High-throughput Sequencing Analysis of Differentially Expressed microRNAs Associated With SREBP1 in Differentiated Thyroid Carcinoma.

BACKGROUND/AIM: MicroRNAs (miRNAs) interact with mRNAs and play important roles in progression and prognosis in multiple cancers. Sterol regulatory element-binding protein 1 (SREBP1) is an important lipid metabolism regulatory gene. The aim of the present study was to analyze the profiles of miRNAs that are associated with SREBP1 expression in differentiated thyroid carcinoma (DTC). MATERIALS AND METHODS: In the present study, a high-throughput small RNA sequencing (miRNA-Seq) method was used to investigate differences in miRNA profiling with versus without interference with SREBP1 expression via small interfering RNA. Real-time qPCR (qRT-PCR) was performed to confirm the results. RESULTS: A total of 1,393 conserved and 84 novel miRNAs were successfully discovered. In two separate batches, a total of 27 differentially expressed miRNAs (11 up-regulated and 16 down-regulated) were observed in BCPAP cells after SREBF1 interference with two distinct siRNA fragments, as compared to the control siRNA treatment. Hsa-miR-941, hsa-miR-27a-5p, hsa-miR-29a-3p, hsa-miR-100-5p, and hsa-miR-21-3p were selected for validation using qRT-PCR. The qRT-PCR results were consistent with the sequencing data. Gene Ontology enrichment showed that the predicted targets of these miRNAs were mainly involved in the regulation of system development, metabolism and protein binding cellular processes, and metabolic processes. Kyoto Encyclopedia of Genes and Genomes pathways analysis showed that the predicted target genes were involved in several signaling pathways, including the Ras, MAPK, insulin, thyroid hormone, and metabolic pathway signaling pathways. CONCLUSION: Differentially expressed miRNAs and their target genes may play an important role in the progression and prognosis of DTC that is associated with SREBP1 expression.

FOXP3 and CEACAM6 expression and T cell infiltration in the occurrence and development of colon cancer.

The transcription factor forkhead box P3 (FOXP3) is involved in immune cell regulation, and carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is an adhesion molecule of the immunoglobulin superfamily. These two genes are associated with cancer progression. In the current study, colon tissue specimens from 78 cases of colon cancer (including 40 of stage I-II and 38 of stage III-IV), 30 cases of colonic adenoma and 12 healthy controls were collected from the First Affiliated Hospital of Soochow University between January 2010 and December 2011. The expression of cluster of differentiation (CD) 3, CD4, CD8, CD45RO, CEACAM6 and FOXP3 in colon tissues was examined by immunohistochemical analysis. In addition, a reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay, based on SYBR Green I, was used to detect CD3, CD4, CD8, CD45RO, CEACAM6 and FOXP3 mRNA levels in the paraffin block specimens. CD3+, CD8+ and CD45RO+ T cell infiltrations in colonic adenoma were significantly higher than in normal colonic mucosa (P<0.001, P=0.001 and P<0.001, respectively). However, CD3+, CD8+ and CD45RO+ lymphocytes in stage III-IV colon cancer tissues were lower than in normal control tissues (P=0.015, P=0.002 and P=0.041, respectively); consistently, CD3+, CD4+, CD8+ and CD45RO+ lymphocytes in stage III-IV tissues were even more markedly lower compared with adenoma (P=0.001, P<0.001, P<0.001 and P<0.001, respectively). Similarly, CD3+, CD8+ and CD45RO+ T cell infiltration was lower in stage I-II cancer tissues compared with adenoma (P=0.001, P<0.001 and P<0.001). CD3+, CD4+, CD8+ and CD45RO+ T cell infiltrations were also significantly higher in stage I-II compared with stage III-IV cancer tissues (P<0.001, P=0.045, P<0.001 and P<0.001, respectively). CEACAM6 was found to gradually increase from normal colon tissue to adenoma and cancer tissue. FOXP3 was expressed more highly in stage I-II compared with normal tissues (P=0.014), and was even higher in stage III-IV (P<0.001). These results were verified using RT-qPCR, which yielded almost identical results. In summary, the current study demonstrates that FOXP3, CEACAM6 and T cell infiltration are significantly associated with the occurrence and progression of colon cancer, and that immune reactions vary between different stages of colon cancer development.

