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N A Sharkey

Publications and source records attributed to N A Sharkey.

At least 73 records · Page 4Linked to original sources
PubMed1984

Mechanism of action of the phorbol ester tumor promoters: specific receptors for lipophilic ligands.

Cells and tissue preparations specifically bind the phorbol ester tumor promoters. The agreement in structure-activity relationships between binding and biological response strongly argues that these binding sites function as phorbol ester receptors. Upon subcellular fractionation, the phorbol ester binding activity is particulate. In addition, a phorbol ester apo-receptor can be detected in cytosol which requires phospholipids for reconstitution. This apo-receptor appears to correspond to protein kinase C. Diacylglycerols, the probable natural activators of protein kinase C, competitively inhibit phorbol ester binding, consistent with their being the postulated endogenous phorbol ester analogs. In certain systems, heterogeneity of phorbol ester binding is found. An outstanding issue therefore is whether protein kinase C is the phorbol ester receptor or whether it is only the most abundant class of receptor. Although this question remains unresolved, we can demonstrate heterogeneity of phorbol ester binding by reconstitution of apo-receptor into a heterogeneous lipid environment.

Animals↗
PubMed1984

Competitive inhibition by diacylglycerol of specific phorbol ester binding.

Emerging evidence suggests that the phorbol ester receptor in brain may be the same as the Ca2+-phospholipid-dependent protein kinase (protein kinase C). Since protein kinase C activity is stimulated by unsaturated diacylglycerol and the phorbol esters can substitute for diacylglycerol in this stimulation, we have examined the effect of diacylglycerols on phorbol ester binding. Assays were carried out with the mouse brain cytosolic phorbol ester aporeceptor, which requires phospholipids for activity. In the presence of phosphatidylserine at 0.96 mg/ml, diolein inhibited specific binding of [3H]phorbol 12,13-dibutyrate ( [3H]PBt2) in a dose-dependent fashion to less than 10% of control levels. The inhibition curves fit the curve expected for a competitive inhibitor and yielded a Ki of 3.6 +/- 0.8 micrograms/ml (n = 5) [0.38% (wt/wt) the concentration of phosphatidylserine]. Scatchard analysis confirmed the competitive nature of the inhibition. At constant phospholipid concentrations, the Ki determined for diolein was independent of the diolein concentrations over the range of 1.5-80 micrograms/ml, suggesting that the inhibition did not arise simply by perturbation of the phospholipid bilayers. The Ki of diolein was approximately proportional to the absolute phospholipid concentration. With phosphatidylserine at 4.8 micrograms/ml, for example, the Ki was 52 ng/ml (1.1% of phosphatidylserine). In addition to diolein, the short-chain saturated diacylglycerol derivatives dicaprylin and dicaproin also inhibited [3H]PBt2 binding, whereas the long-chain saturated derivatives dipalmitin and distearin were much less active. Our results suggest (i) that diacylglycerol may act as an endogenous ligand for the phorbol ester receptor and (ii) that variation in lipid composition provides a mechanism for modulating phorbol ester receptor affinity.

Animals↗
PubMed1984

Analysis of membrane and cytosolic phorbol ester receptors.

Specific phorbol ester receptors are found in the particulate fraction of cells. In addition, cytosol contains a phorbol ester apo-receptor, which requires phospholipids for reconstitution. The apo-receptor corresponds to protein kinase C, and the quantitatively major membrane receptor appears to be a protein kinase C-phospholipid complex. The ability to reconstitute the phorbol ester apo-receptor into different lipid domains now makes it possible to begin elucidation of the role of the lipid domain in phorbol ester action. Studies reviewed here indicate that diglycerides competitively inhibit phorbol ester binding, consistent with their being the postulated endogenous phorbol ester analogues. Highly lipophilic phorbol esters inhibit effectively only if they are incorporated into the lipid phase, indicating that it is the membrane-dissolved form of the ligand which is recognized. The binding affinity of 3H-phorbol 12,13-dibutyrate for holo-receptor depends markedly (greater than 20-fold range) on the phospholipid environment, and heterogeneous phorbol ester binding (i.e., curved Scatchard plots) can be generated by use of heterogeneous lipid environments in the reconstitution. The possible existence of other phorbol ester receptors in addition to protein kinase C-phospholipid complexes remains to be resolved.

