Effects of prenatal testosterone on cumulative markers of oxidative damage to organs of young adult zebra finches (Taeniopygia guttata).

We tested the hypothesis that exposure of avian embryos to androgens in ovo entails long-term costs in the form of oxidative damage to vital cells and organs in adulthood. We injected zebra finch eggs with testosterone (T), monitored postnatal growth, and analyzed markers of oxidative damage in heart and liver in mature birds. We measured 8-oxo-2'-deoxyguanosine and isoprostanes, markers of oxidative damage to DNA and membrane lipids, respectively. T treatment (1) reduced growth rates of female but not male nestlings vs. controls; (2) resulted in less accumulation of 8-oxo-dG, but not IsoPs, in liver tissue of 60-day-old females, but not males; and (3) a trend toward elevated 8-oxo-dG levels in heart tissue of males and females at 60 and 180 days old combined. These results generally support the testosterone oxidative damage hypothesis, in that embryonic exposure to higher T resulted in damage to DNA of heart tissue in both sexes. They also suggest that sex-specific effects of androgens on early growth rates may carry over as differences in some forms of oxidative damage in adults. This supports a basic tenet of evolutionary aging theory that developmental influences early in life can be linked to costs later on.

ToxA Is Present in the U.S. Bipolaris sorokiniana Population and Is a Significant Virulence Factor on Wheat Harboring Tsn1.

ToxA, a necrotrophic effector originally identified from the tan spot fungus Pyrenophora tritici-repentis in 1987, was subsequently identified from Parastagonospora nodorum in 2006. More recently, the ToxA gene was identified in the spot blotch fungus Bipolaris sorokiniana in Australia. Here we show that the ToxA gene is also present in the B. sorokiniana population in the winter wheat region of southcentral Texas. Leaves from 'Duster' wheat showing strong necrotic lesions were collected in Castroville, TX. Fifteen single-spore isolates were collected from separate lesions, and 13 of them harbored the BsToxA gene and secreted ToxA in culture based on sensitivity of BG261, the differential line containing the dominant ToxA sensitivity gene, Tsn1. Four isolates harboring BsToxA and one deficient in BsToxA were used to infiltrate two wheat lines harboring Tsn1 as well as their corresponding tsn1 mutant lines. Culture filtrates of the isolate lacking BsToxA did not induce necrosis on any of the lines. Culture filtrates of the four BsToxA-containing isolates induced necrosis on the wild type (Tsn1) lines but not on the corresponding tsn1 mutant lines. Sensitivity to these culture filtrates also mapped to the previously identified location for Tsn1 in the winter wheat mapping population Arina × Forno. Inoculation of one of these ToxA-producing isolates on the same population showed that the Tsn1 locus accounted for 24.4% of the disease variation. All 13 isolates harbored the same BsToxA nucleotide sequence, which was identical to one of the two haplotypes previously identified in Australia. Sensitivity to ToxA is prevalent in popular hard winter wheat cultivars in the central and southcentral winter wheat regions of the United States, showing the potential of a selective advantage for B. sorokiniana isolates that harbor the ToxA gene.

4''-O-(omega-Quinolylamino-alkylamino)propionyl derivatives of selected macrolides with the activity against the key erythromycin resistant respiratory pathogens.

Four macrolides-6-O-methyl-8a-aza-8a-homoerythromycin, clarithromycin, azithromycin and azithromycin 11,12-cyclic carbonate, have been selected for the construction of a series of new quinolone derivatives. The quinolone moiety is connected to the macrolide scaffold via a diaminoaklyl 4''-O-propionyl ester chain of varying length. At the terminus the linker is attached via one of the nitrogen atoms in the linker at C(6) or C(7) of the quinolone. Many of compounds described, particularly clarithromycin derivative 37, and azithromycin derivatives 48 and 55, exhibited excellent antibacterial activity against a wide range of clinically relevant macrolide-resistant organisms, with profiles superior to that of telithromycin, an enhanced spectrum ketolide.

Comparative and alternative approaches and novel animal models for aging research: introduction to special issue.

