Bacteriophage T4 Mutants Hypersensitive to an Antitumor Agent That Induces Topoisomerase-DNA Cleavage Complexes

Many antitumor agents and antibiotics affect cells by interacting with type II topoisomerases, stabilizing a covalent enzyme-DNA complex. A pathway of recombination can apparently repair this DNA damage. In this study, transposon mutagenesis was used to identify possible components of the repair pathway in bacteriophage T4. Substantial increases in sensitivity to the antitumor agent m-AMSA [4'-(9-acridinyl-amino) methanesulfon-m-anisidide] were found with transposon insertion mutations that inactivate any of six T4-encoded proteins: UvsY (DNA synaptase accessory protein), UvsW (unknown function), Rnh (RNase H and 5' to 3' DNA exonuclease), α-gt (α-glucosyl transferase), gp47.1 (uncharacterized), and NrdB (β subunit of ribonucleotide reductase). The role of the rnh gene in drug sensitivity was further characterized. First, an in-frame rnh deletion mutation was constructed and analyzed, providing evidence that the absence of Rnh protein causes hypersensitivity to m-AMSA. Second, the m-AMSA sensitivity of the rnh-deletion mutant was shown to require a drug-sensitive T4 topoisomerase. Third, analysis of double mutants suggested that uvsW and rnh mutations impair a common step in the recombinational repair pathway for m-AMSA-induced damage. Finally, the rnh-deletion mutant was found to be hypersensitive to UV, implicating Rnh in recombinational repair of UV-induced damage.     

Comparative demographics of a Hawaiian forest bird community

Estimates of demographic parameters such as survival and reproductive success are critical for guiding management efforts focused on species of conservation concern. Unfortunately, reliable demographic parameters are difficult to obtain for any species, but especially for rare or endangered species. Here we derived estimates of adult survival and recruitment in a community of Hawaiian forest birds, including eight native species (of which three are endangered) and two introduced species at Hakalau Forest National Wildlife Refuge, Hawai?i. Integrated population models (IPM) were used to link mark–recapture data (1994–1999) with long‐term population surveys (1987–2008). To our knowledge, this is the first time that IPM have been used to characterize demographic parameters of a whole avian community, and provides important insights into the life history strategies of the community. The demographic data were used to test two hypotheses: 1) arthropod specialists, such as the ‘Akiapōlā‘au Hemignathus munroi, are ‘slower’ species characterized by a greater relative contribution of adult survival to population growth, i.e. lower fecundity and increased adult survival; and 2) a species’ susceptibility to environmental change, as reflected by its conservation status, can be predicted by its life history traits. We found that all species were characterized by a similar population growth rate around one, independently of conservation status, origin (native vs non‐native), feeding guild, or life history strategy (as measured by ‘slowness’), which suggested that the community had reached an equilibrium. However, such stable dynamics were achieved differently across feeding guilds, as demonstrated by a significant increase of adult survival and a significant decrease of recruitment along a gradient of increased insectivory, in support of hypothesis 1. Supporting our second hypothesis, we found that slower species were more vulnerable species at the global scale than faster ones. The possible causes and conservation implications of these patterns are discussed.     

Transformation of differentiated rat hepatocytes with adenovirus and adenovirus DNA

Primary cultures of hepatocytes isolated by collagenase perfusion of adult rats were transformed by infection with adenovirus type 5 or transfection with adenovirus DNA. Total virion DNA or recombinant plasmid DNA containing the adenovirus E1A and E1B genes transformed hepatocytes at comparable frequencies. No foci of replicating hepatocytes were detected after transfection with a plasmid containing the E1A gene alone. The frequency of transformation by the adenovirus E1A and E1B genes was dependent on the composition of the culture medium. Transformation occurred at a low frequency when the transfected hepatocytes were maintained in a chemically defined medium (CDM), but the frequency was enhanced 8- to 10-fold when the cells were maintained in (i) serum-supplemented medium or (ii) CDM supplemented with epidermal growth factor. Cell lines derived from the adenovirus-transformed colonies of hepatocytes expressed adenovirus E1A and E1B RNAs. When hepatocytes were maintained in CDM supplemented with dimethyl sulfoxide and transfected with plasmids containing the E1A and E1B genes, it was possible to derive cell lines that retained the ability to express several liver-specific genes, including albumin, transferrin, hemopexin, and the third component of complement. The amount of albumin secreted per cell varied from 1 to 5 pg per cell per 24 h, and in one cell line it was below detectable levels by passage 9. Adenovirus-transformed hepatocytes were not tumorigenic when inoculated subcutaneously into neonatal syngeneic rats. We conclude that the adenovirus E1A and E1B genes are capable of transforming adult rat hepatocytes, a differentiated epithelial cell type.     

