Dynamics Of Pools Research Articles

Quantitation and immunocytochemical localization of ubiquitin conjugates within rat red and white skeletal muscles.

We employed solid-phase immunochemical methods to probe the dynamics of ubiquitin pools within selected rat skeletal muscles. The total ubiquitin content of red muscles was greater than that of white muscles, even though the fractional conjugation was similar for both types of muscle. The specificity for conjugated ubiquitin in solid-phase applications, previously demonstrated for an affinity-purified antibody against SDS-denatured ubiquitin, was retained when used as a probe for ubiquitin-protein adducts in tissue sections. Immunohistochemical localization revealed that differences in ubiquitin pools derived from the relative content of red (oxidative) vs white (glycolytic) fibers, with the former exhibiting a higher content of ubiquitin conjugates. Subsequent immunogold labeling demonstrated statistically significant enhanced localization of ubiquitin conjugates to the Z-lines in both red and white muscle fiber types.

Simultaneous consumption of bacteria and dissolved organic matter byTetrahymena pyriformis

The addition of ciliated protozoa to aquatic microcosms and bench-scale sewage treatment plants increases decomposition rates. This is surprising, inasmuch as protozoa consume bacteria, which are the primary decomposers. One possible mechanism of the increase in decomposition rate is the direct consumption of dissolved organic matter by protozoa that are feeding primarily on bacteria. This possibility was explored experimentally in two-stage continuous cultures, with glucose limitingEscherichia coli in the first stage andE. coli limitingTetrahymena pyriformis in the second. Glycine and histidine were the test nutrients. The results of adding them to the second stages suggested that direct uptake by ciliates does not affect the dynamics of dissolved amino acids in pelagic environments or activated sludge plants. Ciliates might, however, affect the dynamics of amino acid pools in environments high in nutrients and ciliates, perhaps including some microenvironments near decomposing material or in benthic sediments. Direct uptake of dissolved amino acids by ciliates probably does not affect ciliate or bacterial populations substantially.

Interferon induces a 15-kilodalton protein exhibiting marked homology to ubiquitin.

Immunochemical methods were used to examine the effect of viral infection on the dynamics of intracellular ubiquitin pools. Infection of either the human lung carcinoma line A-549 or the mouse fibroblast line L929 with encephalomyocarditis virus had little effect on either the distribution or fractional level of intracellular ubiquitin conjugates. In contrast, viral infection resulted in a significant decline in the steady state content of the mono-ubiquitin conjugate to histone 2A (uH2A). Prior treatment with interferons protected against this decrease of uH2A. Furthermore, interferons induced the de novo synthesis of a 15-kDa protein immunologically related to ubiquitin. The ubiquitin cross-reactive protein (UCRP) was not constitutively present in control cells but was significantly induced in various cells sensitive to the biological effects of interferons. Induction of UCRP with respect to both time and interferon concentration dependence closely paralleled the appearance of resistance to viral infection and could be blocked by low levels of actinomycin D. Subsequent studies demonstrated that UCRP was identical to an interferon-induced 15-kDa protein whose sequence has recently been reported (Blomstrom, D. C., Fahey, D., Kutny, R., Korant, B. D., and Knight, E. (1986) J. Biol. Chem. 261, 8811-8816). An authentic sample of the 15-kDa protein was found to co-migrate with UCRP and to cross-react with two different anti-ubiquitin antibodies. Using the authentic 15-kDa protein as a standard, UCRP accumulated to 6.2 +/- 0.5 pmol/10(6) cells and 34 +/- 2 pmol/10(6) cells in interferon-treated A-549 and L929 cultures, respectively. Comparison of the primary sequence of the 15-kDa protein to that of ubiquitin indicated that the former is composed of two domains, each of which bears striking homology to ubiquitin. These observations suggest that the 15-kDa protein may represent one example of a functionally distinct family of ubiquitin-like proteins.

The dynamics of ubiquitin pools within cultured human lung fibroblasts.

The dynamics of ubiquitin pools within the cultured human lung fibroblast line IMR-90 were examined using solid phase immunochemical methods to quantitate free and conjugated polypeptide. Fetal calf serum was found to contain a nondialyzable factor that induced a transient accumulation of ubiquitin. During the induction, free and conjugated ubiquitin pools changed in concert so that the fraction conjugated remained constant. The induction of ubiquitin by the serum factor resulted from an enhanced rate of protein synthesis. Within experimental error no change in the first order rate constant for intracellular ubiquitin degradation was observed. Pulse-chase studies revealed ubiquitin to turn over with a half-life of 28-31 h in conditioned and freshly fed cultures. Withdrawal of serum from cultures led to a rapid decline in total ubiquitin during which the fractional level of conjugation remained constant. The accelerated ubiquitin turnover following removal of serum likely involves lysosomal autophagy since 10 mM NH4Cl led to an accumulation of the polypeptide. Since no similar effect of the lysosomotropic compound was observed in conditioned or freshly fed cultures, nonlysosomal processes are probably responsible for ubiquitin turnover under nutritional balance. The dynamics of these intracellular pools suggests that the ubiquitin ligation system is subject to regulatory constraints not previously suspected. The short half-life for ubiquitin is consistent with the apparent ability of cells to alter ubiquitin levels in response to external stimuli and stress.

Ribonucleotide reductase and deoxyribonucleotide pools.

Ribonucleotide reductase plays a crucial role in the maintenance of dNTP pools. A molecular model for the allosteric regulation of the enzyme is substantiated by the effects of nucleosides or inhibitors on pool sizes in intact cells as well as by manifestations of mutations of the reductase and other enzymes involved in deoxynucleoside metabolism. Studies of the dynamics of dNTP pools in 3T6 cells suggest compartmentation of dCTP pools, with perferential DNA synthesis from a pool labeled from cytidine. In contrast, no kinetic evidence for more than one dTTP pool was obtained. Our experiments also suggest that all dTTP formed in growing 3T6 cells is used up for DNA synthesis.