Animals↗
PubMed1983

Phorbol ester receptors-insights into the initial events in the mechanism of action of the phorbol esters.

Specific phorbol ester receptors are found in the particulate fraction of cells. In addition, cytosol contains a phorbol ester apo-receptor, which requires phospholipids for reconstitution. The apo-receptor corresponds to protein kinase C, and the quantitatively major membrane receptor appears to be a protein kinase C-phospholipid complex. The ability to reconstitute the phorbol ester apo-receptor into different lipid domains permits analysis of the role of the lipid domain in phorbol ester receptor function. Studies reviewed here indicate that diacylglycerols competitively inhibit phorbol ester binding, consistent with their being the postulated endogenous phorbol ester analogs. Highly lipophilic phorbol esters only inhibit effectively if incorporated into the lipid phase, indicating that the membrane dissolved form of the ligand can be recognized. The binding affinity of [3H]phorbol 12,13-dibutyrate for holo-receptor depends markedly (greater than 20-fold range) on the phospholipid environment, and heterogeneous phorbol ester binding (i.e., curved Scatchard plots) can be generated by use of heterogeneous lipid environments in the reconstitution. The possible existence of other phorbol ester receptors in addition to protein kinase C-phospholipid complexes remains to be resolved.

Animals↗
PubMed1981

Differential cytotoxicity and DNA cross-linking in normal and transformed human fibroblasts treated with cis-diamminedichloroplatinum(II).

DNA interstrand cross-linking had been found previously to correlate with differences in sensitivity among human cell strains treated with chloroethylnitrosoureas. These differences had been attributed to the presence or absence of a specific DNA repair mechanism. The current work addressed the question of whether another DNA cross-linking agent, cis-diamminedichloroplatinum(II) (cis-Pt), would exhibit analogous differences between cell types. A normal human embryo cell strain (IMR-90) was compared with an SV40-transformed line (VA-13). Interstrand cross-linking and DNA-protein cross-linking were assayed by alkaline elution. As in the case of chloroethylnitrosoureas, the cytotoxicity differences with cis-Pt correlated with differences in interstrand cross-linking. The relative sensitivity of the cell lines to cis-Pt, however, was reversed. Similar DNA-protein cross-linking levels in the two cell lines excluded a difference in cis-Pt uptake or intracellular metabolic drug activation or inactivation prior to DNA interaction. It was concluded that the DNA repair mechanism that prevents interstrand cross-linking by chloroethylnitrosoureas does not prevent interstrand cross-linking by cis-Pt. Interstrand cross-linking by cis-Pt may be prevented by an independent mechanism.

Cell Division↗
PubMed1981

DNA cross-linking and cytotoxicity induced by cis-diamminedichloroplatinum(II) in human normal and tumor cell lines.

We have studied the cytotoxicity and DNA cross-linking induced by cis-diamminedichloroplatinum(II) (cis-Pt) in a normal, a simian virus 40-transformed, and eight tumor cell lines of human origin. These cell strains were chosen on the basis of their Mer phenotype, i.e., their ability to repair DNA containing 6O-alkylated guanine. The cytotoxicity was assayed by measuring the inhibition of cell proliferation after a two-hr treatment of the cell cultures with cis-Pt concentrations ranging from 1 to 50 microM. DNA-protein cross-links and DNA interstrand cross-links were measured by DNA alkaline elution after a two-hr treatment with 10 or 20 microM cis-Pt. The different cell lines differed in sensitivity to cis-Pt. The drug concentration required to produce the same inhibition of growth varied over a 10-fold range. Cell sensitivity correlated with DNA interstrand cross-linking (r = 0.83; p less than 0.01) better than with DNA-protein cross-linking (r = 0.75; p less than 0.05). There was no correlation between cell sensitivity or DNA interstrand cross-linking and the Mer phenotype. The lack of relationship between the extent of DNA interstrand cross-linking by cis-Pt and the Mer phenotype suggests that this repair mechanism does not remove from DNA cis-Pt monoadducts that have the potential to form interstrand cross-links.

Cell Division↗
PubMed1980

DNA cross-linking and monoadduct repair in nitrosourea-treated human tumour cells.

