Isolation of 2000-kDa complexes of N-methyl-D-aspartate receptor and postsynaptic density 95 from mouse brain

Neurotransmitter receptors in vivo are linked to intracellular adaptor proteins and signalling molecules driving downstream pathways. Methods for physical isolation are essential to answer fundamental questions about the size, structure and composition of in vivo complexes and complement the widely used yeast 2-hybrid method. The N-methyl-D-aspartate receptor (NMDAR) binds postsynaptic density 95 (PSD-95) protein; both are required for synaptic plasticity and learning and participate in other important pathophysiological functions. Here we describe the development and optimization of novel methods for large-scale isolation of NMDAR--PSD-95 complexes from mouse brain including immunoaffinity, immunoprecipitation, ligand-affinity and immobilized PSD-95 binding peptides. Short PDZ binding peptides modelled on NMDAR subunits were shown to isolate NMDAR complexes. Gel filtration indicated the native NMDAR--PSD-95 complexes were 2000 kDa, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed a complexity suggesting a huge network of both structural components and signalling enzymes. These methods can be used to define the structure of the complexes at different synapses and in mice carrying gene mutations as well as new tools for drug discovery.     

Tyrosine phosphorylation of the N-methyl-D-aspartate receptor by exogenous and postsynaptic density-associated Src-family kinases

Phosphorylation of the NMDA receptor by Src-family tyrosine kinases has been implicated in the regulation of receptor function. We have investigated the tyrosine phosphorylation of NMDA receptor subunits NR2A and NR2B by exogenous Src and Fyn and compared this to phosphorylation by tyrosine kinases associated with the postsynaptic density (PSD). Phosphorylation of the receptor by exogenous Src and Fyn was dependent upon initial binding of the kinases to PSDs via their SH2-domains. Src and Fyn phosphorylated similar sites in NR2A and NR2B, tryptic peptide mapping identifying seven and five major tyrosine-phosphorylated peptides derived from NR2A and NR2B, respectively. All five tyrosine phosphorylation sites on NR2B were localized to the C-terminal, cytoplasmic domain. Phosphorylation of NR2B by endogenous PSD tyrosine kinases yielded only three tyrosine-phosphorylated tryptic peptides, two of which corresponded to Src phosphorylation sites, and one of which was novel. Phosphorylation-site specific antibodies identified NR2B Tyr1472 as a phosphorylation site for intrinsic PSD tyrosine kinases. Phosphorylation of this site was inhibited by the Src-family-specific inhibitor PP2. The results identify several potential phosphorylation sites for Src in the NMDA receptor, and indicate that not all of these sites are available for phosphorylation by kinases located within the structural framework of the PSD.     

Interaction of Topotecan,DNA Topoisomerase I Inhibitor,with Double-Stranded Polydeoxyribonucleotides. 1. Topotecan Dimerization in Solution

Behavior of topotecan, DNA topoisomerase I inhibitor, was studied in aqueous solutions by optical methods. Topotecan absorption spectra were recorded in the pH range 0.5–11.5 and its pKa were determined. Quantum chemical calculations were made for all charge states of the topotecan molecule in lactone and carboxylate form. The calculated absorption maxima agree well with the experimental data. Protonation of the topotecan D ring (pKa 3.6) was revealed. Comparison of experimental and calculated data showed topotecan structure with a proton at the oxygen atom at C16a rather than N4 to be the most preferable. Topotecan molecules were shown to form dimers at concentrations above 10^–5M. Topotecan dimerization is accompanied by an increase in the pKa of hydroxy group of the A ring from 6.5 ([TPT] = 10^–6M) to 7.1 ([TPT] = 10^–4M), which indicates participation of this group in dimer stabilization, perhaps due to intermolecular hydrogen bonding with N1 of the B ring of a neighboring molecule. Probable dimer structures were proposed. The topotecan dimerization constant was determined, K = (4.0 ± 0.7)·10³M^–1.     

Comparison of the population genetics and densities of five pinanga palm species at kuala belalong, brunei

This study investigated the effect of population density on the population genetic structure of five co-occurring congeneric understorey palm species: Pinanga aristata, P. sp. aff. brevipes, P. dumetosa, P. tenella var. tenella and P. veitchii, all endemic to northern Borneo. The average population densities of the study species varied across a wide spectrum, ranging from 343 (plants per ha) in P. tenella to 10 (plants per ha) in P. veitchii. All species of Pinanga palms studied had quite high levels of genetic diversity (HE: 0.379, 0.256, 0.294, 0.133, 0.352). Genetic diversity (HE) was correlated with population density (D; rs = - 0.433, P < 0.01) and the average distance to the nearest conspecific neighbour (NN; rs = 0.576, P < 0.001) such that the most-dense species had less genetic diversity and the less-dense species had greater genetic diversity. Gene flow (Nm) among populations approximately followed a gradient of increasing species density and abundance, such that the most common species P. dumestosa had the greatest gene flow (Nm = 2.268) between its populations and the rarest, most sparsely distributed species P. sp. aff. brevipes had the lowest (Nm = 0.698). All species of Pinanga were effectively inbred (F: 0.760, 0.856, 0.640, 0.753, 0.674). The amount of homozygosity and inbreeding (HO, F) were not correlated (P > 0.05) with population density (D) or the distance between nearest neighbouring plants of the same species (NN).     

