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Biomedical subjects

A F Borghetti

Publications and source records attributed to A F Borghetti.

At least 55 records · Page 3Linked to original sources
PubMed1986

Association of tyrosine protein kinase activity with mitochondria in human fibroblasts.

A tyrosine protein kinase activity has been detected in the mitochondrial fraction purified from human fibroblasts. By enzymatic and sedimentation analysis this activity appeared to be localized in the mitochondrial outer membrane. Mitochondrial tyrosine phosphorylation was strictly dependent on the presence of Mn2+ ions. An inverse relationship between cell proliferation and mitochondrial protein phosphorylation on tyrosine residues has been found: a marked increase in the mitochondrial tyrosine kinase activity occurred when a significant reduction in the growth rate followed serum step-down. In mitochondria purified from resting cells, a protein band with apparent molecular weight of 50 kd appeared to be phosphorylated on tyrosine.

Electrophoresis, Polyacrylamide Gel↗
PubMed1986

Adaptive response of cultured fibroblasts to hyperosmolarity.

Raising to 0.4 osM the osmolarity of the medium in which chick embryo fibroblasts are incubated quickly increases the internal Na+ concentration, inhibits protein synthesis and also stimulates amino acid transport. On extending the incubation time, cells appear to adapt to the altered environment, as the Na+ content declines toward control values within few hours. Protein synthesis resumes its normal rate within 12-14 h of treatment. Experimental alteration of the monovalent cation content by substituting extracellular Na+ with other osmolites or by using ouabain or the ionophore monensin reveals an impairment of protein synthesis. Analysis by SDS-PAGE reveals an alteration of the polypeptide pattern expressed by hyperosmolarity-exposed cells, resulting in an enhanced synthesis of the 87, 75 and 53 kD proteins and inhibition of a 125 kD band. The previously increased amino acid transport activity also reverts to its normal level, but only after 40-50 h of incubation. The growth rate of these cells does not appear to be significantly affected during the first 3 days of the hyperosmolar treatment. Results presented in this publication identify the alteration of the protein synthesis rate, the change in the intracellular cation content and the increase in amino acid transport activity as plausible parameters of adaptive response, and suggest that the modulation of gene expression observed in cells exposed continuously to hyperosmolarity may be a consequence of the alteration of the intracellular monovalent cation concentration.

Adaptation, Physiological↗
PubMed1986

Induction of stress proteins by hyperosmolarity in normal and transformed cells.

The synthesis of at least three proteins, with molecular weights of approximately 87, 70, and 53 kd, was enhanced following the exposure of chick embryo fibroblasts to hyperosmolar shock of 30 min at 0.6 osM. Two of these proteins, the 87 and 70 kd, comigrated on one-dimensional gel electrophoresis with the stress proteins induced by heat shock after 30 min at 44 degrees C. In 3T3 cells, the hyperosmolar shock enhanced the expression of two proteins of 88 and 52 kd, whereas the heat shock increased the synthesis of several new polypeptides including the 88 and 52 kd mw. In SV40-transformed 3T3 cells the synthesis of two proteins of 72 and 69 kd was enhanced by heat shock, but no change of the protein pattern was recorded after the hyperosmolar shock.

Animals↗
PubMed1985

The effect of the intracellular sodium level on the activity of amino acid transport systems L and A in SV40 3T3 cells.

The rate of transport of phenylalanine and leucine, pertinent amino acids of System L, has been measured in SV40 3T3 cells as a function of the presence of Na+ ions during the reloading phase that precedes the influx determination. The presence of Na+ ions during the reloading phase resulted in an increase of the subsequent substrate influx through System L. This effect was related to the intracellular Na+ level and was found to be independent by the presence of a chemical sodium gradient outside-inside during influx determination; furthermore, this effect could not be ascribed to a difference between control and Na+-treated cells in the internal levels of those amino acids that participate in the exchange phenomena of transport System L. The transport of phenylalanine appeared to have the ability to accept Li+ for Na+ substitution in the 'trans' position. The presence of Na+ ions in the 'trans' position was not required to optimize the transport of System A-reactive substrates, whose influxes are dependent on the presence of the cation in 'cis' position. Analysis of the relationship between influx and substrate concentration indicated that the Na+-dependent increase of substrate influx was associated with an enlarged capacity of the high-affinity component of transport System L.

3-O-Methylglucose↗
PubMed1984

Effect of hyperosmolarity on the activity of amino acid transport system L in avian fibroblasts.

