Characterization of Ser338 phosphorylation for Raf-1 activation

Raf kinases are essential for regulating cell proliferation, survival, and tumorigenesis. However, the mechanisms by which Raf is activated are still incompletely understood. Phosphorylation plays a critical role in Raf activation in response to mitogens. The present study characterizes phosphorylation of Ser338, a crucial event for Raf-1 activation. Here we report that mutation of Lys375 to Met diminishes phosphorylation of Ser338 on both wild type Raf-1 in cells treated with epidermal growth factor (EGF) or 12-O-tetradecanoylphorbol-13-acetate (TPA) and a constitutively active mutant in which Tyr340/Tyr341 are replaced by 2 aspartic acids, a conserved substitution present in natural B-Raf. The loss of Ser338 phosphorylation in these Raf mutants is not engendered by a mutation-induced conformational change, inasmuch as mutation of another site (Ser471 to Ala) in the activation segment also abolishes Ser338 phosphorylation, whereas both the kinase-dead mutants of Raf-1 are phosphorylated well by active Pak1. Furthermore, our data demonstrate that EGF-stimulated phosphorylation of Ser338 is inhibited by Sorafenib, a Raf kinase inhibitor, but not by the MEK inhibitor U0126. Interestingly, a kinase-dead mutation and Sorafenib also markedly reduce phosphorylation of Ser445 on B-Raf, a site equivalent to Raf-1 Ser338. Finally, our data reveal that Ser338 is phosphorylated on inactive Raf-1 by an active mutant of Raf-1 when they are dimerized in cells and that artificial dimerization of Raf-1 causes Ser338 phosphorylation, accompanied by activation of ERK1/2. Altogether, our data suggest that Ser338 on Raf-1 is autophosphorylated in response to mitogens.     

Population genetic structure of the melon fly, <Emphasis Type="Italic">Bactrocera cucurbitae</Emphasis> (Diptera: Tephritidae), from China and Southeast Asia

The melon fly, Bactrocera cucurbitae Coquillett, is a species of fruit flies of significant agricultural interest. Of supposed Indian origin, the melon fly is now widely distributed throughout South East Asia up to China, while it has been recently eradicated from Japan. The population structure of seven geographic populations from coastal China, as well as samples from other regions of South East Asia and Japan, including lab colonies, have been studied using a 782 bp fragment of mitochondrial cytochrome oxidase I (COI) gene sequence. The observed genetic diversity was exceedingly low, considering the geographic scale of the sampling, and one single haplotype was found to be predominant from Sri Lanka to China. We confirm that Bactrocera cucurbitae exists in South East Asia as a single phyletic lineage, that Chinese populations are genetically uniform, and that no apparent genetic differentiation exists between these and three available Japanese melon fly sequences.     

Arginine therapy of transgenic-knockout sickle mice improves microvascular function by reducing non-nitric oxide vasodilators, hemolysis, and oxidative stress

In sickle cell disease, nitric oxide (NO) depletion by cell-free plasma hemoglobin and/or oxygen radicals is associated with arginine deficiency, impaired NO bioavailability, and chronic oxidative stress. In transgenic-knockout sickle (BERK) mice that express exclusively human alpha- and beta(S)-globins, reduced NO bioavailability is associated with induction of non-NO vasodilator enzyme, cyclooxygenase (COX)-2, and impaired NO-mediated vascular reactivity. We hypothesized that enhanced NO bioavailability in sickle mice will abate activity of non-NO vasodilators, improve vascular reactivity, decrease hemolysis, and reduce oxidative stress. Arginine treatment of BERK mice (5% arginine in mouse chow for 15 days) significantly reduced expression of non-NO vasodilators COX-2 and heme oxygenase-1. The decreased COX-2 expression resulted in reduced prostaglandin E(2) (PGE(2)) levels. The reduced expression of non-NO vasodilators was associated with significantly decreased arteriolar dilation and markedly improved NO-mediated vascular reactivity. Arginine markedly decreased hemolysis and oxidative stress and enhanced NO bioavailability. Importantly, arteriolar diameter response to a NO donor (sodium nitroprusside) was strongly correlated with hemolytic rate (and nitrotyrosine formation), suggesting that the improved microvascular function was a response to reduced hemolysis. These results provide a strong rationale for therapeutic use of arginine in sickle cell disease and other hemolytic diseases.     

