Characterization of canthaxanthin isomers isolated from a new soil Dietzia sp. and their antioxidant activities.

Canthaxanthin (cx) is a potent antioxidant that is chemically synthesized at the industrial scale and has imperative applications in the cosmetic and feed industries. An orange pigmented mesophilic bacterium, designated as K44, was isolated from soil samples of Kargil, India. Biochemical tests, 16S rRNA gene sequencing, and FAME analysis of the bacterium indicated it to belong in the genus Dietzia and is distinct from human isolates. The strain showed 98% 16S rRNA gene sequence homology with Dietzia maris DSM 43102. High-performance liquid chromatography profile of the pigments isolated from K44 showed two major peaks absorbing at 465.3 and 475 nm. The liquid chromatography-mass spectrometry (LC-MS) analysis of both these peaks revealed their m/z to be 564. The molecular weights, LC-MS/MS fragmentation patterns, and lambdamax of these fractions corresponded to all-trans- (475 nm) and 9-cis-(465.3 nm) cx isomers. The antioxidant activities of cis- and trans-cx isomers isolated from this bacterium were found to differ, where the cis-isomer showed higher free radical, superoxide radical, and reactive oxygen species scavenging activities than the alltrans- isomer, suggesting that 9-cis-cx is more effective as an antioxidant than the all-trans-cx.

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, 1ß (IL-1ß), 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-1ß, and IL-12 mRNA expressions. Moreover, SBE efficiently suppressed IκB 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.

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.

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.

Silencing of TNF-alpha receptors coordinately suppresses TNF-alpha expression through NF-kappaB activation blockade in THP-1 macrophage.

Persistently elevated level of TNF-alpha has been implicated in several inflammatory disorders, however, its autocrine production through TNF-alpha receptors signaling is poorly understood. Here we report that simultaneous silencing of TNF-receptors, R1 and R2 by DNAzyme or siRNA suppressed TNF-alpha expression more efficiently than silencing them individually in lipopolysaccharides (LPS) stimulated THP-1 macrophages. Co-silencing of TNF-receptors also inhibited TNF-alpha induced NF-kappaB activation to a higher extent. It was further observed that NF-kappaB inhibitor but not c-Jun N-terminal kinase inhibitor (SP600125) suppressed TNF-alpha expression. All these results suggest that TNF-alpha expression is regulated by synergistic signaling of TNF receptors through downstream NF-kappaB activation.

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. METHODS: In vitro cleavage of HCV RNA by IRES specific DNAzyme, CDz and NS5B specific DNAzyme, NDz was carried out using HCV genomic RNA and in vitro synthesized runoff transcripts of core and NS5B genes. Cleavage of core and NS5B mRNAs by DNAzyme (Dz) in HepG2 cells was assessed by reverse transcription polymerase chain reaction (RT-PCR) using RNA from cells co-transfected with cloned core or NS5B gene and its respective DNAzyme. Suppression of core or NS5B protein expression due to mRNA cleavage by Dz in co-transfected cells was determined by Western blot analysis and fluorescence intensity of fluorescent-tagged expressed protein. Reduction of NS5B protein activity in NDz co-transfected cells was determined by enzymatic assays. RESULTS: The designed CDz and NDz cleaved HCV genomic RNA and their respective in vitro generated transcripts. Both mRNA and protein expressions of core or NS5B from their cloned genes reduced substantially when co-transfected with respective Dz. Reduction of RDRP expression by NDz was accompanied with its reduced enzyme activity. Increased RNA cleavage, inhibition of protein expression, and reduction of RDRP activity were observed on increasing Dz concentration. CONCLUSION: Core and NS5B targeted DNAzymes can be used in controlling the replication of HCV RNA.

9-cis-canthaxanthin exhibits higher pro-apoptotic activity than all-trans-canthaxanthin isomer in THP-1 macrophage cells.

All-trans-canthaxanthin (4, 4'-diketo beta-carotene) but not 9-cis-canthaxanthin has been shown to induce apoptosis in some cell lines. In this study apoptotic activity of 9-cis-canthaxanthin on THP-1 macrophage is reported. Comparison of apoptotic activities of the two canthaxanthin isomers on this cell line by annexin V-cy3 and TUNEL assays indicated the higher pro-apoptotic activity of 9-cis-isomer than the all-trans-isomer. Canthaxanthin-induced apoptosis in this cell line was found to be accompanied by increased caspase-3 and caspase-8 activities, indicating its progression via caspase cascade. Induction of both caspase activities was higher by 9-cis-canthaxanthin than that by trans-canthaxanthin. All these results suggest that canthaxanthin stereoisomers differentially induce apoptosis of THP-1 monocyte/macrophage.

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.

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.

Influence of quasispecies on virological responses and disease severity in patients with chronic hepatitis C.