Intercellular adhesion molecule 1 is a sensitive and diagnostically useful immunohistochemical marker of papillary thyroid cancer (PTC) and of PTC-like nuclear alterations in Hashimoto's thyroiditis.

Intercellular adhesion molecule 1 (ICAM-1) is important in the progression of inflammatory responses. Recently, increased levels of ICAM-1 have been reported in a number of types of malignancy. The present study aimed to investigate ICAM-1 expression in papillary thyroid cancer (PTC) and in Hashimoto's thyroiditis (HT) with PTC-like nuclear alterations, and to assess the predictive value of ICAM-1 in thyroid lesions. ICAM-1 expression was retrospectively investigated in 132 consecutive cases of PTC, 72 cases of HT, 10 of follicular cancer, 15 of follicular adenoma, 16 of nodular goiter and 8 samples of normal thyroid tissue using immunohistochemical analyses, and in 42 PTC patients using western blotting. ICAM-1 expression was not detected in normal follicular cells, follicular lesions (adenoma and cancer) and benign nodular hyperplasia, but was frequently overexpressed in PTC cells. ICAM-1 overexpression was associated with extra-thyroidal invasion and lymph node metastasis; no association was found with age, gender, tumor size, multifocality, pathological stage, recurrence or distant metastasis. ICAM-1 expression in HT patients with PTC-like nuclear alterations was significantly higher than that in HT cases with non-PTC-like features. Compared with antibodies against cytokeratin 19, galectin-3 and Hector Battifora mesothelial-1, ICAM-1 was the most sensitive marker for the detection of PTC-like features in HT. These findings demonstrate that ICAM-1 expression is upregulated in PTC and in HT with PTC-like nuclear alterations. This feature may be an important factor in the progression of cancer of the thyroid gland.

Askin's tumor: 11 cases and a review of the literature.

Askin's tumor is a peripheral primitive neruoectodermal tumor within the thoracopulmonary region, which primarily occurs in children and young adults. In addition, Askin's tumor is commonly misdiagnosed, as it is rare and easily mistaken for other small round-cell tumors. The present study aimed to investigate the clinical characteristics, prognostic factors and treatment outcomes of patients diagnosed with Askin's tumor. Computed tomography (CT) scans, histopathology and immunohistochemical analysis were used for diagnosis. Patients were treated with combined (surgery-chemotherapy-radiotherapy) or mono-therapy (chemotherapy or radiotherapy) methods. A total of 11 consecutive patients with Askin's tumor (aged 8-22 years) were treated at the First Affiliated Hospital of Zhengzhou University between April 2010 and June 2013; nine patients underwent combined therapy and two patients were treated using mono-therapy. Chest lumps, swelling and pain were the most common presenting symptoms. Patients were followed up for ≤24 months post surgery and the results revealed that the median survival time of the combined and mono-therapy treatment groups were 15 and 7 months, respectively. Primary tumor size, metastasis, lactate dehydrogenase indicators and tumor stages were found to be important prognostic factors affecting patient outcome. In conclusion, the results of the present study demonstrated that the combination of surgery, chemotherapy and radiotherapy resulted in the optimal outcome for Askin's tumor patients.

Brucella melitensis 16MΔTcfSR as a potential live vaccine allows for the differentiation between natural and vaccinated infection.

Brucellosis is a zoonotic disease that poses a serious threat to public health and safety. Although the live attenuated vaccines targeting brucellosis, such as M5-90, are effective, there are a number of drawbacks to their use. For example, the vaccines are unable to differentiate between the natural and vaccinated forms of the infection, and these vaccines have also been shown to cause abortion in pregnant animals. Therefore, a safer and more potent vaccine is required. In the present study, a B. melitensis 16M TcfSR promoter mutant (16MΔTcfSR) was constructed in an attempt to overcome these drawbacks. A TcfSR mutant was derived from B. melitensis 16M and tested for virulence and protection efficiency. Levels of immuoglobulin G (IgG), and cytokine production were determined. In addition, TcfS was assessed as a diagnostic marker for brucellosis. The survival capacity of the 16MΔTcfSR mutant was shown to be attenuated in the RAW 264.7 murine macrophage cell line and BALB/c mice, and the vaccination was shown to induce a high level of protective immunity in BALB/c mice. In addition, the 16MΔTcfSR vaccination elicited an anti-Brucella-specific IgG response and induced the secretion of interferon-γ. Thus, the TcfS antigen allowed for the serological differentiation between the natural and vaccinated infection in animals. In conclusion, the results demonstrated that the 16MΔTcfSR mutant was attenuated in murine macrophage cells and BALB/c mice; therefore, 16MΔTcfSR is a potential candidate for a live attenuated vaccine against B. melitensis infection.