The N-terminus of PrP is responsible for interacting with tubulin and fCJD related PrP mutants possess stronger inhibitive effect on microtubule assembly in vitro

Microtubule dynamics is essential for many vital cellular processes such as in intracellular transport, metabolism, and cell division. Some evidences demonstrate that PrP may associate with microtubular cytoskeleton and its major component, tubulin. In the present study, the molecular interaction between PrP and tubulin was confirmed using pull-down assays, immunoprecipitation and ELISA. The interacting regions within PrP with tubulin were mapped in the N-terminus of PrP spanning residues 23-50 and 51-91. PrP octapeptide repeats are critical for the binding activity with tubulin, that the binding activity of PrP with tubulin became stronger along with the number of the octapeptide repeats increased. Microtubule assembly assays, sedimental tests and transmission electron microscopy demonstrated that the full-length PrP (aa 23-231) obviously inhibited the microtubule polymerization processes in vitro, whereas the N- (aa 23-91) and C- (aa 91-231) terminal peptides of PrP did not affect microtubule polymerization. Moreover, the familial Cruetzfeldt Jacob disease (fCJD) related PrP mutants with inserted or deleted octapeptide repeats showed much stronger inhibitive capacities on the microtubule dynamics in vitro than wild-type PrP. Our data highlight a potential role of PrP in regulating the microtubule dynamics in neurons.     

海南岛霸王岭热带山地雨林群落结构及树种多样性特征的研究

 以海南岛霸王岭自然保护区1 hm2老龄原始林样地的调查材料为基础,分析了热带山地雨林群落的组成、高度结构、径级结构及有关的树种多样性特征。结果表明：霸王岭热带山地雨林树种较丰富,物种多样性指数较高。树种数和树木的密度都随高度级、径级的增加而呈负指数或负幂函数递减;热带山地雨林不同高度级、不同径级和不同小样方斑块内的树种数都与树木密度呈显著的正相关关系。热带山地雨林经过自然的演替达到老龄顶极群落后,最后进入主林层的只是少部分树种的少数个体。     

警惕外来危险害虫褐纹甘蔗象入侵

褐纹甘蔗象Rhabdosceluslineaticollis (Heller)在菲律宾、日本和台湾 ,严重危害椰子等棕榈科植物和甘蔗。本文对褐纹甘蔗象的分类地位、形态特征、分布、寄主、扩散与危害、生物学特性等进行简要介绍     

人脯氨肽酶活性中心结构的计算机模拟

氨酰基脯氨酸二肽酶 (脯氨肽酶 )为广泛分布于生物界的细胞内二肽水解酶 .它特异性地水解以脯氨酸或羟脯氨酸为羧基端的二肽 (X Pro) ,而且只对反式肽键有催化活性 .此酶与脯氨酸代谢、胶原蛋白合成及细胞生长有密切关系 .文献报道 ,从Alteromonas细菌中提取的脯氨肽酶有水解梭曼的活性 ,其有机磷酸酐水解酶也有脯氨肽酶活性 .用重组基因表达的人肝脯氨肽酶也同时具有脯氨肽酶活性和水解梭曼的活性 .研究脯氨肽酶活性中心的结构具有重要理论意义和潜在实用价值 .但目前尚无人脯氨肽酶晶体结构的报道 .本文采用蛋白质结构模式识别 (threading)方法对脯氨肽酶的高级结构进行模拟 ,以大肠杆菌甲硫氨酸氨肽酶 (1MAT)为模板 ,模建了人脯氨肽酶C端结构域的空间结构 .通过对模建结构的 3D评估及电荷分布分析 ,对人脯氨肽酶活力中心结构进行了预测 .模建的人脯氨肽酶活性中心位于C端结构域 ,为 6条β折叠围成的一个疏水性口袋 ,外面被 5条α螺旋及一些loop包围 ,活力中心位于疏水结构中央 ,其中有 5个保守氨基酸 ,形成 1个较强的负电荷区 ,周围有 3个较弱的正电荷区域 .实验还发现 ,虽然Mn2 + 或Co2 + 对酶的活性极其重要 ,但对酶蛋白结构的贡献很小 .提示它们可能是在催化反应的电荷转移过程中发挥着重要作用     

