Utpal Ghosh

miRNA gene counts in chromosomes vary widely in a species and biogenesis of miRNA largely depends on transcription or post-transcriptional processing of coding genes

MicroRNAs target specific mRNA(s) to silence its expression and thereby regulate various cellular processes. We have investigated miRNA gene counts in chromosomes for 20 different species and observed wide variation. Certain chromosomes have extremely high number of miRNA gene compared with others in all the species. For example, high number of miRNA gene in X chromosome and the least or absence of miRNA gene in Y chromosome was observed in all species. To search the criteria governing such variation of miRNA gene counts in chromosomes, we have selected three parameters- length, number of non-coding and coding genes in a chromosome. We have calculated Pearsons correlation coefficient of miRNA gene counts with length, number of non-coding and coding genes in a chromosome for all 20 species. Major number of species showed that number of miRNA gene was not correlated with chromosome length. Eighty five percent of species under study showed strong positive correlation coefficient (r ≥ 0.5) between the numbers of miRNA gene vs. non-coding gene in chromosomes as expected because miRNA is a sub-set of non-coding genes. 55% species under study showed strong positive correlation coefficient (r ≥ 0.5) between numbers of miRNA gene vs. coding gene. We hypothesize biogenesis of miRNA largely depends on coding genes, an evolutionary conserved process. Chromosomes having higher number of miRNA genes will be most likely playing regulatory roles in several cellular processes including different disorders. In humans, cancer and cardiovascular disease associated miRNAs are mostly intergenic and located in Chromosome 19, X, 14, and 1.

Interaction studies between biosynthesized silver nanoparticle with calf thymus DNA and cytotoxicity of silver nanoparticles.

The interaction of calf thymus DNA (CTDNA) with silver nanoparticles (SNP) has been investigated following spectroscopic studies, analysis of melting temperature (Tm) curves and hydrodynamic measurement. In spectrophotometric titration and thermal denaturation studies of CTDNA it was found that SNP can form a complex with double-helical DNA and the increasing value of Tm also supported the same. The association constant of SNP with DNA from UV-Vis study was found to be 4.1×10(3) L/mol. The fluorescence emission spectra of intercalated ethidium bromide (EB) with increasing concentration of SNP represented a significant reduction of EB intensity and quenching of EB fluorescence. The results of circular dichroism (CD) suggested that SNP can change the conformation of DNA. From spectroscopic, hydrodynamic, and DNA melting studies, SNP has been found to be a DNA groove binder possessing partial intercalating property. Cell cytotoxicity of SNP was compared with that of normal silver salt solution on HeLa cells. Our results show that SNP has less cytotoxicity compared to its normal salt solution and good cell staining property.

Design and synthesis of anthracene-based bispyridinium amides: anion binding, cell staining and DNA interaction studies

The design and synthesis of anthracene labeled bispyridinium amides 1–4 along with their anion binding, cell staining and DNA interaction studies are reported. All the chemosensors exhibit significant response towards H2PO4− in CH3CN. Furthermore, sensors 2 and 3 are quite interesting for the selective sensing of aliphatic dicarboxylates. Among these compounds, 1 is found to be useful in cell staining. Also all of them exhibit significant interaction with DNA. All these properties are found to be dependent on the nature of the spacer that holds the pyridinium binding sites.

Interaction of aurintricarboxylic acid (ATA) with four nucleic acid binding proteins DNase I, RNase A, reverse transcriptase and Taq polymerase

