An affinity labeling of ras p21 protein and its use in the identification of ras p21 in cellular and tissue extracts

We have carried out photoaffinity labeling of the ras p21 protein, a ras oncogene product, with [alpha-32P]GTP. Based on our studies, a sensitive, rapid, and specific assay for the detection of multiple forms of ras p21 has been developed. The specificity of this protocol is shown by (a) sensitivity of affinity labeling of ras p21 to known inhibitors of GTP binding and (b) immunoprecipitation of affinity labeled protein with anti-ras p21 serum. Detection and semiquantitation of ras p21 by this method is accomplished in less than 24 h and requires as little as 100,000 cells or about 5 mg of tissue sample from skin tumor, liver, and mammary tumor tissues. Furthermore, using this approach, we were able to detect the selective loss of one species of ras p21 in transplanted Morris hepatoma cells.     

Concomitant K- and N-ras gene point mutations in clonal murine lymphoma.

We have surveyed a panel of induced murine lymphomas for c-ras gene mutations. The K-ras gene seems to be preferentially activated in our system, and there are at least two examples of concomitant K- and N-ras gene mutations in the same tumor. This indicates that in some cases additional ras mutations may contribute to tumorigenesis and is evidence for a role of ras activation in tumor progression.     

Recombinant expression of different mutant K-ras gene in pancreatic cancer Bxpc-3 cells and its effects on chemotherapy sensitivity

K-ras is a member of ras gene family which is involved in cell survival,proliferation and differentiation.When a mutation occurs in ras gene,the activation of Ras proteins may be prolonged to induce oncogenesis.However,the relationship between K-ras mutation and clinical outcomes in pancreatic cancer patients treated with chemotherapy agents is still under debate.In this study,we constructed five pAcGFP1-C3 plasmids for different types of K-ras gene(WT,G12V,G12R,G12D,and G13D)and stably transfected human pancreatic cancer Bxpc-3 cells with these genes.The wild type and mutant clones showed a comparable growth and expression of K-Ras-GFP fusion protein.The expression of some K-ras mutations resulted in a reduced sensitivity to gefitinib,5-FU,docetaxel and gemcitabine,while showed no effects on erlotinib or cisplatin.Moreover,compared with the wild type clone,K-Ras downstream signals(phospho-Akt and/or phospho-Erk)were increased in K-ras mutant clones.Interestingly,different types of K-ras mutation had non-identical K-Ras downstream signal activities and drug responses.Our results are the first to reveal the relationship between different K-ras mutation and drug sensitivities of these anti-cancer drugs in pancreatic cancer cells in vitro.     

Activated Ras prevents downregulation of Bcl-X(L) triggered by detachment from the extracellular matrix. A mechanism of Ras-induced resistance to anoikis in intestinal epithelial cells

Detachment of epithelial cells from the extracellular matrix (ECM) results in a form of apoptosis often referred to as anoikis. Transformation of intestinal epithelial cells by oncogenic ras leads to resistance to anoikis, and this resistance is required for the full manifestation of the malignant phenotype. Previously, we demonstrated that ras-induced inhibition of anoikis in intestinal epithelial cells results, in part, from the ras-induced constitutive downregulation of Bak, a pro-apoptotic member of the Bcl-2 family. Since exogenous Bak could only partially restore susceptibility to anoikis in the ras-transformed cells, the existence of at least another component of the apoptotic machinery mediating the effect of activated ras on anoikis was suggested. Indeed, here we show that, in nonmalignant rat and human intestinal epithelial cells, detachment from the ECM or disruption of the cytoskeleton results in a significant downregulation of the antiapoptotic effector Bcl-X(L), and that activated H- or K-ras oncogenes completely abrogate this downregulation. In addition, we found that enforced downregulation of Bcl-X(L) in the ras-transformed cells promotes anoikis and significantly inhibits tumorigenicity, indicating that disruption of the adhesion-dependent regulation of Bcl-X(L) is an essential part of the molecular changes associated with transformation by ras. While the ras-induced downregulation of Bak could be reversed by pharmacological inhibition of phosphatidylinositol 3 kinase (PI 3-kinase), the effect of ras on Bcl-X(L) was PI 3-kinase- and mitogen-activated protein kinase (MAP kinase)-independent. We conclude that ras-induced resistance to anoikis in intestinal epithelial cells is mediated by at least two distinct mechanisms: one that triggers downregulation of Bak and another that stabilizes Bcl-X(L) expression in the absence of the ECM.     

