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.

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.

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.

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.

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.

Substrate specificity of endo-polyguluronide lyases from Pseudomonas sp. on the basis of their kinetic properties.

Two endo-alginate lyases [EC 4.2.2.3] differing in their mode of degradation of substrates and practically free of polymannuronide lyase activity were partially purified from Pseudomonas sp. cells. Their substrate specificities were investigated for two different kinds of alginate fragments; a polyguluronide (SG) and a polyuronide consisting of mannuronic (M) and guluronic (G) acid residues (SMG). The effects of various salts and some organic compounds such as EDTA and p-chloromercuribenzoate on the degradation of the two substrates were similar. High concentrations of the substrates similarly inhibited the action ofthe lyases, giving a bell-shaped plot. A polymannuronide alginate fragment (SM) which was a substrate for polymannuronide lyase but was not attacked by these guluronide lyases also inhibited the degradation of SG and SMG. The overall degradation velocities of a mixture of SG and SMG by both lyases coincided with those calculated from the Michaelis-Menten formula. Based on the above results, it was concluded that SG and SMG are attacked by the same endo-polyguluronide lyase.

Multiple components of endo-polyguluronide lyase of Pseudomonas sp.

A lyophilized alginate lyase preparation obtained from dialyzed extract of sonicated Pseudomonas sp. cells was fractionated by gell filtration on a Sephadex G-150 column, and three alginate lyase [EC4.2.2.3] fractions, peaks I, II, and III, were obtained. They were remarkably thermolabile. The lyase fractions degraded two kinds of alginate fragments, a polygluuronide (SG), and a polyuronide consisting of both mannuronic and guluronic acid residues (SMG), as well as commerical alginate, but were virtually inactive toward polymannuronide fragment (SM). The modes of degradation of these substrates by the lyase fractions were endowise with different degrees of randomness. Attack by the peak I fraction was more random than those by peaks II and III. The main lysis products formed from SG and SMG by these layses were identified as mixtures of unsaturated tri- and monouronides. The unsaturated triuronide from SG was deltatugg and SMG yielded a mixture of deltaUGG and a poorly characterized unsaturated trimer, possibly deltaUMG. However, the patterns of monomer and trimer production by theselyase fractions changed in different ways during incubation.

Fine structure of SMG alginate fragment in the light of its degradation by alginate lyases of Pseudomonas sp.

An alginate fragment named SMG, consisting of mannuronic (M) and guluronic acid residues (G)(DP=25), was prepared from the partial acid hydrolysate of a commercial alginate. Two subfractions, SMG-ppt (DP=52) and SMG-sup (DP=18) were obtained from SMG by fractionation with MgC12 and CaC12. The M/G ratios of these alginate fragment were 1.4-1.9. Their lysis products by a pseudomonad alginate lyase [EC 4.2.2.3] preparation were fractionated by gel filtration, giving similar patterns. The major products in their digests were unsaturated monouronides (53-50%) and triuronides (30-35%). The former was identified as a delta4,5-hexuronic acid (deltaU) and the latter was identified as a mixture of delta4,5-hexuronosyl-(1 leads to 4)-beta-D-mannuronosyl-(1 leads to 4)-L-guluronic acid (deltaUMG) and delta4,5-hexuronosyl-(1 leads to 4)-alpha-L-guluronosyl-(1 leads to 4))L-guluronic acid (deltaUGG). The two unsaturated triuronides were present in roughly equal amounts. The presence of 4-O-alpha-L-guluronosyl-L-guluronic acid (GG) and 4-O-beta-D-mannuronosyl-L-guluronic acid (MG) or 4-O-beta-L-guluronosyl-D-mannuronic acid (GM) was also demonstrated inthe digest. Moreover, indirect evidence suggested nonreducing terminal deltaU residue and free deltaU in the digest to be derived more from M than G of the original SMG. Thus, it was concluded that more than one-third of uronic acid residues of SMG molecules may be composed of almost equal amounts of MG and GG sequences, most of which may be connected by M to form MMG and MGG sequences, respectively.

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 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.

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.

Tn5 tagging of the phenol-degrading gene on the chromosome of Pseudomonas putida.

Transposon mutagenesis was performed by the method of conjugational transfer in order to identify and characterize genes encoding enzymes involved in the pathway of phenol utilization as a carbon source. Escherichia coli, which carries the Tn5-132, Was mated with Pseudomonas putida SM25 as a host. We selected a mutant that could not utilize phenol as a carbon source. Chromosomal integration of the transposon was confirmed by Southern analysis, successfully tagging the gene related to a phenol-utilizing pathway. By cell-free enzyme and genetic complementation assays, the inactivated enzyme through the mutation of the corresponding gene was identified as the catB gene, which encodes a cis,cis-muconate lactonizing enzyme.

