Mutually exclusive recombination of wild-type and mutant loxP sites in vivo facilitates transposon-mediated deletions from both ends of genomic DNA in PACs

Recombination of wild-type and mutant loxP sites mediated by wild-type Cre protein was analyzed in vivo using a sensitive phage P1 transduction assay. Contrary to some earlier reports, recombination between loxP sites was found to be highly specific: a loxP site recombined in vivo only with another of identical sequence, with no crossover recombination either between a wild-type and mutant site; or between two different mutant sites tested. Mutant loxP sites of identical sequence recombined as efficiently as wild-type. The highly specific and efficient recombination of mutant loxP sites in vivo helped in developing a procedure to progressively truncate DNA from either end of large genomic inserts in P1-derived artificial chromosomes (PACs) using transposons that carry either a wild-type or mutant loxP sequence. PAC libraries of human DNA were constructed with inserts flanked by a wild-type and one of the two mutant loxP sites, and deletions from both ends generated in clones using newly constructed wild-type and mutant loxP transposons. Analysis of the results provides new insight into the very large co-integrates formed during P1 transduction of plasmids with loxP sites: a model with tri- and possibly multimeric co-integrates comprising the PAC plasmid, phage DNA, and transposon plasmid(s) as intermediates in the cell appears best to fit the data. The ability to truncate a large piece of DNA from both ends is likely to facilitate functionally mapping gene boundaries more efficiently, and make available precisely trimmed genes in their chromosomal contexts for therapeutic applications.     

Essential fatty acids and their role in the treatment of impulsivity disorders

Essential fatty acids (EFAs) have been shown to benefit patients with depression, schizophrenia and dementia. More recently, their role in disorders characterised by impulsivity has attracted some attention. The psychiatric conditions of attention-deficit hyperactivity disorder and borderline personality disorder as well as the phenomena of deliberate self-harm and violence have been ameliorated by the supplementation of EFAs in a number of recent clinical trials. This paper summarises the burgeoning clinical and basic research indicating the existence of significant deficits of EFAs in impulsivity disorders and the supplementation studies of EFAs in these diverse conditions, all of which remain a major therapeutic challenge.     

Artificial selection for high activity favors mighty mini-muscles in house mice

After 14 generations of selection for voluntary wheel running, mice from the four replicate selected lines ran, on average, twice as many revolutions per day as those from the four unselected control lines. To examine whether the selected lines followed distinct strategies in the correlated responses of the size and metabolic capacities of the hindlimb muscles, we examined mice from selected lines, housed for 8 wk in cages with access to running wheels that were either free to rotate ("wheel access" group) or locked ("sedentary"). Thirteen of twenty individuals in one selected line (line 6) and two of twenty in another (line 3) showed a marked reduction ( approximately 50%) in total hindlimb muscle mass, consistent with the previously described expression of a small-muscle phenotype. Individuals with these "mini-muscles" were not significantly smaller in total body mass compared with line-mates with normal-sized muscles. Access to free wheels did not affect the relative mass of the mini-muscles, but did result in typical mammalian training effects for mitochondrial enzyme activities. Individuals with mini-muscles showed a higher mass-specific muscle aerobic capacity as revealed by the maximal in vitro rates of citrate synthase and cytochrome c oxidase. Moreover, these mice showed the highest activities of hexokinase and carnitine palmitoyl transferase. Females with mini-muscles showed the highest levels of phosphofructokinase, and males with mini-muscles the highest levels of pyruvate dehydrogenase. As shown by total muscle enzyme contents, the increase in mass-specific aerobic capacity almost completely compensated for the reduction caused by the "loss" of muscle mass. Moreover, the mini-muscle mice exhibited the lowest contents of lactate dehydrogenase and glycogen phosphorylase. Interestingly, metabolic capacities of mini-muscled mice resemble those of muscles after endurance training. Overall, our results demonstrate that during selection for voluntary wheel running, distinct adaptive paths that differentially exploit the genetic variation in morphological and physiological traits have been followed.     

A mechanosensitive cation channel in endothelial cells and its role in vasoregulation

Ca2+ is an important intracellular second messenger in signal transduction of endothelial cells. It has long been recognized that a mechanosensitive Ca2+-permeable channel is present in vascular endothelial cells. The activity of this channel may increase intracellular Ca2+ level in endothelial cells. A recent finding is that the activity of this channel may be regulated by cGMP through a protein kinase G-dependent pathway. Inhibition of the channel by cGMP abolishes the Ca2+ influx elicited by flow. Several inhibitors of the cation channel including Gd3+, Ni2+, and SK&F-96365 also inhibit the Ca2+ influx due to flow stimulation. These data suggest that a mechanosensitive cation channel is the primary pathway mediating the flow-induced Ca2+ entry in vascular endothelial cells. Another important finding is that the opening of this mechanosensitive channel by KT5823 leads to endothelium-dependent vascular dilation. Therefore, it appears that this channel may play a crucial role in the regulation of vascular tone.     

