Phosphorylation of ATP citrate lyase in response to glucagon.

Incubation of hepatocytes with [32P]orthophosphate resulted in the incorporation of 32P into material that is precipitated by reaction with antibodies to ATP citrate lyase. The amount of radioactivity precipitated was decreased when unlabeled, purified ATP citrate lyase was added to extracts of hepatocytes that had been incubated with [32P]orthophosphate. Addition of glucagon to hepatocytes that had been preincubated with [32P]orthophosphate resulted in a 56% increase in acid-stable 32P in the trichloroacetic acid-insoluble portion of immunoprecipitates. Catalytic phosphate bound to ATP citrate lyase reaction with ATP and Mg2+ is acid-labile; thus, glucagon-dependent phosphorylation is distinguished from the catalytic phosphate. When hepatocytes were incubated in the absence of [32P]orthophosphate and extracted in a medium containing [gamma-32P]ATP, no acid-stable 32P was present in immunoprecipitates. This indicates that the incorporation into ATP citrate lyase of acid-stable phosphate occurs prior to extraction of the enzyme. Preliminary studies, using a procedure that allows for measurement of enzyme activity starting 1 min after beginning the extraction of lyase from hepatocytes, have shown no difference in lyase activity when hepatocytes are treated with or without glucagon.     

Microbial transformation of 14C-labeled 2,4,6-trinitrotoluene in an activated-sludge system

The fate of 14C-labeled 2,4,6-trinitrotoluene (TNT) in an activated-sludge system was investigated. No [14C]TNT could be detected in the contents of an aerated reactor after 3 to 5 days of incubation. No significant 14CO2 was formed, and the radioactivity was about equally divided between the floc and the supernatant. The radioactive carbon present in the microflora was mainly associated with the lipid and protein components, but the characteristic constituents of these compounds (e.g., fatty acids and amino acids) were not radioactive. The major part of the 14C present in the lipid and protein fractions was found in precipitates that formed in both fractions. The solubility properties and infrared spectra of these precipitates suggested that they are macromolecular structures of the polyamide type formed by the reaction of TNT biotransformation products with lipids, fatty acids, and protein constituents of the microbial flora. This hypothesis is further supported by the correspondence of the infrared spectrum of the lipid precipitate with that of a model compound synthesized from TNT transformation products and lipid precursors. The resistance of these macromolecules to further biodegradation was paralleled by the reported resistance to microbial attack of polyamides containing similar linkages.     

Microbial transformation of 14C-labeled 2,4,6-trinitrotoluene in an activated-sludge system.

The fate of 14C-labeled 2,4,6-trinitrotoluene (TNT) in an activated-sludge system was investigated. No [14C]TNT could be detected in the contents of an aerated reactor after 3 to 5 days of incubation. No significant 14CO2 was formed, and the radioactivity was about equally divided between the floc and the supernatant. The radioactive carbon present in the microflora was mainly associated with the lipid and protein components, but the characteristic constituents of these compounds (e.g., fatty acids and amino acids) were not radioactive. The major part of the 14C present in the lipid and protein fractions was found in precipitates that formed in both fractions. The solubility properties and infrared spectra of these precipitates suggested that they are macromolecular structures of the polyamide type formed by the reaction of TNT biotransformation products with lipids, fatty acids, and protein constituents of the microbial flora. This hypothesis is further supported by the correspondence of the infrared spectrum of the lipid precipitate with that of a model compound synthesized from TNT transformation products and lipid precursors. The resistance of these macromolecules to further biodegradation was paralleled by the reported resistance to microbial attack of polyamides containing similar linkages.     

Photohydration of testosterone and 4-androstene-3,17-dione in aqueous solution.

Irradiation of testosterone, 4-androstene-3,17-dione, or their "half-molecule" 4,4a,5,6,7,8-hexahydro-4a-methyl-2(3H)-naphthalenone in dilute aqueous solutions with ultraviolet light of 254 nm wavelength caused rapid addition of water across the olefinic bond with formation of 5,17 beta-dihydroxy-5 alpha-androstan-3-one, 5-hydroxy-5 alpha-androstane-3-17-dione, and 9-hydroxy-10-methyl-2-decalone, respectively. Time-lapse spectrometry in the ultraviolet region showed that the photohydration of the androgenic steroids was extremely efficient and virtually free of the side reactions. Preparative photolytic reactions carried out in water-methanol solutions allowed isolation and characterization of photoproducts.     

