Search PubMed⌕ Search

Biomedical subjects

Y Patel

Publications and source records attributed to Y Patel.

At least 55 records · Page 3Linked to original sources
PubMed1995

Effects of Ca++ and glycine on lipid breakdown and death of ATP-depleted MDCK cells.

The relationships between cytosolic free calcium (Caf), cell associated glycine, phospholipid hydrolysis and cell death were investigated in Madin-Darby canine kidney (MDCK) cells injured by depletion of adenosine triphosphate (ATP). Glucose free incubation for three hours with a mitochondrial uncoupler resulted in progressive loss of glycine from cells. However, they were not lethally injured unless a perturbation of Ca++ homeostasis was also induced. Exposure to a Ca++ ionophore and uncoupler in 1.25 mM Ca++ medium (+Ca) resulted in accelerated cell death. ATP depleted cells with ionophore in 100 nM Ca++ medium (-Ca) were also lethally injured, but after a significant delay. Depletion of glycine preceded death in both groups of cells. Exogenous glycine (5 mM) protected +Ca cells against lethal membrane damage, but the beneficial effects were lost over a period of time. In contrast, -Ca cells were completely protected throughout. Phospholipid mass and radioactive label in lipid fractions of cells prelabeled with 3H-oleic acid were measured. Accelerated death of +Ca cells was accompanied by large decreases of phospholipid mass, loss of phospholipid label, and accumulation of unesterified labeled fatty acid. These changes were greatly decreased by incubation in -Ca medium. On the other hand, protection by glycine could not be attributed to modifications of either the massive breakdown of phospholipids that occurred in +Ca cells, or the modest changes seen in -Ca cells. In +Ca cells, the deleterious effects of increased Caf and phospholipid breakdown ultimately prevailed over protection by the amino acid. Thus, separate pathways of cell death associated with increased Caf and decreased glycine were defined in ATP depleted, Ca(+)- permeabilized MDCK cells. Calcium excess and massive phospholipid loss are features of a damage process that occurs independently of whether cells are protected by glycine or not. Conversely, the glycine sensitive component of injury is expressed regardless of whether intracellular Ca++ is increased, or large phospholipid losses occur. ATP depletion in -Ca medium provides a system to study mechanisms of glycine cytoprotection uncomplicated by Ca++ toxicity.

Adenosine Triphosphate↗
PubMed1995

CT diagnosis of unsuspected traumatic cataracts in patients with complicated eye injuries: significance of attenuation value of the lens.

OBJECTIVE: The purpose of this study was to determine whether the CT attenuation value of the lens is helpful in diagnosing acute traumatic cataracts in patients with complicated eye injuries that preclude evaluation by any other means. SUBJECTS AND METHODS: The CT attenuation values of the lenses of 69 patients with acute orbital trauma, including 24 patients with clinically and surgically diagnosed acute traumatic cataracts, were compared with attenuation values of their own contralateral lens and with the attenuation values of the lenses of 103 consecutive asymptomatic control subjects. The study group was composed of consecutive patients with unilateral orbital trauma who were clinically evaluated and referred for CT examination within 48 hr after their initial injury. In all patients, attenuation measurements of the injured lenses were obtained and compared with those of the contralateral lens as an internal control. All surgically diagnosed cataracts were histologically confirmed. RESULTS: The CT attenuation measurement of a lens in any asymptomatic control subject was identical (within the range of the standard deviation) to the measurement of the contralateral lens of that control patient. In patients with orbital injury, the CT attenuation of the patient's cataractous lens was markedly lower than in the contralateral lens (mean density difference, 30 H, p < .0001). This decreased attenuation corresponds to acute cataract formation with increased fluid within the lens. No patient with normal attenuation values of the lens in the traumatized globe (as compared with the contralateral lens) was found to have an acute traumatic cataract or have a cataract develop within a 1-year follow-up period. CONCLUSION: CT may be useful in the examination of patients with acute traumatic cataracts, unsuspected lens injury, opacification of the anterior chamber, or other injuries of the globe with complications that preclude lens evaluation by any other means. This prompt diagnosis may allow timely removal of the lens in appropriate clinical circumstances, preventing damage to the anterior chamber of the globe and other complications of delayed diagnosis and treatment. Further, normal CT findings at the time of trauma suggest that the lens will not undergo acute traumatic cataract formation.

