Course and extent of variation of equine infectious anemia virus during parallel persistent infections.

Comparisons of peptide and oligonucleotide maps of glycoproteins and RNA from nine isolates of equine infectious anemia virus (EIAV) that were generated during parallel infections of two Shetland ponies revealed that each isolate was structurally unique. Each EIAV isolate contained a unique subset of variant peptides, oligonucleotides, or both, indicating that structural variation in EIAV is a random and noncumulative process and that a large spectrum of possible EIAV variants can be generated in infected animals.     

Action mechanism of Escherichia coli DNA photolyase. I. Formation of the enzyme-substrate complex

Escherichia coli DNA photolyase (photoreactivating enzyme) is a flavoprotein. The enzyme binds to DNA containing pyrimidine dimers in a light-independent step and, upon illumination with 300-600 nm radiation, catalyzes the photosensitized cleavage of the cyclobutane ring thus restoring the integrity of the DNA. We have studied the binding reaction using the techniques of nitrocellulose filter binding and flash photolysis. The enzyme binds to dimer-containing DNA with an association rate constant k1 estimated by two different methods to be 1.4 X 10(6) to 4.2 X 10(6) M-1 S-1. The dissociation of the enzyme from dimer-containing DNA displays biphasic kinetics; for the rapidly dissociating class of complexes k2 = 2-3 X 10(-2) S-1, while for the more slowly dissociating class k2 = 1.3 X 10(-3) to 6 X 10(-4) S-1. The equilibrium association constant KA, as determined by the nitrocellulose filter binding assay and the flash photolysis assay, was 4.7 X 10(7) to 6 X 10(7) M-1, in reasonable agreement with the values predicted from k1 and k2. From the dependence of the association constant on ionic strength we conclude that the enzyme contacts no more than two phosphodiester bonds upon binding; this strongly suggests that the pyrimidine dimer is the main structural determinant of specific photolyase-DNA interaction and that nonspecific ionic interactions do not contribute significantly to substrate binding.     

Testosterone inhibits cAMP-induced de Novo synthesis of Leydig cell cytochrome P-450(17 alpha) by an androgen receptor-mediated mechanism

The regulation of the novo synthesis of the microsomal cytochrome P-450 enzyme, P-450(17 alpha), was studied in mouse Leydig cell cultures. Chronic treatment with 0.05 mM 8-Br-cAMP (cAMP) caused a time-dependent increase in 17 alpha-hydroxylase activity and in the amount of P-450(17 alpha), quantitated by immunoblotting. This increase in both activity and amount was enhanced by inhibiting testosterone production with aminoglutethimide, an inhibitor of cholesterol side-chain cleavage or SU 10603, an inhibitor of 17 alpha-hydroxylase. To examine the mechanism by which cAMP or cAMP plus inhibitors of testosterone production increased the activity and amount of P-450(17 alpha), changes in the rate of de novo synthesis were studied by measuring [35S]methionine incorporation into newly synthesized protein. Treatment with cAMP plus aminoglutethimide or SU 10603 caused a 2-fold or greater increase in the rate of de novo synthesis of P-450(17 alpha) compared to treatment with cAMP only. The addition of exogenous testosterone reversed this increase in the rate of synthesis, indicating that testosterone modulates the extent of cAMP-stimulated induction of P-450(17 alpha). This negative effect of testosterone could be mimicked by the addition of the androgen agonist, mibolerone, and prevented by the addition of the antiandrogen, hydroxyflutamide. Neither estradiol nor dexamethasone had any effect on the synthesis of P-450(17 alpha). Studies on the degradation of newly synthesized P-450(17 alpha) demonstrated that testosterone had no effect on the decay of P-450(17 alpha) during the first 24 h but caused a significant increase in the rate of decay between 24 and 48 h. These data indicate that testosterone produced during cAMP induction of P-450(17 alpha) negatively regulates the amount of this cytochrome P-450 enzyme by two distinct mechanisms: by repressing cAMP-induced synthesis of P-450(17 alpha) by an androgen receptor-mediated mechanism and by increasing the rate of degradation of P-450(17 alpha). A model is proposed for the regulation of P-450(17 alpha) in Leydig cells.     

