Helix–coil transition kinetics in aqueous poly(α,L-glutamic acid)

A polarimetric electric-field-jump relaxation apparatus is described and used to determine the relaxation spectrum for the helix–coil transition of poly(α,L -glutamic acid) in water at 24°C. A maximum relaxation time of 1.7 μc occurs at the transition midpoint (pH = 5.9) yielding a rate constant for helical growth of 6 × 10⁷ sec^?1.     

Definitive characterization of human thymine glycol N-glycosylase activity

An N-glycosylase activity that released cis-[3H]-5,6-dihydroxy-5,6-dihydrothymine (thymine glycol, TG) from chemically oxidized poly(dA-[3H]dT) was unambiguously characterized both in extracts of HeLa cells and in purified Escherichia coli endonuclease III. This was accomplished by use of microderivatization procedure that quantitatively converted cis-TG to 5-hydroxy-5-methylhydantoin (HMH). The reaction products were analyzed by high-pressure liquid chromatography before and after derivatization by using cis-[14C]TG and [14C]HMH, which had been independently synthesized, as reference compounds. This technique facilitated construction of a v/[E]t plot for the enzyme activity in HeLa cells, permitting estimation of its specific activity. The results obtained prove the existence of both human and bacterial N-glycosylase activities that effect removal of TG from DNA.     

Evolutionary divergence of mitochondrial DNA from Paramecium aurelia

Summary Mitochondrial (mt) DNA from four sibling species within the Paramecium aurelia complex, including stocks of different geographic origin and mutants, were analyzed using four 6-bp recognition site and one 4-bp recognition site endonucleases and the sequence divergence was estimated using Upholt's (1977) statistical procedure. All four species were readily distinguishable regardless of the restriction endonuclease employed. With intraspecies comparisons, no differences were observed which could be accounted for on the basis of geographic origin. Except for species 4, each stock (and mutant) gave a species-specific fragment pattern. For species 4, while the patterns were distinct from the other species, two species-specific type of patterns were found, designated A and B. The sequence divergence between these was estimated to be between 1 and 2 percent. With interspecies comparisons, the sequence divergence ranged from 3.9 to 10.3% with the greatest divergence being between species 1 and 4, and the least between species 1 and 5. The similarity between species 1 and 5 is in accord with other criteria for interspecies comparisons. The degree of sequence divergence measured here in Paramecium mt DNA is well within the range reported for rodents and primates. All four species mt DNA were cleaved to many DNA fragments by DPN II, an enzyme which recognizes non-methylated sites, and not by DPNI, the methyl-site specific counterpart of DPN II, suggesting that mt DNA from Paramecium aurelia is not appreciably methylated, if at all.     

Effects of an anthropogenic diet on indicators of physiological challenge and immunity of white ibis nestlings raised in captivity

When wildlife forage and/or live in urban habitats, they often experience a shift in resource availability and dietary quality. Some species even use human handouts, such as bread, as well as human refuse, as a large part of their new diets; yet the influences of this nutritional shift on health and survival remain unclear. American white ibises are increasingly being seen in urban areas in Florida; they collect handouts, such as bread and other food items, from humans in parks, and are also found foraging on anthropogenic sources in trash heaps. We hypothesized that the consumption of these new anthropogenic food sources may trigger increases in indicators of physiological challenge and dampen immune responses. We tested this experimentally by raising 20 white ibis nestlings in captivity, and exposing 10 to a simulated anthropogenic diet (including the addition of white bread and a reduction in seafood content) while maintaining 10 on a diet similar to what ibises consume in more natural environments. We then tested two indicators of physiological challenge (corticosterone and heat shock protein 70), assessed innate immunity in these birds via bactericidal assays and an in vitro carbon clearance assay, and adaptive immunity using a phytohemagglutinin skin test. The anthropogenic diet depressed the development of the ability to kill Salmonella paratyphi in culture. Our results suggest that consuming an anthropogenic diet may be detrimental in terms of the ability to battle a pathogenic bacterial species, but there was little effect on indicators of physiological challenge and other immunological measures.     

Genome Engineering of Drosophila with the CRISPR RNA-Guided Cas9 Nuclease

We have adapted a bacterial CRISPR RNA/Cas9 system to precisely engineer the Drosophila genome and report that Cas9-mediated genomic modifications are efficiently transmitted through the germline. This RNA-guided Cas9 system can be rapidly programmed to generate targeted alleles for probing gene function in Drosophila.     

Benzimidazole-based antibacterial agents against Francisella tularensis

Francisella tularensis is a highly virulent pathogenic bacterium. In order to identify novel potential antibacterial agents against F. tularensis, libraries of trisubstituted benzimidazoles were screened against F. tularensis LVS strain. In a preliminary screening assay, remarkably, 23 of 2,5,6- and 2,5,7-trisubstituted benzimidazoles showed excellent activity exhibiting greater than 90% growth inhibition at 1 μg/mL. Among those hits, 21 compounds showed MIC₉₀ values in the range of 0.35–48.6 μg/mL after accurate MIC determination. In ex vivo efficacy assays, four of these compounds exhibited 2–3 log reduction in colony forming units (CFU) per mL at concentrations of 10 and 50 μg/mL.