Molecular cloning,expression, and functional characterization of a 2Cys-peroxiredoxin in Chinese cabbage

A cDNA (C2C-Prx) corresponding to a 2Cys-peroxiredoxin (2Cys-Prx) was isolated from a leaf cDNA library of Chinese cabbage. The predicted amino acid sequence of C2C-Prx has 2 conserved cysteines and several peptide domains present in most of the 2Cys-Prx subfamily members. It shows the highest sequence homology to the 2Cys-Prx enzymes of spinach (88%) and Arabidopsis (86%). Southern analysis using the cDNA insert of C2C-Prx revealed that it consists of a small multigene family in Chinese cabbage genome. RNA blot analysis showed that the gene was predominantly expressed in the leaf tissue of Chinese cabbage seedlings, but the mRNA was generally expressed in most tissues of mature plant, except roots. The expression of C2C-Prx was slightly induced by treatment with H₂O₂ (100M) or Fe³⁺/O₂/DTT oxidation system, but not by ABA (50 M) or GA₃ (10 M). The C2C-Prx is encoded as a preprotein of 273 amino acids containing a putative chloroplast-targeting signal of 65 amino acids at its N-terminus. The N-terminally truncated recombinant protein (C2C-Prx) migrates as a dimer in a non-reducing SDS-polyacrylamide gel and as a monomer in a reducing condition. The C2C-Prx shows no immuno cross-reactivity to antiserum of the yeast thiol-specific antioxidant protein, and vice versa. The C2C-Prx prevents the inactivation of glutamine synthetase and the DNA cleavage in the metal-catalyzed oxidation system. In the yeast thioredoxin system containing thioredoxin reductase, thioredoxin, and NADPH, the C2C-Prx exhibits peroxidase activity on H₂O₂.     

不同食性甲虫的含能值、含水量及其变动规律研究

以干旱地区的肉食性步甲科(Carabidae)和植食性拟步甲科(Tenebrionidae)昆虫成体为材料,测定鞘翅目甲虫的含能值和含水量,分析其含能值和含水量的相互关系及其变动规律.结果表明,肉食性步甲科昆虫的含能值(22.64 J/mg无灰干重)显著高于植食性拟步甲科昆虫(20.55 J/mg无灰干重)(P<0.05),相反,前者的含水量(54.96%)却显著地低于后者(63.28%)(P<0.05).步甲科和拟步甲科昆虫的含能值都与其含水量成显著负相关(r=-0.5962,P<0.05).步甲科和拟步甲科昆虫的含能值或含水量具有明显的季节变动规律,两科的变动规律基本相同,其中,含能值在秋季最高(22.80 J/mg无灰干重),春季最低(20.42 J/mg无灰干重),含水量则相反,秋季最低(54.93%),春季最高(61.88%)(P<0.05).步甲科和拟步甲科昆虫的头、胸、腹和附肢不同体区的含能值具有显著差异(P<0.05),其中,腹部的含能值最高(22.65 J/mg无灰干重),其次为胸部(21.33 J/mg无灰干重),头部和附肢的含能值最低(20.89 J/mg无灰干重和20.60 J/mg无灰干重).不同体区的含能值也具有同样的季节变动,即含能值在秋季最高(22.30 J/mg无灰干重),春季最低(19.46 J/mg无灰干重)(P<0.05).步甲科和拟步甲科昆虫的个体含能值与其头、胸、腹和附肢各体区的含能值成正相关,相关系数分别为0.718、0.846、0.897和0.604(P<0.05);其中个体含能值与腹部或胸部体区含能值的相关系数显著地高于它们与头部和附肢含能值的相关系数(P<0.05);这些结果说明,脂质在甲虫腹部和胸部的积聚是引起甲虫个体含能值提高的主要原因.本文对不同食性甲虫含能值产生差异的原因,甲虫含能值和含水量变动的影响因素也进行了分析;并结合最佳摄食理论,就甲虫作为猎物,其含能值和含水量的这些变动规律对食虫动物食物选择所可能产生的影响也进行了探讨,就今后进一步检验食虫动物摄食对策所应开展的猎物喂养食虫动物的实验提出建议.     

