Role of resistin in the inflammatory response induced by nicotine plus lipopolysaccharide in human periodontal ligament cells in vitro.

BACKGROUND AND OBJECTIVE: Resistin was recently reported to play a role in inflammation-related diseases such as arthritis. However, the precise role of resistin in chronic inflammatory diseases, such as periodontal disease, remains unclear. The aim of this study was to investigate the combined effects of nicotine and lipopolysaccharide (LPS) on the expression of resistin and to assess whether resistin expression influences the levels of inflammatory cytokines, extracellular matrix (ECM) molecules and MMPs in human periodontal ligament cells (PDLCs) stimulated with both nicotine and LPS. MATERIAL AND METHODS: PDLCs were pretreated with isoproterenol or resistin-specific small interfering RNA (siRNA), stimulated with LPS plus nicotine for 24 h, and then monitored for the production of inflammatory mediators. The concentrations of prostaglandin E2 (PGE2) and nitric oxide (NO) were measured by radioimmunoassay and the Griess method, respectively. RT-PCR and western blot analysis were used to measure the levels of mRNA and protein, respectively. Western blot analysis was also used to assess the activation of various signal-transduction pathways. RESULTS: Treatment with nicotine plus LPS up-regulated the expression of resistin mRNA and the production of resistin protein in PDLCs in a time- and concentration-dependent manner. Isoproterenol-mediated interference with the function of resistin, or siRNA-mediated knockdown of resistin expression, markedly attenuated the LPS plus nicotine-mediated stimulation of PGE2 and NO production, the production of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase proteins and the expression of proinflammatory cytokines [tumor necrosis factor-α, interleukin (IL)-1ß, IL-6 and IL-12] and MMPs (MMP-1, MMP-2 and MMP-9); however, these treatments restored the expression of ECM molecules. Furthermore, pretreatment with isoproterenol or resistin-specific siRNA blocked nicotine plus LPS-induced activation of phosphoinositide-3-kinase, glycogen synthase kinase-3 beta, ß-catenin, p38, ERK, JNK and nuclear factor-κB. CONCLUSION: This is the first study to show that the inhibition of resistin, by either a pharmacological or a genetic silencing approach, has anti-inflammatory effects. These effects include decreased levels of inflammatory cytokines and the prevention of ECM breakdown in a nicotine plus LPS-stimulated PDLC model.

Involvement of SDF-1 and monocyte chemoattractant protein-1 in hydrogen peroxide-induced extracellular matrix degradation in human dental pulp cells.

AIM: To determine whether chemokines such as SDF-1 and monocyte chemoattractant protein-1 (MCP-1) are responsible for hydrogen peroxide (H2 O2 )-induced extracellular matrix (ECM) degradation and to identify the underlying mechanism in human dental pulp cells (HDPCs). METHOD: Human dental pulp cells were exposed to 0.4 mmol H2 O2 for 48 h. mRNA expression and protein expression were examined by RT-PCR and Western blot analysis, respectively. The mRNA expression of chemokine (SDF-1 and MCP-1), their receptors (CXCR4 and CXCR2) and extracellular matrix proteins was evaluated by reverse transcriptase-polymerase chain reaction. The production of SDF-1, MCP-1, CXCR4 and CCR2 in the culture medium was determined by enzyme-linked immunosorbent assay. Signal transduction pathway was examined by Western blotting. RESULTS: Hydrogen peroxide provoked the activation of MCP-1 and SDF-1 mRNA and their respective receptors, CXCR4 and CXCR2. H2 O2 treatment concomitantly downregulated the expression of ECM molecules, such as type I collagen, elastin and fibronectin, and upregulated the mRNA expression of matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-8 and MMP-9. Hydrogen peroxide-induced ECM degradation and MMP upregulation were blocked by neutralizing antibodies and siRNAs directed against SDF-1 and MCP-1. Inhibition of SDF-1 and MCP-1 blocked the H2 O2 -induced activation of Akt, p38, ERK and NF-kB. CONCLUSION: Inhibition of SDF and MCP-1 is a potent component of reducing release reactive oxygen species-induced ECM degradation in HDPCs and may play an important role in pulpal and periapical inflammation.