Abstract In the investigation of interaction of aurintricarboxylic acid (ATA) with four biologically important proteins we observed inhibition of enzymatic activity of DNase I, RNase A, M-MLV reverse transcriptase and Taq polymerase by ATA in vitro assay. As the telomerase reverse transcriptase (TERT) is the main catalytic subunit of telomerase holoenzyme, we also monitored effect of ATA on telomerase activity in vivo and observed dose-dependent inhibition of telomerase activity in Chinese hamster V79 cells treated with ATA. Direct association of ATA with DNase I (K d =9.019μM)), RNase A (K d =2.33μM) reverse transcriptase (K d =0.255μM) and Taq polymerase (K d =81.97μM) was further shown by tryptophan fluorescence quenching studies. Such association altered the three-dimensional conformation of DNase I, RNase A and Taq polymerase as detected by circular dichroism. We propose ATA inhibits enzymatic activity of the four proteins through interfering with DNA or RNA binding to the respective proteins either competitively or allosterically, i.e. by perturbing three-dimensional structure of enzymes.

Induction of apoptosis by the inhibitors of poly (ADP-ribose)polymerase in HeLa cells

To investigate the role of poly(ADP-ribose)polymerase (PARP) in the physiological condition of cell growth, we studied the ability of PARP inhibitors to induce apoptosis. Benzamide (BA) and 4-amino-1,8-naphthalimide (NAP), two well-known inhibitors of PARP, treatment increased nuclear fragmentation and caspase-3 activity in HeLa (Human cervical cancer cell line) cells. The increase of cellular NAD+ level was observed in HeLa cells treated with BA in comparison with untreated control cells. For unrevealing the specific PARP family member responsible for such induction of apoptosis we knocked down and over-expressed PARP-1 gene in HeLa cells. PARP-1 knock down cells were sensitive to BA induced nuclear fragmentation and caspase-3 activation while exogenous expression of PARP-1 rendered cells resistant to BA induced apoptosis. This result indicated that inhibition of PARP-1 resulted in induction of apoptosis.

Synthesis and characterization of two Cu(II) complexes with a new pyrazole-based Schiff base ligand: crystallography, DNA interaction and antimicrobial activity of Ni(II) and Cu(II) complexes

Abstract Reaction of Cu(II) nitrate with a new pyrazole-based Schiff base ligand, 5-methyl-3-formylpyrazole-N-(2′-methylphenoxy)methyleneimine (MPzOA), afforded two types of Cu(II) complexes at different reaction temperatures, [Cu(MPzOA)(NO3)]2 (1) and [Cu(3,7,11,15-tetramethylporphyrin)(H2O)](NO3)2 (2), reported together with a Ni(II) complex, [Ni(MPzOA)2(H2O)2]Br2 (3). The compounds are characterized by single crystal X-ray structure analyses along with several physico-chemical and spectral parameters. Complex 1 is authenticated as a bis(μ-pyrazolato)dicopper(II), while 2 is a porphyrinogen and 3 is a distorted octahedral complex. Structural analyses of the complexes reveal that 1 crystallized in monoclinic P21/n space group while 2 and 3 crystallized in monoclinic C2/c space group. DNA-binding studies of the complexes have shown that the complexes interact with CT-DNA. DNA-cleavage studies with plasmid DNA have shown that 1 and 2 induce extensive DNA cleavage in the presence of H2O2 as an additive, whereas there is no change in degradation of super-coiled DNA by 3 in the presence of additive. The antimicrobial studies of the complexes against Escherichia coli DH5α bacteria strain indicated that all the complexes were capable of killing E. coli with different LD50 values.

Radiosensitivity and Induction of Apoptosis by High LET Carbon Ion Beam and Low LET Gamma Radiation: A Comparative Study

