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The protein CDP, but not CP1, footprints on the CCAAT region of the gamma-globin gene in unfractionated B-cell extracts.

We have identified, by DNase I footprinting, six different factors that interact with the promoter of the human A gamma-globin gene in nuclear extracts of the B-cell line BJA-B. Among them is the vertebrate homologue of the sea-urchin CCAAT displacement protein (CDP) which footprints over the entire duplicated CCAAT region. The CCAAT-binding factor CP1, a potential activator of the gamma-globin promoter, is able to bind to its proximal recognition sequence only once it has been partially enriched and separated from CDP. The factor CDP has an apparent molecular mass of 200 kDa and differs from CP1 by its footprint pattern and competition behavior.

B-Lymphocytes

Genetic diagnosis. Implications for medical practice.

Genetic diagnostic techniques increasingly permit the detection of predisposition to illness long before the onset of the disease process itself. Medicine is on the verge of becoming a predictive science as well as a diagnostic and therapeutic one. Genetic diagnosis could have profound effects on many aspects of our health care system, including the prestige and effectiveness of preventive medicine, the competitive behavior of health care organizations and insurance companies, access to private health insurance, the ability of primary care physicians to serve as gatekeepers, and other matters. This article examines the range of potential effects of the new genetic diagnostics on the organization and financing of health care. For purposes of illustration the authors examine in detail the possible consequences of genetic tests for predisposition to two diseases: Reye's Syndrome and lung cancer in smokers.

Cost-Benefit Analysis

Effects of Ca2+ channel blockers on Ca2+ translocation across synaptosomal membranes.

The binding of [3H]nimodipine to purified synaptic plasma membranes (SPM) isolated from sheep brain cortex was characterized, and the effects of nimodipine, nifedipine, and (+)-verapamil on the [3H]nimodipine binding were compared to the effects on 45Ca2+ translocation under conditions that separate 45Ca2+ fluxes through Ca2+ channels from 45Ca2+ uptake via Na+/Ca2+ exchange. [3H]Nimodipine labels a single class of sites in SPM, with a KD of 0.64 +/- 0.1 nM, a Bmax of 161 +/- 27 fmol X mg-1 protein, and a Hill slope of 1.07, at 25 degrees C. Competition of [3H]nimodipine binding to purified SPM with unlabelled Ca2+ channel blockers shows that: nifedipine and nimodipine are potent competitors, with IC50 values of 4.7 nM and 5.9 nM, respectively; verapamil and (-)-D 600 are partial competitors, with biphasic competition behavior. Thus, (+)-verapamil shows an IC50 of 708 nM for the higher affinity component and the maximal inhibition is 50% of the specific binding, whereas for (-)-verapamil the IC50 is 120 nM, and the maximal inhibition is 30%; (-)-D 600 is even less potent than verapamil in inhibiting [3H]nimodipine binding (IC50 = 430 nM). However, (+)-verapamil, nifedipine, and nimodipine are less potent in inhibiting depolarization-induced 45Ca2+ influx into synaptosomes in the absence of Na+/Ca2+ exchange than in competing for [3H]nimodipine binding. Thus, (+)-verapamil inhibits Ca2+ influx by 50% at about 500 microM, whereas it inhibits 50% of the binding at concentrations 200-fold lower, and the discrepancy is even larger for the dihydropyridines. The Na+/Ca2+ exchange and the ATP-dependent Ca2+ uptake by SPM vesicles are also inhibited by the Ca2+ channel blockers verapamil, nifedipine, and d-cis-diltiazem, with similar IC50 values and in the same concentration range (10(-5)-10(-3) M) at which they inhibit Ca2+ influx through Ca2+ channels. We conclude that high-affinity binding of the Ca2+ blockers by SPM is not correlated with inhibition of the Ca2+ fluxes through channels in synaptosomes under conditions of minimal Na+/Ca2+ exchange. Furthermore, the relatively high concentrations of blockers required to block the channels also inhibit Ca2+ translocation through the Ca2+-ATPase and the Na+/Ca2+ exchanger. In this study, clear differentiation is made of the effects of the Ca2+ channel blockers on these three mechanisms of moving Ca2+ across the synaptosomal membrane, and particular care is taken to separate the contribution of the Na+/Ca2+ exchange from that of the Ca2+ channels under conditions of K+ depolarization.

