Initiation of DNA synthesis by human thrombin: relationships between receptor binding, enzymic activity, and stimulation of 86Rb+ influx

Stimulation of amiloride-sensitive sodium (Na+) influx and the subsequent activation of NA+, K+-ATPase by serum or growth factors have been implicated as early events leading to initiation of cell proliferation. We recently demonstrated that amiloride inhibits thrombin-initiated DNA synthesis not by inhibiting an early event occurring during the first 8 hr, but rather by inhibiting some later event 8 to 12 hr after thrombin addition. To further probe the relationship between stimulation of ion influx and initiation of cell proliferation, human alpha-thrombin was converted to gamma-thrombin, nitro-alpha-thrombin, and diisopropylphospho (DIP)-alpha-thrombin. These derivatives retain either the capacity to bind cell surface alpha-thrombin receptors or thrombin esterase activity, but they do not initiate DNA synthesis. At low concentrations of alpha-thrombin or the various thrombin derivatives, only alpha-thrombin stimulates 86Rb+ influx, suggesting a correlation between stimulation of influx and the ability of these derivatives to initiate DNA synthesis. Concentrations of a DIP-alpha-thrombin that saturate the alpha-thrombin receptors (up to 2 micrograms/ml) do not stimulate either the early or late influx of 86Rb+, indicating that DIP-alpha-thrombin binding alone is not sufficient to stimulate ion fluxes. High concentrations of either gamma-thrombin or nitro-alpha-thrombin, however, stimulate both early and late 86RB+ uptake but do not initiate DNA synthesis. These results demonstrate that events leading to both the early and late stimulation of 86Rb+ influx by themselves are not sufficient to initiate cell proliferation. Thus, initiation may require a combination of events that can be independently regulated by different transmembrane signals.     

Sulfate inhibition of molybdate assimilation by planktonic algae and bacteria: some implications for the aquatic nitrogen cycle

Molybdenum is required for both dinitrogen fixation and nitrate assimilation. In oxic waters the primary form of molybdenum is the molybdate anion. Using radioactive [⁹⁹Mol Na₂MoO₄, we have shown that the transport of molybdate by a natural assemblage of freshwater phytoplankton is light-dependent and follows typical saturation kinetics. The molybdate anion is strikingly similar to sulfate and we present data to show that sulfate is a competitive inhibitor of molybdate assimilation by planktonic algae and bacteria. The ability of freshwater phytoplankton to transport molybdate is inhibited at sulfate concentrations as low as 5% of those in seawater and at sulfate: molybdate ratios as low as 50 to 100 times lower than those found in seawater, Similarly, the growth of both a freshwater bacterium and a saltwater diatom was inhibited at sulfate: molybdate ratios lower than those in seawater.The ratio of sulfate to molybdate is 10 to 100 times greater in seawater than in fresh water. This unfavorable sulfate: molybdate ratio may make molybdate less biologically available in the sea. The sulfate: molybdate ratio may explain, in part, the low rates of nitrogen fixation in N-limited salt waters.     

Amiloride-inhibited Na+ uptake into toad bladder microsomes is Na+-H+ exchange

Amiloride-inhibited Na+ transport into toad urinary bladder microsomes is sensitive to a pH gradient across the vesicular membrane. The magnitude of the gradient was measured directly with acridine orange. Also Na+ could stimulate amiloride-sensitive proton efflux from the microsomes. These results indicated that the transport process was Na+-H+ exchange.     

Partial characterization of chitinolytic enzymes from Streptomyces albidoflavus

Streptomyces albidoflavus NRRL B-16746 secreted three types of chitinolytic enzymes: N -acetyl-glucosaminidase, chitobiosidase and endochitinase. Optimal activity for all three types of enzymes occurred at pH 4–6; however 55–74% of the chitobiosidase and endochitinase activity was detectable at pH 8–10. Chitobiosidase activity originated from two strongly acidic (pI < 3.0) proteins with molecular mass of 27 kDa and 34 kDa, while endochitinase activity originated from five major acidic proteins (pI 5.1, 5.3, 5.75, 5.8–5.9 and 6.4) with molecular mass of 59, 45, 38.5, 27 and 25.5 kDa. Purified chitobiosidases significantly reduced spore germination and germ tube elongation of Botrytis cinerea and Fusarium oxysporum. Chitinolytic enzymes with significant activity at pH 4–10 may be used, transgenically, to reduce the growth and/or development of a broad spectrum of insects and fungi that are major economic pests.     

