Effect of activity duration on recovery and metabolic costs in the desert iguana (Dipsosaurus dorsalis).

The majority of elevated O(2) consumption associated with short and vigorous activity occurs during recovery, thus an assessment of associated metabolic costs should also examine the excess post-exercise oxygen consumption (EPOC). This study examined O(2) uptake during exercise, EPOC and distance traveled during 5-, 15-, 60- and 300-s sprints at maximal treadmill intensity in Dipsosaurus (N=10; 74.3+/-2.1 g). EPOC (0.08, 0.14, 0.23 and 0.18 ml O(2) g(-1), respectively) was large (80-99% of total elevated O(2) consumption) and increased significantly between 5 and 60 s. The cost of activity (C(act); ml O(2) g(-1) x km(-1)), intended to reflect the total net costs associated with the activity, was calculated as the total elevated O(2) consumption per unit distance traveled. C(act) decreased with activity duration due to proportionally larger increases in distance traveled relative to EPOC volume, and is predicted by the equation C(act)=14.7 x activity duration (s)(-0.24). The inclusion of EPOC costs provides an ecologically relevant estimate of the total metabolic cost of locomotor activity. C(act) exceeds standard transport costs at all durations examined due to the addition of obligate recovery costs. The differences are large enough to impact energy budget analyses for ectotherms.     

An integrated framework for discovery and genotyping of genomic variants from high-throughput sequencing experiments

Recent advances in high-throughput sequencing (HTS) technologies and computing capacity have produced unprecedented amounts of genomic data that have unraveled the genetics of phenotypic variability in several species. However, operating and integrating current software tools for data analysis still require important investments in highly skilled personnel. Developing accurate, efficient and user-friendly software packages for HTS data analysis will lead to a more rapid discovery of genomic elements relevant to medical, agricultural and industrial applications. We therefore developed Next-Generation Sequencing Eclipse Plug-in (NGSEP), a new software tool for integrated, efficient and user-friendly detection of single nucleotide variants (SNVs), indels and copy number variants (CNVs). NGSEP includes modules for read alignment, sorting, merging, functional annotation of variants, filtering and quality statistics. Analysis of sequencing experiments in yeast, rice and human samples shows that NGSEP has superior accuracy and efficiency, compared with currently available packages for variants detection. We also show that only a comprehensive and accurate identification of repeat regions and CNVs allows researchers to properly separate SNVs from differences between copies of repeat elements. We expect that NGSEP will become a strong support tool to empower the analysis of sequencing data in a wide range of research projects on different species.     

DNA Sequence Determinants Controlling Affinity,Stability and Shape of DNA Complexes Bound by the Nucleoid Protein Fis

The abundant Fis nucleoid protein selectively binds poorly related DNA sequences with high affinities to regulate diverse DNA reactions. Fis binds DNA primarily through DNA backbone contacts and selects target sites by reading conformational properties of DNA sequences, most prominently intrinsic minor groove widths. High-affinity binding requires Fis-stabilized DNA conformational changes that vary depending on DNA sequence. In order to better understand the molecular basis for high affinity site recognition, we analyzed the effects of DNA sequence within and flanking the core Fis binding site on binding affinity and DNA structure. X-ray crystal structures of Fis-DNA complexes containing variable sequences in the noncontacted center of the binding site or variations within the major groove interfaces show that the DNA can adapt to the Fis dimer surface asymmetrically. We show that the presence and position of pyrimidine-purine base steps within the major groove interfaces affect both local DNA bending and minor groove compression to modulate affinities and lifetimes of Fis-DNA complexes. Sequences flanking the core binding site also modulate complex affinities, lifetimes, and the degree of local and global Fis-induced DNA bending. In particular, a G immediately upstream of the 15 bp core sequence inhibits binding and bending, and A-tracts within the flanking base pairs increase both complex lifetimes and global DNA curvatures. Taken together, our observations support a revised DNA motif specifying high-affinity Fis binding and highlight the range of conformations that Fis-bound DNA can adopt. The affinities and DNA conformations of individual Fis-DNA complexes are likely to be tailored to their context-specific biological functions.     

