Titanium dioxide as a chemo-affinity solid phase in offline phosphopeptide chromatography prior to HPLC-MS/MS analysis

We have developed a new offline chromatographic approach for the selective enrichment of phosphorylated peptides that is directly compatible with subsequent analysis by online nano electrospray ionization tandem mass spectrometry. In this technique, a titanium dioxide (TiO2)-packed pipette tip is used as a phosphopeptide trap that acts as an offline first-dimension separation step in a two-dimensional chromatography system. This is followed by online nano reversed-phase high-performance liquid chromatography. Here, we present suitable methods for enrichment, optimized separately for each step: sample loading, washing and elution from the TiO2-filled tips. To increase the trapping selectivity of the TiO2 column, we used the sodium salt of 1-octanesulfonic acid combined with 2,5-dihydroxybenzoic acid as ion-pairing agents and displacers for acidic peptides. These agents also improve the binding of phosphorylated peptides and block the binding of non-phosphorylated ones. This enrichment procedure takes 30 min, followed by a 100-min HPLC program, including washing and an elution gradient.     

The hr1 and fusion peptide regions of the subgroup B avian sarcoma and leukosis virus envelope glycoprotein influence low pH-dependent membrane fusion

The avian sarcoma and leukosis virus (ASLV) envelope glycoprotein (Env) is activated to trigger fusion by a two-step mechanism involving receptor-priming and low pH fusion activation. In order to identify regions of ASLV Env that can regulate this process, a genetic selection method was used to identify subgroup B (ASLV-B) virus-infected cells resistant to low pH-triggered fusion when incubated with cells expressing the cognate TVB receptor. The subgroup B viral Env (envB) genes were then isolated from these cells and characterized by DNA sequencing. This led to identification of two frequent EnvB alterations which allowed TVB receptor-binding but altered the pH-threshold of membrane fusion activation: a 13 amino acid deletion in the host range 1 (hr1) region of the surface (SU) EnvB subunit, and the A32V amino acid change within the fusion peptide of the transmembrane (TM) EnvB subunit. These data indicate that these two regions of EnvB can influence the pH threshold of fusion activation.     

Identifying and prioritizing greater sage-grouse nesting and brood-rearing habitat for conservation in human-modified landscapes

Background

Balancing animal conservation and human use of the landscape is an ongoing scientific and practical challenge throughout the world. We investigated reproductive success in female greater sage-grouse (Centrocercus urophasianus) relative to seasonal patterns of resource selection, with the larger goal of developing a spatially-explicit framework for managing human activity and sage-grouse conservation at the landscape level.

Methodology/Principal Findings

We integrated field-observation, Global Positioning Systems telemetry, and statistical modeling to quantify the spatial pattern of occurrence and risk during nesting and brood-rearing. We linked occurrence and risk models to provide spatially-explicit indices of habitat-performance relationships. As part of the analysis, we offer novel biological information on resource selection during egg-laying, incubation, and night. The spatial pattern of occurrence during all reproductive phases was driven largely by selection or avoidance of terrain features and vegetation, with little variation explained by anthropogenic features. Specifically, sage-grouse consistently avoided rough terrain, selected for moderate shrub cover at the patch level (within 90 m²), and selected for mesic habitat in mid and late brood-rearing phases. In contrast, risk of nest and brood failure was structured by proximity to anthropogenic features including natural gas wells and human-created mesic areas, as well as vegetation features such as shrub cover.

Conclusions/Significance

Risk in this and perhaps other human-modified landscapes is a top-down (i.e., human-mediated) process that would most effectively be minimized by developing a better understanding of specific mechanisms (e.g., predator subsidization) driving observed patterns, and using habitat-performance indices such as those developed herein for spatially-explicit guidance of conservation intervention. Working under the hypothesis that industrial activity structures risk by enhancing predator abundance or effectiveness, we offer specific recommendations for maintaining high-performance habitat and reducing low-performance habitat, particularly relative to the nesting phase, by managing key high-risk anthropogenic features such as industrial infrastructure and water developments.     

