Culture of large vessel endothelial cells on floating collagen gels promotes a phenotype characteristic of endothelium in vivo

The vascular endothelium in vivo is a remarkably quiescent cell layer that displays a highly differentiated and tissue-specific phenotype. Once established in culture, endothelial cells (EC) are phenotypically different from their in situ counterparts, displaying altered gene expression, increased mitotic index, and decreased cell density. To determine whether manipulating the microenvironment of cells in vitro would lead to a more differentiated phenotype, we cultured bovine aortic EC on floating collagen gels. EC cultured to confluence on floating gels for 24 or 48 hr display mitotic indices nearly identical to those of quiescent endothelium in vivo, nearly two log orders lower than that of EC cultured to confluence on plastic, and cell density on floating gels also resembles that observed for endothelium in vivo. Culture of EC on floating gels leads to decreased expression of platelet-derived growth factor-B, fibronectin, and fibronectin isoform ED-B, and increased levels of connexin40, relative to cells cultured on plastic. We conclude that culture of bovine aortic EC under standard culture conditions results in a phenotype reminiscent of development and/or wound healing, and that culturing them on a floating collagen gel leads to a more differentiated phenotype, reminiscent of that observed for large vessel EC in vivo.     

The kinase activity of DNA-PK is required to protect mammalian telomeres

The kinase activity of DNA-dependent protein kinase (DNA-PK) is required for efficient repair of DNA double-strand breaks (DSB) by non-homologous end joining (NHEJ). DNA-PK also participates in protection of mammalian telomeres, the natural ends of chromosomes. Here we investigate whether the kinase activity of DNA-PK is similarly required for effective telomere protection. DNA-PK proficient mouse cells were exposed to a highly specific inhibitor of DNA-PK phosphorylation designated IC86621. Chromosomal end-to-end fusions were induced in a concentration-dependent manner, demonstrating that the telomere end-protection role of DNA-PK requires its kinase activity. These fusions were uniformly chromatid-type, consistent with a role for DNA-PK in capping telomeres after DNA replication. Additionally, fusions involved exclusively telomeres produced via leading-strand DNA synthesis. Unexpectedly, the rate of telomeric fusions induced by IC86621 exceeded that which occurs spontaneously in DNA-dependent protein kinase catalytic subunit (DNA-PKcs) mutant cells by up to 110-fold. One explanation, that IC86621 might inhibit other, as yet unknown proteins, was ruled out when the drug failed to induce fusions in DNA-PKcs knock-out mouse cells. IC86621 did not induce fusions in Ku70 knock-out cells suggesting the drug requires the holoenzyme to be effective. ATM also is required for effective chromosome end protection. IC86621 increased fusions in ATM knock-out cells suggesting DNA-PK and ATM act in different telomere pathways. These results indicate that the kinase activity of DNA-PK is crucial to reestablishing a protective terminal structure, specifically on telomeres replicated by leading-strand DNA synthesis.     

Cuticle characteristics and volatile emissions of petals in Antirrhinum majus

Floral volatiles, which are small and generally water-insoluble, must move from their intracellular sites of synthesis through the outermost cuticle membrane before release from the flower surface. To determine whether petal cuticle might influence volatile emissions, we performed the first analysis of petal cuticle development and its association with the emission of flower volatiles using Antirrhinum majus L. (snapdragon) as a model system. Petal cuticular wax amount and composition, cuticle thickness and ultrastructure, and the amounts of internal and emitted methylbenzoate (the major snapdragon floral scent compound) were examined during 12 days, from flower opening to senescence. Normal ( n -) alkanes were found to be the major wax class of snapdragon petals (29.0% to 34.3%) throughout the 12 days examined. Besides n -alkanes, snapdragon petals possessed significant amounts of methyl branched alkanes (23.6–27.8%) and hydroxy esters (12.0–14.0%). Hydroxy esters have not been previously reported in plants. Changes in amount of methylbenzoate inside the petals followed closely with levels of methylbenzoate emission, suggesting that snapdragon petal cuticle may provide little diffusive resistance to volatile emissions. Moreover, clear associations did not exist between methylbenzoate emission and the cuticle properties examined during development. Nevertheless, the unique wax composition of snapdragon petal cuticles shows similarities with those of other highly permeable cuticles, suggesting an adaptation that could permit rapid volatile emission by scented flowers.     

