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Biomedical subjects

I Choudhury

Publications and source records attributed to I Choudhury.

14 recordsLinked to original sources
PubMed2006

Matrix metalloproteinase-13 influences ERK signalling in articular rabbit chondrocytes.

OBJECTIVE: Matrix metalloproteinase-13 (MMP-13) is an extracellular MMP that cleaves type II collagen, the major protein component of cartilage, with high specificity and has been implicated in the pathology of osteoarthritis. The present study aimed to characterize the binding and internalization kinetics of MMP-13 in normal rabbit chondrocytes and whether MMP-13 affected cell signalling. METHODS: Rabbit chondrocytes were used in [125I]-MMP-13 binding assays to investigate the MMP-13 binding kinetics and Western analysis allowed for the assessment of intracellular signalling cascades. RESULTS: Rabbit chondrocytes were found to express the cartilage-specific genes aggrecan and type II collagen throughout their in vitro culture period. Appreciable specific cell-association of [125I]-MMP-13 was detected after 10 min of exposure to the ligand and equilibrium was obtained after 2 h. Binding of [125I]-MMP-13 to chondrocytes was specific and approached saturation at 75 nM. Internalization of MMP-13 was evident after 20 min, reached a maximum at 30 min and had returned to baseline by 90 min. Addition of receptor-associated protein (RAP) inhibited the internalization of MMP-13 indicating a likely role for low-density lipoprotein receptor-related protein-1 (LRP1) in this process. Interestingly the presence of MMP-13 induced phosphorylation of the extracellular signal-regulated kinase 1/2 (ERK1/2) protein showing that there is initiation of a signalling process in response to MMP-13 being bound and internalized by rabbit chondrocytes. However, this activation does not involve the MMP-13 internalization receptor LRP1. CONCLUSION: These studies demonstrate and characterize the MMP-13 binding and internalization system in rabbit chondrocytes and indicate that MMP-13 may regulate the phenotype of the chondrocytes through this receptor system.

Aggrecans↗
PubMed1999

Emesis induced by inhibitors of type IV cyclic nucleotide phosphodiesterase (PDE IV) in the ferret.

Emesis induced by inhibitors of type IV cyclic nucleotide phosphodiesterase (PDE IV) has been investigated in the ferret. The PDE IV inhibitors studied were: RS14203, R-rolipram and CT-2450 (i.e. (R)-N-[4-[1-(3-cyclopentyloxy-4-methoxyphenyl)-2-(4-pyridyl)ethyl]phenyl ]N'-ethylurea), in addition to the less active enantiomers S-rolipram and CT-3405. Following oral administrations, different emetic profiles were observed with time. Emesis induced by RS14203 exhibited a dose-response relationship but no such relationship was seen for R-rolipram or CT-2450. The incidence of emesis was positively influenced by the dose of PDE IV inhibitors administered, allowing a rank order of potency: RS14203 > R-rolipram > S-rolipram > CT-2450 > CT-3405. PDE IV inhibitor-induced emesis was abolished by the tachykinin NK1 receptor antagonist, CP-99,994. No peripheral release of substance P by PDE IV inhibitors seems to be involved in triggering the emetic reflex since L-743,310, which only has peripheral NK1 receptor antagonist activity, was without effect. The implication of 5-HT3 receptors in PDE IV inhibitor-induced emesis was variable. Our results suggest that the PDE IV inhibitors studied are mixed peripheral-central emetogens. PDE IV inhibition itself could be plausible mechanism of action of these agents. However, whether emesis is mediated via a specific isoform of PDE IV remains to be established.

Animals↗
PubMed1999

Translational effects of peptide antagonists of Tat protein of human immunodeficiency virus type 1.

The Tat (trans-activator of transcription) regulatory protein of human immunodeficiency virus (HIV-1) acts by interacting with the TAR RNA domain of nascent viral transcripts and with cellular proteins to increase viral transcription. In Jurkat-derived HCLE-D36 cells, which are stably transfected with the chloramphenicol acetyltransferase (CAT) reporter gene expressed from the TAR-encoding long terminal repeat (LTR) of HIV-1, CAT protein expression is dependent on Tat. The Tat9-K-biotin peptide antagonist of Tat binds specifically to TAR RNA and competes with Tat for binding. In the cellular expression system, Tat9-K-biotin reduces Tat-dependent CAT expression. However, while the Tat antagonist greatly reduces CAT protein production and polysome association of CAT mRNA, it has little effect on CAT mRNA levels, suggesting that the antagonist works at the post-transcriptional level.

Anti-HIV Agents↗
PubMed1998

Inhibition of HIV-1 replication by a Tat RNA-binding domain peptide analog.