While K-ras is essential for mouse development, expression of the K-ras 4A splice variant is dispensable

In mammals, the three classical ras genes encode four highly homologous proteins, N-Ras, H-Ras, and the isoforms K-Ras 4A and 4B. Previous studies have shown that K-ras is essential for mouse development and that while K-ras 4A and 4B are expressed during development, K-ras 4A expression is regulated temporally and spatially and occurs in adult kidney, intestine, stomach, and liver. In the present study, the pattern of K-ras 4A expression was examined in a wide range of wild-type adult mouse tissues, and gene targeting was used to generate K-ras 4A-deficient mice to examine its role in development. It was found that K-ras 4A is also expressed in uterus, lung, pancreas, salivary glands, seminal vesicles, bone marrow cells, and cecum, where it was the major K-Ras isoform expressed. Mating between K-ras(tmDelta4A/+) mice produced viable K-ras(tmDelta4A/tmDelta4A) offspring with the expected Mendelian ratios of inheritance, and these mice expressed the K-ras 4B splice variant only. K-ras(tmDelta4A/tmDelta4A) mice were fertile and showed no histopathological abnormalities on inbred (129/Ola) or crossbred (129/Ola x C57BL/6) genetic backgrounds. The results demonstrate that K-Ras 4A, like H- and N-Ras, is dispensable for normal mouse development, at least in the presence of functional K-Ras 4B.     

Neutron radiation can activate K-ras via a point mutation in codon 146 and induces a different spectrum of ras mutations than does gamma radiation.

Neutron radiation is known to produce tumors in animals and cause cell transformation. We have developed a protocol to efficiently induce thymic lymphomas in RF/J mice by a single acute dose of neutron irradiation. Activated ras genes were detected in 17% (4 of 24) of the tumors analyzed. One of the tumors contained a K-ras gene activated by a point mutation in codon 146. Activating ras mutations at position 146 have not been previously detected in any known human or animal tumors. The spectrum of ras mutations detected in neutron radiation-induced thymic lymphomas was different from that seen in thymic lymphomas induced by gamma radiation in the same strain of mice. These results may have important implications for the mechanisms by which different types of radiation damage DNA.     

新型冠状病毒RBD蛋白原核表达及多克隆抗体的制备

为原核表达严重急性呼吸综合征冠状病毒2（简称新型冠状病毒,severe acute respiratory syndrome-coronavirus 2,SARS-CoV-2）S蛋白受体结合域（receptor binding domain, RBD）并制备多克隆抗体,利用基因克隆技术将RBD基因连接到原核表达载体pGEX-6p-1和pET-32a(+)上,电转化至大肠杆菌XL1-Blue感受态细胞,利用优化后的表达条件大量表达重组蛋白,经亲和层析纯化后通过SDS-PAGE检测蛋白的表达情况。利用GST-RBD融合蛋白作为免疫抗原免疫小鼠制备多克隆抗体,ELISA和Western blot分析抗血清的效价和特异性。PCR鉴定和序列测定结果显示,成功构建了重组载体pGEX-RBD和pET-RBD,在大肠杆菌中实现了GST-RBD和RBD-His融合蛋白的可溶性高效表达。研究获得的多克隆抗体的滴度达到约1∶3 000,并具有良好的结合特异性。原核表达的可溶性新型冠状病毒RBD重组蛋白具有良好的免疫原性,为后续制备基因工程抗体奠定了实验基础。     

The role of signal-transducing events in the proliferative response of cells to a mitogenic viral K-ras protein

Activated oncogenic ras proteins are powerful mitogenic agents which by themselves can initiate and maintain the proliferation of quiescent cells in the absence of any exogenous growth factors. In an attempt to understand how ras proteins induce proliferation we examined the early events in the G0 to G1 transition caused by the activation of a thermolabile K-ras protein in quiescent, serum-starved tsKSV-transformed NRK cells. We show that ras reactivation, in the absence of exogenous growth factors, triggered a rapid surge in free cytosolic Ca2+ and diacylglycerol production, which led to a transient increase in membrane-associated protein kinase C (PKC) activity which was necessary for G1 transit. Unlike TPA-stimulated PKC activity, the ras-induced increase in PKC was readily extracted from membranes by EGTA. These signal transducing events occurred despite the fact that ras activation did not induce the tyrosine phosphorylation of any known surface receptor. The results indicate that the K-ras protein triggers the G0 to G1 transition by an intracellular mechanism and not indirectly via autocrine stimulation.     

