PubMed Health⌕ Search

Biomedical subjects

J S Cook

Publications and source records attributed to J S Cook.

At least 55 records · Page 3Linked to original sources
PubMed1982

Recycling of surface sialoglycoconjugates in HTC and HeLa cells.

Surface sialoglycoconjugates of HeLa and HTC cells were labeled with NaB[3H]4 after oxidation of NaIO4. The labeling procedure cleaves the sialic acids to a neuraminidase-sensitive 7-carbon derivative, 5-acetamido-3,5-dideoxy-L-arabino-heptulosonic acid, termed AcNeu7 (Van Lenten, L., and Ashwell, G. (1971) J. Biol. Chem. 246, 1889-1894). After labeling, the radioactivity is lost from both cell types with biphasic kinetics. The half-time for the fast phase is about 4-5 h; the slow phase has a half-time of 100-200 h. About 30 h after labeling and at later times, approximately 30% of the cell-associated radioactivity is susceptible to removal by external neuraminidase, suggesting an exchange with an internal pool that is twice the size of the surface pool. An internal pool of relatively high specific activity compared to the surface was generated by labeling as above, followed by a period of time to allow internalization and enzymatic removal of external neuraminidase-sensitive radioactivity. During subsequent reincubation in growth medium, the surface became relabeled from the internal pool, again reaching a 30% neuraminidase-sensitive plateau. The relabeling of the surface was confirmed by radioactivity measurements on isolated plasma membranes. [3H]AcNeu7 cannot be reutilized by these cells in the de novo membrane biosynthetic pathway. The argument is made that the labeled sialoglycoconjugates are recycling intact through the internal pool.

Animals↗
PubMed1982

Effect of 4-hydroxypyrazole on tryptophan and formate metabolism in isolated rat liver cells.

1. 4-Hydroxypyrazole inhibits flux through tryptophan 2.3-dioxygenase in cells. The inhibition is apparently non-competitive with Ki = 0.15 mM. 2. Hydroxypyrazole inhibits the oxidation of formate to CO2 in liver cells. 3. Glycollate, which generates H2O2, stimulates formate oxidation. This process is inhibited by 4-hydroxypyrazole. 4. Methionine stimulates formate oxidation in cells and this stimulation is insensitive to 4-hydroxypyrazole. 5. It is concluded that, in freshly isolated liver cells, formate oxidation proceeds by a pathway involving catalase. In vivo, or when methionine is added to cell incubations, the pathway of oxidation involves tetrahydrofolate, and is insensitive to catalase inhibitors. 6. Methionine at physiological concentrations inhibits the activity of tryptophan 2,3-dioxygenase in isolated liver cells.

Animals↗
PubMed1982

Uptake of [3H]ouabain from the cell surface into the lysosomal compartment of HeLa cells.

[3H]Ouabain specifically bound at sublethal concentrations to Na,K-ATPase on the surface of HeLa cells is taken up (internalized) by the cells at a rate of three membrane equivalents of labeled sites per generation. Immediately following a pulse label with the glycoside, codistribution of radioactivity with the surface marker 5'-nucleotidase is found in both conventional sucrose-gradient fractionation and in fractionation following a digitonin treatment. At appropriate concentrations digitonin increases the buoyant density of the HeLa surface membrane and solubilizes the lysosomal marker beta-hexosaminidase (Tulkens et al., 1974). After internalization, [3H]ouabain is also solubilized by digitonin. A shear analysis is described which shows internalized ouabain and beta-hexosaminidase to be codistributed in a particulate fraction that is homogeneous with respect to shear; extrapolation to zero-shear shows that little or none of either marker is found in the soluble fraction of the cytosol. Both markers are coreleased from the particulate fraction by osmotic shock. Although internalized ouabain is subsequently released from these cells with a half-time of about 70 hr, apparently by exocytosis, the shear sensitivity of the remaining cell-associated ouabain does not change for up to 72 hr. Thus ouabain (together with Na,K-ATPase?) appears to be taken up from the surface into a lysosomal compartment and, by at least one criterion, this compartment does not change its physical properties with time, i.e., does not "age."

Biological Transport↗
PubMed1982

Development of Na+-dependent hexose transport in a cultured line of porcine kidney cells.

LLC-PK1 cells in culture do not concentrate alpha-methylglucoside (alpha-meG) during their early growth phase but develop the capacity to concentrate this hexose as the growth rate decreases in confluent cultures. The concentrating ability is dependent on the Na+ electrochemical gradient and is inhibited by phlorizin with KI,0.5 approximately 0.2 microM. The development of the concentrative capacity can be accelerated by the Friend cell inducer hexamethylene bisacetamide (HMBA) and by the phosphodiesterase inhibitors dibutyryl cAMP, theophylline, and 1-methyl-3-isobutylxanthine (MIX). In cultures treated with any of these differentiation-accelerating chemicals, the development of alpha-meG concentrating capacity is severely inhibited by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) but not by inactive (in tumor promotion) analogs of TPA. In all cases, an early event in the development of alpha-meG accumulating capacity is an elevated intracellular cAMP concentration; however the results suggest that this increase in cAMP may be necessary but not sufficient to induce the differentiated hexose-accumulating capacity.