Outbreak of brucellosis in domestic elk in Korea.

Seven of 18 elk on a deer farm were found by the official Rose-Bengal agglutination test (RBT) and tube agglutination test to be brucellosis reactors/suspects. Evaluation with the competitive ELISA (C-ELISA) and the fluorescence polarization assay (FPA) tests revealed that six and five sera were positive respectively. The seven reactors/ suspects were slaughtered and their blood and tissues were collected. Brucella species could be isolated from three of the slaughtered animals, with nine isolates being obtained from the popliteal, supramammary and submandibular lymph nodes, vaginal discharge, mammary tissue and spleen. Brucella genus-specific PCR based on 16S rRNA and AMOS-PCR, which is specific for differential Brucella species, revealed that all nine isolates were Brucella abortus. These nine were further confirmed to be B. abortus biovar 1 by classical biotyping scheme assays. This is the first report of an outbreak of brucellosis in domestic elk in Korea. Our observations suggest that deer should be included in the routine Brucella surveillance programme for the effective control and prevention of brucellosis in Korea.

Reevaluation of the National Institutes of Health criteria for classification and scoring of chronic GVHD.

We used the National Institutes of Health (NIH) criteria for the diagnosis, classification and scoring of chronic GVHD (cGVHD) to reevaluate patients with cGVHD originally diagnosed using classic criteria. We retrieved data from 236 patients diagnosed with cGVHD on the basis of classic criteria. Excluding 20 'liver-alone' patients, we re-categorized 216 patients in keeping with the NIH criteria. Twenty patients were reclassified as having acute GVHD and 196 patients as having cGVHD (170 'classic chronic' (Cl-Ch) and 26 'overlap chronic' (Ov-Ch)). The 5-year GVHD-specific survival (GSS) was significantly different between the two cGVHD subtypes, specifically 87.3% for Cl-Ch vs 70.2% for Ov-Ch (P=0.006). The NIH severity criteria were effective in expecting 5-year GSS rates at both the onset (93.5, 81.3 and 79.7% (P=0.047)) and peak intensity of the disease (100, 89.7 and 78.7% (P=0.004) for the mild, moderate and severe grade, respectively). Multivariate analysis showed that NIH severity criteria were independently significant prognostic factors for GSS (mild vs moderate, HR 4.35, P=0.036; mild vs severe, HR 5.25, P=0.020). Our results support the role of the NIH criteria in classifying cGVHD and in assessing the severity of the disease to predict patient prognosis of cGVHD.

Allogeneic hematopoietic cell transplantation for myelodysplastic syndrome: prognostic significance of pre-transplant IPSS score and comorbidity.

We analyzed the clinical significance of pre-transplant International Prognostic Scoring System (IPSS) score and comorbidity in 68 patients who underwent allogeneic hematopoietic cell transplantation (HCT) for myelodysplastic syndrome (MDS) (n=48) or acute myeloid leukemia evolved from MDS (n=20) between December 1995 and January 2008 in a single institute. During a median follow-up period of 41.0 months (range, 3.2-132.0 months), 27 patients died, and 7 relapsed. The 5-year probabilities of overall survival (OS) and event-free survival (EFS) were 60.0 and 57.4%, respectively, and the 5-year cumulative incidences of non-relapse mortality (CINRM) and relapse were 32.7 and 9.9%, respectively. OS, EFS, and CINRM were significantly different according to pre-transplant IPSS score and presence of pre-transplant comorbidity, which were independent risk factors along with Karnofsky performance score in multivariate analyses. In conclusion, pre-transplant IPSS score and comorbidity may stratify the risk of post transplant outcomes in MDS.

A new steroidal alkaloid from the roots of Cynanchum caudatum.

