-regions.

One of the mechanisms underlying the induction of human inducible nitric oxide synthase (hiNOS) by cytokines is transcriptional regulation. However, previous reports suggest that in human cells, the cytokine activation of the hiNOS expression is accompanied by a drastic increase in cellular mRNA and protein content, but by little increment in transcriptional activity. We designed luciferase reporter constructs containing the hiNOS 5'-flanking region and 3'-untranslated region (UTR). The construct containing both 5'-flanking region and 3'-UTR plus its downstream canceled the constitutive activity of the reporter gene, and was more responsive to the cytokines. This is an initial attempt to highlight the important role played by the 3'-region of the hiNOS gene. Our studies provide the evidence that the cooperative interaction between the 5'-promoter and the 3'-region conveys marked induction of the gene in the presence of cytokines.

Deletion analysis of promoter elements of the Aspergillus oryzae agdA gene encoding alpha-glucosidase.

The nucleotide sequence of a 1.5-kb fragment of the promoter region of the Aspergillus oryzae agdA gene encoding alpha-glucosidase was determined. A comparison with the promoter regions of other Aspergillus amylase genes indicated that there are three highly conserved sequences, designated Regions I, II and III, located at -670 nt, -596 nt and -544 nt relative to the start codon, respectively. The function of these consensus sequences in the agdA promoter was investigated by deletion analysis of a promoter fusion with the Escherichia coli uidA gene, using the niaD homologous-transformation system. Deletion of the upstream half of Region III (IIIa; -544 to -529) resulted in a more than 90% reduction in GUS activity and abolished maltose induction, suggesting that Region IIIa is a functionally essential element for high-level expression and maltose induction. Deletion of Region I and the downstream half of Region III (IIIb; -521 to -511) resulted in a significant reduction in GUS activity, but did not affect maltose induction. This suggested that these two elements most likely contain sequences involved in efficient expression in cooperation with Region IIIa. In addition, deletion of a 340-bp region between Region IIIb and the putative TATA box resulted in a 2-fold increase in activity.

Human inducible nitric oxide synthase gene is transcriptionally regulated by nuclear factor-kappaB dependent mechanism.

We previously showed a sequence of the 5'-flanking region of the human inducible nitric oxide synthase (hiNOS) gene that included putative cis-acting elements. A reported plasmid containing the 5'-flanking region of the hiNOS gene upstream from its reporter gene was constructed, and then transiently or stably transfected into human cells known to express the hiNOS gene following cytokine stimulation. The transfected cells showed the inducibility of the reporter activity following interleukin-1beta stimulation. Reporter inducibility disappeared in cells transfected with a plasmid mutated in the putative nuclear factor (NF)-kappaB binding region. In addition the induction was inhibited by a treatment of anti-oxidant, pyrrolidinedithiocarbamate, known as an NF-kappaB inhibitor. Our results demonstrate that the promotor including the NF-kappaB region is functional and that the hiNOS gene is transcriptionally regulated via NF-kappaB activation in human cells.

Construction of a promoter probe vector autonomously maintained in Aspergillus and characterization of promoter regions derived from A. niger and A. oryzae genomes.

We used a plasmid carrying a sequence for autonomous maintenance in Aspergillus (AMA1) and the E. coli uidA gene as a reporter gene to search the A. oryzae and A. niger genomes for DNA fragments having strong promoter activity. Beta-glucuronidase (GUS)-producing A. oryzae transformants containing the No. 8AN derived from A. niger, or the No. 9AO derived from A. oryzae, were constitutive for the expression of the uidA gene when cultivated in the presence of a variety of carbon and nitrogen sources. When the GUS-producing transformants were grown in liquid culture, the No. 8AN showed an increase of approximately 3-fold in GUS activity compared to the amyB (alpha-amylase encoding gene) promoter. There was also a corresponding increase in the amount of GUS gene-specific mRNA. When these transformants were grown as rice-koji, the No. 8AN showed an increase of approximately 6-fold compared to the amyB promoter, and the amount of GUS protein produced also increased. These strong promoter regions might be applicable to the production of other heterologous proteins in Aspergillus species.

A method for the re-isolation of an autonomously replicating plasmid from Aspergillus transformants.