The peptidic compound, N-acetyl-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Cys(biotin)-NH2 (Tat10-biotin), contains the 9-amino acid sequence from the basic domain of the Tat protein responsible for specific interaction with TAR RNA. The cysteine residue provides an attachment site for biotin, which acts as a cellular uptake enhancer. Tat10-biotin binds a fragment of TAR RNA (deltaTAR) avidly and specifically, as measured in an electrophoretic gel shift assay. Tat10-biotin inhibited tat gene-induced expression of a stably transfected chloramphenicol acetyl transferase (CAT) reporter gene linked to the HIV-1 long terminal repeat (LTR) in a model cell assay, but did not inhibit phorbol ester-induced expression of CAT, thereby demonstrating a Tat-dependent mechanism of inhibition. Inhibition of HIV-1 replication after acute infection of MT2 cells was demonstrated by absence of HIV-induced syncytium formation and cytotoxicity, as well as by suppression of reverse transcriptase production. These results suggest that a peptide or peptide mimetic capable of competing with the TAR RNA-binding domain of Tat protein might be useful as a therapeutic agent for AIDS.

Anti-HIV Agents↗
PubMed1995

Use of a polyethylene glycol-peptide conjugate in a competition gel shift assay for screening potential antagonists of HIV-1 Tat protein binding to TAR RNA.

Interference of binding of Tat protein to TAR RNA in HIV-1-infected cells may be a useful therapeutic strategy for AIDS. An electrophoretic assay to screen potential low-molecular-weight (< 2 kDa) Tat antagonists has been established. A radiolabeled TAR RNA fragment (delta TAR) is retarded in mobility when bound by a Tat peptide-polyethylene glycol conjugate (Tat-PEG), which is used in place of the Tat protein. The assay determines the ability of a potential antagonist to compete with Tat-PEG for binding to delta TAR, as measured by interference with the gel shift of delta TAR. To discriminate between specific and nonspecific interactions, the assay is done in the absence or the presence of a 250-fold molar excess of tRNA.

Amino Acid Sequence↗
PubMed1989

Metabolism of small RNAs in cultured human cells.

There are gaps in what is known about the metabolism of some mammalian small RNA species. Our present observations can be summarized as follows. The level of radiolabeled mature U1 RNA doubled between 2 and 24 hr of label chase, while that of all other small RNA species tested did not change. These results are compatible with the possibility that the nucleotide precursor pool for U1 RNA transcription may be partly segregated, or that there may be a second pathway of U1 RNA formation. Precursors of nucleolar U3 RNA were detected whose electrophoretic mobilities are equivalent to those of transcripts approximately 14 and approximately 8 nucleotides longer than the mature species, and which are apparently cytoplasmic. The ladder of U2 RNA precursors showed a gap, suggesting that some of the cleavages during U2 RNA processing are endonucleolytic. We detected an apparent U5 RNA precursor whose electrophoretic mobility is equivalent to that of a species approximately 1 nucleotide longer than mature U5 RNA. There was a predominant band in the middle of the ladder of U4 RNA precursors (apparently approximately 3 nucleotides longer than mature U4 RNA) which suggests that U4 RNA maturation may pause briefly at that intermediate. There are apparently two additional species of mature hY3 RNA, which are less abundant and are about 1 and 2 bases longer than the major hY3 RNA species. An apparent hY3 RNA precursor was detected, which may be approximately 2-3 nucleotides longer than the main mature hY3 RNA species.

Cells, Cultured↗
PubMed1988

Effect of ultraviolet light on the expression of genes for human U1 RNA.

Two types of UV-light-induced inhibitions of the synthesis of small nuclear RNA species U1, U2, U3, U4, and U5 were described previously: an immediate inhibition and a separate, delayed suppression that requires 1-2 hr of postirradiation cell incubation and UV doses that are about tenfold lower. In the present report, U1 RNA transcription in isolated nuclei from HeLa cells, assayed by RNAase T1 protection, reproduced the delayed inhibition. The sizes of the protected RNA fragments suggest that it is the initiation of U1 RNA transcription that is blocked during this inhibition. Transient expression of a marked human U1 RNA gene that contains 425 and 92 nucleotides of the 5' and 3' flanking sequences, respectively, showed delayed, but not immediate inhibition (while the endogenous U1 RNA genes exhibited immediate suppression). This indicates that continuity of the U1 gene flanking sequences beyond those segments and/or chromosomal integration of the U1 gene are not needed for the delayed inhibition, but may be required for the immediate inhibition. Irradiation of a U1 RNA gene, followed by its injection into Xenopus laevis oocyte nuclei, did not reproduce the immediate or delayed inhibitions. This suggests that direct UV radiation damage to DNA in the U1 RNA gene region is not the critical lesion in either the immediate or delayed UV-light-induced inhibitions of U1 RNA synthesis. In addition, the RNAase T1 protection pattern of transcripts synthesized in isolated nuclei from nonirradiated HeLa cells suggests that these cells may produce small amounts of U1 RNA molecules with variant nucleotide sequences in the mature region of the transcript.

