Enhancement by Cytidine of Membrane Phospholipid Synthesis

Cytidine, as cytidine 5'-diphosphate choline, is a major precursor in the synthesis of phosphatidylcholine in cell membranes. In the present study, we examined the relationships between extracellular levels of cytidine, the conversion of [14C]choline to [14C]phosphatidylcholine, and the net syntheses of phosphatidylcholine and phosphatidylethanolamine by PC12 cells. The rate at which cytidine (as [3H]cytidine) was incorporated into the PC12 cells followed normal Michaelis-Menten kinetics (Km = 5 microM; Vmax = 12 x 10(-3) mmol/mg of protein/min) when the cytidine concentrations in the medium were below 50 microM; at higher concentrations, intracellular [3H]cytidine nucleotide levels increased linearly. Once inside the cell, cytidine was converted mainly into cytidine triphosphate. In pulse-chase experiments, addition of cytidine to the medium caused a time- and dose-dependent increase (by up to 30%) in the incorporation of [14C]choline into membrane [14C]-phosphatidylcholine. When the PC12 cells were supplemented with both cytidine and choline for 14 h, small but significant elevations (p less than 0.05) were observed in their absolute contents of membrane phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine, all increasing by 10-15% relative to their levels in cells incubated with choline alone. Exogenous cytidine, acting via cytidine triphosphate, can thus affect the synthesis and levels of cell membrane phospholipids.     

Increased Neopterin Levels in Brains of Patients with Human Immunodeficiency Virus Type 1 Infection

Postmortem levels of native neopterin (D-erythro-neopterin) were measured in cerebral cortical samples from 44 human immunodeficiency virus type 1-infected and eight uninfected, nonneurological control patients. Cerebral cortical gray and white matter neopterin levels for the controls ranged from 0.5 to 7.2 pmol/mg of protein in contrast to neopterin levels in brains of the virus-infected patients, which frequently were more than threefold and occasionally more than 30-fold higher than mean control levels. Cortical neopterin levels did not correlate with severity of the acquired immunodeficiency syndrome dementia complex, but subcortical levels correlated with the presence of active human immunodeficiency virus type 1 infection, as reflected by pathological evidence of multinucleated giant cell encephalitis. Evidence of opportunistic cytomegalovirus infections in approximately 25% of the human immunodeficiency virus type 1-infected patients was associated with enhanced levels of neopterin in frontal cortex.     

Multiple components of a complex androgen-dependent enhancer.

Sex-limited protein (Slp) is expressed in adult male mice. A 160-basepair fragment 2 kilobases upstream of the gene serves as an androgen-dependent enhancer of chloramphenicol acetyltransferase expression in transient transfection assays in cells with endogenous or cotransfected androgen receptor. One element that is necessary, but not sufficient, for induction is a consensus glucocorticoid (or hormone) response element (HRE). This element binds to the mouse androgen receptor in vitro, but with apparent weak affinity. Induction by the HRE is greatly augmented by an accessory sequence within the 160 basepairs, suggesting that cooperative interactions confer strong response to androgen. Additional elements within the enhancer modulate induction, positively or negatively, and exhibit cell-specific behavior. Of particular interest are two degenerate HREs that are adjacent to the consensus sequence; they show no independent activity, but are functionally significant in conjunction with other elements. The complexity of this enhancer may reflect biological mechanisms that ensure specificity of hormonal response and allow gene expression to respond to changes in hormone concentration.     

Expression of a group II phospholipase A2 from the venom of Agkistrodon piscivorus piscivorus in Escherichia coli: Recovery and renaturation from bacterial inclusion bodies

A synthetic gene encoding the Group 11 phospholipase A₂ (PLA₂) from the venom of Agkistrodon piscivorus piscivorus has been constructed and expressed with high efficiency in Escherichia coli. No enzymatic activity was recovered when the polypeptide contained the initiator Met residue. Replacement of an Asn residue penultimate to the initiator Met with Ser or Gly permitted removal of the initiator Met by the endogenous methionine aminopeptidase. The amino-terminal serine (N-Ser) and amino-terminal glycine PLA₂'s were isolated from intracellular inclusion bodies and were renatured with 25% recovery. Automated Edman degradation confirmed the removal of the initiator Met and confirmed the sequence of the first 40 residues of N-Ser PLA₂. The recombinant proteins were purified to apparent homogeneity and showed the same specific activity as the wild-type protein. N-Ser PLA₂ demonstrated the same kinetics of activation as the wild type enzyme on large vesicles of zwitterionic lipid.     