Obligate anaerobic Salmonella typhimurium strain YB1 treatment on xenograft tumor in immunocompetent mouse model.

The present authors have previously reported a novel approach to genetically engineer Salmonella typhimurium for the medically important therapeutic strategy of using bacterial agents to target malignant tumors in a breast cancer tumor-bearing nude mouse model. However, studying an immunocompromised mouse model for cancer therapy is insufficient, as certain crucial information about the influence of the immune system may be missing. In the present study, inoculation of the Salmonella strain, YB1, into a colon cancer tumor-bearing immunocompetent mouse model was investigated. The present study determined the tumor targeting efficiency, antitumor potential, the effects of multiple treatments and the systemic toxicity. Intravenous inoculation of YB1 in BALB/c mice exhibited high antitumor effects and also greatly increased the tumor targeting ability and safety compared with the previously-reported nude mouse model. In addition, repeated administration of YB1 further enhanced this effect. Furthermore, no marked toxicity was observed with YB1 treatment, while the VNP20009 and SL7207 strains demonstrated certain adverse effects. The findings of the present study indicate that the YB1 strain is effective and safe in targeting a colon cancer tumor in an immunocompetent mouse model.

Use of a correlation analysis model in the optimization of intensity-modulated radiotherapy of prostate cancer.

The aim of the present study was to develop a statistical model-based method for the optimization of intensity-modulated radiotherapy (IMRT). A prostate cancer IMRT plan was redesigned while retaining the same beam orientation and prescribed dose as the regular plan. A series of dosimetric parameters were generated, and a 4-step protocol was performed to analyze the data: i) The tumor control probability of the target was ensured by setting a number of strict constraint parameters so that much of the target was covered by the 95% isodose line; ii) the parameters for optimization [weight ratio, equivalent characteristic parameter a and maximum equivalent uniform dose of the organ at risk (OAR)] were adjusted; iii) the overall optimization space (OOS) was determined via analysis of the dose-parameter tables based on the correlation factor (CF) and optimization efficiency factor analysis; iv) the OOS in the Pinnacle V7.6 treatment planning system with IMRT function was transposed. A selected optimization phenomenon existed when different optimization methods were used to optimize dose distribution to the targets and OARs, which demonstrates a wide variation in the CFs between the percentage of planning target volume receiving 95% of the prescribed dose and the maximum dose of the bladder, rectum and femur. The OOS used to optimize the randomly selected plan exhibited relatively high efficiency, with benefits for the optimization of IMRT plans. For patients with prostate cancer who require complex IMRT plan optimization, the obtained OOS from the two core analysis techniques is likely to have relatively high efficiency in achieving an optimized plan. These results suggest that the correlation analysis model is a novel method for the optimization of IMRT for prostate cancer.

Liquid chromatography-mass spectrometry and liquid chromatography-NMR characterization of in vitro metabolites of a potent and irreversible peptidomimetic inhibitor of rhinovirus 3C protease.

In vitro metabolism of AG7088 [trans-(4S,2'R,5'S,3"'S)-4-[2'-4-(4-fluorobenzyl)-6'-methyl-5'-[(5"-methylisoxazole-3"-carbonylamino]-4-oxoheptanoylamino]-5-(2"'-oxopyrrolidin-3-"'-yl)pent-2-enoic acid ethyl ester] was studied in liver microsomes isolated from mice, rats, rabbits, dogs, monkeys, and humans. The structures of the metabolites were characterized by liquid chromatography (LC)-tandem mass spectrometry and LC-NMR methods. Hydrolysis of the ethyl ester to produce metabolite M4 (AG7185) is the predominant pathway in all species, with the greatest activity observed in rodents and rabbits, followed by monkeys, dogs, and humans. Several hydroxylation products were identified as minor metabolites, including diastereomers M1 and M2, with a hydroxy group at the P1-lactam moiety, and M3, with a hydroxy group at the methyl position of the methylisoxazole ring. Rodent and rabbit liver microsomes formed almost exclusively the acid metabolite M4 (AG7185), with very little hydroxylated metabolites, whereas monkey liver microsomes formed more secondary metabolites (i.e., acid analogs of the hydroxylated metabolites). The overall metabolic profile of AG7088 formed in dog liver microsomes closely resembled that of human liver microsomes; therefore, this species may be the most appropriate animal model relative to humans for exposure to AG7088 and its metabolites.

