Two-dimensional and epitaxial crystallization of a mutant form of yeast RNA polymerase II

A mutant form of yeast RNA polymerase II that lacks the fourth and seventh largest subunits, referred to as pol II delta 4/7, crystallized on positively charged lipid layers. Both single-layered (two-dimensional) crystals and several multi-layered crystal forms were obtained. The two-dimensional crystals, preserved in negative stain, diffracted strongly to about 1/20 A-1 and more weakly to 1/13 A-1 resolution. A projection map computed from averaged Fourier transforms revealed four pol II delta 4/7 complexes per unit cell and further revealed a cleft on the surface of the complex similar to that previously observed in the structure of Escherichia coli RNA polymerase. One of the multi-layered crystal forms, preserved in negative stain, diffracted strongly beyond 1/15 A-1 resolution. Coherent diffraction from the multi-layered crystal is indicative of protein-protein interactions between layers and ordering in the third dimension.     

Development of simple sequence repeat (SSR) markers for the assessment of gene flow and genetic diversity in pigeonpea (Cajanus cajan)

Pigeonpea (Cajanus cajan) is an important subsistence crop in India where traditional landraces and improved hybrids are grown alongside each other. Gene flow may result in genetic erosion of these landraces and their wild relatives, whilst transgene escape from future genetically engineered varieties is another potential hazard. To assess the impact of these factors gene flow needs to be measured. A set of 10 simple sequence repeat markers have been developed, which exhibit polymorphism across a range of pigeonpea varieties. Use of these markers also offers an efficient system for the assessment of genetic diversity within populations of pigeonpea.     

The effects of cultural conditions on growth and secondary metabolism in Streptomyces thermoviolaceus

Abstract Granaticin, an isochromate quinone antibiotic is synthesized as a secondary metabolite by Streptomyces thermoviolaceus . Antibiotic productivity was investigated under a variety of cultural conditions, including complex and defined media, mesophilic and thermophilic temperatures and a variety of sole carbon sources. In a defined medium growth was supported, to varying extents, by different carbon sources and in most cases granaticin production was observed. Highest biomass and granaticin yields were obtained when cultures were grown in the presence of xylan, fructose, glutamate or proline as carbon source. Changes in pH during growth affected both the timing and extent of granaticin production.     

Incidence and Predictors of End Stage Renal Disease among Low-Income Blacks and Whites

We evaluated whether black race is associated with higher incidence of End Stage Renal Disease (ESRD) among a cohort of blacks and whites of similar, generally low socioeconomic status, and whether risk factor patterns differ among blacks and whites and explain the poorly understood racial disparity in ESRD. Incident diagnoses of ESRD among 79,943 black and white participants in the Southern Community Cohort Study (SCCS) were ascertained by linkage with the United States Renal Data System (USRDS) from 2002 through 2009. Person-years of follow up were calculated from date of entry into the SCCS until date of ESRD diagnosis, date of death, or September 1, 2009, whichever occurred first. Cox proportional hazards models were used to estimate hazard ratios (HRs) and 95% confidence intervals (CI) for incident ESRD among black and white participants in relation to baseline characteristics. After 329,003 person-years of follow-up, 687 incident cases of ESRD were identified in the cohort. The age-adjusted ESRD incidence rate was 273 (per 100,000) among blacks, 3.5-fold higher than the rate of 78 among whites. Risk factors for ESRD included male sex (HR = 1.6; 95% CI 1.4–1.9), low income (HR = 1.5; 95% CI 1.2–1.8 for income below vs. above $15,000), smoking (HR = 1.2; 95% CI 1.02–1.4) and histories of diabetes (HRs increasing to 9.4 (95% CI 7.4–11.9) among those with ≥20 years diabetes duration) and hypertension (HR = 2.9; 95% CI 2.3–3.7). Patterns and magnitudes of association were virtually identical among blacks and whites. After adjustment for these risk factors, blacks continued to have a higher risk for ESRD (HR = 2.4; 95% CI = 1.9–3.0) relative to whites. The black-white disparity in risk of ESRD was attenuated but not eliminated after control for known risk factors in a closely socioeconomically matched cohort. Further research characterizing biomedical factors, including CKD progression, in ESRD occurrence in these two racial groups is needed.     