An autonomously replicating plasmid is expected to increase the frequency of Aspergillus transformation. To construct this type of plasmid, we developed a rapid method of re-isolating autonomously replicating plasmids from Aspergillus transformants. Transformants grown in MM medium under selective pressure for 1-2 days were converted to protoplasts with a cell wall lytic enzyme (e.g. Yatalase). The protoplasts were lysed with phenol/chloroform followed by precipitation with ethanol. The total DNA was treated with RNaseA, re-precipitated with PEG, and then used to transform E. coli. These re-isolated plasmids were mainly the plasmid monomer.

Transformation of intact Aspergillus niger by electroporation.

A new rapid transformation system for Aspergillus niger that uses electroporation to render intact germinating conidia permeable to DNA is described. The transformant colonies appeared earlier than transformants obtained by the protoplast-forming method. Without pretreatment of the conidia the transformation frequencies were 1.2 colonies per micrograms of integrative vector and 100 colonies per micrograms of plasmid DNA. When the conidia were treated with a dilute solution of fungal cell wall lytic enzyme, the frequency of transformation was increased by approx. 2-fold when using two vectors. Southern blot analysis of genomic DNA and restriction endonuclease-digested DNA from a random sample of transformants showed homologous and nonhomologous integration of the integrative vector into the genome, as is also observed with the protoplast-forming method. In transformation with the plasmid vector, the transformant DNA was shown to be mostly maintained in free form with minimal integration into the chromosome when transformed by either intact electroporation or the conventional method.

Promoter analysis of human inducible nitric oxide synthase gene associated with cardiovascular homeostasis.

We previously showed that interferon (IFN)-gamma inhibited the proliferation of rat vascular smooth muscle cells (VSMC) by generation of nitric oxide (NO) through the induction of an NO synthase (NOS) and cloned the rat inducible NOS cDNA in VSMC (VSM-NOS). To study the regulation of human inducible NOS (hiNOS) transcription in VSMC, we now cloned and sequenced a 2.9-kb fragment for the hiNOS gene containing a putative promoter, exon 1 and exon 2. The 5'-flanking region contains several consensus sequences for the binding of transcription factors involved in the inducibility of other genes by cytokines. These include IFN-gamma responsive element and NF-IL6 and NF-kappa B binding consensus sequences. Interestingly, hiNOS gene contains a shear-stress responsive element (GAGACC) which was also found to exist in human endothelial-type NOS but not in murine inducible NOS.

Evaluation of serum amyloid A protein as an acute-phase reactive protein in horses.

Serum amyloid A protein (SAA) was isolated from equine acute-phase serum by repeating Sephadex G-75 gel filtration 3 times. Quantitative measurement of equine SAA was performed by the single radial immunodiffusion technique with rabbit anti-equine SAA serum. In clinically normal horses, the SAA concentration remained relatively high from immediately after birth up to 1 week of age. After this the concentration showed periodic fluctiation in the range of approximately 13 to 30 micrograms/ml. The mean (+/- SD) concentration of SAA in foals (< or = 12 months old) and in adult horses (> or = 18 months old) was 19.37 +/- 9.41 and 21.53 +/- 9.81 micrograms/ml, respectively. In mares during the perinatal period, the SAA concentration remained stable and within the normal range for 4 months before parturition. After foaling, it increased quickly and reached a peak value of 136.78 +/- 56.74 micrograms/ml on day 3 postpartum, and then began to decrease at 2 weeks postpartum returning to within the normal range by 1 month postpartum. In horses with experimentally induced inflammation, the SAA concentration increased quickly, and reached the highest value, approximately 4 to 20 times higher than pre-treatment values, on day 2 after treatment. It then returned to the base line values within 10 days to 4 weeks, concurrent with the disappearance of local inflammatory signs. The SAA concentration was very high in most horses with clinical signs of inflammation. It was concluded from these data that equine SAA was a sensitive acute-phase reactive protein which increased in the early phase of various acute inflammations.

Cloning of inducible nitric oxide synthase in rat vascular smooth muscle cells.

We previously showed that interferon(IFN)-gamma inhibited the proliferation of rat vascular smooth muscle cells(VSMC) by generation of nitric oxide(NO) through the induction of an NO synthase(NOS). To identify the NOS in the VSMC at molecular level, we analyzed messenger RNA(mRNA) levels and primary structure of the novel NOS by cDNA cloning with application of polymerase chain reaction(PCR). mRNA of the NOS was induced and the level of induction was significantly increased by IFN-gamma in VSMC within a few hours. The amino acid sequence deduced from the cloned NOS cDNA was distinct from that of the previously reported constitutive types of NOSs, while highly similar to that of macrophage NOS. Cofactor binding regions were highly conserved among these NOSs. These findings show that the NOS is inducible and could regulate the proliferation of VSMC. Besides, this is the first report of cloning of the NOS in VSMC.