Circulating metabolites of the human immunodeficiency virus protease inhibitor nelfinavir in humans: structural identification, levels in plasma, and antiviral activities.

Nelfinavir mesylate (Viracept, formally AG1343) is a potent and orally bioavailable human immunodeficiency virus (HIV) type 1 (HIV-1) protease inhibitor (K(i) = 2 nM) and is being widely prescribed in combination with HIV reverse transcriptase inhibitors for the treatment of HIV infection. The current studies evaluated the presence of metabolites circulating in plasma following the oral administration of nelfinavir to healthy volunteers and HIV-infected patients, as well as the levels in plasma and antiviral activities of these metabolites. The results showed that the parent drug was the major circulating chemical species, followed in decreasing abundance by its hydroxy-t-butylamide metabolite (M8) and 3'-methoxy-4'-hydroxynelfinavir (M1). Antiviral assays with HIV-1 strain RF-infected CEM-SS cells showed that the 50% effective concentrations (EC50) of nelfinavir, M8, and M1 were 30, 34, and 151 nM, respectively, and that the corresponding EC50 against another HIV-1 strain, IIIB, in MT-2 cells were 60, 86, and 653 nM. Therefore, apparently similar in vitro antiviral activities were demonstrated for nelfinavir and M8, whereas an approximately 5- to 11-fold-lower level of antiviral activity was observed for M1. The active metabolite, M8, showed a degree of binding to human plasma proteins similar to that of nelfinavir (ca. 98%). Concentrations in plasma of nelfinavir and its metabolites in 10 HIV-positive patients receiving nelfinavir therapy (750 mg three times per day) were determined by a liquid chromatography tandem mass spectrometry assay. At steady state (day 28), the mean plasma nelfinavir concentrations ranged from 1.73 to 4.96 microM and the M8 concentrations ranged from 0.55 to 1.96 microM, whereas the M1 concentrations were low and ranged from 0.09 to 0.19 microM. In conclusion, the findings from the current studies suggest that, in humans, nelfinavir forms an active metabolite circulating at appreciable levels in plasma. The active metabolite M8 may account for some of the antiviral activity associated with nelfinavir in the treatment of HIV disease.

The absorption, distribution, metabolism and excretion of rofecoxib, a potent and selective cyclooxygenase-2 inhibitor, in rats and dogs.

Absorption, distribution, metabolism, and excretion studies were conducted in rats and dogs with rofecoxib (VIOXX, MK-0966), a potent and highly selective inhibitor of cyclooxygenase-2 (COX-2). In rats, the nonexponential decay during the terminal phase (4- to 10-h time interval) of rofecoxib plasma concentration versus time curves after i.v. or oral administration of [(14)C]rofecoxib precluded accurate determinations of half-life, AUC(0-infinity) (area under the plasma concentration versus time curve extrapolated to infinity), and hence, bioavailability. After i.v. administration of [(14)C]rofecoxib to dogs, plasma clearance, volume of distribution at steady state, and elimination half-life values of rofecoxib were 3.6 ml/min/kg, 1.0 l/kg, and 2.6 h, respectively. Oral absorption (5 mg/kg) was rapid in both species with C(max) occurring by 0.5 h (rats) and 1.5 h (dogs). Bioavailability in dogs was 26%. Systemic exposure increased with increasing dosage in rats and dogs after i.v. (1, 2, and 4 mg/kg), or oral (2, 5, and 10 mg/kg) administration, except in rats where no additional increase was observed between the 5 and 10 mg/kg doses. Radioactivity distributed rapidly to tissues, with the highest concentrations of the i.v. dose observed in most tissues by 5 min and by 30 min in liver, skin, fat, prostate, and bladder. Excretion occurred primarily by the biliary route in rats and dogs, except after i.v. administration of [(14)C]rofecoxib to dogs, where excretion was divided between biliary and renal routes. Metabolism of rofecoxib was extensive. 5-Hydroxyrofecoxib-O-beta-D-glucuronide was the major metabolite excreted by rats in urine and bile. 5-Hydroxyrofecoxib, rofecoxib-3',4'-dihydrodiol, and 4'-hydroxyrofecoxib sulfate were less abundant, whereas cis- and trans-3,4-dihydro-rofecoxib were minor. Major metabolites in dog were 5-hydroxyrofecoxib-O-beta-D-glucuronide (urine), trans-3, 4-dihydro-rofecoxib (urine), and 5-hydroxyrofecoxib (bile).

