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K C Gupta

Publications and source records attributed to K C Gupta.

At least 19 recordsLinked to original sources
PubMed2006

Imidazolyl-PEI modified nanoparticles for enhanced gene delivery.

The derivatives of polyethylenimine (PEI 25 and 750kDa) were synthesized by partially substituting their amino groups with imidazolyl moieties. The series of imidazolyl-PEIs thus obtained were cross-linked with polyethylene glycol (PEG) to get imidazolyl-PEI-PEG nanoparticles (IPP). The component of hydrophobicity was introduced by grafting the lauryl groups in the maximal substituted IPP nanoparticles (IPPL). The nanoparticles were characterized with respect to DNA interaction, hydrodynamic diameter, zeta potential, in vitro cytotoxicity and transfection efficiency on model cell lines. The IPP and IPPL nanoparticles formed a loose complex with DNA compared to the corresponding native PEI, leading to more efficient unpackaging of DNA. The DNA loading capacity of IPP and IPPL nanoparticles was also lower compared to PEI. The imidazolyl substitution improved the gene delivery efficiency of PEI (750kDa) by nine- to ten-fold and PEI (25kDa) by three- to four-fold. At maximum transfection efficiency, the zeta potential of nanoparticles was positive after forming a complex with DNA. The maximum level of reporter gene expression was mediated by IPPL nanoparticles in both the series. The cytotoxicity, another pertinent problem with cationic polymers, was also negligible in case of IPP and IPPL nanoparticles.

Active Transport, Cell Nucleus↗
PubMed2006

An efficient and versatile approach for the construction of oligonucleotide microarrays.

A new immobilization chemistry for covalent attachment of phosphorylated oligonucleotides on epoxy-activated glass surface via opening of oxirane ring is described. The proposed strategy results in excellent immobilization efficiency, spot homogeneity, and morphology. The constructed microarray was successfully demonstrated for discrimination of nucleotide mismatches.

Base Pair Mismatch↗
PubMed2006

N-(3-Triethoxysilylpropyl)-6-(N-maleimido)-hexanamide: An efficient heterobifunctional reagent for the construction of oligonucleotide microarrays.

Synthesis of a new heterobifunctional reagent, N-(3-triethoxysilylpropyl)-6-(N-maleimido)-hexanamide (TPMH), for the preparation of oligonucleotide microarrays is described. Its triethoxysilyl function is specific toward virgin glass surface and maleimide function undergoes conjugate addition to 3'- or 5'-thiol-modified oligonucleotides. The construction of microarrays using TPMH has been realized via two routes. In Route A, TPMH was reacted first with 3'- or 5'-thiol-modified oligonucleotide under microwaves, thereby producing oligonucleotide-triethoxysilyl conjugate in 15min, which was then brought in contact with virgin glass microslide, resulting in immobilization of an oligonucleotide sequence. In Route B, immobilization involves generation of maleimide functions on virgin glass surface by the reaction with TPMH, followed by coupling with thiol-modified oligonucleotides under microwaves in 15 min to produce surface-bound oligonucleotides. The microarrays constructed using both routes were analyzed by hybridizing with tetrachlorofluorescein-labeled complementary oligonucleotide. Subsequently, these microarrays were successfully used in the discrimination of single and double nucleotide mismatches based on fluorescence intensity.

Amides↗
PubMed2006

PEI-alginate nanocomposites as efficient in vitro gene transfection agents.

The positive charge on PEI was partially shielded by forming ionic nanocomposites with a polysaccharide, alginic acid, in aqueous solution, bypassing tedious chemical synthesis. The content of alginic acid was varied systematically to obtain a series of nanocomposites. The nanocomposites were first characterized by assessing the surface charge (zeta potential), size (DLS) and morphology (AFM) followed by evaluation for their DNA interaction ability, cytotoxicity and transfection efficiency on various cell lines. The transfection efficiency of PEI-alginate (6.26%) nanocomposites improved dramatically (2-16-fold over native PEI) in all the cell lines studied. However, a decrease in transfection efficiency was observed on deviating from this optimal concentration of alginic acid in nanocomposites. Cytotoxicity of PEI-alginate/DNA complexes was nearly abolished on increasing the concentration of alginic acid in nanocomposites. PEI-alginate (6.26%) nanocomposites also delivered SiRNAs efficiently into mammalian cells, resulting in 80% suppression of GFP expression. The cellular uptake and endosomal escape of PEI-alginate nanocomposites and PEI were found to follow a similar route when transfection was carried out in presence of chloroquine, bafilomycin A1, cytochalasin B and methyl-beta-cyclodextrin. The results demonstrate a versatile vector that can be used for efficient cytoplasmic delivery of a broad range of nucleic acids.