The 1-(2-chloroethyl)-1-nitrosoureas are potent anti-cancer drugs which produce DNA inter-strand cross-links in a two-step reaction sequence. The first step was proposed to be an addition of a chloroethyl group to a guanine-O6 position of DNA; the second step, which occurs over a period of several hours in the absence of free drug, could then form an interstrand cross-link by the slow reaction of the bound chloroethyl group with a nucleophilic site on the opposite DNA strand. The delay between the formation of chloroethyl monoadducts and the formation of inter-strand cross-links allows time for a DNA repair mechanism, capable of removing the monoadducts, to prevent the cross-linking. We recently proposed this mechanism to account for a difference in inter-strand cross-linking between a normal and a transformed human cell strain. Day and his coworkers (see refs 7, 8 and previous paper) found that some human tumour cell strains (designated Mer- phenotype) are deficient in the ability to repair O6-methylguanine lesions in DNA. We therefore hypothesized that the repair function that removes O6-methylguanine residues from DNA would also remove chloroethyl monoadducts and hence prevent chloroethylnitrosourea-induced inter-strand cross-linking. We now present evidence that supports this hypothesis and indicates also that the O6-methylguanine repair confers resistance to cell killing by chloroethylnitrosourea.

Cell Line↗
PubMed1979

Mutagenicity, cytotoxicity and DNA crosslinking in V79 Chinese hamster cells treated with cis- and trans-Pt(II) diamminedichloride.

The mutagenicity and cytotoxicity of cis- and trans-Pt(II) diamminedichloride (PDD) were examined in V79 Chinese hamster lung cells and compared with effects on DNA measured by alkaline elution. DNA--protein crosslinks and DNA interstrand crosslinks were detected following doses of cis-PDD which reduced cell survival 80--90% and which produced a mutant frequency of 3 X 10(-4) at the HGPRT locus. Equitoxic doses of trans-PDD were much less mutagenic than cis-PDD. At equitoxic doses, trans-PDD produced more DNA-protein crosslinking than did cis-PDD, but interstrand crosslinking for the two isomers was comparable. Hence, the interstrand crosslink could be the cytotoxic lesion produced by these Pt compounds whereas neither of these DNA lesions are necessarily mutagenic. The mutagenesis produced by cis-PDD could be due to crosslinks of a different type than those produced by trans-PDD or it may be due to monofunctional damage.

Animals↗
PubMed1977

Differential cytotoxicity between transformed and normal human cells with combinations of aminonucleoside and hydroxyurea.

Normal human WI-38 cells can be protected from killing by hydroxyurea if proliferation is arrested during drug treatment. This protection was demonstrated both in cells arrested by density-dependent inhibition and by 3'-amino-3'deoxy-N6,N6-dimethyladenosine (puromycin aminonucleoside). In contrast, VA-13 cells (a simian virus 40-transformed clone of WI-38) were not arrested under these conditions, and continued to be sensitive to hydroxyurea. These results suggest that a search for agents that selectively and reversibly inhibit normal cycling human cells might lead to an enhancement of differential tumor toxicity.

Cell Division↗
PubMedDate not supplied

Blood distribution of nickel, cobalt, and chromium following intramuscular injection into hamsters.

In vivo experiments were undertaken to study the transport of metal ions away from the intramuscular (IM) injection site and to study the binding of the metals to blood cells. Hamsters were injected intramuscularly with metal salts or with corrosion products generated by fretting corrosion of 316 LVM stainless steel or MP-35N plates and screws. The animals were bled at 0 time, and 2, 4, 6, 24, 48, and 96 h after injection. The concentration of nickel, cobalt, and chromium in the serum, attached to red cells, and attached to white cells was determined. This study showed that metals are rapidly transported from the intramuscular site with high levels in the blood by 2 h. The level of metal in the blood varied considerably with nickel being transported in high concentration to the blood, chromium with a valence of 6+ being transported to the blood, and cobalt and chromium with a valence of 3+ being transported less to the blood. The highest amount of cell binding was observed with chromium 6+. Cobalt showed negligible binding to blood cells. When all the metal salts injected together were compared with the individual salts injected alone, there was no difference. This indicated that the each metal behaves independently of the presence of the others.

Animals↗
PubMedDate not supplied

The binding of metal salts and corrosion products to cells and proteins in vitro.