Metabolism of A dopamine D(4)-selective antagonist in rat, monkey, and humans: formation of A novel mercapturic acid adduct.

3-([4-(4-Chlorophenyl)piperazin-1-yl]-methyl)-1H-pyrrolo-2, 3-beta-pyridine (L-745,870) is a dopamine D(4) selective antagonist that has been studied as a potential treatment for schizophrenia, with the expectation that it would not exhibit the extrapyramidal side effects often observed with the use of classical antipsychotic agents. The metabolism of L-745,870 in vivo was investigated in the rat, rhesus monkey, and human using liquid chromatography-tandem mass spectrometry and/or NMR techniques in conjunction with radiochemical detection. In all three species, two major metabolic pathways were identified, namely N-dealkylation at the substituted piperazine moiety and the formation of a novel mercapturic acid adduct. It is proposed that the latter biotransformation process involves the formation of an electrophilic imine methide intermediate, analogous to that produced from 3-methyl indole. This report appears to represent the first example of metabolic activation of a 3-alkyl-7-azaindole nucleus.

Pharmacokinetics of intravenous alendronate.

Alendronate is a potent bisphosphonate that has been studied for the treatment of osteoporosis and Paget's disease of the bone. To examine the pharmacokinetics of this drug, several groups of postmenopausal women were dosed intravenously in several studies. Twelve patients with metastatic bone disease were administered an intravenous dose of 10 mg of 14C-labeled alendronate (approximately 26 muCi), and plasma, feces, and urine samples were collected for 72 hours. Radioactivity was excreted almost exclusively in urine, and all of it was accounted for by alendronate. Overall recovery accounted for 47% of dose, with the remainder presumed to be retained in bone. Metabolism of alendronate was not observed. Renal clearance of alendronate was 71 mL/min. An additional 10 subjects were given repeated i.v. administrations of alendronate to demonstrate that previous exposure does not alter the pharmacokinetic behavior of the drug. Examination of the findings from these and other studies in which alendronate was administered intravenously revealed that disposition of single doses is linear in the range of 0.125 to 10 mg. With the possible exception of a somewhat greater skeletal retention of a systemically administered dose, the pharmacokinetics of i.v. alendronate were found to be similar to those of other bisphosphonates.

Microsomal metabolism of the 5-lipoxygenase inhibitor L-739,010: evidence for furan bioactivation.

The novel 5-lipoxygenase inhibitor [1S,5R]-3-cyano-1-(3-furyl)-6-(6-[3-(3 alpha-hydroxy-6,8-dioxabicyclo[3.2.1]octanyl)]pyridin-2-yl- methoxyl)naphthalene (L-739,010), when administered to rats and rhesus monkeys, was found to produce metabolites which appeared to be covalently bound to plasma proteins. Incubation of [14C]L-739,010 with rat liver microsomes did not yield appreciable amounts of soluble metabolites but resulted in covalent binding to microsomal proteins. The covalent binding was NADPH-dependent and was enhanced by 1.5- and 2-fold in liver microsomes from rats, pretreated with phenobarbital and dexamethasone, respectively. Addition of triacetyloleandomycin and diethyldithiocarbamate to the incubation mixture inhibited the covalent binding by 60% and 46%, respectively. These findings suggest that the cytochrome P450 3A family of enzymes play an important role in the bioactivation of L-739,010. The presence of GSH attenuated the covalent binding by 50%, while methoxylamine, an aldehyde trapping agent, blocked the covalent binding completely and, concurrently, produced several soluble metabolic adducts. Subsequently, major methoxylamine adducts were identified by LC-MS/MS and NMR as O-methyloximes of the ring-opened furan moiety of L-739,010. Incubation of L-739,010 with methoxylamine and hepatic microsomes from dog, rhesus monkey, and human produced similar metabolic adducts as those formed by rat liver microsomes. Therefore, under these experimental conditions, the furan moiety, which undergoes oxidative cleavage to the highly reactive 2-butene-1,4-dialdehyde, represents the major site of L-739,010 biotransformation. This putative reactive intermediate could react with microsomal proteins in vitro and physiological proteins in vivo. Since furan bioactivation is believed to be responsible for the toxicity of many furan-containing compounds, the furan moiety of L-739,010 may be regarded as undesirable.