Alginates↗
PubMed2006

Preparation and characterization of sodium hexameta phosphate cross-linked chitosan microspheres for controlled and sustained delivery of centchroman.

The cross-linked microspheres using chitosan with different molecular weights and degree of deacetylation have been prepared in presence of sodium hexameta polyphosphate (SHMP) as physical cross-linker. The degree of cross-linking through electrostatic interactions in chitosan microspheres has been evaluated by varying the charge density on chitosan and varying degree of dissociation of sodium hexameta polyphosphate by solution pH. The degree of deacetylation and molecular weight of chitosan has controlled electrostatic interactions between hexameta polyphosphate anions and chitosan, which played significant role in swelling, loading and release characteristics of chitosan microspheres for centchroman. The microspheres prepared by hexameta polyphosphate anions cross-linker were compact and more hydrophobic than covalently cross-linked microspheres, which has been attributed to the participation of all amino groups of chitosan in physical cross-linking with added hexameta polyphosphate anions. The microspheres prepared under different experimental conditions have shown an initial step of burst release, which was followed by a step of controlled release for centchroman. The extent of drug release in these steps has shown dependence on properties of chitosan and degree of cross-linking between chitosan and added polyanions. The degree of swelling and release characteristics of microspheres was also studied in presence of organic and inorganic salts, which shown significant effect on controlled characteristics of microspheres due to variations in ionic strength of the medium. The initial step of drug release has followed first order kinetics and become zero order after attaining an equilibrium degree of swelling in these microspheres. The microspheres prepared using chitosan with 62% (w/w) degree of deacetylation and molecular weight of 1134kgmol(-1) have shown a sustained release for centchroman for 50h at 4% (w/w) degree of cross-linking with SHMP.

Anions↗
PubMed2006

Glutaraldehyde and glyoxal cross-linked chitosan microspheres for controlled delivery of centchroman.

Glutaraldehyde and glyoxal cross-linked microspheres were prepared using chitosan with different molecular weights (MWs) and degrees of deacetylation (DDAs) for sustained release of centchroman under physiological conditions. The DDA in chitosan was determined by different methods, and the samples were categorized as chitosan with low (48%), medium (62%), and high (75%) DDA. The size and shape of the microspheres were determined by scanning electron microscopy (SEM), and hydrophobicity was determined by adsorption of Rose Bengal dye on microspheres cross-linked with glutaraldehyde or glyoxal. The effect of MW, DDA, and degree of cross-linking in microspheres was studied on the degree of swelling, as well as by the loading and release of centchroman. The glyoxal cross-linked microspheres were more compact and hydrophobic and showed better sustained release in companion to chitosan microspheres and glutaraldehyde cross-linked microspheres. The linear fractional release of centchroman with the square root of time indicated a Fickian behavior of centchroman, and the microspheres also showed zero-order release kinetics for centchroman.

Acetylation↗
PubMed2006

A new synthetic protocol for labeled oligonucleotides, using a chemically cleavable universal linker.

A two-step general method for labeling of synthetic oligonucleotides is described. The protocol employs a cleavable universal linker, 5'-O-(4,4'-dimethoxytrityl)-3'-O-benzoyl-2'-O-(2-cyanoethyl-N,N-diisopropyl)-uridine phosphoramidite, to effect coupling to polymer-bound oligonucleotide chains. Sequentially, coupling with commercially available phosphoramidite reagent of an appropriate label (Biotin, HEX etc.) in an automated DNA synthesizer is carried out. The labeled oligomers, obtained after cleavage and deprotection reactions, are analyzed on RP-HPLC. A distinctive feature of this protocol is the recovery of free oligomers from their labeled analogs under mild conditions. The oligomers obtained are comparable to the corresponding standard oligonucleotides (HPLC).