Escherichia coli tryptophan synthase: synthesis of catalytically competent alpha subunit in a cell-free system containing preacylated tRNAs

A cell-free protein biosynthesizing system prepared from Escherichia coli CF300 was found to synthesize E. coli tryptophan synthase alpha subunit in a time-dependent manner when programmed with pBN69 plasmid DNA. This plasmid contains the trp promoter from Serratia marcescens adjacent to the coding region of E. coli tryptophan synthase alpha protein [Nichols, B.P., & Yanofsky, C. (1983) Methods Enzymol. 101, 155-164]. The synthesized tryptophan synthase alpha subunit was found to be indistinguishable from authentic alpha subunit protein when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and to have the same specific activity for catalyzing the conversion of indole----L-tryptophan by tryptophan synthase beta 2 subunit, as well as the conversion of indole + glyceraldehyde 3-phosphate to indole-3-glycerol phosphate. In the absence of exogenously added phenylalanine, admixture of E. coli phenylalanyl-tRNAPhe to the protein biosynthesizing system stimulated the production of functional alpha protein; the analogous result was obtained when valine was replaced by E. coli valyl-tRNAVal. The ability of a misacylated tRNA to participate in alpha protein synthesis in this system was established by the use of E. coli phenylalanyl-tRNAVal in the absence of added valine. Protein biosynthesis proceeded normally and gave a product having the approximate molecular weight of tryptophan synthase alpha subunit; as expected, this polypeptide lacked catalytic activity.     

Transformation ofAspergillus flavus to study aflatoxin biosynthesis

Aflatoxin contamination of agricultural commodities continues to be a serious problem in the United States. Breeding for resistant genotypes has been unsuccessful and detoxification of food sources is not economically feasible. New strategies for control may become apparent once more is known about the biosynthesis and regulation of aflatoxin. Although the biosynthetic pathway of aflatoxin has been extensively studied, little is known about the regulation of the individual steps in the pathway. We have developed a genetic transformation system forAspergillus flavus that provides a new and expedient approach to studying the biosynthesis of aflatoxin and its regulation. Through the use of this genetic transformation system, genes for aflatoxin biosynthesis can be identified and isolated by the complementation of aflatoxin negative mutants. In this paper we discuss molecular strategies for studying the regulation and biosynthesis of aflatoxin.     

Genetic transformation system for the aflatoxin-producing fungus Aspergillus flavus.

A heterologous transformation system was developed for Aspergillus flavus with efficiencies greater than 20 stable transformants per micrograms of DNA. Protoplasts of uracil-requiring strains of the fungus were transformed with plasmid and cosmid vectors containing the pyr-4 gene of Neurospora crassa. Transformants were selected for their ability to grow and sporulate on medium lacking uracil. Vector DNA appeared to integrate randomly into the genome of A. flavus with a tendency for multiple, tandem insertion. Transformants with single or multiple insertions were stable after five consecutive transfers on medium containing uracil. Uracil-requiring recipient strains were obtained either by UV-irradiating conidia and selecting colonies resistant to 5-fluoroorotic acid or by transferring the mutated pyr locus to strains by parasexual recombination. This is the first report of a transformation system for an aflatoxin-producing fungus. The transformation system and the availability of aflatoxin-negative mutants provide a new approach to studying the biosynthesis and regulation of aflatoxin.     