Adolescent↗
PubMed1994

Calpain-induced down-regulation of protein kinase C inhibits dense-granule secretion in human platelets. Inhibition of platelet aggregation or calpain activity preserves protein kinase C and restores full secretion.

The relationship between platelet aggregation, calpain activation, PKC activities and the secretory response have been examined in PMA-and ionomycin-stimulated platelets. Co-addition of PMA and ionomycin resulted in a maximal synergistic secretion of [14C]5-hydroxytryptamine ([14C]5-HT) from platelet dense granules. However, prior addition of PMA for 5 or 10 min resulted in a reduction of this secretory response. Inclusion of either RGDS (to inhibit platelet aggregation) or E64-d (to inhibit calpain activity) resulted in full restoration of the secretory response. In experiments to determine the activity status of PKC, PMA was found to induce a loss in cytosolic and total PKC activity without an increase in membrane-associated activities during this time period. Inhibition of either platelet aggregation or calpain activity resulted in preservation of total and cytosolic activities with a measurable increase in membrane translocated activity. PMA-induced phosphorylation of a number of PKC substrates was measured in 32P-labelled platelets. PMA induced potent phosphorylation of the 45 and 20 kDa species and also proteins of the molecular masses 66, 80, 97 and 119 kDa. Phosphorylation was maximal at either 1 or 2 min after which dephosphorylation occurred. Inclusion of either RGDS or E64-d resulted in a reduction of the dephosphorylation rates, and sustained phosphorylation of the 66, 80, 97 and 119 kDa proteins. These studies suggest that the activity status of PKC is an important factor in the level of secretion obtained and that platelet aggregation is involved in calpain-initiated down-regulation of PKC.

Blood Platelets↗
PubMed1994

Purification of 14-3-3 protein and analysis of isoforms in chicken brain.

14-3-3 proteins are apparently ubiquitous eukaryotic proteins that comprise a large number of isoforms. We have used specific antibodies raised against each mammalian isoform to probe for 14-3-3 isoforms in adult hen brains. The results suggest that there is a remarkable degree of similarity in primary structure (at least in the regions containing the epitopes). Reverse-phase HPLC of the purified avian 14-3-3 proteins indicates a high overall degree of similarity in sequence and levels of expression of each isoform that are remarkably similar to their mammalian counterparts.

14-3-3 Proteins↗
PubMed1994

Mechanism of inhibition of protein kinase C by 14-3-3 isoforms. 14-3-3 isoforms do not have phospholipase A2 activity.

The ability of individual members of the 14-3-3 protein family to inhibit protein kinase C (PKC) has been studied by using a synthetic peptide based on the specific 80 kDa substrate for PKC (MARCKS protein) in two different assay systems. Recombinant 14-3-3 and isoforms renatured by a novel method after separation by reverse-phase h.p.l.c. were studied. The detailed effects of diacylglycerol and the phorbol ester phorbol 12-myristate 13-acetate on the inhibition were also investigated. This suggests that one of the sites of interaction of 14-3-3 may be the cysteine-rich (C1) domain in PKC. Since a region in secreted phospholipase A2 (PLA2) shares similarity with this domain, the ability of 14-3-3 to interact with mammalian PLA2 was studied. Cytosolic PLA2 has some similarity to the C2 region of PKC, and the effect of 14-3-3 on this class of PLA2 was also analysed. In contrast with a previous report, no PLA2 activity was found in brain 14-3-3, nor in any of the recombinant proteins tested. These include zeta 14-3-3 isoform, on which the original observation was made.

14-3-3 Proteins↗
PubMed1994

Specific binding sites for inositol 1,3,4,5-tetrakisphosphate are located predominantly in the plasma membranes of human platelets.

