Synthesis and Hybridization Studies of Oligonucleotide Sequences with Modified Fluorescent Nucleoside Analogs

Abstract

Two complementary oligodeoxynucleotide hexamers CATGAA and TTCATG and a pentamer with a fluorescent nucleoside analog viz. 9-N-(2′-deoxy-β-D-ribofuranosyl) carbazole (C*) incorporated into it, TTC*ATG were synthesized and characterised by spectroscopic and chromatographic studies. The comparative fluorescent studies of the two nucleoside analogs viz. 9-N-(2′-deoxy-β-D-ribofuranosyl) acridone and its carbazole analog (C*) have been carried out under different experimental conditions. The effect on fluorescence by incorporation of (C*) into the sequence and its subsequent hybridization with the complementary sequence have been studied.     

Evidence of Unique and Generalist Microbes in Distantly Related Sympatric Intertidal Marine Sponges (Porifera: Demospongiae)

The diversity and specificity of microbial communities in marine environments is a key aspect of the ecology and evolution of both the eukaryotic hosts and their associated prokaryotes. Marine sponges harbor phylogenetically diverse and complex microbial lineages. Here, we investigated the sponge bacterial community and distribution patterns of microbes in three sympatric intertidal marine demosponges, Hymeniacidon perlevis, Ophlitaspongia papilla and Polymastia penicillus, from the Atlantic coast of Portugal using classical isolation techniques and 16S rRNA gene clone libraries. Microbial composition assessment, with nearly full-length 16S rRNA gene sequences (ca. 1400 bp) from the isolates (n = 31) and partial sequences (ca. 280 bp) from clone libraries (n = 349), revealed diverse bacterial communities and other sponge-associated microbes. The majority of the bacterial isolates were members of the order Vibrionales and other symbiotic bacteria like Pseudovibrio ascidiaceiocola, Roseobacter sp., Hahellaceae sp. and Cobetia sp. Extended analyses using ecological metrics comprising 142 OTUs supported the clear differentiation of bacterial community profiles among the sponge hosts and their ambient seawater. Phylogenetic analyses were insightful in defining clades representing shared bacterial communities, particularly between H. perlevis and the geographically distantly-related H. heliophila, but also among other sponges. Furthermore, we also observed three distinct and unique bacterial groups, Betaproteobactria (∼81%), Spirochaetes (∼7%) and Chloroflexi (∼3%), which are strictly maintained in low-microbial-abundance host species O. papilla and P. penicillus. Our study revealed the largely generalist nature of microbial associations among these co-occurring intertidal marine sponges.     

Induction and establishment of somatic embryogenesis in elite Indian cotton cultivar (Gossypium hirsutumL. cv Khandwa-2)

Embryogenesis in cotton is a difficult task due its genome dependency. We used 3 cotton cultivars (Khandwa-2, G. Cot. 10, and BC-68–2) and Coker-312 as control for regeneration. Efficient somatic embryogenesis was induced in agronomically important Indian cotton cultivars, Khandwa-2 and G. Cot. 10. For callusing in all the cultivars, different media combinations were tried. Embryogenesis was initiated on a hormone-free MS medium (MSB). For embryo maturation and recovery excess of L-glutamine and l-asparagine were used. Khandwa-2 somatic embryos were successfully regenerated into plants. However, no plantlet was obtained in case of G. Cot. 10. Callus induction was also observed in BC-68–2 but there was no embryogenesis observed. The study indicated that the medium and genotype significantly effects embryogenesis. An efficient protocol is described here for regenerating plants via somatic embryogenesis in an elite Indian cotton cultivar Khandwa-2.     

Growth and alkaloid production along with expression profiles of biosynthetic pathway genes in two contrasting morphotypes of prickly and prickleless Solanum viarum Dunal

Protoplasma - Growth and production kinetics of three important glycoalkaloids viz. α-solanine, solanidine, and solasodine in two contrasting prickly and prickleless plants of Solanum viarum...     

