Rational and computational design of stabilized variants of cyanovirin-N that retain affinity and specificity for glycan ligands

Cyanovirin-N (CVN) is an 11 kDa pseudosymmetric cyanobacterial lectin that has been shown to inhibit infection by the human immunodeficiency virus by binding to high-mannose oligosaccharides on the surface of the viral envelope glycoprotein gp120. In this work, we describe rationally designed CVN variants that stabilize the protein fold while maintaining high affinity and selectivity for their glycan targets. Poisson-Boltzmann calculations and protein repacking algorithms were used to select stabilizing mutations in the protein core. By substituting the buried polar side chains of Ser11, Ser20, and Thr61 with aliphatic groups, we stabilized CVN by nearly 12 °C against thermal denaturation, and by 1 M GuaHCl against chemical denaturation, relative to a previously characterized stabilized mutant. Glycan microarray binding experiments confirmed that the specificity profile of carbohydrate binding is unperturbed by the mutations and is identical for all variants. In particular, the variants selectively bound glycans containing the Manα(1→2)Man linkage, which is the known minimal binding unit of CVN. We also report the slow denaturation kinetics of CVN and show that they can complicate thermodynamic analysis; in particular, the unfolding of CVN cannot be described as a fixed two-state transition. Accurate thermodynamic parameters are needed to describe the complicated free energy landscape of CVN, and we provide updated values for CVN unfolding.     

Gastrointestinal peptides and the adaptation to extrauterine nutrition

The adaptation to extrauterine nutrition involves complex physiological changes at birth which may be regulated by genetic endowment; enteral nutrients, secretions, and bacteria; and endogenous hormones and exogenous hormones in breast milk. The hypothesis is explored that enteral feeding after birth may trigger key adaptations in the gut and in metabolism partly through the mediation of gastrointestinal hormone secretion. Gut peptides are found in the early human fetal gut and by the second trimester some are found in high concentrations in the fetal circulation and amniotic fluid. Major plasma hormonal surges occur during the neonatal period in term and preterm infants: notably in enteroglucagon, gastrin, motilin, neurotensin, gastrointestinal peptide, and pancreatic polypeptide. These events do not occur in neonates deprived of enteral feeding. A progressive development of dynamic gut hormonal responses to feeding is observed. The pattern of gut endocrine changes after birth is influenced by the type and route of feeding. Potential pathophysiological effects of depriving high risk neonates of enteral feeding are considered. It is speculated that infants committed to prolonged total parenteral nutrition from birth may benefit from the biological effects of intraluminal nutrients used in subnutritional quantities.     

Effect of vascular cannulation on serum concentrations of LH, FSH, prolactin and cortisol in prepubertal gilts

Two experiments were conducted to determine whether cannulation of the jugular vein in gilts alters serum concentrations of LH, FSH, prolactin (PRL) or cortisol (C). In Experiment 1, 12 crossbred prepubertal gilts weighing 95 +/- 1.3 kg were immobilized by snaring, and tygon tubing was threaded into the anterior vena cava through a 12-gauge needle inserted into the jugular vein. Five hours later, blood samples were drawn at 20-min intervals for 4 h (Day 0). Samples were also drawn at 20-min intervals for 4-h periods 24 h (Day 1) and 48 h (Day 2) after cannulation. Serum concentrations of LH were similar (P=0.26) among Day 0 (0.40 ng/ml), Day 1 (0.39 ng/ml) and Day 2 (0.34 ng/ml). Serum PRL was similar (P=0.07) among Day 0 (4.10 ng/ml), Day 1 (3.87 ng/ml) and Day 2 (3.43 ng/ml). Serum concentrations of C were greater (P < 0.001) on Day 0 (8.32 ng/ml) than Day 1 (4.48 ng/ml) or Day 2 (3.54 ng/ml). In Experiment 2, cannulas were placed in 29 prepubertal gilts. Two days after initial cannulation, six blood samples were drawn at 20-min intervals. Gilts were then immobilized by snaring, and a second cannulae was inserted into the contralateral vein. Five blood samples were taken at 2-min intervals during the second cannulation and then six samples were drawn at 20-min intervals. Serum LH and FSH were not altered by cannulation or elevated during the subsequent 2-h sampling period (P>0.05). In contrast, serum concentrations of PRL rose slowly (P<0.05) during cannulation and remained elevated for 60 min before returning to baseline. Serum concentrations of C rose within 6 min of cannulation, remained elevated for 30 min, and then declined over the next 90 min. From these two experiments, it appears that secretory patterns of LH and FSH can be accurately assessed immediately after cannulation in prepubertal gilts. Measurements of serum PRL and C that reflect nonstressed conditions, however, cannot be obtained until at least 2 h or 1 d after cannulation, respectively.     