In the present study we describe the characterization and localization of Ins(1,3,4,5)P4-binding sites in human platelet membranes. Specific binding sites for Ins(1,3,4,5)P4 have been identified on mixed, plasma and intracellular membranes from neuraminidase-treated platelets using highly purified carrier-free [32P]Ins(1,3,4,5)P4. The displacement of Ins(1,3,4,5)P4 from these sites by Ins(1,4,5)P3 and InsP6 occurs at greater than two orders of magnitude higher concentrations and with Ins(1,3,4,5,6)P5 at about 40-fold higher concentrations than with Ins(1,3,4,5)P4. The membranes were further separated by free-flow electrophoresis into plasma and intracellular membranes. The Ins(1,3,4,5)P4-binding sites separated with plasma membranes, and showed similar affinities and specificities as mixed membranes, whereas Ins(1,4,5)P3-binding sites were predominantly in the intracellular membranes. These results suggest a predominantly plasma membrane location for putative Ins(1,3,4,5)P4 receptors in human platelets.

Animals↗
PubMed1994

Subcellular localisation of 14-3-3 isoforms in rat brain using specific antibodies.

The 14-3-3 protein family, which is present at particularly high concentrations in mammalian brain, is known to be involved in various cellular functions, including protein kinase C regulation and exocytosis. Despite the fact that most of the 14-3-3 proteins are cytosolic, a small but significant proportion of 14-3-3 in brain is tightly and selectively associated with some membranes. Using a panel of isoform-specific antisera we find that the epsilon, eta, gamma, beta, and zeta isoforms are all present in purified synaptic membranes but absent from mitochondrial and myelin membranes. In addition, the eta, epsilon, and gamma isoforms but not the beta and zeta isoforms are associated with isolated synaptic junctions. When different populations of synaptosomes were fractionated by a nonequilibrium Percoll gradient procedure, the epsilon and gamma isoforms were present and the beta and zeta isoforms were absent from the membranes of synaptosomes sedimenting in the more dense parts of the gradient. The finding that these proteins are associated with different populations of synaptic membranes suggests that they are selectively expressed in different classes of neurones and raises the possibility that some or all of them may influence neurotransmission by regulating exocytosis and/or phosphorylation.

14-3-3 Proteins↗
PubMed1994

Expression and function of FGF-4 in peri-implantation development in mouse embryos.

One of the earliest events in mammalian embryogenesis is the formation of the inner cell mass (ICM) and the subsequent delamination of primitive endoderm. We have found that mRNA for fibroblast growth factor (FGF)-4, but not FGF-3, is expressed in preimplantation mouse blastocysts and that the FGF-4 polypeptide is present in ICM cells. ICM-like embryonal carcinoma cells and embryonic stem cells also express FGF-4. Conversely, differentiated embryonal carcinoma cells in the endoderm lineage express FGF-3, but not FGF-4 mRNA. Although mouse embryos expressed FGF-4 mRNA from the 1-cell stage, embryos cultured from the 2-cell through the blastocyst stage in the presence of recombinant FGF-4 did not respond mitogenically. However, when ICMs that were isolated by immunosurgery were cultured with FGF-4, the number of morphologically distinct, differentiated parietal endoderm cells growing out onto the coverslip increased, without an increase in the number of undifferentiated ICM cells. ICM outgrowths cultured with FGF-4 increased their secretion of 92 x 10(3) M(r) gelatinase and tissue plasminogen activator, a hallmark of migrating cells. Receptors for FGF-4 (FGFR-3 and FGFR-4) are expressed in all cells of the mouse blastocyst. These findings indicate that FGF-4 produced by undifferentiated ICM cells acts in the peri-implantation period of embryogenesis to influence the production and behavior of endoderm cells derived from them.

Animals↗
PubMed1993

Antibodies against the major brain isoforms of 14-3-3 protein. An antibody specific for the N-acetylated amino-terminus of a protein.