Biotin↗
PubMed2006

Spectrophotometric estimation of functional groups on microslides for preparation of biochips.

A universal reagent 1-O-(4,4'-dimethoxytrityl)-6-aminohexanol (DTAH) is described for the estimation of surface-bound functionalities (epoxy, aldehyde, and carboxyl) required for preparation of oligonucleotide arrays (biochips). The method involves the reaction of universal reagent DTAH with surface-bound functionality under microwaves for 10 min, followed by washings to remove the excess reagent. In the subsequent step, a weighed amount of DTAH-treated surface is exposed to acid to liberate 4,4'-dimethoxytrityl cation, which is measured at 505 nm to determine the functional group loading on the surface.

Aldehydes↗
PubMed2005

The studies of density, apparent molar volume, and viscosity of bovine serum albumin, egg albumin, and lysozyme in aqueous and RbI, CsI, and DTAB aqueous solutions at 303.15 K.

Density (rho), apparent molar volume (V(phi)), and viscosity (eta) of 0.0010 to 0.0018% (w/v) of bovine serum albumin (BSA), egg albumin, and lysozyme in 0.0002, 0.0004, and 0.0008 M aqueous RbI and CsI, and (dodecyl)(trimethyl)ammonium bromide (DTAB) solutions were obtained. The experimental data were regressed against composition, and constants are used to elucidate the conformational changes in protein molecules. With salt concentration, the density of proteins is found to decrease, and the order of the effect of additives on density is observed as CsI > RbI > DTAB. The trend of apparent molar volume of proteins is found as BSA > egg-albumin > lysozyme for three additives. In general, eta values of BSA remain higher for all compositions of RbI than that of egg-albumin for CsI and DTAB. These orders of the data indicate the strength of intermolecular forces between proteins and salts, and are helpful for understanding the denaturation of proteins.

Animals↗
PubMed2004

Construction of oligonucleotide arrays on a glass surface using a heterobifunctional reagent, N-(2-trifluoroethanesulfonatoethyl)-N-(methyl)-triethoxysilylpropyl-3-amine (NTMTA).

A rapid method for construction of oligonucleotide arrays on a glass surface, using a novel heterobifunctional reagent, N-(2-trifluoroethanesulfonatoethyl)-N-(methyl)-triethoxysilylpropyl-3-amine (NTMTA), has been described. The heterobifunctional reagent, NTMTA, carries two different thermoreactive groups. The triethoxysilyl group on one end is specific towards silanol functions on the virgin glass surface, while the trifluoroethanesulfonyl (tresyl) group on the other end of the reagent reacts specifically with aminoalkyl- or mercaptoalkyl- functionalized oligonucleotides. Immobilization of oligonucleotides on a glass surface has been realized via two routes. In the first one (A), 5'- aminoalkyl- or mercaptoalkyl-functionalized oligonucleotides were allowed to react with NTMTA to form a oligonucleotide-triethoxysilyl conjugate which, in a subsequent reaction with unmodified (virgin) glass microslide, results in surface-bound oligonucleotides. In the second route (B), the NTMTA reagent reacts first with a glass microslide whereby it generates trifluoroethanesulfonate ester functions on it, which in a subsequent step react with 5'-aminoalkyl or mercaptoalkyl oligonucleotides to generate support-bound oligonucleotides. Subsequently, the oligonucleotide arrays prepared by both routes were analyzed by hybridization experiments with complementary oligonucleotides. The constructed microarrays were successfully used in single and multiple nucleotide mismatch detection by hybridizing these with fluorescein-labeled complementary oligonucleotides. Further more, the proposed method was compared with the existing methods with respect to immobilization efficiency of oligonucleotides.

Base Pair Mismatch↗
PubMed2004

A new heterobifunctional reagent for immobilization of biomolecules on glass surface.

