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Biomedical subjects

P Saha

Publications and source records attributed to P Saha.

At least 37 records · Page 2Linked to original sources
PubMed1997

p21CIP1 and Cdc25A: competition between an inhibitor and an activator of cyclin-dependent kinases.

Cdc25A, a phosphatase essential for G1-S transition, associates with, dephosphorylates, and activates the cell cycle kinase cyclin E-cdk2. p21CIP1 and p27 are cyclin-dependent kinase (cdk) inhibitors induced by growth-suppressive signals such as p53 and transforming growth factor beta (TGF-beta). We have identified a cyclin binding motif near the N terminus of Cdc25A that is similar to the cyclin binding Cy (or RR LFG) motif of the p21CIP1 family of cdk inhibitors and separate from the catalytic domain. Mutations in this motif disrupt the association of Cdc25A with cyclin E- or cyclin A-cdk2 in vitro and in vivo and selectively interfere with the dephosphorylation of cyclin E-cdk2. A peptide based on the Cy motif of p21 competitively disrupts the association of Cdc25A with cyclin-cdks and inhibits the dephosphorylation of the kinase. p21 inhibits Cdc25A-cyclin-cdk2 association and the dephosphorylation of cdk2. Conversely, Cdc25A, which is itself an oncogene up-regulated by the Myc oncogene, associates with cyclin-cdk and protects it from inhibition by p21. Cdc25A also protects DNA replication in Xenopus egg extracts from inhibition by p21. These results describe a mechanism by which the Myc- or Cdc25A-induced oncogenic and p53- or TGF-beta-induced growth-suppressive pathways counterbalance each other by competing for cyclin-cdks.

Animals↗
PubMed1997

Rapid screening of marketed paracetamol tablets: use of thin-layer chromatography and a semiquantitative spot test.

Evaluated is the use of thin-layer chromatography (TLC) to determine the quality of paracetamol tablets marketed in Bangladesh. The procedure was carried out using a cheap and rapid TLC method developed by the U.S. Food and Drug Administration. Reported also is a semiquantitative specific spot test for screening paracetamol tablets. The results obtained indicate that, of the 38 brands tested, three were spurious, while 11 were of borderline quality. In some cases, the results were also verified using the British Pharmacopoeia method. The simplified tests described in this article cannot replace the British Pharmacopoeia or U.S. Pharmacopoeia methods but can be employed as initial screening tests.

Acetaminophen↗
PubMed1996

p21Cip1/Waf1 disrupts the recruitment of human Fen1 by proliferating-cell nuclear antigen into the DNA replication complex.

Fen1 or maturation factor 1 is a 5'-3' exonuclease essential for the degradation of the RNA primer-DNA junctions at the 5' ends of immature Okazaki fragments prior to their ligation into a continuous DNA strand. The gene is also necessary for repair of damaged DNA in yeast. We report that human proliferating-cell nuclear antigen (PCNA) associates with human Fen1 with a Kd of 60 nM and an apparent stoichiometry of three Fen1 molecules per PCNA trimer. The Fen1-PCNA association is seen in cell extracts without overexpression of either partner and is mediated by a basic region at the C terminus of Fen1. Therefore, the polymerase delta-PCNA-Fen1 complex has all the activities associated with prokaryotic DNA polymerases involved in replication: 5'-3' polymerase, 3'-5' exonuclease, and 5'-3' exonuclease. Although p21, a regulatory protein induced by p53 in response to DNA damage, interacts with PCNA with a comparable Kd (10 nM) and a stoichiometry of three molecules of p21 per PCNA trimer, a p21-PCNA-Fen1 complex is not formed. This mutually exclusive interaction suggests that the conformation of a PCNA trimer switches such that it can either bind p21 or Fen1. Furthermore, overexpression of p21 can disrupt Fen1-PCNA interaction in vivo. Therefore, besides interfering with the processivity of polymerase delta-PCNA, p21 also uncouples Fen1 from the PCNA scaffold.

Binding Sites↗
PubMed1996

A 39 amino acid fragment of the cell cycle regulator p21 is sufficient to bind PCNA and partially inhibit DNA replication in vivo.

