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S D Skaper

Publications and source records attributed to S D Skaper.

At least 163 records · Page 9Linked to original sources
PubMed1979

Sodium dependence of the nerve growth factor--regulated hexose uptake in chick embryo ganglionic cells.

Embryonic dorsal root ganglionic cells, when incubated in vitro in the absence of nerve growth factor (NGF) undergo a general metabolic degeneration which is preceded by certain changes in permeation properties. Previous studies demonstrated that NGF can rapidly modulate permeation properties which regulate the availability to the cell of an important energy source, glucose. Hexose uptake was determined by measuring the ability of the cells to accumulate [3H]labeled 2-deoxy-D-glucose. The work reported here shows that the NGF-dependent portion (about one-third) of the total specific hexose uptake was also dependent on the presence of Na+, with the apparent uptake constant (Kt) for deoxyglucose varying inversely with an external Na+ concentration of 70-140 mM; Vmax was unaffected in this range. Preincubation of ganglionic cells with 10 mM ouabain for 15-60 min, followed by a pulse with [3H]-deoxyglucose, also resulted in 50-95% reduction of the NGF-sensitive uptake. A similar pretreatment of cells with veratridine gave a 25-50% reduction in uptake. The NGF-controlled hexose uptake was also energy dependent, being diminished 50-95% after a 30-90 min preincubation with 2 mM 2,4-dinitrophenol. Uptake activities for other substrates (alpha-aminoisobutyric acid, uridine) which exhibited NGF regulation were likewise Na+-sensitive. These results indicate that availability of major energy substrates to NGF-dependent dorsal root ganglionic neurons is controlled by sodium gradients across their membranes. It is conceivable that NGF provides for maintenance and development of its target neurons by acting on such sodium gradients and, consequently, regulating the intake of essential nutrients.

Amino Acids↗
PubMed1979

Nerve growth factor action on 2-deoxy-D-glucose transport in dorsal root ganglionic dissociates from chick embryo.

Dorsal root ganglionic cells, when incubated in vitro in the absence of nerve growth factor (NGF), undergo a general metabolic degeneration which is preceded by loss of certain permeation properties. To determine in which ways an absence of NGF can also affect the capacity of these cells to take up an important energy source, namely glucose, experiments were carried out in which cells were incubated with or without NGF for varying times, and then presented with the factor and tested for the ability to take up 3H-labeled 2-deoxy-D-glucose. As with exogenous uridine, hexose transport in DRG cells was reduced by NGF deprivation and restored by delayed NGF administration (up to 6 h). Both the initial rate and equilibrium level were affected in an NGF dose-dependent fashion. Calculation of apparent Kt and V max in NGF-deprived and NGF-supported cells showed about two-fold differences between NGF-controlled and NGF-independent hexose transports, suggesting corresponding differences between NGF-dependent and other ganglionic cells. Restoration of hexose transport by delayed NGF administration took place within minutes of presentation of the factor. The delay before onset of restoration and the speed with which restoration was achieved have been found also to be dependent on the NGF concentration, suggesting that they reflect equilibration kinetics between NGF and its binding sites rather then the development of the response within cells. Thus, NGF can rapidly modulate permeation properties which regulate the availability of major energy substrates for the cell. This effect of NGF is discussed in the content of current views on the mode of action of the factor.

Animals↗
PubMed1979

A procedure for purifying neuron-like cells in cultures from central nervous tissue with a defined medium.

A serum-free medium (N1) containing the supplements insulin, transferrin, progesterone, putrescine and selenium was used to culture cells from a variety of embryonic chick central nervous system tissues, namely, optic lobe, neural retina, spinal cord and telencephalon. The N1 medium supported the survival of fiber-bearing cells (features typical of cultured neurons) as well as or better than horse serum, while permitting no, or almost no flat cells. Survival and growth of chick flat cells required fetal calf, but not horse serum. Cultivation of newborn mouse telencephalon under these conditions yielded similar results, except that either fetal calf or horse serum supported flat cells.

Animals↗
PubMed1978

The influence of ammonia on purine and pyrimidine nucleotide biosynthesis in rat liver and brain in vitro.

1. The effect of ammonia on purine and pyrimidine nucleotide biosynthesis was studied in rat liver and brain in vitro. The incorporation of NaH(14)CO(3) into acid-soluble uridine nucleotide (UMP) in liver homogenates and minces was increased 2.5-4-fold on incubation with 10mm-NH(4)Cl plus N-acetyl-l-glutamate, but not with either compound alone. 2. The incorporation of NaH(14)CO(3) into orotic acid was increased 3-4-fold in liver homogenate with NH(4)Cl plus acetylglutamate. 3. The 5-phosphoribosyl 1-pyrophosphate content of liver homogenate was decreased by 50% after incubation for 10min with 10mm-NH(4)Cl plus acetylglutamate. 4. Concomitant with this decrease in free phosphoribosyl pyrophosphate was a 40-50% decrease in the rates of purine nucleotide synthesis, both de novo and from the preformed base. 5. Subcellular fractionation of liver indicated that the effects of NH(4)Cl plus acetylglutamate on pyrimidine and purine biosynthesis required a mitochondrial fraction. This effect of NH(4)Cl plus acetylglutamate could be duplicated in a mitochondria-free liver fraction with carbamoyl phosphate. 6. A similar series of experiments carried out with rat brain demonstrated a significant, though considerably smaller, effect on UMP synthesis de novo and purine base reutilization. 7. These data indicate that excessive amounts of ammonia may interfere with purine nucleotide biosynthesis by stimulating production of carbamoyl phosphate through the mitochondrial synthetase, with the excess carbamoyl phosphate in turn increasing pyrimidine nucleotide synthesis de novo and diminishing the phosphoribosyl pyrophosphate available for purine biosynthesis.

