Murine virus susceptibility of cell cultures of mouse, rat, hamster, monkey, and human origin.

Studies were initiated to determine the practicality of using various tissue cultures for the propagation of murine viruses isolated from laboratory animals. The cytopathogenic effects of 10 murine viruses known to cause disease in laboratory rodents were compared in monolayer cultures of L929, BHK-21, WI-38, BSC-1, and Vero cells. The susceptibility of primary hamster embryo, hamster kidney, mouse embryo, mouse kidney, and rat embryo cell cultures was also tested. Seven of the viruses produced effects in at least 1 of the cell substrates. The remaining 3 viruses, namely H-1, K, and mouse hepatitis, produced no effects in the cell cultures tested.     

Hepatitis C virus-encoded NS2-3 protease: cleavage-site mutagenesis and requirements for bimolecular cleavage.

Cleavage at the 2/3 site of hepatitis C virus (HCV) is thought to be mediated by a virus-encoded protease composed of the region of the polyprotein encoding NS2 and the N-terminal one-third of NS3. This protease is distinct from the NS3 serine protease, which is responsible for downstream cleavages in the nonstructural region. Site-directed mutagenesis of residues surrounding the 2/3 cleavage site showed that cleavage is remarkably resistant to single-amino-acid substitutions from P5 to P3' (GWRLL decreases API). The only mutations which dramatically inhibited cleavage were the ones most likely to alter the conformation of the region, such as Pro substitutions at the P1 or P1' position, deletion of both amino acids at P1 and P1', or simultaneous substitution of multiple Ala residues. Cotransfection experiments were done to provide additional information on the polypeptide requirements for bimolecular cleavage. Polypeptides used in these experiments contained amino acid substitutions and/or deletions in NS2 and/or the N-terminal one-third of NS3. Polypeptides with defects in either NS2 or the N-terminal portion of NS3 but not both were cleaved when cotransfected with constructs expressing intact versions of the defective region. Cotransfection experiments also showed that certain defective NS2-3 constructs partially inhibited cleavage of wild-type polypeptides. Although these results show that inefficient cleavage can occur in a bimolecular reaction, they suggest that both molecules must contribute a functional subunit to allow formation of a protease which is capable of cleavage at the 2/3 site. This reaction may resemble the cis cleavage thought to occur at the 2/3 site during processing of the wild-type HCV polyprotein.     

Comparison of enzyme-linked immunomagnetic chemiluminescence with U.S. Food and Drug Administration's Bacteriological Analytical Manual method for the detection of Escherichia coli O157:H7

Escherichia coli O157:H7, a major foodborne pathogen, has been associated with numerous cases of foodborne illnesses. Rapid methods have been developed for the screening of this pathogen in foods in order to circumvent timely plate culture techniques. Unfortunately, many rapid methods are presumptive and do not claim to confirm the presence of E. coli O157:H7. The previously developed method, enzyme-linked immunomagnetic chemiluminescence (ELIMCL), has been improved upon to allow for fewer incidences of false positives when used to detect E. coli O157:H7 in the presence of mixed cultures. The key feature of this assay is that it combines the highly selective synergism of both anti-O157 and anti-H7 antibodies in the sandwich immunoassay format. This work presents application of a newly semi-automated version of ELIMCL to the detection of E. coli O157:H7 in pristine buffered saline yielding detection limits of approximately 1 x 10(5) to 1 x 10(6) of live cells/mL. ELIMCL was further demonstrated to detect E. coli O157:H7 inoculated into artificially contaminated ground beef at ca. 400 CFU/g after a 5 h enrichment and about 1.5 h assay time for a total detection time of about 6.5 h. Finally, ELIMCL was compared with USFDA's Bacteriological Analytical Manual method for E. coli O157:H7 in a double-blind study. Using McNemar's treatment, the two methods were determined to be statistically similar for the detection of E. coli O157:H7 in ground beef inoculated with mixed cultures of select bacteria.     

