Hypoxia-inducible factor 1α promotes interleukin 1ß and tumour necrosis factor α expression in lipopolysaccharide-stimulated human dental pulp cells.

AIM: To elucidate the role of HIF1α in pro-inflammatory cytokine mRNA expression from lipopolysaccharide (LPS)-stimulated human dental pulp cells (hDPCs). METHODOLOGY: mRNA expression of interleukin (IL) 1ß and tumour necrosis factor (TNF) α in LPS-stimulated hDPCs was determined by quantitative RT-PCR. Expression of nuclear factor kappa B (NFκB) p65 and phospho-NFκB p65 was analysed by Western blotting. Activation of NFκB signalling was measured by luciferase assay using a reporter vector containing an NFκB response element. Enforced expression of HIF1α was induced by transfection of expression vectors with native or constitutively active forms of HIF1α. Expression of HIF1α protein in hDPCs was evaluated by immunocytochemistry and Western blotting. One-way analysis of variance and the Tukey-Kramer test were performed to determine a significant difference (P < 0.05). RESULTS: mRNA expression of IL1ß and TNFα, protein expression of phospho-NFκB p65 and LPS-induced NFκB signalling activity were promoted in low oxygen conditions (1% O2 ; P < 0.05). These findings were replicated following enforced expression and stabilization of HIF1α in hDPCs. Dimethyloxalylglycine, an inhibitor of prolyl hydroxylase (a HIF1α degrading enzyme), promoted IL1ß and TNFα mRNA expression and NFκB signalling in LPS-stimulated hDPCs (P < 0.05). HIF1α expression was detected in hDPCs cultured in low oxygen conditions (1% O2 ). LPS stimulation further enhanced HIF1α expression in hDPCs, especially within their nuclei. CONCLUSION: HIF1α promoted mRNA expression of IL1ß and TNFα via NFκB signalling in LPS-stimulated hDPCs, suggesting that HIF1α is involved in the progress of inflammation in dental pulp.

Mineral trioxide aggregate suppresses pro-inflammatory cytokine expression via the calcineurin/nuclear factor of activated T cells/early growth response 2 pathway in lipopolysaccharide-stimulated macrophages.

AIM: To elucidate mechanisms by which mineral trioxide aggregate (MTA) suppresses pro-inflammatory cytokine mRNA expression in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. METHODOLOGY: Mineral trioxide aggregate extracts were prepared by immersing set ProRoot MTA in culture medium. RAW264.7 cells were cultured in the presence of LPS and MTA extracts. mRNA expression levels of interleukin (IL)-1α, IL-6, early growth response 2 (Egr2), suppressor of cytokine signalling 3 (Socs3) and IL-10 were quantified with reverse transcription-quantitative polymerase chain reaction. Phosphorylation of nuclear factor-kappa B (NF-κB) p65 in RAW264.7 cells was analysed by Western blotting. Intracellular calcium imaging was performed with Fluo-4 AM. The activity of nuclear factor of activated T cells (NFAT) was determined by luciferase assays. Enforced expression and silencing of Egr2 in RAW264.7 cells were carried out using an expression vector and specific RNAi, respectively. In vivo kinetics of Egr2+ cells in MTA-treated rat molar pulp tissues were examined using immunohistochemistry. Data were analysed by one-way analysis of variance, followed by the Tukey-Kramer test (P < 0.05). RESULTS: Exposure to MTA extracts resulted in reduced mRNA expression levels of IL-1α and IL-6, as well as reduced expression of phosphorylated NF-κB, in LPS-stimulated RAW264.7 cells. Exposure to MTA extracts induced Ca2+ influx, which was blocked by NPS2143, an antagonist of calcium-sensing receptor (CaSR); Ca2+ influx then triggered activation of calcineurin/NFAT signalling and enhanced mRNA expression of Egr2. Enforced expression of Egr2 in RAW264.7 cells promoted the expression of both IL-10 and Socs3. In vivo application of MTA onto rat molar pulp tissue resulted in the appearance of Egr2-expressing cells that coexpressed CD163, a typical M2 macrophage marker. CONCLUSIONS: Mineral trioxide aggregate extracts induced downregulation of IL-1α and IL-6 in LPS-stimulated RAW264.7 cells via CaSR-induced activation of calcineurin/NFAT/Egr2 signalling and subsequent upregulation of IL-10 and Socs3.

