Molecular dynamics of the FixJ receiver domain: movement of the beta4-alpha4 loop correlates with the in and out flip of Phe101

FixJ is a two-domain response regulator involved in nitrogen fixation in Sinorhizobium meliloti. Recent X-ray characterization of both the native (unphosphorylated) and the active (phosphorylated) states of the protein identify conformational changes of the beta4-alpha4 loop and the conserved residue Phe101 as the key switches in activation. These structures also allowed investigation of the transition between conformations of this two-component regulatory receiver domain by molecular dynamics simulations. The path for the conformational change was studied with a distance constraint directing the system from one state to the other. The simulations provide evidence for a correlation between the conformation of the beta4-alpha4 loop and the orientation of the residue Phe101. A model presenting the sequence of events during the activation/deactivation process is discussed.     

Quantification of the influence of HPrSer46P on CcpA-cre interaction

Carbon catabolite repression (CCR) of the Bacillus megateriumxyl operon is dependent on the catabolite responsive element cre, the catabolite control protein (CcpA) and the histidine-containing phosphocarrier protein phosphorylated at the serine 46 residue (HPrSer46P). The latter is formed in the presence of glucose and mediates CCR via CcpA. We present evidence for the presence of HPrSer46P in a ternary complex with CcpA and cre. We also demonstrate increased stability of this complex compared to the CcpA-cre complex by electrophoretic mobility shift analysis (EMSA). This stabilization by HPrSer46P is the same for the xyl cre and an improved cre. Thus, HPrSer46P is a co-repressor for CcpA. In addition, surface plasmon resonance (SPR) experiments yielded binding constants of CcpA and the CcpA-HPrSer46P complex with cre. HPrSer46P stimulated CcpA binding to cre 50-fold. The binding constant is 4.9(+/- 0.5) x 10(6) M(-1). Non-phosphorylated HPr did not affect the complex formation between CcpA and cre. Previously proposed effects by glucose-6-phosphate, fructose-1,6-diphosphate and NADP on CcpA-cre or CcpA-HPrSer46P-cre formation were not found in EMSA and SPR experiments.     

The human histamine H2-receptor couples more efficiently to Sf9 insect cell Gs-proteins than to insect cell Gq-proteins: limitations of Sf9 cells for the analysis of receptor/Gq-protein coupling

The human histamine H2-receptor (hH2R) couples to Gs-proteins to activate adenylyl cyclase and to Gq-proteins to activate phospholipase C, but phospholipase C activation has not consistently been observed. The aim of this study was to compare coupling of hH2R to insect and mammalian Gs- and Gq-proteins in Spodoptera frugiperda (Sf9) cells. Interaction of hH2R with mammalian G proteins was assessed with coexpressed proteins or receptor-Galpha fusion proteins that enhance coupling efficiency. hH2R efficiently coupled to insect Gs-proteins to activate adenylyl cyclase. However, hH2R poorly coupled to insect Gq-proteins as assessed by the lack of enhancement of histamine-stimulated steady-state GTP hydrolysis by regulators of G protein signaling (RGS proteins). In contrast, RGS-proteins efficiently enhanced GTP hydrolysis stimulated by the human platelet-activating factor receptor (PAFR) and the histamine H1-receptor (H1R) from man and guinea pig. The measurement of intracellular free Ca2+ concentration was not useful for studying receptor/Gq-protein coupling. hH2R also efficiently interacted with mammalian Gs-proteins, specifically with fused Gsalpha as assessed by guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS)-sensitive high-affinity agonist binding, agonist-stimulated [35S]GTPgammaS binding and adenylyl cyclase activation. In contrast, coupling of hH2R to coexpressed and fused mammalian Gqalpha was poor. However, our inability to reconstitute efficient coupling of PAFR and H1R to mammalian Gqalpha indicated that a large portion of the expressed G protein was functionally inactive. Taken together, our data show that hH2R couples more efficiently to insect cell Gs-proteins than to insect cell Gq-proteins. Unfortunately, there are significant limitations in the usefulness of Sf9 cells for comparing the coupling of receptors to mammalian Gs- and Gq-proteins and assessing Gq-mediated activation of effector systems.     

Membrane potential and bicarbonate secretion in isolated interlobular ducts from guinea-pig pancreas

The interlobular duct cells of the guinea-pig pancreas secrete HCO(3)(-) across their luminal membrane into a HCO(3)(-)-rich (125 mM) luminal fluid against a sixfold concentration gradient. Since HCO(3)(-) transport cannot be achieved by luminal Cl-/HCO(3)(-) exchange under these conditions, we have investigated the possibility that it is mediated by an anion conductance. To determine whether the electrochemical potential gradient across the luminal membrane would favor HCO(3)(-) efflux, we have measured the intracellular potential (V(m)) in microperfused, interlobular duct segments under various physiological conditions. When the lumen was perfused with a 124 mM Cl- -25 mM HCO(3)(-) solution, a condition similar to the basal state, the resting potential was approximately -60 mV. Stimulation with dbcAMP or secretin caused a transient hyperpolarization (approximately 5 mV) due to activation of electrogenic Na+-HCO(3)(-) cotransport at the basolateral membrane. This was followed by depolarization to a steady-state value of approximately -50 mV as a result of anion efflux across the luminal membrane. Raising the luminal HCO(3)(-) concentration to 125 mM caused a hyperpolarization (approximately 10 mV) in both stimulated and unstimulated ducts. These results can be explained by a model in which the depolarizing effect of Cl- efflux across the luminal membrane is minimized by the depletion of intracellular Cl- and offset by the hyperpolarizing effects of Na+-HCO(3)(-) cotransport at the basolateral membrane. The net effect is a luminally directed electrochemical potential gradient for HCO(3)(-) that is sustained during maximal stimulation. Our calculations indicate that the electrodiffusive efflux of HCO(3)(-) to the lumen via CFTR, driven by this gradient, would be sufficient to fully account for the observed secretory flux of HCO(3)(-).     