The cell cycle regulator p21 interacts with and inhibits the DNA replication and repair factor proliferating cell nuclear antigen (PCNA). We have defined a 39 amino acid fragment of p21 which is sufficient to bind PCNA with high affinity (Kd 10-20 nM). This peptide can inhibit DNA replication in vitro and microinjection of a GST fusion protein containing this domain inhibited S phase in vivo. Despite its high affinity for PCNA, the free 39 amino acid peptide does not have a well-defined structure, as judged from circular dichroism and nuclear magnetic resonance measurements, suggesting an induced fit mechanism for the PCNA-p21 interaction. The association of the small peptide with PCNA was thermolabile, suggesting that portions of p21 adjoining the minimal region of contact stabilize the interaction. In addition, a domain containing 67 amino acids from the N-terminus of PCNA was defined as both necessary and sufficient for binding to p21.

Amino Acid Sequence↗
PubMed1996

Cyclin-binding motifs are essential for the function of p21CIP1.

The cyclin-dependent kinase (Cdk) inhibitor p21 is induced by the tumor suppressor p53 and is required for the G1-S block in cells with DNA damage. We report that there are two copies of a cyclin-binding motif in p21, Cy1 and Cy2, which interact with the cyclins independently of Cdk2. The cyclin-binding motifs of p21 are required for optimum inhibition of cyclin-Cdk kinases in vitro and for growth suppression in vivo. Peptides containing only the Cy1 or Cy2 motif partially inhibit cyclin-Cdk kinase activity in vitro and DNA replication in Xenopus egg extracts. A monoclonal antibody which recognizes the Cy1 site of p21 specifically disrupts the association of p21 with cyclin E-Cdk2 and with cyclin D1-Cdk4 in cell extracts. Taken together, these observations suggest that the cyclin-binding motif of p21 is important for kinase inhibition and for formation of p21-cyclin-Cdk complexes in the cell. Finally, we show that the cyclin-Cdk complex is partially active if associated with only the cyclin-binding motif of p21, providing an explanation for how p21 is found associated with active cyclin-Cdk complexes in vivo. The Cy sequences may be general motifs used by Cdk inhibitors or substrates to interact with the cyclin in a cyclin-Cdk complex.

Amino Acid Sequence↗
PubMed1996

A primary culture model of rabbit conjunctival epithelial cells exhibiting tight barrier properties.

PURPOSE: The present study was conducted to develop and characterize a functional primary culture of pigmented rabbit conjunctival epithelial cells on permeable support exhibiting tight barrier properties. METHODS: Conjunctival epithelial cells were isolated by 0.2% protease treatment, cultured at 0.5-1.8 x 10(6) cells/cm2 onto collagen-treated Transwell filters, and were maintained either in the presence of 1% fetal bovine serum throughout or serum-free media from day 3 onwards. Transepithelial potential difference (PD) and transepithelial electrical resistance (TEER) were measured and equivalent short-circuit current (Ieq = PD/TEER) estimated. RESULTS: There appears to be a critical plating density of 1.5 x 10(6) cells/cm2 for functional development of tight epithelial cell cultures. The culture conditions as noted above did not affect either the time when peak bioelectric parameters were attained (days 8-10) or the magnitude of these parameters at a plating density of 1.5 x 10(6) cells/cm2. Specifically, cells grown in a serum-free media showed a peak TEER of 1.9 +/- 0.2 k omega.cm2, a PD of 14.2 +/- 1.6 V (apical side negative), and and Ieq of 8.0 +/- 0.4 microA/cm2 (mean +/- SEM, n = 45). Electron microscopy of serum-weaned cultures revealed a multilayered epithelium with numerous microvilli on the outermost layer of cells, while sporadic positive Periodic Acid Schiff (PAS) staining under light microscopy suggested the presence of mucin-secretory goblet cells. CONCLUSIONS: A functional, tight, epithelial barrier of the pigmented rabbit conjunctiva on a permeable support has been developed, which may be useful for mechanistic studies of ion and drug transport at the cellular level.

Animals↗
PubMed1996

Permeability characteristics of primary cultured rabbit conjunctival epithelial cells to low molecular weight drugs.

