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Biomedical subjects

K C Gupta

Publications and source records attributed to K C Gupta.

At least 37 records · Page 2Linked to original sources
PubMed2000

Drug release behavior of beads and microgranules of chitosan.

Beads and microgranules carriers have important potential applications for the administration of therapeutic molecules. A novel approach for the preparation of chitosan beads and microgranules is presented. The present work is an investigation of the in vitro release kinetics of diclofenac sodium (DFS) from chitosan beads and microgranules. The in vitro release profiles of DFS from chitosan beads and microgranules are monitored using Shimadzu 1601 UV-VIS spectrophotometer. Drug release behavior of beads and microgranules has been compared. The release rate of DFS from the beads has been found to be slower in comparison to the microgranules. It may also be noted that the percent and amount of the drug release were much higher in acidic solution than in basic solution, probably due to the swelling properties of the matrix at acidic pH.

Administration, Oral↗
PubMed1999

Role of primary constitutive phosphorylation of Sendai virus P and V proteins in viral replication and pathogenesis.

Functional analysis of the primary constitutive phosphorylation of Sendai virus P and V proteins was performed using both in vitro and in vivo systems. Sendai virus minigenome transcription and replication in transfected cells were not significantly affected in the presence of primary phosphorylation deficient P protein (S249A, S249D, P250A) as measured by either the luciferase activity or the Northern blot analysis. Similarly, recombinant Sendai viruses lacking the primary phosphorylation in P grew to titers close to the wild-type virus in cell cultures and in the natural host of Sendai virus, the mouse. Mutant viruses showed no altered pathogenesis in mice lungs. Oligomerization of P by binding WT P or mutant P to GST-P (WT) Sepharose beads revealed that the primary phosphorylation was not crucial for P protein oligomerization. Similar to P protein primary phosphorylation, the V protein primary phosphorylation at serine249 was not essential for minigenome transcription and replication, as both WT and mutant V proteins were found equally inhibitory to the minigenome replication. These results show that the primary phosphorylation of P protein has no essential role in Sendai virus transcription, replication, and pathogenesis.

Animals↗
PubMed1999

Rapid conditions for the cleavage of oligodeoxyribonucleotides from cis-diol-bearing universal polymer supports and their deprotection.

Two sets of deprotection conditions have been evolved for the deprotection of oligodeoxyribonucleotides and their cleavage from commercially available cis -diol group-bearing universal polymer supports. In the first case, oligodeoxyribonucleotides anchored on the universal support were subjected to one of the standard deprotection conditions followed by treatment with aqueous 0.5 M sodium chloride + 0.2 M sodium hydroxide solution for 30 min at room temperature. In the second case, oligonucleotides bound to the universal support were treated with methanolic sodium hydroxide solution under microwave radiation to obtain fully deprotected oligomers within 4 min. Under both conditions, the cleavage of oligonucleotides from the support and their deprotection occurred quantitatively without any side product formation. The cleaved oligonucleotides were found to be identical in all respects (retention time on HPLC and biological activity in PCR) to the corresponding standard oligo-nucleotides.

Chromatography, High Pressure Liquid↗
PubMed1999

Human parainfluenza virus type 1 phosphoprotein is constitutively phosphorylated at Ser-120 and Ser-184.

RNA-dependent RNA polymerases of single-stranded, negative-sense RNA viruses comprise a phosphoprotein (P) and a large protein. The constitutive phosphorylation of the P protein in these viruses is highly conserved, yet the functional significance of phosphorylation is enigmatic. To approach this problem, phosphorylation sites were determined in two closely related paramyxovirus P proteins. Sendai virus (SV) is a prototypic paramyxovirus. Previously, using a phosphopeptide mapping technique, the primary constitutive phosphorylation site of SV P protein was mapped to Ser-249. Phosphorylation at Ser-249 is dependent on the presence of Pro-250. Human parainfluenza virus type 1 (HPIV-1) P protein has 66% similarity to SV P protein and its predicted secondary structure is highly similar to that of SV P protein. However, there is no obvious conserved phosphorylation site in HPIV-1 P protein. Using the phosphopeptide mapping strategy, the constitutive phosphorylation sites of HPIV-1 P protein were mapped. The HPIV-1 P protein is primarily phosphorylated at Ser-120. Phosphorylation at Ser-120 is dependent on the presence of Pro-121. It also has a minor phosphorylation site at Ser-184. The sequence at Ser-184 does not match any consensus phosphorylation target site for the known kinases. Significantly, the P proteins from both viruses are constitutively and primarily phosphorylated at one serine and the phosphorylation of that serine is dependent on the presence of a proline on its carboxyl side.

Amino Acid Sequence↗
PubMed1997

Microwave-assisted rapid deprotection of oligodeoxyribonucleotides.