Impaired Autophagy Flux is Associated with Proinflammatory Microglia Activation Following Japanese Encephalitis Virus Infection

Neurochemical Research - Role of autophagy in Japanese encephalitis viral (JEV) infection is not well known. In the present study, we reported the role of autophagy flux in microglia activation,...     

Molecular characterisation of Fusarium oxysporum causing rot diseases in small cardamom (Elettaria cardamomum Maton)

Incidence of root rot and foliar yellowing, rhizome rot, panicle wilt and stem rot diseases of small cardamom (Elettaria cardamomum Maton) are caused by Fusarium oxysporum Schlecht., and were surveyed in the high ranges of Idukki district, Kerala during 2010–2011. The diseases were noticed in different areas to varying degrees. Root rot was found to be most severe, followed by pseudostem rot, rhizome rot and panicle wilt. The Fusarium infections were prevalent throughout the year (January–December) and varied from 1.5 to 10.6%. Even though the pathogen was isolated from different plant parts, during pathogenicity studies, all the isolates could cross-infect other plant parts too. Twenty different isolates of F. oxysporum were obtained from diseased samples, and five morphologically distinct isolates were analysed with Randomly Amplified Polymorphic DNA (RAPD) markers to study the genetic variability, if any, among them. PCR amplification of total genomic DNA with random oligonucleotide primers generated unique banding patterns, depending upon primers and isolates. Nine oligunucleotide primers were selected for the RAPD assays, which resulted in 221 bands for the five isolates of F. oxysporum. The number of bands obtained was entered into an NTSYS, and the results showed moderate genetic variability among F. oxysporum isolates causing root rot, rhizome rot, panicle wilt and pseudostem rot, collected from different locations. The dendrogram of different isolates into groups resulted in one major cluster at 0.61 similarity index comprising of four isolates (CRT 3, CRR 3, CPW 2 and CSR 1) and one isolate (CRT 5) formed in a separate cluster. Among the five isolates of F. oxysporum, CRT 5 was entirely different from the other four isolates. The isolates also differ according to the geographical area, as revealed from the genetic variability observed in different root rot isolates (CRT 3 and CRT 5). It is inferred that despite moderate variability, F. oxysporum, infecting small cardamom in Idukki district of Kerala, consists of a single clonal lineage.     

Association of peroxisome proliferator activated receptor-γ gene with non-alcoholic fatty liver disease in Asian Indians residing in north India

Background

Genetics of non-alcoholic fatty liver (NAFLD) in Asian Indians has been inadequately studied. We investigated the association of polymorphisms C161T and Pro12Ala of peroxisome proliferator-activated receptor gamma (PPARγ) with clinical and biochemical parameters in Asian Indians with NAFLD.

Methods

In this case–control study, 162 NAFLD cases and 173 controls were recruited. Abdominal ultrasound, clinical and biochemical profiles, fasting insulin levels and value of homeostasis model assessment of insulin resistance were determined. Polymerase chain reaction–restriction fragment length polymorphisms of two polymorphisms were performed. The association of these polymorphisms with clinical and biochemical parameters was analysed.

Results

Higher frequency of Ala and T alleles of PPARγ was obtained in cases. Ala/Ala genotype of PPARγ (Pro12Ala) was associated with significantly higher serum triglycerides (TG), alkaline phosphatase (ALK) and waist–hip ratio in cases as compared to controls. In C161T polymorphism, TT genotype was significantly increased TG (p = 0.04), total cholesterol (p = 0.01), ALK (p = 0.04) and gamma-glutamyl transpeptidase (p = 0.007) in cases. The linkage disequilibrium for these two single-nucleotide polymorphisms of PPARγ was differed in cases (D1 = 0.1; p = 0.006) and controls (D1 = 0.07; p = 0.1). Using a multivariate analysis after adjusting for age, sex and body mass index, the presence of NAFLD was linked to these two polymorphisms (odds ratio 1.64 (95% CI: 1.09–2.45, p = 0.05)].