Global Positioning System (GPS) location accuracy improvement due to selective availability removal

Global Positioning System (GPS) is an important new technology for spatio-temporal behaviour studies of animals. Differential correction improves location accuracy. Previously, it mostly removed partially the influence of Selective Availability (SA). SA was deactivated in May 2000. The aim of this study was to quantify the influence of SA cancellation on location accuracy of various GPS receivers. We tested the accuracy of locations obtained from non-differential and differential GPS animal collars before and after SA removal. We found a significant improvement in accuracy for both types of GPS collars. However, differential GPS still provides more accurate locations.     

陆地生态系统类型转变与碳循环

 土地利用变化引起的陆地生态系统类型转变对于全球碳循环有着极其重要的作用。　通过总结国内外有关森林砍伐以及森林、草地转变成农田对于碳循环的影响,阐述了可能引起全球“未知汇”现象的重要原因,强调未来中国陆地生态系统碳循环研究应充分重视陆地生态系统类型转变对于全球碳循环的影响研究,包括研究陆地生态系统的不同发展阶段(自然与退化生态系统)、利用方式的改变(森林转化为人工林或农田,草地转化为农田、退耕还林草等)所引起的碳库类型转换的增汇机理及其对全球变化响应,并指出了建立统一观测方法与规范的陆地生态系统碳通量观测网     

Cloning and characterization of the fur gene from Moraxella bovis

A homologue of the ferric uptake regulator gene (fur) was isolated from Moraxella bovis by degenerate polymerase chain reaction and cloning. Fur protein of M. bovis exhibited 72.1% amino acid identity with Acinetobacter calcoaceticus Fur. Western blot analysis showed a decrease of Fur expression in response to sufficient-iron conditions compared with deficient-iron conditions. An electrophoretic mobility-shift assay indicated that Fur protein binds to DNA fragments containing a putative Fur-box derived from the upstream region of the M. bovis fur gene. Fur of M. bovis may regulate the expression of iron transport systems in response to iron limitation in the environment.     

High copper levels improve callus induction and plant regeneration in <Emphasis Type="Italic">Sorghum bicolor</Emphasis> (L.) Moench

Summary A highly efficient protocol for callus induction and plant regeneration in Sorghum bicolor was developed by varying the concentrations of copper (0.1, 0.3, 0.5, 0.7, 1, 1.5, 2.5 μM) in Murashige and Skoog (MS) medium. The mature embryos of Sorghum bicolor were cultured on MS medium containing 2,4-dichlorophenoxyacetic acid (9μM), kinetin (2.3 μM), and 3% (w/v) sucrose for embryogenic callus induction. Plant regeneration from this callus occurred on MS medium containing kinetin (9.2 μM) and indole-3-acetic acid (2.85 μM). A much greater response was noted on these media with higher levels of copper. Frequency of plant regeneration and number of regenerants dramatically increased with an optimal amount of copper (2 μM) in the MS medium. Rooting of the regenerated shoots readily occurred on half-strength MS medium supplemented with α-naphthaleneacetic acid (10.7 μM) and 3% (w/v) sucrose. Well-developed plantlets were transferred to the field where 100% survival and normal seed setting was noted.     

Scaled marginal models for multiple continuous outcomes

In studies that involve multivariate outcomes it is often of interest to test for a common exposure effect. For example, our research is motivated by a study of neurocognitive performance in a cohort of HIV-infected women. The goal is to determine whether highly active antiretroviral therapy affects different aspects of neurocognitive functioning to the same degree and if so, to test for the treatment effect using a more powerful one-degree-of-freedom global test. Since multivariate continuous outcomes are likely to be measured on different scales, such a common exposure effect has not been well defined. We propose the use of a scaled marginal model for testing and estimating this global effect when the outcomes are all continuous. A key feature of the model is that the effect of exposure is represented by a common effect size and hence has a well-understood, practical interpretation. Estimating equations are proposed to estimate the regression coefficients and the outcome-specific scale parameters, where the correct specification of the within-subject correlation is not required. These estimating equations can be solved by repeatedly calling standard generalized estimating equations software such as SAS PROC GENMOD. To test whether the assumption of a common exposure effect is reasonable, we propose the use of an estimating-equation-based score-type test. We study the asymptotic efficiency loss of the proposed estimators, and show that they generally have high efficiency compared to the maximum likelihood estimators. The proposed method is applied to the HIV data.