AIM: To elucidate the influence of quasispecies on virological response and disease severity in patients with chronic hepatitis C. METHODS: Forty seven patients with hepatitis C [32 with chronic active hepatitis (CAH), 9 with cirrhosis, and 6 with hepatocellular carcinoma (HCC)] were screened for the presence of quasispecies by single stranded conformational polymorphism (SSCP) analysis in the hypervariable region (HVR) and non-structural 5B (NS5B) viral genes of hepatitis C virus. The 41 patients excluding those with HCC were on therapy and followed up for a year with the determination of virological response and disease severity. Virus isolated from twenty three randomly selected patients (11 non-responders and 12 showing a sustained virological response) was sequenced for the assessment of mutations. RESULTS: The occurrence of quasispecies was proportionately higher in patients with HCC and cirrhosis than in those with CAH, revealing a significant correlation between the molecular evolution of quasispecies and the severity of disease in patients with hepatitis C. The occurrence of complex quasispecies has a significant association (P < 0.05) with the non-responders, and leads to persistence of infection. Significant differences (P < 0.05) in viral load (log10 IU/mL) were observed among patients infected with complex quasispecies (CQS), those infected with simple quasispecies (SQS) and those with no quasispecies (NQS), after 12 wk (CQS-5.2 +/- 2.3, SQS-3.2 +/- 1.9, NQS-2.8 +/- 2.4) and 24 wk (CQS-3.9 +/- 2.2, SQS-3.0 +/- 2.2, NQS-2.1 +/- 2.3) in the HVR region. However, a statistically significant difference (P < 0.05) was observed between the viral loads of patients infected with CQS and those infected with NQS in NS5B viral gene after 24 wk (CQS-3.9 +/- 2.2, SQS-3.0 +/- 2.2, and NQS-2.1 +/- 2.3) and 48 wk (CQS-3.1 +/- 2.7, SQS-2.3 +/- 2.4, NQS-2.0 +/- 2.3) of therapy. Disease severity was significantly associated with viral load during therapy. The strains isolated from non-responders showed close pairing on phylogeny based on the NS5B gene, but dissimilar HVR regions. This revealed the possibility of the selection of resistant strains during the evolution of quasispecies in NS5B. CONCLUSION: Viral quasispecies may be an important predictor of virological responses to combination therapy in patients with chronic hepatitis C. Complex quasispecies and resistant strains may lead to high viral loads during therapy, with a concerted effect on disease severity.

Response of combination therapy on viral load and disease severity in chronic hepatitis C.

Influence of genotypes on viral kinetics and disease severity in chronic hepatitis C (CH-C) patients has been implicated, but requires further investigation. The 41 HCV patients were genotyped by restriction fragment length polymorphism and included for 48 weeks of combination therapy on the basis of clinical (alanine amino transferase > or =60 IU/l) and histological features (histological activity index > or =3). A significant number (30/41) of patients (6/9 of genotype 1, 23/30 of genotype 3 and 1/2 of mixed genotype) attain sustained virological response despite high viral load at baseline. More aggressive treatment was required in genotype 1 than in genotype 3 due to slow response rate. Significant (P < 0.05) difference in the viral load of sustained virological responder and non-responder (NR) was observed after 12 weeks of therapy. Severe course of liver disease was observed in 81.81% (9/11) of NR patients at baseline. Data indicate that genotype 1 was a slow responder compared to genotype 3 on combination therapy. Response to combination therapy was almost independent of baseline viral load. However, a positive correlation of viral load with disease severity was observed. The viral kinetics at 12 weeks is an important tool for determination of virological response.

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.

Serine/Threonine kinase dependent transcription from the polyhedrin promoter of SpltNPV-I.

Polyhedrin (polh) and p10 are the two hyper-expressed very late genes of nucleopolyhedroviruses. Alpha amanitin resistant transcription from Spodoptera litura nucleopolyhedrovirus (SpltNPV-I) polyhedrin promoter was observed with virus infected nuclear extract of NIV-HA-197 cells but not with that from uninfected nuclear extract. Anti-protein kinase-1 (pk1) antibody inhibited the transcription and the inhibition reversed on addition of pk1, however, pk1 mutant protein, K50M having no phosphorylation activity did not overcome the transcription inhibition. Chromatin immuno-precipitation assays with viral anti-pk1 antibody showed the interaction of pk1 with the polh while electrophoretic mobility shift assays indicated the strong binding affinity (K(d) approximately 5.5x10(-11)) of purified pk1 with the polh promoter. These results suggested that the viral coded pk1 acts as a transcription factor in transcribing baculovirus very late genes.

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.

A pathogenesis related protein, AhPR10 from peanut: an insight of its mode of antifungal activity.

A pathogenesis related protein (AhPR10) is identified from a clone of 6-day old Arachis hypogaea L. (peanut) cDNA library. The clone expressed as a approximately 20 kDa protein in E. coli. Nucleotide sequence derived amino acid sequence of the coding region shows its homology with PR10 proteins having Betv1 domain and P loop motif. Recombinant AhPR10 has ribonuclease activity, and antifungal activity against the peanut pathogens Fusarium oxysporum and Rhizoctonia solani. Mutant protein AhPR10-K54N where lys54 is mutated to asn54 loses its ribonuclease and antifungal activities. FITC labeled AhPR10 and AhPR10-K54N are internalized by hyphae of F. oxysporum and R. solani but the later protein does not inhibit the fungal growth. This suggests that the ribonuclease function of AhPR10 is essential for its antifungal activity. Energy and temperature dependent internalization of AhPR10 into sensitive fungal hyphae indicate that internalization of the protein occurs through active uptake.

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.

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.

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.

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.