Trizbenzim, Cu-Trizbenzim and Zn-Trizbenzim as G-Quadruplex Inducing and Stabilizing Compounds on Human Telomeric Sequence and their Anticancer Properties.

BACKGROUND: The benzimidazole and their derivatives have rich biological relevance with respect to available natural amino acids and their role in protein folding and quaternary conformations. Thus the ligand trizbenzim and their Cu(II) and Zn(II) metal complexes were prepared to induce G-quadruplex conformation even under no-salt conditions with remarkable anticancer activities. METHODS: The ligand N,N',N''-Tris-(1H-benzoimidazol-2-ylmethyl)-[1,3,5]triazine-2,4,6-triamine ( trizbenzim) and its Cu and Zn complexes (Cu-trizbenzim, Zn-trizbenzim) were synthesized and characterized by IR, NMR, and MALDI-TOF techniques. The pure ligand and its complexes interacted with human telomere DNA sequence d(TTAGGG), HTelo8and HTelo20and the interactions were followed by circular dichroism spectroscopy, FID assay, and molecular docking techniques. The compounds were tested for anticancer activity towards selected cell lines. RESULTS: All the three compounds stabilized the HTelo8 and HTelo20 in parallel and antiparallel G-quadruplex conformations with salt conditions. Under no-salt conditions, the compounds induce and stabilize the G-quadruplex conformation in antiparallel topology selectively. The pure ligand, Cu-trizbenzim, and Zn-trizbenzim were involved in partial or classical intercalation and some backbone interactions on the strand. The FID assay using thiazole orange intercalator supports the proposed intercalation mode of binding for the three compounds, especially for the pure ligand and the Cu-complex. The MOE docking experiments using X-ray and NMR derived G-quadruplex models with the title compounds extensively support the G-quadruplex induction and stabilization of the telomere sequence by these compounds. The guanines bases involved in the G-tetrad formation interact well with the triazine and the benzimidazole part of the ligand through strong π-π interactions. The primary mode of binding is described as end stacking and intercalation of the compounds to the G-quadruplex structures. The Cu-trizbenzim exhibited more anticancer property in comparison to the pure ligand and the Zntrizbenzim complex. The IC50 values were in the nanomolar range from 50 to 150nM in concentration. CONCLUSION: This novel self-induction of G-quadruplex is novel without the presence of alkali metal ions.

Diversity of Glutathione S-Transferases (GSTs) in Cyanobacteria with Reference to Their Structures, Substrate Recognition and Catalytic Functions.

Glutathione S-Transferases (GSTs) comprise a diverse group of protein superfamily involved in cellular detoxification of various harmful xenobiotics and endobiotics. Cyanobacteria, being the primordial photosynthetic prokaryotes, served as an origin for the evolution of GSTs with diversity in their structures, substrate recognition, and catalytic functions. This study analysed the diversity of GSTs in cyanobacteria for the first time. Based on the sequence alignment and phylogenetic tree analysis, 12 GST classes were identified, which are distributed variedly within cyanobacterial orders such as four in Pleurocapsales, eight in Chroococcales, seven in Oscillatoriales, five in Stigonematales, and nine in Nostocales. Detailed evolutionary analysis of cyanobacterial GSTs suggested that the order Pleurocapsales served as the ancestry for GST evolution. The analysis also identified a conserved motif S[GLNTARS][ADE]I[LAI] with signature residues, cysteine, serine, and tyrosine at the N-terminal end that serves as the initiating residue for detoxification. Alternatively, the grouping of cyanobacterial GSTs and their unique signature residues were located, which serve as a possible discriminating factor. The study also described the mode of glutathione binding between the identified cyanobacterial GST groups highlighting the differences among the GST classes. New GST sequence data may improve further our understanding on GST evolution and other possible divergences in cyanobacteria.

Lipid membrane modulation and pigmentation: A cryoprotection mechanism in Arctic pigmented bacteria.

The present study aims to address the effect of gradual change in temperature (15-4 °C) followed by freeze-thaw on pigmented bacterial strains - Leeuwenhoekiella aequorea, Pseudomonas pelagia, Halomonas boliviensis, Rhodococcus yunnanensis, and Algoriphagus ratkwoskyi, isolated from Kongsfjorden (an Arctic fjord) to understand their survival in present climate change scenario. The total cell count and retrievability of the isolates were not affected despite the variation in temperature. In all the isolates, the saturated fatty acids, particularly stearic and palmitic acid were predominant at higher temperature, while at 4 °C, the unsaturated fatty acids, primarily cis-10-pentadecenoic, palmitoleic, and oleic acid, were major constituents, confirming homeoviscous adaptation. Even after freeze-thaw, the unsaturated fatty acid composition was retained in all the isolates except A. ratkwoskyi. The increase in unsaturated fatty acids was at the expense of their saturated analogs, probably by desaturase activity. The major pigment in the isolates resembled Zeaxanthin, whose concentration was found to be 26-65% higher after freeze-thaw, suggesting its vital role as a cryoprotective agent in regulating membrane fluidity. Such experimental simulations related to freeze-thaw in polar bacterial isolates are helpful in understanding the physiological plasticity adaptations, which could be critical for survival in harsh and rapidly changing polar environments.