A novel method for the deprotection of oligodeoxyribonucleotides under microwave irradiation has been developed. The oligodeoxynucleotides having base labile, phenoxyacetyl (pac), protection for exocyclic amino functions were fully deprotected in 0. 2 M sodium hydroxide (methanol:water : : 1:1, v/v) = A and 1 M sodium hydroxide (methanol:water : : 1:1, v/v) = B using microwaves in 4 and 2 min, respectively. The deprotection of oligodeoxyribonucleotides carrying conventional protecting groups, dAbz, dCbzand dGpac, for exocyclic amino functions was achieved in 4 min in B without any side product formation. The deprotected oligonucleotides were compared with the oligomers deprotected using standard deprotection conditions (29% aq. ammonia, 16 h, 55 degrees C) with respect to their retention time on HPLC and biological activity.

Kinetics↗
PubMed1997

Stimulation of Sendai virus C' protein synthesis by cycloheximide.

The polycistronic Sendai virus P/C mRNA is translated into five proteins (P, C', C, Y1 and Y2) from distinct start sites in virus-infected cells. The translation mechanism(s) of these proteins from two overlapping open reading frames in the P/C mRNA are poorly understood [Gupta, Ono and Xu (1996) Biochemistry 35, 1223-1231]. While investigating the initiation mechanism of C' from an ACG start site, we found that C' synthesis was resistant to inhibitors of peptide chain elongation such as cycloheximide (CHX) and anisomycin, but not to pactamycin (an inhibitor of chain initiation) or puromycin (a peptide chain terminator). Moreover, low levels (less than 30 microg/ml) of CHX significantly stimulated C' synthesis. Whereas C' synthesis was stimulated, synthesis of the P and C proteins, which are translated from the same mRNA, decreased by more than 95%. Stimulation of C' synthesis by CHX is not related to its initiation at an ACG codon. Mutation of ACG to alternative start sites had no effect on the CHX-stimulated C' synthesis. Similarly, C' synthesis was preferentially stimulated when Sendai virus-infected cells were exposed to hypotonic growth medium. These results suggest that the P/C mRNA may exist in at least two reversible conformations: whereas one conformation allows synthesis of the P and C proteins, the alternative conformation allows synthesis of the C' protein. It might be that low concentrations of CHX somehow increase the alternative conformation, which increases C' synthesis. The C' protein synthesis is reminiscent of the synthesis of stress-related proteins. Perhaps Sendai virus has evolved a novel mechanism to express both non-stress-related and stress-related proteins from the same mRNA.

Animals↗
PubMed1997

Efficient hammerhead ribozymes targeted to the polycistronic Sendai virus P/C mRNA. Structure-function relationships.

The Sendai virus polycistronic P/C mRNA encodes the P and C proteins from alternate overlapping reading frames. To determine the functions of these proteins in virus replication, hammerhead ribozymes were targeted to cleave the 5'-untranslated region of the P/C mRNA. Both cell-free and intracellular assays were employed to determine ribozyme efficacy. To appropriately compare activities between cell-free and intracellular assays, identical ribozymes were synthesized in vitro as well as expressed in cells. Ribozyme parameters, namely hybridization arm length (HAL) and nonhybridizing extraneous sequences (NES), were found to have rate-determining properties. In cell-free reactions, ribozymes with 13-mer HAL were up to 10-fold more efficient than those with 9-mer HAL. Ribozymes with 9-mer HAL were relatively ineffective in transfected cells. Minimizing the number of NES increased ribozyme efficiency in vitro. However, ribozymes with minimal NES were essentially inert intracellularly. The NES at the termini of the most effective intracellular ribozyme, Rz13st ( approximately 95% inhibition of the p gene expression), were predicted to fold into stem-loop structures. These structures most likely increase ribozyme stability as evidenced by the 8-fold higher resistance to ribonuclease T2 digestion of Rz13st compared with Rz13B. Our results suggest that when designing effective intracellular ribozymes, parameters that enhance formation of productive ribozyme:substrate duplexes and that increase RNA stability should be optimized.

Base Sequence↗
PubMed1997

Evaluation of eye irritation potential: statistical analysis and tier testing strategies.

Eye irritation testing, specifically the Draize test, has been the centre of controversy for many reasons. Several alternatives, based on the principles of reduction, refinement and replacement, have been proposed and are being used by the industry and government authorities. However, no universally applicable, validated non-animal alternative(s) is currently available. This report presents a statistical analysis and two testing approaches: the partial least squares multivariate statistical analysis of de Silva and colleagues from France, the tier-testing approach for regulatory purposes described by Gerner and colleagues from Germany, and the three-step tier-testing approach of the US Interagency Regulatory Alternatives Group described by Gupta and Hill. These approaches were presented as three separate papers at the November 1993 Interagency Regulatory Alternatives Group (IRAG) Workshop on Eye Irritation Testing; they have been summarized and combined into the following three-part report. The first part (de Silva et al.) presents statistical techniques for establishing test batteries of in vitro alternatives to the eye irritation test. The second (Gerner et al.) and third (Gupta and Hill) parts are similar in that they stage assessment of information by using a combination of screening information and animal testing to effect reductions in animal use and distress.