(6R)-5,6,7,8-tetrahydro-L-biopterin modulates nitric oxide-associated soluble guanylate cyclase activity in the rat cerebellum.

(6R)-5,6,7,8-Tetrahydro-L-biopterin (R-THBP) is a cofactor not only for aromatic amino acid hydroxylases in mammalian tissues but also for nitric oxide synthase (NOS) induced by endotoxins or cytokines in some kinds of cells. Recently it has been reported that nitric oxide (NO) has biological activity in endothelium and in brain as well. NO activates soluble guanylate cyclase (sGC). Superoxide reacts with NO easily and shortens the half-life of NO actions. We found, in a study using rat cerebellar cytosol fraction, that R-THBP itself did not directly activate sGC, but activated sGC at concentrations ranging from 0.1 to 10 microM only under NO generating conditions of activated NOS and in the presence of sodium nitroprusside. In addition, R-THBP (1 microM) did not alter the NOS activity, which was determined by L-citrulline formation. These results suggest that R-THBP may regulate sGC activity associated with NO formation in the central nervous system.

Interferon-gamma inhibits proliferation of rat vascular smooth muscle cells by nitric oxide generation.

Interferon (IFN)-gamma inhibited the proliferation of rat vascular smooth muscle cells (VSMC) and increased the cyclic GMP (cGMP) concentration in the cells. The dose dependencies of the two effects were similar (IC50 = 4 U/ml for the anti-proliferation and EC50 = 3 U/ml for cGMP formation) and the effect of IFN-gamma was enhanced by tumor necrosis factor-alpha treatment. Furthermore, NG-nitro-L-arginine, a nitric oxide (NO) synthase inhibitor, inhibited both activities induced by IFN-gamma. These findings show that the anti-proliferation and cGMP formation are closely related and that IFN-gamma inhibits the proliferation of rat VSMC by generation of NO through the induction of an NO synthase.

Regulation of serum glycosaminoglycan sulfotransferase activities: inhibition by sulfated glycosaminoglycans and activation by polyamines and basic peptides including a polylysine-containing segment of the c-Ki-ras 2 protein.

The regulatory mechanisms for the glycosaminoglycan sulfotransferases in fetal calf serum were investigated. The enzymes examined were those which transfer sulfate from 3'-phosphoadenosine 5'-phosphosulfate to 1) position 6 of the internal N-acetylgalactosamine units of chondroitin, 2) position 6 of galactose units of keratan sulfate, and 3) position 2 (an amino group) of glucosamine units of heparan sulfate. The former two enzymes were activated by spermidine, spermine, protamine, and poly L-lysine. All the enzymes were strongly inhibited by heparin and dextran sulfate, whereas only the chondroitin 6-O-sulfotransferase was inhibited by sulfated galactosaminoglycans. The inhibition of this enzyme by the sulfated glycosaminoglycans was abolished by polylysine, indicating that the activation by polylysine is partly due to the neutralization of endogenous acidic inhibitors, including sulfated glycosaminoglycans. Affinity chromatographic studies demonstrated that heparin specifically binds to the three enzymes, which have anionic isoelectric points, and that chondroitin 6-sulfate, spermine, and polylysine bind to the former two enzymes under physiological conditions. Thus, the activation by spermine and polylysine as well as the inhibition by sulfated glycosaminoglycans also appears to occur through their binding to the enzymes. Studies with synthetic lysine oligomers and an affinity-purified (approximately 700-fold) fraction containing the former two enzymes indicated that the pentamer is the minimum unit required for the activation. A synthetic peptide, containing six consecutive lysines at the carboxy terminus of the human c-Ki-ras 2 protein, also regulated the two enzyme activities at micromolar concentrations. The possible physiological implications of the observed effects of these regulatory substances on the glycosaminoglycan sulfotransferases are discussed in relation to glycosaminoglycan synthesis during the proliferation, differentiation, and transformation of cells. The possibility of sulfated glycosaminoglycans being enzyme regulators is also discussed.