A new steroidal alkaloid, 12-O-nicotinoylsarcostin, gagamine (1), was isolated from the roots of Cynanchum caudatum Max. (Asclepiadaceae), together with a known alkaloid, gagaminine (2). Their structures were established using spectroscopic methods, some 13C-NMR data of 2 have to be revised.

New multimeric magnetic resonance imaging agents.

RATIONALE AND OBJECTIVES: The authors investigated the effect of multimerization on the relaxivity of macrocyclic gadolinium (Gd) chelates. The objective was to develop more sensitive magnetic resonance imaging (MRI) contrast agents to study biochemical processes. METHODS: Covalently linked nonionic, macrocyclic, multimeric lanthanide chelates that belong to the classes of dimers, trimers, tetramers, hexamer, and octamer, in the molecular weight range approximately 1 to 5 KDa, were synthesized. The chemical linkage was based on either the amide bond or the 2-hydroxypropylidene bond. Relaxivity values, 20r1, on Gd3+ chelates and hydration numbers, Q, on Tb3+ chelates were determined. RESULTS: Relaxivity values increased with molecular weight and Q values were not affected, the increase in r1 in attributable to the expected increase in the overall rotational correlation time, tau r with an increase in molecular weight. The rigidity of the linkers, which is expected to affect the intrachelate rotational correlation time tau r* that makes a contribution to the overall correlation time, tau r, exerted a noticeable effect. The hydroxyl-based chelates generally had lower r1 values than the amide-based chelates. This is rationalized as arising from the longer and thereby rate-limiting effect of the tau m value for the hydroxyl chelates compared with that reported of the amide-based chelates. This rate limiting effect of tau m becomes a dominant factor controlling attainable enhanced relaxivity when multimers based on traditional chelate designs are used for MRI applications. CONCLUSIONS: Approaches aimed at enhancing relaxivity by modulating the water relaxation time, tau m, will be important for the future development of functional MRI contrast agents for the imaging of biochemical processes.

Antimicrobial activity of 8-alkyl- and 8-phenyl-substituted berberines and their 12-bromo derivatives.

The 8-alkyl- (3-6), 8-phenyl- (7), 12-bromo- (8), 8-alkyl-12-bromo- (9-12), and 12-bromo-8-phenyl- (13) berberine derivatives were prepared and tested for their antimicrobial activity in vitro to evaluate structure-activity relationships. Introduction of the alkyl or phenyl group and the bromine atom into the C-8 and C-12 positions of berberine (1), respectively, led to significant increases of the antimicrobial activity. In both the 8-alkyl- and 8-alkyl-12-bromo-berberines (3-6 and 9-12, respectively), the antibacterial activity increased as the length of the aliphatic chain increased. The exception was the activity against Candida albicans and Escherichia coli, which did not always increase as the alkyl side chain lengthened. Among the compounds tested, 12-bromo-8-n-hexylberberine (12) was 64, 256, 128, 16, and 32 times more active against Staphylococcus aureus, Bacillus subtilis, Salmonella enteritidis, E. coli, and C. albicans, respectively, in comparison to the clinically used berberine. Compound 12 was also found to be 8, 16, and 128 times more active against S. aureus, S. enteritidis, and C.albicans, respectively, than kanamycin sulfate, but was of the same order of activity against B. subtilis, and only one-fourth as active against E. coli.

Structure-activity relationships of protoberberines having antimicrobial activity.

13-Alkyl derivatives (2-6 and 8-12) of berberine (1) and palmatine (7) were subjected to in vitro antibacterial activity tests against Bacillus subtilis and Salmonella enteritidis. Antibacterial activity increased as the length of the C-13 aliphatic side chain increased. The effects of the oxygen-substituents on aromatic rings A, C, and D of protoberberinium salts 13-20 on the antimicrobial activity against Staphylococcus aureus, B. subtilis, S. enteritidis, Escherichia coli, and Candida albicans are also discussed. The change in lipophilicity of the protoberberinium salts caused by modification of the substituents appears to influence the antibacterial activity. 13-Hexylberberine (6) and 13-hexylpalmatine (12) exhibited the greatest antibacterial activity.