Requirement for transglutaminase in the activation of latent transforming growth factor-beta in bovine endothelial cells.

A hitherto unknown function for transglutaminase (TGase; R-glutaminyl-peptide: amine gamma-glutamyltransferase, EC 2.3.2.13) was found in the conversion of latent transforming growth factor-beta (LTGF-beta) to active TGF-beta by bovine aortic endothelial cells (BAECs). The cell-associated, plasmin-mediated activation of LTGF-beta to TGF-beta induced either by treatment of BAECs with retinoids or by cocultures of BAECs and bovine smooth muscle cells (BSMCs) was blocked by seven different inhibitors of TGase as well as a neutralizing antibody to bovine endothelial cell type II TGase. Control experiments indicated that TGase inhibitors and/or a neutralizing antibody to TGase did not interfere with the direct action of TGF-beta, the release of LTGF-beta from cells, or the activation of LTGF-beta by plasmin or by transient acidification. After treatment with retinoids, BAECs expressed increased levels of TGase coordinate with the generation of TGF-beta, whereas BSMCs and bovine embryonic skin fibroblasts, which did not activate LTGF-beta after treatment with retinoids, did not. Furthermore, both TGase inhibitors and a neutralizing antibody to TGase potentiated the effect of retinol in enhancing plasminogen activator (PA) levels in cultures of BAECs by suppressing the TGF-beta-mediated enhancement of PA inhibitor-1 (PAI-1) expression. These results indicate that type II TGase is a component required for cell surface, plasmin-mediated LTGF-beta activation process and that increased expression of TGase accompanies retinoid-induced activation of LTGF-beta.

Involvement of innate immunity in the pathogenesis of intestinal Behçet's disease.

The involvement of excessive T helper 1 (Th1) cell functions in the pathogenesis of Behçet's disease (BD) has been reported. We therefore studied Toll-like receptor (TLR)-expressing cells, which play important roles in innate immunity in patients with BD. Peripheral blood mononuclear cells (PBMC) of BD and healthy controls, and tissue specimens of intestinal BD and Crohn's disease (CD) were analysed for messenger RNA (mRNA) and protein expressions by reverse transcription-polymerase chain reaction and immunostaining respectively. PBMC of BD expressed TLR-2 and TLR-4 mRNA almost comparable with healthy controls. Intestinal lesions of BD expressed TLR-2 and TLR-4 mRNA consistently. In contrast, TLR-4 mRNA was expressed preferentially and TLR-2 mRNA was expressed less frequently in CD lesions. In intestinal samples of BD, TLR-2 and TLR-4 mRNA were detected in ileocaecal ulcer lesions, but not in unaffected sites of the same sample, indicating the association of the TLR expression with the disease manifestation of intestinal BD. TLR-2-expressing cells which were simultaneously cluster of distribution (CD)68-positive produced interleukin (IL)-12 in the lesions, indicating the participation of TLR-2-expressing cells in the Th1 skewed responses in vivo. As a possible ligand of TLR-2, in BD self-heat shock protein 60 was expressed in peripheral blood lymphocytes and intestinal tissues. Collectively, TLR-2-expressing cells as well as TLR-4-expressing cells accumulated in the intestinal lesions of BD. IL-12 produced by TLR-2-expressing cells may contribute to the induction of Th1-dominant immune responses in intestinal BD.

Highly elevated ultraviolet-induced mutation frequency in isolated Chinese hamster cell lines defective in nucleotide excision repair and mismatch repair proteins.

