Plasma volume restoration with salt tablets and water after bed rest prevents orthostatic hypotension and changes in supine hemodynamic and endocrine variables

Head-down bed rest changes the values of many cardiovascular and endocrine variables and also elicits significant hypovolemia. Because previous studies had not controlled for hypovolemia, it is unknown whether the reported changes were primary effects of bed rest or secondary effects of bed rest-induced hypovolemia. We hypothesized that restoring plasma volume with salt tablets and water after 12 days of head-down bed rest would result in an absence of hemodynamic and endocrine changes and a reduced incidence of orthostatic hypotension. In 10 men, we measured changes from pre-bed-rest to post-bed-rest in venous and arterial pressures; heart rate; stroke volume; cardiac output; vascular resistance; plasma norepinephrine, epinephrine, vasopressin, renin activity (PRA), and aldosterone responses to different tilt levels (0 degrees, -10 degrees, 20 degrees, 30 degrees, and 70 degrees); and plasma volume and platelet alpha2- and lymphocyte beta2-adrenoreceptor densities and affinities (0 degrees tilt only). Fluid loading at the end of bed rest restored plasma volume and resulted in the absence of post-bed-rest orthostatic hypotension and changes in supine hemodynamic and endocrine variables. Fluid loading did not prevent post-bed-rest increases in beta2-adrenoreceptor density or decreases in the aldosterone-to-PRA ratio (P = 0.05 for each). Heart rate, epinephrine, and PRA responses to upright tilt after bed rest were increased (P < 0.05), despite the fluid load. These results suggest that incidents of orthostatic hypotension and many of the changes in supine hemodynamic and endocrine variables in volume-depleted bed-rested subjects occur secondarily to the hypovolemia. Despite normovolemia after bed rest, beta2-adrenoreceptors were upregulated, and heart rate, epinephrine, and PRA responses to tilt were augmented, indicating that these changes are independent of volume depletion.     

Development of solution phase hybridisation PCR-ELISA for the detection and quantification of Enterococcus faecalis and Pediococcus pentosaceus in Nurmi-type cultures

Nurmi-type cultures (NTCs), derived from the fermentation of caecal contents of specifically pathogen-free (SPF) birds, have been used successfully to control salmonella colonisation in chicks. These cultures are undefined in nature and, consequently, it is difficult to obtain approval from regulatory agencies for their use as direct fed microbials (DFMs) for poultry. Progress towards the generation of effective defined probiotics requires further knowledge of the composition of these cultures. As such, species-specific, culture-independent quantification methodologies need to be developed to elucidate the concentration of specific bacterial constituents of NTCs. Quantification of specific bacterial species in such ill-defined complex cultures using conventional culturing methods is inaccurate due to low levels of sensitivity and reproducibility, in addition to slow turnaround times. Furthermore, these methods lack selectivity due to the nature of the accompanying microflora. This study describes the development of a rapid, sensitive, reliable, reproducible, and species-specific culture-independent, solution phase hybridisation PCR-ELISA procedure for the detection and quantification of Enterococcus faecalis and Pediococcus pentosaceus in NTCs. In this technique, biotin-labelled primers were designed to amplify a species-specific fragment of a marker gene of known copy number, in both species. Resulting amplicons were hybridised with a dinitrophenol (DNP)-labelled oligonucleotide probe in solution and were subsequently captured on a streptavidin-coated microtitre plate. The degree of binding was determined by the addition of IgG (anti-DNP)-horseradish peroxidase conjugate, which was subsequently visualised using a chromogenic substrate, tetramethylbenzidine. This novel quantitative method was capable of detecting E. faecalis and P. pentosaceus at levels as low as 5 CFU per PCR reaction.     

