Rakha H. Das

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-α (TNF-α). However, only G. robusta and Q. amara extracts significantly inhibited (by 50%) IL-1β and IL-12 secretions. Furthermore, all these plant extracts were shown to prevent the LPS-mediated nuclear translocation of nuclear factor-κB (NF-κB). All the above observations indicate the anti-inflammatory potential of these plant extracts.

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

AbstractSingle 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=6.35×10−6) lower in RA patients. We replicated this association (P=1.78×10−5) 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.

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 Sephadex 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 dodecyl sulphate-polyacrylamide 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.

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 ∼32 kDa and ∼62 kDa) showed an alteration in expression while an altered monosaccharide profile (high mannose) was observed in the ∼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.

Serine/threonine kinase (pk-1) is a component of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) very late gene transcription complex and it phosphorylates a 102 kDa polypeptide of the complex.

The baculovirus gene, protein kinase-I (pk-1) encodes a serine/threonine kinase that is essential for very late gene expression. Late and very late genes of the baculoviruses are transcribed by an alpha-amanitin resistant RNA polymerase. The very late gene promoter transcription initiation complex was isolated from nuclei of Autographa californica multiple nucleopolyhedrovirus (AcMNPV)-infected Sf9 cells by DNA affinity purification and found to contain 4 major polypeptides of sizes approximately 102, 38, 32, and 18 kDa. The 32 kDa polypeptide was immunoreactive to AcMNPV anti-pk-1 antibody and phosphorylated the 102 kDa polypeptide, earlier reported as late gene expression factor LEF-8. Electrophoretic mobility shift assays with anti-pk-1 antibody indicated the binding of promoter DNA with recombinant AcMNPV-pk-1 and transcription initiation complex proteins. All these results suggested AcMNPV-pk-1 to be a component of the viral very late gene transcription initiation complex.

Site-specific cleavage of HCV genomic RNA and its cloned core and NS5B genes by DNAzyme

Background and Aims:  The 9600 nt hepatitis C virus (HCV) genomic RNA has only one internal ribosome entry site (IRES) for translation to a single polyprotein. In search of nucleic acid‐based antiviral agents, two 10‐23 DNAzymes were designed to cleave the RNA in IRES and RNA dependent RNA polymerase (RDRP/NS5B) regions to prevent translation and replication of HCV RNA.

Restriction endonuclease analysis of a Spodoptera litura nuclear polyhedrosis virus (NPV) isolate

The DNA of a nuclear polyhedrosis virus (NPV) propagated in the larvae of S. litura was analysed with AluI, ApaI, BamHI, BglII, BstEII, BstNI, ClaI, EcoRI, EcoRV, HindIII, HinfI, KpnI, MspI, PstI, PvuII, SalI, Sau3AI, SmaI, TaqI, XbaI, and XhoI. The unique restriction endonuclease profiles of the virus indicate it to be a distinct NPV isolate. The size of the viral genome was estimated to 132 kbp; the genome was mapped with HindIII and PstI.

Inhibition of Autographa californica nucleopolyhedrovirus (AcNPV) polyhedrin gene expression by DNAzyme knockout of its serine/threonine kinase (pk1) gene.

DNAzyme is known to selectively cleave RNA at predetermined site. Transfection of Autographa californica nucleopolyhedrovirus (AcNPV) infected Sf9 cells with serine/threonine kinase (pk1) mRNA specific DNAzymes, DZ1 and DZ2 to cleave the viral coded (pk1) mRNA in between 87th and 88th, and 250th and 251st nucleotide, respectively inhibited the pk1 mRNA and its protein expressions. Interestingly, polh mRNA and protein expressions were also inhibited by these DNAzymes despite their inability to cleave polh mRNA. The polyhedrin promoter driven green fluorescent protein (GFP) mRNA and protein expressions were also inhibited by these pk1 specific DZs. Surprisingly the extents of inhibition of polyhedrin and GFP at different concentrations of both DZs were higher than that of pk1 mRNA and protein expressions. These results suggested that pk1 regulates polyhedrin promoter driven transcription of AcNPV, and the effect of one gene expression on that of other can be studied by DNAzyme knockdown.

Anti-inflammatory effects of shea butter through inhibition of iNOS, COX-2, and cytokines via the Nf-κB pathway in LPS-activated J774 macrophage cells.

Shea butter is traditionally used in Africa for its anti-inflammatory and analgesic effects. In this study we explored the anti-inflammatory activities of the methanolic extract of shea butter (SBE) using lipopolysaccharide (LPS)-induced murine macrophage cell line J774. It was observed that SBE significantly reduced the levels of LPS-induced nitric oxide, Tumor necrosis factor-α (TNF-α), interleukins, 1β (IL-1β), and -12 (IL-12) in the culture supernatants in a dose dependent manner. Expression of pro-inflammatory enzymes, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) were also inhibited by SBE. These anti-inflammatory effects were due to an inhibitory action of SBE on LPS-induced iNOS, COX-2, TNF-α, IL-1β, and IL-12 mRNA expressions. Moreover, SBE efficiently suppressed IκBα phosphorylation and NF-κB nuclear translocation induced by LPS. These findings explain the molecular bases of shea butter’s bioactivity against various inflammatory conditions and substantiate it as a latent source of novel therapeutic agents.

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.