Surya S. Singh

Efficient pH Dependent Drug Delivery to Target Cancer Cells by Gold Nanoparticles Capped with Carboxymethyl Chitosan

Doxorubicin (DOX) was immobilized on gold nanoparticles (AuNPs) capped with carboxymethyl chitosan (CMC) for effective delivery to cancer cells. The carboxylic group of carboxymethyl chitosan interacts with the amino group of the doxorubicin (DOX) forming stable, non-covalent interactions on the surface of AuNPs. The carboxylic group ionizes at acidic pH, thereby releasing the drug effectively at acidic pH suitable to target cancer cells. The DOX loaded gold nanoparticles were effectively absorbed by cervical cancer cells compared to free DOX and their uptake was further increased at acidic conditions induced by nigericin, an ionophore that causes intracellular acidification. These results suggest that DOX loaded AuNPs with pH-triggered drug releasing properties is a novel nanotheraputic approach to overcome drug resistance in cancer.

Phosphatidylinositol 3‐kinase binds to profilin through the p85α subunit and regulates cytoskeletal assembly

Phosphatidylinositol 3‐kinase (PI3‐K), endowed with catalytic (110kDa) and regulatory (85kDa) subunits co‐precipitates with anti‐tyrosine antibodies in mitogen‐activated cells. Association of PI3‐K with cytoskeleton activates its catalytic activity through undeciphered mechanisms. Recently Singh et al., (Biochemistry, 35, 16544‐16549, 1996) have shown that profilin activates PI3‐K activity in a concentration‐ dependent manner. Consequently, we investigated the interaction between the PI3‐K and profilin employing the GSTp85α fusion protein and the results indicate a specific interaction between pofilin and p85α. The effect of p85α/profilin complex on polymerization of actin monomers was monitored fluorimetrically employing pyrene‐labelled actin monomers. It was noted that p85α/profilin complex inhibits actin polymerization suggesting that profilin can simultaneously bind to actin as well as to p85α. The affinity of p85α/profilin complex to actin increases in the presence of p85α subunit of PI3‐K as compared to profilin itself.

Cellular uptake, cytotoxicity, apoptosis and DNA-binding investigations of Ru(II) complexes.

Three new compounds, [Ru(Hdpa)2PyIP](ClO4)2·2H2O (1) [Ru(Hdpa)2FyIP](ClO4)2·2H2O (2) and [Ru(Hdpa)2IIP](ClO4)2·2H2O (3) have been synthesized and characterized by spectroscopic techniques such as elemental analysis, UV/Vis, FT-IR, (1)H NMR, (13)C NMR and mass spectra. The CT-DNA binding properties of 1-3 have been investigated by absorption, emission spectroscopy and viscosity measurements. Experimental results suggested that they can interact with DNA through intercalative mode with different binding strengths. These were found to promote the cleavage of plasmid DNA. Cell viability results indicated that all compounds showed significant dose dependent cytotoxicity in selected cell lines and 1 shown higher cytotoxicity than cisplatin on HeLa cells. Cellular uptake studies were studied by flow cytometry and confocal microscopy.

Protein kinase C isozyme-specific phosphorylation of profilin.

Profilin, a cytoskeletal protein, is emerging as an important link between signal transduction pathways and cytoskeletal dynamics. Profilin is phosphorylated on its C-terminal serine by protein kinase C (PKC). The protein kinase used for the in vitro phosphorylation studies reported earlier was a mixture of isozymes, and therefore, attempts were made to address the isozyme specificity on profilin phosphorylation under in vitro conditions. Profilin was subjected to phosphorylation by PKCalpha, PKCepsilon, and PKCzeta isozymes individually, and it was observed that profilin phosphorylation is cofactor-independent. PKCzeta phosphorylates profilin to a higher extent, but exhibits cofactor dependency with respect to phosphoinositides. The stoichiometry of phosphorylation was measured in the presence of these different isozymes, and a maximum stoichiometry of 0.8 (mole phosphate incorporated/mole profilin) was obtained in the presence of PKCzeta. Phosphorylation of profilin by PKCzeta was maximal in the presence of phosphatidylinositol4,5-bisphosphate (PI4,5-P2) when compared to the other phosphoinositides studied.

MrdH, a Novel Metal Resistance Determinant of Pseudomonas putida KT2440, Is Flanked by Metal-Inducible Mobile Genetic Elements

We report here the identification and characterization of mrdH, a novel chromosomal metal resistance determinant, located in the genomic island 55 of Pseudomonas putida KT2440. It encodes for MrdH, a predicted protein of approximately 40 kDa with a chimeric domain organization derived from the RcnA and RND (for resistance-nodulation-cell division) metal efflux proteins. The metal resistance function of mrdH was identified by the ability to confer nickel resistance upon its complementation into rcnA mutant (a nickel- and cobalt-sensitive mutant) of Escherichia coli. However, the disruption of mrdH in P. putida resulted in an increased sensitivity to cadmium and zinc apart from nickel. Expression studies using quantitative reverse transcription-PCR showed the induction of mrdH by cadmium, nickel, zinc, and cobalt. In association with mrdH, we also identified a conserved hypothetical gene mreA whose encoded protein showed significant homology to NreA and NreA-like proteins. Expression of the mreA gene in rcnA mutant of E. coli enhanced its cadmium and nickel resistance. Transcriptional studies showed that both mrdH and mreA underwent parallel changes in gene expression. The mobile genetic elements Tn4652 and IS1246, flanking mrdH and mreA were found to be induced by cadmium, nickel, and zinc, but not by cobalt. This study is the first report of a single-component metal efflux transporter, mrdH, showing chimeric domain organization, a broad substrate spectrum, and a location amid metal-inducible mobile genetic elements.

