Risk factors for multi-joint disease in patients with glucocorticoid-induced osteonecrosis.

This study investigated risk factors for osteonecrosis involving multiple joints (MJON) among glucocorticoid-treated patients. The best predictor of MJON was cumulative oral glucocorticoid dose. Risk of MJON was 12-fold higher in patients who had a second risk factor for osteonecrosis. Further research is needed into strategies for prevention of MJON. INTRODUCTION: Osteonecrosis (ON) is a debilitating musculoskeletal condition in which bone cell death can lead to mechanical failure. When multiple joints are affected, pain and disability are compounded. Glucocorticoid treatment is one of the most common predisposing factors for ON. This study investigated risk factors for ON involving multiple joints (MJON) among glucocorticoid-treated patients. METHODS: Fifty-five adults with glucocorticoid-induced ON were prospectively enrolled. MJON was defined as ON in ≥ three joints. Route, dose, duration, and timing of glucocorticoid treatment were assessed. RESULTS: Mean age of enrolled subjects was 44 years, 58% were women. Half had underlying conditions associated with increased ON risk: systemic lupus erythematosus (29%), acute lymphoblastic leukemia (11%), HIV (9%), and alcohol use (4%). Mean daily oral dose of glucocorticoids was 29 mg. Average cumulative oral dose was 30 g over 5 years. The best predictor of MJON was cumulative oral glucocorticoid dose. For each increase of 1,000 mg, risk of MJON increased by 3.2% (95% CI 1.03, 1.67). Glucocorticoid exposure in the first 6 months of therapy, peak dose (oral or IV), and mean daily dose did not independently increase risk of MJON. The risk of MJON was 12-fold in patients who had a second risk factor (95% CI 3.2, 44.4). CONCLUSIONS: Among patients with glucocorticoid-induced ON, cumulative oral dose was the best predictor of multi-joint disease; initial doses of IV and oral glucocorticoids did not independently increase risk. Further research is needed to better define optimal strategies for prevention and treatment of MJON.

Association between interleukin-27 polymorphisms and pulmonary tuberculosis.

OBJECTIVE: To investigate the effect of interleukin (IL) 27 -964A/G, 2095T/G, 4603G/A and 4730T/C gene polymorphisms on the development of pulmonary tuberculosis (PTB), radiographic characteristics and severity. DESIGN: Differences in the allele and genotype distributions of the -964A/G, 2095T/G, 4603G/A and 4730T/C polymorphisms between 224 PTB patients and 233 healthy controls, between patients with single- and multi-lobe involvement, and between patients with and without cavitation, were investigated. Serum IL-27 concentration was measured using an enzyme-linked immunosorbent assay. RESULTS: There were no significant differences in the allele or genotype distributions between PTB patients and healthy controls. However, the -964A/A genotype was more prevalent in patients with single-lobe involvement than the -964A/G or -964G/G genotype in patients with multi-lobe involvement (50.0% vs. 31.3%, P = 0.01). There was no difference between patients with and without cavitation (P > 0.05). Serum median IL-27 concentration was significantly higher in patients with single-lobe involvement than in those with multi-lobe involvement (P = 0.03) and in those with -964A/A genotypes than in those with -964A/G or -964G/G genotypes (P = 0.02). CONCLUSIONS: In terms of serum IL-27 levels, the -964 A/A genotype may be associated with a protective role that prevents the intrapulmonary spread of PTB rather than its development.

Phosphoinositide 3-kinase δ inhibitor suppresses interleukin-17 expression in a murine asthma model.

Phosphoinositide 3-kinases (PI3Ks) contribute to the pathogenesis of asthma by regulating the activation of inflammatory mediators, inflammatory cell recruitment and immune cell function. Recent findings have indicated that PI3Ks also regulate the expression of interleukin (IL)-17, which has been recognised as an important cytokine involved in airway inflammation. In the present study, we investigated a role of PI3Kδ in the regulation of IL-17 expression in allergic airway disease using a murine model of asthma. After ovalbumin inhalation, administration of a selective p110δ inhibitor, IC87114, significantly attenuated airway infiltration of total cells, lymphocytes, neutrophils and eosinophils, as well as airway hyperresponsiveness, and attenuated the increase in IL-17 protein and mRNA expression. Moreover, IC87114 reduced levels of IL-4, -5 and -13, expression of keratinocyte chemoattractant protein and mRNA, and nuclear factor (NF)-κB activity. In addition, a NF-κB inhibitor, BAY 11-7085 substantially reduced the increase in IL-17 protein levels. Our results also showed that inhibition of IL-17 activity with an anti-IL-17 antibody remarkably reduced airway inflammation and hyperresponsiveness. These findings suggest that inhibition of the p110δ signalling pathway suppresses IL-17 expression through regulation of NF-κB activity and, thus, has therapeutic potential in asthma.