The binding of metal ions from salts and from corrosion products of 316 LVM stainless steel and MP-35 to blood cells and serum proteins was studied in vitro. In the first series of experiments, metal salts were added to whole blood and then the blood separated into red cells, white cells, and serum. Nickel from nickel chloride or corrosion products of stainless steel bound in very small quantities to blood cells. Cobalt from cobalt chloride bound to both red cells and white cells. Chromium from chromic chloride (Cr3+) bound to cells in very small quantities whereas chromium from potassium dichromate (Cr6+) and corrosion products showed very high to binding to red cells and some binding to white cells. In a second series of experiments the blood was separated into its components and then the metal salts were added and the binding pattern was identical. In a third series of experiments serum which had interacted with the metal salts or corrosion products was separated into its components by isoelectric focusing on polyacrylamide gels. Almost all of the metal, whatever the source, was detected in the albumin region of the gels indicating strong binding to albumin. These studies on the cell and protein binding of the metals help to explain the dissemination of corrosion products from the site of the implant and subsequent systemic responses by some individuals.

Blood Cells↗
PubMedDate not supplied

Biomechanical evaluation of a low anterior wall fracture: correlation with the CT subchondral arc.

OBJECTIVE: To measure the effect of a simulated low anterior wall fracture of the acetabulum on load transmission in the hip joint. DESIGN: We measured the contact areas and pressure between the acetabulum and the femoral head of cadaveric pelves in three different conditions: intact, with an operatively created fracture of the anterior wall, and after anatomic reduction and internal fixation of the fracture. SETTING: Hips were loaded in simulated single-limb stance. Pressure and area measurements were made with Fuji pressure-sensitive film. SPECIMENS: Seven hip joints in seven whole pelves were tested. INTERVENTION: Anterior wall fractures were anatomically reduced and fixed. MAIN OUTCOME MEASUREMENTS: Contact area, load, and mean and maximum pressures were measured. RESULTS: Anterior wall fractures in our specimens entered the hip joint an average of 9.7 millimeters from the vertex of the acetabulum, corresponding to a 45-degree roof arc measurement. Peripheral loading seen in the intact acetabulum was disrupted after fracture. The loading pattern was not restored to preinjury levels with anatomic reduction and fixation. There was no significant change in the contact area (p = 0.43), force (p = 0.06), or mean (p = 0.57) or maximum (p = 0.20) pressures in the superior aspect of the acetabulum after creation of the anterior wall fracture. CONCLUSIONS: These results differ from those of previous studies with posterior wall acetabulum fractures, where significant increases in force and mean and maximum pressures were noted in the superior acetabulum after fracture. The lack of significant increases in superior acetabular pressures is discussed in relation to the mean computed tomographic subchondral arc of approximately ten millimeters in our specimens.

Acetabulum↗
PubMedDate not supplied

Biomechanical consequences of anterior column fracture of the acetabulum.

OBJECTIVES: To measure biomechanical consequences of a high anterior column acetabular fracture. DESIGN: A benchtop biomechanical model using quasi-static loading of the hip joint in a simulated single-leg stance. Pressure-sensitive prescale (Fuji) film was used to determine hip joint loading parameters. PARTICIPANTS: Six cadaveric hemipelvi with one hip tested in each specimen. Three right and three left hips were tested. INTERVENTION: Creation of an anterior column fracture with anatomic reduction and fixation, followed by gap malreduction/fixation, and subsequently step malreduction/fixation. MAIN OUTCOME MEASUREMENTS: Contact pressure, contact area, and load distribution throughout the hip joint in each experimental condition. RESULTS: There were significant increases in load (p<0.01) and peak pressures (p<0.01) in the superior acetabular region after gap malreduction and in peak contact pressures after step malreduction (p<0.01) as compared with the intact acetabulum. Anatomic reduction was not associated with increased mean or peak contact pressures (in any region). CONCLUSIONS: Both step and gap malreductions of a high anterior column fracture resulted in significantly increased peak contact pressures in the superior acetabular region. These biomechanical data cannot be directly extrapolated to clinical applications, but these data suggest that anatomic reduction of anterior column fracture affords the best opportunity to restore contact pressures, contact area, and load distribution within the hip to levels similar to those seen in the intact acetabulum.

Acetabulum↗