Cholesterol stimulation of penetration of unilamellar liposomes by hydrophobic compounds

Summary The incorporation of cholesterol into unilamellar liposomes greatly increased the transmembranous movement of hydrophobic ionophores such as nigericin. In reconstituted liposomes containing rhodopsin as the only protein, the presence of cholesterol lowers by 10-fold or more the amount of nigericin required to eliminate the light-driven proton gradient. These effects are seen both above and below the transition temperature of the phospholipid used for reconstitution.Cholesterol similarly increases the ability of A-23187, 1799, or NH₄SCN to collapse the proton gradient of bacteriorhodopsin vesicles. Cholesterol also lowers the concentration of nigericin or valinomycin required for a rapid translocation of Rb⁺ into protein-free liposomes. It also lowers the concentration of A-23187 required for the release of Ca⁴⁵ trapped in protein-free liposomes. In contrast to these observations and in confirmation of previous findings, we observed that cholesterol decreased the permeability of liposomes for glucose. Thus the effects of cholesterol on the permeability of the membrane vary with the chemical nature of the permeating compounds. We have also confirmed that in multilamellar liposomes cholesterol decreases the permeability of Rb⁺ in the presence of valinomycin. It therefore appears that the effect of cholesterol changes with the size and structural features of the model membranes.     

Influence of estuarine sediment on virus survival under field conditions.

The survival of poliovirus 1 (LSc) and echovirus 1 (Farouk) in estuarine water and sediment was studied in Galveston Bay, Texas. Viruses were suspended in estuarine water and sediment both in dialysis tubing and in chambers constructed with polycarbonate membrane walls. Virus inactivation rates in seawater were similar in both types of chambers. Virus adsorption to sediment greatly increased survival time. The time required to inactivate 99% (T-99) of poliovirus increased from 1.4 days in seawater alone to 6.0 days for virus adsorbed to sediment at a relatively nonpolluted site. At a more polluted site, poliovirus T-99 was increased from approximately 1 h to 4925 days by virus adsorption to sediment. This study demonstrates that under field conditions virus association with estuarine sediment acts to prolong its survival in the marine environment.     

31P NMR studies of metabolism in Acanthamoebacastellanii: Polyphosphate release from encysted cells

${}^{31}\text{P NMR}$ studies of $\text{Acanthamoeba}\text{castellanii}$ have shown that encysting cells release polyphosphate into the encystment medium. Mature cysts contain low levels of polyphosphate, as do vegetative cells. Young cysts (7 days) show detectable levels of nucleotide diphosphates and triphosphate similar to those observed in vegetative cells. Mature cysts (90 days) show only excreted polyphosphate as well as a component which has a chemical shift of a phosphodiester. The inorganic phosphate peak in the cyst shows that the cyst milieu is liquid-like and that the intracellular environment maintains a pH between 6 and 7.5 in the presence of extracellular values from 4 to 9.     

Genome sequence of the clover-nodulating Rhizobium leguminosarum bv. trifolii strain TA1

Rhizobium leguminosarum bv. trifolii strain TA1 is an aerobic, motile, Gram-negative, non-spore-forming rod that is an effective nitrogen fixing microsymbiont on the perennial clovers originating from Europe and the Mediterranean basin. TA1 however is ineffective with many annual and perennial clovers originating from Africa and America. Here we describe the features of R. leguminosarum bv. trifolii strain TA1, together with genome sequence information and annotation. The 8,618,824 bp high-quality-draft genome is arranged in a 6 scaffold of 32 contigs, contains 8,493 protein-coding genes and 83 RNA-only encoding genes, and is one of 20 rhizobial genomes sequenced as part of the DOE Joint Genome Institute 2010 Community Sequencing Program.