The transport of selected neutral amino acids known as good substrates of amino acid transport System L has been studied in chick embryo fibroblasts exposed for 4 hours to hyperosmolar culture medium. The activity of the L system, as measured by initial rates of L-phenylalanine uptake, increased in hyperosmolarity treated cells when determined before any cell depletion of intracellular amino acids. This effect was lost after depletion but reappeared after reloading the cells with pertinent substrates of System L. This transport activity appeared to be related to the internal level of amino acids capable of exchange through System L. In hyperosmolarity-treated chick embryo fibroblasts a higher level of System L substrates was obtained during the reloading phase in comparison to control cells. This expanded amino acid pool reflected an increased activity of transport System A, an agency of amino acid mediation known to enlarge its capacity following a hyperosmolar treatment of chick embryo fibroblasts (see Tramacere et al., 1984). L-Methionine, a preferred substrate of both A and L systems, appeared to be involved in the coupling between the activity of amino acid transport Systems A and L in these cells.

Amino Acids↗
PubMed1984

Early events during the activation of human lymphocytes by the mitogenic monoclonal antibody OKT3.

The effects of the mitogenic monoclonal antibody OKT3 on the metabolic changes preceding DNA synthesis during the activation of human peripheral blood mononuclear cells were compared with those induced by PHA. The aspects studied included uridine transport, the incorporation of inositol into phospholipids, Na+-dependent amino acid uptake, and protein synthesis. All four parameters were increased in response to the ligation of the T lymphocyte receptor recognized by OKT3. These changes were apparent as early as the corresponding changes induced by PHA. However, the increases in uridine uptake and inositol incorporation were disproportionately reduced when compared to those caused by PHA, and no evidence of high-dose inhibition was seen in cells activated by OKT3. This suggests that at least some lectin-induced changes in metabolism are mediated through additional mechanisms, probably involving distinct receptors.

Alanine↗
PubMed1984

Osmoregulation of amino acid transport activity in cultured fibroblasts.

The effect of exposure of chick embryo cells to increasing concentrations of Na+ in the culture medium on the subsequent amino acid transport as determined at physiological osmolarity was investigated in detail. It was found that the hyperosmolar treatment stimulated amino acid transport in a dose-dependent manner up to 200 mM Na+. Changes were measurable as early as 1 h after altering Na+ and reached a maximum after 4 h, remaining constant thereafter. The maintenance of this effect required continuous exposure of the cell to high Na+ in the culture medium. Hyperosmolarity-mediated increases in amino acid transport activity by system A have been detected with L-proline and L-alanine. Transport activities of systems ASC and L did not change appreciably after exposure of the cells to high Na+. Inhibition of protein synthesis by cycloheximide or RNA synthesis by actinomycin D (actD) prevented these uptake changes. Kinetic analysis indicated that the stimulation of the activity of transport system A by high Na+ treatment occurred through a mechanism affecting Vmax rather than Km.

Amino Acids↗
PubMed1983

Effect of cyclosporin A on lymphocyte activation by the calcium ionophore A23187.

The activation of pig lymphocytes by the divalent cation ionophore A23187 is more sensitive to inhibition by cyclosporin A than is activation by plant lectins such as concanavalin A. Complete inhibition of A23187-induced activation was seen at a cyclosporin A concentration of 0.3 micrograms/ml. The very early stimulation of nucleoside uptake induced by A23187 was not affected by cyclosporin A, but other early metabolic changes occurring during the first few hours after activation were inhibited.

Alanine↗
PubMed1982

The regulation by cell density of amino acid transport system L in SV40 3T3 cells.

The rate of transport of phenylalanine by System L has been measured in SV40 3T3 cells at various cell densities. When the activity of the L system was determined before any cell depletion of intracellular amino acids, a density-dependent increase in transport paralleled the decrease in cell density. This regulation was lost after cell depletion but reappeared after reloading the cells with pertinent substrates of System L. The phenylalanine transport activity modulated by cell density appeared to be related to the internal level of amino acids capable of exchange up to a definite concentration, beyond which transport activity by System L did not parallel a further increase of internal substrate level. Analysis of the relationship between influx and substrate concentration suggested that two saturable components contribute to entry of phenylalanine and leucine in depleted and in reloaded cells: a low-affinity and a high-affinity component. Both kinetic parameters of the high-affinity component appeared to be modulated by the loading treatment, but only V changed markedly. Activation energies for the high-affinity component of the amino acid transport reaction were calculated from an Arrhenius plot in reloaded cells, and were found to be different for low- and high-density cultures. This result is consistent with the interpretation that cell density modulated the rates at which the amino acid-carrier complex can move within the cell membrane.

Amino Acids↗
PubMed1982

Effect of cell density on growth rate and amino acid transport in simian virus 40-transformed 3T3 cells.

Rate of proliferation and amino acid transport were assessed in simian virus 40 (SV40)-transformed 3T3 cells by measurements of growth rate quotient and L-serine uptake via System ASC, respectively. Growth rate and cell density of the cultures were varied by modifying: (a) the number of cells initially plated; and (b) the period spent by the cells in culture. The growth rate quotient of SV40 3T3 cells was not correlated with cell density. Sparse cultures exhibited marked fluctuations in their growth rate as a function of time, whereas, under comparable conditions, crowded cultures retained some form of growth control by density. Transport activity by System ASC decreased as a function of increased cell density following a complex trend described by a double-exponential equation. The density-dependent decrease in amino acid transport was not accompanied by a parallel change in the rate of cell proliferation. These results indicate that alterations in amino acid transport are not linked with cell growth and suggest that an increase in transport activity is not a prerequisite for an optimal rate of proliferation in SV40-transformed 3T3 cells.

