Scaling oxygen uptake to body size and several practical applications

Oxygen uptake (VO(2)) has typically been expressed in milliliters per kilogram per minute to equate people of different body masses. However, research suggests that VO(2) increases in proportion to body mass raised to a power between 0.6 and 0.75, rather than in proportion to body mass raised to a power of 1. The potential for several errors arises when using a body mass exponent of 1 (ml x kg(-1).min(-1)), and these include the following: (a) penalizing larger subjects and inflating the scores of lighter subjects when comparing maximal aerobic capacity scores, (b) inaccurately estimating aerobic capacity via submaximal tests, causing misinterpretations of those scores, and (c) reducing accuracy in the assessment of movement economy and the energy cost of physical activity. Expressions of VO(2) scaled to body mass to a power less than 1 tend to minimize the bias and errors associated with linear-relative expressions (ml x kg(-1) x min(-1)). The purpose of this article is to briefly review recent literature related to the scaling of VO(2) to body size and to consider some of the practical applications suggested by the review. These implications appear to be important for fitness professionals, strength and conditioning coaches, and exercise scientists.     

Arrayed adenoviral expression libraries for functional screening

With the publication of the sequence of the human genome, we are challenged to identify the functions of an estimated 70,000 human genes and the much larger number of proteins encoded by these genes. Of particular interest is the identification of gene products that play a role in human disease pathways, as these proteins include potential new targets that may lead to improved therapeutic strategies. This requires the direct measurement of gene function on a genomic scale in cell-based, functional assays. We have constructed and validated an individually arrayed, replication-defective adenoviral library harboring human cDNAs, termed PhenoSelect library. The adenoviral vector guarantees efficient transduction of diverse cell types, including primary cells. The arrayed format allows screening of this library in a variety of cellular assays in search for gene(s) that, by overexpression, induce a particular disease-related phenotype. The great majority of phenotypic assays, including morphological assays, can be screened with arrayed libraries. In contrast, pooled-library approaches often rely on phenotype-based isolation or selection of single cells by employing a flow cytometer or screening for cell survival. An arrayed placental PhenoSelect library was screened in cellular assays aimed at identifying regulators of osteogenesis, metastasis, and angiogenesis. This resulted in the identification of known regulators, as well as novel sequences that encode proteins hitherto not known to play a role in these pathways. These results establish the value of the PhenoSelect platform, in combination with cellular screens, for gene function discovery.     

The permeability of gap junction channels to probes of different size is dependent on connexin composition and permeant-pore affinities

Gap junctions have traditionally been characterized as nonspecific pores between cells passing molecules up to 1 kDa in molecular mass. Nonetheless, it has become increasingly evident that different members of the connexin (Cx) family mediate quite distinct physiological processes and are often not interchangeable. Consistent with this observation, differences in permeability to natural metabolites have been reported for different connexins, although the physical basis for selectivity has not been established. Comparative studies of different members of the connexin family have provided evidence for ionic charge selectivity, but surprisingly little is known about how connexin composition affects the size of the pore. We have employed a series of Alexa dyes, which share similar structural characteristics but range in size from molecular weight 350 to 760, to probe the permeabilities and size limits of different connexin channels expressed in Xenopus oocytes. Correlated dye transfer and electrical measurements on each cell pair, in conjunction with a three-dimensional mathematical model of dye diffusion in the oocyte system, allowed us to obtain single channel permeabilities for all three dyes in six homotypic and four heterotypic channels. Cx43 and Cx32 channels passed all three dyes with similar efficiency, whereas Cx26, Cx40, and Cx45 channels showed a significant drop-off in permeability with the largest dye. Cx37 channels only showed significant permeability for the smaller two dyes, but at two- to sixfold lower levels than other connexins tested. In the heterotypic cases studied (Cx26/Cx32 and Cx43/Cx37), permeability characteristics were found to resemble the more restrictive parental homotypic channel. The most surprising finding of the study was that the absolute permeabilities calculated for all gap junctional channels in this study are, with one exception, at least 2 orders of magnitude greater than predicted purely on the basis of hindered pore diffusion. Consequently, affinity between the probes and the pore creating an energetically favorable in-pore environment, which would elevate permeant concentration within the pore and hence the flux, is strongly implicated.     

