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

Whole plant methanolic extracts of 14 traditionally used medicinal herbs were evaluated for their anti-inflammatory 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-alpha (TNF-alpha). However, only G. robusta and Q. amara extracts significantly inhibited (by 50%) IL-1beta and IL-12 secretions. Furthermore, all these plant extracts were shown to prevent the LPS-mediated nuclear translocation of nuclear factor-kappaB (NF-kappaB). All the above observations indicate the anti-inflammatory potential of these plant extracts.

Enhanced expression of alpha1-acid glycoprotein and fucosylation in hepatitis B patients provides an insight into pathogenesis.

Altered glycosylation and concentration of alpha1-acid glycoprotein has been known to be related to the pathogenesis of the hepatic diseases. The present study investigated enhanced fucosylation of AGP in the sera of chronic hepatitis B (HBV-CH) and hepatitis B cirrhosis (HBV-LC) patients by high performance anion exchange chromatography and by ELISA using fucose binding Aleuria aurantia lectin. The concentration of AGP determined by ELISA using monoclonal anti-human AGP (mAb-AGP) showed high level of AGP in HBV-CH and HBV-LC patients. This was further judged by association constant (K (A)) measured by surface plasmon resonance analysis. There was no apparent linkage variation of sialic acid among different patient groups when tested with two sialic acid binding lectins viz., Maackia amurensis agglutinin (MAA, NeuAc alpha2-3-) and Sambucus nigra agglutinin (SNA, NeuAc alpha2-6-) respectively. There was no change of oligosaccharide branching in HBV-CH in comparison to controls whereas a slight change was observed in HBV-LC using ConA. The above results suggest that the changes in concentration of AGP and fucosylation have a prognostic value of hepatitis diseases and it could be possible to use AGP as diagnostic marker besides clinical examination and routine laboratory investigation.

S-nitrosylation of mannose binding lectin regulates its functional activities and the formation of autoantibody in rheumatoid arthritis.

The possibility of post-translational modifications of mannose binding lectin (MBL) leading to functional impairment of the MBL pathway and the presence of anti-MBL autoantibodies were reported earlier in rheumatoid arthritis (RA). MBL was observed to be S-nitrosylated (S-nitrosated) in vitro. HepG2 cells were stimulated with 10% synovial fluid from RA patients to produce increased levels of MBL and nitric oxide. Under these experimental conditions MBL was observed to be S-nitrosated using biotin switch assay. The plasma of RA patients was also found to contain higher levels of S-nitrosylated MBL (SNO-MBL) in comparison to the healthy controls. Functional activities of SNO-MBL were compared with normal MBL. Mannan binding and C4 deposition ability of MBL was found to decrease after S-nitrosylation. It was also observed that S-nitrosylation of MBL leads to a decrease in the bacterial phagocytosis and apoptotic cell binding as measured by fluorescence microscopy and FACS analysis. These results indicate that the carbohydrate binding ability of MBL was affected by S-nitrosylation (S-nitrosation). High levels of anti-MBL autoantibodies were detected against SNO-MBL in plasma of RA patients in comparison to normal MBL suggesting a role of SNO-MBL in generation of autoantibodies in RA patients.

Suppression of inducible nitric oxide synthase by 10-23 DNAzymes in murine macrophage.

iNOS mRNA of J774 murine macrophage cells was cleaved by 10-23 DNAzymes. DNAzyme target site I or translation initiation site and site II have computer predicted (MFOLD) secondary structures but site III has no secondary structure. All the three DNAzymes cleaved the short transcripts generated from cloned DNA almost with equal efficiency while cleavage efficiency is higher at site III than the other two sites on isolated iNOS mRNA. Interestingly, at intracellular level, DNAzyme targeted at translation initiation codon (site I) having secondary structure cleaved iNOS mRNA, and suppressed its activity and protein expression more efficiently than that targeted at sites II and III.

A novel coumarin derivative, 8-methoxy chromen-2-one alleviates collagen induced arthritis by down regulating nitric oxide, NFκB and proinflammatory cytokines.

