Tetradecyltrimethylammonium Inhibits <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> Hemolytic Phospholipase C Induced by Choline

Pseudomonas aeruginosa expresses hemolytic phospholipase C (PlcH) with choline or under phosphate-limiting conditions. PlcH from these conditions were differently eluted from the Celite-545 column after application of an ammonium sulfate linear reverse gradient. The PlcH from supernatants of bacteria grown in the presence of choline was eluted with 30% ammonium sulfate and was more than 85% inhibited by tetradecyltrimethylammonium. PlcH from supernatants of bacteria grown with succinate and ammonium ions in a low-phosphate medium was eluted as a peak with 10% of salt and was less than 10% inhibited by tetradecyltrimethylammonium. PlcH from low phosphate was purified associated with a protein of 17 kDa. This complex was dissociated and separated on a Sephacryl S-200 column with 1% (w/v) sodium dodecyl sulfate. After this dissociation, the resulting protein of 70 kDa, corresponding to PlcH, was inhibited by tetradecyltrimethylammonium, showing a protection effect of the accompanying protein. RT-PCR analyses showed that in choline media, the plcH gene was expressed independently of plcR. In low-phosphate medium, the plcH gene was expressed as a plcHR operon. Because plcR encodes for chaperone proteins, this result correlates with the observation that PlcH from supernatants of bacteria grown in the presence of choline was purified without an accompanying protein. The consequence of the absence of this chaperone was that tetradecyltrimethylammonium inhibited the PlcH activity.     

Intestinotrophic effects of exogenous IGF-I are not diminished in IGF binding protein-5 knockout mice

IGF binding protein-5 (IGFBP-5) modulates the availability of IGF-I to its receptor and potentiates the intestinotrophic action of IGF-I. Our aim was to test the hypothesis that stimulation of intestinal growth due to coinfusion of IGF-I with total parenteral nutrition (TPN) solution is dependent on increased expression of IGFBP-5 through conducting our studies in IGFBP-5 knockout (KO) mice. IGFBP-5 KO, heterozygote (HT) and wild type (WT) male and female mice were maintained with TPN or TPN plus coinfusion of IGF-I [recombinant human (rh)IGF-I; 2.5 mg x kg(-1) x day(-1)] for 5 days. The concentration of IGF-I in serum was 73% greater (P < 0.0001) in mice given TPN + IGF-I infusion compared with TPN alone. IGF-I attenuated the 2-3 g loss of body weight associated with TPN in WT mice, whereas KO and HT mice did not show improvement in body weight with IGF-I treatment. KO and HT mice had significantly greater levels of circulating IGF-I binding proteins (IGFBPs) compared with WT mice. Intestinal growth due to IGF-I was observed in all groups treated with IGF-I based on greater concentrations of protein and DNA in small intestine and colon and significantly greater crypt depth and muscularis thickness in jejunum. Jejunal expression of IGFBP-5 mRNA was greater in WT mice, whereas IGFBP-3 mRNA was greater in KO mice treated with IGF-I. In summary, the absence of the IGFBP-5 gene did not block the ability of IGF-I to stimulate intestinal growth, possibly because greater jejunal expression of IGFBP-3 compensates for the absence of IGFBP-5.     

Thin-fiber mechanoreceptors reflexly increase renal sympathetic nerve activity during static contraction

The renal vasoconstriction induced by the sympathetic outflow during exercise serves to direct blood flow from the kidney toward the exercising muscles. The renal circulation seems to be particularly important in this regard, because it receives a substantial part of the cardiac output, which in resting humans has been estimated to be 20%. The role of group III mechanoreceptors in causing the reflex renal sympathetic response to static contraction remains an open question. To shed some light on this question, we recorded the renal sympathetic nerve responses to static contraction before and after injection of gadolinium into the arterial supply of the statically contracting triceps surae muscles of decerebrate unanesthetized and chloralose-anesthetized cats. Gadolinium has been shown to be a selective blocker of mechanogated channels in thin-fiber muscle afferents, which comprise the afferent arm of the exercise pressor reflex arc. In decerebrate (n = 15) and chloralose-anesthetized (n = 12) cats, we found that gadolinium (10 mM; 1 ml) significantly attenuated the renal sympathetic nerve and pressor responses to static contraction (60 s) after a latent period of 60 min; both responses recovered after a latent period of 120 min. We conclude that thin-fiber mechanoreceptors supplying contracting muscle are involved in some of the renal vasoconstriction evoked by the exercise pressor reflex.     

Exercise training normalizes beta-adrenoceptor expression in dogs susceptible to ventricular fibrillation