Post-translational modifications of arabinogalactan-peptides of Arabidopsis thaliana. Endoplasmic reticulum and glycosylphosphatidylinositol-anchor signal cleavage sites and hydroxylation of proline

We have developed a method for separating the deglycosylated protein/peptide backbones of the small arabinogalactan (AG)-peptides from the larger classical arabinogalactan-proteins (AGPs). AGPs are an important class of plant proteoglycans implicated in plant growth and development. Separation of AG-peptides enabled us to identify eight of 12 AG-peptides from Arabidopsis thaliana predicted from genomic sequences. Of the remaining four, two have low abundance based on expressed sequence tag databases and the other two are only present in pollen (At3g20865) or flowers (At3g57690) and therefore would not be detected in our analysis. Characterization of AG-peptides was performed using matrix-assisted laser desorption ionization-time of flight mass spectrometry and tandem mass spectrometry protein sequencing. These data provide (i) experimental evidence that AG-peptides are processed in vivo for the addition of a glycosylphosphatidylinositol (GPI) anchor, (ii) cleavage site information for both the endoplasmic reticulum secretion signal and the GPI-anchor signal for eight of the 12 AG-peptides, and (iii) experimental evidence that the Gly-Pro motif is hydroxylated in vivo. Furthermore, we show that AtAGP16 is GPI-anchored despite its unusually long hydrophobic C-terminal GPI-signal sequence. Prior to this work, the GPI-anchor cleavage site for only two plant proteins, NaAGP1 from Nicotiana alata and PcAGP1 from Pyrus communis, had been determined experimentally. Characterization of the post-translational modifications of AG-peptides contributes toward obtaining the complete primary structure of this class of biologically important plant proteoglycans and provides a greater understanding of post-translational modifications of plant proteins.     

Caspase-specific and nonspecific in vivo protein processing during Fas-induced apoptosis

We generated a comprehensive picture of protease substrates in anti-Fas-treated apoptotic human Jurkat T lymphocytes. We used combined fractional diagonal chromatography (COFRADIC) sorting of protein amino-terminal peptides coupled to oxygen-16 or oxygen-18 differential labeling. We identified protease substrates and located the exact cleavage sites within processed proteins. Our analysis yielded 1,834 protein identifications and located 93 cleavage sites in 71 proteins. Indirect evidence of apoptosis-specific cleavage within 21 additional proteins increased the total number of processed proteins to 92. Most cleavages were at caspase consensus sites; however, other cleavage specificities suggest activation of other proteases. We validated several new processing events by immunodetection and by an in vitro assay using recombinant caspases and synthetic peptides containing presumed cleavage sites. The spliceosome complex appeared a preferred target, as 14 of its members were processed. Differential isotopic labeling further revealed specific release of nucleosomal components from apoptotic nuclei.     

Preparation of protein extracts from recalcitrant plant tissues: an evaluation of different methods for two-dimensional gel electrophoresis analysis

This study focuses on the specific problems of protein extraction from recalcitrant plant tissues and evaluates several methods to bypass them. Sample preparation is a critical step in a two-dimensional gel electrophoresis proteome approach and is absolutely essential for good results. We evaluated four methods: the classical trichloroacetic acid (TCA)/acetone precipitation, TCA/acetone precipitation and fractionation, an alternative based on fractionation and without precipitation, and phenol extraction methanol/ammonium acetate precipitation. We optimized the phenol extraction protocol for small amounts of tissue, which is essential when the study material is limited. The protocol was optimized for banana (Musa spp.) and was subsequently applied to two other plant species: apple (Malus domestica L.) and potato (Solanum tuberosum L.). Banana (Musa spp.) is a good representative of a "difficult" plant species since it contains many interfering metabolites. Only classical TCA/acetone precipitation and phenol extraction methods proved useful as standard methods. Both methods are associated with a minor but reproducible loss of proteins. Every extraction method and the subsequent analytical procedure have their physicochemical limitations; both methods should be investigated before selecting an appropriate protocol. The study, which is presented in this paper, is useful for guiding the experimental setup of many other nonmodel species, containing various interfering elements.     

