Genetical and biochemical comparisons of alcohol dehydrogenase isozymes from Anastrepha fraterculus and A. obliqua (Diptera: Tephritidae): Evidence for gene duplication

The electrophoretic patterns of the enzyme alcohol dehydrogenase (ADH) from Anastrepha fraterculus and A. obliqua were studied. Two loci were found to code for the enzyme in A. fraterculus, and three in A. obliqua. In both species, all isozymes were active in third-instar larvae. A cationic isozyme (Adh-1) was active mainly in the visceral fat body of both species. In A. fraterculus, the locus had an anionic polymorphic isozyme (Adh-3) that was detected in the parietal fat body. In addition to these two loci, a third locus for an anionic isozyme (Adh-2), which was active in the digestive tube of larvae, was present in A. obliqua and probably resulted from gene duplication. For both species, multiple forms of the isozymes are formed by binding of an NAD-carbonyl compound, as in Drosophila melanogaster. Both larvae and early pupae of A. obliqua had almost twice the specific ADH activity as A. fraterculus. The ethanol content of the host fruit infested with A. obliqua (red mombim) was also higher than that of the host fruit infested with A. fraterculus (guava).This research was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnologico (CNPq-PIG 40.2486/82).     

Automated detection and mapping of electrical activation when electrogram morphology is complex

BackgroundMapping of cardiac electrical activity can be difficult when electrogram morphology is complex. Complex morphology (multiple and changing deflections) causes activation maps to vary when constructed by different analysts, particularly at areas with spatially varying conduction pattern. An algorithm was developed to automatically detect electrogram activation time which is robust to complex morphology.MethodElectrograms, many of which were complex, were collected from 320 canine epicardial border zone sites in five experiments. A library of electrogram activation times were manually marked a priori by two expert analysts. Then an algorithm which combined correlation and error functions was used to compare each input electrogram to library electrogram patterns. The closest match of input to library electrogram was used to estimate activation time. Once activation times at 320 sites were determined, activation maps were automatically constructed on a computerized grid. The algorithm was validated by comparison with activation times determined by the analysts.ResultsThe mean difference between manual and automated marking of activation time in electrograms acquired during reentrant ventricular tachycardia was 2.1 ± 3.9 ms. The mean sensitivity and positive predictive value were 95.9% and 83.8% respectively. The computer-automated marking process was completed within a few seconds and was robust to fractionated electrograms. Measurement error was mostly attributable to 60 Hz noise, which can be rectified with filtering.ConclusionsThe automated algorithm is useful for rapid and accurate automatic marking of multichannel electrograms, some of which may be fractionated, as well as for real-time display of activation maps in clinical electrophysiology or research studies.     

Enumeration of an Extremely High Particle-to-PFU Ratio for Varicella-Zoster Virus

Varicella-zoster virus (VZV) is renowned for its low titers. Yet investigations to explore the low infectivity are hampered by the fact that the VZV particle-to-PFU ratio has never been determined with precision. Herein, we accomplish that task by applying newer imaging technology. More than 300 images were taken of VZV-infected cells on 4 different samples at high magnification. We enumerated the total number of viral particles within 25 cm² of the infected monolayer at 415 million. Based on these numbers, the VZV particle:PFU ratio was approximately 40,000:1 for a cell-free inoculum.A precise ratio of particles to PFU of varicella-zoster virus (VZV) has never been determined, even though VZV was first isolated in cell culture by the Nobel laureate T. H. Weller in 1952 (21). His group determined that VZV replicated in a few embryonic tissues and in amnion cells. Subsequently, Taylor-Robinson and Caunt found that VZV replication was restricted to a small number of mainly embryonic cells by testing more than 20 primary and continuous cell lines (19). A decade later, VZV was propagated in melanoma cell lines, which are derived from the neural crest (8). In all of these cultured cells, the titer was found to be low, particularly when compared with that of the closely related herpes simplex type 1 virus (HSV-1). Again, in sharp contrast with HSV-1, the virus remained strongly cell associated.The term particle/PFU ratio refers to the number of viral particles required to form one plaque in a plaque assay. It is a measure of the efficiency by which a virus infects cultured cells. Early in the 1960s, investigators began using negative staining electron microscopy to count viral particles in inoculum material and compare those counts to the measured titer, thereby measuring ratios for a few animal viruses (6). For example, the ratio for HSV-1 is around 10:1 (10, 20). Due to the strong cell association of VZV infection of cultured cells, no precise VZV particle/PFU ratio has ever been determined. The lack of any widely accepted VZV ratio severely limits our ability to assess whether mutated or recombinant viruses produce more or fewer complete infectious particles in cultured cells (4, 5, 15, 17). In other words, if an attenuated virus has a lower titer, we do not know whether fewer viral particles are produced per square centimeter of cellular monolayer (without a change in the particle/PFU ratio) or alternatively fewer infectious viral particles are produced overall (with a higher particle/PFU ratio).In this report, we successfully define a VZV particle/PFU ratio by imaging viral particles with advanced scanning electron microscopic (SEM) technology not available during our earlier investigations of viral structure (12). We demonstrate that the VZV ratio is much higher than that for other common human viruses grown in cultured cells and remarkably higher than that for HSV. Finally, this report documents evidence of an ever-widening difference between HSV and VZV replication and assembly in cultured cells (7, 13, 18).     