Inhibition of VEGF blocks TGF-beta1 production through a PI3K/Akt signalling pathway.

Vascular endothelial growth factor (VEGF) is a mediator of airway inflammation and remodelling in asthma. Transforming growth factor (TGF)-beta(1) plays pivotal roles in diverse biological processes, including tissue remodelling and repair in a number of chronic lung diseases. However, there are few studies elucidating the interactions between VEGF and TGF-beta(1) in allergic airway disease. A murine model of allergic airway disease was used to define the mechanism by which VEGF induces subepithelial fibrosis and to investigate a potential relationship between VEGF and TGF-beta(1) and the mechanisms by which VEGF signalling regulates TGF-beta(1) expression in allergic airway disease. The ovalbumin (OVA)-inhaled murine model revealed the following typical pathophysiological features of allergic airway disease in the lungs: increased numbers of inflammatory cells of the airways, airway hyperresponsiveness, increased peribronchial fibrosis, and increased levels of VEGF and TGF-beta(1). Administration of VEGF inhibitors reduced the pathophysiological signs of allergic airway disease and decreased the increased TGF-beta(1) levels and peribronchial fibrosis, including phosphoinositide 3-kinase (PI3K) activity after OVA inhalation. In addition, the increased TGF-beta(1) levels and collagen deposition after OVA inhalation were decreased by administration of PI3K inhibitors. These results suggest that inhibition of vascular endothelial growth factor attenuates peribronchial fibrosis, at least when mediated by regulation of transforming growth factor-beta(1) expression through phosphoinositide 3-kinase/Akt pathway in a murine model of allergic airway disease.

Cloning and sequence analyses of a 2,3-dihydroxybiphenyl 1,2-dioxygenase gene ( bphC) from Comamonas sp. SMN4 for phylogenetic and structural analysis.

A genomic library of biphenyl-degrading Comamonas sp. SMN4 for isolating fragments containing the 2,3-dihydroxybiphenyl 1,2-dioxygenase (23DBDO) gene was constructed. The smallest subclone (pNPX9) encoding 23DBDO activity was sequenced and analyzed. The C-terminal domain of 23DBDO from Comamonas sp. SMN4 had five catalytically essential residues and was more highly conserved than the N-terminal domain. Phylogenetic and structural relationships of 23DBDO from Comamonas sp. SMN4 were analyzed.

Enhanced IL-18 expression in common skin tumors.

Interleukin-18 (IL-18) has been found to have multiple effects upon various cells involved in inflammatory response. Recently we reported that B16 murine melanoma cells are able to produce IL-18, which is involved in the regulation of intracellular reactive oxygen intermediates (ROI) and Fas-ligand expression, indicating that IL-18 plays key role in the tumor activity of melanoma. In this study, we investigated the pattern of IL-18 expression in the human system. IL-18 production was tested by enzyme linked immunosorbent assay (ELISA) assay in various tumor cell lines, including Raji (Burkitt's lymphoma), IM-9 (B lymphoblast), Jurkat (acute T cell leukemia), SK-MES-1 (squamous cell carcinoma (SCC) cell line), SK-MEL-2, G-361, DM-4, and DX-3 (melanoma cell lines). ELISA tests showed that IL-18 was highly expressed in malignant skin tumors such as SK-MES-1, SK-MEL-2, G-361, DM-4, and DX-3 cell lines, thus suggesting that IL-18 production may be associated with the malignancy of skin tumors. Here, we report that enhanced IL-18 expression is positively correlated with malignant skin tumors such as SCC and melanoma, suggesting the importance role of IL-18 in malignancy of skin tumors. Taken together, expression of IL-18 by tumor cells in human skin tissue may provide an important clue to understand the pathogenesis of malignant skin tumors.

Molecular cloning of the nahG gene encoding salicylate hydroxylase from Pseudomonas fluorescens.

