Biomedical subjects

N Sahyoun

Publications and source records attributed to N Sahyoun.

At least 37 records · Page 2Linked to original sources

PubMed1990

Sequence analysis and DNA-protein interactions within the 5' flanking region of the Ca2+/calmodulin-dependent protein kinase II alpha-subunit gene.

The 5' flanking region of the brain Ca2+/calmodulin-dependent protein kinase II alpha-subunit gene was identified and characterized. A total of 430 bases was sequenced upstream from the translation initiation codon, and the site of transcription initiation was located at -149 or -147 bases as determined by primer extension and S1 nuclease protection analysis, respectively. TATA and CAAT boxes were absent from their standard positions; however, the 5' flanking region was rich in G + C and contained a GGGCG and a TATATAA sequence 76 and 160 bases upstream from the transcription initiation site, respectively. Moreover, the sequence CAACGG was found 85 and 146 bases upstream from this site, indicating presumptive binding sites for the Myb protein. Gel-mobility shift assays revealed that a 120-base-pair fragment, which included the G + C-rich, TATA, and CAACGG sequences bound nuclear proteins specifically. DNA-protein complexes with similar gel mobilities were obtained with nuclear extracts from rat forebrain or cerebellum and from neonatal or adult brains. Extracts from rat liver, kidney, and spleen generated specific DNA-protein complexes with different electrophoretic mobilities, suggesting the occurrence of different nuclear proteins that bind to 5' regulatory elements of the Ca2+/calmodulin-dependent kinase II alpha-subunit gene.

Amino Acid Sequence↗

PubMed1989

Effect of vitamin D intake on seasonal variations in parathyroid hormone secretion in postmenopausal women.

Vitamin D intake should be sufficient to maintain calcium absorption and prevent increased parathyroid secretion throughout the year. To determine the level of intake that achieved the latter in elderly women, we studied the interrelations among vitamin D intake, serum 25-hydroxyvitamin D (25(OH)D) levels, and parathyroid hormone concentrations in a cross-sectional study of 333 healthy, white, postmenopausal women with low median calcium (408 mg a day) and vitamin D (112 IU a day) intakes who lived in Massachusetts. The overall inverse relation between serum parathyroid hormone and 25(OH)D levels was found to be dependent on vitamin D intake. In women whose estimated intake of vitamin D was less than or equal to 220 IU a day, the mean (+/- SD) serum parathyroid hormone values were lowest in those studied between August and October (30 +/- 11 ng per liter; n = 72) and highest in those studied between March and May (37 +/- 16 ng per liter; n = 54); the respective serum 25(OH)D levels were 93 +/- 32 and 63 +/- 21 nmol per liter. At vitamin D intakes of more than 220 IU a day, the mean serum parathyroid hormone and 25(OH)D levels did not vary with the season. The correlation between vitamin D intake and serum 25(OH)D concentration, although significant in all women (r = 0.29; P less than 0.001), was highest in those studied between March and May (r = 0.65; P less than 0.001) and lowest in those studied between August and October (r = 0.13; P greater than 0.10). The estimated serum 25(OH)D level associated with a vitamin D intake of 220 IU a day between March and May was 95 nmol per liter. Mean serum calcium values were similar at all times in both groups. We conclude that the dietary intake of more than 220 IU of vitamin D a day by postmenopausal women in Massachusetts may be sufficient to maintain constant serum 25(OH)D and parathyroid hormone concentrations throughout the year. Such an intake prevents a seasonal increase in parathyroid hormone secretion, with its possible deleterious skeletal effects.

Calcium↗

PubMed1988

The growth cone cytoskeleton. Glycoprotein association, calmodulin binding, and tyrosine/serine phosphorylation of tubulin.

Cytoskeletons were prepared from the growth cones of neonatal rat forebrains and were utilized to explore several aspects of growth cone function. The cytoskeletal fraction retained about 50% of total growth cone protein, was highly enriched in tubulin, and constituted an interconnected lattice of 10-25 nm homogeneous particles. The cytoskeleton appeared to be a target for Ca2+ signaling since it contained the majority of growth cone calmodulin-binding polypeptides which featured prominently an Mr 135,000 component. Most of the growth cone glycoproteins were at least partially associated with the cytoskeleton, thus suggesting the possibility of a transmembrane coupling mechanism which allows for communication between the cytoskeleton and the external surface of the growth cone. The cytoskeleton was also endowed with one or more protein kinases which phosphorylated endogenous and exogenous tubulin achieving a stoichiometry of 9-13 mol of phosphate/mol of substrate dimer. Interestingly, the site of tubulin phosphorylation included tyrosine as well as serine residues providing a possible target for the action of neuronal tyrosine kinases such as growth factor receptors or oncogene products. Finally, a comparison between cytoskeletal preparations from growth cones and from mature synaptosomes revealed several differences in glycoprotein association, calmodulin binding, and protein phosphorylation, evidently reflecting maturational events which might underlie relevant aspects of synaptogenesis.

