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Biomedical subjects

N Salem

Publications and source records attributed to N Salem.

At least 19 recordsLinked to original sources
PubMed1997

Molecular species analysis of phospholipids.

The elucidation of phospholipid molecular species composition provides detailed structural information concerning various lipids and thus offers descriptions of crucial determinants of membrane physical and biological properties. Various methods differing in labor intensity, mode of separation and detection, type of calibration, as well as other factors, have been published. Thus precision and accuracy are expected to vary considerably between methods. Qualitative and quantitative aspects of different procedures for molecular species analysis of individual phospholipid classes are discussed in this review. Special emphasis has been given to the characterization of biological tissue samples.

Animals↗
PubMed1997

Effect of mutations in vesicle-associated membrane protein (VAMP) on the assembly of multimeric protein complexes.

The assembly of multimeric protein complexes that include vesicle-associated membrane protein 2 (VAMP-2) and the plasma membrane proteins syntaxin 1A and synaptosome-associated protein of 25 kDa (SNAP-25) are thought to reflect the biochemical correlates of synaptic vesicle targeting, priming, or fusion. Using a variety of protein-protein interaction assays and a series of deletion and point mutations, we have investigated the domains of VAMP-2 required for the formation of binary complexes with either syntaxin 1A or SNAP-25 and ternary complexes with both syntaxin 1A and SNAP-25. Deletions within the central conserved domain of VAMP-2 eliminated binding to either syntaxin 1A or both syntaxin 1A and SNAP-25. Although all of the deletion mutants were able to form ternary complexes, only some of these complexes were resistant to denaturation in sodium dodecyl sulfate. These results demonstrate that cooperative interactions result in the formation of at least two biochemically distinct classes of ternary complex. Two point mutations previously shown to have effects on the intracellular trafficking of VAMP-2 (M46A, reduced endocytosis and sorting to synaptic vesicles; N49A, enhanced sorting to synaptic vesicles) lie within a domain required for both syntaxin 1A and SNAP-25 binding. Syntaxin 1A and SNAP-25 binding was reduced by the M46A mutation and enhanced by the N49A mutation, suggesting that a correlation exists between the membrane-trafficking phenotype of the two VAMP-2 point mutants and their competence to form complexes with either syntaxin 1A or SNAP-25.

Animals↗
PubMed1997

Retinal and brain accretion of long-chain polyunsaturated fatty acids in developing felines: the effects of corn oil-based maternal diets.

A study was carried out in domestic felines to determine whether corn oil-based maternal diets are an adequate source of essential fatty acids to support normal accumulation of long-chain polyunsaturated fatty acids in the brains and retinas of offspring and whether these diets have any subsequent effect on visual function. Female domestic felines were acclimated to one of six different defined diets 1 mo before mating and maintained on the diets throughout pregnancy and lactation. Four diets contained only corn and hydrogenated coconut oils as their source of fat in ratios of 1:9, 3:7, 6:4, and 9:1, respectively. Two reference diets also contained the long-chain polyunsaturated fatty acids arachidonate (20:4n-6) and docosahexaenoate (22:6n-3). When the offspring were 8 wk old, electroretinograms were obtained and the a- and b-wave implicit times were determined. The results showed that animals raised in litters in which the maternal diets were devoid of 20:4n-6 and 22:6n-3 had an increase in a- and b-wave implicit times compared with the controls. In the rod outer segments and brains of these animals, there were lower amounts of 22:6n-3 and higher amounts of long-chain n-6 polyunsaturated fatty acids compared with control animals. These findings showed that although corn oil-based diets were capable of maintaining 20:4n-6 concentrations in the developing brain and retina, only those diets containing 22:6n-3 could support a high accumulation of docosahexaenoic acid in these tissues. Moreover, low amounts of 22:5n-6 in the brains of animals in all of the corn oil-diet groups suggested that young felines have a low biosynthetic capacity to produce this fatty acid or 22:6n-3. These findings suggest that in juvenile felines, maintenance of 22:6n-3 status in the nervous system is important for optimal retinal function.

Animals↗
PubMed1996

Cell lines expressing an acetylcholine release mechanism; correction of a release-deficient cell by mediatophore transfection.

Several neuronal and non-neuronal cell lines express a Ca(2+)-dependent mechanism of transmitter release that can be demonstrated after loading the cells with acetylcholine during culture. In contrast, a particular cell line, the neuroblastoma N18TG-2, was found to be deficient for release. We transfected N18TG-2 cells with a plasmid encoding Torpedo mediatophore, a protein able to translocate acetylcholine in response to calcium. The N18TG-2 cells expressed the Torpedo protein which reached their plasma membrane. At the same time, these cells acquired a Ca(2+)-dependent quantal release mechanism similar to the one naturally expressed by other cell lines. Hence, the presence of mediatophore in the plasma membrane seems essential for quantal release.