The dynamics of phosphate pools and intracellular pH in yeasts as studied by 31P nuclear magnetic resonance

The dynamics of phosphate pools and intracellular pH in yeasts as studied by 31P nuclear magnetic resonance

In vivo 31P NMR studies on the role of the vacuole in phosphate metabolism in yeasts.

31P NMR was used to study the dynamics of phosphate pools during substrate utilization by aerobic and anaerobic suspensions of the yeast Candida utilis and by aerobic suspensions of the yeast Brettanomyces intermedius. In both yeast, the cytoplasmic pH was monitored; in C. utilis also the vacuolar pH could be measured. When glucose was used as a substrate for C. utilis, the vacuolar store of inorganic phosphorus (both orthophosphate and polyphosphate) was mobilized to replenish cytoplasmic phosphate which had become very low due to the build-up of high sugar phosphate levels. The hydrolysis of polyphosphate was glucose-dependent; it did not occur with ethanol as the substrate. After glucose depletion resynthesis of polyphosphate occurred only under aerobic conditions; anaerobic C. utilis cells continued to hydrolyze vacuolar polyphosphate. This difference between the aerobic and anaerobic suspension could be related to differences in cellular ATP levels. When ethanol was employed as a substrate, both Candida utilis and Brettanomyces intermedius exhibited a substantial increase in polyphosphate levels. These observations suggested a dual role for polyphosphate in yeasts both as a phosphate and an energy store. The cytoplasmic pH in C. utilis displayed characteristic responses to metabolic changes during glucose degradation. B. intermedius experienced a strong cytoplasmic acidification upon ethanol utilization due to acetic acid formation. The mechanism of transport of Pi across the vacuolar membrane in C. utilis appeared to be different from that reported for the plasma membrane.

Dynamics of pyrimidine deoxynucleoside triphosphate pools in relationship to DNA synthesis in 3T6 mouse fibroblasts.

The 3H-labeled nucleosides cytidine, deoxycytidine, and thymidine are rapidly incorporated into DNA via dCTP or dTTP pools. Between 30 and 60 min after addition of tracer amounts of a labeled nucleoside to the medium of rapidly growing 3T6 cells, dNTP pools attained a constant specific activity resulting from a steady-state equilibrium between incorporation of nucleoside, de novo synthesis, and linear incorporation of isotope into DNA. Removal of labeled deoxycytidine or thymidine depleted the dNTP pools of isotope within a few minutes and incorporation into DNA stopped. When de novo synthesis of dTTP was blocked with amethopterin, the intracellular dTTP pool rapidly reached the specific activity of thymidine of the medium and isotope incorporation into DNA then measured absolute rates of DNA synthesis. In experiments with and without amethopterin, we found no kinetic evidence for the existence of more than one dTTP pool and the decay of the pool suggested that all dTTP served as precursor of DNA. In contrast, experiments with deoxycytidine and cytidine suggested the presence of separate dCTP pools with preferential DNA synthesis from the pool labeled from cytidine.

Proton NMR detection of acetylcholine status in synaptic vesicles.

The storage granules of cholinergic nerve terminals, the synaptic vesicles, have a central role in the mechanism of cholinergic transmission, and direct evidence as to whether acetylcholine is stored in them in a free or partially immobilized form is therefore of interest. In some secretory systems it has become evident that low molecular weight secretion products are stored within the granules as complexes with proteins or proteoglycans. The application of high resolution NMR spectroscopy to isolated granules has recently elucidated the internal structure of secretory granules. We report here that proton NMR analysis of isolated cholinergic synaptic vesicles from Torpedo marmorata allows the identification of acetylcholine within the organelle. The spectrum shows that all the acetylcholine is dissolved in an essentially fluid phase within the core. Vesicular and uncompartmentalized acetylcholine can be distinguished from each other and in addition, any hydrolysis to choline and acetate can be monitored. The results open the possibility of studying dynamics of acetylcholine pools and their breakdown in cholinergic tissue by a direct and non-perturbing method.

A Simulation Model of Primary Production and Carbon Allocation in the Creosotebush (Larrea Tridentata [DC] COV.)

A preliminary simulation model of primary productivity and carbon allocation in creosotebush (Larrea tridentata [DC] Cov.) is described. The model utilizes a systems approach in which movement of assimilate within the plant is in response to changes in source—sink strengths of leaves, stems, roots, early reproductive buds, maturing reproductive buds, flowers, and fruits. Two distinct compartments are defined per organ or developmental stage to separate assimilate into a pool fraction (labile or translocatable) and a structural fraction (nonlabile). The changes in magnitude (within upper and lower limits) of a pool compartment during the course of a simulation (i.e., growth and development of the plant) are a function of the rates of maintenance respiration and growth as well as a priority scheme governing allocation of assimilates; the increases and decreases in dry weight of a structural compartment are a function of aging and the magnitude of its associated pool (which determines structural growth and physiological death). A 1—yr simulation of a hypothetical Larrea plant shows that the model exhibits a reasonable behavior, although no validation is attempted at this stage in its development. The heuristic value of the model is illustrated in the sensitivity analysis, which shows the need for detailed knowledge of priority carbon movement during both vegetative and reproductive growth periods, the importance of substrate controlled respiration rates, and the need for further studies of the dynamics of labile pools in the plant. The model has been an excellent tool in our initial attempt to integrate the voluminous information on Larrea into a complete functional description of the autecology of the species. Further refinement of this model as data from our current research and that of other investigators become available should lead us to be better understanding of the ecological role of Larrea in desert ecosystems.