Animals↗

PubMed1988

Nutrient supplement use by healthy elderly.

As part of a nutritional status survey of 691 non-institutionalized men and women aged 60 years and older, supplement use was reported by 45% of the males and 55% of the females. Supplement use was more prevalent in females than males at each age decade. Vitamins C and E were the most commonly used supplements. Mean dietary nutrient intakes were calculated from a 3-day food diary. The percentage of dietary intakes falling below 2/3 1980 Recommended Dietary Allowance (RDA) was comparable for users and non-users of supplements. Use of supplements markedly decreased the proportion of subjects with inadequate nutrient intake (using a 2/3 RDA criterion), particularly for vitamins B6, B12, and D, folic acid, and calcium. However, for both males and females, potentially excessive intake levels (10 times the RDA) of thiamin, vitamin A, and vitamin E supplementation were observed.

Aged↗

PubMed1987

Protein kinase C binding to isolated nuclei and its activation by a Ca2+/phospholipid-independent mechanism.

The direct interaction of protein kinase C with the nucleus was examined utilizing endogenous protein phosphorylation and [3H]PDBu binding to detect the enzyme. Rat brain nuclei were relatively rich in phorbol ester receptors whereas liver nuclei contained less than 10% of their brain counterpart. Purified protein kinase C from rat brain could bind to purified rat liver nuclei at 4 degrees C or at 24 degrees C reaching apparent equilibrium by 20 min. The binding was linearly dependent on protein kinase C concentration and required free Ca2+ with an EC50 of 0.5 microM. Chelation of Ca2+ with EGTA resulted in rapid loss of phorbol ester receptors from nuclei. Differential extraction experiments with Triton X-100 and NaCl suggested that about 50% of the acquired phorbol ester receptors were bound to chromatin and 25% were associated with the nuclear matrix. Protein Kinase C bound to nuclei was also able to phosphorylate several endogenous nuclear substrates in a Ca2+/phospholipid-independent reaction. These data suggest that protein kinase C can associate with nuclear components leading to the phosphorylation of nuclear substrates.

Animals↗

PubMed1987

Characterization of a neuronal subtype of insulin-like growth factor I receptor.

Primary neuronal cultures from fetal rat brain were utilized to investigate the possible role of insulin-like growth factor I (IGF-I) in neuronal growth and differentiation. 125I-IGF-I binding to intact cultured neurons was specific and saturable with an apparent Kd of 7.0 +/- 1.2 nM and a Bmax of 1.8 +/- 0.3 pmol/mg protein. Binding of 125I-IGF-I to neurons was inhibited by IGF-I, followed by IGF-II and insulin. 7 S nerve growth factor, but not beta-nerve growth factor, also inhibited 125I-IGF-I binding. A similar binding site was detected on brain membranes. Affinity cross-linking of 125I-IGF-I to intact cultured neurons revealed, under reducing conditions, a major binding moiety with an Mr of 115,000 and a minor component at Mr 260,000. The former represents a neuronal type of the IGF-I receptor alpha subunit, whereas the latter probably represents an alpha dimer. The Mr = 115,000 binding component for 125I-IGF-I was also present in membranes prepared from postnatal whole brain. In contrast, the binding moiety in cultured glial cells was of Mr = 135,000, which was identical to the IGF-I receptor alpha subunit of placenta. Thus mature brain, despite its cellular heterogeneity, expresses a structural subtype of IGF-I receptor which appears to be unique to differentiated neurons. Moreover, glial and neuronal cultures secreted a polypeptide which specifically bound IGF-I; the apparent Mr of this binding protein was determined by affinity cross-linking to be approximately 35,000. The presence of neuronal IGF-I receptors and binding proteins suggested that IGF-I may exert neurotrophic effects on developing neurons. This possibility was supported by the observation that IGF-I markedly stimulated neuronal RNA synthesis.

Animals↗

PubMed1987

Purification of phosphatidylinositol kinase from bovine brain myelin.

