Expression of TNF-α and Related Signaling Molecules in the Peripheral Blood Mononuclear Cells of Rheumatoid Arthritis Patients

We examined the role of tumor necrosis factor (TNF-α) and its related signaling intermediates leading to apoptosis/proliferation in the peripheral blood mononuclear cells (PBMCs) of RA patients. The constitutive expression of mRNA for TNF-α receptors (TNFR-I and TNFR-II) and the adapter molecules, such as the TNF receptor-associated death domain protein (TRADD), Fas-associated death domain protein (FADD), receptor interacting protein (RIP), and TNF receptor-associated factor 2 (TRAF-2) were analyzed by reverse transcriptase-PCR (RT-PCR) in PBMCs from control and RA cases. PBMCs of RA patients showed a significant increase in TNF-α and TNFR-I expression as compared with that from control subjects along with significantly increased constitutive expression of TRADD, RIP, and TRAF-2 mRNA. There was a decrease in expression of FADD in RA patients, but the difference was not significant as compared to controls. These data suggested enhanced signaling by the TNFR-I-TRADD-RIP-TRAF-2 pathway and suppressed signaling by the TNFR-I-TRADD-FADD pathway in PBMCs of RA patients. However, the regulatory mechanisms for TNF-α induced signaling may not be explained only by these pathways

<span style="mso-bidi-language:HI">Rhizobial lipopolysaccharide as the receptor in lectin<i>-Rhizobium </i>interaction </span>

89-95Rhizobial specificity was examined on the basis of interaction between legume lectins (peanut, pea and soybean) and different rhizobial species (various bradyrhizobia specific for peanut, P 14-93 and SB16). Legume lectins showed higher affinity towards host-specific Rhizobium and lipopolysaccharides (LPS) isolated from those particular rhizobia. Two LPS mutants of peanut-specific Bradyrhizobium sp. (Arachis) strain GN17 were isolated by Tn5 mutagenesis. These mutants (GN17MI and GNI7M2) were characterized by their higher hydrophobicity with respect to the parent cells. The hexose content in exopolysaccharides. (EPS) and LPS of the mutants was found reduced significantly, whereas 2-keto-3-deoxyoctulosonic acid (Kdo) and uronic acid in LPS were less by 20-times and thrice, respectively in the mutants. Glucose was the major sugar in LPS from all the strains. However, glucosamine appeared only in the mutants. Spectrofluorimetric analysis showed that LPS from GN17Ml mutant interacted most significantly with peanut root agglutinin or lectin (PRA II). The results indicate that LPS on the surface of rhizobial cells is the possible receptor for lectin. </span

Isolation and characterization of a lectin from peanut roots

A glucose-specific lectin has been purified to apparent homogeneity from 7-day-old peanut (Arachis hypogaea) roots by affinity chromatography on a Sephades G-50. The lectin has a 66 kDa native molecular mass and a 33 kDa subunit molecular mass as revealed by native and denaturing sodium dedecyl sulphate-polycrylamide gel electrophoresis, respectively. The purified lectin, gives a single precipitin line with the antiserum produced against 7-day-old root extract and shows 5 bands in the pH range of 4.4–5.4 in the isoelectric focusing gel. The glucose-specific lectin activity in the peanut roots appears from the fourth day onwards. Lipopolysaccharides isolated from the host specific Rhizobium strain are a 68-fold more potent inhibitor of the lectin as compared to glucose

Evaluation of anti-inflammatory activity of <i style="mso-bidi-font-style:normal">Dracaena cinnabari</i> Balf. f. resin

215-222Dracaena cinnabari Balf. f. (Family- Dracaenaceae) resin is a traditional medicine since ancient times in many cultures. Most of the pharmacological investigations of this resin have been addressed to its anti-microbial, anti-viral and antioxidant activities. However, anti-inflammatory activity of <i style="mso-bidi-font-style: normal">D. cinnabari resin is not evaluated so far. The present study indicates that methanol extract of D. cinnabari resin (MEDC) and one of its components, 4'-hydroxy-7,8-methylenedioxyhomoisoflavan (MHF) inhibited nitrite, TNF-α and IL-6 productions in lipopolysaccharide-stimulated mouse macrophage cell line RAW 264.7 with increase in their concentrations. Anti-inflammatory activities of these treatments were further confirmed by the reduction of rat hind paw edema. These results suggest that MEDC and MHF have potential anti-inflammatory activity at the selected doses