Disposition of DMP 811 (L-708,404), a potent angiotensin II receptor antagonist, in the rat, monkey, and chimpanzee.

DMP 811 {4-ethyl-2-n-propyl-1-[(2'-(1H-tetrazole-5-yl)biphenyl-4-yl) methyl]imidazole-5-carboxylic acid; L-708,404} is a highly potent angiotensin II receptor antagonist. The physiological disposition of DMP 811 was examined in the Sprague-Dawley rat, rhesus monkey, and pan troglodytes chimpanzee. Plasma concentrations of DMP 811 were determined by an HPLC assay with fluorescence detection. After intravenous administration of DMP 811 to rats (1 mg/kg), monkeys (0.5 mg/kg), and chimpanzees (0.5 mg/kg), the plasma clearance was 1.3, 1.8, and 0.3 ml/min/kg, respectively. The corresponding volumes of distribution were 0.16, 0.10, and 0.10 liters/kg, and the value for the terminal half-life was 3.0, 2.4, and 10.1 hr in the respective species. After oral administration to rats (5 mg/kg), monkeys (2 mg/kg), and chimpanzees (2 mg/kg), DMP 811 was 8.4%, 10.2%, and 8.0% bioavailable, respectively. The mass balance of [14C]DMP 811 was investigated in rats and monkeys. In rats, the radiolabeled dose was excreted primarily in feces (79% intravenous; 99% oral) with <1% of the dose in urine. In monkeys, the intravenous radiolabeled dose was excreted in both urine (48%) and feces (42%), whereas the oral dose was excreted largely in feces (79%), with an additional 6% in urine. In summary, DMP 811 was cleared slowly in all three species. The oral bioavailability of DMP 811 was low, but consistent across species. Pharmacokinetic data suggest that the low oral bioavailability was not caused by first-pass metabolism, but probably caused by limited absorption.

Regulated and constitutive activity by CDC25Mm (GRF), a Ras-specific exchange factor.

Serum stimulates cells to increase their proportion of Ras protein in the active GTP-bound state. We have recently identified four types (I to IV) of apparently full-length cDNAs from a single mammalian gene, called CDC25Mm or GRF, which is homologous to the Ras-specific exchange factor CDC25 of S. cerevisiae. The largest cDNA (type IV) is brain specific, with the other three classes, although they have distinct 5' ends, essentially representing progressive N-terminal deletions of this cDNA. When placed in a retroviral expression vector, all four types of cDNAs induced morphologic transformation of NIH 3T3 cells and an increase in the basal level of GTP.Ras. Serum stimulation of these transformants lead to a further increase in GTP.Ras only in cells expressing the type IV cDNA. Each type of GRF protein was found in cytosolic and membrane fractions, and the protein in each fraction could stimulate guanine nucleotide release from GDP.Ras in vitro. When NIH 3T3 cells and cells expressing the type IV protein were transfected with two versions of a mutant ras gene, one encoding membrane-associated Ras protein and the other encoding a cytosolic Ras protein, the basal levels of GTP bound to both forms of the mutant Ras protein were significantly higher in the cells expressing the type IV protein. However, serum increased the level of GTP bound to the membrane-associated mutant Ras protein in NIH 3T3 cells and in cells expressing the type IV protein but not in cells expressing the cytosolic version of the Ras protein. We conclude that each type of CDC25Mm induces cell transformation via the ability of its C terminus to stimulate guanine nucleotide exchange on Ras, the presence of N-terminal sequences is associated with a serum-dependent change in GTP.Ras, and the serum-dependent increase in GTP.Ras by exogenous CDC25Mm or by endogenous exchange factors probably requires membrane association of both Ras and the exchange factor.