A membrane-bound phosphatidylinositol (PI) kinase (EC 2.7.1.67) was purified by affinity chromatography from bovine brain myelin. This enzyme activity was solubilized with non-ionic detergent and chromatographed on an anion-exchange column. Further purification was achieved by affinity chromatography on PI covalently coupled to epoxy-activated Sepharose, which was eluted with a combination of PI and detergent. The final step in the purification was by gel filtration on an Ultrogel AcA44 column. This procedure afforded greater than 5500-fold purification of the enzyme from whole brain myelin. The resulting activity exhibited a major silver-stained band on SDS/polyacrylamide-gel electrophoresis with an apparent Mr 45,000. The identity of this band as PI kinase was corroborated by demonstration of enzyme activity in the gel region corresponding to that of the stained protein. The purified enzyme exhibited a non-linear dependence on PI as substrate, with two apparent kinetic components. The lower-affinity component exhibited a Km similar to that observed for the phosphorylation of phosphatidylinositol 4-phosphate by the enzyme.

1-Phosphatidylinositol 4-Kinase↗

PubMed1986

Postsynaptic densities contain a subtype of protein kinase C.

Protein kinase C or an isoenzyme thereof appears to be a significant component of postsynaptic densities (PSDs) from rat brain. This cytoskeletal organelle binds 4 beta-phorbol 12,13-dibutyrate (PDBu) with a Bmax of about 20 pmol/mg protein and an apparent Kd of 3.3 nM. Ca2+ and phosphatidyl serine (PS) stimulated the endogenous phosphorylation of a subset of PSD polypeptides with Mr values between 16,000 and 22,000. Finally, a monospecific protein kinase C antibody reacted with a Mr 70,000 PSD polypeptide which migrated on SDS-PAGE slightly ahead of the Mr 77,000 purified enzyme. These data suggest that protein kinase C or a similar enzyme can be integrated into a cytoskeletal system and may play an important role in postsynaptic function.

Animals↗

PubMed1986

Protein kinase C and phosphatidylserine bind to Mr 110,000/115,000 polypeptides enriched in cytoskeletal and postsynaptic density preparations.

The selective binding of protein kinase C to nitrocellulose-immobilized polypeptides from rat brain and human erythrocytes was investigated. Bound enzyme was detected immunochemically with a monospecific protein kinase C antibody, or by using radiolabeled enzyme. Two polypeptides from erythrocyte membranes with Mr values of 110,000 and 115,000 bound protein kinase C in the presence of phosphatidylserine (PS) and were highly enriched in the cytoskeletal fraction. A prominent protein kinase C-binding polypeptide at Mr about 115,000 was also evident in brain cytoplasm, postsynaptic densities, and nuclei. Overlays of electrophoretic blots with 14C-phospholipids revealed that the protein kinase C-binding polypeptides also bound PS but not other phospholipids. The binding of both protein kinase C and PS was markedly inhibited after phosphorylation of the Mr 110,000/115,000 polypeptides with the kinase itself. The relevance of the results to the binding of protein kinase C to membranes and to phospholipid-cytoskeletal interactions is discussed.

Animals↗

PubMed1986

Specific postsynaptic density proteins bind tubulin and calmodulin-dependent protein kinase type II.

Cytoskeletal interactions which contribute to the assembly of the postsynaptic density (PSD) were investigated. PSDs bound 125I-tubulin specifically with an apparent Km of 2 X 10(-7) M and a Bmax of about 1 nmol/mg of protein. 125I-Tubulin blots revealed that a group of polypeptides between Mr 135,000 and 147,000 (P-140) was a major tubulin-binding PSD component. The P-140 polypeptides were highly enriched in the PSD fraction of purified synaptosomes and could not be detected in crude brain cytoplasm preparations. These polypeptides were subject to phosphorylation by endogenous calmodulin-dependent protein kinase type II, with a concomitant reduction in 125I-tubulin binding. The tubulin-binding polypeptides could also associate with the radiolabeled alpha- and beta-subunits of the calmodulin-dependent protein kinase. These observations are consistent with a role for the P-140 polypeptides in organizing the molecular structure of the PSD. The data also suggest that this structure may be modified by Ca2+-sensitive phosphorylation, thus permitting neuronal activity to modulate the cytoskeletal interactions of the PSD.

Animals↗

PubMed1986

Determination of calmodulin by competitive binding assay.