We have isolated N-methyl-N'-nitro-N-nitrosoguanidine-resistant cell lines from 43-3B Chinese hamster ovary cells, which are deficient in the ERCC1 gene involved in nucleotide excision repair. By Western blotting analysis, we found cell lines that are deficient or decreased in the amount of MSH6, or PMS2, or MSH2 proteins. Cell extracts of these cell lines show reduced efficiency of G:T mismatch repair activity. Compared with 43-3B, these cell lines exhibit highly elevated UV-induced mutation rates, indicating that mammalian mismatch repair can suppress UV-induced mutagenesis and may play a role in the fidelity of DNA replication at the sites of UV damage.

Disseminated Metastatic Tissue Calcification After Orthotopic Liver Transplantation: A Case Report.

BACKGROUND: Hypercalcemia has been observed in patients after liver transplantation. However, it is rare that the hypercalcemia induced disseminated tissue calcification and heart failure. CASE REPORT: We report a rare case of heart failure caused by disseminated metastatic tissue calcification that involved extensive progressive myocardial calcification after liver transplantation. A 20-year-old man with end-stage liver disease due to biliary atresia underwent ABO-incompatible living donor liver transplantation. After successful transplantation, he suffered from antibody-mediated rejection. Subsequently, ABO-matched cadaveric liver retransplantation was successfully performed. Hypercalcemia developed gradually following the second transplantation. His serum calcium level increased to 18.3 mg/dL with sudden onset of ventricular tachycardia. Although he was resuscitated with a cardiopulmonary support device, he died of heart and liver failure. Histopathologic examination revealed systemic disseminated metastatic tissue calcification, including massive myocardial calcification. CONCLUSION: Progressive worsening of hypercalcemia resulted in disseminated metastatic tissue calcification and massive metastatic myocardial calcification, which led to heart failure after liver transplantation. Because hypercalcemia after liver transplantation can cause fatal tissue calcification, early intervention for hypercalcemia should be considered.

Up-regulation of elafin/SKALP gene expression in psoriatic epidermis.

The expression of mRNA for elafin/SKALP, an inhibitor of leukocyte elastase and proteinase 3, in human normal and psoriatic epidermis was examined by in situ hybridization. In normal epidermis, elafin/SKALP mRNA was detected in the granular layer, but not in the spinous or basal layers. In fully developed psoriatic lesions, elafin/SKALP mRNA was found in the suprabasal layers of the ridges, and in the upper two thirds of the stratum malpighii at the elongated rete ridges. Intense staining was noted near the subcorneal microabscess in psoriasis vulgaris and under the subcorneal pustule in localized pustular psoriasis. In the marginal psoriatic epidermis, elafin/SKALP mRNA was expressed from the middle or upper spinous layer to the subcorneal layer, and the cells expressing elafin/SKALP mRNA increased especially under the parakeratotic corneal layer intermingled with pyknotic nuclei of neutrophils. These findings suggest that the induction of elafin/SKALP gene expression is related closely to the infiltration of neutrophils into the epidermis in psoriasis and plays an important role in protecting the skin components against the tissue damage caused by the infiltrated leukocytes.

HNF-1 regulates the liver-specific transcription of the chipmunk HP-20 gene.

The chipmunk hibernation-specific protein HP-20 is a component of the 140 kDa complex that drastically decreases in the blood during hibernation, and its gene is expressed specifically in the liver. To reveal molecular mechanisms underlying the liver-specific transcription of the HP-20 gene, we isolated chipmunk HP-20 genomic clones. The HP-20 gene spans approximately 6 kb, and consists of three exons. The transcription start site, as determined by 5' RACE-PCR analysis, was found to be 160 bp upstream of the translation initiation codon. Transient transfection studies in HepG2 cells revealed that the 57 bp 5' flanking sequence was sufficient for the liver-specific promoter activity. A database search revealed that this region contains a potential binding site for hepatocyte nuclear factor-1 (HNF-1). In a gel retardation assay, in vitro-synthesized HNF-1 bound to the 5' flanking sequence from -52 to -26. A similar shifted band was also observed with HepG2 nuclear extracts, and this complex was super-shifted by an anti-(HNF-1) Ig. When transfected into COS-7 cells, HNF-1 transactivated transcription from the HP-20 gene promoter, and this activity was abolished by a mutation of the HNF-1 binding site, indicating that HNF-1 plays an important role in HP-20 gene expression.