A gene encoding the salicylate hydroxylase was cloned from the genomic DNA of Pseudomonas fluorescens SME11. The DNA fragment containing the nahG gene for the salicylate hydroxylase was mapped with restriction endonucleases and sequenced. The DNA fragment contained an ORF of 1,305 bp encoding a polypeptide of 434 amino acid residues. The nucleotide and amino acid sequences of the salicylate hydroxylase revealed several conserved regions with those of the enzyme encoded in P. putida PpG7: The homology of the nucleotide sequence is 83% and that of amino acid sequence is 72%. We found large conserved regions of the amino acid sequence at FAD and NADH binding regions. The FAD binding site is located at the amino terminal region and a lysine residue functions as a NADH-binding site.

Pimarane cyclooxygenase 2 (COX-2) inhibitor and its structure-activity relationship.

The structure-activity relationship and molecular modelings of a novel pimarane COX-2 inhibitor are reported. Particularly, a series of linker extended analogues designed on the basis of these studies exhibited significantly enhanced COX-2 inhibitory activities and selectivities.

The Bradyrhizobium japonicum hsfA gene exhibits a unique developmental expression pattern in cowpea nodules.

The Bradyrhizobium japonicum host-specific fixation gene hsfA was identified as essential for nitrogen fixation on cowpea, but not required for nitrogen fixation on soybean or siratro. The DNA sequence of the hsfA promoter contains a consensus RpoN, -24/-12 binding site, suggesting the involvement of a regulatory protein that binds to an upstream activating sequence (UAS). To further explore the regulation of this interesting gene, serial deletions of the hsfA promoter were made and fused with the beta-glucuronidase (GUS) gene. The HsfA3 deletion, containing 60 bp 5' of the -24/-12 sequence, showed a similar level of GUS expression to that shown by the longest fusion construct (HsfA1), containing 464 bp of upstream sequence. In contrast, the HsfA4-GUS fusion, containing only 20 bp 5' of the -24/-12 region, showed no GUS activity, delimiting the location of a putative UAS to a 40-bp region. During nodule development, GUS expression first appeared in nodules 12 days postinoculation (dpi) and reached a maximum level of expression in approximately 17-day-old nodules. By 28 dpi, HsfA-GUS expression had returned to a low, basal level. These data were consistent with the detection of hsfA mRNA by in situ hybridization in 17-day-old nodules, but not in 28-day-old nodules. In contrast to the stage-specific expression in cowpea, HsfA-GUS expression increased with nodule development in HsfA3-inoculated soybean. These data indicate that HsfA expression is regulated in cowpea in a unique developmental manner and that the DNA regulatory regions that control this expression are confined to a short, promoter-proximal region.

Isolation of a potent anti-MRSA sesquiterpenoid quinone from Ulmus davidiana var. japonica.

A highly potent anti-MRSA sesquiterpenoid has been isolated from Ulmus davidiana var. japonica, which has been traditionally used to treat infectious diseases in Korea. This naturally occurring antibiotic was identified as mansonone F (1). This compound has been found to be highly active specifically against MRSA and showed an MIC range of 0.39-3.13 microg/ml which is comparable to that of vancomycin.

Cloning and nucleotide sequence analysis of xylE gene responsible for meta-cleavage of 4-chlorocatechol from Pseudomonas sp. S-47.

Pseudomonas sp. S-47 expresses catechol 2,3-dioxygenase (C230) catalyzing the conversion of 4-chlorocatechol (4CC) as well as catechol to 5-chloro-2-hydroxymuconic semialdehyde and 2-hydroxymuconic semialdehyde, respectively, through meta-ring cleavage. The xylE gene encoding C230 for meta-cleavage was cloned from strain S-47 and its nucleotide sequence was analyzed. The pRES101 containing the xylE gene exhibited high C230 activity toward catechol and 4CC without altering the substrate specificity from natural strain. The xylE gene was composed of 924 bp and encoded polypeptide of molecular mass 35 kDa containing 307 amino acids. A deduced amino acid sequence of the C230 from strain S-47 exhibited over 80% identity with those of Pseudomonas putida mt-2, Pseudomonas putida G7, and Pseudomonas sp. CF600. However, it shows below 45% identity with those of Pseudomonas cepacia LB400 and Pseudomonas sp. KKS102. The C230 of strain S-47 was conserved in the amino acids (His150, His214, Glu261) for metal binding ligands and those (His199, His242, and Tyr251) for catalytic sites. Therefore, Pseudomonas sp. S-47 can be explained as acting by degrading catechol as well as 4CC by xylE-encoding C230 which was fused by N domain of nahH and C domain of dmpB from other Pseudomonas strains.