Ruta graveolens (Rue) is a well-known medicinal plant having anti-inflammatory and other healing properties. This contains many active phytochemicals such as coumarins which possess anti-inflammatory and anti-cancer activities. The present study was carried out to evaluate the therapeutic potential of a newly isolated coumarin derivative from rue plant, 8-methoxy-chromen-2-one (MCO) in the collagen induced arthritic (CIA) rat model. MCO showed inhibition of cytokines and NF-κB in LPS stimulated J774 cells which prompted its possible use in animal. In CIA, arthritic index and arthritic score reduced markedly within 15days of MCO treatment at doses of 2mg and 20mg per kg body weight. Alleviation of joint damage in CIA animals on treatment with MCO was evident from radiographic and histological data. Behavioral studies by open field tests also showed convalescence in the MCO treated CIA rats. Further, escalated plasma levels of pro-inflammatory cytokines TNF-α, IL-1ß and IL-6, and also nitric oxide reduced significantly with the treatment. All these results indicate the therapeutic efficacy of MCO and its possible use as an anti-arthritic drug.

Cell surface characteristics of two halotolerant strains of Sinorhizobium meliloti.

The halotolerant Sinorhizobium meliloti strain Rmd201 and its variant Rmd201 a were examined for their cell surface properties. The variant strain formed rough colonies and was found to be more hydrophobic. Growth of the variant strain was not affected appreciably when NaCl concentration of the medium was increased from 2 mM to 700 mM. Exopolysaccharide (EPS) and the lipopolysaccharide (LPS) content of the variant strain was found to be 7 and 14 times less, respectively, than the parent strain. However, enhanced synthesis of high molecular weight LPS bands were observed in SDS-PAGE analysis in the variant strain when the NaCl concentration was raised from 2 mM to 700 mM. Ribose and glucosamine were present in the variant LPS only. Mannose appeared as a major LPS constituent of the variant when grown in high salt containing medium. All these cell surface characteristics indicated that there were significant differences between the halotolerant strains of S. meliloti. The changes in the cell surface of the variant strain indicated the possible mutation in the gene(s) of cell surface polysaccharide biosynthesis.

Rhizobial lipopolysaccharide as the receptor in lectin-Rhizobium interaction.

Rhizobial 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 Bradyrhizobiumn sp. (Arachis) strain GN17 were isolated by Tn5 mutagenesis. These mutants (GN17M1 and GN17M2) 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 GN17M1 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.

Enhanced expression and fucosylation of ficolin3 in plasma of RA patients.

OBJECTIVES: The aim of this work was to detect low abundant proteins, which may be potential biomarkers of rheumatoid arthritis (RA) at the early stage. We compared plasma protein profiles of RA patients with healthy individuals in two dimensional gel electrophoresis after removal of abundant proteins (albumin and IgG) using depletion kit and Aleuria aurantia lectin (AAL) affinity chromatography. DESIGN AND METHODS: Forty plasma samples each from healthy control individuals and RA patients were used in this study. RESULTS: We found ficolin 3, haptoglobin alpha chain, IgM chain, alpha-1-antitrypsin and hemopexin precursor to be up regulated in the plasma of RA patients. These proteins were identified by matrix assisted laser desorption ionization-time of flight (MALDI-TOF) from several reproducible 2D gels. Ficolin 3, which was not at all visible in albumin and IgG depleted gels, but detected in AAL bound fractions, was further verified by immunobloting and enzyme immunoassay. Elevated fucosylation in ficolin 3 was detected using high performance anion exchange chromatography-pulse amperometric detection (HPAEC-PAD), lectin blotting and enzyme linked lectin binding assay. CONCLUSIONS: Altered fucosylation and elevated level of Ficolin 3 might be exploited to be a potential marker for diagnosis of RA.

Identification of novel autoantigen in the synovial fluid of rheumatoid arthritis patients using an immunoproteomics approach.