Behavioral stimulation without alteration of beta and 5-HT receptors and adenylate cyclase activity in rat brain after chronic sertraline administration.

Effects of chronic treatment with selective 5-HT reuptake inhibitors (SSRIs) on the monoaminergic functions have not been much investigated in compared with tricyclic antidepressants. Therefore, we compared the effects of 3-week treatment with sertraline, a potent SSRI, to those of imipramine (10 mg/kg, IP, twice a day), on monoamine receptors and adenylate cyclase (AC) activity in rat brain. Two-week treatment with both sertraline and imipramine reduced immobility in the water wheel test to the comparable extent. Sertraline treatment did not affect Kd and Bmax of [3H]CGP12177 and [3H]ketanserin bindings or cAMP, accumulation by norepinephrine, isoproternol, 5'-guanylylimidodiphosphate [Gpp(NH)p] and forskolin in the cortical membrane compared with vehicle-treated rats. On the other hand, imipramine treatment decreased Bmax of both bindings and norepinephrine- or isoproternol-stimulated cAMP accumulation. Treatment with either antidepressant induced no apparent changes in [3H]8-OH-DPAT [2-(N, N-dipropylamino)-8-hydroxy-1,2,3,4-tetrahydronaphthalene] binding in the hippocampal membrane. These results suggested that chronic treatment of sertraline induced little effect on monoamine receptors and AC activity in the brain and that the alteration of these functions may not be primarily involved in antidepressive effects of antidepressants, at least of SSRIs.

The molecular cloning and expression of alpha 2,8-sialyltransferase (GD3 synthase) in a rat brain.

We have cloned the cDNA for a GD3 synthase (alpha-2,8-sialyltransferase, [EC 2.4.99.8]) from a rat embryonic brain cDNA library. Mammalian cells transfected with the cloned cDNA expressed GD3 on the cell surface and showed GD3 synthase activity. The deduced protein (342 amino acid residues) was predicted to have a type II membrane topology containing the "sialyl motif" and was found to be 91% similar to its human homologue. Analysis of the acceptor specificity of GD3 synthase protein indicated that this enzyme catalyzed the biosynthesis of GT1a and GQ1b as well as GD3. Northern blot analyses showed that the GD3 synthase gene is preferentially transcribed in the brain and the spleen. The expression of GD3 synthase mRNA was developmentally regulated, with the highest level in the brain during embryonic days 15 to 18. In situ hybridization analyses demonstrated that the GD3 synthase is strongly expressed in the ventricular/subventricular zone of the embryonic rat brain and retina. In the adult rat, GD3 synthase mRNA was detected in the cerebral cortex, hippocampus, thalamus, and cerebellum. These studies show that the spatio- and stage-restricted expression of GD3 in the developing rat brain may be regulated in part by the level of GD3 synthase mRNA.

The aconitase of yeast. II. Crystallization and general properties of yeast aconitase.

Yeast aconitase [citrate (isocitrate) hydro-lyase, ED 4.2.1.3], inductively formed by Candida iipolytica in the presence of fluoroacetate, was purified approximately 100-fold by Sephadex G-100 gel filtration and DEAE-Sephadex column chromatography, yielding dark-brown needle crystals. The crystalline aconitase was homogenious as judged by polyacrylamide gel electrophoresis and sedimentation by ultracentrifugation. The enzyme showed maximal activity at pH 8.0 and at 55 degrees. It has an S20, W of 5.03 S, a molecular weight of 68,500 and an isolectric point of pH 4.2. The presence of 2.10 moles of iron per mole of the enzyme was demonstrated by atomic absorption spectroscopy.

Elastase inhibitor elafin is a new type of proteinase inhibitor which has a transglutaminase-mediated anchoring sequence termed "cementoin".