Reactive oxygen species function as second messenger during ischemic preconditioning of heart

The mechanisms involved in the hypolipidemic effects of fish oil have not been clearly established. This study shows that supplementation of 10% menhaden oil to the chick diet for 7 days produced a significant hypocholesterolemia and hypotriglyceridemia. Fatty acid composition of chick plasma drastically changed by the same dietary manipulation. Percentages of 20:5 and 22:6 n-3 fatty acids strongly increased, while percentages of 20:4 n-6, 18:2 n-6, and 18:1 n-9 significantly decreased. Changes observed in the relative percentages were parallel to those obtained in the amount of each fatty acid. Ratio of n-3/n-6 clearly decreased in plasma by fish oil feeding. Total cholesterol and triacylglycerol contents decreased in high density lipoprotein (HDL) but did not change in low density lipoprotein (LDL). All chemical constituents of very low density lipoprotein (VLDL) significantly decreased after the first week of menhaden oil supplementation to the diet. Similar modifications in fatty acid composition of the three lipoprotein fractions were also found. Our results suggest that the hypocholesterolemic effects of fish oil may be mediated by the depletion in VLDL synthesis and secretion into the chick plasma. On the other hand, the strong decrease found in the arachidonic acid (AA) content of chick plasma and lipoproteins may contribute to the beneficial effects of fish oil consumption by lowering the production of its derived eicosanoids.     

Canine teeth of bats (Microchiroptera): size, shape and role in crack propagation

Upper canines in microchiropteran bats show a variety of cross-sectional shapes. A consistent feature of all species studied here is that the tooth is edged and not simply round or oval. Prominent sharp edges are positioned in several directions but particularly antero-medially toward the incisors and posteriorly toward the premolars. These edges appear to direct the cracks made in food items to the incisors or to the premolars. A continuous cutting edge is apparent in the occlusal view of the palate running from tip of canine to the ectoloph of the molars. Size and shape analysis indicates that larger bats have slender, rather than stouter, canines for their height, a condition that may be attributable to the nature of the prey. Most bats take prey that have little hard substance imbedded within. The compromises in tooth shape may vary between that of a terrestrial predator with short, conical canines for processing endoskeletal prey to that of a small flying predator with long, slender, edged canines for capturing and processing exoskeletal prey. Unicuspid teeth and how they might function in food break-up have been overlooked to the literature; such a study could lead to an understanding of how more complex teeth function.     

Functional design space of single-veined leaves: role of tissue hydraulic properties in constraining leaf size and shape

BACKGROUND AND AIMS: Morphological diversity of leaves is usually quantified with geometrical characters, while in many cases a simple set of biophysical parameters are involved in constraining size and shape. One of the main physiological functions of the leaf is transpiration and thus one can expect that leaf hydraulic parameters can be used to predict potential morphologies, although with the caveat that morphology in turn influences physiological parameters including light interception and boundary layer thickness and thereby heat transfer and net photosynthesis. METHODS: An iterative model was used to determine the relative sizes and shapes that are functionally possible for single-veined leaves as defined by their ability to supply the entire leaf lamina with sufficient water to prevent stomatal closure. The model variables include the hydraulic resistances associated with vein axial and radial transport, as well as with water movement through the mesophyll and the leaf surface. KEY RESULTS: The four parameters included in the model are sufficient to define a hydraulic functional design space that includes all single-veined leaf shapes found in nature, including scale-, awl- and needle-like morphologies. This exercise demonstrates that hydraulic parameters have dissimilar effects: surface resistance primarily affects leaf size, while radial and mesophyll resistances primarily affect leaf shape. CONCLUSIONS: These distinctions between hydraulic parameters, as well as the differential accessibility of different morphologies, might relate to the convergent evolutionary patterns seen in a variety of fossil lineages concerning overall morphology and anatomical detail that frequently have evolved in linear and simple multi-veined leaves.     

Variation in floral sex allocation in Polygonatum odoratum (Liliaceae)

BACKGROUND AND AIMS: It is well known that resource allocation to male and female functions can be highly variable in hermaphroditic plants. The purpose of this study was to investigate variations in sexual investment at different levels (flower, plant and population) in Polygonatum odoratum, a plant with sequentially opening flowers. METHODS: Pollen and ovule production in base, middle and top flowers of P. odoratum flowering shoots from two natural populations were quantified. Plant measurements of phenotypic and functional gender were calculated in both populations. Total leaf number was used to investigate the relationship between gender assessments and plant size. KEY RESULTS: Pollen and ovule production varied depending on flower position, although the precise pattern differed between both studied populations; only investment in female floral function decreased markedly from base to top flowers in both populations. The frequency distribution of phenotypic gender and their relationship with plant size differed between populations. Phenotypic and functional gender were correlated in both populations. CONCLUSIONS: Sexual investment in P. odoratum has shown a marked variability within plants, among plants, and between populations, which confirms the importance of analysing sex expression in plants of this type. Differences in relative investment in male and female components (phenotypic gender) are reflected in the functional gender and it would be expected that the evolution of sexual specialization in Polygonatum odoratum would be promoted.