Rheumatoid arthritis (RA) is a chronic, autoimmune and inflammatory joint disease with a poorly understood etiology. Despite widespread diagnostic use of anti-citrullinated protein antibodies and rheumatoid factor proteins there is a strong demand for novel serological biomarkers to improve the diagnosis this disease. The present study was aimed to identify novel autoantigens involved in rheumatoid arthritis (RA) pathogenesis through immune-proteomic strategy. Synovial fluid samples from clinically diagnosed RA patients were separated on two-dimensional gel electrophoresis (2-DE). Samples from patients with non-RA rheumatisms (osteoarthritis and trauma) were used as controls. Immunoreactive proteins were spotted by Western blotting followed by identification through Q-TOF mass spectrometer analysis. Forty Western blots were generated using plasma from ten individual RA patients and 33 reactive spots were identified, 20 from the high molecular weight (HMW) gel and 13 from the low molecular weight (LMW) gel. Among the 33 common immunogenic spots, 18 distinct autoantigens were identified, out of which 14 are novel proteins in this context. Expression analysis of five important proteins, vimentin, gelsolin, alpha 2 HS glycoprotein (AHSG), glial fibrillary acidic protein (GFAP), and α1B-glycoprotein (A1BG) by Western blot analysis using their specific antibodies revealed their higher expression in RA synovial fluid as compared to non-RA samples. Recombinantly expressed GFAP and A1BG protein were used to develop an in-house ELISA to quantify the amount of autoantibodies in the RA patients. RA patients revealed an increase in the expression of GFAP and A1BG in the plasma as compared to osteoarthritis patients. Therefore, GFAP and A1BG can be proposed as potential new autoantigens of diagnostic importance for RA subjects. Further characterization of these proteins in rheumatoid arthritis will be helpful in understanding the role of these proteins in the disease pathogenesis providing new diagnostic tool with better specificity and accurate detection of the disease.

Jacalin bound plasma O-glycoproteome and reduced sialylation of alpha 2-HS glycoprotein (A2HSG) in rheumatoid arthritis patients.

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 (p<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.

Suramin ameliorates collagen induced arthritis.

Suramin, a polysulfonated polyaromatic symmetrical urea is known for multiple therapeutic effects including antineoplastic activity. It is known as an antagonist of ATP at P2X purinergic receptors. Suramin is also found to inhibit protein synthesis affecting both initiation and elongation of the polypeptide chain. As a growth factor blocker, it is reported to suppress experimental myocardial inflammation. Here, we describe the anti-arthritic property of suramin in the collagen induced arthritic (CIA) rat, a model of human rheumatoid arthritis (RA). Intraperitoneal (i.p) injection of suramin (10 mg/kg/day) for 3 weeks was found to reduce inflammation and repair joint destruction in CIA rats. Recovery of body weight (p<0.0001), reduction in splenic (p<0.05) and arthritic indices (p<0.0001) and reappearance of smooth synovial lining after suramin treatment to CIA rats were found to be significant. Levels of pro-inflammatory cytokines such as TNF-α, IL-1ß and IL-6 in plasma and joint extracts were reduced (p<0.0001) significantly in response to suramin treatment. Several acute phase proteins were normalized after suramin administration.

Altered glycosylation and expression of plasma alpha-1-acid glycoprotein and haptoglobin in rheumatoid arthritis.

Altered glycosylation patterns in plasma proteins are found to be associated with the pathogenesis of various malignancies and autoimmune disorders. Our previous studies demonstrated the occurrence of some differentially glycosylated plasma proteins in rheumatoid arthritis (RA) patients. The current study was conducted to evaluate the alterations in expression and glycosylation of major acute phase proteins from wheat germ agglutinin enriched RA patients' plasma. Immunoblotting studies revealed a significant enhancement in the plasma levels of alpha-1 acid glycoprotein (AGP) and haptoglobin (Hp) in RA patients with respect to healthy controls. Monosaccharide analysis by high performance anion exchange-chromatography with pulse amperometric detection showed significant variations in the relative percentage of galactose, glucosamine and mannose in AGP and of mannose in Hp in RA patients. Altered patterns of mannosylation in AGP and Hp were also established by enzyme linked immunosorbent assay and Western blotting using Concanavalin-A lectin. These results could give information for understanding the disease pathogenesis and may provide an insight into the development and progression of the disease.

A Mesorhizobium lipopolysaccharide (LPS) specific lectin (CRL) from the roots of nodulating host plant, Cicer arietinum.