Comparison of resistance and conduit vessel nitric oxide-mediated vascular function in vivo: effects of exercise training.

Exercise training improves vascular function in subjects with cardiovascular disease and risk factors, but there is mounting evidence these vascular adaptations may be vessel bed specific. We have therefore examined the hypothesis that exercise-induced improvements in conduit vessel function are related to changes in resistance vessel function. Endothelium-dependent and -independent conduit vessel function were assessed by using wall-tracking of high-resolution brachial artery ultrasound images of the response to flow-mediated dilation (FMD) and nitroglycerine [glyceryl trinitrate (GTN)] administration. Resistance vessel endothelium-dependent and -independent function were assessed using intrabrachial administration of acetylcholine (ACh) and nitroprusside (SNP). Randomized crossover studies of 8-wk exercise training were undertaken in untreated hypercholesterolemic (n = 10), treated hypercholesterolemic (n = 10), coronary artery disease (n = 8), and Type 2 diabetic subjects (n = 15). Exercise training significantly enhanced responses to ACh (P < 0.05) and FMD (P < 0.0001). There were no significant changes in either SNP or GTN responses. The correlation between ACh and FMD responses at entry was not significant (r = 0.186; P = 0.231), and training-induced changes in the ACh did not correlate with those in FMD (r = -0.022; P = 0.890). Similarly, no correlation was evident between the SNP and GTN responses at entry (r = -0.010; P = 0.951) or between changes in these variables with training (r = -0.211; P = 0.191). We conclude that, although short-term exercise training improves endothelium-dependent nitric oxide-mediated vascular function in both conduit and resistance vessels, the magnitude of these improvements are unrelated.     

Demographic mechanism of a historical bird population collapse reconstructed using museum specimens

Long-term studies of demographic rates provide clues about the external causes of animal population declines, but systematic monitoring is rarely in place until after the decline has occurred. This study evaluates alternative hypotheses about the demographic mechanisms underlying the historical collapse of corncrake (Crexcrex) populations in Britain and Ireland in the late nineteenth and early twentieth centuries using characteristics of museum specimens. The proportion of adult corncrakes that are 1-year old was estimated from feather characteristics of birds collected before, during and after the population decline and showed a marked transitory reduction during the decline. This pattern would be expected if the decline was caused by a large reduction in the recruitment of young birds to the breeding population and is the opposite of what would be expected if a change in adult survival had caused the decline. These results are consistent with previous suggestions that the corncrake population decline was caused by adverse effects on breeding productivity caused by the mechanization of the harvesting of hay crops.     

Effects of seed ingestion and herbivory by waterfowl on seedling establishment: a field experiment with wigeongrass Ruppia maritima in Doñana,south-west Spain

The ingestion of seeds by vertebrates usually affects the viability and/or germination rate of seeds. Increases in germination rate following passage through the vertebrate gut have often been assumed to be favourable for seedling survival and plant fitness, but this assumption has never been tested experimentally. Given that numbers of herbivorous waterfowl are higher in winter in Mediterranean wetlands, herbivory pressure there will be higher for early growing plants. In a factorial experiment we investigated the effects of seed ingestion by ducks (shoveler, Anas clypeata) on the survival of wigeongrass Ruppia maritima seedlings in the field in Doñana (south-west Spain), under differing exposures to herbivory by waterfowl and fish. We planted ingested and non-ingested seeds in December, using exclosures to protect half of them from herbivores. When they were protected inside exclosures, there was no difference between ingested and non-ingested seeds in the number of plants that survived until June-July. However, fewer plants survived from ingested seeds when exposed to natural levels of herbivory because they were exposed for longer than plants germinating from non-ingested seeds. In conclusion, increases in germination rate after ingestion are not necessarily beneficial for the plant, and the final outcome depends on complex interactions with other factors such as herbivore abundance.