High-throughput fluorescence assay for small-molecule inhibitors of autophagins/Atg4

Autophagy is an evolutionarily conserved process for catabolizing damaged proteins and organelles in a lysosome-dependent manner. Dysregulation of autophagy may cause various diseases, such as cancer and neurodegeneration. However, the relevance of autophagy to diseases remains controversial because of the limited availability of chemical modulators. Herein, the authors developed a fluorescence-based assay for measuring activity of the autophagy protease, autophagin-1(Atg4B). The assay employs a novel reporter substrate of Atg4B composed of a natural substrate (LC3B) fused to an assayable enzyme (PLA(2)) that becomes active upon cleavage by this cysteine protease. A high-throughput screening (HTS) assay was validated with excellent Z' factor (>0.7), remaining robust for more than 5 h and suitable for screening of large chemical libraries. The HTS assay was validated by performing pilot screens with 2 small collections of compounds enriched in bioactive molecules (n = 1280 for Lopac? and 2000 for Spectrum? library), yielding confirmed hit rates of 0.23% and 0.70%, respectively. As counterscreens, PLA(2) and caspase-3 assays were employed to eliminate nonspecific inhibitors. In conclusion, the LC3B-PLA(2) reporter assay provides a platform for compound library screening for identification and characterization of Atg4B-specific inhibitors that may be useful as tools for interrogating the role of autophagy in disease models.     

Modification of the tetrodotoxin receptor in Electrophorus electricus by phospholipase A2

The effects of phospholipase A2 treatment on the tetrodotoxin receptors in Electrophorus electricus was studied. (1) The binding of [3H]tetrodotoxin to electroplaque membranes was substantially reduced by treatment of the membranes with low concentrations of phospholipase A2 from a number of sources, including bee venom, Vipera russelli and Crotalus adamanteus and by beta-bungarotoxin. (2) Phospholipase A2 from bee venom and from C. adamanteus both caused extensive hydrolysis of electroplaque membrane phospholipids although the substrate specificity differed. Analysis of the phospholipid classes hydrolyzed revealed a striking correlation between loss of toxin binding and hydrolysis of phosphatidylethanolamine but not of phosphatidylserine. (3) The loss of toxin binding could be partially reversed by treatment of the membranes with bovine serum albumin, conditions which are known to remove hydrolysis products from the membrane. (4) Equilibrium binding studies on the effects of phospholipase A2 treatment of [3H]tetrodotoxin binding showed that the reduction reflected loss of binding sites and not a change in affinity. (5) These results are interpreted in terms of multiple equilibrium states of the tetrodotoxin-receptors with conformations determined by the phospholipid environment.     

Drosophila Kelch regulates actin organization via Src64-dependent tyrosine phosphorylation

The Drosophila kelch gene encodes a member of a protein superfamily defined by the presence of kelch repeats. In Drosophila, Kelch is required to maintain actin organization in ovarian ring canals. We set out to study the actin cross-linking activity of Kelch and how Kelch function is regulated. Biochemical studies using purified, recombinant Kelch protein showed that full-length Kelch bundles actin filaments, and kelch repeat 5 contains the actin binding site. Two-dimensional electrophoresis demonstrated that Kelch is tyrosine phosphorylated in a src64-dependent pathway. Site-directed mutagenesis determined that tyrosine residue 627 is phosphorylated. A Kelch mutant with tyrosine 627 changed to alanine (KelY627A) rescued the actin disorganization phenotype of kelch mutant ring canals, but failed to produce wild-type ring canals. Electron microscopy demonstrated that phosphorylation of Kelch is critical for the proper morphogenesis of actin during ring canal growth, and presence of the nonphosphorylatable KelY627A protein phenocopied src64 ring canals. KelY627A protein in ring canals also dramatically reduced the rate of actin monomer exchange. The phenotypes caused by src64 mutants and KelY627A expression suggest that a major function of Src64 signaling in the ring canal is the negative regulation of actin cross-linking by Kelch.     