Ammonia↗
PubMed1977

Reversion in expression of hypoxanthine-guanine phosphoribosyltransferase in 6-thioguanine resistant neuroblastoma: evidence for reduced enzyme levels associated with unaltered catalytic activity.

Five clones of mouse neuroblastoma cells able to grow in hypoxanthine-aminopterin-thymidine containing medium were isolated from a hypoxanthine-guanine phosphoribosyltransferase (HGPRT; EC 2.4.2.8; IMP: pyrophosphate phosphoribosyltransferase) deficient cell line. These hypoxanthine-aminopterin-thymidine resistant revertant clone had 45-55% of wild-type cell HGPRT activity. Kinetic studies indicated that the HGPRT in revertant clones had a reduced maximal velocity as compared to wild type cells based on cell protein. Apparent Km values of HGPRT for hypoxanthine and 5-phosphoribosyl-1-pyrophosphate were similar in wild-type and revertant cells. Heat inactivation studies demonstrated a similar heat lability for HGPRT in revertant and wild-type cells. An antibody fraction prepared from serum of rabbits immunized with HGPRT partially purified from mouse liver was used to measure the amount of cross-reacting material in normal and revertant clones. The revertant clones had one-half the amounth of cross-reacting material present in wild-type cells, based on a given amount of cell protein. These data indicate that the revertant cells may contain fewer HGPRT molecules with unaltered catalytic activity.

Adenine Phosphoribosyltransferase↗
PubMed1976

Maple syrup urine disease: branched-chain amino acid concentrations and metabolism in cultured human lymphoblasts.

The intracellular concentration of free leucine, isoleucine, and valine and their metabolism were studied in lymphoblast cultures established from peripheral blood of an individual with maple syrup urine disease (MSUD) and a control subject. Branched-chain alpha-keto acid decarboxylase activity in the MSUD cells was 10% or less of the control value as measured by the ability of the cells to release 14CO2 from the corresponding [1-14C]labeled branched-chain amino acid. The intracellular concentrations of free leucine and isoleucine were increased three-fold in MSUD lymphoblasts as compared to control cells. Free valine was present in only trace amounts of less than 0.1 mM in both cell lines. Exposure of normal and mutant cells to a 10 mM load of leucine, isoleucine, and valine revealed in a comparable concentration within cells after 24 hr. Concentrations returned to base values in normal cells 12 hr after removal of load, but leucine remained elevated in MSUD cells after 3 days. Leucine and its keto acid, alpha-ketoisocaproic acid, added to the culture medium gave significant growth inhibition of MSUD lymphoblasts but not of normal cells, in the millimolar range. Isoleucine, valine, and their keto acids had no effect.

Adult↗
PubMed1976

Metabolism of biogenic amines in neuroblastoma and glioma cells in culture.

The kinetic parameters of monoamine oxidase (MAO; E.C 1.4.3.4) and catechol-O-methyltransferase (COMT; EC 2.1.1.6) were evaluated in extracts of adrenergic and non-adrenergic mouse neuroblastoma cells and in rat glioma cells. Using the naturally-occurring substrates tyramine, tryptamine, serotonin and norepinephrine, the affinity of MAO for a given substrate was independent of the presence of the catecholaminergic pathway or cell type used, with apparent Km values ranging from 8-14 microM for tryptamine to 510-580 microM for norepinephrine. The MAO activity in glioma cells was substantially greater than in either neuroblastoma clone, but Vmax values varied little with substrate among cell lines. Both the neuronal and glial COMT had a similar Km for 1-norepinephrine (200 microM); the corresponding Vmax values were also similar among the different cell lines, but represented only 2-10% of the maximal MAO activity. Neuroblastoma and glioma cells, when grown from early logarithmic to stationary phase, showed no significant changes in specific activity of either MAO or COMT. Growth of cells for 3 days with 1 mM-N6,O2'-dibutyryl adenosine-3',5'-cyclic monophosphate resulted in no marked change in either MAO or COMT activity. These results suggest that in neurons neither MAO nor COMT plays a major role in the type of transmitter inactivation that is analogous to that of acetylcholinesterase in cholinergic synapses. The occurrence of considerable MAO and acetylcholinesterase activities in glioma cells may indicate a role for these cells in neurotransmitter inactivation.

Acetylcholinesterase↗
PubMedDate not supplied

Purification and characterization of Fab fragments from anti-mouse NGF polyclonal antibodies.

A functional role for Nerve Growth Factor (NGF) in the peripheral nervous system is well-documented, but a similar case for NGF in the central nervous system remains to be established. One approach to answering this question would be the availability of high-affinity monospecific Fab fragments obtained against NGF. In the present studies we describe the preparation and characterization of such Fab fragments from anti-mouse NGF polyclonal antibodies. Following their purification by the use of a NGF Sepharose-coupled affinity column, the Fab fragments were examined for biological competence in several ways. In vitro, the anti-Fab fragments blocked the neuronotrophic activity of NGF, as measured by the survival of chicken embryonic day 8 dorsal root ganglion neurons. In vivo, these Fab fragments, when administered systemically to neonatal rats, produced a decrease of noradrenaline levels in two sympathetically innervated organs, the heart and the spleen. These findings suggest that affinity purified Fab fragments of anti-NGF antibodies can be a useful tool for studying the physiological function of NGF in the nervous system.

Animals↗