Ras Behari’s guide to buying fish seed

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Regulación de las GTPasas de la familia Ras y la viabilidad de neuronas dopaminérgicas en respuesta a la señalización purinérgica

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Homologs of eukaryotic Ras superfamily proteins in prokaryotes and their novel phylogenetic correlation with their eukaryotic analogs

Ras superfamily proteins are key regulators in a wide variety of cellular processes. Previously, they were considered to be specific to eukaryotes, and MglA, a group of obviously different prokaryotic proteins, were recognized as their only prokaryotic an

Ras 超家族蛋白的起源演化研究

Ras 超家族蛋白是真核生物中普遍存在的一类小分子GTP 结合蛋白。它们 具有高度保守的GTP 结合结构域，根据序列结构和细胞功能被分为七个家族： Sar1、Arf、SRβ、Ran、Rab、Rho 和Ras。这些蛋白分别行使着真核生物特有的 细胞功能，诸如运输小泡的形成和转运（Sar1、Arf、Rab），胞质骨架的建成（Rho）， 细胞核-胞质运输及核膜重建（Ran）等，其起源演化和真核细胞的起源密切相关。 本文利用生物信息学手段和分子生物学实验调查研究了原核生物和原生生物中 Ras 超家族蛋白同源物的存在情况，并进行了分子系统分析，对Ras 超家族蛋白 的起源演化问题进行了较为深入、系统的探讨。获得了以下结果和结论： 1）通过原核生物基因组的搜索和序列结构分析，在一些真细菌中首次鉴定 出了高度相似于真核生物Ras 超家族蛋白的原核生物同源物，且实验证明它们的 基因具有表达活性；在原细菌中的产甲烷菌和热原体中也发现有序列分歧较大的 同源物。并在更多的真细菌种类中鉴定出了更多的前人已报道的另一种小分子 GTP 结合蛋白—MglA。序列比对分析表明MglA 蛋白具有自己独特的序列特征， 与真核生物的Ras 超家族蛋白序列差异较大。进一步的分子系统分析显示：真核 生物Ras 超家族蛋白的七个家族中，Ran、Rab、Rho 和Ras 等四个家族聚在一 起，上述我们所鉴定的真细菌的Ras 超家族蛋白同源物则紧聚在其外围；真核生 物的另三个家族（Sar1、Arf、SRβ）聚成另一枝，并接着与产甲烷原细菌的的同 源物及真细菌的MglA 蛋白聚在一起。这些结果表明：Ras 超家族蛋白不是前人 所认为的为真核生物所特有，实际上在一些原核生物中就已产生；真核生物Ras 超家族蛋白的祖先也不太可能是前人所认为的为真细菌的MglA；真核生物Ras 超家族蛋白的七个家族可能有两种不同的起源：Ran、Rab、Rho 和Ras 等可能 来源于蓝细菌或蛋白菌，或二者的共同祖先，而Sar1、Arf 和SRβ 可能来源于产 甲烷原细菌，这也可能反映了真核细胞“融合起源”的历史。 2）通过搜索一些较为低等的单细胞真核生物——原生生物基因组中Ras 超 家族蛋白，并结合一系列其他处在不同进化地位真核生物的Ras 超家族蛋白进行 分析，发现Sar1、Arf、Rab 和Ran 家族的蛋白在真核生物中普遍存在，而SRβ、 Rho 和Ras 家族蛋白在有些真核生物中未找到。根据各家族蛋白在真核生物中的分布情况推测在真核生物的最近共同祖先中存在的Ras 超家族蛋白可能有下列 两种情况：（1）最近的共同祖先已经具有了所有七个家族的蛋白，并且至少有 11 个成员：1 个Sar1、1 个SRβ、3 个Arf（Arf1、Arl1、Arl2）、3 个Rab（Rab1、 Rab6、Rab11）、1 个Ran、1 个Rho（Rac）和1 个Ras（RheB）。因而，部分真 核生物中缺少SRβ、Rho 和Ras 家族蛋白很可能是因基因丢失所致。植物中Ras 家族蛋白的缺少应该是由于在进化早期，其祖先绿藻丢失了单个Ras 家族蛋白基 因所致；（2）根据Cavalier-Smith 的真核生物划分为单鞭毛（变形虫类、真菌和 后生动物）和双鞭毛（藻类、植物和除变形虫外的原生动物）两大类的分类观点， 真核生物最近的共同祖先可能只具有除Ras 家族而外的六个家族的成员，而Ras 家族蛋白则是在此两大类群分化以后在单鞭毛类生物中才产生的，多数双鞭毛类 生物如原生动物、绿藻和植物中没有Ras 的情况应该是一种祖征，而个别双鞭毛 类生物如红藻具有的Ras 家族蛋白则很可能是从单鞭毛类生物那里水平基因转 移而来的。至于SRβ 和Rho 家族蛋白在部分物种中的缺少，则还是可能因为基 因丢失所致。此外，变形虫类生物中大量的Ras 超家族蛋白提示基因组的大小或 进化地位的高低并不是Ras 超家族蛋白成员多少的决定性因素，而细胞相应生理 活动的需求才是家族成员增多的关键。

Detection of K-ras exon 1 mutations by constant denaturant capillary electrophoresis

Among various mutation detection methods, constant denaturant capillary electrophoresis (CDCE) is one of the most common techniques for rapid identification of known or unknown mutations. In this report, a CDCE analysis method with homemade linear polyacrylamide (LPA) kit was developed on ABI 310 genetic analyzer, the effect and relationship of various denaturing factors in CDCE analysis were investigated and K-ras gene mutations of 31 coloerctal cancer patients were detected. Results indicate that, with the increase of chemical danaturant concentration, the optimum temperature was lowered, and when the concentration of urea (formamide) was higher than 7 M (40%), the homoduplex and heteroduplex of mutant samples were separated with difficulty. Detection results of K-ras gene in colorectal samples indicated that mutations were present in eight (26%) of 31 patients; most mutations were localized in codon 12, which is thought to be a critical step and plays an important role in human colorectal carcinogenesisas. Copyright (C) 2004 John Wiley Sons, Ltd.

