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E J Neufeld

Publications and source records attributed to E J Neufeld.

61 records · Page 4Linked to original sources
PubMed1983

Arachidonate release and phosphatidic acid turnover in stimulated human platelets.

We have examined the temporal relationship between arachidonate release and phosphatidic acid (PA) metabolism in human platelets stimulated with thrombin. Within 1 min of stimulation at 37 degrees C, platelets released up to 16 nmol of arachidonate/10(9) cells from membrane phospholipids and exhibited an increase of severalfold in PA mass. At 23 degrees C, arachidonate release was half-maximal by 10-30 s, but PA remained near control levels for 10-30 s, indicating that increased PA is not necessary for release. [3H]Glycerol-labeled platelets synthesized phospholipids from [3H]PA at a rate of 0.08-0.3 nmol/min/10(9) cells at 37 degrees C. This rate of [3H]glycerol-PA turnover was not enhanced by thrombin stimulation; thus, PA did not turn over rapidly enough to be an intermediate in the release of several nanomoles of arachidonate from phosphatidylinositol in the first few seconds of stimulation. When aspirin-treated platelets were prelabeled with [14C]- or [3H]arachidonate, the specific activity of phospholipid-bound arachidonate pools remained constant after thrombin stimulation. Released arachidonate had specific activity intermediate to that in phosphatidylcholine and phosphatidylinositol. [14C]arachidonate in phosphatidylethanolamine had specific activity 6-fold less than released [14C]arachidonate; this phospholipid could therefore contribute only a small fraction of released fatty acid.

Arachidonic Acid↗
PubMed1983

High affinity esterification of eicosanoid precursor fatty acids by platelets.

We have examined the relative rates of uptake of several fatty acids into washed, human platelets by measuring incorporation into cellular phospholipids. In the presence of 15 microM fatty acid-free albumin and with radioactive fatty acid concentrations of 5-500 nM, esterification into phospholipid was linear with time and platelet concentration and saturable with respect to fatty acid concentration. Two distinct classes of uptake rate were observed. Arachidonate and 5,8,11,14,17-eicosapentaenoate exhibited high affinity, relatively rapid incorporation into platelet phospholipids at pH 6.5: apparent Michaelis constant (Km) = 30 nM, apparent maximum velocity (Vmax) = 28 pmol/min per 10(9) platelets. Two other eicosanoid precursors, 5,8,11-eicosatrienoate and 8,11,14-eicosatrienoate, exhibited the same Vmax, but Km of 85 and 60 nM, respectively. Under the same conditions, stearate, oleate, and linoleate were incorporated into phospholipids much less efficiently (Vmax approximately 8 pmol/10(9) cells per min, apparent Km greater than or equal to 170 nM). Qualitatively similar results were found at pH 7.4. Uptake of radiolabeled, rapid-uptake fatty acids was not diminished by the presence of excess, unlabeled, slow-uptake fatty acids. Thus, the specificity of this esterification system resembles that of the arachidonate-specific, long-chain acyl-CoA synthetase present in platelets. It may represent the expression in vivo of the synthetase, although the apparent affinity of the synthetase for fatty acid is much less. This esterification system probably represents the physiologic mechanism for platelet arachidonate uptake, whereby arachidonate is collected from plasma, despite the fact that its concentration is considerably lower than that of other plasma fatty acids.

8,11,14-Eicosatrienoic Acid↗
PubMed1980

Mobility, clustering, and transport of nerve growth factor in embryonal sensory cells and in a sympathetic neuronal cell line.

We have prepared a fluorescent conjugate of nerve growth factor (NGF) containing 8--10 rhodamine molecules attached to free carboxyl groups of the protein. This analogue retained full binding capacity toward NGF receptors, full antigenic properties, and the potency to stimulate the differentiation of embryonal chicken sensory ganglia cells in vitro. We have used this analogue to study the mobility and distribution of NGF receptors on embryonal chicken sensory cells from dorsal root ganglia and on a pheochromocytoma cell line (PC-12) that responds to NGF by differentiating along a neuronal pathway. The rhodamine conjugate of nerve growth factor (R-NGF) binds initially to diffusely distributed mobile receptors (D approximately 8 X 10(-10) cm2/sec) on immature sensory and PC-12 cells. At 37 degrees C, the NGF receptor complexes cluster and form immobile visible patches. These patches undergo endocytosis in a process that consumes metabolic energy. Methylamine blocks the formation of visible patches of NGF and the receptors remain dispersed and mobile at 37 degrees C. On differentiated chicken sensory cells, R-NGF binds to diffusely distributed mobile receptors and to aggregated immobile binding sites. These clusters are localized at the tip of the axon, along the axon, and in the main body. The NGF molecules that are internalized at the tip of the axon are transported retrogradely from the peripherey to the cell body.

Animals↗
PubMedDate not supplied

Thiamine-responsive megaloblastic anemia syndrome: a disorder of high-affinity thiamine transport.

Thiamine-responsive megaloblastic anemia (TRMA) syndrome (OMIM No. 249270) comprises a distinctive triad of clinical features: megaloblastic anemia with ringed sideroblasts, diabetes mellitus, and progressive sensorineural deafness. The TRMA gene has been mapped and cloned. Designated "SLC19A2" as a member of the solute carrier gene superfamily, this gene is mutated in all TRMA kindreds studied to date. The product of the SLC19A2 gene is a membrane protein which transports thiamine (vitamin B1) with sub-micromolar affinity. Cells from TRMA patients are uniquely sensitive to thiamine depletion to the nanomolar range, while pharmacologic doses of vitamin B1 ameliorate the anemia and diabetes. Here we review the current status of studies aimed at understanding the pathophysiology of this unique transport defect.

Anemia, Megaloblastic↗
PubMedDate not supplied

Characterization of a murine high-affinity thiamine transporter, Slc19a2.

Thiamine-responsive megaloblastic anemia with deafness and diabetes (TRMA) is a rare autosomal recessive disorder of thiamine transport. Previous studies have demonstrated that the disease is caused by mutations in the SLC19A2 gene encoding a high-affinity thiamine transporter. We hypothesize that thiamine transport, mediated by SLC19A2, plays a role in the development and or maintenance of several organ systems, in particular the erythropoietic, auditory, and glucose homeostasis systems. To investigate the transporter further, we cloned the murine Slc19a2 locus and characterized the resulting protein. Murine Slc19a2 is a 498 amino acid protein, with 12 predicted transmembrane domains. The gene spans approximately 13kb with 6 exons, structurally identical to that of the human homolog. We localized the Slc19a2 gene to mouse chromosome 1, a region syntenic to human chromosome 1q23 that contains the TRMA locus. Transient expression of Slc19a2 in HEK293T cells resulted in specific uptake of [3H] thiamine, confirming a thiamine transporter function. Western blot analysis of mouse tissues reveals a wide distribution of Slc19a2 protein. Immunohistochemistry studies indicate that Slc19a2 is expressed on the cell surface and intracellularly, and is specifically localized to a subpopulation of cells in cochlea, small intestine, and pancreas.

Animals↗