Acetamides↗
PubMed1982

Regulation by turnover of Na,K-ATPase in HeLa cells.

As in most if not all animal cells, HeLa Na,K-ATPase is an essential enzyme of the cell surface. The three ways, referred to in the Introduction, in which it is regulated may be summarized as follows: 1) The activity of the enzyme under normal conditions responds almost linearly to small perturbations in internal (Na+); this is short-term regulation. 2) In the normal steady state, the enzyme is one of the rapidly turning over components of the cell surface; this is part of long-term regulation. The functional importance of turnover to cell homeostasis depends on the reversibility of any event that may inactive the enzyme. Thus turnover is an important, but not the only, means of recovery from ouabain intoxication, and for an inactivating ligand that dissociates more readily than ouabain turnover may be relatively unimportant. Conversely, turnover may be the only means of recovery from thermal inactivation at physiological temperatures. 3) Under conditions of prolonged stress, turnover itself may be modulated so as to enhance the number of active enzymes on the cell surface. Regulation by turnover has the advantage of permitting a prompt response to a changing cell environment, whereas regulation by synthesis would introduce a delay in response at least equal to the transit time. Broadman et al [1974] have suggested that the signal for the increase in Na,K-ATPase is elevated cellular (Na+), but how this is translated into a mechanism for altering the specific clearance of this enzyme from the membrane is not known.

HeLa Cells↗
PubMed1981

Na+, K+-ATPase in HeLa cells after prolonged growth in low K+ or ouabain.

Effects of long-term, subtotal inhibition of Na+-K+ transport, either by growth of cells in sublethal concentrations of ouabain or in low-K+ medium, are described for HeLa cells. After prolonged growth in 2 X 10(-8) M ouabain, the total number of ouabain molecules bound per cell increases by as much as a factor of three, mostly due to internalization of the drug. There is only about a 20% increase in ouabain-binding sites on the plasma membrane, representing a modest induction of Na+, K+-ATPase. In contrast, after long-term growth in low K+ there can be a twofold or greater increase in ouabain binding per cell, and in this case the additional sites are located in the plasma membrane. The increase is reversible. To assess the corresponding transport changes, we have separately estimated the contributions of increased intracellular [Na+] and of transport capacity (number of transport sites) to transport regulation. During both induction and reversal, short-term regulation is achieved primarily by changes in [Na+]i. More slowly, long-term regulation is achieved by changes in the number of functional transporters in the plasma membrane as assessed by ouabain binding Vmax for transport, and specific phosphorylation. Parallel exposure of cryptic Na+,K+-ATPase activity with sodium dodecyl sulfate in the plasma membranes of both induced and control cells showed that the induction cannot be accounted for by an exposure of preexisting Na+,K+-ATPase in the plasma membrane. Analysis of the kinetics of reversal indicates that it may be due to a post-translational event.

Binding Sites↗
PubMed1981

Turnover and regulation of Na-K-ATPase in HeLa cells.

HeLa cells in log growth have 10(6) surface Na-K-ATPase molecules as estimated by the specific binding of [3H]-ouabain. Studies utilizing ouabain as a label show that the ligand is internalized at a rate corresponding to the turnover of three sets of Na-K-ATPase enzymes per generation. The label is taken up exclusively into a particulate cell compartment where it is codistributed with beta-hexosaminidase, identifying the internal compartment as lysosomal. Turnover is an important parameter in the recovery of the cells from glycoside intoxication. The unmetabolized glycoside is subsequently released by exocytosis. 13C-density-labeled Na-K-ATPase has been identified by specific phosphorylation of its catalytic subunit with [32P]ATP or [33P]ATP, and the rate of turnover of the density label is shown to be the same as the internalization of the ouabain-labeled site. There is a transit time of about 4 h from the onset of synthesis of the catalytic subunit to its insertion in the surface membrane; 2,800 catalytic subunits are synthesized per minute per cell, and 2,100 are turned over K+-starved cells respond to the stress in 24-30 h with modulation of the surface density of Na-K-ATPase the synthetic rate remains constant; the number of functional enzymes per cell is controlled by change in the rate constant for turnover.

Cell Division↗
PubMed1981

Effect of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate on alpha-aminoisobutyrate uptake in friend Erythroleukemic cells.

In Friend erythroleukemic cells, the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) increases the rate of influx as well as the steady-state accumulation of the amino acid transport probe alpha-aminoisobutyric acid (AIB). This nonmetabolizable analog is concentrated in these cells by utilizing the energy of the Na+ electrochemical gradient. The effect of TPA is to increase the ouabain-sensitive component of uptake of AIB (and of the K+ analog 86Rb+). Transport changes are not observed until 30 min after application of the promoter and reach a maximum in about 6 hr. The effects are blocked by cycloheximide. The flux results are consistent with a model in which the membrane potential is increased and, with it, the driving force on the coupled Na+-AIB movement. The effect is possibly mediated by a change in electrogenicity of the Na:K pump. TPA raises the steady-state accumulation levels of AIB. By altering external K+ in the presence of valinomycin, the membrane potential may be adjusted and, with it, the accumulation of AIB. Over a wide range of membrane potentials, TPA-treated cells accumulate more AIB than do controls, suggesting a possible change in the internal Na+ activity.

