A competition smFRET assay to study ligand-induced conformational changes of the dengue virus protease

Ligand binding to proteins often is accompanied by conformational transitions. Here, we describe a competition assay based on single molecule Förster resonance energy transfer (smFRET) to investigate the ligand-induced conformational changes of the dengue virus (DENV) NS2B-NS3 protease, which can adopt at least two different conformations. First, a competitive ligand was used to stabilize the closed conformation of the protease. Subsequent addition of the allosteric inhibitor reduced the fraction of the closed conformation and simultaneously increased the fraction of the open conformation, demonstrating that the allosteric inhibitor stabilizes the open conformation. In addition, the proportions of open and closed conformations at different concentrations of the allosteric inhibitor were used to determine its binding affinity to the protease. The K_D value observed is in accordance with the IC₅₀ determined in the fluorometric assay. Our novel approach appears to be a valuable tool to study conformational transitions of other proteases and enzymes.     

Effects of several insecticide formulations on oothecal drop and hatchability in German cockroaches (Dictyoptera: Blattellidae)

Formulations used for control of German cockroaches, Blattella germanica (L.), were investigated. The insecticides tested on gravid females were bendiocarb, chlorpyrifos, cyfluthrin, cypermethrin, fenvalerate, hydramethylnon, malathion, propetamphos, propoxur, and pyrethrins. Exposure to each insecticide increased the frequency of oothecal drop and reduced the percentage oothecal hatch. The percentage of oothecae that were dropped increased as a curvilinear function of insecticide concentration. An LD50 of propoxur to female German cockroaches resulted in the greatest oothecal drop (83.50%); fenvalerate caused the least drop (22.60%). LD50's of cypermethrin, propetamphos, and propoxur resulted in 29.60, 31.807, and 37.30% hatch from dropped oothecae, respectively. The smallest percentage hatch from retained oothecae was caused by LD50's of propoxur (1.5%) and cyfluthrin (7.70%). Retained oothecae from females treated with an LD50 of fenvalerate (68.70%) or pyrethrins (68.70%) had the greatest percentage hatch. Total percentage hatch (dropped and retained oothecae) declined exponentially as the insecticide concentration increased. An LD50 of cypermethrin limited total oothecal hatch to 24.50%, whereas LD50's of fenvalerate, malathion, and pyrethrins resulted in 53.60, 52.20, and 58.90% hatch, respectively. Mean time to oothecal hatch increased linearly with increasing insecticide concentration for all insecticides tested.     

Low concentrations of hydrogen sulphide alter monoamine levels in the developing rat central nervous system.

The central nervous system is one of the primary target organs for hydrogen sulphide (H2S) toxicity; however, there are limited data on the neurotoxic effects of low-dose chronic exposure on the developing nervous system. Levels of serotonin and norepinephrine in the developing rat cerebellum and frontal cortex were determined following chronic exposure to 20 and 75 ppm H2S during perinatal development. Both monoamines were altered in rats exposed to 75 ppm H2S compared with controls; serotonin levels were significantly increased at days 14 and 21 postnatal in both brain regions, and norepinephrine levels were significantly increased at days 7, 14, and 21 postnatal in cerebellum and at day 21 in the frontal cortex. Exposure to 20 ppm H2S significantly increased the levels of serotonin in the frontal cortex at day 21, whereas levels of norepinephrine were significantly reduced in the frontal cortex at days 14 and 21, and at day 14 in the cerebellum.     

X-ray solution scattering reveals conformational changes upon iron uptake in lactoferrin, serum and ovo-transferrins.

X-ray solution scattering has been used for studying the structural changes that take place upon uptake and release of iron from serum and chicken ovo-transferrin and human lactoferrin. In the case of chicken ovo-transferrin, data have been obtained for both the intact protein and the isolated N and C-lobes with and without iron. These studies reveal that both lobes undergo a change that is consistent with an opening of the inter-domain cleft when iron is removed from the protein. We suggest that the conformational change of the protein increases the specificity of receptor binding and that the closed configuration of the iron-loaded protein is one, or perhaps the, decisive step in the mechanism for receptor-mediated endocytosis.     

Diseases and parasites of red foxes, gray foxes, and coyotes from commercial sources selling to fox-chasing enclosures.