14-3-3 proteins are apparently ubiquitous eukaryotic proteins that comprise a large number of isoforms. They have been implicated in the regulation of a wide range of biological processes [reviewed in Aitken et al. (1992) Trends Biochem. Sci. 17, 498-501]. We have raised specific antibodies against each mammalian brain isoform of 14-3-3 employing peptides synthesised from the amino-terminal regions. The peptides were, like the proteins from mammalian brain, N-acetylated. The antiserum specific for the epsilon isoform did not recognise the recombinant form of this protein (lacking the N-acetyl co-translational modification) expressed in E. coli until it was chemically acetylated.

14-3-3 Proteins↗
PubMed1993

Ca2+ release from platelet intracellular stores by thapsigargin and 2,5-di-(t-butyl)-1,4-benzohydroquinone: relationship to Ca2+ pools and relevance in platelet activation.

The effects of the Ca(2+)-ATPase inhibitors thapsigargin (Tg) and 2,5-di-(t-butyl)-1,4-benzohydroquinone (tBuBHQ) were examined by using Ca(2+)-regulatory systems of platelet mixed membranes, saponin-permeabilized and intact platelets. Both agents inhibit Ca(2+)-ATPase activities of platelet mixed membranes, without any effect on the basal Mg(2+)-ATPase activity. Tg is more effective (EC50 = 35 nM) than tBuBHQ (EC50 = 580 nM). The effect of the two inhibitors on 45Ca2+ release from saponin-permeabilized platelets has also been characterized. 45Ca2+ uptake into non-mitochondrial intracellular stores occurs via an ATP-dependent mechanism, and if added at equilibrium the second messenger Ins(1,4,5)P3 releases 50% of the accumulated 45Ca2+. Maximally effective concentrations of Tg (1 microM) and tBuBHQ (50 microM) release 77% and 68% of the accumulated 45Ca2+. Addition of Ins(1,4,5)P3 together with either Tg or tBuBHQ resulted in a non-additive release which was the same as with either Tg or tBuBHQ alone, indicating that the Ins(1,4,5)P3-sensitive Ca2+ pool was a subset of the pool that is sensitive to the Ca(2+)-ATPase inhibitors. Release of 45Ca2+ by either Tg or tBuBHQ was not affected by heparin, which totally blocked Ins(1,4,5)P3-induced Ca2+ release, and Tg was found not to affect [32P]Ins(1,4,5)P3 binding to its receptor on mixed membranes. Thus both Tg and tBuBHQ release Ca2+ from a pool that totally overlaps the Ins(1,4,5)P3-sensitive pool without affecting Ins(1,4,5)P3 function. In intact indomethacin-treated Fura 2-loaded platelets, Tg and tBuBHQ cause Ca2+ elevation, arising from release from intracellular stores and influx from the outside. Both Tg and tBuBHQ elevated Ca2+ to similar levels, which were less and slower than those observed with thrombin. Addition of thrombin to cells already treated with Tg or tBuBHQ produced further elevation of Ca2+, indicating agonist utilization of a Ca(2+)-ATPase inhibitor-insensitive pool. In aggregation experiments Tg and tBuBHQ showed different functional effects. In indomethacin-treated cells Tg induces slow aggregation and secretion responses, whereas tBuBHQ only induces shape change. Both agents show synergistic secretory responses with the protein kinase C activator dioctanoylglycerol (DiC8). Tg also showed greater ability than tBuBHQ to release [3H]arachidonic acid (AA) from [3H]AA-labelled platelets. Additionally, in [32P]Pi-labelled platelets both Tg and tBuBHQ induced phosphorylation of myosin light chain, a 27 kDa protein and the 45 kDa protein pleckstrin, but Tg showed a greater ability than tBuBHQ to cause phosphorylation of pleckstrin. These studies indicate that Tg and tBuBHQ are effective in releasing the Ins(1,4,5)P3-sensitive Ca2+ pool in platelets.(ABSTRACT TRUNCATED AT 400 WORDS)

Binding Sites↗
PubMed1992

Novel isoforms of CaBP 33/37 (annexin V) from mammalian brain: structural and phosphorylation differences that suggest distinct biological roles.