Age and growth-related changes in cyclopiazonic acid-potentiated lipophilic cation accumulation by cultured cells and binding to freeze-thaw lysed cells

In a previous study (1) we demonstrated that increased tetraphenylphosphonium (TPP) uptake by renal epithelial cells (LLC-PK₁) exposed to the fungal metabolite cyclopiazonic acid (CPA) was not a result of hyperpolarization across the plasma membrane even though CPA-potentiated TPP uptake could be totally inhibited by the depolarizing agent carbonylcyanide-m-chlorophenylhydrazone (CCCP). We now demonstrate that CPA potentiates TPP accumulation by proliferating skeletal muscle (L6) and LLC-PK₁ cells but not by nonproliferating primary rat hepatocytes. In LLC-PK₁ cells, CPA-potentiated TPP accumulation is observed in cells at all ages. In s cells, CPA-potentiated TPP accumulation is maximal soon after subculturing, and as the cells age they become less sensitive to CPA until TPP accumulation by CPA-treated cells approaches that of untreated cells. The temporal change in sensitivity of L6 cells to CPA may be related to biochemical and/or metabolic changes which occur as the cells age in culture. Hepatocytes, LLC-PK₁ cells, and L6 cells permeabilized by freeze-thaw lysis, all exhibit CPA-potentiated TPP partitioning, even in the presence of CCCP. This result indicates that both TPP and CPA must have access to the intracellular space in order for potentiated TPP partitioning to be observed. We hypothesize that the site of interaction between CPA and TPP is intracellular and probably associated with the cytoplasmic side of the plasma membrane and possibly the mitochondria.     

Thermal inhibition of repair of methylmethane sulfonate-damaged DNA in chick embryo fibroblasts

Chick embryo fibroblasts were treated with the monofunctional alkylating agent methylmethane sulfonate at various concentrations for 1 h at 42°C, rinsed and then incubated post-treatment at various temperatures at which the kinetics of alkali-labile bond disappearance was followed. Growth experiments showed that these cells grew similarly at temperatures of either 37°C or 42°C. Repair as assessed by removal of alkali-labile bond was also similar for postincubation in the temperature range 37–42°C for damage due to methylmethane sulfonate treatment at concentrations less than 1.5 mM. When the postincubation temperature was raised higher than 42.5–43°C, this type of repair was stopped. The normal internal body temperature of adult chickens is about 41.6°C. Hence the present finding indicates that chick cells are much more severely restricted in DNA repair at temperatures above normal than are mammalian cells, which can function in this respect for several deg. C above 37°C.     

SWiP-1: novel SOCS box containing WD-protein regulated by signalling centres and by Shh during development.

We describe a novel chick WD-protein, cSWiP-1, expressed in somitic mesoderm and developing limb buds as well as in other embryonic structures where Hedgehog signalling has been shown to play a role. Using embryonic manipulations we show that in somites cSWiP-1 expression integrates two signals originating from structures adjacent to the segmental mesoderm: a positive signal from the notochord and a negative signal from intermediate and/or lateral mesoderm. In explant cultures of somitic mesoderm, Shh protein induces cSWiP-1, while a blocking antibody to Shh inhibits the induction of cSWiP-1 by the notochord. These results show that the positive signal from the notochord is mediated by Shh. We also show that in limb buds cSWiP-1 is upregulated by ectopic Shh. This occurs in about the same time period as upregulation of BMP2, placing cSWiP-1 among the earliest markers for the change of limb pattern caused by ectopic Shh. We also describe a human homologue of cSWiP-1 and a mouse gene, mSWiP-2, that is more distantly related to SWiP-1, suggesting that SWiP-1 belongs to a novel subfamily of WD-proteins.