Telomere loss in relation to age and early environment in long-lived birds

Shortening of telomeres, specific nucleotide repeats that cap eukaryotic chromosomes, is thought to play an important role in cellular and organismal senescence. We examined telomere dynamics in two long-lived seabirds, the European shag and the wandering albatross. Telomere length in blood cells declines between the chick stage and adulthood in both species. However, among adults, telomere length is not related to age. This is consistent with reports of most telomere loss occurring early in life in other vertebrates. Thus, caution must be used in estimating annual rates of telomere loss, as these are probably not constant with age. We also measured changes within individuals in the wild, using repeat samples taken from individual shags as chicks and adults. We found high inter-individual variation in the magnitude of telomere loss, much of which was explained by circumstances during growth. Individuals laying down high tissue mass for their size showed greater telomere shortening. Independently of this, individuals born late in the season showed more telomere loss. Early conditions, possibly through their effects on oxidative stress, appear to play an important role in telomere attrition and thus potentially in the longevity of individuals.     

Maternal condition, yolk androgens and offspring performance: a supplemental feeding experiment in the lesser black-backed gull (Larus fuscus)

It has been proposed that the maternal androgens in avian egg yolk enhance offspring fitness by accelerating growth and improving competitive ability. Because egg quality is strongly influenced by maternal condition, we predicted that females in good condition would produce high-quality eggs with relatively high androgen content. We experimentally enhanced maternal condition by supplementary feeding lesser black-backed gulls (Larus fuscus) during egg formation and compared the concentrations of androstenedione (A4), 5alpha-dihydrotestosterone (DHT) and testosterone (T) in their eggs with those in eggs laid by control females. We also measured circulating levels of T in females immediately after laying. Egg androgens could affect offspring performance directly through chick development and/or indirectly through changes in the competitive ability of a chick relative to its siblings. To avoid confounding these two routes, and to separate effects operating through the egg itself with those operating through experimental changes in parental chick rearing capacity, we fostered eggs from both maternal treatment groups singly into the nests of unmanipulated parents. Contrary to expectation, mothers with experimentally enhanced body condition laid eggs with lower levels of androgens, while exhibiting higher circulating T concentrations post-laying. Despite these lower levels of egg androgen, offspring hatched from eggs laid by mothers in good condition did not show reduced growth or survival when reared in the absence of sibling competition. Our results demonstrate that yolk androgen concentrations vary with the body condition of the female at the time of egg formation and that females in good condition reduced the yolk androgen content of their eggs without altering offspring performance.     

Escherichia coli--a model system that benefits from and contributes to the evolution of proteomics

The large body of knowledge about Escherichia coli makes it a useful model organism for the expression of heterologous proteins. Proteomic studies have helped to elucidate the complex cellular responses of E. coli and facilitated its use in a variety of biotechnology applications. Knowledge of basic cellular processes provides the means for better control of heterologous protein expression. Beyond such important applications, E. coli is an ideal organism for testing new analytical technologies because of the extensive knowledge base available about the organism. For example, improved technology for characterization of unknown proteins using mass spectrometry has made two-dimensional electrophoresis (2DE) studies more useful and more rewarding, and much of the initial testing of novel protocols is based on well-studied samples derived from E. coli. These techniques have facilitated the construction of more accurate 2DE maps. In this review, we present work that led to the 2DE databases, including a new map based on tandem time-of-flight (TOF) mass spectrometry (MS); describe cellular responses relevant to biotechnology applications; and discuss some emerging proteomic techniques.     

The inotropic effect of nitric oxide on mammalian papillary muscle is dependent on the level of beta1-adrenergic stimulation

We tested the hypothesis that nitric oxide has a positive inotropic effect on mammalian cardiac muscle contractility and that this effect sums with the positive inotropic effect of beta1-adrenergic agonists when both are present. Feline right ventricular papillary muscles were stimulated to contract isometrically at 0.2 Hz in Krebs-Henseleit bicarbonate buffer (KREBS) gassed with 95% O2 and 5% CO2 (26 degrees C; pH 7.34). The nitric oxide (NO) donor, S-nitroso-N-acetylpenicillamine (SNAP, 10(-5) M), and the membrane permeable cGMP analog 8-bromoguanosine-3',5'-cyclophosphate sodium (Br-cGMP, 10(-5) M), significantly increased developed force by 13.3+/-1.5% (n = 11) and 7.8+/-2.8% (n = 7), respectively. SNAP, at 10(-5) M, significantly increased the force developed by papillary muscle treated with 10(-11) M or 10(-9) M dobutamine hydrochloride (a beta1-adrenergic agonist) (n = 25, 11.3+/-2.9% and 10.0+/-3.6%, respectively) when compared with the addition of KREBS (n = 27, 2.6+/-0.9% and 5.5+/-0.9%), but the increase was less than predicted by the sum of inotropic effects of SNAP and dobutamine. SNAP at 10(-5) M did not change developed force in muscles treated with 10(-7) M dobutamine but it significantly decreased developed force in muscles challenged with 10(-5) M dobutamine (n = 18, 29.3+/-5.0%) when compared with KREBS (n = 10, 41.5+/-6.8%). Similarly, 10(-4) M 8-bromo-adenosine cyclic 3',5'-hydrogen phosphate monosodium (a membrane permeable cAMP analog) increased developed force 14.9+/-3.3% and the addition of 10(-5) M Br-cGMP to those muscles significantly reduced developed force by 3.5%+/-1.1% (n = 7). Thus, the positive inotropic effect of NO decreased and ultimately became an attenuation as the level of beta1-adrenergic stimulation increased due at least in part, to an interaction between the cAMP and cGMP second messenger pathways.     