Adenosine Triphosphatases

A behavioral classification of welfare children from survey data.

From survey data on 1000 urban Welfare AFDC children aged six to eighteen, a hierarchical cluster analysis yielded six distinct behavioral types of Welfare children. Characteristics of each type, and its relationship to treatment, ethnicity, and other variables are discussed. Advantages of this system of behavioral classification for research and population assessment are outlined.

Achievement

An expanded function for superoxide dismutase.

alpha-Hydroxyalkylperoxyl radicals were generated from the primary and secondary alcohols methanol, ethanol and 2-propanol in N2O/O2-saturated aqueous solutions by pulse radiolysis. These radicals reduced a ferric iron porphyrin complex, tetrakis-(4-N-methylpyridyl)porphine, with diffusion-controlled rate constants. The extreme sensitivity of the shift of the Soret absorption band in this reaction was used to determine, by competition kinetics, the reactivity of the peroxyl radicals with different proteins. Only native Cu,Zn-superoxide dismutase and metallothionein showed competitive behavior, with SOD exhibiting rate constants close to the dismutation rate for O2-. Metallothionein was slower by a factor of 30 with hydroxymethylperoxyl radicals. We propose, that SOD has unique properties of the protein surface in addition to the prosthetic copper site, having possibly evolved as a 'general-purpose radical-scavenging protein'.

Alcohols

[An analysis of the joint activities of primates in a situation of delayed spatial choice].

A leader-dominant baboon of a couple took practically all the food in the situation of spatial delayed choice. The subdominant baboon managed to get some food, too, using fast changes of behavioral tactics during the experimental session. The obvious negative state of the subdominant baboon resulted in a severe neurotic breakdown. Long delays in the experimental scheme enabled the subdominant to get up to 50% of the food reinforcement due to the dominant baboons mistakes. The role of changes in the tactics of the monkeys is discussed.

Animals

Modulation of the mitochondrial megachannel by divalent cations and protons.

In patch-clamp experiments on rat liver mitoplasts, the 1.3 nanosiemens (in 150 mM KCl) mitochondrial megachannel was activated by Ca2+ and competitively inhibited by Mg2+, Mn2+, Ba2+, and Sr2+. Cyclosporin A, which inhibits the megachannel, also showed a competitive behavior versus Ca2+. The pore is regulated by pH in the physiological range; lower pH values cause its closure in a Ca(2+)-reversible manner. The modulating sites involved in these effects are located on the matrix side of the membrane. As illustrated in the companion paper (Bernardi, P., Vassanelli, S., Veronese, P., Colonna, R., Szabó, I., and Zoratti, M. (1992) J. Biol. Chem. 267, 2934-2939), the calcium-induced permeability transition of mitochondria is affected by these various agents in a similar manner. The results support the identification of the megachannel with the pore believed to be involved in the permeabilization process. The kinetic characteristics of the single channel events support the idea that the megachannel is composed of cooperating subunits.

Animals

Characterization of Na+/H+ exchange in platelets.

Acidification of the cytoplasm of human blood platelets leads to activation of Na+/H+ exchange. As a result, alkalinization occurs that is detectable by an intracellular fluorescent pH indicator. The activity of the exchanger can also be measured by the swelling of platelets suspended in Na-propionate medium using a Coulter Counter: the rapid entry of propionic acid leads to acidification and activation of Na+/H+ exchange with the parallel entry of Na+ and propionic acid leading to osmotic swelling. The Na+/H+ exchanger is sensitive to amiloride and to derivatives that are reported to be more specific inhibitors; it is specific for Na+ and Li+ with no measurable transfer of K+, Rb+ or Cs+; its Km for Na+ is 75 to 90 mM; it displays competitive behavior between Na+ and amiloride; its activity is decreased in cells loaded with Na+ by prolonged ouabain treatment; and it has a high temperature coefficient. These properties are in general similar to those of the exchanger in other cells. It is suggested that the Na+/H+ exchanger plays a role in platelet pH regulation.