Peroxidase-catalyzed oxidation of capsaicinoids: steady-state and transient-state kinetic studies

Capsaicinoids are the pungent compounds in Capsicum fruits (i.e., "hot" peppers). Peroxidases catalyze capsaicinoid oxidation and may play a central role in their metabolism. However, key kinetic aspects of peroxidase-catalyzed capsaicinoid oxidation remain unresolved. Using transient-state methods, we evaluated horseradish peroxidase compound I and II reduction by two prominent capsaicinoids (25 degrees C, pH 7.0). We determined rate constants approaching 2 x 10(7) and 5 x 10(5)M(-1)s(-1) for compound I and compound II reduction, respectively. We also determined k(app) values for steady-state capsaicinoid oxidation approaching 8 x 10(5)M(-1)s(-1) (25 degrees C, pH 7.0). Accounting for stoichiometry, these are in excellent agreement with constants for compound II reduction, suggesting that this reaction governs capsaicinoid-dependent peroxidase turnover. Ascorbate rapidly reduced capsaicinoid radicals, assisting in the determination of the kinetic constants reported. Because ascorbate accumulates in Capsicum fruits, it may also be an important determinant for capsaicinoid content and preservation in Capsicum fruits and related products.     

Evolutionary transitions in parental care and live bearing in vertebrates

We provide the first review of phylogenetic transitions in parental care and live bearing for a wide variety of vertebrates. This includes new analyses of both numbers of transitions and transition probabilities. These reveal numerous transitions by shorebirds and anurans toward uniparental care by either sex. Whereas most or all of the shorebird transitions were from biparental care, nearly all of the anuran transitions have been from no care, reflecting the prevalence of each form of care in basal lineages in each group. Teleost (bony) fishes are similar to anurans in displaying numerous transitions toward uniparental contributions by each sex. Whereas cichlid fishes have often evolved from biparental care to female care, other teleosts have usually switched from no care to male care. Taxa that have evolved exclusive male care without courtship-role reversal are characterized by male territoriality and low costs of care per brood. Males may therefore benefit from care through female preference of parental ability in these species. Primates show a high frequency of transitions from female care to biparental care, reflecting the prevalence of female care in basal lineages. In the numerous taxa that display live bearing by females, including teleosts, elasmobranchs, squamate reptiles and invertebrates, we find that live bearing has always evolved from a lack of care. Although the transition counts and probabilities will undoubtedly be refined as phylogenetic information and methodologies improve, the overall biases in these taxa should help to place adaptive hypotheses for the evolution of care into a stronger setting for understanding directions of change.     

Life-history correlates of the evolution of live bearing in fishes

Selection for live bearing is thought to occur when the benefits of increasing offspring survival exceed the costs of reduced fecundity, mobility and the increased metabolic demands of carrying offspring throughout development. We present evidence that live bearing has evolved from egg laying 12 times in teleost (bony) fishes, bringing the total number of transitions to 21 to 22 times in all fishes, including elasmobranchs (sharks and rays). Live bearers produce larger offspring than egg layers in all of 13 independent comparisons for which data were available. However, contrary to our expectation there has not been a consistent reduction in fecundity; live bearers have fewer offspring in seven out of the 11 available comparisons. It was predicted that live bearers would have a larger body size, as this facilitates accommodation of developing offspring. This prediction was upheld in 14 out of 20 comparisons. However, this trend was driven by elasmobranchs, with large live bearers in seven out of eight comparisons. Thus, while the evolution of live bearing in elasmobranchs is correlated with predicted increases in offspring size and adult size, teleost live bearers do not have such a consistent suite of life-history correlates. This suggests that constraints or selection pressures on associated life histories may differ in live-bearing elasmobranchs and teleost fishes.     

Dose responses for chromosome aberrations produced in noncycling primary human fibroblasts by alpha particles,and by gamma rays delivered at sublimiting low dose rates

As the total dose of X or gamma rays is delivered at lower and lower rates, the yield of chromosome aberrations progressively diminishes. Simultaneously, the shape of the dose response changes from one exhibiting pronounced upward curvature at high dose rates to one approaching linearity at low dose rates. Although the maximum sparing effect caused by lowering the dose rate can be predicted from classical cytogenetic theory, it has yet to be verified experimentally. Here, noncycling normal human fibroblasts were exposed to graded doses of (137)Cs gamma rays at chronic dose rates of 6.3 and 2.8 cGy h(-1), dose rates that we reasoned should be lower than those required to achieve maximal sparing. This was indeed shown to be the case, after it was determined that the two chronic dose rates produced identical linear dose responses of 0.05 total aberrations per cell Gy(-1). Consistent with cytogenetic theory, this value was statistically indistinguishable from the linear coefficient derived from a fit to aberration frequencies produced by high-dose-rate exposure. Exposure to (238)Pu alpha particles also produced a linear dose response for total aberrations, whose slope-with respect to (137)Cs gamma rays as a reference radiation-implied a maximum RBE of 35 +/- 2.