Discovery stage pharmacokinetics using dried blood spots

Early in the discovery stage, the measurement of drug candidates in biological fluids as a function time provides important information used in decision making for lead optimization. The detection methodology primarily used is liquid chromatography coupled to triple quadrupole mass spectrometry (LC-MS). Sample preparation is an important aspect of these experiments and robotic-based automation is commonly used. The often overlooked aspect of these experiments is the sample collection itself. Typically, several hundred microliters of whole blood is collected and the plasma fraction separated for each time-point. The plasma is then transferred to an appropriate vessel for subsequent aliquoting and processing. We describe a method for performing discovery stage pharmacokinetic analysis using whole blood dried onto filter paper. The use of dried blood spots is a well established technique for neo-natal screening, and its application to early screening of drug candidates proves to be robust, reliable and reproducible.     

Remotely Sensed Interannual Variations and Trends in Terrestrial Net Primary Productivity 1981–2000

Spatial and temporal variations in net primary production (NPP) are of great importance to ecological studies, natural resource management, and terrestrial carbon sink estimates. However, most of the existing estimates of interannual variation in NPP at regional and global scales were made at coarse resolutions with climate-driven process models. In this study, we quantified global NPP variation at an 8 km and 10-day resolution from 1981 to 2000 based on satellite observations. The high resolution was achieved using the GLObal Production Efficiency Model (GLO-PEM), which was driven with variables derived almost entirely from satellite remote sensing. The results show that there was an increasing trend toward enhanced terrestrial NPP that was superimposed on high seasonal and interannual variations associated with climate variability and that the increase was occurring in both northern and tropical latitudes. NPP generally decreased in El Niño season and increased in La Niña seasons, but the magnitude and spatial pattern of the response varied widely between individual events. Our estimates also indicate that the increases in NPP during the period were caused mainly by increases in atmospheric carbon dioxide and precipitation. The enhancement of NPP by warming was limited to northern high latitudes (above 50°N); in other regions, the interannual variations in NPP were correlated negatively with temperature and positively with precipitation.     

Vitrification of pronuclear-stage mouse embryos on solid surface (SSV) versus in cryotube: comparison of the effect of equilibration time and different sugars in the vitrification solution

The cryopreservation of pronuclear-stage embryos has particular importance in transgenic technology and human assisted reproductive technology (ART). The objective of this study was to improve the efficiency of cryopreservation of pronuclear-stage mouse embryos. Two vitrification methods (solid surface vitrification (SSV) vs. vitrification in cryotube) have been compared with special emphasis on the effect of the exposure of the embryos to the solutions for various times and the sugar content (trehalose, sucrose, or raffinose) of the vitrification solutions. Pronuclear-stage embryos were either exposed to 1 M dimethyl sulfoxide (DMSO) + 1 M propylene-glycol (PG) solution for 2, 5, 10, or 15 min or not exposed to this "equilibration" solution. The vitrification solutions consisted of 2.75 M DMSO and 2.75 M PG in M2 medium supplemented with 1 M trehalose (DPT), 1 M sucrose (DPS), or 1 M raffinose (DPR). In the cryotube method, groups of 15-25 embryos were transferred into a 1.8 ml cryotube containing 30 microl of DPT, DPS, or DPR. After 30 sec, the cryotubes were directly plunged into liquid nitrogen (LN(2)) and stored for 1 day to 1 month. Vitrified samples were warmed by immersing the cryotubes in a 40 degrees C water bath and then immediately diluted with 300 microl of 0.3 M trehalose, sucrose, or raffinose in M2. In the SSV method, after equilibration 15-20 embryos were exposed to DPT, DPS, or DPR solutions for around 20 sec before being dropped in 2-microl drops onto a pre-cooled (-150 to -180 degrees C) metal surface. Vitrified droplets were stored in cryovials in LN(2). Warming was performed by transferring the vitrified droplets into 0.3 M solutions of trehalose, sucrose, or raffinose at 37 degrees C, respectively. Results showed that both SSV and cryotube vitrification methods can result in high rates of in vitro blastocyst development (up to 58.3 and 68.5% with DPR, respectively), not statistically different from that of the controls (58.3 and 64.4%). Even without the equilibration step prior to vitrification, relatively high-survival rates have been achieved, except for the DPS solution. In conclusion, vitrification of pronuclear-stage mouse embryos can result in high rates of in vitro development to blastocyst, and the use of raffinose in the vitrification solution is advantageous to improve cryosurvival.     

