Abscisic acid and 14-3-3 proteins control K channel activity in barley embryonic root

Germination of seeds proceeds in general in two phases, an initial imbibition phase and a subsequent growth phase. In grasses like barley, the latter phase is evident as the emergence of the embryonic root (radicle). The hormone abscisic acid (ABA) inhibits germination because it prevents the embryo from entering and completing the growth phase. Genetic and physiological studies have identified many steps in the ABA signal transduction cascade, but how it prevents radicle elongation is still not clear. For elongation growth to proceed, uptake of osmotically active substances (mainly K(+)) is essential. Therefore, we have addressed the question of how the activity of K(+) permeable ion channels in the plasma membrane of radicle cells is regulated under conditions of slow (+ABA) and rapid germination (+fusicoccin). We found that ABA arrests radicle growth, inhibits net K(+) uptake and reduces the activity of K(+) (in) channels as measured with the patch-clamp technique. In contrast, fusicoccin (FC), a well-known stimulator of germination, stimulates radicle growth, net K(+) uptake and reduces the activity of K(+) (out) channels. Both types of channels are under the control of 14-3-3 proteins, known as integral components of signal transduction pathways and instrumental in FC action. Intriguingly, 14-3-3 affected both channels in an opposite fashion: whereas K(+) (in) channel activity was fully dependent upon 14-3-3 proteins, K(+) (out) channel activity was reduced by 14-3-3 proteins by 60%. Together with previous data showing that 14-3-3 proteins control the activity of the plasma membrane H(+)-ATPase, this makes 14-3-3 a prime candidate for molecular master regulator of the cellular osmo-pump. Regulation of the osmo-pump activity by ABA and FC is an important mechanism in controlling the growth of the embryonic root during seed germination.     

Advances in the structural biology, design and clinical development of Bcr-Abl kinase inhibitors for the treatment of chronic myeloid leukaemia

The constitutively activated Abl tyrosine kinase domain of the chimeric Bcr-Abl oncoprotein is responsible for the transformation of haematopoietic stem cells and the symptoms of chronic myeloid leukaemia (CML). Imatinib targets the tyrosine kinase activity of Bcr-Abl and is a first-line therapy for this malignancy. Although highly effective in chronic phase CML, patients who have progressed to the advanced phase of the disease frequently fail to respond to imatinib or develop resistance to therapy and relapse. This is often due to the emergence of clones expressing mutant forms of Bcr-Abl, which exhibit a decreased sensitivity towards inhibition by imatinib. Considerable progress has recently been made in understanding the structural biology of Abl and the molecular basis for resistance, facilitating the discovery and development of second generation drugs designed to combat mutant forms of Bcr-Abl. The first of these compounds to enter clinical development were BMS-354825 (BristolMyersSquibb) and AMN107 (Novartis Pharma) and, from Phase I results, both of these promise a breakthrough in the treatment of imatinib-resistant CML. Recent advances with these and other promising classes of new CML drugs are reviewed.     

Avian influenza (H5N1) viruses isolated from humans in Asia in 2004 exhibit increased virulence in mammals

The spread of highly pathogenic avian influenza H5N1 viruses across Asia in 2003 and 2004 devastated domestic poultry populations and resulted in the largest and most lethal H5N1 virus outbreak in humans to date. To better understand the potential of H5N1 viruses isolated during this epizootic event to cause disease in mammals, we used the mouse and ferret models to evaluate the relative virulence of selected 2003 and 2004 H5N1 viruses representing multiple genetic and geographical groups and compared them to earlier H5N1 strains isolated from humans. Four of five human isolates tested were highly lethal for both mice and ferrets and exhibited a substantially greater level of virulence in ferrets than other H5N1 viruses isolated from humans since 1997. One human isolate and all four avian isolates tested were found to be of low virulence in either animal. The highly virulent viruses replicated to high titers in the mouse and ferret respiratory tracts and spread to multiple organs, including the brain. Rapid disease progression and high lethality rates in ferrets distinguished the highly virulent 2004 H5N1 viruses from the 1997 H5N1 viruses. A pair of viruses isolated from the same patient differed by eight amino acids, including a Lys/Glu disparity at 627 of PB2, previously identified as an H5N1 virulence factor in mice. The virus possessing Glu at 627 of PB2 exhibited only a modest decrease in virulence in mice and was highly virulent in ferrets, indicating that for this virus pair, the K627E PB2 difference did not have a prevailing effect on virulence in mice or ferrets. Our results demonstrate the general equivalence of mouse and ferret models for assessment of the virulence of 2003 and 2004 H5N1 viruses. However, the apparent enhancement of virulence of these viruses in humans in 2004 was better reflected in the ferret.     

Role of PYK2 in the development of obesity and insulin resistance

Non-receptor proline-rich tyrosine kinase-2 (PYK2), which is activated by phosphorylation of one or more of its tyrosine residues, has been implicated in the regulation of GLUT4 glucose transporter translocation and glucose transport. Some data favor a positive role of PYK2 in stimulating glucose transport, whereas other studies suggest that PYK2 may participate in the induction of insulin resistance. To ascertain the importance of PYK2 in the setting of obesity and insulin resistance, we (1) evaluated the regulation of PYK2 in mice fed a high-fat diet and (2) characterized body and glucose homeostasis in wild type (WT) and PYK2(-/-) mice on different diets. We found that both PYK2 expression and phosphorylation were significantly increased in liver and adipose tissues harvested from high-fat diet fed mice. Wild type and PYK2(-/-) mice were fed a high-fat diet for 8 weeks to induce insulin resistance/obesity. Surprisingly, in response to this diet PYK2(-/-) mice gained significantly more weight than WT mice (18.7+/-1.2g vs. 9.5+/-0.6g). Fasting serum leptin and insulin and blood glucose levels were significantly increased in high-fat diet fed mice irrespective of the presence of PYK2 protein. There was a close correlation between serum leptin and body weight. Intraperitoneal glucose tolerance tests revealed that as expected, the high-fat diet resulted in increased blood glucose levels following glucose administration in wild type mice compared to those fed normal chow. An even greater increase in blood glucose levels was observed in PYK2(-/-) mice compared to wild type mice. These results demonstrate that a lack of PYK2 exacerbates weight gain and development of glucose intolerance/insulin resistance induced by a high-fat diet, suggesting that PYK2 may play a role in slowing the development of obesity, insulin resistance, and/or frank diabetes.     