Calmodulin levels in tissue or cellular extracts can be determined by competition with 125I-calmodulin in a filtration-based direct binding assay. The method is rapid, uses readily available stable components, and possesses a selectivity and sensitivity comparable to that observed with immunoassay and phosphodiesterase activation. This assay provides a tool to readily probe changes in calmodulin levels in cells and tissues as a function of pathophysiologic state.

Animals↗

PubMed1986

Protein kinase C phosphorylates topoisomerase II: topoisomerase activation and its possible role in phorbol ester-induced differentiation of HL-60 cells.

DNA topoisomerase II from Drosophila was phosphorylated effectively by protein kinase C. With a Km of about 100 nM, the reaction was rapid, occurring at 4 degrees C as well as at 30 degrees C and requiring as little as 0.6 ng of the protein kinase per 170 ng of topoisomerase. About 0.85 mol of phosphate could be incorporated per mol of topoisomerase II, with phosphoserine as the only phospho amino acid produced. The reaction was dependent on Ca2+ and phosphatidylserine and was stimulated by phorbol esters. Calmodulin-dependent protein kinase II, but not cyclic AMP-dependent protein kinase, was also able to phosphorylate the topoisomerase. Phosphorylation of topoisomerase II by protein kinase C resulted in appreciable activation of the topoisomerase, suggesting that it may represent a possible target for the regulation of nuclear events by protein kinase C. This possibility is supported by the finding that the phorbol ester-induced differentiation of HL-60 cells was blocked by the topoisomerase II inhibitors novobiocin and 4'-(9-acridinylamino)methanesulfon-m-anisidide(m-AMSA), but not by the inactive analog o-AMSA.

Aminoacridines↗

PubMed1986

Phorbol ester receptors and protein kinase C in primary neuronal cultures: development and stimulation of endogenous phosphorylation.

Embryonic rat neurons cultured in defined medium, essentially in the absence of glia, were highly enriched in phorbol ester receptors. The neurons displayed a single class of phorbol 12,13-dibutyrate binding sites with a maximum binding capacity, after 10 d in culture, of 18.6 pmol/mg protein and an apparent dissociation constant of 7.1 nM. Phorbol ester binding sites were associated with protein kinase C, which represented a major protein kinase activity in primary neuronal cultures. Ca2+-phosphatidylserine-sensitive phosphorylation of endogenous substrates was more marked than that observed in the presence of cyclic AMP or Ca2+ and calmodulin. Phorbol ester receptors and protein kinase C levels were critically dependent on the culture age. Thus, about a 20-fold increase in binding sites occurred during the first week in culture and was accompanied by a corresponding increase in Ca2+-phosphatidylserine-sensitive protein phosphorylation in soluble neuronal extracts. These changes largely paralleled a similar rise in phorbol ester binding during fetal development in vivo. The apparent induction of phorbol ester receptors was specific relative to other cellular proteins and could be inhibited by cycloheximide or Actinomycin D. Phosphorylation of endogenous substrates in intact cultured neurons paralleled the age-dependent increase in protein kinase C. Furthermore, 32P incorporation into several major phosphoproteins was markedly augmented by treating the neuronal cultures with phorbol esters. Such phosphorylation events may provide a clue to the significance of protein kinase C in developing neurons.

Animals↗

PubMed1985

Interaction of protein kinase C with membranes is regulated by Ca2+, phorbol esters, and ATP.

Physiologic regulation of protein kinase C activity requires its interaction with cellular membranes. We have recently shown that binding of the enzyme to plasma membranes is controlled by Ca2+, whereas enzyme activators, like phorbol esters, regulate both membrane binding and enzyme activity. Here we describe the factors which control the dissociation of protein kinase C from the plasma membrane. In the absence of phorbol esters, the dissociation reaction is rapid and is determined by varying the Ca2+ concentration between 0.1 and 1 microM. However, the presence of 4-beta-phorbol 12,13-dibutyrate greatly reduces enzyme release in response to Ca2+ depletion; removal of the phorbol ester itself permits efficient membrane-enzyme dissociation. The stabilization of the membrane-protein kinase C complex by phorbol esters can be reversed by ATP with an apparent Km for the nucleotide of 6.5 microM. The ATP effect requires MgCl2 and cannot be reproduced by other nucleotides or by a nonhydrolyzable analogue, suggesting that an ATP-dependent phosphorylation reaction may be involved. 4-beta-Phorbol 12,13-dibutyrate appears to stabilize membrane-enzyme association by reducing the apparent Km for Ca2+ to about 15 nM, whereas ATP reverses the phorbol ester effect by increasing the Km for Ca2+ to about 760 nM. Furthermore, the strong degree of negative cooperativity displayed by the Ca2+-dependent enzyme-membrane dissociation is consistent with the presence of multiple interacting Ca2+-binding sites on protein kinase C.