Facile and green synthesis of fluorescent carbon dots from onion waste and their potential applications as sensor and multicolour imaging agents

Here, we report a novel, facile and green approach for the synthesis of highly fluorescent carbon dots (CDs) with 28% quantum yield by utilizing onion waste as a precursor and by employing a simple autoclave. Optical and physicochemical properties of as synthesized CDs were studied using transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), dynamic light scattering (DLS), X-ray diffraction (XRD), Raman, thermogravimetric analysis (TGA), Fourier transform infrared (FT-IR), UV-visible, fluorescence spectroscopy and elemental analysis. These CDs exhibited high aqueous dispersibility, excitation dependent fluorescence emission and excellent stability to various effects like pH, high ionic strength and continuous irradiation. It was observed that the presence of Fe3+ ions could result in a strong fluorescence quenching, hence these CDs were applied as a fluorescent probe for the selective & sensitive detection of Fe3+ ions and a good linear correlation (R2 = 0.996) in the range of 0–20 μM with a detection limit of 0.31 μM was obtained. Cytotoxicity studies performed on both normal (HEK-293 cells) and cancerous (HeLa) cells revealed their excellent biocompatibility and they were further employed as fluorescent probes for multicoloured (blue, green and red) imaging of HeLa cells. Impressed by their high selectivity, photo stability and excellent biocompatibility, the Fe3+ detection capability of these CDs was further evaluated in HeLa cells and applied for real water samples.

In vitro Activation of Protein Kinase C by β-N-oxalyl-l-α, β-diaminopropionic acid, the Lathyrus sativus Neurotoxin

Abstractβ-N-oxalyl-l-α,β-diaminopropionic acid (l-ODAP) toxicity has been associated with lathyrism; a spastic paraparesis caused by excessive dietary intake of the pulse Lathyrus sativus. We investigated the effect of Lathyrus neurotoxin l-ODAP on protein kinase C (PKC) activity under in vitro conditions. l-ODAP activated phosphorylation activity of purified chick brain PKC. Both lysine-rich (histone III-S) and arginine-rich (protamine sulfate) substrate phosphorylation was enhanced in the presence of l-ODAP. The activation is concentration dependent, and maximal activation is observed at 100 μM concentration. Protamine sulfate phosphorylation was enhanced by 47%, whereas histone III-S phosphorylation was enhanced by 50% over PS/PDBu/Ca2+ dependent activity. The nontoxic d-isomer (d-ODAP) did not affect both histone III-S and protamine sulfate phosphorylation activity. These results indicate that l-ODAP taken up by neuronal cells could also contribute to PKC activation and so be associated with toxicity.

Association of Taq I, Fok I and Apa I polymorphisms in Vitamin D Receptor (VDR) gene with leprosy

BACKGROUND Vitamin D Receptor (VDR) is a transacting transcription factor which mediates immunomodulatory function and plays a key role in innate and adaptive immune responses through its ligand and polymorphisms in VDR gene may affect its regulatory function. OBJECTIVE To investigate the association of three VDR gene polymorphisms (TaqI rs731236, FokI rs2228570 and ApaI rs7975232) with leprosy. METHODS The study group includes 404 participants of which 222 were leprosy patients (paucibacillary=87, multibacillary=135) and 182 healthy controls. Genotyping was done using PCR-RFLP technique. Statistical analysis was performed using SNP Stats and PLINK software. RESULTS The VDR FokI (rs2228570) ff genotype, ApaI (rs7975232) AA, Aa genotype and haplotype T-f-a, T-F-A were positively associated with leprosy when compared to healthy controls. CONCLUSION The two variants at Fok and Apa positions in VDR gene are significantly associated with leprosy. Genotypes at FokI (ff), ApaI (aa) and haplotype (T-F-a, T-f-a) may contribute to the risk of developing leprosy by altering VDR phenotype/levels subsequently modulation of immune response.

Understanding perspectives of signalling mechanisms regulating PEBP1 function

Phosphatidylethanolamine‐binding protein 1 (PEBP1), also known as Raf kinase inhibitor protein, belongs to PEBP family of proteins. It is known to interact with many proteins that are mainly involved in pathways that monitor cell proliferation and differentiation. PEBP1 in many cells interacts with several pathways, namely MAPK, GRK2, NF‐кB, etc. that keeps the cell proliferation and differentiation in check. This protein is expressed by many cells in humans, including neurons where it is predominantly involved in production of choline acetyltransferase. Deregulated PEBP1 is known to cause cancer, diabetic nephropathy and neurodegenerative diseases like Alzheimers and dementia. Recent research led to the discovery of many drugs that mainly target the interaction of PEBP1 with its partners. These compounds are known to bind PEBP1 in its conserved domain which abrogate its association with interacting partners in several different pathways. We outline here the latest developments in understanding of PEBP1 function in maintaining cell integrity. Copyright

Profilin oligomerization and its effect on poly (L-proline) binding and phosphorylation.

Profilin is a cytoskeletal protein that interacts specifically with actin, phosphoinositides and poly (l-proline). Experimental results and in silico studies revealed that profilin exists as dimer and tetramer. Profilin oligomers possess weak affinity to poly (l-proline) due to unavailability of binding sites in dimers and tetramers. Phosphorylation studies indicate that profilin dimers are not phosphorylated while teramers are preferentially phosphorylated over monomers. In silico studies revealed that PKC phosphorylation site, S137 is buried in dimer while it is accessible in tetramer.