Schlemm’s Canal and Trabecular Meshwork in Eyes with Primary Open Angle Glaucoma: A Comparative Study Using High-Frequency Ultrasound Biomicroscopy

We investigated in vivo changes in Schlemm’s canal and the trabecular meshwork in eyes with primary open angle glaucoma (POAG). Relationships between Schlemm’s canal diameter, trabecular meshwork thickness, and intraocular pressure (IOP) were examined. Forty POAG patients and 40 normal individuals underwent 80-MHz Ultrasound Biomicroscopy examinations. The Schlemm’s canal and trabecular meshwork were imaged in superior, inferior, nasal and temporal regions. Normal individuals had an observable Schlemm’s canal in 80.3% of sections, a meridional canal diameter of 233.0±34.5 μm, a coronal diameter of 44.5±12.6 μm and a trabecular meshwork thickness of 103.9±11.1 μm, in POAG patients, Schlemm’s canal was observable in 53.1% of sections, a meridional canal diameter of 195.6±31.3 μm, a coronal diameter of 35.7±8.0 μm, and a trabecular meshwork thickness of 88.3±13.2 μm, which significantly differed from normal (both p <0.001). Coronal canal diameter (r = -0.623, p < 0.001) and trabecular meshwork thickness (r = -0.663, p < 0.001) were negatively correlated with IOP, but meridional canal diameter was not (r = -0.160, p = 0.156). Schlemm’s canal was observable in 50.5% and 56.6% of POAG patients with normal (<21 mmHg) and elevated (>21 mmHg) IOP, respectively (χ = 1.159, p = 0.282). Coronal canal diameter was significantly lower in the elevated IOP group (32.6±4.9 μm) than in the normal IOP group (35.7±8.0 μm, p < 0.001). This was also true of trabecular meshwork thickness (81.9±10.0 μm vs. 97.1±12.0 μm, p < 0.001). In conclusion, eyes with POAG had fewer sections with an observable Schlemm’s canal. Canal diameter and trabecular meshwork thickness were also lower than normal in POAG patients. Schlemm’s canal coronal diameter and trabecular meshwork thickness were negatively correlated with IOP.     

Murine recombinant IL-4 is a bifunctional regulator of macrophage growth induced by colony-stimulating factors.

Murine peritoneal exudate macrophage (PEM) coexpress receptors for both granulocyte-macrophage CSF (GM-CSF) and macrophage CSF (M-CSF) and can be induced by both factors, either alone or in combination, to undergo extensive proliferation in vitro. In this study the effect of murine rIL-4 (MurIL-4) on the proliferation of PEM was examined. MurIL-4 alone did not support macrophage proliferation but prolonged their survival in vitro. When MurIL-4 was combined with human (Hu)rM-CSF, it enhanced the proliferative response of PEM to rHuM-CSF in a dose-dependent manner, reaching a maximum at approximately 10 ng/ml. Contrarily, MurIL-4 suppressed the proliferative response of PEM to MurGM-CSF. Receptor binding assays using radiolabeled ligands showed that MurIL-4 selectively enhanced the expression of M-CSF receptors; suggesting that at least part of the synergistic effect of MurIL-4 is mediated at the receptor level. Of relevance to this effect is the finding that MurIL-4 greatly promoted the responsiveness of PEM to low concentrations of HurM-CSF. Unlike M-CSF receptors, however, MurIL-4 treatment failed to modulate the levels of GM-CSF receptors in PEM. The proliferative responses of PEM to both MurGM-CSF and HurM-CSF could be inhibited by MurIFN-gamma with similar sensitivity. This inhibitory effect of MurIFN-gamma was partially neutralized by MurIL-4 in cultures containing HurM-CSF but not those containing MurGM-CSF. This study demonstrates that IL-4 is involved directly in the regulation of macrophage production by modulating their responsiveness to various cytokines.     

Elaborate differences between trees and understory plants in the deployment of fine roots

Plant and Soil - Leaf-litter decomposition rate (k L ) regulates nutrient dynamics and is affected at microsite level by species traits, soil biota and microclimate conditions. Fallen fruits form...