Glutamine kinetics and protein turnover in end-stage renal disease

Alanine and glutamine constitute the two most important nitrogen carriers released from the muscle. We studied the intracellular amino acid transport kinetics and protein turnover in nine end-stage renal disease (ESRD) patients and eight controls by use of stable isotopes of phenylalanine, alanine, and glutamine. The amino acid transport kinetics and protein turnover were calculated with a three-pool model from the amino acid concentrations and enrichment in the artery, vein, and muscle compartments. Muscle protein breakdown was more than synthesis (nmol.min(-1).100 ml leg(-1)) during hemodialysis (HD) (169.8 +/- 20.0 vs. 125.9 +/- 21.8, P < 0.05) and in controls (126.9 +/- 6.9 vs. 98.4 +/- 7.5, P < 0.05), but synthesis and catabolism were comparable pre-HD (100.7 +/- 15.7 vs. 103.4 +/- 14.8). Whole body protein catabolism decreased by 15% during HD. The intracellular appearance of alanine (399.0 +/- 47.1 vs. 243.0 +/- 34.689) and glutamine (369.7 +/- 40.6 vs. 235.6 +/- 27.5) from muscle protein breakdown increased during dialysis (nmol.min(-1).100 ml leg(-1), P < 0.01). However, the de novo synthesis of alanine (3,468.9 +/- 572.2 vs. 3,140.5 +/- 467.7) and glutamine (1,751.4 +/- 82.6 vs. 1,782.2 +/- 86.4) did not change significantly intradialysis (nmol.min(-1).100 ml leg(-1)). Branched-chain amino acid catabolism (191.8 +/- 63.4 vs. -59.1 +/- 42.9) and nonprotein glutamate disposal (347.0 +/- 46.3 vs. 222.3 +/- 43.6) increased intradialysis compared with pre-HD (nmol.min(-1).100 ml leg(-1), P < 0.01). The mRNA levels of glutamine synthase (1.45 +/- 0.14 vs. 0.33 +/- 0.08, P < 0.001) and branched-chain keto acid dehydrogenase-E2 (3.86 +/- 0.48 vs. 2.14 +/- 0.27, P < 0.05) in the muscle increased during HD. Thus intracellular concentrations of alanine and glutamine are maintained during HD by augmented release of the amino acids from muscle protein catabolism. Although muscle protein breakdown increased intradialysis, the whole body protein catabolism decreased, suggesting central utilization of amino acids released from skeletal muscle.     

Cyclic mechanical strain increases reactive oxygen species production in pulmonary epithelial cells

Overdistention of lung tissue during mechanical ventilation may be one of the factors that initiates ventilator-induced lung injury (VILI). We hypothesized that cyclic mechanical stretch (CMS) of the lung epithelium is involved in the early events of VILI through the production of reactive oxygen species (ROS). Cultures of an immortalized human airway epithelial cell line (16HBE), a human alveolar type II cell line (A549), and primary cultures of rat alveolar type II cells were cyclically stretched, and the production of superoxide (O2-) was measured by dihydroethidium fluorescence. CMS stimulated increased production of O2- after 2 h in each type of cell. 16HBE cells exhibited no significant stimulation of ROS before 2 h of CMS (20% strain, 30 cycles/min), and ROS production returned to control levels after 24 h. Oxidation of glutathione (GSH), a cellular antioxidant, increased with CMS as measured by a decrease in the ratio of the reduced GSH level to the oxidized GSH level. Strain levels of 10% did not increase O2- production in 16HBE cells, whereas 15, 20, and 30% significantly increased generation of O2-. Rotenone, a mitochondrial complex I inhibitor, partially abrogated the stretch-induced generation of O2- after 2 h CMS in 16HBE cells. NADPH oxidase activity was increased after 2 h of CMS, contributing to the production of O2-. Increased ROS production in lung epithelial cells in response to elevated stretch may contribute to the onset of VILI.     

Phylogenetics of the australasian mudfishes: evolution of an eel-like body plan

The mudfish genus Neochanna (Osmeriformes: Galaxiidae) contains six species that exhibit varying degrees of morphological and ecological adaptation to life in swampy conditions. Here, we present the first molecular phylogenetic analysis (16S rRNA+cytochrome b; 1681bp) of the entire genus to (1) test for monophyly of Australian and New Zealand taxa and (2) elucidate morphological character evolution. In addition, we analyse a matrix of 21 morphological characters to test for congruence between mitochondrial DNA and morphology, and to examine total evidence under a Bayesian framework. Molecular data indicate that the diadromous Tasmanian mudfish, N. cleaveri, is sister to a clade of five non-diadromous New Zealand mudfishes. Mapping of morphological characters onto the molecular phylogeny suggests an evolutionary transition from a plesiomorphic "stream" galaxiid morphotype to a more specialised "anguilliform" galaxiid morphotype. Pelvic fins have become increasingly reduced and dorsal, anal, and caudal fins are increasingly confluent. Associated with these changes are elongated nostrils, reduced eyes, and increased anterior cranial ossification. Morphological and total evidence analyses yield similar or identical topologies, respectively. The phylogenetic distribution of diadromy in Neochanna is consistent with a single loss of this character state in New Zealand. However, the strong sister relationship (3.6% divergent; 100% bootstrap support) detected between non-diadromous N. burrowsius (South Island, NZ) and N. rekohua (Chatham Islands) indicates geologically recent dispersal across 850km of ocean. Diadromy may therefore have been retained in the common ancestor of these two mudfish species, and subsequently lost from both lineages.     

Molecular recognition with designed peptides and proteins

The design of proteins and peptides as molecular receptors is a rapidly growing area of research. Two primary approaches have been utilized, involving the minimization of known protein binding motifs or the de novo design of binding pockets within well-folded protein structures. These approaches are complementary and help define the minimum requirements necessary for biomolecular recognition. Recent advances in this area include the design of cavities within helix bundles for the binding of anesthetics, the design of beta-hairpins for the recognition of nucleotides and oligonucleotides, the redesign of protein binding sites for unique ligands, and the design of mini-proteins via protein grafting for the recognition of proteins and DNA. These advances provide exciting new opportunities to develop novel biosensors, de novo designed catalysts, exogenously triggered synthetic signal transduction cascades, and novel approaches to therapeutic treatments.     