PURPOSE: To evaluate the permeability characteristics of primary cultured rabbit conjunctival epithelial cell (RCEC) layers to low molecular weight drugs of varying lipophilicity. METHODS: 3H-mannitol; hydrophilic sotalol and atenolol; moderately lipophilic metoprolol, timolol, propranolol; and highly lipophilic betaxolol were used as model compounds. RESULTS: The conjunctival apparent permeability coefficient (Papp) of mannitol (1 x 10(-7) cm/s) was 2.4 times lower than that of the most hydrophilic beta-blocker, sotalol (Papp = 2.4 x 10(-7) cm/s). Differences in the degree of tightness of the epithelial cell layers brought about a 30-fold difference in the transport of atenolol in favor of the leaky cell layers, while not affecting the transport of the lipophilic drug, propranolol. Within the log partition coefficient (PC) range of -0.62 (sotalol) and 3.44 (betaxolol), there was a hundred-fold difference in the Papp. A sigmoidal curve was used to depict the influence of lipophilicity on solute permeation across conjunctival epithelial cell layers. An effective half-maximal Papp was observed at a log PC value of 1.2. CONCLUSIONS: These findings on the lipophilicity effect on drug transport are generally similar to those reported for the isolated rabbit conjunctiva, suggesting the utility of cultured rabbit conjunctival epithelial cell layers as an in vitro model for evaluating drug transport.

Adrenergic beta-Antagonists↗
PubMed1995

Characterization of a winged bean (Psophocarpus tetragonolobus) protein kinase with calmodulin-like domain: regulation by autophosphorylation.

A soluble protein kinase purified from winged bean (Psophocarpus tetragonolobus) shoots, has been assessed as a monomeric enzyme with an approximate M(r) of 60,000 in spite of the presence of two polypeptides of 61 and 58 kDa determined by SDS/PAGE. Immunoblot analyses using either of the two antisera raised individually against the polypeptides, detect both of them in purified preparations and a single larger polypeptide (62 kDa) in freshly prepared tissue homogenates, clearly indicating the likelihood of the doublet being formed from the larger one by proteolysis. Histone H1, syntide 2 and a synthetic myosin light chain-related peptide (MLC-peptide) have been identified as exogenous substrates of the enzyme. Complete Ca(2+)-dependence for substrate phosphorylation, a drastic inhibition of the reaction by a calmodulin (CaM) antagonist which can be partially reversed by a heterologous CaM and direct 45Ca(2+)-binding on blot, form compelling evidence in favour of a CaM-like domain of the enzyme. Both the polypeptides of the purified enzyme undergo intramolecular autophosphorylation on serine residue(s). Unlike the substrate phosphorylation reaction, autophosphorylation is Ca(2+)-independent and is not inhibited by the CaM antagonist. Down-regulation of substrate phosphorylation by auto-phosphorylation, and stimulation of the autophosphorylation by histone H1 and MLC-peptide, are novel regulatory features of the enzyme.

Amino Acid Sequence↗
PubMed1992

Autologous blood donation in nonorthopaedic surgical procedures as a blood conservation strategy.

Autologous blood donation in many nonorthopaedic procedures is controversial. Our study of 408 consecutive such procedures could be divided into two groups. In group I, the anticipated probability for homologous blood transfusion was very low (less than 5%): vaginal hysterectomy and miscellaneous gynecologic procedures, obstetrical delivery, mammoplasty and cholecystectomy. In group II, the anticipated probability for homologous blood transfusion was high (greater than 5%): open heart and vascular surgery, neurosurgery, mastectomy, abdominal and radical hysterectomy, and extensive urologic procedures. We conclude that for procedures in which the blood transfusion probability is very low, autologous blood donation should not be encouraged; this practice should be promoted in procedures in which the blood transfusion probability is 'high' (i.e. greater than 5%), with emphasis on maximizing autologous blood collection in order to minimize homologous blood transfusion.

Blood Transfusion, Autologous↗
PubMed1991

Extensive CT scan abnormality in Wilson's disease.

A case of Wilson's disease with extensive white matter hypodensity, including in the basal ganglia, on CT scan is presented. Such extensive CT scan abnormality has not been described in Wilson's disease.

Adolescent↗