Community-level physiological profiling performed with an oxygen-sensitive fluorophore in a microtiter plate

Community-level physiological profiling based upon fluorometric detection of oxygen consumption was performed on hydroponic rhizosphere and salt marsh litter samples by using substrate levels as low as 50 ppm with incubation times between 5 and 24 h. The rate and extent of response were increased in samples acclimated to specific substrates and were reduced by limiting nitrogen availability in the wells.     

Chemisorptions of bacterial receptors for hydrophobic amino acids and sugars on gold for biosensor applications: a surface plasmon resonance study of genetically engineered proteins

This paper demonstrates potential applications of two periplasmic receptor proteins from E. coli as sensing elements for biosensors using the surface plasmon resonance (SPR) technique. These molecules, namely the aspartate to cysteine mutant of the leucine-specific receptor (LS-D1C) and the glutamine to cysteine mutant of the D-glucose/D-galactose receptor (GGR-Q26C) proteins, are chemisorbed on a thin (approximately 40 nm) Au film in neutral K2HPO4 buffers. Using angle and time resolved SPR measurements; we show that adsorption behaviors of both proteins are dominated by diffusion-free second order Langmuir kinetics. We also show that the protein-modified Au films exhibit measurable SPR shifts upon binding to their respective target ligands. According to these SPR data, the kinetics of ligand binding for both LS-D1C and GGR-Q26C are governed by irreversible first order diffusion limited Langmuir model. The utility of the SPR technique for studying reactions of biological molecules is further illustrated in this work.     

Properties of smooth muscle hyperpolarization and relaxation to K+ in the rat isolated mesenteric artery

Smooth muscle membrane potential and tension in rat isolated small mesenteric arteries (inner diameter 100-200 microm) were measured simultaneously to investigate whether the intensity of smooth muscle stimulation and the endothelium influence responses to exogenous K+. Variable smooth muscle depolarization and contraction were stimulated by titration with 0.1-10 microM phenylephrine. Raising external K+ to 10.8 mM evoked correlated, sustained hyperpolarization and relaxation, both of which were inhibited as the smooth muscle depolarized and contracted to around -38 mV and 10 mN, respectively. At these higher levels of stimulation, raising the K+ concentration to 13.8 mM still hyperpolarized and relaxed the smooth muscle. Relaxation to endothelium-derived hyperpolarizing factor, released by ACh, was not altered by the level of stimulation. In endothelium-denuded arteries, the concentration-relaxation curve to K+ was shifted to the right but was not depressed. In denuded arteries, relaxation to K+ was unaffected by the extent of prior stimulation and was blocked with 0.1 mM ouabain but not with 30 microM Ba2+. The ability of K+ to stimulate simultaneous hyperpolarization and relaxation in the mesenteric artery is consistent with a role as an endothelium-derived hyperpolarizing factor activating inwardly rectifying K+ channels on the endothelium and Na+-K+-ATPase on the smooth muscle cells.     

3D Modeling for TM Receptors: Algorithms and Validations

This paper outlines the basic strategy to build 3D models of transmembrane G-protein coupled receptors (GPCRs) starting from their amino acid sequences in a block-by-block manner: (i) locate possible TM helical fragments in the GPCR sequence; (ii) build 3D structures for these helices; (iii) arrange isolated helices across the membrane; (iv) calculate all pairwise helix-helix interactions; (v) assemble helical bundle(s); (vi) restore interhelical loops and N- and C-termini; and (vii) refine the entire 3D structure(s). Computer algorithms and preliminary results for most of the steps are discussed.     

Effects of voluntary activity and genetic selection on aerobic capacity in house mice (Mus domesticus)

Swallow, John G., Theodore Garland, Jr., Patrick A. Carter,Wen-Zhi Zhan, and Gary C. Sieck. Effects of voluntary activity andgenetic selection on aerobic capacity in house mice(Mus domesticus). J. Appl. Physiol. 84(1): 69-76, 1998.An animal model was developed to study effects on components ofexercise physiology of both "nature" (10 generations of geneticselection for high voluntary activity on running wheels) and"nurture" (7-8 wk of access or no access to running wheels,beginning at weaning). At the end of the experiment, mice from bothwheel-access groups were significantly lighter in body mass than micefrom sedentary groups. Within the wheel-access group, a statisticallysignificant, negative relationship existed between activity and finalbody mass. In measurements of maximum oxygen consumption during forcedtreadmill exercise (O_{2 max}), mice withwheel access were significantly more cooperative than sedentary mice;however, trial quality was not a significant predictor of individualvariation in O_{2 max}.Nested two-way analysis of covariance demonstrated that both geneticselection history and access to wheels had significant positive effects on O_{2 max}.A 12% difference inO_{2 max} existedbetween wheel-access selected mice, which had the highestmass-correctedO_{2 max}, andsedentary control mice, which had the lowest. The respiratory exchangeratio at O_{2 max} wasalso significantly lower in the wheel-access group. Our results suggestthe existence of a possible genetic correlation between voluntaryactivity levels (behavior) and aerobic capacity (physiology).