This special issue of AGE showcases powerful alternative or unconventional approaches to basic aging research, including the use of exceptionally long-lived animal model species and comparative methods from evolutionary biology. In this opening paper, we introduce several of these alternative aging research themes, including the comparative phylogenetic approach. This approach applies modern inferential methods for dissecting basic physiological and biochemical mechanisms correlated with phenotypic traits including longevity, slow aging, sustained somatic maintenance, and repair of molecular damage. Comparative methods can be used to assess the general relevance of specific aging mechanisms--including oxidative processes--to diverse animal species, as well as to assess their potential clinical relevance to humans and other mammals. We also introduce several other novel, underexploited approaches with particular relevance to biogerontology, including the use of model animal species or strains that retain natural genetic heterogeneity, studies of effects of infectious disease and parasites on aging and responses to caloric restriction, studies of reproductive aging, and naturally occurring sex differences in aging. We emphasize the importance of drawing inferences from aging phenomena in laboratory studies that can be applied to clinically relevant aging syndromes in long-lived, outbred animals, including humans.

Performance data indicate summation for pictorial depth-cues in slanted surfaces.

Over recent years much has been learned about the way in which depth cues are combined (e.g. Landy et al.. 1995). The majority of this work has used subjective measures, a rating scale or a point of subjective equality, to deduce the relative contributions of different cues to perception. We have adopted a very different approach by using two interval forced-choice (21FC) performance measures and a signal processing framework. We performed summation experiments for depth cue increment thresholds between pairs of pictorial depth cues in displays depicting slanted planar surfaces made from arrays of circular 'contrast' elements. Summation was found to be ideal when size-gradient was paired with contrast-gradient for a wide range of depth-gradient magnitudes in the null stimulus. For a pairing of size-gradient and linear perspective, substantial summation (> 1.5 dB) was found only when the null stimulus had intermediate depth gradients; when flat or steeply inclined surfaces were depicted, summation was diminished or abolished. Summation was also abolished when one of the target cues was (i) not a depth cue, or (ii) added in conflict. We conclude that vision has a depth mechanism for the constructive combination of pictorial depth cues and suggest two generic models of summation to describe the results. Using similar psychophysical methods. Bradshaw and Rogers (1996) revealed a mechanism for the depth cues of motion parallax and binocular disparity. Whether this is the same or a different mechanism from the one reported here awaits elaboration.

Birds as long-lived animal models for the study of aging.

Despite their high lifetime energy expenditures, most birds can be characterized as long-lived homeotherms with moderately slow aging. A growing body of research confirms the prediction that birds have special adaptations for preventing aging-related oxidative and glycoxidative damage. Nonetheless, biogerontologists have been slow to develop avian laboratory models. A number of domestic poultry and cage bird species represent either established or very promising animal models for studies of basic aging processes and their prevention, including degenerative neurobiological, behavioral and reproductive processes. Several kinds of birds have also been used in studies of cellular resistance to oxidative stressors in vitro. Results of preliminary studies on chickens and quail suggest that caloric restriction may extend the reproductive life span of hens, but its long-term effects on life span remain unstudied. Birds' innate anti-aging mechanisms may actually make them more suitable in some respects as models of longevity than short-lived laboratory rodents, and bird studies may ultimately reveal routes for therapeutic intervention in diseases of human aging and infertility.

Novel targets for the future development of antibacterial agents.

Recent advances in DNA sequencing technology have made it possible to elucidate the entire genomes of pathogenic bacteria, and advancements in bioinformatic tools have driven comparative studies of these genome sequences. These evaluations are dramatically increasing our ability to make valid considerations of the limitations and advantages of particular targets based on their predicted spectrum and selectivity. In addition, developments in gene knockout technologies amenable to pathogenic organisms have enabled new genes and gene products critical to bacterial growth and pathogenicity to be uncovered at an unprecedented rate. Specific target examples in the areas of cell wall biosynthesis, aromatic amino acid biosynthesis, cell division, two component signal transduction, fatty acid biosynthesis, isopreniod biosynthesis and tRNA synthetases illustrate how aspects of the above capabilities are impacting on the discovery and characterization of novel antibacterial targets. An example of a novel inhibitor of bacterial fatty acid biosynthesis discovered from high throughput screening processes is described, along with its subsequent chemical optimization. Furthermore, the application and importance of technologies for tracking the mode of antibacterial action of these novel inhibitors is discussed.