Genome sequence of Thermotoga sp. strain RQ2, a hyperthermophilic bacterium isolated from a geothermally heated region of the seafloor near Ribeira Quente, the Azores

Thermotoga sp. strain RQ2 is probably a strain of Thermotoga maritima. Its complete genome sequence allows for an examination of the extent and consequences of gene flow within Thermotoga species and strains. Thermotoga sp. RQ2 differs from T. maritima in its genes involved in myo-inositol metabolism. Its genome also encodes an apparent fructose phosphotransferase system (PTS) sugar transporter. This operon is also found in Thermotoga naphthophila strain RKU-10 but no other Thermotogales. These are the first reported PTS transporters in the Thermotogales.     

Are deception-pollinated species more variable than those offering a reward?

In most pollination systems, animals transfer pollen among plants of a given species. Pollinator visitations do not come without cost, so plants usually offer a reward. However, the flowers of some plant species, mostly orchids, lack rewards and deceive animals into visiting their flowers. Deceptive species are thought to have high levels of variation in traits associated with advertisement and pollinator attraction, which have been attributed to genetic drift, or disruptive selection due to pollinator behavior. Rewarding species are assumed to have less variation due to stabilizing selection. We compared variability in floral morphology and fragrance composition between deceptive and rewarding species. Because both suites of traits are often linked with floral advertisement and pollinator attraction, we expected variation to be greater in species with deceptive pollination systems than in those offering rewards. We obtained floral morphology metrics for 20 deceptive species and 41 rewarding species native or naturalized in Puerto Rico, Venezuela, and Ecuador. Floral fragrances were sampled from eight deceptive species and four rewarding species. We found that the amplitude of variation in floral morphology and fragrance composition covaries significantly. Comparison of coefficients of variation for morphology indicated that, overall, deceptive species show significantly higher variation than rewarding species, and this pattern was also found among just orchids or just nonorchids. There were no statistical differences in morphological variation between orchids and nonorchids within a functional pollination group. Fragrance variation, measured by Jaccard distance, tended to be greater for deceptive species than for rewarding species. Although overlap in measures of variation occurs between the two groups, the data support the hypothesis that populations of deception-pollinated species are more variable than rewarding species in traits associated with pollinator attraction.     

Prevention of the normal expansion of maternal plasma volume: a model for chronic fetal hypoxaemia

The effects of inadequate expansion of maternal blood volume on uterine blood flow, fetal oxygen levels and vasoactive mediators during the third trimester were studied in 8 pregnant sheep. Results were compared to those obtained during 15 normal pregnancies. Prevention of the normal (20 ml/day) increase in maternal plasma volume was achieved by repeated haemorrhage and injections of furosemide. These treatments also reduced the rise in blood flow to the pregnant uterine horn that normally occurs during this period of gestation: at term flow was only 508 +/- 61 (SEM) compared to 838 +/- 83 ml/min in the control group (P greater than 0.01). This reduction in uterine blood flow caused a gradual fall in fetal PaO2, and rise in fetal levels of plasma renin activity, vasopressin, catecholamines and angiotensin II without change in pHa or base excess. Four to 5 days prior to delivery, the difference from control in PaO2 was -3.9 +/- 0.5 mmHg, plasma renin activity +2.9 +/- 1.7 ng/ml.h, vasopressin +4.2 +/- 1.1 pg/ml, catecholamines +957 +/- 145.3 pg/ml and angiotensin II +243 +/- 108.2 pg/ml. Furthermore, the fall in PaO2 and rise in vasoactive mediators that normally occur 3-5 days prior to the onset of labour was either absent (PaO2 and plasma renin activity) or blunted. Thus when expansion of blood volume during pregnancy is inadequate, blood flow to the uterus is adversely affected. This leads to various degrees of chronic fetal hypoxaemia and stimulation of vasoactive mediator systems. However, the normal stimulation of vasoactive mediator systems that occurs 3-5 days before delivery appears to be blunted. Experimental prevention of blood volume expansion during pregnancy produces an excellent model for the study of chronic mild fetal hypoxaemia.     