Assessment of the Correlation Between Two Defining Criteria for Bidirectional Isthmic Block in the Ablation of Typical Atrial Flutter

Background

A complete, bidirectional conduction block in the cavotricuspid isthmus (CTI) represents the end-point of the typical atrial flutter ablation. We investigated the correlation between two criteria for successful ablation, one based on the atrial bipolar electrogram morphology before and after complete CTI conduction block, compared to the standard criteria of differential pacing and reversal in the right atrial depolarization sequence during coronary sinus (CS) pacing.

Method

We conducted a retrospective study in 111 patients (81 males, average age 62±10 years) who underwent an atrial flutter ablation during September 2007 - July 2009 in the Cardiology - Rehabilitation Hospital, UMF Cluj-Napoca. We assessed the presence of a bidirectional block at the end of the procedure using the standard criteria. We then analyzed the morphology of the bipolar atrial electrograms adjacent to the ablation line, before and after CTI conduction block.

Results

A change from a qRs morphology to a rSr'' morphology when pacing from the coronary sinus and from a rsr'' morphology to a QRS morphology when pacing from the low-lateral right atrium was associated with a CTI conduction block. Sensitivity (Se), specificity(Sp), positive predictive value (PPV), negative predictive value (NPV) were 96%, 89%, 99% and 67% respectively.

Conclusion

Our study suggests that the analysis of the atrial bipolar electrogram next to the ablation line before and after CTI ablation may be used as a reliable criterion to validate CTI conduction block due to its high sensitivity, specificity and positive predictive value.     

Spatial and temporal regulation of tenascin-R glycosylation in the cerebellum

The cellular adhesion molecule tenascin-R is a multifunctional extracellular matrix component expressed exclusively in the central nervous system. The expression of tenascin-R by oligodendrocytes and small interneurons in the hippocampus and cerebellum is highly regulated during development of these regions. This complex glycoprotein displays both adhesive and anti-adhesive properties that contribute to the formation and maintenance of synapses. We have determined that tenascin-R associated with Purkinje cell bodies and their dendrites in the molecular layer of the cerebellum bears N-linked oligosaccharides terminating with beta1,4-linked GalNAc-4-SO(4), whereas tenascin-R in other regions of the cerebellum does not bear this modification. Expression of this unique sulfated carbohydrate structure is also temporally regulated, increasing throughout cerebellar development. The most dramatic increase in GalNAc-4-SO(4) occurs between postnatal days 14 and 21, corresponding to a period of Purkinje cell dendrite extension and synaptogenesis. The spatially and temporally regulated addition of this unique sulfated carbohydrate to tenascin-R may serve to modulate its adhesive/anti-adhesive or other biological properties in vivo.     

Characterization of proteins in flagellates and growing amebae of Naegleria fowleri.

Polypeptides of whole-cell extracts of Naegleria fowleri flagellates and growing amebae were resolved by two-dimensional polyacrylamide gel electrophoresis. Autoradiograms of the [35S]methionine-labeled polypeptides of amebae and flagellates were analyzed by two dimensional densitometry to determine whether there were correlations between intracellular concentration of a protein and subunit size or charge. The majority of the polypeptides of amebae and flagellates had molecular sizes in the range of 20 to 60 kilodaltons. The radioactivity per polypeptide species in the size range of 20 to 60 kilodaltons was greater in amebae than in flagellates. The greatest number of polypeptides detected in amebae and flagellates was in the isoelectric focusing range of pH 6 to 7. The radioactivity per polypeptide species in the isoelectric focusing gradient below 6.3 was greater in amebae than in flagellates. Polypeptides in the size range of 20 to 60 kilodaltons had a median isoelectric point below pI 6.3, whereas those larger than 60 kilodaltons had a median pI value above 6.3. These data indicated that molecular size and charge were not entirely independent variables and that the size and charge of a polypeptide might have an important influence in determining its intracellular concentration in both amebae and flagellates. Autoradiograms were also compared so that changes in intracellular protein complement and concentrations occurring during differentiation could be recognized. The relative amounts of a limited number of polypeptides increased markedly, and others decreased markedly, during enflagellation.     

Power Estimation for Two-Sample Tests Using Importance and Antithetic Resampling

Collings and Hamilton (1988), described a uniform bootstrap method that is applied on observed or pilot data in order to approximate the power of the two-sample Wilcoxon test for location shift alternatives. In this paper we demonstrate how importance and antithetic resampling can be used to substantially reduce the amount of computation needed to approximate the power of the two-sample tests for location shift and scale alternatives. Importance and antithetic bootstrap resampling methods are applied to simulated data of different sample sizes from a variety of distributions as well as to data from the Iowa 65+ Rural Health Study. Also, a suggestion is given for using a combination of importance and antithetic resampling for approximating the power of two-sample tests.