Precise recapitulation of methylation change in early cloned embryos

Change of DNA methylation during preimplantation development is very dynamic, which brings this term to the most attractive experimental target for measuring the capability of cloned embryo to reprogram its somatic genome. However, one weak point is that the preimplantation stage carries little information on genomic sequences showing a site-specific re-methylation after global demethylation; these sequences, if any, may serve as an advanced subject to test how exactly the reprogramming/programming process is recapitulated in early cloned embryos. Here, we report a unique DNA methylation change occurring at bovine neuropeptide galanin gene sequence. The galanin gene sequence in early bovine embryos derived by in vitro fertilization (IVF) maintained a undermethylated status till the morula stage. By the blastocyst, certain CpG sites became methylated specifically, which may be an epigenetic sign for the galanin gene to start a differentiation programme. The same sequence was moderately methylated in somatic donor cell and, after transplanted into an enucleated oocyte by nuclear transfer (NT), came rapidly demethylated to a completion, and then, at the blastocyst stage, re-methylated at exactly the same CpG sites, as observed so in normal blastocysts. The precise recapitulation of normal methylation reprogramming and programming at the galanin gene sequence in bovine cloned embryos gives a cue for the potential of cloned embryo to superintend the epigenetic states of foreign genome, even after global demethylation.     

AGE-breakers cleave model compounds,but do not break Maillard crosslinks in skin and tail collagen from diabetic rats

Advanced glycation end products (AGE), formed by nonenzymatic Maillard reactions between carbohydrate and protein, contribute to the increase in chemical modification and crosslinking of tissue proteins with age. Acceleration of AGE formation in collagen during hyperglycemia, with resultant effects on vascular elasticity and basement membrane permeability, is implicated in the pathogenesis of diabetic complications. AGE-breakers, such as N-phenacylthiazolium (PTB) and N-phenacyl-4,5-dimethylthiazolium (PMT) halides, have been proposed as therapeutic agents for reversing the increase in protein crosslinking in aging and diabetes. We have confirmed that these compounds, as well as the AGE-inhibitor pyridoxamine (PM), cleave the model AGE crosslink, phenylpropanedione, and have studied the effects of these compounds in reversing the increased crosslinking of skin and tail collagen isolated from diabetic rats. Crosslinking of skin collagen, measured as the half-time for solubilization of collagen by pepsin in 0.5M acetic acid, was increased approximately 5-fold in diabetic, compared to nondiabetic rats. Crosslinking of tail tendon collagen, measured as insolubility in 0.05 N acetic acid, was increased approximately 10-fold. Collagen preparations were incubated in the presence or absence of AGE-breakers or PM in phosphate buffer, pH 7.4, for 24h at 37 degrees C. These treatments did not decrease the half-time for solubilization of diabetic skin collagen by pepsin or increase the acid solubility of diabetic tail tendon collagen. We conclude that, although AGE-breakers and PM cleave model crosslinks, they do not significantly cleave AGE crosslinks formed in vivo in skin collagen of diabetic rats.     

Membrane protein folding: beyond the two stage model

The folding of alpha-helical membrane proteins has previously been described using the two stage model, in which the membrane insertion of independently stable alpha-helices is followed by their mutual interactions within the membrane to give higher order folding and oligomerization. Given recent advances in our understanding of membrane protein structure it has become apparent that in some cases the model may not fully represent the folding process. Here we present a three stage model which gives considerations to ligand binding, folding of extramembranous loops, insertion of peripheral domains and the formation of quaternary structure.     