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.

The biochemical basis for the distinction between the two Cryptococcus neoformans varieties with CGB medium.

The biochemical basis for the reaction to canavanine-glycine-bromthymol blue (CGB) agar by Cryptococcus neoformans var. gattii and C. neoformans var. neoformans was investigated. All of the var. gattii isolates tested were found to utilize glycine as the sole source of carbon and nitrogen and were resistant to L-canavanine. Only 11% of the serotype D isolates of var. neoformans utilized glycine as the sole source of carbon and nitrogen, but these were all sensitive to canavanine. Nineteen percent of the serotype A isolates of var. neoformans were able to assimilate glycine, and 81% of the glycine users were resistant to canavanine. However, these canavanine-resistant, glycine-assimilating, var. neoformans isolates failed to grow when they were cultured on a medium containing glycine and canavanine. Unlike the var. neoformans isolates, all of the var. gattii isolates tested grew on a medium that contained both of these compounds. Glycine-utilizing isolates exhibited good uptake of the amino acid, and a glycine-cleaving enzyme was discernable in the isolate. The isolates that fail to utilize glycine accumulated the amino acid at a rate which was barely 15% of that seen in the glycine users, and no glycine-cleaving enzyme was apparent within the 48-hr incubation period. When a cell-free extract (which had been derived from a glycine-utilizing isolate), was incubated with 14C-labeled glycine, ammonia, radiolabeled CO2, and serine were produced. The glycine decarboxylase activity of the cell-free extract was found to be enhanced by the addition of dithiothreitol, tetrahydrofolate, pyridoxal phosphate, and nicotinamide adenine dinucleotide (NAD). The ammonia released during glycine cleavage seems to be responsible for the positive reaction on CGB medium.

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.

Characterization of the gene encoding catechol 2,3-dioxygenase of Alcaligenes sp. KF711: overexpression, enzyme purification, and nucleotide sequencing.

Catechol 2,3-dioxygenase (C23O) is an extradiol-type dioxygenase that catalyzes the aromatic ring fission of catechol to form 2-hydroxymuconic semialdehyde. The C23O gene of Alcaligenes sp. KF711 was overexpressed in Escherichia coli HB101 by using the lac promoter of pUC18, and its gene product was purified by using immunoaffinity chromatography. The purified C23O exhibited a 35-kDa single band on an SDS-polyacrylamide gel, and its ring-fission activity on dihydroxylated aromatics was 4-methylcatechol > 4-chlorocatechol > catechol > 3-methylcatechol >> 2,3-dihydroxybiphenyl. Nucleotide sequence analysis of the C23O gene revealed an open reading frame of 927 bp, which can encode a polypeptide of 308 amino acid residues. The predicted molecular mass of 35 kDa is in agreement with that of purified C23O on an SDS-polyacrylamide gel. The amino acid sequence of the C23O was compared with those of nine other extradiol-type dioxygenases, including 2,3-dihydroxybiphenyl dioxygenase (2,3-DHBD) and 1,2-dihydroxynaphthalene dioxygenase (1,2-DHND). The C23O of Alcaligenes sp. KF711 exhibited 80 to 94% identity in amino acid sequence with other C23Os, and 20 to 25% identity with 1,2-DHND and 2,3-DHBDs. Furthermore, sequence comparison of 10 extradiol-type dioxygenases has led to identifying 19 evolutionarily conserved amino acid residues whose possible catalytic roles are proposed.

n-Alkane dissimilation by Rhodopseudomonas sphaeroides transferred OCT plasmid.

The OCT plasmid from Pseudomonas maltophilia N246-1 was transferred to Rhodopseudomonas sphaeroides M1 with very low frequency (1.4-1.9 × 10(-5) per recipient cell at pH 7-8 for a 3-hour reaction time). P. maltophilia N246-1 was able to utilize C8-C14 of n-alkanes, whereas R. gas-liquid chromatography determined that the broad range of carbon numbers of n-alkanes in crude oil was remarkably degraded by the transconjugant, R. sphaeroides M1-C1, compared with donor strain N246-1. The fact that donor and transconjugant strains simultaneously lost the capacity to utilize n-alkanes on L-broth medium suggests that the OCT plasmids are unstable. It was found that the OCT plasmid of P. maltophilia N246 was incompatible with the IncP-2 group of P. aeruginosa KCTC 11245.

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.