Plasmodium falciparum Transfected with Ultra Bright NanoLuc Luciferase Offers High Sensitivity Detection for the Screening of Growth and Cellular Trafficking Inhibitors

Drug discovery is a key part of malaria control and eradication strategies, and could benefit from sensitive and affordable assays to quantify parasite growth and to help identify the targets of potential anti-malarial compounds. Bioluminescence, achieved through expression of exogenous luciferases, is a powerful tool that has been applied in studies of several aspects of parasite biology and high throughput growth assays. We have expressed the new reporter NanoLuc (Nluc) luciferase in Plasmodium falciparum and showed it is at least 100 times brighter than the commonly used firefly luciferase. Nluc brightness was explored as a means to achieve a growth assay with higher sensitivity and lower cost. In addition we attempted to develop other screening assays that may help interrogate libraries of inhibitory compounds for their mechanism of action. To this end parasites were engineered to express Nluc in the cytoplasm, the parasitophorous vacuole that surrounds the intraerythrocytic parasite or exported to the red blood cell cytosol. As proof-of-concept, these parasites were used to develop functional screening assays for quantifying the effects of Brefeldin A, an inhibitor of protein secretion, and Furosemide, an inhibitor of new permeation pathways used by parasites to acquire plasma nutrients.     

Epitopes of glycoprotein G of equine herpesviruses 4 and 1 located near the C termini elicit type-specific antibody responses in the natural host.

Specific serological diagnosis of equine herpesvirus 4 (EHV4; equine rhinopneumonitis virus) and EHV1 (equine abortion virus) hitherto has not been possible because of extensive antigenic cross-reactivity between these two closely related but distinct viruses. Recently, we identified EHV4 glycoprotein G (gG) and characterized it as a type-specific, secreted glycoprotein (B. S. Crabb, H. S. Nagesha, and M. J. Studdert, Virology 190:143-154, 1992). This paper shows that EHV1 gG also possesses type-specific epitopes and describes the localization of strong, type-specific epitopes to the apparently corresponding and highly variable regions comprising amino acids 287 to 382 of EHV4 gG and 288 to 350 of EHV1 gG. Fusion proteins expressing these variable regions reacted strongly and type specifically with sera from four foals, three of which were colostrum-deprived, specific-pathogen-free foals, whose history with respect to exposure to EHV4 or EHV1 was well-defined. These antigens provided the basis for the development of a single-well diagnostic enzyme-linked immunosorbent assay to distinguish horses infected with EHV4, EHV1, or both. Such a type-specific test provides for the first time the opportunity to differentiate antibodies to these viruses, and it has, therefore, important implications for understanding the epidemiology of these equine pathogens. Evidence for the existence of EHV1 in Australia 10 years prior to the first confirmed case of EHV1 abortion is presented.     

Voltage dependent membrane conductances in cultured renal distal cells.

Cultured Na(+)-transporting epithelia from amphibian renal distal tubule (A6) were impaled with microelectrodes and analyzed at short-circuit and after transepithelial voltage perturbation to evaluate the influence of voltage on apical and basolateral membrane conductances. For equivalent circuit analysis, amiloride was applied at each setting of transepithelial potential. At short-circuit, apical and basolateral membrane conductances averaged 88 and 497 microS/cm2, respectively (n = 10). Apical membrane conductance, essentially due to Na(+)-specific pathways, decreased after depolarization of the apical membrane. The drop was considerably larger than predicted by the Goldman-Hodgkin-Katz (GHK) constant-field equation. This suggests decrease in permeability of the apical Na+ channels upon depolarization. Basolateral membrane conductance, preferentially determined by K+ channels, increased after hyperpolarization of the basolateral membrane. This behavior is contrary to the prediction of the GHK constant field equation and reflects inward rectification of the K+ channels. The observed rectification patterns can be valuable for maintenance of cellular homeostasis.     

Purification of rabbit and human serum paraoxonase.

Rabbit serum paraoxonase/arylesterase has been purified to homogeneity by Cibacron Blue-agarose chromatography, gel filtration, DEAE-Trisacryl M chromatography, and preparative SDS gel electrophoresis. Renaturation (Copeland et al., 1982) and activity staining of the enzyme resolved by SDS gel electrophoresis allowed for identification and purification of paraoxonase. Two bands of active enzyme were purified by this procedure (35,000 and 38,000). Enzyme electroeluted from the preparative gels was reanalyzed by analytical SDS gel electrophoresis, and two higher molecular weight bands (43,000 and 48,000) were observed in addition to the original bands. This suggested that repeat electrophoresis resulted in an unfolding or other modification and slower migration of some of the purified protein. The lower mobility bands stained weakly for paraoxonase activity in preparative gels. Bands of each molecular weight species were electroblotted onto PVDF membranes and sequenced. The gas-phase sequence analysis showed that both the active bands and apparent molecular weight bands had identical amino-terminal sequences. Amino acid analysis of the four electrophoretic components from PVDF membranes also indicated compositional similarity. The amino-terminal sequences are typical of the leader sequences of secreted proteins. Human serum paraoxonase was purified by a similar procedure, and ten residues of the amino terminus were sequenced by gas-phase procedures. One amino acid difference between the first ten residues of human and rabbit was observed.     

The flaA locus of Bacillus subtilis is part of a large operon coding for flagellar structures, motility functions, and an ATPase-like polypeptide.