Characterization of a guanine nucleotide dissociation stimulator for a ras-related GTPase.

ras-related GTPases participate in signaling for a variety of cellular processes. The GTPases cycle between a GTP-bound active state and a GDP-bound inactive state. This cycling is partially controlled by guanine nucleotide dissociation stimulators (GDS, also known as exchange factors). We report on the molecular cloning of cDNAs encoding a new mammalian GDS protein, using sequences derived from the yeast ras GDS proteins as probes. The encoded protein stimulates the dissociation of guanine nucleotides from the ras-related ralA and ralB GTPases at a rate at least 30-fold faster than the intrinsic nucleotide dissociation rate. This new GDS, ralGDS, is at least 20-fold more active on the ralA and ralB GTPases than on any other GTPase tested, including other members of the ras family (H-ras, N-ras, K-ras, R-ras, rap1a and rap2), members of the rho family (rhoA, rhoB and CDC42-Hs) and members of the rab family (rab3a and ypt1). While the ralGDS protein is phosphorylated on serine residues, we find no evidence that phosphorylation affects the activity of insect cell-expressed ralGDS towards the ralA or ralB GTPase. The 3600 nucleotide ralGDS mRNA and the 115 kDa protein were found in all tissues and cell lines examined.     

Deregulation of hamster fibroblast proliferation by mutated ras oncogenes is not mediated by constitutive activation of phosphoinositide-specific phospholipase C

Stable expression of high levels of activated forms of Haras (T24) or v-Ki-ras by transfection of Chinese hamster lung fibroblasts (CCL39) yielded cells highly tumorigenic in nude mice. Two classes of transformed cells were distinguished, one with moderate p21 expression (10-fold increased) had retained growth factor dependency, the second with higher level of p21 (greater than 50-fold) appeared autonomous for growth. Neither class of transformants expressing Ki-ras or Ha-ras displayed a significant basal activity of polyphosphoinositide-specific phospholipase C, measured either in serum-starved cells or during exponential growth in the presence of growth factors of the tyrosine kinase family (EGF, FGF, insulin). In the growth-factor-dependent class of T24-Ha-ras-transfected cells (clone 39THaB), phospholipase C could be stimulated normally by serum, thrombin and AlF-4. In the more growth autonomous class (clones 39THaC and 39Ki9), release of inositol phosphates after stimulation with thrombin or serum was drastically reduced. This desensitization, apparently at the receptor level since the response to AlF-4 persisted, is, however, not specific to ras expression. We observed it to the same degree in polyoma virus-transformed CCL39 cells. Finally, expression of mutated forms of p21 ras did not abrogate the sensitivity of phospholipase C activation to pertussis toxin. We conclude that the transforming potential of activated forms of p21ras does not result from persistent activation of phospholipase C and that ras GTP-binding proteins cannot substitute for Gp.     

原核表达MurA蛋白并制备其多克隆抗体用于免疫磁珠快速检测单增李斯特菌

【目的】制备MurA多抗,结合免疫磁珠与选择平板进行单增李斯特菌的快速检测,建立单增李斯特菌的免疫磁珠快速检测方法。【方法】构建MurA的原核表达载体,转化大肠杆菌进行优化表达。镍柱纯化表达产物,质谱鉴定重组蛋白,再免疫小鼠,制备其多克隆抗体。用所获多抗制备免疫磁珠,建立单增李斯特菌免疫磁珠-选择性培养基检测方法,并对人工污染牛奶样品进行检测。【结果】在大肠杆菌中高效表达了分子量约为72 kD的可溶性融合蛋白,质谱鉴定其为MurA蛋白;免疫小鼠获得的抗血清效价达1:10 000,与伤寒沙门氏菌、副溶血弧菌、大肠杆菌及属内其它病原菌均无交叉;所建立的免疫磁珠-选择性培养基检测法可检出浓度为103 CFU/mL及以上的单增李斯特菌,仅与英诺克李斯特菌存在一定交叉反应;牛奶样品单次仅需9 h增菌就能被检出,较常规增菌时间缩短39 h;检测限为0.4 CFU/mL。【结论】表达并纯化得到高纯度的单增李斯特菌MurA蛋白,制备的鼠源多克隆抗体亲和力高,特异性好;建立了快速检测单增李斯特菌的免疫磁珠联合选择性培养基法,在灵敏度不变的情况下,实现24 h内成功对牛奶样品的检测,较国标法减少42 h以上。     

K-ras codon 12 mutations may be detected in serum of patients suffering from adeno- and large cell lung carcinoma. A preliminary report

We investigated the presence of K-ras mutations in the serum of 40 patients with respectable stages of adeno- and large cell lung carcinomas. Mutations in codon 12 of the K-ras gene were examined by enriched PCR method in DNA extracts from surgical specimens and serum samples. K-ras mutations were detected in 20 (51%) of 39 analyzed tumours, and in 7 (35%) of 20 patients with K-ras gene mutation positive tumours, a mutation was found in the serum DNA. We also found K-ras mutation in two (10.5%) of 19 serum samples obtained from patients whose tumours were not found to harbor mutation and in one serum sample from patient without tumour sample available for investigation. All of the 14 control healthy persons were negative for serum DNA K-ras mutation assay. Although our results are preliminary they show that K-ras mutation may be detected in serum of patients suffering from adeno- and large cell lung carcinomas and confirm the suggestion that at least a part of a free-cell extracellular blood DNA in cancer patients has neoplastic origin and may become a noninvasive target for genetic investigations of lung cancer patients.     