Calcineurin initiates smooth muscle differentiation in neural crest stem cells

The process of vascular smooth muscle cell (vSMC) differentiation is critical to embryonic angiogenesis. However, despite its importance, the vSMC differentiation program remains largely undefined. Murine gene disruption studies have identified several gene products that are necessary for vSMC differentiation, but these methodologies cannot establish whether or not a factor is sufficient to initiate the differentiation program. A gain-of-function system consisting of normal vSMC progenitor cells would serve as a useful complement to whole animal loss-of-function studies. We use such a system here, namely freshly isolated rat neural crest stem cells (NCSCs), to show that activation of the calcineurin signaling pathway is sufficient to drive these cells toward a smooth muscle fate. In addition, we present data suggesting that transforming growth factor (TGF)-beta1, which also causes NCSCs to differentiate into smooth muscle, activates calcineurin signaling in NCSCs, leading to a model in which activation of calcineurin signaling is the mechanism by which TGF-beta1 causes SMC differentiation in these cells.     

Role of Oxidative Phosphorylation in Histatin 5-Induced Cell Death in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The succeptibility of Saccharomyces cerevisiae to the anti-microbial peptide, histatin 5, was tested after pre-growth in fermentable and non-fermentable carbon sources and in the absence or presence of the uncoupler of oxidative phosphorylation, carbonyl cyanide m-chlorophenylhydrazone (CCCP). S. cerevisiae was more resistant to histatin 5 when grown on a fermentable carbon source compared to growth on a non-fermentable carbon source, indicating an important role for oxidative phosphorylation in histatin 5-induced cell death. Oxidative phosphorylation is a pre-requisite for histatin 5-induced cell death in Candida albicans but this is not the case in S. cerevisiae. Incubation of CCCP-treated S. cerevisiae cells with histatin 5 still resulted in cell death. These results suggest that histatin 5-induced cell death in S. cerevisiae differs from that in C. albicans.Revisions received 28 September 2004     

DNA vaccines: designing strategies against parasitic infections

The complexity of parasitic infections requires novel approaches to vaccine design. The versatility of DNA vaccination provides new perspectives. This review discusses the use of prime-boost immunizations, genetic adjuvants, multivalent vaccines and codon optimization for optimal DNA vaccine design against parasites.     

Aggressiveness during monogamous pairing in the sleepy lizard, <Emphasis Type="Italic">Tiliqua rugosa</Emphasis>: a test of the mate guarding hypothesis

The Australian sleepy lizard, Tiliqua rugosa, maintains monogamous associations for an average of 6 weeks before mating each spring. One hypothesis to explain this prolonged partnership is that males are guarding their female partners from rival males. This hypothesis has three predictions, that males are more aggressive than females to conspecific males, that male aggression will increase as the time of mating gets closer, and that males will be more aggressive towards conspecific males when they are with their partner than when they are alone. We tested those predictions with indirect evidence of aggression, using counts of scale damage on randomly encountered lizards, and with direct observations of their responses to approaches by conspecific and heterospecific models. As predicted by the mate guarding hypothesis, males showed more evidence of aggression towards conspecifics than did females. However, in contrast to the hypothesis, males did not become more aggressive as the time of mating came closer, and males in pairs were less aggressive than males on their own. Mate guarding cannot be the only process that has led to the prolonged monogamous associations in this species. Parental care is also unknown in these lizards, and we suggest that monogamy may be maintained through some form of female coercion, allowing females to gain additional fitness from the enhanced vigilance that results from male proximity.