Nitrite reduction in Bacteroids of Rhizobium “hedysari” strain HCNT 1

Ex planta, bacteroids of the sulla-symbiont Rhizobium hedysari strain HCNT 1 terminated reduction of nitrite at nitrous oxide irrespective of the presence or absence of acetylene. Nitrate was not reduced during the experimental period, but slight nitrate reductase activity occurred if incubation with nitrate was prolonged (up to 15 h). As was observed in free-living cells, exposure of the bacteroids to the metal chelator, diethyldithiocarbamate, prevented reduction of nitrite, indicating the presence of a copper-containing nitrite reductase. Pulses of 10–75 M nitrite transiently impeded O₂ uptake in bacteroids, which resumed consumption of O₂ when the nitrite had been reduced. Exposure to >1.0 mM nitrite for 24h greatly inhibited nitrogenase activity (assayed as acetylene reduction activity) of bacteroids in planta. Exposure to the same concentrations of nitrite after 1h of incubation in the presence of acetylene almost completely stopped ongoing ethylene production in bacteroids of strain HCNT 1 extracted from nodules. Free cells of the non-nitrite-reducing R. hedysari strain CC 1335 were lacking in nitrogenase (acetylene-reduction) activity, whereas identically cultured (low-oxygen) strain HCNT 1 cells reduced both nitrite and acetylene.Abbreviations PMS phenazine methosulfate - DDC diethyldithiocarbamate     

Did medicated salt hasten the spread of chloroquine resistance in Plasmodium falciparum?

With acknowledged difficulties in achieving satisfactory compliance rates for the large-scale delivery of many antiporositic drugs, the use of medicated salt has often been seen as a useful way to improve the level o f treatment in target populations. Iodinated salt is said to have contributed to a decline in endemic goitre, and salt medicated with chloroquine and/or other antimalorials, or with diethylcarbomazine, has been widely used in public health programmes against malaria and filariosis respectively. In this article, however, David Payne suggests that chloroquinized salt programmes may have been a major factor in promoting chloroquine resistance in Plasmodium falciparum.     

Comparative permeability of canine visceral and parietal pleura

To determine the permeability of canine pleural mesothelium, visceral and intercostal parietal pleura from mongrel dogs was carefully stripped from the underlying tissue and mounted as a planar sheet in a Ussing-type chamber. The hydraulic conductivity (Lp) was determined from the rate of volume flux in response to hydrostatic pressure gradients applied to either the mucosal or serosal surface of the pleural membrane. The diffusional permeability (Pd) of radiolabeled water, sucrose, inulin, and albumin was determined under equilibrium conditions from the unidirectional tracer flux. The Lp of the visceral pleura was 0.39 +/- 0.032 (SE) X 10(-4) ml.s-1.cmH2O-1.cm-2 and that Lp of parietal pleura was 1.93 +/- 0.93 X 10(-4) ml.s-1.cmH2O-1.cm-2 (P less than 0.001). The Pd of the visceral pleura ranged from 12.21 +/- 0.45 X 10(-4) cm/s for 3H2O to 0.34 +/- 0.03 X 10(-4) cm/s for [3H]albumin. The Pd of the parietal pleura for water and sucrose was similar to that of the visceral membrane, whereas its Pd for the larger inulin and albumin molecules was greater than that of visceral pleura (P less than 0.01). A spontaneous potential difference could not be detected across either membrane. The relatively higher parietal pleural Lp and Pd for larger solutes is probably due to the presence of stomata in this membrane. These results indicate that both the parietal and the visceral pleura are extremely permeable tissues which offer little resistance to water and solute flux.     

The localization of calcium release by inositol trisphosphate in Limulus photoreceptors and its control by negative feedback

Microvillar photoreceptors of invertebrates exhibit a light-induced rise in the intracellular concentration of free calcium (Cai) that results in part from release of calcium from an intracellular compartment. This light-induced release of calcium appears to result from a cascade of reactions that involve rhodopsin, a GTP-binding protein and a phospholipase-C which releases inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) from the plasma membrane; the Ins(1,4,5)P3 acts to release calcium from smooth endoplasmic reticulum. In the ventral photoreceptor of the horseshoe crab Limulus polyphemus not all of the endoplasmic reticulum is subject to calcium release by Ins(1,4,5)P3. Only endoplasmic reticulum in the light-sensitive region of the cell is competent to release calcium in response to Ins(1,4,5)P3. The release of calcium by Ins(1,4,5)P3 in ventral photoreceptors appears to be subject to feedback inhibition through elevated Cai. We suggest that this feedback inhibition contributes to sensory adaptation in the photoreceptor and may account for oscillatory membrane responses sometimes observed with large injections of Ins(1,4,5)P3.