Animals↗
PubMed1981

Amino acid transport in pig lymphocytes. Enhanced activity of transport system asc following mitogenic stimulation.

Changes in neutral amino acid transport activity caused by addition of phytohaemagglutinin-P to quiescent peripheral pig lymphocytes have been evaluated by measurements of 14C-labelled neutral and analogue amino acids under conditions approaching initial entry rates. Utilizing methylaminoisobutyric acid, the best model substrate of System A, we confirmed our previous report (Borghetti, A.F., Kay, J.E. and Wheeler, K.P. (1979) Biochem. J. 182, 27-32) on the absence of this transport system in quiescent cells and its emergence following stimulation. Furthermore, we demonstrated the presence in quiescent cells of an Na+-dependent transport system for neutral amino acids that has been characterized as System ASC by several criteria including intolerance to methylaminoisobutyric acid, strict Na+-dependence, the property of transtimulation and specificity for pertinent substrates such as alanine, serine, cysteine and threonine. Analysis of the relationship between influx and substrate concentration revealed that two independent saturable components contribute to entry of alanine in quiescent cells: a low affinity (Km = approximately 4 mM) and a high affinity (Km = approximately 0.2 mM) component. The high affinity component could be inhibited in a competitive way by serine, cysteine and threonine, but methylaminoisobutyric acid did not change appreciably its constants. The enhanced activity of alanine transport through the ASC system observed in activated cells resulted from a large increase in the capacity (V) of the high affinity component without any substantial change in the apparent affinity constant (Km).

Alanine↗
PubMed1980

Cell density and amino acid transport in 3T3, SV3T3, and SV3T3 revertant cells.

The transport of selected neutral and cationic amino acids has been studied in Balb/c 3T3, SV3T3, and SV3T3 revertant cell lines. After properly timed preincubations to control the size of internal amino acid pools, the activity of systems A, ASC, L, and Ly+ has been discriminated by measurements of amino acid uptake (initial entry rate) in the presence and absence of sodium and of transport-specific model substrates. L-Proline, 2-aminoisobutyric acid, and glycine were primarily taken up by system A; L-alanine and L-serine by system ASC; L-phenylalanine by system L; and L-lysine by system Ly+ in SV3T3 cells. L-Proline and L-serine were also preferential substrates of systems A and ASC, respectively, in 3T3 and SV3T3 revertant cells. Transport activity of the Na+-dependent systems A and ASC decreased markedly with the increase of cell density, whereas the activity of the Na+-independent systems L and Ly+ remained substantially unchanged. The density-dependent change in activity of system A occurred through a mechanism affecting transport maximum (Vmax) rather than substrate concentration for half-maximal velocity (Km). Transport activity of systems A and ASC was several-fold higher in transformed SV3T3 cells than in 3T3 parental cells at all the culture densities that could be compared. In SV3T3 revertant cells, transport activity by these systems remained substantially similar to that observed in transformed SV3T3 cells. The results presented here add cell density as a regulatory factor of the activity of systems A and ASC, and show that this control mechanism of amino acid transport is maintained in SV40 virus-transformed 3T3 cells that have lost density-dependent inhibition of growth, as well as in SV3T3 revertant cells that have resumed it.

Amino Acids↗
PubMed1979

Enhanced transport of natural amino acids after activation of pig lymphocytes.

Na+-dependent uptake of the amino acids L-proline and L-methionine was greatly accelerated when pig lymphocytes were activated with phytohaemagglutinin or other mitogens. The increased influx was apparent after incubation with phytohaemagglutinin for 1 h, and reached a maximum after 24 h. The lymphocytes appear to possess at least three different transport systems for neutral amino acids with properties similar to, but not identical with, those described for other cells. The activity of a system resembling the A system of other cells was increased most dramatically after activation, its activity in unstimulated lymphocytes being extremely low or absent. A second Na+-dependent system, which has properties similar to those of the ASC system in other cells, but with a broader specificity for amino acids, was more active in unstimulated lymphocytes, and uptake by this system was also accelerated after incubation with phytohaemagglutinin. The activity of a third system, very similar to the L system in other cells, was increased to a much smaller extent after lymphocyte activation.

Amino Acids↗
PubMed1979

Enhanced stability of the Na+-dependent amino acid-transport system after lymphocyte activation.

The Na+-dependent transport system for the amino acid proline in unstimulated lymphocytes decays with a half-life of about 4 h when protein synthesis is inhibited by cycloheximide, but the system in mitogen-activated lymphocytes is more stable. However, the early increase in activity of this transport system after mitogen addition is not due to a direct effect on its stability, but is a secondary effect requiring protein synthesis.

Animals↗