Novel targets for the future development of antibacterial agents.

Recent advances in DNA sequencing technology have made it possible to elucidate the entire genomes of pathogenic bacteria, and advancements in bioinformatic tools have driven comparative studies of these genome sequences. These evaluations are dramatically increasing our ability to make valid considerations of the limitations and advantages of particular targets based on their predicted spectrum and selectivity. In addition, developments in gene knockout technologies amenable to pathogenic organisms have enabled new genes and gene products critical to bacterial growth and pathogenicity to be uncovered at an unprecedented rate. Specific target examples in the areas of cell wall biosynthesis, aromatic amino acid biosynthesis, cell division, two component signal transduction, fatty acid biosynthesis, isopreniod biosynthesis and tRNA synthetases illustrate how aspects of the above capabilities are impacting on the discovery and characterization of novel antibacterial targets. An example of a novel inhibitor of bacterial fatty acid biosynthesis discovered from high throughput screening processes is described, along with its subsequent chemical optimization. Furthermore, the application and importance of technologies for tracking the mode of antibacterial action of these novel inhibitors is discussed.

Adaptation and gain pool summation: alternative models and masking data.

Foley [J. Opt. Soc. Am. A 11 (1994) 1710] has proposed an influential psychophysical model of masking in which mask components in a contrast gain pool are raised to an exponent before summation and divisive inhibition. We tested this summation rule in experiments in which contrast detection thresholds were measured for a vertical 1 c/deg (or 2 c/deg) sine-wave component in the presence of a 3 c/deg (or 6 c/deg) mask that had either a single component oriented at -45 degrees or a pair of components oriented at +/-45 degrees. Contrary to the predictions of Foley's model 3, we found that for masks of moderate contrast and above, threshold elevation was predicted by linear summation of the mask components in the inhibitory stage of the contrast gain pool. We built this feature into two new models, referred to as the early adaptation model and the hybrid model. In the early adaptation model, contrast adaptation controls a threshold-like nonlinearity on the output of otherwise linear pathways that provide the excitatory and inhibitory inputs to a gain control stage. The hybrid model involves nonlinear and nonadaptable routes to excitatory and inhibitory stages as well as an adaptable linear route. With only six free parameters, both models provide excellent fits to the masking and adaptation data of Foley and Chen [Vision Res. 37 (1997) 2779] but unlike Foley and Chen's model, are able to do so with only one adaptation parameter. However, only the hybrid model is able to capture the features of Foley's (1994) pedestal plus orthogonal fixed mask data. We conclude that (1) linear summation of inhibitory components is a feature of contrast masking, and (2) that the main aftereffect of spatial adaptation on contrast increment thresholds can be assigned to a single site.

Exceptional cellular resistance to oxidative damage in long-lived birds requires active gene expression.

Previous studies indicated that renal tubular epithelial cells from some long-lived avian species exhibit robust and/or unique protective mechanisms against oxidative stress relative to murine cells. Here we extend these studies to investigate the response of primary embryonic fibroblast-like cells to oxidative challenge in long- and short-lived avian species (budgerigar, Melopsittacus undulatus, longevity up to 20 years, vs Japanese quail, Coturnix coturnix japonica, longevity up to 5 years) and short- and long-lived mammalian species (house mouse, Mus musculus, longevity up to 4 years vs humans, Homo sapiens, longevity up to 122 years). Under the conditions of our assay, the oxidative-damage resistance phenotype appears to be associated with exceptional longevity in avian species, but not in mammals. Furthermore, the extreme oxidative damage resistance phenotype observed in a long-lived bird requires active gene transcription and translation, suggesting that specific gene products may have evolved in long-lived birds to facilitate resistance to oxidative stress.

Lipid modification of prelipoproteins is dispensable for growth in vitro but essential for virulence in Streptococcus pneumoniae.