Cancer treatment with high LET heavy ion beam, especially, carbon ion beam (12C), is becoming very popular over conventional radiotherapy like low LET gamma or X-ray. Combination of Poly(ADP-ribose) polymerase (PARP) inhibitor with xenotoxic drugs or conventional radiation (gamma or X-ray) is the newer approach for cancer therapy. The aim of our study was to compare the radiosensitivity and induction of apoptosis by high LET 12C and low LET gamma radiation in HeLa and PARP-1 knocked down cells. We did comet assay to detect DNA breaks, clonogenic survival assay, and cell cycle analysis to measure recovery after DNA damage. We measured apoptotic parameters like nuclear fragmentation and caspase-3 activation. DNA damage, cell killing, and induction of apoptosis were significantly higher for 12C than gamma radiation in HeLa. Cell killing and apoptosis were further elevated upon knocking down of PARP-1. Both 12C and gamma induced G2/M arrest although the 12C had greater effect. Unlike the gamma, 12C irradiation affects DNA replication as detected by S-phase delay in cell cycle analysis. So, we conclude that high LET 12C has greater potential over low LET gamma radiation in killing cells and radiosensitization upon PARP-1 inhibition was several folds greater for 12C than gamma.

Insight into the binding of a non-toxic, self-assembling aromatic tripeptide with ct-DNA: Spectroscopic and viscositic studies

The report describes the synthesis, self-association and DNA binding studies of an aromatic tripeptide H-Phe-Phe-Phe-OH (FFF). The peptide backbone adopts β—sheet conformation both in solid and solution. In aqueous solution, FFF self-assembles to form nanostructured aggregates. Interactions of this peptide with calf-thymus DNA (ct-DNA) have been studied using various biophysical techniques including ultraviolet (UV) absorption spectroscopy, fluorescence spectroscopy and circular dichroism (CD) spectroscopy. The value of mean binding constant calculated from UV and fluorescence spectroscopic data is (2.914 ± 0.74) x 103 M−1 which is consistent with an external binding mode. Fluorescence intercalator displacement (FID) assay, iodide quenching study, viscosity measurement and thermal denaturation study of DNA further confirm the groove binding mode of peptide, FFF with ct-DNA. MTT cell survival assay reveals very low cytotoxicity of the peptide toward human lung carcinoma cell line A549.

Green synthesis of gold nanoparticles for staining human cervical cancer cells and DNA binding assay.

Gold nanoparticles have been functionalized by non-ionic surfactants (polysorbates) used in pharmaceutical formulations. This results in the formation of more well-dispersed gold nanoparticles (GNPs) than the GNPs formed in neat water. The synthesized GNPs show good temporal stability. The synthesis conditions are mild and environmentally benign. The GNPs can bind to ct-DNA and displace bound dye molecules. The DNA-binding assay is significant as it preliminarily indicated that DNA-GNP conjugates can be formed. Such conjugates are extremely promising for applications in nanobiotechnology. The GNPs can also stain the human cervical cancer (HeLa) cells over a wide concentration range while remaining non-cytotoxic, thus providing a non invasive cell staining method. This result is very promising as we observe staining of HeLa cells at very low GNP concentrations (1 μM) while the cell viability is retained even at 10-fold higher GNP concentrations.

Expression of Telomere-Associated Proteins is Interdependent to Stabilize Native Telomere Structure and Telomere Dysfunction by G-Quadruplex Ligand Causes TERRA Upregulation

Telomere DNA can form specialized nucleoprotein structure with telomere-associated proteins to hide free DNA ends or G-quadruplex structures under certain conditions especially in presence of G-quadruplex ligand. Telomere DNA is transcribed to form non-coding telomere repeat-containing RNA (TERRA) whose biogenesis and function is poorly understood. Our aim was to find the role of telomere-associated proteins and telomere structures in TERRA transcription. We silenced four [two shelterin (TRF1, TRF2) and two non-shelterin (PARP-1, SLX4)] telomere-associated genes using siRNA and verified depletion in protein level. Knocking down of one gene modulated expression of other telomere-associated genes and increased TERRA from 10q, 15q, XpYp and XqYq chromosomes in A549 cells. Telomere was destabilized or damaged by G-quadruplex ligand pyridostatin (PDS) and bleomycin. Telomere dysfunction-induced foci (TIFs) were observed for each case of depletion of proteins, treatment with PDS or bleomycin. TERRA level was elevated by PDS and bleomycin treatment alone or in combination with depletion of telomere-associated proteins.