Animal Testing Alternatives↗
PubMed1996

Sendai virus P protein is constitutively phosphorylated at serine249: high phosphorylation potential of the P protein.

Previously we showed that the Sendai virus P protein (568 aa) in virus-infected cells and in virions was primarily and constitutively phosphorylated on serine(s) in a single tryptic phosphopeptide TP1. By two-dimensional thin-layer electrophoresis and chromatography analysis of tryptic phosphopeptides of several deletion and point mutants of the P protein, we now show that the sole phosphorylation site in TP1 is serine249. Interestingly, when serine249 was deleted or mutagenized alternate potential serine sites were more heavily phosphorylated. A similar effect was observed when the deletion was very close to serine249 (delta 208-236). Mutagenesis of proline250 to alanine abrogated phosphorylation at serine249 suggesting that proline250 is essential for the primary phosphorylation of the P protein. Conceivably, serine249 phosphorylation is mediated by a proline-directed protein kinase. This finding is unusual because a majority of the P proteins from other negative-strand RNA viruses have been shown to be phosphorylated primarily by casein kinase II. Our results demonstrate that the P protein has a strong potency to remain phosphorylated. Based on our previous and present results, we suggest that the phosphorylation sites on P are dependent on the accessibility of phosphatases rather than kinases as all potential sites are about equally competent for phosphorylation. We propose that phosphorylation is important for maintaining the structural integrity of the Sendai virus P protein.

Amino Acid Sequence↗
PubMed1996

Lack of correlation between Sendai virus P/C mRNA structure and its utilization of two AUG start sites from alternate reading frames: implications for viral bicistronic mRNAs.

The polycistronic P/C mRNA of Sendai virus encodes five proteins (C', P, C, Y1, and Y2) each of which initiates from a distinct start site. Two major proteins, P and C, are expressed in approximately equimolar amounts from two consecutive AUGs in overlapping reading frames. To better understand the mechanism of expression of the C protein from a downstream AUG, site-directed mutants of the P/C mRNA were created and expressed in COS1 cells. The secondary structure of the mRNA was examined to determine whether the mRNA structure played any role in the synthesis of the C protein. Our results ruled out any significant involvement of the 5' UTR, sequence contexts, secondary structure, distance between the start sites, and sequences downstream to the C-AUG. However, they are consistent with the concept that the synthesis of the C protein is primarily dependent on the orientation of its reading frame, i.e., +1 in relation to the upstream P reading frame. The downstream reading frame was translated poorly when it occurred in +2 orientation in relation to the upstream reading frame. Interestingly, all the known functional bicistronic mRNAs with overlapping reading frames from cytoplasmic RNA viruses have their downstream reading frame in +1 orientation relative to the upstream frame. We propose that the evolutionary conservation of the downstream reading frame in +1 orientation in these bicistronic mRNAs is important for its efficient translation.

Base Sequence↗
PubMed1996

Evaluation of serum lipids in patients of acute stroke.

Stroke, though considered a thromboembolic disorder, is known to be associated with hyperlipidaemia. In Western country, some workers have performed studies exploring the role of lipids in stroke in their country. Such a study is lacking in Indian population. This study was therefore conducted to observe the role of lipids in stroke by evaluating 13 parameters of lipids in 48 patients of non haemorrhagic cerebral stroke hospitalised in acute condition and compared with those of 70 age matched normal subjects. Results revealed that phospholipids and arachidonic acid were significantly altered in patients of acute stroke.

Acute Disease↗
PubMed1995

Intracellular phosphorylation of the Sendai virus P protein.

Phosphorylation status of the Sendai virus P protein was examined during virus infection and compared with cell-free phosphorylation. P protein from Sendai virus-infected (VI) and P/C gene-transfected (PT) mammalian cells and from purified virions (PV) was phosphorylated at only serine residues. In contrast, cell-free phosphorylation of the P protein with virion-associated protein kinase (VAPK) occurred at both threonine and serine. Tryptic phosphopeptide maps of the P protein from VI, PT, and PV showed that the phosphorylation was primarily localized on one peptide (TP1), while VAPK phosphorylated the P protein on several peptides. There was no change in the steady-state phosphopeptide map of the P protein during virus replication, indicating that the TP1 is constitutively phosphorylated. Inhibition of cellular phosphatases (PP1 and PP2A) by okadaic acid (OA) in virus-infected cells caused a sixfold increase in the P protein phosphorylation, solely at serine residues. The phosphopeptide map of the OA-P protein revealed that phosphorylation occurred on several peptides, but the OA-P map was significantly different from the VAPK-P map. However, additional phosphorylation of the P protein did not block its association with nucleocapsids. These results suggest that the Sendai virus P protein is constitutively phosphorylated primarily at one locus but has the potential for phosphorylation at additional sites. Further, our results do not show any correlations between the intracellular and cell-free phosphorylation of the P protein and, therefore, question the validity of cell-free phosphorylations.