缙云山马尾松种群生物量生殖配置研究

 对缙云山亚热带常绿阔叶林不同演替阶段马尾松(Pinus massoniana)种群生殖器官的生物量配置进行了系统研究。结果显示从蕾期、花期到果熟期,马尾松种群生殖器官的生物量配置逐渐增加,但不同演替阶段的生殖配置格局各异。马尾松纯林各生殖阶段的生殖配置分别为1.31％,7.61％,23.25％;针阔混交林各生殖阶段的生殖配置分别为0.6％,3.29％,15.14％;林缘旷地各生殖阶段的生殖配置分别为0.76％,3.78％,18.44％。一年中缙云山马尾松种群的生殖配置动态变化呈现低一渐高一高一低的规律性。缙云山马尾松种群的生殖年龄大致可分为4个时期,即幼龄生殖期、过渡生殖期、稳定生殖期和衰退生殖期。种群密度和群落透光度与其生殖配置显著相关。     

Scavenger receptors for oxidized and glycated proteins

Summary. Our present knowledge on chemically modified proteins and their receptor systems is originated from a proposal by Goldstein and Brown in 1979 for the receptor for acetylated LDL which is involved in foam cell formation, one of critical steps in atherogenesis. Subsequent extensive studies using oxidized LDL (OxLDL) as a representative ligand disclosed at least 11 different scavenger receptors which are collectively categorized as scavenger receptor family. Advanced glycation endproducts (AGE) and their receptor systems have been studied independently until recent findings that AGE-proteins are also recognized as active ligands by scavenger receptors including class A scavenger receptor (SR-A), class B scavenger receptors such as CD36 and SR-BI, type D scavenger receptor (LOX-1) and FEEL-1/FEEL-2. Three messages can be summarized from these experiments; (i) endocytic uptake of OxLDL and AGE-proteins by macrophages or macrophage-derived cells is mainly mediated by SR-A and CD36, which is an important step for foam cell formation in the early stage of atherosclerosis, (ii) selective uptake of cholesteryl esters of high density lipoprotein (HDL) mediated by SR-BI is inhibited by AGE-proteins, suggesting a potential pathological role of AGE in a HDL-mediated reverse cholesterol transport system, (iii) a novel scavenger receptor is involved in hepatic clearance of plasma OxLDL and AGE-proteins.     

Evaluation of major genetic loci contributing to inbreeding depression for survival and early growth in a selfed family of Pinus taeda

The magnitude of fitness effects at genetic loci causing inbreeding depression at various life stages has been an important question in plant evolution. We used genetic mapping in a selfed family of loblolly pine (Pinus taeda L.) to gain insights on inbreeding depression for early growth and viability. Two quantitative trait loci (QTLs) were identified that explain much of the phenotypic variation in height growth through age 3 and may account for more than 13% inbreeding depression in this family. One of these QTLs maps to the location of cad-nl, a lignin biosynthesis mutation. Both QTLs show evidence of overdominance, although evidence for true versus pseudo-overdominance is inconclusive. Evidence of directional dominance for height growth was noted throughout the genome, suggesting that additional loci may contribute to inbreeding depression. A chlorophyll-deficiency mutation, spf did not appear to be associated with growth effects, but had significant effects on survival through age 3. Previously identified embryonic viability loci had little or no overall effect on germination, survival, or growth. Our results challenge, at least in part, the prevailing hypothesis that inbreeding depression for growth is due to alleles of small effect. However, our data support predictions that loci affecting inbreeding depression are largely stage specific.     

束长蝽科若虫记述及其系统学意义(半翅目:异翅亚目)

有关束长蝽科若虫期的报道很少,其中束长蝽亚科尤其如此,迄今只有Ｍａｌｃｕｓ ｆｌａｖｉｄｉｐｅｓ Ｓｔａｌ一种曾有详细描述。本文首次记述Ｍａｌｃｕｓ ｓｉｎｉｃｕｓ Ｓｙｔｓ（５龄）,Ｍ．ｅｌｏｎｇａｔｕｓ Ｓｔｙｓ（５龄和４龄）以及Ｃｈａｕｌｉｏｐｓ ｂｉｓｏｎｏｔｕｌａ Ｂａｎｋｓ（５龄）的若虫,并记录寄主植物。对于腺毛系统及其系统学意义进行了讨论。