Fifty-six red foxes (Vulpes vulpes), 18 gray foxes (Urocyon cinereoargenteus), and 13 coyotes (Canis latrans) obtained by the South Carolina Wildlife and Marine Resources Department during an investigation of suspected illegal wildlife translocation were examined for diseases and parasites. Red foxes and coyotes were confiscated from an animal dealer based in Ohio (USA), and gray foxes were purchased from an animal dealer in Indiana (USA). Emphasis was placed on detection of pathogens representing potential health risks to native wildlife, domestic animals, or humans. All animals were negative for rabies; however, 15 gray foxes were incubating canine distemper at necropsy. Serologic tests disclosed antibodies to canine parvovirus, canine distemper virus, canine adenovirus, canine coronavirus, canine herpesvirus, and canine parainfluenza virus in one or more host species. Twenty-three species of parasites (two protozoans, three trematodes, four cestodes, eleven nematodes, and three arthropods) were found, including species with substantial pathogenic capabilities. Echinococcus multilocularis, a recognized human pathogen not enzootic in the southeastern United States, was found in red foxes. Based on this information, we conclude that the increasingly common practice of wild canid translocation for stocking fox-chasing enclosures poses potential health risks to indigenous wildlife, domestic animals, and humans and, therefore, is biologically hazardous.     

Expression of reg/PSP, a pancreatic exocrine gene: relationship to changes in islet beta-cell mass

A cDNA termed reg was recently isolated by differential screening of a library prepared from regenerating islets isolated from pancreatic remnants of rats subjected to 90% pancreatectomy and nicotinamide treatment. This led to speculation that this gene may be involved in expansion of beta-cell mass. In the current study we have measured reg expression after implantation and resection of a solid insulinoma tumor into rats, maneuvers known, respectively, to reduce and reexpand the volume of beta-cells in the islet. Animals with an implanted insulinoma tumor became profoundly hypoglycemic. Islet beta-cells declined from the normal 75% of total islet volume to less than 30%, in concert with a marked reduction in the reg mRNA level. Removal of the tumor resulted in a sharp increase in beta-cell replication, as measured by [3H]thymidine incorporation and a return to normal beta-cell volume within 4 days of tumor resection. This was associated with a transient induction in reg expression compared to that in tumor-bearing animals, effectively returning the amount of reg mRNA to the levels found in normal animals within 48 h; at later time points after tumor removal (3-7 days) reg expression declined, but then rose toward normal. In situ hybridization analysis localized the initial induction in reg mRNA expression to the exocrine pancreas. Continuous infusion of insulin into normal rats for 4 days, a maneuver that does not significantly reduce beta-cell mass, resulted in dramatically reduced insulin mRNA in islets, but no change in the levels of reg mRNA. We conclude that the diminution in pancreatic beta-cell mass caused by subcutaneous implantation of an insulinoma is associated with reduced reg gene expression and that the increase in beta-cell replication after resection of the tumor is preceded by return of reg gene expression toward normal.     

The 5S RNA genes of Schizosaccharomyces pombe.

The genomic arrangement and sequences of S. pombe 5S RNA genes are reported here. The 5S gene sequences appear to be dispersed within the genome, and are found independently of other rRNA genes. The sequences of two 5S genes examined show identical coding regions of 119 base pairs but have widely varying flanking sequences. A tRNAAsp gene is found in the 3' flanking region of one of the 5S genes. The tRNAAsp gene is faithfully transcribed in an X. laevis in vitro system, while the 5S genes are not transcribed in this system. The phylogenetic position of S. pombe is examined through comparison of 5S RNA sequences.     

The role of acetylcholine receptor antibodies in myasthenia gravis.

Myasthenia gravis is an autoimmune disease of man characterized by remitting and relapsing muscle fatigability. Although the etiology and pathogenesis are incompletely understood, the presence of circulating antibodies directed against the nicotinic acetylcholine (ACh) receptor in 80--90% of patients with myasthenia gravis and the identification of immune complexes at their neuromuscular junction have helped explain the altered neuromuscular transmission. The ACh receptor antibodies do not block access of ACh to the receptor, but do decrease the number of receptors by accelerating their degradation both in rat myotube cultures and in vivo models. In vitro these antibodies play a major role in myasthenia gravis. However, correlations of antibody titers with the clinical state following thymectomy or in neonatal myasthenia suggest that host factors may be equally important in determining whether the ACh receptor antibodies will result in clinical myasthenia.     

Determination of base pairing in Escherichia coli and Bacillus stearothermophilus 5S RNAs by infrared spectroscopy.

The extent of base pairing in Escherichia coli and Bacillus stearothermophilus 5S RNAs was determined by infrared spectroscopy. From the infrared spectra taken at 20 degrees and 52 degrees C it is concluded that E. coli and B. stearothermophlius 5S RNAs possess a large number of base pairs (Table I). Comparison of our results with those previously published using other methods leads to the conclusion that the structures of prokaryotic 5S RNAs involve a large number of tertiary interactions, in which the base pairing is not necessarily solely of the Watson-Crick type.