Affinity purification of the voltage-sensitive sodium channel from electroplax with resins selective for sialic acid

The voltage-sensitive sodium channel present in the eel (Electrophorus electricus) has an unusually high content of sialic acid, including alpha-(2----8)-linked polysialic acid, not found in other electroplax membrane glycopeptides. Lectins from Limax flavus (LFA) and wheat germ (WGA) proved the most effective of 11 lectin resins tried. The most selective resin was prepared from IgM antibodies against Neisseria meningitidis alpha-(2----8)-polysialic acid which were affinity purified and coupled to Sepharose 4B. The sodium channel was found to bind to WGA, LFA, and IgM resins and was readily eluted with the appropriate soluble carbohydrates. Experiments with LFA and IgM resins demonstrated binding and unbinding rates and displacement kinetics, which suggest highly specific binding at multiple sites on the sodium channel protein. In preparative-scale purification of protein previously fractionated by anion-exchange chromatography, without stabilizing TTX, high yields were reproducibly obtained. Further, when detergent extracts were prepared from electroplax membranes fractionated by low-speed sedimentation, a single step over the IgM resin provided a 70-fold purification, yielding specific activities of 3200 pmol of [3H]TTX-binding sites/mg of protein and a single polypeptide of approximately 285,000 Da on SDS-acrylamide gels. No small peptides were observed after this 5-h isolation. We further describe a cation-dependent stabilization with millimolar levels of monovalent and micromolar levels of divalent species.     

Effects of manganese and other divalent cations on calcium uptake and catecholamine secretion by primary cultures of bovine adrenal medulla cells

Primary cultures of bovine adrenal medullary chromaffin cells were used to examine the effect of replacing divalent cations in the extracellular media on secretion. When calcium was replaced by manganese, nicotine-stimulated secretion was delayed in onset for 3 to 5 minutes, but continued for approximately 60 minutes. In contrast, calcium-supported secretion began immediately on stimulation and plateaued by 10 minutes. ⁵⁴Mn²⁺ uptake occurred on stimulation but at a lower rate than ⁴⁵Ca²⁺ uptake. There was no delay of ⁵⁴Mn²⁺ uptake upon stimulation and ⁵⁴Mn²⁺ uptake was considerably prolonged compared to ⁴⁵Ca²⁺ uptake. Replacement of calcium with strontium gave results similar to those with calcium, and, in addition, strontium was able to bring about secretion by itself in a manner similar to barium. Inhibition experiments showed that the potency for inhibiting calcium uptake was Cd²⁺>Mn²⁺>Ca²⁺>Sr²⁺.     

Hyperglycemic Damage to Mitochondrial Membranes During Cerebral Ischemia: Amelioration by Platelet-Activating Factor Antagonist BN 50739

Abstract: The Pulsinelli-Brierley four-vessel occlusion model was used to study the consequences of hyperglycemic ischemia and reperfusion. Rats were subjected to either 30 min of normo- or hyperglycemic ischemia or 30 min of normo- or hyperglycemic ischemia followed by 60 min of reperfusion. In some animals, 2 mg/kg BN 50739, a platelet-activating factor receptor antagonist, was administered intraarterially either before or after the ischemic insult. The changes in mitochondrial membrane free fatty acid levels, phosphatidylcholine fatty acyl composition, and thiobarbituric acid-reactive material (TBAR) content plus the mitochondrial respiratory control ratio (RCR) were monitored. When the platelet-activating factor antagonist was present during normoglycemia, (a) the mitochondrial free fatty acid release both during and after ischemia was slowed, (b) reacylation of phosphatidylcholine following ischemia was promoted, and (c) TBAR accumulation during and following ischemia was decreased. The detrimental effects of hyperglycemia were muted when BN 50739 was present during ischemia. The RCR was preserved and phosphatidylcholine hydrolysis during ischemia was decreased. TBAR levels were consistently higher in hyperglycemic brain mitochondria both during and after ischemia. The RCR correlated directly with mitochondrial phosphatidylcholine polyunsaturated fatty acid content during ischemia and reperfusion. BN 50739 protection of mitochondrial membranes in brain may be influenced by tissue pH.