Aminoisobutyric Acids↗
PubMed1981

Remote, semiautomated production of F-18-labeled 2-deoxy-2-fluoro-D-glucose.

A routine production system for 2-deoxy-2-[18F]fluoro-D-glucose (2-18FDG) for medical use is presented. The method involves the preparation of F-18-labeled F2 by the 20Ne(d, alpha) 18F reaction, subsequent fluorination of 3,4,6-tri-o-acetyl-D-glucal, purification, and hydrolysis of the appropriate diastereoisomer to obtain 2-18FDG. These operations are performed using a completely remote, semiautomatic system. There have been no failures in more than 220 consecutive production runs. Currently, two preparations per day of 15-25 mCi of 2-18FDG are routinely performed, with a radiochemical purity, measured by TLC, in excess of 95% and with less than 1 mR radiation exposure to the chemist. Reaction conditions, operational variables, and systems are described in detail.

Deoxy Sugars↗
PubMed1980

Indoleamine 2,3-dioxygenase. A new, rapid, sensitive radiometric assay and its application to the study of the enzyme in rat tissues.

A simple and convenient assay for indoleamine 2,3-dioxygenase has been developed. This depends on the conversion of D-[ring-2-14C]tryptophan to [14C]formate, excess substrate is removed by adsorption onto charcoal. This assay, which is 20-fold more sensitive than previous procedures, is applicable both to crude extracts and to large numbers of samples. Activity in rat tissues is very much lower than in those of the rabbit; measureable activity is found only in the stomach, spleen, intestine and kidney. Enzyme activity in the rat intestine was increased by 50% in rats pretreated with L-tryptophan.

Adsorption↗
PubMed1978

Nosocomial infection: the hidden cost in health care.

Excessive length of stay due to nosocomial infections is contributing enormously to the high cost of health care. The authors suggest ways to reduce the incidence of infection and, thereby, decrease hospital lengths of stay and lower patient bills while improving the quality of patient care.

Anti-Bacterial Agents↗
PubMed1978

Quercetin inhibits hexose transport in a human diploid fibroblast.

The flavonol quercetin, a phloretin analog, inhibits transport of 2-deoxyglucose and 3-O-methylglucose in a cultured human diploid fibroblast. This inhibition is related to transport itself and not to the reported effects of flavonoids on membrane-bound ATPases. From concentration-inhibition curves at several pH's we conclude that uncharged (acid) quercetin (pK = 7.65) is the inhibitory form of the molecule (K1 = 10micron). Quercetin, unlike phloretin, is rapidly degraded in 0.1 N NaOH; the degradation products are weakly inhibitory to hexose transport.

Biological Transport, Active↗
PubMed1978

Characterization of HeLa 5'-nucleotidase: a stable plasma membrane marker.

5'-Nucleotidase, assayed as 5'-AMPase, has been extensively characterized and established as a stable, quantitative plasma membrane marker in HeLa S3 cells. The membrane 5'-AMPase has a Km of 7.0 microM. Relative affinities of the other 5'-mononucleotides for the enzyme are 5'-GMP > 5'-TMP > 5'-UMP > 5'-CMP. There are activity optima at pH7 and 10; the latter is Mg(2+)-dependent. The membrane preparations have a small amount of acid phosphatase activity that is distinct from 5'-AMPase activity but no alkaline phosphatase. AOPCP, ADP, and ATP are strongly inhibitory. Mg2+, Ca2+, or Co2+ additions do not affect the pH 7.0 activity; Mn2+ activates slightly, whereas Zn2+, Cu2+, and Ni2+ are inhibitory. EDTA slowly inactivates, but removal of the EDTA without the addition of divalent cations restores activity. The inactivation is also substantially reversed by Co2+ or Mn2+, but reactivability by divalent cations decreases with time in EDTA. ConA strongly inhibits, and alpha-methyl-D-mannoside or glucose (the latter much less efficiently) relieves the inhibition, indicating that the 5'-AMPase is a glycoprotein. Histidine is also inhibitory. Ouabain, phloretin, cytochalasin B, cysteine, phenyl-alanine, MalNEt, and IAA are without effect. 5'-AMPase activity codistributes with pulse-bound [3H]ouabain when either of two cell fractionation procedures are used. The 5'-AMPase activity per cell is constant at different cell densities in exponentially growing cells, and activity per unit cell volume remains constant throughout the cell cycle. These properties, together with its absence in other organelles, its stability to storage, its insensitivity to certain experimental manipulations, and its general insensitivity to inhibitors of specific transport systems, make 5'-AMPase a useful quantitative marker in studies on the regulation of HeLa membrane transport systems. Key Words: HeLa, 5'-nucleotidase, plasma membrane marker, non-specific phosphatases, divalent ions, ConA, AOPCP, cell cycle, mitochondria, transport inhibitors.

5'-Nucleotidase↗