A 30 kDa rabbit erythrocyte agglutinating glycoprotein isolated and characterized from the roots of Cicer arietinum and designated as cicer root lectin (CRL). Hemagglutination activity of CRL is strongly inhibited by cell surface LPS of nodulating cicer specific Rhizobium. CRL agglutinates mesorhizobial cells and not Escherichia coli or yeast cells. It binds to immobilized LPS of cicer specific Rhizobium only. The primary structure of CRL as predicted by peptide mass fingerprinting by MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight) indicated ~54% amino acid sequence homology with C. arietinum seedling lectin (Accession no. gi/3204123) and ~26% with C. arietinum (Accession no. gi/110611256), and Pisum sativum (Accession nos. gi/230612, gi/6729956, gi/126148) lectins. These suggested CRL to be a member of vegetative tissue lectin. Circular dichroism analysis indicated that the secondary structure of CRL consists of 48% ß-sheets, 26% random coils, and 11% α-helix. CRL has six isoforms of closely associated molecular mass with differential acidic pI of 5.30, 5.20, 5.15, 5.05, 5.00, 4.80. Identity of these isoforms was confirmed from their binding with cicer specific Rhizobium LPS. All the isoforms of CRL are differentially glycosylated as identified by deglycosylation and monosaccharide analysis using high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). All these results suggest that unlike other plant lectins CRL is a LPS-binding lectin.

iNOS-targeted 10-23 DNAzyme reduces LPS-induced systemic inflammation and mortality in mice.

Sepsis and/or systemic inflammatory response syndrome are leading causes of death in intensive care unit patients. NO is a critical player in the pathogenesis of bacterial sepsis. Several studies demonstrate elevation of iNOS in LPS-induced acute inflammatory responses and mortality; however, the effectiveness of its therapeutic suppression in systemic inflammation is largely controversial. Earlier, we have reported that DNAzymes specific to iNOS mRNA efficiently suppress iNOS expression in LPS-stimulated J774 murine macrophages. In the present study, we explored the effects of two of these DNAzymes in BALB/c mice model of LPS-induced lethal systemic inflammation. Experimental animal groups receiving previous injections of iNOS-specific DNAzyme (100 microg, i.p.) showed significantly reduced mortality. Total cell counts of peritoneal lavage and histopathological studies of tissues demonstrated substantial reduction in the leukocytic infiltration and edema in DNAzyme-treated mice. In addition, DNAzyme-injected animals displayed significantly decreased IL-12 serum level, whereas the levels of IL-1[beta], IFN-[gamma], and TNF-[alpha] also declined to a great extent. DNAzyme treatment resulted in significantly reduced NO levels in serum and peritoneal lavage, confirming functional suppression of iNOS gene in LPS-injected mice. These DNAzymes were also able to limit excessive NO production by cytokine and LPS co-challenges in cultured peritoneal macrophages from DNAzyme-treated mice. Estimation of iNOS mRNA and protein expression in the peritoneal macrophages of DNAzyme-administered animals further confirmed the iNOS gene knockdown. All these results indicated that iNOS-specific DNAzymes reduce inflammatory responses and enhance survival in murine model of LPS-induced lethal systemic inflammation.

Purification and mass spectrometric characterization of Sesbania aculeata (Dhaincha) stem lectin.

A glucose specific lectin (STA) was isolated from Sesbania aculeata stem by using Sephadex G-50 affinity column chromatography. The lectin is a glycoprotein having 29 kDa subunit molecular weight. Two dimensional gel electrophoresis analysis revealed that the lectin existed in two isomeric forms with varied carbohydrate content as analyzed by high performance anion exchange chromatography-pulsed amperometric detector (HPAEC-PAD). Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) and N-terminal sequence (LDSLSFTYNNFE) analysis of this lectin showed 95% homology with stem lectin SL-I (accession no. AJ585523) from peanut plant. The nucleotide sequence of the lectin (STA) was submitted to the gene bank (accession no. EU263636).

Isolation and characterization of a novel cross-infective rhizobia from Sesbania aculeata (Dhaincha).

The Sesbania has been widely used as green manure to improve the productivity of several crops. Sinorhizobium saheli strain (SB2) was isolated from the root nodule of Sesbania aculeata. The Tn5 mutants (300) of SB2 were generated and studied for their nodulation efficiencies in its specific and cross-infective host plants. The mutant, SB2M3, was found to have two- and four fold higher nodulation efficiency than wild type in parent host and nonspecific host plant, respectively. SB2M3 differed from SB2 in exopolysaccharide and lipopolysaccharide content. SB2M3 was halotolerant and could grow in alkaline pH at comparatively high temperatures. Hence, it may find an application in agritechnology.