Measurements of tumor tissue oxygen tension using a time-resolved luminescence-based optical oxylite probe: comparison with a paired survival assay

Recently, a system that measures tissue oxygen tension using time-resolved luminescence-based optical sensors has become available commercially (Oxford Optronix, Oxford, England). Two experiments were conducted using this system. First, the oxygen tension distribution was measured in two tumor lines: a spontaneous mouse fibrosarcoma, FSa-II, and a human squamous cell carcinoma xenograft, FaDu. The area in which the pO(2) was equal to or lower than 2.5 mmHg was defined as the hypoxic lesion, and the hypoxic cell fraction was taken as the fraction of these measurements in a tumor. The measured hypoxic cell fractions were compared with those determined by the paired cell survival assay for tumors of various sizes. Second, the tumor tissue pO(2) was measured continuously after administration of two different anesthetics to evaluate the effect of these drugs on tissue pO(2). Results indicated a good agreement between the hypoxic cell fractions measured by this system and those determined by the paired cell survival curve assay for tumors smaller than approximately 500 mm(3). For tumors larger than approximately 500 mm(3), the hypoxic cell fractions measured by the oxygen probe system were higher than those measured by the paired cell survival assay. This may suggest that the hypoxic cell fraction measured by the oxygen probes included both hypoxic and necrotic areas in large tumors where necrotic lesions occupied a significant portion of the tumor. Continuous measurements of pO(2) after anesthesia (Nembutal, or ketamine plus xylazine) showed a consistent rise in the pO(2) during the first 20-30 min of measurement. Subsequently, the pO(2) values became constant or continued to rise slowly. For comparison, the tumor cell survivals were assayed after a dose of 20 Gy given in air at 5, 20 and 60 min after anesthesia. The result showed a decrease in cell survival only in tumors irradiated 20 min after an injection of Nembutal.     

Isolation and characterization of endophytic colonizing bacteria from agronomic crops and prairie plants

Endophytic bacteria reside within plant hosts without causing disease symptoms. In this study, 853 endophytic strains were isolated from aerial tissues of four agronomic crop species and 27 prairie plant species. We determined several phenotypic properties and found approximately equal numbers of gram-negative and gram-positive isolates. In a greenhouse study, 28 of 86 prairie plant endophytes were found to colonize their original hosts at 42 days postinoculation at levels of 3.5 to 7.7 log(10) CFU/g (fresh weight). More comprehensive colonization studies were conducted with 373 corn and sorghum endophytes. In growth room studies, none of the isolates displayed pathogenicity, and 69 of the strains were recovered from corn or sorghum seedlings at levels of 8.3 log(10) CFU/plant or higher. Host range greenhouse studies demonstrated that 26 of 29 endophytes were recoverable from at least one host other than corn and sorghum at levels of up to 5.8 log(10) CFU/g (fresh weight). Long-range dent corn greenhouse studies and field trials with 17 wild-type strains and 14 antibiotic-resistant mutants demonstrated bacterial persistence at significant average colonization levels ranging between 3.4 and 6.1 log(10) CFU/g (fresh weight) up to 78 days postinoculation. Three prairie and three agronomic endophytes exhibiting the most promising levels of colonization and an ability to persist were identified as Cellulomonas, Clavibacter, Curtobacterium, and Microbacterium isolates by 16S rRNA gene sequence, fatty acid, and carbon source utilization analyses. This study defines for the first time the endophytic nature of Microbacterium testaceum. These microorganisms may be useful for biocontrol and other applications.     

Genomics of steroid hormones: in silico analysis of nucleotide sequence variants (polymorphisms) of the enzymes involved in the biosynthesis and metabolism of steroid hormones

Alterations of steroid hormone biosynthesis and metabolism are suspected to be involved in the pathogenesis of several diseases. Several polymorphisms of the enzymes involved in these processes have already been described and some could be associated with certain diseases. We attempted to examine the sequence variants of these genes in order to find novel variants by an in silico analysis. We analyzed the known human nucleotide sequences of the enzymes p450 side-chain cleavage enzyme, steroid 17-alpha-hydroxylase/17,20-lyase, 3-beta-hydroxysteroid dehydrogenase types 1 and 2, 21-hydroxylase, 11-beta-hydroxylase, aldosterone synthase, aromatase, 11-beta-hydroxysteroid dehydrogenase types 1 and 2, steroid 5-alpha-reductase types 1 and 2, steroid 5-beta-reductase, dehydroepiandrosterone sulfotransferase, 17-beta-hydroxysteroid dehydrogenase types 1–3. The analysis was performed using the National Center for Biotechnology Information Database by the search tool blastn. We found numerous sequence variants in both coding and non-coding sequences. The majority of these sequence variants have already been described, nevertheless, some appear as novel variants. Some of these may also have functional significance. We hypothesize over the possible significance of these findings and briefly review the available literature.     

Validation of single nucleotide polymorphism quantification in pooled DNA samples with SNaPIT. A glycosylase-mediated methods for polymorphism detection method

Association studies using genome scans to identify quantitative trait loci for multifactorial disorders, with anything approaching reasonable power, have been compromised by the need for a very dense array of genetic markers and large numbers of affected individuals. These requirements impose enormous burdens on the genotyping capacity for most laboratories. DNA pooling has been proposed as a possible approach to reduce genotyping costs and effort. We report on the application of the SNaPIT™ technology to evaluate allele frequencies in pooled DNA samples and conclude that it offers a cost effective, efficient and accurate estimator and provides several advantages over competing technologies in this regard.