A Deltalgt (Lgt, lipoprotein diacylglyceryl transferase) isogenic mutant was obtained which indicates that lgt is not essential for cell growth in vitro, like in the Gram-positive bacterium Bacillus subtilis, but unlike in the proteobacteria Escherichia coli and Salmonella typhimurium. The mutation was transduced to a virulent strain. A 5 log attenuation was observed in a respiratory tract model of infection. Metabolic labeling by [U-14C]palmitate revealed the presence of eight to ten lipoproteins in the wild-type strain only, with molecular masses between 15 and 80 kDa. Our findings suggest a major difference in the role of lipoproteins in Gram-positive bacteria versus the proteobacteria.

Bacterial fatty-acid biosynthesis: a genomics-driven target for antibacterial drug discovery.

In this review we demonstrate how the interplay of genomics, bioinformatics and genomic technologies has enabled an in-depth analysis of the component enzymes of the bacterial fatty-acid biosynthesis pathway as a source of novel antibacterial targets. This evaluation has revealed that many of the enzymes are potentially selective, broad-spectrum antibacterial targets. We also illustrate the suitability of some of these targets for HTS. Furthermore, we discuss how the availability of a robust selectivity assay, mode-of-action assays and numerous crystal structures provide an excellent set of tools with which to initiate integrated programs of research to identify novel antibiotics targeted at these enzymes.

Comparative biology of aging in birds: an update.

The long life spans and slow aging rates of birds relative to mammals are paradoxical in view of birds' high metabolic rates, body temperatures and blood glucose levels, all of which are predicted to be liabilities by current biochemical theories of aging. Available avian life-table data show that most birds undergo rapid to slow "gradual" senescence. Some seabird species exhibit extremely slow age-related declines in both survival and reproductive output, and even increase reproductive success as they get older. Slow avian senescence is thought to be coupled evolutionarily with delayed maturity and low annual fecundity. Recent research in our lab and others supports the hypothesis that birds have special adaptations for preventing age-related tissue damage caused by reactive oxygen species (ROS) and advanced glycosylation endproducts, or AGEs, as well as an unusual capacity for neurogeneration in brain. Much of this work is in its early stages, however, and reliable biomarkers for comparing avian and mammalian aging need more thorough development.

Delivering novel targets and antibiotics from genomics.

Recent advances in DNA sequencing technology have made it possible to elucidate the sequences of the entire genomes of pathogenic bacteria and concomitant advancements in bioinformatic tools have driven comparative studies of these genome sequences. These evaluations are significantly increasing our ability to make valid considerations of the limitations and advantages of particular targets based on their predicted spectrum and selectivity. In addition, developments in gene-essentiality technologies amenable to pathogenic organisms liave enabled new genes and gene products critical to bacterial growth and pathogenicity to be uncovered at an unprecedented rate. This review will describe how aspects of the above capabilities are impacting the discovery and characterization of known and novel antibacterial targets using specific examples taken from a variety of important, diverse bacterial processes.

Genetic characterization of gram-positive homologs of the XerCD site-specific recombinases.

Homologs of the XerCD enzymes, which in Escherichia coli have been shown to be responsible for resolving chromosomal multimers prior to chromosome segregation, were identified in the genomes of Staphylococcus aureus and Streptococcus pneumoniae. Phylogenetic and conservation pattern analysis suggests that the S. aureus gene products are orthologs of XerC and D. A S. aureus xerC null mutant displayed in vitro characteristics consistent with the segregation defect reported for E. coli xer mutants, and was found to be attenuated in a murine infection model. Strikingly, the S. aureus xerD gene appears to be absolutely required for viability, and may therefore be the first example of an essential gene of the lambda integrase family. In contrast, phylogenetic and conservation pattern analysis show that the S. pneumoniae gene products are more closely related to phage integrases than to XerCD. S. pneumoniae xer1, 2 and 3 null mutants were each found to be attenuated in a murine infection model, suggesting that they may control processes which affect virulence.

Two active forms of UDP-N-acetylglucosamine enolpyruvyl transferase in gram-positive bacteria.