Synthesis of a new heterobifunctional reagent, [N-(2-trifluoroethanesulfonatoethyl)-N-(methyl)-triethoxysilylpropyl-3-amine] (NTMTA) is described for the immobilization of a variety of biomolecules on glass surface. Its triethoxysilyl group reacts with glass surface and trifluoroethanesulfonate ester structure reacts selectively with aminoalkyl/mercaptoalkyl function in biomolecules. The immobilization can be achieved by two ways involving two steps. The first route involves the reaction of NTMTA with glass beads followed by attachment of aminoalkyl- or mercaptoalkylated biomolecules. The second one involves the reaction of biomolecules, viz., oligonucleotides, proteins, etc., with NTMTA via their aminoalkyl or mercaptoalkyl functions to form a biomolecule conjugate, which is then reacted with glass beads (unmodified) to complete immobilization process. This has been demonstrated by successful immobilization of 5'-mercaptoalkyl- or aminoalkylated oligonucleotides and some commonly used enzymes on glass beads using NTMTA reagent.

Glass↗
PubMed2002

Polyamine-assisted rapid and clean cleavage of oligonucleotides from cis-diol bearing universal support.

Polyamine-assisted deprotection conditions have been developed for the rapid and clean cleavage of oligonucleotide chains from a cis-diol group bearing universal polymer support, making it compatible with modern oligonucleotide synthesis via all types of phosphoramidite synthons, including base labile protecting group bearing synthons as well. The synthesized oligonucleotides were found to be comparable with the corresponding standard oligomers with respect to their retention time on HPLC, mass on MALDI-TOF and biological activity in PCR amplification.

Amines↗
PubMed2001

Graft copolymerization of acrylonitrile and ethyl methacrylate comonomers on cellulose using ceric ions.

The graft copolymerization of acrylonitrile and ethyl methacrylate from their binary mixtures onto cellulose has been carried out in the presence of Ce(IV) ions in acidic media at 30 +/- 0.1 degrees C. The graft copolymerization has shown an increasing trend on increasing the feed molarity of the comonomers and on increasing the reaction time. The analysis of grafted cellulose prepared with different feed compositions (f(AN)) has clearly indicated the existence of the synergistic effect of ethyl methacrylate on acrylonitrile which normally shows poor affinity for grafting on cellulose while used individually. The elemental analysis and IR data were used to determine the composition of grafted copolymers and reactivity ratios (r(1), r(2)) of the monomers using the Mayo and Lewis method. The reactivity ratios for acrylontrile and ethyl methacrylate have been found to be 0.68 and 1.15, respectively, which supported the alternate arrangement of the monomer blocks of individual monomers in the grafted chains. The rate of grafting has shown a square dependence on the concentration of the comonomers within the studied range of feed molarity from 0.5 to 2.5 mol dm(-3). The grafting data were used to calculate the number of grafted chains (N(g)), frequency of grafting (G(F)) per molecule of the cellulose and efficiency of grafting (G(E)). The variation in these parameters as a function of feed molarity, feed composition, and ceric ion concentration has been determined. The rate of ceric ion disappearance as a function of feed molarity and reaction time has provided strong support to consider the participation of ceric ions in the formation of free radicals at the backbone of the cellulose. On the basis of the experimental observations, reaction steps for grafting of monomers on cellulose have been proposed.

Acrylonitrile↗
PubMed2001

pH dependent hydrolysis and drug release behavior of chitosan/poly(ethylene glycol) polymer network microspheres.

Semi-interpenetrating polymer network (IPN) microspheres of chitosan and poly(ethylene glycol) PEG were prepared for controlled release of drugs. A new method for the chemical crosslinking of chitosan microspheres containing isoniazid (INH) as a model drug is proposed and evaluated. The method consists of the exposure of microspheres to the vapor of crosslinking agent that act in gaseous phase under mild conditions. The structural analysis of the microspheres was carried out by FTIR-analysis. The swelling behavior, hydrolytic degradation, structural changes of the microspheres and loading capacity (LC) of the microspheres for INH were investigated. The prepared microspheres have shown 93% drug loading capacity, which suggested that these semi-IPN microspheres are suitable for controlled release of drugs in an oral sustained delivery system.

Journal Article↗
PubMed2001

Polyvalent envelope glycoprotein vaccine elicits a broader neutralizing antibody response but is unable to provide sterilizing protection against heterologous Simian/human immunodeficiency virus infection in pigtailed macaques.