We cloned and sequenced 8.3 kb of Bacillus subtilis DNA corresponding to the flaA locus involved in flagellar biosynthesis, motility, and chemotaxis. The DNA sequence revealed the presence of 10 complete and 2 incomplete open reading frames. Comparison of the deduced amino acid sequences to data banks showed similarities of nine of the deduced products to a number of proteins of Escherichia coli and Salmonella typhimurium for which a role in flagellar functioning has been directly demonstrated. In particular, the sequence data suggest that the flaA operon codes for the M-ring protein, components of the motor switch, and the distal part of the basal-body rod. The gene order is remarkably similar to that described for region III of the enterobacterial flagellar regulon. One of the open reading frames was translated into a protein with 48% amino acid identity to S. typhimurium FliI and 29% identity to the beta subunit of E. coli ATP synthase.     

Mechanism of rhodopsin kinase activation

The role of the cytoplasmic loops and C-terminal region of bovine rhodopsin (Rho) in binding and activating rhodopsin kinase was investigated. The ability of various enzymatically truncated forms of photolyzed rhodopsin (Rho*) to stimulate rhodopsin kinase activity was quantified. Following endopeptidase Asp-N cleavage of all phosphorylation sites on the C-terminal, the resulting truncated Rho* (329G-Rho*) was not phosphorylated by rhodopsin kinase. This suggests that rhodopsin kinase only phosphorylates C-terminal sites of Rho*. However 329G-Rho* could bind rhodopsin kinase and stimulate phosphorylation of exogenous peptide. Kinase stimulation was investigated for other truncated forms of Rho* in which the C-terminal region was either partially or completely eliminated, and the V-VI loop was either cleaved or left intact (339K-Rho*, 341E239E-Rho*, 329G239E-Rho*, 327P240S-Rho*). Results suggest that the V-VI loop is crucial for kinase binding (similar to the binding of GT). Mastoparan, a model peptide for G-protein-coupled receptors, was found to stimulate rhodopsin kinase in a mechanism similar to that of truncated Rho*. We conclude that rhodopsin kinase binds to the cytoplasmic loops of Rho* to cause a stimulation of its catalytic activity.     

Effects of aldosterone on (Na+ + K+)-ATPase of amphibian sodium-transporting epithelial cells (A6) in culture

Stimulation by aldosterone of sodium reabsorption can be reproduced on a cell line, A6, derived from the renal tissue of Xenopus laevis. These cells organize themselves as a polarized epithelium carrying out unidirectional sodium transport, reflected by the short-circuit current (Isc). Isc response to aldosterone starts to be apparent after a latency period of 2-3 h; the full hormonal effect takes much longer. On the other hand, (Na+ + K+)-ATPase activity and density in ouabain binding sites did not increase before several hours of treatment. At that stage, while Isc more than trebled, Na+ pump activity and density went up by less than 50%. A significant influence of aldosterone on the way the Na+ pump operates is considered unlikely, since cell interaction with ouabain remained unchanged (Kd approximately 18 nM). Furthermore, the close correspondence of hormonal effect, in relative terms, on (Na+ + K+)-ATPase activity vs density, argues against a significant degree of recruitment of spare pump units. Thus aldosterone effect on Na+ pump probably results from increased biosynthesis of the enzyme. The aldosterone dependent Na+ pump stimulation is apparently unrelated to sodium available for transport. The hormone seems to act on Na+ pump directly.     

Rapid detection of tetM in Mycoplasma hominis and Ureaplasma urealyticum by PCR: tetM confers resistance to tetracycline but not necessarily to doxycycline

Tetracycline resistance in Mycoplasma hominis and Ureaplasma urealyticum has been associated with the tetM determinant and has recently been increasing in incidence. We report here a rapid method for detection of the tetM determinant based on the use of the polymerase chain reaction (PCR) to amplify a 397-bp DNA fragment from the tetM gene and verification of specificity using the restriction enzyme TaqI. Analysis of 42 U. urealyticum and 49 M. hominis isolates indicates that the PCR method may be clinically useful for determination of tetracycline sensitivity, as tetM is presently the only known determinant associated with tetracycline resistance in these two organisms. All of the tetM-positive M. hominis isolates were sensitive to doxycycline, indicating that tetM does not necessarily confer resistance to this antibiotic.     

Expression and immunochemical analysis of rat and human fibroblast growth factor receptor (flg) isoforms.

Potentially 96 splice variants among four genes that code for the human heparin-binding fibroblast growth factor receptor family complicate study of structure, metabolism, and function of single isoforms in mammalian cells. As an alternative, we expressed structural subdomains and isoforms of the flg receptor gene in bacteria and baculoviral-infected insect cells. We developed and characterized a panel of 16 isoform and domain-specific polyclonal and monoclonal antibodies. The panel of antibodies was used to distinguish mature glycosylated ligand-binding and kinase-active and -inactive recombinant isoforms in baculoviral insect cells and transfected mammalian cells and natural isoforms in rat prostate and human liver cells. The results revealed a cell type-specific expression of the flg gene and isoforms that result from combinations of splice variations. Reactive epitopes of monoclonal antibodies against both the three (alpha) and two (beta) immunoglobulin-like disulfide loop extracellular domain isoforms were mapped by cross-reactivity with synthetic polypeptide sequences and deletion mutants expressed in bacteria. The native alpha and beta receptor isoforms differed in display of shared epitopes and suggested that the NH2-terminal Loop I and COOH-terminal Loops II and III of the alpha isoform are interactive. Although the common Loops II and III appear qualitatively sufficient for ligand binding, the results suggest that tertiary relationships among loops in the three and two loop isoforms are distinct and, therefore, the two isoforms may have distinct activities. Spatial models for arrangement of immunoglobulin-like loops in the extracellular domain of the two isoforms are presented.