Gene sequences encoding the enzymes UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) from many bacterial sources were analyzed. It was shown that whereas gram-negative bacteria have only one murA gene, gram-positive bacteria have two distinct genes encoding these enzymes which have possibly arisen from gene duplication. The two murA genes of the gram-positive organism Streptococcus pneumoniae were studied further. Each of the murA genes was individually inactivated by allelic replacement. In each case, the organism was viable despite losing one of its murA genes. However, when attempts were made to construct a double-deletion strain, no mutants were obtained. This indicates that both genes encode active enzymes that can substitute for each other, but that the presence of a MurA function is essential to the organism. The two genes were further cloned and overexpressed, and the enzymes they encode were purified. Both enzymes catalyzed the transfer of enolpyruvate from phosphoenolpyruvate to UDP-N-acetylglucosamine, confirming they are both active UDP-N-acetylglucosamine enolpyruvyl transferases. The catalytic parameters of the two enzymes were similar, and they were both inhibited by the antibiotic fosfomycin.

Identification, evolution, and essentiality of the mevalonate pathway for isopentenyl diphosphate biosynthesis in gram-positive cocci.

The mevalonate pathway and the glyceraldehyde 3-phosphate (GAP)-pyruvate pathway are alternative routes for the biosynthesis of the central isoprenoid precursor, isopentenyl diphosphate. Genomic analysis revealed that the staphylococci, streptococci, and enterococci possess genes predicted to encode all of the enzymes of the mevalonate pathway and not the GAP-pyruvate pathway, unlike Bacillus subtilis and most gram-negative bacteria studied, which possess only components of the latter pathway. Phylogenetic and comparative genome analyses suggest that the genes for mevalonate biosynthesis in gram-positive cocci, which are highly divergent from those of mammals, were horizontally transferred from a primitive eukaryotic cell. Enterococci uniquely encode a bifunctional protein predicted to possess both 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and acetyl-CoA acetyltransferase activities. Genetic disruption experiments have shown that five genes encoding proteins involved in this pathway (HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, and mevalonate diphosphate decarboxylase) are essential for the in vitro growth of Streptococcus pneumoniae under standard conditions. Allelic replacement of the HMG-CoA synthase gene rendered the organism auxotrophic for mevalonate and severely attenuated in a murine respiratory tract infection model. The mevalonate pathway thus represents a potential antibacterial target in the low-G+C gram-positive cocci.

A genomic analysis of two-component signal transduction in Streptococcus pneumoniae.

A genomics-based approach was used to identify the entire gene complement of putative two-component signal transduction systems (TCSTSs) in Streptococcus pneumoniae. A total of 14 open reading frames (ORFs) were identified as putative response regulators, 13 of which were adjacent to genes encoding probable histidine kinases. Both the histidine kinase and response regulator proteins were categorized into subfamilies on the basis of phylogeny. Through a systematic programme of mutagenesis, the importance of each novel TCSTS was determined with respect to viability and pathogenicity. One TCSTS was identified that was essential for the growth of S. pneumoniaeThis locus was highly homologous to the yycFG gene pair encoding the essential response regulator/histidine kinase proteins identified in Bacillus subtilis and Staphylococcus aureus. Separate deletions of eight other loci led in each case to a dramatic attenuation of growth in a mouse respiratory tract infection model, suggesting that these signal transduction systems are important for the in vivo adaptation and pathogenesis of S. pneumoniae. The identification of conserved TCSTSs important for both pathogenicity and viability in a Gram-positive pathogen highlights the potential of two-component signal transduction as a multicomponent target for antibacterial drug discovery.

Cultured renal epithelial cells from birds and mice: enhanced resistance of avian cells to oxidative stress and DNA damage.

Current mechanistic theories of aging would predict that many species of birds, given their unusually high metabolic rates, body temperatures, and blood sugar levels, should age more rapidly than mammals of comparable size. On the contrary, many avian species display unusually long life spans. This finding suggests that cells and tissues from some avian species may enjoy unusually robust and/or unique protective mechanisms against fundamental aging processes, including a relatively high resistance to oxidative stress. We therefore compared the sensitivities of presumptively homologous epithelial somatic cells derived from bird and mouse kidneys to various forms of oxidative stress. When compared to murine cells, we found enhanced resistance of avian cells from three species (budgerigars, starlings, canaries) to 95% oxygen, hydrogen peroxide, paraquat, and gamma-radiation. Differential resistance to 95% oxygen was demonstrated with both replicating and quiescent cultures. Hydrogen peroxide was shown to induce DNA single-strand breaks. There were fewer breaks in avian cells than in mouse cells when similarly challenged.