Evaluation of anti-inflammatory activity of Dracaena cinnabari Balf. f. resin

Dracaena cinnabari Balf. f. (Family- Dracaenaceae) resin is a traditional medicine since ancient times in many cultures. Most of the pharmacological investigations of this resin have been addressed to its anti-microbial, anti-viral and antioxidant activities. However, anti-inflammatory activity of D. cinnabari resin is not evaluated so far. The present study indicates that methanol extract of D. cinnabari resin (MEDC) and one of its components, 4'-hydroxy-7,8-methylenedioxyhomoisoflavan (MHF) inhibited nitrite, TNF-α and IL-6 productions in lipopolysaccharide-stimulated mouse macrophage cell line RAW 264.7 with increase in their concentrations. Anti-inflammatory activities of these treatments were further confirmed by the reduction of rat hind paw edema. These results suggest that MEDC and MHF have potential anti-inflammatory activity at the selected doses

Evaluation of inhibitory activities of plant extracts on production of LPS-stimulated pro-inflammatory mediators in J774 murine macrophages. Mol Cell Biochem.,

Abstract Whole plant methanolic extracts of 14 traditionally used medicinal herbs were evaluated for their antiinflammatory activity. Extracts of Grindelia robusta, Salix nigra, Arnica montana, and Quassia amara showed up to 4.5-fold inhibition of nitric oxide (NO) production in the J774 murine macrophage cells challenged with LPS without cytotoxicity. These four selected extracts significantly reduced the protein levels of inducible NO synthase (iNOS) and the cyclooxygenase-2 (COX-2) as observed by Western blot analysis. Culture supernatants from cells treated with these extracts indicated 3-5-fold reduction of tumor necrosis factor-a (TNF-a). However, only G. robusta and Q. amara extracts significantly inhibited (by 50%) IL-1b and IL-12 secretions. Furthermore, all these plant extracts were shown to prevent the LPS-mediated nuclear translocation of nuclear factor-jB (NF-jB). All the above observations indicate the anti-inflammatory potential of these plant extracts

Expression, purification and characterization of peanut (Arachis hypogaea) agglutinin (PNA) from baculovirus infected insect cells

Peanut (Arachis hypogaea) seed lectin, PNA is widely used to identify tumor specific antigen (T-antigen), Galβ1-3GalNAc on the eukaryotic cell surface. The functional amino acid coding region of a cDNA clone, pBSH-PN was PCR amplified and cloned downstream of the polyhedrin promoter in the Autographa californica nucleopolyhedrovirus (AcNPV) based transfer vector pVL1393. Co-transfection of Spodoptera frugiperda cells (Sf9) with the transfer vector, pAcPNA and AcRP6 (a recombinant AcNPV having B-gal downstream of the polyhedrin promoter) DNAs produced a recombinant virus, AcPNA which expresses PNA. Infection of suspension culture of Sf9 cells with plaque purified AcPNA produced as much as 9.8 mg PNA per liter (2.0×106 cells/ml) of serum-free medium. Intracellularly expressed protein (re-PNA) was purified to apparent homogeneity by affinity chromatography using ECD-Sepharose. Polyclonal antibodies against natural PNA (n-PNA) cross-reacted with re-PNA. The subunit molecular weight (30kDa), hemagglutination activity, and carbohydrate specificity of re-PNA were found to be identical to that of n-PNA, thus confirming the abundant production of a functionally active protein in the baculovirus expression system

Jacalin Bound Plasma O-Glycoproteome and Reduced Sialylation of Alpha 2-HS Glycoprotein (A2HSG) in Rheumatoid Arthritis Patients

<div><p>Glycosylation studies of plasma proteins can reveal information about the onset and progression of diseases, where in the glycan biosynthetic pathways are disturbed as in rheumatoid arthritis (RA). The present study was focused on analysis of O-linked glycoproteins of plasma in RA patients. Two dimensional gel electrophoresis of jacalin bound plasma of RA patients revealed a number of differentially expressed protein spots as compared to healthy controls. Eighteen protein spots were found to have statistically significant (<em>p</em><0.05) difference in their expression level from four sets of gels and were identified by MALDI-TOF MS. Most of the identified proteins were predicted to be O-glycosylated proteins by Net–O-Gly 3.1 algorithm. Among these the alpha 2HS glycoprotein (A2HSG) was found to be down regulated whereas inter alpha trypsin inhibitor H4 (ITIH4) was up regulated and this was validated by Western blotting. The glycosylation studies showed the reduced N-linked sialylation of A2HSG in RA patients. Altered glycoprotein expression and functional as well as structural studies of glycans might help in the diagnosis of RA and understanding the disease pathogenesis.</p> </div