Identification and characterization of an intracellular lectin, calnexin, from Aspergillus oryzae using N-glycan-conjugated beads

Recently, asparagine-linked oligosaccharides (N-glycans) have been found to play a pivotal role in glycoprotein quality control in the endoplasmic reticulum (ER). In order to screen proteins interacting with N-glycans, we developed affinity chromatography by conjugating synthetic N-glycans on sepharose beads. Using the affinity beads with the dodecasaccharide Glc(1)Man(9)GlcNAc(2), one structure of the N-glycans, a 75-kDa protein, was isolated from the membranous fraction including the ER in Aspergillus oryzae. By LC-MS/MS analysis using the A. oryzae genome database, the protein was identified as one (AO090009000313) sharing similarities with calnexin. Further affinity chromatographic experiments suggested that the protein specifically bound to Glc(1)Man(9)GlcNAc(2), similarly to mammalian calnexins. We designated the gene AoclxA and expressed it as a fusion gene with egfp, revealing the ER localization of the AoClxA protein. Our results suggest that our affinity chromatography with synthetic N-glycans might help in biological analysis of glycoprotein quality control in the ER.     

Glutathione-S-transferase-green fluorescent protein fusion protein reveals slow dissociation from high site density beads and measures free GSH.

BACKGROUND: Glutathione, a ubiquitous tripeptide, is an important cellular constituent, and measurement of reduced and oxidized glutathione is a measure of the redox state of cells. Glutathione-S-transferase (GST) fusion proteins bind naturally to beads derivatized with glutathione, and elution of such bead-bound fusion proteins with buffer containing millimolar glutathione is a commonly used method of protein purification. Many protein-protein interactions have been established by using GST fusion proteins and measuring binding of fusion protein binding partners by GST pulldown assays, usually monitored by Western blot methodology. METHODS: Dextran beads suitable for flow cytometry were derivatized with glutathione. A fusion protein of GST and green fluorescent protein was used to define kinetic and equilibrium binding characteristics of GST fusion proteins to glutathione beads. Free glutathione competes with this binding, and this competition was used to measure free glutathione concentration. RESULTS: A 10 microl assay can measure 5 microl of 20 microM glutathione (100 pmol glutathione) in 2 h by flow cytometry. This concentration is two orders of magnitude lower than cellular glutathione concentrations, and three orders of magnitude lower than affinity chromatography eluates. One important result is that by generating high site density, the GST fusion proteins can be constrained to the surface of one bead without hopping to the next bead in multiplex assays. CONCLUSIONS: Glutathione in cellular lysates and GST-fusion protein affinity chromatography eluates can be measured by flow cytometry. Many interactions between GST fusion proteins and their fluorescent binding partners should be quantifiable by flow cytometry. Although a system may have the disadvantage that it has a low affinity and a correspondingly quick off-rate in solution, it may remain on beads if the site density can be increased to offer a slow apparent off rate.     

A CAAX or a CAAL motif and a second signal are sufficient for plasma membrane targeting of ras proteins.

Mutational analysis of p21ras has shown that plasma membrane targeting requires the combination of a CAAX motif with a polybasic domain of six lysine residues or a nearby palmitoylation site. However, it is not known from these studies whether these signals alone target p21ras to the plasma membrane. We now show that these C-terminal sequences are sufficient to target a heterologous cytosolic protein to the plasma membrane. Interestingly, the key feature of the p21K-ras(B) polybasic domain appears to be a positive charge, since a polyarginine domain can function as a plasma membrane targeting motif in conjunction with the CAAX box and p21K-ras(B) with the polylysine domain replaced by arginines is biologically active. Since some ras-related proteins are modified by geranylgeranyl rather than farnesyl we have investigated whether modification of p21ras with geranylgeranyl affects its subcellular localization. Geranylgeranyl can substitute for farnesyl in combining with a polybasic domain to target p21K-ras(B) to the plasma membrane, but such geranylgeranylated proteins are more tightly bound to the membrane. This increased avidity of binding is presumably due to the extra length of the geranylgeranyl alkyl chain.