Simultaneous genotyping of multiplex single nucleotide polymorphisms of the K-ras gene with a home-made kit

Accurate and fast genotyping of single nucleotide polymorphisms (SNPs) is important in the human genome project. Here an automated fluorescent method that can rapidly and accurately genotype multiplex known SNPs was developed by using a homemade kit, which has lower cost but higher resolution than commercial kit. With this method, oncogene K-ras was investigated, four known SNPs of K-ras gene exon 1 in 31 coloerctal cancer patients were detected. Results indicate that mutations were present in 8(26%) of 31 patients, and most mutations were localized in codon 12. The presence of these mutations is thought to be a critical step and plays an important role in human colorectal carcinogenesisas. (C) 2003 Elsevier B.V. All rights reserved.

Ligation of cell surface GRP78 with antibody directed against the COOH-terminal domain of GRP78 suppresses Ras/MAPK and PI 3-kinase/AKT signaling while promoting caspase activation in human prostate cancer cells.

We have previously shown that treatment of prostate cancer and melanoma cells expressing GRP78 on their cell surface with antibody directed against the COOH-terminal domain of GRP78 upregulates and activates p53 causing decreased cell proliferation and upregulated apoptosis. In this report, we demonstrate that treatment of 1-LN prostate cancer cells with this antibody decreases cell surface expression of GRP78, Akt(Thr308) and Akt(Ser473) kinase activities and reduces phosphorylation of FOXO, and GSK3beta. This treatment also suppresses activation of ERK1/2, p38 MAPK and MKK3/6; however, it upregulates MKK4 activity. JNK, as determined by its phosphorylation state, is subsequently activated, triggering apoptosis. Incubation of cells with antibody reduced levels of anti-apoptotic Bcl-2, while elevating pro-apoptotic BAD, BAX and BAK expression as well as cleaved caspases-3, -7, -8 and -9. Silencing GRP78 or p53 gene expression by RNAi prior to antibody treatment abrogated these effects. We conclude that antibody directed against the COOH-terminal domain of GRP78 may prove useful as a pan suppressor of proliferative/survival signaling in cancer cells expressing GRP78 on their cell surface.

Ras controls melanocyte expansion during zebrafish fin stripe regeneration.

Regenerative medicine for complex tissues like limbs will require the provision or activation of precursors for different cell types, in the correct number, and with the appropriate instructions. These strategies can be guided by what is learned from spectacular events of natural limb or fin regeneration in urodele amphibians and teleost fish. Following zebrafish fin amputation, melanocyte stripes faithfully regenerate in tandem with complex fin structures. Distinct populations of melanocyte precursors emerge and differentiate to pigment regenerating fins, yet the regulation of their proliferation and patterning is incompletely understood. Here, we found that transgenic increases in active Ras dose-dependently hyperpigmented regenerating zebrafish fins. Lineage tracing and marker analysis indicated that increases in active Ras stimulated the in situ amplification of undifferentiated melanocyte precursors expressing mitfa and kita. Active Ras also hyperpigmented early fin regenerates of kita mutants, which are normally devoid of primary regeneration melanocytes, suppressing defects in precursor function and survival. By contrast, this protocol had no noticeable impact on pigmentation by secondary regulatory melanocyte precursors in late-stage kita regenerates. Our results provide evidence that Ras activity levels control the repopulation and expansion of adult melanocyte precursors after tissue loss, enabling the recovery of patterned melanocyte stripes during zebrafish appendage regeneration.

Tyrosine phosphorylated SOCS-3 inhibits STAT activation but binds p120-RasGAP and activates Ras

Suppressors of cytokine signalling (SOCS, also known as CIS and SSI) are encoded by immediate early genes that act in a feedback loop to inhibit cytokine responses and activation of 'signal transducer and activator of transcription' (STAT). Here we show that SOCS-3 is strongly tyrosine-phosphorylated in response to many growth factors, including interleukin-2 (IL-2), erythropoietin (EPO), epidermal growth factor (EGF) and platelet-derived growth factor (PDGF). The principal phosphorylation sites on SOCS-3 are residues 204 and 221 at the carboxy terminus, and upon phosphorylation tyrosine 221 interacts with the Ras inhibitor p120 RasGAP. After IL-2 stimulation, phosphorylated SOCS-3 strongly inhibits STAT5 activation but, by binding to RasGAP, maintains activation of extracellular-signal-regulated kinase (ERK). A tyrosine mutant of SOCS-3 still blocks STAT phosphorylation, but also strongly inhibits IL-2-dependent activation of ERK and cell proliferation. Moreover, it also inhibits EPO- and PDGF-induced proliferation and ERK activation. Therefore, although SOCS proteins inhibit growth-factor responses, tyrosine phosphorylation of SOCS-3 can ensure cell survival and proliferation through the Ras pathway.