O-raffinose crosslinked hemoglobin lacks site-specific chemistry in the central cavity: structural and functional consequences of beta93Cys modification

Reacting human deoxyHbA0 with oxidized raffinose (O-raffinose), a trisaccharide, results in a low oxygen affinity "blood substitute," stabilized in a noncooperative T-conformation and possesses readily oxidizable rhombic heme. In this study, we fractionated the O-raffinose-modified HbA0 heterogeneous polymer (O-R-PolyHbA0) into six distinct fractions with a molecular weight distribution ranging from 64 to approximately 600 kDa using size-exclusion chromatography (SEC). Oxygen equilibrium and kinetics binding parameters of all fractions were nearly identical, reflecting a lack of heterogeneity in ligand binding properties among O-R-PolyHbA0 species (Hill coefficient n equal to 1.0). Several mass spectrometry techniques were used to evaluate undigested and digested HbA0, O-R-PolyHbA0, and O-R-PolyHbA0 fractions. Proposed sites of intramolecular crosslinking (i.e., beta1Lys82, beta2Lys82, and beta1Val1) were not found to be the predominant site of crosslinking within the central cavity. Intermolecular crosslinking with O-raffinose results in no discernible site of amino acids modifications with the exception of beta93Cys and alpha104Cys. Based on accessible surface area (ASA) calculations in intact deoxyHbA0, slight conformational changes are required to allow for the S on alpha104Cys to be modified during the reaction with O-raffinose or its partially oxidized product(s). The stabilization of HbA0 in the T-conformation may not be a direct correlate of O-raffinose induced changes, but an indirect consequence of changing hydration in the water-filled central cavity and/or the distal heme pocket leading in the latter case to accelerated iron oxidation. Structural data presented here when taken together with the oxidative instability of O-R-PolyHbA0 may provide some basis for the reported toxicity of this oxygen carrier.     

古田山常绿阔叶林凋落量时间动态及冰雪灾害的影响

2006年10月至2009年12月期间,我们通过对古田山24 ha常绿阔叶林动态样地169个种子雨收集器的凋落物进行烘干、分类、称量和数据分析,研究了凋落量的组成特征和时间动态,以及受2008年2月特大冰雪灾害的影响.2007年和2009年凋落量分别为532.05 g/m2和375.17 g/m2,年际变化显著,这与2008年的冰雪灾害有关.2007年古田山常绿阔叶林各组分在凋落量中所占比例依次为:叶(78.99%)>枝(14.69%)>皮(3-33%)>其他(2.99%);叶凋落量中各组分所占比例依次为:常绿阔叶树种叶(78.70%)>落叶阔叶树种叶(12.37%)>针叶树种叶(8.92%),其中甜槠(Castanopsis eyrei)、木荷(Schima superba)、马尾松(Pinus massoniana)和短柄袍(Quercus serrata vat.brevipetiolata)4个优势种年凋落量合计占叶凋落总量的71.36%,它们的动态直接影响着凋落总量的变化格局.凋落量高峰发生在春季(4月)和秋冬季(10月末至12月初),其中总凋落量、叶凋落量动态呈双峰型,枝条凋落量和树皮凋落量动态为不规则峰型,其他凋落量动态为单峰型.冰雪灾害后总凋落量、叶凋落量、枝条凋落量显著减少(P<0.05),其中甜槠、杨梅叶蚊母树(Distylium myricoides)叶凋落最显著减少(P<0.05)、木荷叶凋落量减少达到边缘显著水平(P<0.1),这也反映了冰亏灾害期间森林群落的受损情况.其他凋落量(主要成分为虫粪)在4B份增加极其显著(P<0.01),说明灾后植物的枝叶出现了补偿性生长.     

蚯蚓纤溶酶的分离纯化及部分序列的测定

以新鲜蚯蚓为原料,经过保温抽提、乙醇沉淀、ＤＥＡＥ－ＳｅｐｈａｒｏｓｅＦａｓｔＦｌｏｗ离子交换层析、Ｌｙｓｉｎｅ－Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析以及ＳＤＳ－ＰＡＧＥ制备电泳等纯化步骤,得到一种纯度达９５％以上的蝗蚓纤溶酶,该酶具有强烈的溶解纤维蛋白折作用及蛋白酶活性,平板法测得其比活性为９００ＵＫ单位／毫克蛋白,ＴＡＭＥ法测得其比活性为２５０００单位／毫克蛋白,酶学性质研究表明其最适反应温度为６５     

睫状神经营养因子对听觉损伤的保护作用

本研究以耳廓反射、听觉脑干诱发电位、耳蜗生物电和耳蜗铺片组织学检测为指标,观察重组人睫状神经营养因子对豚鼠庆大霉素耳毒性的防治作用。实验结果表明,睫状神经营养因子能减轻庆大霉素对耳蜗及听神经的损害,具有保护听觉功能的作用。