Acetylcholine↗
PubMed1996

High-performance liquid chromatography method with light-scattering detection for measurements of lipid class composition: analysis of brains from alcoholics.

A high-performance liquid chromatographic method with evaporative light-scattering detection was developed for the analysis of intact lipid classes in nervous tissue. The method had the ability to resolve plasmalogen-phosphatidyl-ethanolamine and diacyl-phosphatidylethanolamine along with other major phospholipid classes in a single run. This technique was employed for the investigation of the effects of chronic alcohol consumption on the membrane lipid class composition of human brains (alcoholics, n = 13; controls, n = 11). Measurements were performed on cholesterol, cerebrosides, sulfatides, phospholipids and sphingolipids in total lipid extracts of white matter, gray matter and cerebellar regions of human brains. No significant differences in the lipid class composition between the groups were observed.

Alcoholism↗
PubMed1996

Quantal acetylcholine release induced by mediatophore transfection.

Mediatophore is a protein of approximately 200 kDa able to translocate acetylcholine in response to calcium. It was purified from the presynaptic plasma membranes of the electric organ nerve terminals. Mediatophore is a homooligomer of a 16-kDa subunit, homologous to the proteolipid of V-ATPase. Cells of the N18TG-2 neuronal line are not able to produce quantal acetylcholine release. We show here that transfection of N18TG-2 cells with a plasmid encoding the mediatophore subunit restored calcium-dependent release. The essential feature of such a release was its quantal nature, similar to what is observed in situ in cholinergic synapses from which mediatophore was purified.

Acetylcholine↗
PubMed1996

Arachidonic and docosahexaenoic acids are biosynthesized from their 18-carbon precursors in human infants.

It is becoming clear that an adequate level of long-chain highly unsaturated fatty acids in the nervous system is required for optimal function and development; however, the ability of infants to biosynthesize long-chain fatty acids is unknown. This study explores the capacity of human infants to convert 18-carbon essential fatty acids to their elongated and desaturated forms, in vivo. A newly developed gas chromatography/negative chemical ionization/mass spectrometry method employing 2H-labeled essential fatty acids allowed assessment of this in vivo conversion with very high sensitivity and selectivity. Our results demonstrate that human infants have the capacity to convert dietary essential fatty acids administered enterally as 2H-labeled ethyl esters to their longer-chain derivatives, transport them to plasma, and incorporate them into membrane lipids. The in vivo conversion of linoleic acid (18:2n6) to arachidonic acid (20:4n6) is demonstrated in human beings. All elongases/desaturases necessary for the conversion of linolenic acid (18:3n3) to docosahexaenoic acid (22:6n3) are also active in the first week after birth. Although the absolute amounts of n-3 fatty acid metabolites accumulated in plasma are greater than those of the n-6 family, estimates of the endogenous pools of 18:2n6 and 18:3n3 indicate that n-6 fatty acid conversion rates are greater than those of the n-3 family. While these data clearly demonstrate the capability of infants to biosynthesize 22:6n3, a lipid that is required for optimal neural development, the amounts produced in vivo from 18:3n3 may be inadequate to support the 22:6n3 level observed in breast-fed infants.

Arachidonic Acid↗
PubMed1996

Artificial rearing of infant rats on milk formula deficient in n-3 essential fatty acids: a rapid method for the production of experimental n-3 deficiency.

Research into the function of docosahexaenoic acid (DHA; 22:6n-3), the predominant polyunsaturated fatty acid (PUFA) in the central nervous system (CNS), is often hindered by the difficulty in obtaining dramatic experimental decreases in DHA in the brain and retina of laboratory rats. In this study, the artificial rearing procedure, whereby infant rats are removed from their mothers, gastrostomized, and fed synthetic formula, was used in an attempt to produce rapid changes in CNS levels of DHA. Female rats were raised, from day 4-5 of life, on one of two formulas-one containing the essential fatty acids of both the n-6 and n-3 series in proportions approximately equal to those of rat milk, and the other containing high levels of 18:2n-6 but very little n-3 fatty acid. At weaning, both groups were given AIN-76A diets modified so that the PUFA content resembled that of the preweaning formula. At eight weeks of age, the n-3-deficient group exhibited decreases of more than 50% in total DHA content in the brain, accompanied by increases in arachidonic acid (AA) (20:4n-6) and, especially, docosapentaenoic acid (22:5n-6). Other artificially-reared rats were mated and their offspring were also maintained on the respective diets. In spite of the fact that they had been reared artificially, the rats mated successfully and reared litters with no obvious abnormalities. At both ten days of age and again at eight weeks, offspring of the n-3-deficient mothers exhibited decreases of more than 90% in total DHA content. Again, the long-chain n-6 PUFA increased proportionately so that total PUFA levels in the brain were not lower. As these differences are greater than those commonly reported, even after 2-3 generations of normal dietary deprivation in rodents, this procedure may be an important tool in the study of the effects of n-3 deficiency on neural development and, subsequently, of the function of DHA in nervous tissue.