Purification and Properties of Inulin Fructotransferase (DFA III-producing) from Bacillus sp. snu-7.

Inulin fructotransferase (DFA III-producing) [EC 2.4.1.93] secreted from Bacillus sp. snu-7 was purified 60.3-fold with a yield of 11.6% from a culture supernatant by ammonium sulfate precipitation, preparative isoelectrofocusing, anion exchange chromatography and preparative polyacrylamide gel electrophoresis. The purified enzyme gave a single band on polyacrylamide gel electrophoresis. The molecular mass of the enzyme was estimated to be 62 kDa on SDS-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence was found to be Ala-Asp-Gly-Gln-Asp-Gly-Ala-Pro-Leu-Asn-Gln-Val-Asn-Thr-Tyr-Asp. The optimal pH and temperature for the enzyme reaction were 6.0 and 40°C, respectively. The enzyme was stable with a pH range of 4.0 to 7.0 and at up to 60°C. As the production of di-D-fructose 1,2':2,3' dianhydride increased in the course of enzyme reaction, the Km of the purified enzyme was estimated to be 5.4 mM. One mM each of Cu(2+), Fe(2+) and Hg(2+) inhibited the enzyme activity strongly. Exhaustive enzymatic digestion of inulin produced 1-kestose, 1-nystose, and 1-F-fructofuranosylnystose as well as di-D-fructose 1,2':2,3' dianhydride.

Effects of mastoparan on a vascular contractility in rabbit aorta.

Mastoparan is an amphiphilic tetradecapeptide derived from wasp venom which activates G-proteins. Several secondary effects have been attributed to this peptide, including activation of phospholipase and phosphatidylinositol kinase. The aim of the present study was to investigate the effects of mastoparan on vascular contractility. Rabbit aortic rings were cut and mounted on a force transducer to record isometric tension on a polygraph. The effects of mastoparan were then investigated on the contractile responses in the isolated rabbit aorta with or without endothelium. The results were summarized as follows; 1. Mastoparan caused biphasic response, a transient relaxation followed by a further contraction, in norepinephrine (NE)-precontracted ring with endothelium. These effects were not observed in the aorta in the absence of endothelium. 2. Mastoparan-induced transient relaxation was significantly inhibited by treatment with a N-omega-nitro-L-arginine or methylene blue. 3. When an inhibitor of phospholipase C, neomycin was added to the precontracted aortic ring with NE, the transient relaxation induced by mastoparan was inhibited, but sustained contraction was not inhibited. 4. When an inhibitor of phospholipase A2, quinacrine and inhibitor of the cyclooxygenase pathway, indomethacin, were added to a precontracted ring with NE, the transient relaxation induced by mastoparan was not inhibited, but sustained contraction was inhibited. 5. Mastoparan induced a contraction of the aorta either with or without endothelium. Indomethacin and nifedipine inhibited mastoparan-induced contraction. From the above results, we concluded that mastoparan acts on the endothelium and modifies the release of endothelium-derived relaxing factors such as nitric oxide and also endothelium-derived contracting factors such as metabolites of arachidonic acid.

Synthesis and biological activities of novel 5-(2-acylethynyl)uracils.

5-(2-Acylethynyl)-2,4-dimethoxypyrimidines (3-6) were synthesized in excellent yields from 2,4-dimethoxy-5-[2-(trimethylsilyl)ethynyl]pyrimidine (2) by treatment with acid chlorides in the presence of anhydrous aluminum chloride. Compounds 3-6 were deblocked with chlorotrimethylsilane and sodium iodide in acetonitrile to the corresponding 5-[(2-acyl-1-iodo)vinyl]uracils (7-10), which on treatment with potassium hydroxide in dioxane yielded the corresponding 5-(2-acylethynyl)uracils (11-14). The 5-(2-acylethynyl)uracils were found to be active against Ehrlich ascites carcinoma (EAC) cells in vivo, the most active compounds being 5-(2-benzoylethynyl)uracil (11) and 5-(2-p-toluoylethynyl)uracil (12). The T/C values of 281 and 300 were obtained for compounds 11 and 12, respectively, in the case of mice bearing EAC cells. The 5-(2-acylethynyl)uracils have also shown in vitro activity against CCRF-CEM and L1210/0 tumor cell lines. The lead compound 5-(2-p-toluoylethynyl)uracil effectively inhibited thymidylate synthetase.