Conclusion

Asian Indians in north India carrying the alleles Ala and T of PPARγ (Pro12Ala and C161T) polymorphisms are predisposed to develop NAFLD.     

ParA encoded on chromosome II of Deinococcus radiodurans binds to nucleoid and inhibits cell division in Escherichia coli

Bacterial genome segregation and cell division has been studied mostly in bacteria harbouring single circular chromosome and low-copy plasmids. Deinococcus radiodurans, a radiation-resistant bacterium, harbours multipartite genome system. Chromosome I encodes majority of the functions required for normal growth while other replicons encode mostly the proteins involved in secondary functions. Here, we report the characterization of putative P-loop ATPase (ParA2) encoded on chromosome II of D. radiodurans. Recombinant ParA2 was found to be a DNA-binding ATPase. E. coli cells expressing ParA2 showed cell division inhibition and mislocalization of FtsZ-YFP and those expressing ParA2-CFP showed multiple CFP foci formation on the nucleoid. Although, in trans expression of ParA2 failed to complement SlmA loss per se, it could induce unequal cell division in slmAminCDE double mutant. These results suggested that ParA2 is a nucleoid-binding protein, which could inhibits cell division in E. coli by affecting the correct localization of FtsZ and thereby cytokinesis. Helping slmAminCDE mutant to produce minicells, a phenotype associated with mutations in the ‘Min’ proteins, further indicated the possibility of ParA2 regulating cell division by bringing nucleoid compaction at the vicinity of septum growth.     

Protection against peroxynitrite-induced DNA damage by mesalamine: implications for anti-inflammation and anti-cancer activity

Mesalamine (5-aminosalicylic acid, 5-ASA) is known to be the first-line medication for treatment of patients with ulcerative colitis. Studies have demonstrated that ulcerative colitis patients treated with 5-ASA have an overall decrease in the risk of developing colorectal carcinoma. However, the mechanisms underlying 5-ASA-mediated anti-inflammatory and anti-cancer effects are yet to be elucidated. Because peroxynitrite has been critically involved in inflammatory stress and carcinogenesis, this study was undertaken to investigate the effects of 5-ASA in peroxynitrite-induced DNA strand breaks, an important event leading to peroxynitrite-elicited cytotoxicity. Incubation of φX-174 plasmid DNA with the peroxynitrite generator 3-morpholinosydnonimine (SIN-1) led to the formation of both single- and double-stranded DNA breaks in a concentration-dependent manner. The presence of 5-ASA at 0.1 and 1.0 mM was found to significantly inhibit SIN-1-induced DNA strand breaks in a concentration-dependent manner. The consumption of oxygen induced by SIN-1 was found to not be affected by 5-ASA at 0.1–50 mM, indicating that 5-ASA at these concentrations is not involved in the auto-oxidation of SIN-1 to form peroxynitrite. It is observed that 5-ASA at 0.1–1 mM showed considerable inhibition of peroxynitrite-mediated luminol chemiluminescence in a dose-dependent fashion, suggesting that 5-ASA is able to directly scavenge the peroxynitrite. Electron paramagnetic resonance (EPR) spectroscopy in combination with spin-trapping experiments, using 5,5-dimethylpyrroline-N-oxide (DMPO) as spin trap resulting in the formation of DMPO-hydroxyl radical adduct from peroxynitrite, and 5-ASA only at higher concentration (1 mM) inhibited the hydroxyl radical adduct while shifting EPR spectra, indicating that 5-ASA at higher concentrations may generate a more stable free radical species rather than acting purely as a hydroxyl radical scavenger. Taken together, these studies demonstrate for the first time that 5-ASA can potently inhibit peroxynitrite-mediated DNA strand breakage, scavenge peroxynitrite, and affect peroxynitrite-mediated radical formation, which may be responsible, at least partially, for its anti-inflammatory and anti-cancer effects.