The great difficulty in eliciting broadly cross-reactive neutralizing antibodies (NAbs) against human immunodeficiency virus type 1 (HIV-1) isolates has been attributed to several intrinsic properties of their viral envelope glycoprotein, including its complex quaternary structure, extensive glycosylation, and marked genetic variability. Most previously evaluated vaccine candidates have utilized envelope glycoprotein from a single virus isolate. Here we compare the breadth of NAb and protective immune response following vaccination of pigtailed macaques with envelope protein(s) derived from either single or multiple viral isolates. Animals were challenged with Simian/human immunodeficiency virus strain DH12 (SHIV(DH12)) following priming with recombinant vaccinia virus(es) expressing gp160(s) and boosting with gp120 protein(s) from (i) LAI, RF, 89.6, AD8, and Bal (Polyvalent); (ii) LAI, RF, 89.6, AD8, Bal, and DH12 (Polyvalent-DH12); (iii) 89.6 (Monovalent-89.6); and (iv) DH12 (Monovalent-DH12). Animals in the two polyvalent vaccine groups developed NAbs against more HIV-1 isolates than those in the two monovalent vaccine groups (P = 0.0054). However, the increased breadth of response was directed almost entirely against the vaccine strains. Resistance to SHIV(DH12) strongly correlated with the level of NAbs directed against the virus on the day of challenge (P = 0.0008). Accordingly, the animals in the Monovalent-DH12 and Polyvalent-DH12 vaccine groups were more resistant to the SHIV(DH12) challenge than the macaques immunized with preparations lacking a DH12 component (viz. Polyvalent and Monovalent-89.6) (P = 0.039). Despite the absence of any detectable NAb, animals in the Polyvalent vaccine group, but not those immunized with Monovalent-89.6, exhibited markedly lower levels of plasma virus than those in the control group, suggesting a superior cell-mediated immune response induced by the polyvalent vaccine.

AIDS Vaccines↗
PubMed2001

Compensatory hyperparathyroidism following high fluoride ingestion - a clinico - biochemical correlation.

OBJECTIVE: To evaluate the effect of varying ingestion of drinking water containing high fluorides and its effect on serum parathyroid hormone. DESIGN: Cross sectional clinical study. SETTING: S.M.S. Medical College, Jaipur. SUBJECT: 200 children were selected from four areas (50 from each area) consuming water containing 2.4, 4.6, 5.6 and 13.5 mg/l of fluoride. All children were in an age group of 6 to 12 years. METHODS: All children were graded for clinical, radiological and dental fluorosis and biochemical estimations were made for serum calcium, serum and urinary fluoride and serum parathyroid hormone. RESULTS: Serum calcium levels were well within normal range in the patients of all areas but an increase in serum parathyroid levels (S. PTH) was noted. The increased S. PTH was well correlated with increase in fluoride ingestion. The severity of clinical and skeletal fluorosis was observed to increase with increase in S. PTH concentration. CONCLUSIONS: High Fluoride ingestion has a definite relationship with increased parathyroid hormone secretion, which may be responsible for maintaining serum calcium levels and may have a role in toxic manifestations of fluorosis.

Child↗
PubMed2000

Functional significance of alternate phosphorylation in Sendai virus P protein.

Phosphorylation of the negative-sense RNA virus phosphoproteins is highly conserved, implying functional significance. Sendai virus (SV) phosphoprotein (P) is constitutively phosphorylated at S249. Abrogation of the SV P primary phosphorylation causes phosphorylation of P at alternate sites, creating a problem in determining the function of phosphorylation. We have now identified the alternate phosphorylation sites using two-dimensional phosphopeptide analysis of several deletion and point mutants of the P protein. The alternate phosphorylation sites were mutagenized to create P with (S249combo) or without (combo) primary phosphorylation. The combo protein has less than 10% phosphorylation compared with the wild-type P or S249combo. Functional analysis of the mutant proteins using a Sendai virus minigenome replication system showed that the combo P protein was as proficient in supporting minigenome replication as the wild-type P in cell cultures. These studies suggest that like the primary, the alternate phosphorylation of the P protein is also dispensable for virus replication in cell cultures. Interestingly, the ability of the multiple site mutant of P (combo mutant has eight serine residues changed to alanine residues) to support efficient virus RNA synthesis suggests that the P protein has a high flexibility at least in its sequence and perhaps also in structure.

Animals↗