Cloning of the histidine biosynthetic genes from Corynebacterium glutamicum: organization and analysis of the hisG and hisE genes.

The physically linked hisG and hisE genes, encoding for ATP-phosphoribosyltransferase and phosphoribosyl-ATP-pyrophosphohydrolase were isolated from the Corynebacterium glutamicum gene library by complementation of Escherichia coli histidine auxotrophs. They are two of the nine genes that participate in the histidine biosynthetic pathway. Molecular genetics and sequencing analysis of the cloned 9-kb insert DNA showed that it carries the hisG and hisE genes. In combining this result with our previous report, we propose that all histidine biosynthetic genes are separated on the genome by three unlinked loci. The coding regions of the hisG and hisE genes are 279 and 87 amino acids in length with a predicted size of about 30 and 10 kDa, respectively. Computer analysis revealed that the amino acid sequences of the hisG and hisE gene products were similar to those of other bacteria.

Direct activation of capsaicin receptors by products of lipoxygenases: endogenous capsaicin-like substances.

Capsaicin, a pungent ingredient of hot peppers, causes excitation of small sensory neurons, and thereby produces severe pain. A nonselective cation channel activated by capsaicin has been identified in sensory neurons and a cDNA encoding the channel has been cloned recently. However, an endogenous activator of the receptor has not yet been found. In this study, we show that several products of lipoxygenases directly activate the capsaicin-activated channel in isolated membrane patches of sensory neurons. Among them, 12- and 15-(S)-hydroperoxyeicosatetraenoic acids, 5- and 15-(S)-hydroxyeicosatetraenoic acids, and leukotriene B(4) possessed the highest potency. The eicosanoids also activated the cloned capsaicin receptor (VR1) expressed in HEK cells. Prostaglandins and unsaturated fatty acids failed to activate the channel. These results suggest a novel signaling mechanism underlying the pain sensory transduction.

Incidence and characterization of Listeria monocytogenes from domestic and imported foods in Korea.

A total of 1,537 domestic and imported food products were examined for the incidence of Listeria monocytogenes between 1993 and 1997 in Korea. L. monocytogenes was detected using the U.S. Department of Agriculture isolation method. Isolated L. monocytogenes was confirmed by polymerase chain reaction with hly1 and hly2 primers designed from the listeriolysin O. Overall, 122 samples (7.9%) contained L. monocytogenes. The rate of isolation was 4.3% for beef, 19.1% for pork, 30.2% for chicken, 1.2% for shellfish, 4.4% for raw milk, 4.4% for frozen smoked mussels, and 6.1% for ice cream. No L. monocytogenes was found in pasteurized milk, pasteurized processed cheese, saltwater fish, dried seafoods, or ham. The overall incidence was lower than that reported in previous studies from other countries. Most isolates were serotype 1/2b except for chicken, in which serotype 1/2a was predominant. The serotyping results might imply the presence of food or geography-specific L. monocytogenes strains.

Role of hsfA gene on host-specificity by Bradyrhizobium japonicum in a broad range of tropical legumes.

Bradyrhizobium japonicum mutant strain NAD163, containing a 30-kb deletion mutant encompassing the hsfA gene, was inoculated onto a broad range of legume species to test host-specificity. Most legume species formed ineffective nodules except Vigna angularis var. Chibopat and Glycine max var. Pureunkong. A hsfA insertion mutant, BjjC211, gave similar results to strain NAD163, implying that many legume species require HsfA for host-specific nitrogen fixation. To determine whether other genes in the deleted region of NAD163 are also necessary, the hsfA gene was conjugally transferred into the NAD163 mutant. The transconjugant formed effective nodules on the host legume plants, which earlier had formed ineffective nodules with mutant NAD163. Thus, we conclude that the hsfA gene in the 30-kb region is the only factor responsible for host-specific nitrogen fixation in legume plants.

Cloning and characterization of the UDP-sugar hydrolase gene (ushA) of Enterobacter aerogenes IFO 12010.