Global coral disease prevalence associated with sea temperature anomalies and local factors

Coral diseases are taking an increasing toll on coral reef structure and biodiversity and are important indicators of declining health in the oceans. We implemented standardized coral disease surveys to pinpoint hotspots of coral disease, reveal vulnerable coral families and test hypotheses about climate drivers from 39 locations worldwide. We analyzed a 3 yr study of coral disease prevalence to identify links between disease and a range of covariates, including thermal anomalies (from satellite data), location and coral cover, using a Generalized Linear Mixed Model. Prevalence of unhealthy corals, i.e. those with signs of known diseases or with other signs of compromised health, exceeded 10% on many reefs and ranged to over 50% on some. Disease prevalence exceeded 10% on 20% of Caribbean reefs and 2.7% of Pacific reefs surveyed. Within the same coral families across oceans, prevalence of unhealthy colonies was higher and some diseases were more common at sites in the Caribbean than those in the Pacific. The effects of high disease prevalence are potentially extensive given that the most affected coral families, the acroporids, faviids and siderastreids, are among the major reef-builders at these sites. The poritids and agaricids stood out in the Caribbean as being the most resistant to disease, even though these families were abundant in our surveys. Regional warm temperature anomalies were strongly correlated with high disease prevalence. The levels of disease reported here will provide a much-needed local reference point against which to compare future change.     

Comparative study of the cellular replication kinetics of rat mammary epithelial cells in vivo and in vitro

Ts-131b, one of the temperature-sensitive (ts) mutants isolated from mouse FM3A cells, was found to be defective in DNA replication at a non-permissive temperature. After the cells were transferred to 39.5 °C, the cell number increased by only 10% and the rate of incorporation of precursors into cellular DNA decreased rapidly. Cell cycle analysis by a flow cytometric method with the cells incubated at 39.5 °C revealed that progression of the cells through the S phase was inhibited and most of the cells were arrested in the S phase. To study the defect in DNA replication of this ts-mutant at 39.5 °C, DNA-fiber autoradiography was performed to measure the rate of DNA-chain elongation. The results showed that the rate of DNA-chain elongation was decreased at 6 h after the temperature shift. However, since the decrease in the rate of DNA-chain elongation was not sufficient to account for the decrease in the rate of incorporation of the precursors, it was suggested that there was also a decrease in the rate of initiation of DNA replication at some of the replicon origins.     

In silico protein fragmentation reveals the importance of critical nuclei on domain reassembly

Protein complementation assays (PCAs) based on split protein fragments have become powerful tools that facilitate the study and engineering of intracellular protein-protein interactions. These assays are based on the observation that a given protein can be split into two inactive fragments and these fragments can reassemble into the original properly folded and functional structure. However, one experimentally observed limitation of PCA systems is that the folding of a protein from its fragments is dramatically slower relative to that of the unsplit parent protein. This is due in part to a poor understanding of how PCA design parameters such as split site position in the primary sequence and size of the resulting fragments contribute to the efficiency of protein reassembly. We used a minimalist on-lattice model to analyze how the dynamics of the reassembly process for two model proteins was affected by the location of the split site. Our results demonstrate that the balanced distribution of the “folding nucleus,” a subset of residues that are critical to the formation of the transition state leading to productive folding, between protein fragments is key to their reassembly.