Preemption of space can lead to intransitive coexistence of competitors

Intransitive competition has the potential to be a powerful contributor to species coexistence, but there are few proposed biological mechanisms that could create intransitivities in natural communities. Using a three‐species model of competition for space, we demonstrate a mechanism for coexistence that combines a colonization–competition tradeoff between two species with the ability of a third species to preempt space from the other competitors. The combination of differential abilities to colonize, preempt, and overtake space creates a community where no single species can exclude both of its competitors. The dynamics of this kind of community are analogous to rock‐paper‐scissors competition, and the three‐species community can persist even though not all pairs of species can coexist in isolation. In distinction to prior results, this is a mechanism of intransitivity that does not require nonhierarchical local interference competition. We present parameter estimates from a subtidal marine community illustrating how documented competitive traits can lead to preemption‐based intransitivities in natural communities, and we describe methods for an empirical test of the occurrence of this mechanism.     

Sputum neutrophils as a biomarker in COPD: findings from the ECLIPSE study

Introduction

The percentage of neutrophils in sputum are increased in COPD patients, and may therefore be a biomarker of airway inflammation. We studied the relationships between sputum neutrophils and FEV₁, health status, exacerbation rates, systemic inflammation and emphysema, and long term variability at 1 year.

Methods

Sputum samples were obtained from 488 COPD patients within the ECLIPSE cohort. 359 samples were obtained at baseline, and 297 after 1 year. 168 subjects provided samples at both visits. Serum interleukin-6 (IL-6), IL-8, surfactant protein D and C-reactive protein levels were measured by immunoassays. Low-dose CT scans evaluated emphysema.

Results

Sputum neutrophil % increased with GOLD stage. There was a weak association between % sputum neutrophils and FEV₁ % predicted (univariate r² = 0.025 and 0.094 at baseline and year 1 respectively, p < 0.05 after multivariate regression). Similar weak but significant associations were observed between neutrophil % and health status measured using the St Georges Respiratory Questionairre. There were no associations between neutrophils and exacerbation rates or emphysema. Associations between sputum neutrophils and systemic biomarkers were non-significant or similarly weak. The mean change over 1 year in neutrophil % was an increase of 3.5%.

Conclusions

Sputum neutrophil measurements in COPD are associated weakly with FEV₁ % predicted and health status. Sputum neutrophil measurements were dissociated from exacerbation rates, emphysema and systemic inflammation.     

Interactions of piperidine derivatives with the nicotinic cholinergic receptor complex from Torpedo electric organ

The interactions of eight piperidine derivatives with nicotinic receptor complexes fromTorpedo californica electric organ were studied using [¹²⁵I]alpha-bungarotoxin ([¹²⁵I]BGT) as a probe for the acetylcholine binding site and [³H]perhydrohistrionicotoxin ([³H]H₁₂-HTX) as a probe for a site associated with the receptor-gated ion channel.Cis- andtrans-2-methyl-6-n-undecanyl piperidines (MUP), major constituents of fire ant venom, had a high-affinity for [³H]H₁₂-HTX binding sites (K_i=0.08–0.24 M), but had no affect on receptor binding. MUP affinity for [³H]H₁₂-HTX binding sites was approximately doubled in the presence of 1 M carbamylcholine. Introduction of a 2-hydroxyl group to the undecanyl side channel had little effect on activity of the alkaloid. The analog 2,6- (but not 3,5-) dimethylpiperidine was a moderately active inhibitor of [³H]H₁₂-HTX binding (K _i-8.8 M). 2-Methylpiperidine was considerably less active (K _i=600 M), although it was more potent than either 3- or 4-methylpiperidine. The affinities of 2,6-dimethylpiperidine and 2-methylpiperidine for [³H]H₁₂-HTX binding sites were decreased in the presence of 1 M carbamylcholine. Carbamylcholine affinity for the receptor was increased by up to 7 fold in the presence of 10 and 32 M MUP, but was decreased in the presence of 2,6-dimethylpiperidine and 2-methylpiperidine. Thecis- andtrans-isomers of MUP were equipotent in producing each of its effects. In these actions, MUP resembles a variety of other compounds derived from 2,6-disubstituted piperidines, including histrionicotoxins, gephyrotoxins and pumiliotoxins. These studies establish the importance of alkyl substitutions in theortho position of the piperidine ring in conferring ion channel specificity, and the importance of substantial alkyl side chains in conferring the ability of channel blockers to stabilize the nicotinic receptor complex in high affinity, desensitized conformations.     