Acute prooxidant effects of vitamin C in EDTA chelation therapy and long-term antioxidant benefits of therapy

Chelation therapy is thought to not only remove contaminating metals but also to decrease free radical production. EDTA chelation therapy, containing high doses of vitamin C as an antioxidant, is often used in the treatment of diseases such as diabetes and cardiovascular diseases but the effectiveness of this treatment may be variable and its efficacy has not been demonstrated conclusively. The objective of this work was to determine if the vitamin C added to standard chelation therapy cocktails was prooxidant. We administered a standard EDTA cocktail solution with or without 5 g of sodium ascorbate. One hour following the standard chelation therapy, there were highly significant prooxidant effects on lipids, proteins, and DNA associated with decreased activities of RBC glutathione peroxidase and superoxide dismutase while in the absence of sodium ascorbate, there were no acute signs of oxidative damage. After 16 sessions of standard chelation therapy, the acute prooxidant effects of vitamin C remained, but, even in the absence of nutrient supplements, there were beneficial long-term antioxidant effects of chelation therapy and plasma peroxide levels decreased. In conclusion, multiple sessions of EDTA chelation therapy protect lipids against oxidative damage. However, standard high amounts of vitamin C added to EDTA chelation solutions also display short term prooxidant effects. The added benefits of lower levels of vitamin C in chelation therapy need to be documented.     

c-Src is involved in regulating signal transmission from PDGFbeta receptor-GPCR(s) complexes in mammalian cells

We have reported that the platelet-derived growth factor receptor-beta (PDGFbeta) forms a novel signaling complex with G protein-coupled receptors (GPCR) (e.g. S1P(1) receptor) that enables more efficient activation of p42/p44 mitogen-activated protein kinase (MAPK) in response to PDGF and sphingosine 1-phosphate (S1P). We now demonstrate that c-Src participates in regulating the endocytosis of PDGFbeta receptor-GPCR complexes in response to PDGF. This leads to association of cytoplasmic p42/p44 MAPK with the receptor complex in endocytic vesicles. c-Src is regulated by G protein betagamma subunits and can interact with beta-arrestin. Indeed, the PDGF-dependent activation of p42/p44 MAPK was reduced by over-expression of the C-terminal domain of GRK2 (sequesters Gbetagamma subunits), the clathrin-binding domain of beta-arrestin and by inhibitors of c-Src and clathrin-mediated endocytosis. Moreover, PDGF and S1P induce the recruitment of c-Src to the PDGFbeta receptor-S1P(1) receptor complex. This leads to a G protein/c-Src-dependent tyrosine phosphorylation of Gab1 and accumulation of dynamin II at the plasma membrane, a step required for endocytosis of the PDGFbeta receptor-GPCR complex. These findings provide important information concerning the molecular organisation of novel receptor tyrosine kinase (RTK)-GPCR signal relays in mammalian cells.     

cDNA microarray analysis of developing grape (Vitis vinifera cv. Shiraz) berry skin

Microarray analysis of Vitis vinifera cv. Shiraz developing berries has revealed the expression patterns of several categories of genes. Microarray slides were constructed from 4,608 PCR-amplified cDNA clones derived from a ripening grape berry cDNA library. The mRNA expression levels of the genes represented by these cDNAs were measured in flowers, week 2 post-flowering whole berries, week 5, week 8, week 10 (véraison, green berries), week 12 and week 13 berry skin. In addition, a comparison of RNA expression in pigmented and unpigmented berry skin at véraison (week 10) was undertaken. Image and statistical analysis revealed four sets of genes with distinctive and similar expression profiles over the course of berry development. The first set was composed of genes which had maximum RNA expression in flowers, followed by a steady decrease in expression. The most prominent group within this set were genes which have a role in photosynthesis. The second set of cDNAs was dominated by genes involved in flavonoid biosynthesis and had a peak of expression week 2 post-flowering. The data indicate co-ordinate regulation of flavonoid biosynthetic genes which code for the enzymes 4-coumarate-CoA ligase, chalcone synthase, chalcone isomerase, flavonone hydroxylase, anthocyanidin reductase and cytochrome b5. The third set of cDNAs exhibited maximum expression week 5 post-flowering, midway between flowering and véraison, a period of rapid berry growth. This set of cDNAs is dominated by genes which code for structural cell wall proteins. The fourth set of genes was dramatically up-regulated at véraison and remained up-regulated until 13 weeks post-flowering. This set of genes was composed of a diverse range of genes, a reflection of the complexity of ripening, most with no known function.