Animals↗
PubMed1988

Metabolism of U6 RNA species in nonirradiated and UV-irradiated mammalian cells.

We observed a series of rapidly labeled U6 RNA bands, which were hybrid selected with U6 DNA, in nonirradiated human cells. The electrophoretic mobility of these bands in denaturing gels was lower than that of the known mature U6 RNA species, and was equivalent to transcripts up to approximately 7 nucleotides longer. These multiple U6 RNA species lost their label during a chase without a proportional increase in radioactivity in the known mature U6 RNA, which suggests that a substantial fraction is not processed into the major mature U6 RNA. During a label chase, the multiple U6 RNA bands appeared first in the cytoplasmic fraction and later in nuclei. One of the major rapidly labeled U6 RNA bands had the electrophoretic mobility of an RNA species one nucleotide shorter than the known mature U6 RNA. UV light induced a UV dose-dependent, preferential disappearance of recently synthesized molecules of the U6 RNA species of higher gel electrophoretic mobility, including the known mature U6 RNA. Since this effect was seen in cells pulse-labeled immediately before or after irradiation, it suggests that UV radiation induces the specific degradation of the electrophoretically faster moving species of U6 RNA, which are apparently shorter chains. The effect of UV light was RNA species-specific, was not seen in molecules synthesized long (e.g., 22 hr) before irradiation, and occurred in human and mouse cells.

Animals↗
PubMed1984

Action of polyamines on ribonucleic acid and protein synthesis during ontogeny of human fetal liver.

A higher rate of RNA synthesis in human fetal liver cells was found during the early age of gestation, after a drop, it gradually increases to a maximum value between 18 and 22 weeks, followed by a sharp decrease at later period of gestation. Total polyamine content of fetal liver tissue shows a similar trend. However, when liver cell suspension was incubated with exogenous spermine, spermidine and putrescine there is a dose-dependent inhibition in RNA synthesis. Protein synthesis in human fetal liver cells was stimulated with lower doses of spermine, spermidine and putrescine and inhibited at higher doses. The optimum dose for stimulation and the degree of stimulation was, however, not the same for fetuses of different gestational ages.

Fetus↗
PubMed1983

Ontogeny of polyamines in relation to nucleic acids in brain and spinal cord of the developing human fetus.

The polyamines, spermine and spermidine, and the diamine, putrescine, were estimated in relation to the distribution of RNA and DNA in cerebral cortex, cerebellum and spinal cord of the human CNS during prenatal development. Polyamines were found to be maximally located in the spinal cord, minimum being in cerebellum. Relation between polyamines and nucleic acids was region specific.

Brain↗
PubMed1982

Studies on superoxide dismutase activities in virulent and avirulent strains of Agrobacterium tumefaciens and also in normal and crown gall tumor cells of Bryophyllum calycinum.

Superoxide dismutase activity in virulent strains of Agrobacterium tumefaciens was found to be higher than that in avirulent strains. Polyacrylamide gel electrophoresis revealed two isoenzymes in both these strains. These isoenzymes are suggested to be iron and manganese containing superoxide dismutases. Crown gall tumor cells of the plant Bryophyllum calycinum were found to have higher superoxide dismutase activity than the normal plant cells. Polyacrylamide gel electrophoresis revealed two isoenzymes in both normal and crown gall tumor cells. Advantages of the higher superoxide dismutase activities in respect of the survival of virulent strains of A. tumefaciens and crown gall tumor growth have been discussed.

Iron↗
PubMedDate not supplied

A polyethylene glycol copolymer for carrying and releasing multiple copies of cysteine-containing peptides.

Two different methods were developed to prepare an adduct of a poly(ethylene glycol)-lysine copolymer with either cysteamine or 1-amino-2-methyl-2-propanethiol. Cysteine-containing peptides could then be disulfide-linked to the thiol groups on the polymer in a facile manner. In the described procedures, a coupling ratio of about 8 peptides/molecule of poly(ethylene glycol)-lysine copolymer (Mw = 27 000) was typically attained. The products were stable at neutral pH, but the peptides could be released from the polymer in a physiologically relevant reducing environment. The release rate was highly dependent on the linker used for forming the disulfide bond. To illustrate the potential biomedical usefulness of this polymer carrier, a Tat peptide-PEG conjugate was shown to inhibit expression of a reporter gene fused to the TAR element of human immunodeficiency virus in a model cell assay.

Biotinylation↗