Expression of TNF-alpha and related signaling molecules in the peripheral blood mononuclear cells of rheumatoid arthritis patients.

We examined the role of tumor necrosis factor (TNF-alpha) 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-alpha 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-alpha 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-alpha induced signaling may not be explained only by these pathways.

Molecular cloning, expression, and cytokinin (6-benzylaminopurine) antagonist activity of peanut (Arachis hypogaea) lectin SL-I.

Isolation and purification of a alpha-methyl-mannoside specific lectin (SL-I) of peanut was reported earlier [Singh and Das (1994) Glycoconj J 11:282-285]. Native SL-I is a glycoprotein having approximately 31 kDa subunit molecular mass and forms dimer. The gene encoding this lectin is identified from a 6-day old peanut root cDNA library by anti-SL-I antibody and N-terminal amino acid sequence homology to the native lectin. Nucleotide sequence derived amino acid sequence of the re-SL-I shows amino acid sequence homology with the N-terminal and tryptic digests' amino acid sequence of the native SL-I (nSL-I). Presence of a putative glycosylation (QNPS) site and a hydrophobic adenine-binding (VLVSYDANS) site is also identified in SL-I. Homology modeling of the lectin suggests it to be an archetype of legume lectins. It is expressed as a approximately 30 kDa apoprotein in E. coli and has the carbohydrate specificity and secondary structure identical to its natural counterpart. The lectin SL-I inhibits cytokinin 6-benzylaminopurine (BA)-induced "delayed leaf senescence" and "cotyledon expansion". Equilibrium dialysis revealed a single high-affinity binding site for adenine (7.6 x 10(-6 )M) and BA (1.09 x 10(-5 )M) in the SL-I dimer and thus suggesting that the cytokinin antagonist effect of SL-I is mediated by the direct interaction of SL-I with BA.

Altered expression and glycosylation of plasma proteins in rheumatoid arthritis.

Altered glycosylation of plasma proteins has been directly implicated in the pathogenesis of rheumatoid arthritis (RA). The present study investigated the changes in the Concanavalin-A (Con-A)-bound plasma proteins in the RA patients in comparison to that of the healthy controls. Two proteins (MW approximately 32 kDa and approximately 62 kDa) showed an alteration in expression while an altered monosaccharide profile (high mannose) was observed in the approximately 62 kDa protein in the samples collected from RA patients. The 2-dimensional polyacrylamide gel electrophoresis analysis of the Con-A-bound plasma samples showed a large number of protein spots, a few of which were differentially expressed in the RA patients. Some unidentified proteins were detected in the RA patients which were absent in the control samples. The present study, therefore, enunciates the role of carbohydrates as well as that of the acute phase response in the disease pathogenesis.

Association of mannose-binding lectin gene (MBL2) polymorphisms with rheumatoid arthritis in an Indian cohort of case-control samples.

Single nucleotide polymorphisms in the mannose-binding lectin (MBL2) gene, as well as the serum MBL2 level, have been associated with various autoimmune diseases. We investigated whether such polymorphisms and/or the serum MBL2 level were associated with rheumatoid arthritis (RA) in an Indian population. The frequency of the B variant (codon 54) of the MBL2 gene was quite frequent in the healthy Indian population and was significantly (P=6.35x10(-6)) lower in RA patients. We replicated this association (P=1.78x10(-5)) in an independent cohort of control individuals. Promoter polymorphism at -550 nt showed a significant overrepresentation (P=0.003) of the minor allele G in severe RA patients compared with the less severe group. Haplotype LYA frequency was significantly (P=0.03) high in the less severe group, while the frequency of the HYA haplotype was significantly (P=0.04) increased in the severe RA patients. No statistically significant difference in serum MBL2 was observed as a whole, but the individuals homozygous for the LYA haplotype had significantly lower (P=0.017) serum MBL2 levels compared with individuals homozygous for the HYA haplotype. Therefore, the B variant of the MBL2 gene may be associated with protection from RA in our study population, and the promoter polymorphism (-550 nt) seems to have some role in disease progression.