Composting of explosives and propellant contaminated soils under thermophilic and mesophilic conditions

Summary Composting was investigated as a bioremediation technology for clean-up of sediments contaminated with explosives and propellants. Two field demonstrations were conducted, the first using 2,4,6-trinitrotoluene (TNT), octahydro-1,3,5,7-tetranitro-1,3,5,7-tetraazocine (HMX), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), and N-methyl-N,2,4,6-tetranitroaniline (tetryl) contaminated sediment, and the second using nitrocellulose (NC) contaminated soil. Tests were conducted in thermophilic and mesophilic aerated static piles. Extractable TNT was reduced from 11840 mg/kg to 3 mg/kg, and NC from 13090 mg/kg to 16 mg/kg under thermophilic conditions. Under mesophilic conditions, TNT was reduced from 11 190 mg/kg to 50 mg/kg. The thermophilic and mesophilic half-lives were 11.9 and 21.9 days for TNT, 17.3 and 30.1 days for RDX, and 22.8 and 42.0 days for HMX, respectively. Known nitroaromatic transformation products increased in concentration over the first several weeks of the test period, but decreased to low concentrations thereafter.     

Presence and binding characteristics of calcitriol receptors in human fetal gut

In the present study, we show for the first time the presence of calcitriol-specific binding sites in hypertonic extracts of cells isolated from human fetal small intestine and colon from 13-21 weeks of gestation. Woolf plot analysis of the binding characteristics revealed the presence of a single class of high affinity receptors. The presence of specific receptors for calcitriol in fetal intestine and colon opens interesting possibilities as to the role of this hormone in human gut development.     

Effect of crab effluent and scent of damaged conspecifics on feeding,growth, and shell morphology of the Atlantic dogwhelk Nucella lapillus (L.)

Juvenile Nucella lapillus of two different shell phenotypes, exposed shore and protected shore, were maintained in running seawater under each of three experimental conditions for 94 d: a) laboratory control, b) exposed to the effluent of crabs (Cancer pagurus) fed frozen fish (fish-crab), and c) exposed to the effluent of crabs fed live conspecific snails (snail-crab). Rates of barnacle consumption and rates of body weight change varied significantly between phenotypes and among experimental conditions. Individuals from the protected-shore consumed consistently fewer barnacles and grew consistently less than those from the exposed shore. Body weight increases in the fish-crab treatments were from 25 to 50% less than those in the controls and body weights in the snail-crab treatment either did not change or actually decreased. The perceived risk of predation thus appears to have a dramatic effect on the rates of feeding and growth of N. lapillus.At the end of the experiment, size-adjusted final shell weights for both phenotypes were consistently higher than controls (no crab) in both the fish-crab and snail-crab treatments. In addition, apertural tooth height, thickness of the lip, and retractability (i.e. the extent to which a snail could withdraw into its shell), with few exceptions all varied in an adaptive manner in response to the various risk treatments. Similar changes in the shell form of starved snails exposed to the same stimuli suggest very strongly that the morphological responses of both phenotypes were not just due to differences in rates of growth. These differences, at least in part, represented a direct cueing of the shell form of Nucella lapillus to differences in the perceived risk of predation. Somewhat surprisingly, the extent of phenotypic plasticity appeared to differ between the populations examined. Both field and laboratory evidence suggest that the exposed-shore population was much more labile morphologically than the protected-shore population.In many instances, particularly among starved snails, the development of antipredatory shell traits was greater in the fish-crab treatment than in the snail-crab treatment. Because the scent of crabs was present in both treatments, these results suggest a) that, at the frequency/concentration used in the experiments, the scent of damaged conspecifics may have been a supernormal stimulus and b) that the morphological response in these treatments might have been greater if the stimulus had been provided at a lower level.     

Inhibition and killing of fungi by the polyamine oxidase-polyamine system

Both components of the polyamine oxidase (PAO)-polyamine system are known to be present in phagocytes and have thus been postulated to contribute to the antimicrobial activity of these cells. Therefore, the effects of the PAO-polyamine system on three medically important opportunistic fungi were examined. Yeasts of Cryptococcus neoformans, but not Candida albicans blastoconidia or Aspergillus fumigatus conidia, were efficiently killed by the system. Two putative end products of the system, hydrogen peroxide and acrolein, both killed C. neoformans at concentrations attainable with the whole system. However, catalase failed to inhibit activity of the whole system, making hydrogen peroxide an unlikely mediator of killing. Although C. albicans blastoconidia and A. fumigatus conidia were not killed by the PAO-polyamine system, germ tube formation by the former, and hyphal growth by the latter, were markedly inhibited. These data establish that the PAO-polyamine system possesses antifungal activity.