Previous studies demonstrated an enhanced beta(2)-adrenoceptor (AR) responsiveness in animals susceptible to ventricular fibrillation (VF) that was eliminated by exercise training. The present study investigated the effects of endurance exercise training on beta(1)-AR and beta(2)-AR expression in dogs susceptible to VF. Myocardial ischemia was induced by a 2-min occlusion of the left circumflex artery during the last minute of exercise in dogs with healed infarctions: 20 had VF [susceptible (S)] and 13 did not [resistant (R)]. These dogs were randomly assigned to either 10-wk exercise training [treadmill running; n = 9 (S) or 8 (R)] or an equivalent sedentary period [n = 11 (S) or 5 (R)]. Left ventricular tissue beta-AR protein and mRNA were quantified by Western blot analysis and RT-PCR, respectively. Because beta(2)-ARs are located in caveolae, caveolin-3 was also quantified. beta(1)-AR gene expression decreased ( approximately 5-fold), beta(2)-AR gene expression was not changed, and the ratio of beta(2)-AR to beta(1)-AR gene expression was significantly increased in susceptible compared with resistant dogs. beta(1)-AR protein decreased ( approximately 50%) and beta(2)-AR protein increased (400%) in noncaveolar fractions of the cell membrane in susceptible dogs. Exercise training returned beta(1)-AR gene expression to levels seen in resistant animals but did not alter beta(2)-AR protein levels in susceptible dogs. These data suggest that beta(1)-AR gene expression was decreased in susceptible dogs compared with resistant dogs and, further, that exercise training improves beta(1)-AR gene expression, thereby restoring a more normal beta-AR balance.     

Aminopyrrolidineamide inhibitors of site-1 protease

The discovery and efficacy of a series of potent aminopyrrolidineamide-based inhibitors of sterol regulatory element binding protein site-1 protease is described.     

Brain responses in 4-month-old infants are already language specific

Language is the most important faculty that distinguishes humans from other animals. Infants learn their native language fast and effortlessly during the first years of life, as a function of the linguistic input in their environment. Behavioral studies reported the discrimination of melodic contours [1] and stress patterns [2, 3] in 1-4-month-olds. Behavioral [4, 5] and brain measures [6-8] have shown language-independent discrimination of phonetic contrasts at that age. Language-specific discrimination, however, has been reported for phonetic contrasts only for 6-12-month-olds [9-12]. Here we demonstrate language-specific discrimination of stress patterns in 4-month-old German and French infants by using electrophysiological brain measures. We compare the processing of disyllabic words differing in their rhythmic structure, mimicking German words being stressed on the first syllable, e.g., pápa/daddy[13], and French ones being stressed on the second syllable, e.g., papá/daddy. Event-related brain potentials reveal that experience with German and French differentially affects the brain responses of 4-month-old infants, with each language group displaying a processing advantage for the rhythmic structure typical in its native language. These data indicate language-specific neural representations of word forms in the infant brain as early as 4 months of age.     

Efficient optimization of crystallization conditions by manipulation of drop volume ratio and temperature

An efficient optimization method for the crystallization of biological macromolecules has been developed and tested. This builds on a successful high-throughput technique for the determination of initial crystallization conditions. The optimization method takes an initial condition identified through screening and then varies the concentration of the macromolecule, precipitant, and the growth temperature in a systematic manner. The amount of sample and number of steps is minimized and no biochemical reformulation is required. In the current application a robotic liquid handling system enables high-throughput use, but the technique can easily be adapted in a nonautomated setting. This method has been applied successfully for the rapid optimization of crystallization conditions in nine representative cases.     

Quantifying the risk from ovine BSE and the impact of control strategies

Although no naturally infected sheep with bovine spongiform encephalopathy (BSE) has ever been discovered, it remains possible that BSE once infected the UK sheep population, has been transmitted between sheep, and is still present today. We constructed a mathematical model to assess the current maximum theoretical exposure to consumers from BSE-infected ovine material and to estimate the risk reduction that could be achieved by abattoir-based control options if BSE-infected sheep were ever found in the national flock. We predict that, if present, the exposure to consumers from a single BSE-infected sheep would be high: one sheep, close to the end of its incubation period, is likely to contribute 10-1000 times more infectious material than a fully infectious cow. Furthermore, 30% of this exposure comes from infectivity residing in lymphatic and peripheral tissue that cannot be completely removed from a carcass.We are 95% confident that throughout Great Britain, no more than four sheep flocks currently harbour an ongoing BSE epidemic. However, since the exposure from a single infected sheep is high, the annual human exposure from four 'typical' BSE-infected flocks could be considerable. Small reductions in exposure could be achieved by strategies based on tissue testing, a 12-month age restriction or expanded definitions of high-risk tissues. A six-month age restriction is likely to be more effective and genotype-based strategies the most effective.     

Survival against the odds: ontogenetic changes in selective pressure mediate growth-mortality trade-offs in a marine fish

For organisms with complex life cycles, variation among individuals in traits associated with survival in one life-history stage can strongly affect the performance in subsequent stages with important repercussions on population dynamics. To identify which individual attributes are the most influential in determining patterns of survival in a cohort of reef fish, we compared the characteristics of Pomacentrus amboinensis surviving early juvenile stages on the reef with those of the cohort from which they originated. Individuals were collected at hatching, the end of the planktonic phase, and two, three, four, six and eight weeks post-settlement. Information stored in the otoliths of individual fish revealed strong carry-over effects of larval condition at hatching on juvenile survival, weeks after settlement (i.e. smaller-is-better). Among the traits examined, planktonic growth history was, by far, the most influential and long-lasting trait associated with juvenile persistence in reef habitats. However, otolith increments suggested that larval growth rate may not be maintained during early juvenile life, when selective mortality swiftly reverses its direction. These changes in selective pressure may mediate growth-mortality trade-offs between predation and starvation risks during early juvenile life. Ontogenetic changes in the shape of selectivity may be a mechanism maintaining phenotypic variation in growth rate and size within a population.