Anadromy,potamodromy and residency in brown trout Salmo trutta: the role of genes and the environment

Brown trout Salmo trutta is endemic to Europe, western Asia and north-western Africa; it is a prominent member of freshwater and coastal marine fish faunas. The species shows two resident (river-resident, lake-resident) and three main facultative migratory life histories (downstream–upstream within a river system, fluvial–adfluvial potamodromous; to and from a lake, lacustrine–adfluvial (inlet) or allacustrine (outlet) potamodromous; to and from the sea, anadromous). River-residency v. migration is a balance between enhanced feeding and thus growth advantages of migration to a particular habitat v. the costs of potentially greater mortality and energy expenditure. Fluvial–adfluvial migration usually has less feeding improvement, but less mortality risk, than lacustrine–adfluvial or allacustrine and anadromous, but the latter vary among catchments as to which is favoured. Indirect evidence suggests that around 50% of the variability in S. trutta migration v. residency, among individuals within a population, is due to genetic variance. This dichotomous decision can best be explained by the threshold-trait model of quantitative genetics. Thus, an individual's physiological condition (e.g., energy status) as regulated by environmental factors, genes and non-genetic parental effects, acts as the cue. The magnitude of this cue relative to a genetically predetermined individual threshold, governs whether it will migrate or sexually mature as a river-resident. This decision threshold occurs early in life and, if the choice is to migrate, a second threshold probably follows determining the age and timing of migration. Migration destination (mainstem river, lake, or sea) also appears to be genetically programmed. Decisions to migrate and ultimate destination result in a number of subsequent consequential changes such as parr–smolt transformation, sexual maturity and return migration. Strong associations with one or a few genes have been found for most aspects of the migratory syndrome and indirect evidence supports genetic involvement in all parts. Thus, migratory and resident life histories potentially evolve as a result of natural and anthropogenic environmental changes, which alter relative survival and reproduction. Knowledge of genetic determinants of the various components of migration in S. trutta lags substantially behind that of Oncorhynchus mykiss and other salmonines. Identification of genetic markers linked to migration components and especially to the migration–residency decision, is a prerequisite for facilitating detailed empirical studies. In order to predict effectively, through modelling, the effects of environmental changes, quantification of the relative fitness of different migratory traits and of their heritabilities, across a range of environmental conditions, is also urgently required in the face of the increasing pace of such changes.     

A role for exon sequences and splice-site proximity in splice-site selection

Analysis of the in vitro splicing products of RNA precursors containing tandem duplications of the 5' or 3' splice sites of human beta-globin IVS 1 revealed that exon sequences play an important role in the relative use of the duplicated sites. These studies also show that the proximity of the 5' and 3' splice sites is an important determinant in the selection of splice-sites. Deletion, substitution, or even subtle changes of exon sequences can alter the pattern of splice-site selection, and in many cases the splice site adjacent to the altered exon is not used. The relative use of the duplicated splice sites can also be altered by diluting the splicing extract, suggesting that factors involved in splice-site selection are limiting.     

Dual regulation of the yeast CDC28-p40 protein kinase complex: cell cycle, pheromone, and nutrient limitation effects

A 40 kd polypeptide that coprecipitates with the CDC28 gene product in immune complexes is specifically phosphorylated by the CDC28 protein kinase. Using this reaction, we detect activity only in extracts from dividing G1 phase cells. Exit from G1 by entry into S phase or the preconjugatory state induced by mating pheromone correlates with loss of p40 phosphorylation activity. Inactive extracts from cdc28 mutants complement extracts from cells arrested in S or M phase, suggesting that non-G1 cells are deficient in an exchangeable activating factor. Stationary and pheromone-treated cultures are rich in this exchangeable factor, but possess an inactive kinase that is not activated by complementation. cAMP-deficient mutants resemble stationary cells.