The glutamine commute: take the N line and transfer to the A

The transfer of glutamine between cells contributes to signaling as well as to metabolism. The recent identification and characterization of the system N and A family of transporters has begun to suggest mechanisms for the directional transfer of glutamine, and should provide ways to test its physiological significance in diverse processes from nitrogen to neurotransmitter release.     

The isolation and partial characterization of the plasma membrane from Trypanosoma brucei.

Whole sheets of plasma membrane, each with their attached flagellum, were purified from Trypanosoma brucei. The method devised for their isolation included a new technique of cell breakage that used a combination of osmotic stress followed by mechanical sheer and avoided the problem of extreme vesiculation as well as the trapping of organelles in cell 'ghosts'. The purified membranes all contained the pellicular microtubular array. The antigenic surface coat was completely released from the plasma membrane during the isolation procedure. The membranes had a very high cholesterol/phospholipid ratio (1.54). A large proportion (42%) of the cellular DNA was recovered in the plasma-membrane fraction unless a step involving deoxyribonuclease treatment, which decreased the DNA content to less than 13%, was included before secrose-density gradient centrifugation. This step also aided the separation of plasma membranes from other cellular components. The ouabain-sensitive Na+ + K+-stimulated adenosine triphosphatase and adenylate cyclase co-purified with the plasma membranes. Although 5'-nucleotidase was thought to be a plasma-membrane component, it was easily detached from the membrane. The purified membranes were essentially free of L-alanine-alpha-oxoglutarate aminotransferase, L-asparte-alpha-oxoglutarate aminotransferase, malate dehydrogenase, oligomycin-sensitive adenosine triphosphatase, glucose 6-phosphatase, Mg2+-stimulated p-nitrophenyl phosphatase and catalase.     

Control of Photosynthesis and Stomatal Conductance in Ricinus communis L. (Castor Bean) by Leaf to Air Vapor Pressure Deficit

Castor bean (Ricinus communis L.) has a high photosynthetic capacity under high humidity and a pronounced sensitivity of photosynthesis to high water vapor pressure deficit (VPD). The sensitivity of photosynthesis to varying VPD was analyzed by measuring CO₂ assimilation, stomatal conductance (g_s), quantum yield of photosystem II (_II), and nonphotochemical quenching of chlorophyll fluorescence (q_N) under different VPD. Under both medium (1000) and high (1800 micromoles quanta per square meter per second) light intensities, CO₂ assimilation decreased as the VPD between the leaf and the air around the leaf increased. The g_s initially dropped rapidly with increasing VPD and then showed a slower decrease above a VPD of 10 to 20 millibars. Over a temperature range from 20 to 40°C, CO₂ assimilation and g_s were inhibited by high VPD (20 millibars). However, the rate of transpiration increased with increasing temperature at either low or high VPD due to an increase in g_s. The relative inhibition of photosynthesis under photorespiring (atmospheric levels of CO₂ and O₂) versus nonphotorespiring (700 microbars CO₂ and 2% O₂) conditions was greater under high VPD (30 millibars) than under low VPD (3 millibars). Also, with increasing light intensity the relative inhibition of photosynthesis by O₂ increased under high VPD, but decreased under low VPD. The effect of high VPD on photosynthesis under various conditions could not be totally accounted for by the decrease in the intercellular CO₂ in the leaf (C_i) where C_i was estimated from gas exchange measurements. However, estimates of C_i from measurements of _II and q_N suggest that the decrease in photosynthesis and increase in photorespiration under high VPD can be totally accounted for by stomatal closure and a decrease in C_i. The results also suggest that nonuniform closure of stomata may occur in well-watered plants under high VPD, causing overestimates in the calculation of C_i from gas exchange measurements. Under low VPD, 30°C, high light, and saturating CO₂, castor bean (C₃ tropical shrub) has a rate of photosynthesis (61 micromoles CO₂ per square meter per second) that is about 50% higher than that of tobacco (C₃) or maize (C₄) under the same conditions. The chlorophyll content, total soluble protein, and ribulose-1,5-bisphosphate carboxylase/oxygenase level on a leaf area basis were much higher in castor bean than in maize or tobacco, which accounts for its high rates of photosynthesis under low VPD.