Animals↗
PubMed1996

Essential fatty acid uptake and metabolism in the developing rodent brain.

Studies were carried out to determine whether the brain takes up and metabolizes essential fatty acids during early postnatal development in rodents. Rats and mice were dosed with deuterium-labeled linoleic and linolenic acids either by intraperitoneal injection or by gavage. Animals were killed at different times thereafter, and organs were removed. Brains, livers, and blood were analyzed by gas chromatography--negative-ion-mass spectrometry for labeled fatty acids. To determine whether fatty acids were present in the brain apart from cerebral blood, a subset of animals was exsanguinated by perfusion with buffered saline, and the brain was then fractionated into subcellular components. Results demonstrated that the brain took up both labeled essential fatty acids within 8 h from the time of dosing. There was on average a greater uptake of linolenic acid into the cerebellum than into the cerebral cortex during the first 8 d of life in rats. The amount of linoleic acid taken into either region was similar, however. Docosahexaenoic acid intermediates, 20:5n-3 and 22:5n-3, were also found labeled in the brain. Time-course labeling experiments indicated that these intermediates may be converted to 22:6n-3 within the brain. A rise of labeled 22:6n-3 in the brain at 24 h appeared to be due to uptake of this fatty acid from the blood. The amount of labeled 22:6n-3 in the brain continued to increase beyond 24 h, and this did not appear to be correlated with its blood concentration. These results suggest that, during development in the rodent, different regions within the brain may vary in their capacity to synthesize 22:6n-3, and this may be correlated with regional growth rates.

Administration, Oral↗
PubMed1996

Losses of arachidonic acid in rat liver after alcohol inhalation.

This paper presents an animal model of alcoholism in which rats were exposed to alcohol by inhalation and were fed a diet that simulated the poor diet of some alcoholics. It is hypothesized that some of the pathophysiological effects of alcohol are related to its effects on essential fatty acid metabolism and composition of vital organs. A diet that contains no 20- and 22-carbon essential fatty acids and has low levels of 18-carbon essential fatty acids was used as a dietary challenge. Addition of a second metabolic challenge, i.e., alcohol, led to loss of tissue polyunsaturates, particularly liver arachidonate. A method of cycling alcohol inhalation for 12 h/d was also presented, which was also shown to lower liver arachidonic acid content.

Administration, Inhalation↗
PubMed1996

The structure-activity relationship of lipoxygenase products of long-chain polyunsaturated fatty acids: effects on human platelet aggregation.

The effect of hydroperoxy and hydroxy derivatives of various fatty acids on human platelet aggregation was determined to delineate potencies and structure-activity function. In this regard, the 22-carbon n-3 fatty acids are the most potent inhibitors in comparison to the n-6 lipoxygenase derivatives. Submicromolar levels of the docosapentaenoic (22:5) and especially docosahexaenoic (22:6) n-3 hydroperoxy and hydroxy derivatives specifically antagonize the platelet aggregating effect to arachidonic acid (AA, 20:4n-6) but not that of ADP or collagen. Chain length (22-C > 20-C), double-bond position (n-3 > n-6), and double-bond number (6 > 5 > 4) influence the degree of inhibition of AA-induced aggregation of human platelets. Moreover, significant differences in potency were associated with specific structural aspects of 22:6n-3 lipoxygenase derivatives of 22:6n-3 as follows: functional group (OOH > OH) and positional isomer (14-OOH, 14-OH, 20-OOH > 11-OOH, 17-OOH > 10-OOH > 11-OH, 8-OOH, 7-OOH > 4-OOH).

Arachidonic Acid↗
PubMed1996

Docosahexaenoic acid block of neuronal voltage-gated K+ channels: subunit selective antagonism by zinc.