Elafin was shown to be a new type of proteinase inhibitor which has an anchoring sequence. Human elafin, a potent inhibitor specific for elastase and proteinase 3, has a unique repeating sequence in its prosegment that is rich in Gln and Lys residues. The prosegment, termed "cementoin," exhibits high homology with the repetitive element of seminal vesicle clotting protein, which is known as a good substrate for prostate transglutaminase. The cross-linking of cementoin by tissue transglutaminase showed that the cementoin moiety is indeed a preferable substrate for transglutaminase. In addition, transglutaminase-mediated cross-linking between cementoin and laminin was observed in vitro, suggesting that cementoin has the ability to covalently attach to other extracellular matrix proteins. To determine whether or not this type of covalent gluing of elafin through the cementoin moiety occurs in vivo, we determined the molecular size of cementoin-elafin in the trachea mucous epithelium by Western blotting; the rationale of this approach is that (i) the trachea is the richest source of cementoin-elafin, as shown below, and (ii) if cementoin-elafin is covalently associated with other proteins, it should migrate as a higher M(r) species on SDS-polyacrylamide gel electrophoresis; cementoin-elafin immunoreactivity was indeed detected at a position corresponding to 50 kDa, a value much higher than that of its monomeric form. RNase protection analysis and immunohistochemical staining revealed that cementoin-elafin is densely distributed in the skin and trachea, and moderately in the stomach, duodenum and small intestine. These sites of localization are consistent with the locations where elastic fibers are abundant.(ABSTRACT TRUNCATED AT 250 WORDS)

Dopamine D3 receptor gene polymorphism and alcohol dependence: relation to personality rating.

Hereditary dopaminergic mechanisms have been implicated in the aetiology of alcoholism. For this study, the distribution of a dopamine D3 receptor gene polymorphism (Ball) has been investigated in patients suffering from alcohol dependence, and compared with non-dependent controls. The allele A1 occurred significantly more frequently among patients compared to controls. Patients with the genotype A1/A2 showed significantly higher novelty seeking (NS) scores in the tridimensional personality questionnaire (TPQ) than patients with the genotype A1/A1. The distribution of patients with high and low NS scores in heterozygotes (A1/A2) did not follow a random distribution. There were significantly more individuals with higher NS scores, and fewer individuals with lower NS scores than expected. The results of this study support the hypothesis of a genetically determined involvement of the dopaminergic system in alcohol dependence. This is probably related to the modulation of personality traits. The observed effects are relatively small, but statistically significant. Thus, the genetics of the dopaminergic neurotransmitter system alone cannot explain the aetiopathogenesis of alcoholism.

Free radicals in Parkinson's disease.

Although there are a number of hypotheses to explain the pathobiochemistry of Parkinson's disease (PD), the one on oxidative stress (OS) has gained major interest. The evidence for OS participation as a cause of PD can be summarized as follows: 1) OS is involved in physiological aging, 2) there is ample evidence that OS is significantly enhanced in PD compared to age-matched healthy persons, 3) OS is an early feature of PD because OS-dependent aggregation of proteins in the form of advanced glycation end products can be imaged in Lewy bodies at a time in a person's life, when no phenotype of a neurodegenerative disorder is evident, 4) Experimental models of PD show OS and degeneration of dopaminergic neurons. The toxin-induced neurodegeneration can be blocked by antioxidants, and 5) Activated microglia, known to release free radicals and inflammatory cytokines, are present in brains of Parkinsonian patients. In conclusion, a great body of evidence points to the view that OS is a major component underlying the pathobiochemistry of PD. Together a genetic disposition and endogenous/exogenous toxic events of various origins result in a synergistic cascade of toxicity which leads to dysfunction and finally to cell death of dopaminergic neurons. Again, OS plays a significant role in generating cell death signals including apoptosis.

Induction of cysteine string protein after chronic antidepressant treatment in rat frontal cortex.

We have previously identified 204 partial cDNA fragments (ADRG1-204) as antidepressant related genes/expressed sequence tags. Then, we developed our original cDNA microarrays, on which the 194 clones out of ADRG1-204 were spotted. With this ADRG microarray, we found that the expression of a spot, ADRG55, which representing cysteine string protein (CSP), was significantly increased in rat brain after chronic treatment with a selective serotonin reuptake inhibitor, sertraline. In the present study, reverse transcription-polymerase chain reaction analysis confirmed the induction of CSP at mRNA levels in rat frontal cortex after chronic treatment with two different classes of antidepressants, imipramine or sertraline. Western blot analysis also revealed that CSP-immunoreactivity was increased after antidepressant treatment. In conclusion, our data suggest that CSP is one of the common functional molecules induced after chronic antidepressant treatment.