Animals↗
PubMed1995

A highly efficient procedure for site-specific mutagenesis of full-length plasmids using Vent DNA polymerase.

Careful titration of Vent polymerase activity allows efficient amplification of full-length plasmids (12 kb). The high processivity and fidelity of this enzyme made oligonucleotide-directed site-specific mutagenesis of plasmids a straight-forward process. Using only two primers, a mutagenic and a complementary, single-base mutants of recombinant plasmids were obtained consistently with > 90% efficiency from a single round of PCR. This procedure also made site-specific deletion, insertion, and several bases mutagenesis facile and efficient.

Base Sequence↗
PubMed1995

Multiple elements in the 5' untranslated region down-regulate c-sis messenger RNA translation.

Expression of the platelet derived growth factor (PDGF) B-chain, the product of the c-sis proto-oncogene, is regulated both at the transcriptional and translational level. Previous studies have shown that the long 5' untranslated region (UTR) of the c-sis mRNA strongly inhibits synthesis of the PDGF-B chain. However, the assignments of down-regulatory regions within the 5' UTR were ambiguous. Expression of several site-directed point and deletion mutants of the 5' UTR of the c-sis mRNA in COS1 cells revealed that the UTR inhibited PDGF-B chain synthesis in a more complex manner than indicated by the previous studies. Abrogation of the three upstream short open reading frames by mutating each of the AUGs did not have any effect on the synthesis of the PDGF-B chain. Expression of deletion mutants revealed two partially overlapping regions, nucleotides 1-651 and 475-1022, each of which independently inhibited c-sis mRNA translation as effectively as the entire 5' UTR. Each of these regions contains a potentially strong stem-loop structure and a GC-rich element. These elements of the alternate down-regulatory regions could interact within the same region and/or with the elements of the other regulatory region to block c-sis mRNA translation. We show, in contrast to the previous reports, that the inhibition of c-sis mRNA translation cannot be attributed exclusively to any particular predicted secondary structure or a GC-rich element within the 5' UTR.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Translation initiation from non-AUG codons in COS1 cells is mRNA species dependent.

Sendai virus P/C mRNA, human erythrocyte membrane protein 4.1 mRNA and PDGF-B chain mRNA were used to test whether translation initiation from non-AUG codons in COS1 cells was mRNA species dependent. Site-directed mutants of the authentic translation start sites of these mRNAs to alternate start codons showed that while P/C mRNA is capable of initiating translation from non-AUG start sites the other two mRNAs are not. Our study shows that translation initiation from non-AUG codons is mRNA species dependent and suggests that higher order structure of an mRNA determines the non-AUG translation start site.

Animals↗
PubMed1994

Effect of gugulipid on bioavailability of diltiazem and propranolol.

The effect of single oral dose of 1 gm gugulipid was studied on bioavailability of single oral dose of propranolol (40 mg) and diltiazem (60 mg) in 10 and 7 normal healthy male volunteers respectively. It was a randomised within group crossover study. Blood samples were collected at hourly intervals upto 8 hrs. Gugulipid significantly reduced (P < .01) peak plasma concentration (Cmax) and area under curve (AUC 0-8 hrs) of both the drugs in normal volunteers. Such interaction in patients receiving propanolol or diltiazem with gugulipid may lead to diminished efficacy or nonresponsiveness due to significant reduction in bioavailability.

Administration, Oral↗
PubMed1993

Phosphorylation of the Sendai virus C proteins.

We show that the Sendai virus C' and C proteins exist as both phosphorylated and nonphosphorylated forms, while the Y1 and Y2 proteins are not phosphorylated in virus-infected cells. Phosphorylation occurs primarily on serine residues, most likely at the N-terminus of the C' and C proteins. Phosphatases PP1 and PP2A significantly modulate the phosphorylation status of the C proteins as evidenced by okadaic acid inhibition of these phosphatases. The other Sendai virus proteins including the cotranslationally expressed P protein are not necessary for the appropriate phosphorylation of the C' and C proteins. Differential phosphorylation and potential for the modulation of phosphorylation suggests regulatory functions for the C proteins.

Animals↗