The omega-3 polyunsaturated fatty acid docosahexaenoic acid is highly enriched in neuronal membranes, and several studies suggest that DHA is critical for neuronal development. We have investigated the effects of exogenously applied DHA on voltage-gated K+ channels using patch-clamp techniques. DHA produced a concentration-dependent inhibition of the sustained outward current in isolated neocortical neurons. This blocking action was examined in more detail with two cloned neuronal K+ channels (Kv1.2 and Kv3.1a) expressed in mammalian fibroblasts. DHA produced a potent inhibition of depolarization-activated K+ currents from cells expressing these channels (Kd values, 1.8 +/- 0.1 muM and 690 +/- 60 nM, for Kv1.2 and Kv3.1a, respectively, at +40 mV). The DHA block of both channel types was rapidly reversed (approximately 2 sec) by bovine serum albumin, which binds the fatty acid. Micromolar concentrations of extracellular Zn2+ non-competitively antagonized DHA inhibition of Kv1.2 channels, whereas there was little effect on DHA block of Kv3.1a channels. Experiments with membrane patches from Kv1.2 transfected cells demonstrated that the DHA block occurred from the outside, suggesting that the fatty acid interacts directly with an external domain of the ion channel. DHA may serve as a local messenger molecule that selectively modulates the activity of certain voltage-gated K+ channels in a Zn2(+)-dependent fashion.

Animals↗
PubMed1996

Anandamide, an endogenous cannabinoid, inhibits Shaker-related voltage-gated K+ channels.

Anandamide has been identified in porcine brain as an endogenous cannabinoid receptor ligand and is believed to be a counterpart to the psychoactive component of marijuana, delta 9-tetrahydrocannabinol (delta 9-THC). Here we report that anandamide directly inhibits (IC50, 2.7 muM) Shaker-related Kv1.2 K+ channels that are found ubiquitously in the mammalian brain. Delta 9-THC also inhibited Kv1.2 channels with comparable potency (IC50, 2.4 muM), as did several N-acyl-ethanolamides with cannabinoid receptor binding activity. Potassium current inhibition occurred through a pertussis toxin-insensitive mechanism and was not prevented by the cannabinoid receptor antagonist SR141716A. Utilizing excised patches of Kv1.2 channel-rich membrane as a rapid and sensitive bioassay, we found that phospholipase D stimulated the release of an endogenous anandamide-like K+ channel blocker from rat brain slices. Structure-activity studies were consistent with the possibility that the released blocker was either anandamide or another N-acyl-ethanolamide.

Animals↗
PubMed1996

Evoked acetylcholine release expressed in neuroblastoma cells by transfection of mediatophore cDNA.

Transmitter release was elicited in two ways from cultured cells filled with acetylcholine: (a) in a biochemical assay by successive addition of a calcium ionophore and calcium and (b) electrophysiologically, by electrical stimulation of individual cells and real-time recording with an embryonic Xenopus myocyte. Glioma C6-Bu-1 cells were found to be competent for Ca(2+)-dependent and quantal release. In contrast, no release could be elicited from mouse neuroblastoma N18TG-2 cells. However, acetylcholine release could be restored when N18TG-2 cells were transfected with a plasmid coding for mediatophore. Mediatophore is a protein of nerve terminal membranes purified from the Torpedo electric organ on the basis of its acetylcholine-releasing capacity. The transfected N18TG-2 cells expressed Torpedo mediatophore in their plasma membrane. In response to an electrical stimulus, they generated in the myocyte evoked currents that were curare sensitive and calcium dependent and displayed, discrete amplitude levels, like in naturally occurring synapses.

Acetylcholine↗
PubMed1996

A gene responsible for a sensorineural nonsyndromic recessive deafness maps to chromosome 2p22-23.

The recessive mode of transmission accounts for approximately 75% of inherited non syndromic deafness cases. We have previously designed the conditions for linkage studies of this highly heterogeneous disorder [Guilford et al. (1994) Nature Genet. 6, 24-28]. Here, using a similar approach, we have studied the segregation of a gene responsible for congenital, profound and fully penetrant sensorineural deafness in a consanguineous family living in an isolated region of Lebanon. A maximum lod score of 8.03 (theta = 0.00) was detected with a new polymorphic marker, AFMa052yb5 (D2S2144). Observed recombinants and homozygosity mapping define a maximum interval of 2 cM for this gene, DFNB6, which lies between AFMb346ye5 (a new polymorphic marker) (D2S2303) and AFM254vc9 (D2S174) on chromosome 2p22-23.

Base Sequence↗