Time course of manganese superoxide dismutase concentrations in serum of alcohol-dependent patients during abstinence.

Mean manganese superoxide dismutase (Mn-SOD) concentrations in the serum of patients suffering from alcohol dependence is almost twice as high as in serum of non-dependent controls. In order to investigate the time course of this parameter during abstinence, we determined it at different time points. Patients had mean Mn-SOD serum concentrations (+/-S.D.) of 150.4 +/- 76.3, 121.1 +/- 40.7, 94.6 +/- 37.8 micrograms/ml at 1, 10 and 40 days after abstinence compared to 76.3 +/- 16.9 micrograms/ml as mean Mn-SOD value in the control group. Although the Mn-SOD concentration tended to normalise during abstinence, the differences between index and control group remained significant up to the last measurement at day 40.

New marine steroids, yonarasterols, isolated from the okinawan soft coral, Clavularia viridis.

Six new marine steroids, yonarasterols A through F, were isolated from the Okinawan soft coral, Clavularia viridis. Their structures were determined based on the results of spectroscopic analysis.

Three new chlorinated marine steroids, yonarasterols G, H and I, isolated from the okinawan soft coral, Clavularia viridis.

Three new chlorinated marine steroids, yonarasterols G, H and I, were isolated from the Okinawan soft coral, Clavularia viridis. Their structures including the absolute configuration were determined based on the results of spectroscopic analysis and chemical conversion.

Existence of lipid vesicles containing platelet-activating factor in endothelial cell lysate.

Platelet aggregation activity due to platelet-activating factor (PAF) was detected at high molecular weight (HMW) and low molecular weight fractions after gel-filtration chromatography of cell lysate of endothelial cells. [3H]PAF added to the cell lysate was similarly distributed after chromatography. The radioactivity associated with HMW fraction was not reduced by digesting the lysate with trypsin, suggesting that PAF was not making complexes with proteins but was included in lipid vesicles in cell lysate. Further evidence showed that an unknown specific factor(s) was needed to form these PAF-containing lipid vesicles. Radioactivity was not found in HMW fraction when [3H]PAF was mixed with cell lysate of vascular smooth muscle cells. When monomeric PAF was added to endothelial cell lysate, the specific activity of aggregation decreased to the level exerted by endogenous PAF-containing lipid vesicles due to incorporation into lipid vesicles. PAF in the form of lipid vesicles was more stable in plasma than monomeric form.

Dimethylsulfoxide potentiates the suppressive effects of lidocaine on synaptic transmission in bullfrog sympathetic ganglion.

The effects of dimethylsulfoxide used as a solvent on the suppressive effects of lidocaine on synaptic transmission were examined in the bullfrog sympathetic ganglion. Compound action potentials (CAP) in response to orthodromic stimulation of the presynaptic nerve trunk were recorded extracellularly from the bullfrog sympathetic ganglion. Application of lidocaine dissolved in normal Ringer's solution significantly decreased the amplitude of CAP. This suppressive effect of lidocaine was markedly potentiated when lidocaine was dissolved in 0.1% DMSO solution. A similar potentiating effect of DMSO was observed on the suppressive action of lidocaine on acetylcholine (ACh)-induced depolarizing response recorded intracellularly from the sympathetic ganglion cell. On the other hand, 0.1% DMSO solution alone had no effect on synaptic transmission, resting membrane potential, resting membrane resistance or ACh receptor activity. The present findings suggest that DMSO as solvent should be used carefully because of its potentiating effect on drug actions. Since the effect of lidocaine is due to the suppression of nicotinic ACh receptor at the postsynaptic membrane, DMSO may allosterically act on the nicotinic ACh receptor causing an increase in efficacy of lidocaine.