Adult movement of the native holly leafminer,Phytomyza ilicicola Loew (Diptera: Agromyzidae): consequences for host choice within and between habitats

Summary Phytomyza ilicicola is a specialist herbivore, the larvae of which mine, and the adults of which feed and oviposit on the developing leaves of American Holly, Ilex opaca. Adult Phytomyza have been shown to discriminate among host plants on the basis of several host and habitat factors. Such discrimination may explain, in part, the observed density variation of larval Phytomyza among individual trees and between forest and suburban habitats. In order for discrimination to influence density, adult Phytomyza must be sufficiently vagile to move among habitats and to encounter many hosts. We experimentally monitored the movement of adults to ask if this were so. In addition we asked whether inter-host movements were altered by resource concentration, and whether vagility offered an escape from parasitism. To answer these questions, three isolated holly trees to serve as colonization sources were selected in early spring, prior to adult emergence. Potted target trees were then placed singly and in groups of four at 3 compass directions around and at 3 different distances (10, 25, and 50 m) from each of the source trees. Six weeks after exposure to adult Phytomyza, the number of feeding punctures and first instar larvae were counted. Clones were then overwintered in cold frames and in early spring, just prior to adult emergence, all pupae were examined for parasitism by the dominant parasitoid Opius striatriventris. Feeding and parasitism were similar at all 3 distances from the source trees, indicating that Opius was at least as vagile as Phytomyza. However, first instar larvae decreased with distance. There was no evidence that resource concentration affected adult movement. Our results suggest that Phytomyza are sufficiently vagile to choose among hosts within and between habitats. However, movement does not translate into equally high oviposition at all distances from a source, nor does it provide an escape from parasitism by Opius.     

Amphitropic proteins: regulation by reversible membrane interactions (review).

What do Src kinase, Ras-guanine nucleotide exchange factor, cytidylyltransferase, protein kinase C, phospholipase C, vinculin, and DnaA protein have in common? These proteins are amphitropic, that is, they bind weakly (reversibly) to membrane lipids, and this process regulates their function. Proteins functioning in transduction of signals generated in cell membranes are commonly regulated by amphitropism. In this review, the strategies utilized by amphitropic proteins to bind to membranes and to regulate their membrane affinity are described. The recently solved structures of binding pockets for specific lipids are described, as well as the amphipathic alpha-helix motif. Regulatory switches that control membrane affinity include modulation of the membrane lipid composition, and modification of the protein itself by ligand binding, phosphorylation, or acylation. How does membrane binding modulate the protein's function? Two mechanisms are discussed: (1) localization with the substrate, activator, or downstream target, and (2) activation of the protein by a conformational switch. This paper also addresses the issue of specificity in the cell membrane targetted for binding.     

Second virial coefficient studies of cosolvent-induced protein self-interaction

Protein self-interaction is important in protein crystal growth, solubilization, and aggregation, both in vitro and in vivo, as with protein misfolding diseases, such as Alzheimer's. Although second virial coefficient studies can supply invaluable quantitative information, their emergence as a systematic approach to evaluating protein self-interaction has been slowed by the limitations of traditional measurement methods, such as static light scattering. Comparatively, self-interaction chromatography is an inexpensive, high-throughput method of evaluating the osmotic second virial coefficient (B) of proteins in solution. In this work, we used self-interaction chromatography to measure B of lysozyme in the presence of various cosolvents, including sucrose, trehalose, mannitol, glycine, arginine, and combinations of arginine and glutamic acid and arginine and sucrose in an effort to develop a better fundamental understanding of protein self-interaction in complex cosolvent systems. All of these cosolvents, alone or in combination, increased B, indicating a reduction in intermolecular attraction. However, the magnitude of cosolvent-induced changes in B was found to be largely dependent on the ability to control long-range electrostatic repulsion. To the best of our knowledge, this work represents the most comprehensive virial coefficient study to date focusing on complex cosolvent-induced effects on the self-interaction of lysozyme.     

Orientation dependence of NMR relaxation time, T(1rho), in lipid bilayers

The orientation dependence of the low frequency NMR relaxation time, T(1rho), of protons in aligned phospholipid bilayers was measured using 13C cross polarisation and direct proton experiments. The contribution of intra- and inter-molecular interactions to proton T(1rho) was determined by using dimyristoyl phosphatidylcholine (DMPC) with one hydrocarbon chain deuterated and dispersed in perdeuterated DMPC. The results indicated that intramolecular motions on the kHz timescale were the major cause of T(1rho) relaxation in phospholipid bilayers.