A bacterial alkaline phosphatase (BAP, the phoA gene product) is primarily responsible for the hydrolysis of the substrates 5-bromo-4-chloro-3-indolylphosphate-p-toluidine (XP) and p-nitrophenyl phosphate (pNPP). Using these substrates and an E. coli phoA mutant, we have cloned Enterobacter aerogenes genes conferring an XP(+) phenotype. Two types of clones were identified based on phenotypic tests and DNA sequences. One of them is a E. aerogenes phoA gene (XP(+), pNPP(+)) as expected; surprisingly the other one was found to be a ushA gene (XP(+), pNPP(-)), which encodes an UDP (uridine 5'-diphosphate)-sugar hydrolase. The E. aerogenes ushA gene shares high sequence identity with ushA of E. coli and the mutationally silent ushA0 gene of Salmonella typhimurium at both the nucleotide (over 79%) and amino acid (over 93%) levels. Expression of the E. aerogenes ushA gene in E. coli produced high level of UDP-sugar hydrolase, as confirmed by TLC (thin layer chromatography) analysis together with a presence of a strong band due to a XP hydrolysis on a polyacrylamide gel.

Functional interactions between conserved motifs of the hepatitis C virus RNA helicase protein NS3.

The hepatitis C virus NS3 gene encodes a RNA helicase with several sequence motifs conserved among the members of the DExH box protein family. The contributions of the sequence motifs to enzyme activity were assessed in this study by substitution of alanine for the Lys in the ATP binding motif GxGK (referred to as K1236A mutation), or for the Asp in the DExH motif (D1316A), or for the Arg in the middle of the QRxGRxGR motif known for RNA binding (R1490A). Histidine-tagged recombinant proteins of Mr 54,000 were expressed in Escherichia coli and purified by chromatography on nickel agarose. All three mutants were severely defective in ATPase and RNA helicase activities, but loss of the ATPase activity was not dependent on polynucleotide cofactors. With the exception of R1490A mutant, a stable complex was formed between dsRNA substrates and recombinant proteins, indicating that the arginine-rich motif is required for efficient RNA binding. Complex formation was not affected by omission of ATP or substitution by a non-hydrolyzable analog AMP-PCP, suggesting that neither binding nor hydrolysis of ATP is required for RNA binding. Moreover, the K1236A mutant which was defective in binding ATP exhibited an unusually strong affinity for RNA duplex. These results suggest that the conserved motifs cooperatively constitute a large functional domain rather than act as individual domains with strictly independent functions, and that alteration of one motif affects functions of other motifs in a mutually interactive fashion.

Cloning and sequencing of the fcbB gene encoding 4-chlorobenzoate-coenzyme A dehalogenase from Pseudomonas sp. DJ-12.

Pseudomonas sp. DJ-12 degrades 4-chlorobenzoate through hydrolytic dechlorination to produce 4-hydroxybenzoate and a chloride ion. The fcbB gene encoding the 4-chlorobenzoate-coenzyme A (4CBA-CoA) dehalogenase which catalyzes the nucleophilic substitution reaction to convert 4CBA-CoA to 4-hydroxybenzoate-coenzyme A (4HBA-CoA) in the consecutive steps of dechlorination was cloned from the chromosome of the organism. A nucleotide sequence analysis of the gene showed an open reading frame consisting of 810 nucleotides, which can encode for a polypeptide of molecular mass 30 kDa, containing 269 amino acid residues. A promoter-like sequence (-35 and -10 region) and a putative ribosome-binding sequence were identified. A deduced amino acid sequence of the 4CBA-CoA dehalogenase showed 86%, 50%, and 50% identity with those of corresponding enzymes in the Pseudomonas sp. CBS3, Arthrobacter sp. SU, and Arthrobacter sp. TM1, respectively.

Cloning and analysis of the aroB gene encoding dehydroquinate synthase from Corynebacterium glutamicum.

The aroB gene encoding dehydroquinate synthase of Corynebacterium glutamicum has been cloned by complementation of an aro auxotrophic mutant of Escherichia coli with the genomic DNA library. The recombinant plasmid contained a 1.4-kb fragment that complemented the Escherichia coli dehydroquinate-synthase-deficient mutant. The nucleotide sequences of the subcloned DNA has been determined. The sequences contain an open reading frame of 360 codons, from which a protein with a molecular mass of about 38 kDa could be predicted. This is consistent with the size of the AroB protein expressed in E. coli. Alignment of different prokaryotic and eukaryotic aroB gene products reveals an overall identity ranging from 29 to 57% and the presence of several highly conserved regions.