Adult

Reduced nicotinamide adenine dinucleotide phosphate, a structural and conformational probe of chicken liver fatty acid synthetase.

Structural and conformational organization of chicken liver fatty acid synthetase has been probed using its fluorescent coenzyme, NADPH. Three NADPH binding sites per mole of the enzyme complex, of apparently identical dissociation constant (KD = 0.6 muM) can be titrated at temperatures above 12 degrees. These results are in disagreement with the earlier studies of Hsu and Wagner (Hsu, R. Y., and Wagner, B. J. (1970) Biochemistry, 9, 245-251) in which four such sites could be titrated. At 12 degrees, the composite sites split into two subsets: a pair of sites with a KD of 0.3 muM and a third site with a Kd of 1.1 muM. At lower temperatures (5 degrees or 2 degrees), the site with weak affinity disappears, leaving a pair of sites with a Kd of 0.5 muM. Similar observations were made when the enzyme was modified with phenylmethylsulfonyl fluoride, a specific and selective inhibitor of fatty acyl-CoA deacylase (s) of the pigeon liver enzyme complex (Kumar, S. (1975) J. Biol. Chem. 250, 5150-5158). Partial modification with phenylmethylsulfonyl fluoride elicits a NADPH binding response similar to the binding observed at 12 degrees, i.e. two sets of binding sites with nonidentical dissociation constants. Further modification corresponding to the complete loss of deacylase function results in a set of two apparently identical binding sites, and the third site is not available for titration. The modified enzyme retains the two reductase functions as measured by the model substrates, acetoacetyl-N-acetylcysteamine and crotonyl-CoA. Furthermore, the addition of acetyl- and malonyl-CoA (100 muM each) to the modified enzyme lowers the NADPH binding affinity by a factor of 3. Other observations show that the quantum yield, as measured by the ratio of fluorescence intensity of bound and free NADPH, changes with temperature and ionic strength. Lowering the temperature from 30 degrees to 2 degrees increases the enhancement ratio by 50%, whereas increase in ionic strength from 0.05 to 0.2 M potassium phosphate lowers it to 50% of the original level. Measurement of NADPH binding in the presence of NADP+, NADH, NAD+ and adenosine-2'-monophospho-5'-diphosphoribose demonstrates that NADP+ shows competitive behavior for NADPH sites (KD = 10.6 muM), whereas NADH and NAD+ show noncompetitive (KD (apparent) = nearly 600 muM) and rather complicated interactions implicating nonspecific conformational alteration of the enzyme complex. The behavior of adenosine 2'-monophospho-5'-diphosphoribose is intermediate between NADP+ and NADH. These data are discussed in terms of substrate-mediated conformational changes and the moles of each of the reductase enzymes per mole of the enzyme complex, the polarity of the NADPH binding region, and the probable structure of the nicotinamide moiety when bound to the enzyme.

Animals

The organization of agonistic relations within two captive groups of Java-monkeys (Macaca fascicularis).

The paper offers a detailed quantitative descripition of the distribution of agonistic activities over the members of two groups of Java-monkeys (Macaca fascicularis). These groups lived in captivity and were well-established: i.e. they had an extensive network of genealogical relationships. The study pays special attention to agonistic interactions with three or more participants. Its main purpose is an analysis of the way dyadic agonistic relations (e.g. dominance relations) are affected by third group members and the relations among these. The paper presents data on the ontogeny of 'dependent dominance', the 'control role' of the alpha-male, and the functions of different types of alliances.

Age Factors