Adenosine Triphosphate↗

PubMed1985

Competitive binding assay for drugs that interfere with calmodulin function.

A direct 125I-calmodulin-binding system utilizing a particulate cytoskeletal preparation of the neuronal calmodulin-dependent protein kinase type II provides a simple, rapid filtration-based method for assessing anti-calmodulin drug activity. The binding assay avoids potential artifacts due to direct drug effects on the standard phosphodiesterase enzyme assay while obtaining comparable IC50 values for a spectrum of drugs. It also provides a tool to probe mechanisms of action of putative anticalmodulin agents which may help elucidate selective pharmacologic modifications of pathophysiological processes.

Animals↗

PubMed1985

Early postnatal development of calmodulin-dependent protein kinase II in rat brain.

The postnatal levels of the alpha- (Mr 50,000) and beta- (Mr 58,000/60,000) subunits of type II calmodulin-dependent protein kinase were investigated in cytosolic, cytoskeletal and nuclear subfractions of rat brain. Substantial amounts of the beta-subunit were present neonatally, especially in the cytoplasm; both subunits increased markedly during the subsequent three weeks with alpha-preponderance in the cortex and midbrain. The development of the alpha-subunit was attenuated in the cerebellum and brain stem, resulting in a relative excess of the beta-subunit. Primary neuronal cultures from fetal brain initially displayed low enzyme content, followed by an increase in the levels of the beta- but not alpha-subunit. Regulation of the cellular content of type II calmodulin-dependent protein kinase may be relevant to neuronal differentiation.

Animals↗

PubMed1985

Endogenous dephosphorylation of synaptosomal calmodulin-dependent protein kinase type II.

Calmodulin-dependent protein kinase Type II autophosphorylation in synaptosomes is localized to the cytoskeleton (synaptic junction), while a potent dephosphorylating activity is present in the lipid bilayer. The dephosphorylating activity is operative in intact synaptosomes and in a reconstitution system comprised of the cytoskeletal and Triton X-100 - soluble fractions. Dephosphorylation is inhibited by EDTA and pyrophosphate, but not by EGTA or NaF. The present characterization of endogenous synaptosomal dephosphorylating activity completes the regulatory cycle operating on this enzyme in which phosphorylation of calmodulin-dependent protein kinase type II inhibits its response to Ca+2 and calmodulin.

Calcium↗

PubMed1985

Mechanism of phorbol diester-induced regulation of surface transferrin receptor involves the action of activated protein kinase C and an intact cytoskeleton.

Phorbol diesters are tumor-promoting agents that cause differentiation of HL60 human leukemic cells and concomitantly regulate surface transferrin receptors. Regulation of transferrin receptors by phorbol diesters involves receptor internalization in association with increased receptor phosphorylation (hyperphosphorylation). The intracellular mechanism of action of phorbol diester involves binding to and activation of the Ca2+-phospholipid-dependent protein kinase (protein kinase C). Present studies comparing results obtained with whole cells and those from a cell-free system reconstituted from purified protein kinase C and transferrin receptor components have revealed that the transferrin receptor is phosphorylated by protein kinase C activated by phorbol esters. Following tryptic digestion and two-dimensional separation of phosphopeptides of phosphorylated transferrin receptors, two major and several minor phosphoserine-containing fragments are resolved. These fragments are identical whether transferrin receptor is phosphorylated in whole cells incubated with phorbol diesters or following phosphorylation of affinity immobilized transferrin receptor in the in vitro reconstitution system. Phosphoamino acid analysis of these fragments indicates that serine is the only amino acid phosphorylated in whole cells or in the cell-free system. In addition, colchicine is shown to inhibit in a dose-dependent manner phorbol diester-induced internalization but not hyperphosphorylation of the surface transferrin receptor in whole cells. This inhibition is specific for colchicine since inactive beta- and gamma-Lumicolchicine have no such effect, while taxol reverses the inhibition. These results indicate that the phorbol diester-mediated process of down-regulation of the surface transferrin receptor is associated with phosphorylation of the receptor by activated protein kinase C and requires an intact cytoskeleton to affect receptor internalization.

Cell Line↗