Analysis and elucidation of phosphoenolpyruvate carboxylase in cyanobacteria.

Phosphoenolpyruvate carboxylase (PEPC) a cytosolic enzyme of higher plants is also found in bacteria and cyanobacteria. Genetic and biochemical investigations have indicated that there are several isoforms of PEPC belonging to C3; C3/C4 and C4 groups but, the evolution of PEPC in cyanobacteria is not yet understood. The present study opens up an opportunity to understand the isoforms and functions of PEPC in cyanobacteria. The variations observed in PEPC among lower and higher orders of cyanobacteria, suggests convergent evolution of PEPC. There is a specific PEPC phosphorylation residue 'serine' at the N-terminus and PEPC determinant residue 'serine' at the C-terminal that facilitates high affinity for substrate binding. These residues were unique to higher orders of cyanobacteria, but, not in lower orders and other prokaryotes. The different PEPC forms of cyanobacteria were investigated for their kinetic properties with phosphoenolpyruvate as the substrate and the findings corroborated well with the in silico findings. In vitro enzymatic study of cyanobacteria belonging to three different orders demonstrated the role of aspartate as an allosteric effector, which inhibited PEPC by interacting with the highly conserved residues in the active site. The differences in mode of inhibition among the different order, thus, give a fair picture about the cyanobacterial PEPCs. The higher orders appear to possess the sequence coordinates and functionally conserved residues similar to isoforms of C4 type higher plants, whereas isoforms of PEPC of the lower orders did not resemble either that of C3 or C4 plants.

Siderophore mediated uranium sequestration by marine cyanobacterium Synechococcus elongatus BDU 130911.

Four different marine cyanobacterial morphotypes were tested for their efficacy to produce siderophores in Fe minus [Fe(-)], Fe minus Uranium dosed [Fe(-)U(+)], and Fe dosed Uranium dosed [Fe(-)U(+)] media. Of the four organisms tested, Synechococcus elongatus BDU 130911 produced the highest amount of siderophore of 58µgmg(-1) dryweight. The results clearly indicate that uranium induces siderophore production in marine cyanobacteria even in the presence of iron [Fe(-)U(+)] condition. The type of siderophore revealed by FeCl(3), Tetrazolium and Atkin's tests is a hydroxamate; and thin layer chromatogram also authenticates our finding. Uranium siderophore complexation was confirmed through modified Chrome Azurol S (CAS) assay as well as based on residual uranium presence. In silico docking studies further validate siderophore complexation with uranium.

Ability to use the diazo dye, C.I. Acid Black 1 as a nitrogen source by the marine cyanobacterium Oscillatoria curviceps BDU92191.

Ten different strains of marine cyanobacteria were tested for their ability to decolourise and degrade a recalcitrant diazo dye, C.I. Acid Black 1. Of them, Oscillatoria curvicepsBDU92191 was able to grow up to a tested concentration of 500 mG L(-1). The organism degraded 84% of the dye at 100 mG L(-1) in 8 days in a medium free of combined nitrogen. The dye degrading ability is attributed to the activities of the enzymes: laccase, polyphenol oxidase and azoreductase. The absence of the doublet amine peak in addition to the overall reduction of absorption in the IR spectra confirmed the mineralisation of the tested azo dye. The nitrogen assimilating enzyme studies along with nitrogenase assay strongly suggested the ability of the non-heterocystous, filamentous marine cyanobacterium, O. curvicepsBDU92191 to use C.I. Acid Black 1 as a nitrogen source in an oligotrophic environment.

Characterization of manganese superoxide dismutase from a marine cyanobacterium Leptolyngbya valderiana BDU20041.

BACKGROUND: Cyanobacteria are recognized as the primordial organisms to grace the earth with molecular oxygen ~3.5 billion years ago as a result of their oxygenic photosynthesis. This laid a selection pressure for the evolution of antioxidative defense mechanisms to alleviate the toxic effect of active oxygen species (AOS) in cyanobacteria. Superoxide dismutases (SODs) are metalloenzymes that are the first arsenal in defense mechanism against oxidative stress followed by an array of antioxidative system. Unlike other living organisms, cyanobacteria possess multiple isoforms of SOD. Hence, an attempt was made to demonstrate the oxidative stress tolerance ability of marine cyanobacterium, Leptolyngbya valderiana BDU 20041 and to PCR amplify and sequence the SOD gene, the central enzyme for alleviating stress. RESULT: L. valderiana BDU 20041, a filamentous, non-heterocystous marine cyanobacterium showed tolerance to the tested dye (C.I. Acid Black 1) which is evident by increased in biomass (i.e.) chlorophyll a. The other noticeable change was the total ROS production by culture dosed with dye compared to the control cultures. This prolonged incubation showed sustenance, implying that cyanobacteria maintain their antioxidant levels. The third significant feature was a two-fold increase in SOD activity of dye treated L. valderiana BDU20041 suggesting the role of SOD in alleviating oxidative stress via Asada-Halliwell pathway. Hence, the organism was PCR amplified for SOD gene resulting in an amplicon of 550 bp. The sequence analysis illustrated the presence of first three residues involved in motif; active site residues at H4, 58 and D141 along with highly conserved Mn specific residues. The isolated gene shared 63.8% homology with MnSOD of bacteria confirmed it as Mn isoform. This is the hitherto report on SOD gene from marine cyanobacterium, L. valderiana BDU20041 of Indian subcontinent. CONCLUSION: Generation of Reactive Oxygen Species (ROS) coupled with induction of SOD by marine cyanobacterium, L. valderiana BDU20041 was responsible for alleviating stress caused by an azo dye, C. I. Acid Black 1. The partial SOD gene has been sequenced and based on the active site, motif and metal specific residues; it has been identified as Mn metalloform.