Structure-activity studies on organoselenium alkylating agents.

A variety of organoselenium alkylating agents were synthesized, using 2-hydroxyethyl and 3-hydroxypropyl selenocyanate intermediates, and studied to determine their chemical reactivities with 4-(4-nitrobenzyl)pyridine (NBP) and cytotoxicities against CCRF-CEM, L1210/0, and L1210/L-PAM cells. The comparison between the 2-chloroethyl sulfides and selenides 1-4 revealed the markedly enhanced nucleophilicity of selenium (Se) over sulfur (S) by two or more orders of magnitude. This finding indicates that a major consideration in the design of antitumor alkylating organoselenides is the reactivity of selenium. A Taft plot of the experimental first-order rate constant, knbp, and sigma* in a series of 2-chloroethylseleno compounds gave a slope of -1.73 (rho*), with the exception of 2-chloroethyl 2-nitrophenyl selenide (10). The anomalous behavior of 10 is explained in terms of the ortho-nitro stabilization effect directly interacting with the selenium atom of ethyleneselenonium ion to form a 5-membered cyclic intermediate. In the same series, a 5000-fold difference in alkylating reactivity offered only a sixfold variation in cytotoxicity against CCRF-CEM cells. Increasing the alkylating chain length from ethlene to propylene units markedly reduced alkylating reactivities. In the CH3Se(CH2)n Cl series, 16 (n = 3) was 1.5 X 10(5) times slower than 2 (n = 2) in NBP alkylation, revealing that 3-chloro-n-propyl selenides are not chemically reactive enough to be biological alkylating agents despite the presence of the highly nucleophilic selenium atom. Replacement of chloride with mesylate in 3-substituted propyl selenides, such as 17 and 20, restored desirable reactivities and cytotoxicities.

Linear free energy relationships and cytotoxicities of para-substituted 2-haloethyl aryl selenides and bis(2-chloroethyl) selenides.

Examples of a new class of alkylating agents, selenium mustards, were prepared for study of their chemical kinetic properties and cytotoxicities against human lymphoblastoid CCRF-CEM cells. In a series of para-substituted aryl 2-chloroethyl selenides, a linear free energy relationship between the first-order rate constant, k'nbp and sigma p gave a rho value of -1.3, indicating that formation of a cyclic ethylene selenonium ion is the rate-controlling step for alkylation of 4-(4-nitrobenzyl)pyridine (NBP). Consistent with the ethyleneselenonium ion pathway, rates of solvolyses were extremely sensitive to increasing water content, and a positive correlation was found between reactivity with NBP and nucleophilic selectivity (Swain-Scott s constant). The s constant, which predicts for variation in intracellular product spread, varied from 0.53 up to 0.95, equal to aliphatic nitrogen mustards. Alkylating activities based on extent of NBP alkylation, however, showed relatively low values, 8-23% of that of mechlorethamine, possibly due to hydrolysis occurring by a separate pathway from nucleophilic substitution. Reactivities and nucleophilic selectivities both showed positive correlations with cytotoxicities, suggesting that the rate and extent of alkylation of relatively strong nucleophilic centers mediate the biologic effects of these compounds. Two bifunctional selenium mustards were substantially more cytotoxic than monofunctional aromatic selenides. No additional cytotoxicity due to the selenium atom was observed, with the exception of diselenide (-SeSe-) compounds. Thus, selenium alkylating agents kinetically and biologically resemble classical, mustard-type alkylating agents.