Two calcium-dependent phospholipid- and membrane-binding proteins have been purified from bovine brain. These are termed CaBP33 and CaBP37. Complete sequence analysis has revealed that these two proteins are isoforms of annexin V. Despite an apparent difference of 4 kDa between the two proteins on SDS-PAGE, only two amino-acid substitutions were found. These are, in CaBP33, Ser-36 and Lys-125 and in CaBP37, Thr-36 and Glu-125. This corresponds to a mass difference of 15 Da. This was confirmed by electrospray mass spectrometric analysis. Both isoforms can be phosphorylated substoichiometrically in vitro by protein kinase C at residue Thr-22.

Amino Acid Sequence↗
PubMed1992

Multiple isoforms of a protein kinase C inhibitor (KCIP-1/14-3-3) from sheep brain. Amino acid sequence of phosphorylated forms.

A potent inhibitor of protein kinase C (PKC), inhibitor protein-1 (KCIP-1), isolated from sheep brain has been shown to consist of eight isoforms by reverse-phase HPLC. Direct protein sequence analysis has revealed these to be the same as those of 14-3-3 protein, described as an activator of tyrosine and tryptophan hydroxylases involved in neurotransmitter biosynthesis. The N-termini of KCIP-1 isoforms were shown to be acetylated, and secondary structure predictions revealed a high degree of alpha-helix with an amphipathic nature. KCIP-1 showed no inhibitory activity towards protein kinase M (the catalytic fragment of PKC) and had no effect on the activities of three other protein kinases, cAMP-dependent protein kinase, Ca2+/calmodulin-dependent protein kinase II and casein kinase 2. Four forms of KCIP-1 were shown to be substrates for PKC in vitro, but none were phosphorylated by the other protein kinases mentioned above.

14-3-3 Proteins↗
PubMed1991

Identification of a gene, MLL, that spans the breakpoint in 11q23 translocations associated with human leukemias.

Recurring chromosomal translocations involving chromosome 11, band q23, have been observed in acute lymphoid leukemias and especially in acute myeloid leukemias. We recently showed that breakpoints in four 11q23 translocations, t(4;11)(q21;q23), t(6;11)(q27;q23), t(9;11)(p22;q23), and t(11;19)(q23;p13.3), were contained within a yeast artificial chromosome clone bearing the CD3D and CD3G gene loci. We have identified within the CD3 yeast artificial chromosome a transcription unit that spans the breakpoint junctions of the 4;11, 9;11, and 11;19 translocations, and we describe two other, related transcripts that are upregulated in the RS4;11 cell line. We have named this gene MLL (myeloid/lymphoid, or mixed-lineage, leukemia.

Adult↗
PubMed1991

Venous insufficiency is not the cause of leg ulcers in sickle cell disease.

Leg ulcers are a well recognized complication of sickle cell disease that has been attributed to venous insufficiency. We studied 16 patients with sickle cell disease and active ulcers using venous pulse volume recordings and photoplethysmography (Doppler studies). Based on hemodynamic monitoring, all 16 patients exhibited rapid refilling times, findings that imply venous insufficiency but are also compatible with high-output syndrome or arteriovenous shunting. Direct invasive venous pressure measurements of these patients demonstrated normal pressures in all of the four patients tested. A different set of four patients underwent venography, which also failed to demonstrate venous incompetence. We hypothesize that anemia results in an increase in peripheral arteriovenous shunting in the extremities and that this, together with the high-output syndrome of sickle cell disease, produces plethysmography readings that may be confused with findings observed in venous insufficiency. We conclude that measurements of vascular stasis, as recorded by plethysmography, are usually misinterpreted in sickle cell disease. Normal manometric pressure readings and normal venographic studies suggest that venous insufficiency is not a primary factor in sickle cell leg ulcer formation.

Blood Pressure↗