Comparative analysis of cyanobacterial superoxide dismutases to discriminate canonical forms.

BACKGROUND: Superoxide dismutases (SOD) are ubiquitous metalloenzymes that catalyze the disproportion of superoxide to peroxide and molecular oxygen through alternate oxidation and reduction of their metal ions. In general, SODs are classified into four forms by their catalytic metals namely; FeSOD, MnSOD, Cu/ZnSOD and NiSOD. In addition, a cambialistic form that uses Fe/Mn in its active site also exists. Cyanobacteria, the oxygen evolving photosynthetic prokaryotes, produce reactive oxygen species that can damage cellular components leading to cell death. Thus, the co-evolution of an antioxidant system was necessary for the survival of photosynthetic organisms with SOD as the initial enzyme evolved to alleviate the toxic effect. Cyanobacteria represent the first oxygenic photoautotrophs and their SOD sequences available in the databases lack clear annotation. Hence, the present study focuses on structure and sequence pattern of subsets of cyanobacterial superoxide dismutases. RESULT: The sequence conservation and structural analysis of Fe (Thermosynechococcus elongatus BP1) and MnSOD (Anabaena sp. PCC7120) reveal the sharing of N and C terminal domains. At the C terminal domain, the metal binding motif in cyanoprokaryotes is DVWEHAYY while it is D-X-[WF]-E-H-[STA]-[FY]-[FY] in other pro- and eukaryotes. The cyanobacterial FeSOD differs from MnSOD at least in three ways viz. (i) FeSOD has a metal specific signature F184X3A188Q189.......T280......F/Y303 while, in Mn it is R184X3G188G189......G280......W303, (ii) aspartate ligand forms a hydrogen bond from the active site with the outer sphere residue of W243 in Fe where as it is Q262 in MnSOD; and (iii) two unique lysine residues at positions 201 and 255 with a photosynthetic role, found only in FeSOD. Further, most of the cyanobacterial Mn metalloforms have a specific transmembrane hydrophobic pocket that distinguishes FeSOD from Mn isoform. Cyanobacterial Cu/ZnSOD has a copper domain and two different signatures G-F-H-[ILV]-H-x-[NGT]-[GPDA]-[SQK]-C and G-[GA]-G-G-[AEG]-R-[FIL]-[AG]-C-G, while Ni isoform has an nickel containing SOD domain containing a Ni-hook HCDGPCVYDPA. CONCLUSION: The present analysis unravels the ambiguity among cyanobacterial SOD isoforms. NiSOD is the only SOD found in lower forms; whereas, Fe and Mn occupy the higher orders of cyanobacteria. In conclusion, cyanobacteria harbor either Ni alone or a combination of Fe and Ni or Fe and Mn as their catalytic active metal while Cu/Zn is rare.

Molecular characterization of marine Cyanobacteria from the Indian subcontinent deduced from sequence analysis of the phycocyanin operon (cpcb-IGS-cpcA) and 16S-23S ITS region.

Molecular characterization of ten marine cyanobacterial isolates belonging to the order Oscillatoriales was carried out using the phycocyanin locus (cpcBA-IGS) and the 16S-23S internally transcribed spacer region. DNA sequences from the phycocyanin operon discriminated ten genotypes, which corresponded to seven morphotypes identified by traditional microscopic analysis. The cpcB coding region revealed 17 % nucleotide variation, while cpcA exhibited 29 % variation across the studied species. Phylogenetic analyses support the conclusion that the Phormidium and Leptolyngbya genera are not monophyletic. The nucleotide variations were heterogeneously distributed with no or minimal informative nucleotides. Our results suggest that the discriminatory power of the phycocyanin region varies across the cyanobacterial species and strains. The DNA sequence analysis of the 16S-23S internally transcribed spacer region also supports the polyphyletic nature of the studied oscillatorian cyanobacteria. This study demonstrated that morphologically very similar strains might differ genotypically. Thus, molecular approaches comprising different gene regions in combination with morphological criteria may provide better taxonomical resolution of the order Oscillatoriales.