[Expression and significance of INSM1 and SOX11 in pancreatic neuroendocrine tumor and solid pseudopapillary neoplasm].

OBJECTIVE: To investigate the expression and significance of insulinoma associated protein 1 (INSM1) and SRY-related high-mobility group box 11 (SOX11) in pancreatic neuroendocrine tumor (PNET) and solid pseudopapillary neoplasm (SPN). METHODS: To detect the expression of INSM1, SOX11, Syn, CgA, CD56, ß-catenin, and CD99 in 56 cases of PNET, 42 cases of SPN, 16 cases of ductal adenocarcinoma (DACC) and 8 cases of acinar cell carcinoma (ACC) by immunohistochemistry. The application value of combination of INSM1 and SOX11 was compared with conventional markers (Syn, CgA, CD56, ß-catenin, and CD99) in diagnosis and differential diagnosis of PNET and SPN. RESULTS: (1) In the 56 cases of PNET, the positive signals of INSM1 were located in the tumor and islet nucleus, the positive expression rate in the tumor tissues was 91.07% (51/56), whereas the signal was absent in 42 cases of SPN, 16 cases of DACC and 8 cases of ACC, and there were significant statistical difference between PNET with SPN, DACC, and ACC respectively (P < 0.001). (2) The positive signals of SOX11 were located in the tumor nucleus, with the positive expression rate was 92.86% (39/42) in SPN, however, the positive expression rate of SOX11 was 8.93% (5/56) in PNET, which included 3 cases of G1 and 2 cases of G3 types of PNET, the SOX11 positive signal was absent in 16 cases of DACC, 8 cases of ACC and peritumoral nomal pancreatic tissue, and the differences were statistically significant of positive rate between SPN with PNET, DACC and ACC, respectively (P < 0.001). (3) The sensitivity of INSM1(+)/SOX11(-) immunophenotype for PNET was 85.71%, vs. CD56 (57.14%), the difference was statistically significant (P=0.001); vs. Syn (80.36%) and CgA (71.43%), the difference was no statistically significant (P>0.05). The specificity of INSM1(+)/SOX11(-) for PNET was 100.00%, vs. Syn (42.86%) and CD56 (47.62%), the difference was statistically significant (P < 0.001); vs. CgA (92.86%), the difference was no statistically significant (P>0.05). The sensitivity of INSM1(-)/SOX11(+) immunophenotype for SPN was 92.86%, vs. ß-catenin (90.48%) and CD99 (85.71%), the difference was no statistically significant (P>0.05). The specificity of INSM1(-)/SOX11(+) for SPN was 96.43%, vs. CD99 (48.21%), the difference was statistically significant (P < 0.001); vs. ß-catenin (100.00%), the difference was no statistically significant (P>0.05). (4) The positive expression of INSM1 and SOX11 in PNET and SOX11 were not correlated with clinicopathological parameters (age, gender, tumor size, location, grade, and metastasis) (P>0.05). CONCLUSION: The positive expression patterns of INSM1 and SOX11 in PNET and SPN respectively are conductive to distinguish the both tumors. The combination of both take precedence over some corresponding conventional immunohistochemical markers in terms of sensitivity and specificity.

Evidence for causal effects of sleep disturbances on risk for osteoarthritis: a univariable and multivariable Mendelian randomization study.

OBJECTIVE: To disentangle whether sleep disturbances have a causal effect on the risk of osteoarthritis (OA) using genetically based approaches. METHOD: We performed univariable and multivariable Mendelian randomization (MR) analyses using publicly released genome-wide association studies summary statistics to estimate the causal associations of sleep disturbances with OA risk. The inverse-variance weighted (IVW) method was utilized as primary MR analysis, whereas complementary methods including weighted median, weighted mode, MR-Egger regression, and MR pleiotropy residual sum and outlier (MR-PRESSO) were applied to detect and correct for the presence of pleiotropy. RESULTS: There were 228 independent instrumental variables (IVs) for insomnia and 78, 27 and 8 IVs for sleep duration, short sleep duration and long sleep duration, respectively. Univariable MR analysis suggested that genetically determined insomnia or short sleep duration exerted a causal effect on overall OA in an unfavorable manner (Insomnia: OR = 1.22, 95%CI = 1.15-1.30, P = 8.05 × 10-10; Short sleep duration: OR = 1.04, 95%CI = 1.02-1.07, P = 2.20 × 10-3). More compelling, increasing genetic liability to insomnia or short sleep duration was also associated with OA risk, after accounting for effects of insomnia or short sleep duration on body mass index, type 2 diabetes and depression individually, and in a combined model considering all three confounders. CONCLUSIONS: Findings suggested consisted evidence for an adverse effect of increased insomnia or short sleep duration on OA risk. Strategies to mitigate sleep disturbances may be one of the cornerstones protects against OA.

[Bendamustine monotherapy for Chinese patient treatment with relapsed or refractory B cell non-Hodgkin lymphoma: a phase â ¡, prospective, multicenter, single-arm study].

Objective: To evaluate the efficacy and safety of bendamustine monotherapy in Chinese patients with relapsed/refractory (R/R) B cell non-Hodgkin lymphoma (B-NHL) . Methods: This prospective, multicenter, open label, single-arm, phase Ⅱ study investigated bendamustine's efficacy and safety in Chinese patients with R/R B-NHL. A total of 78 patients with B-NHL in 11 hospitals in China from March 2012 to December 2016 were included, and their clinical characteristics, efficacy, and survival were analyzed. Results: The median age of all patients was 58 (range, 24-76) years old, and 69 (88.4% ) patients had stage Ⅲ/Ⅳ disease. 61 (78.2% ) patients were refractory to previous treatments. Patients received a median of 4 (range, 1-10) cycles of bendamustine treatment. The overall response rate was 61.5 (95% CI 49.8-72.3) % , the median response duration was 8.3 (95% CI 5.5-14.0) months, and the complete remission (CR) rate was 5.1 (95% CI 1.4-12.6) % . In the full analysis set, median progression-free survival (PFS) and median OS were 8.7 (95% CI 6.7-13.2) months and 25.5 months (95% CI 14.2 months to not reached) , respectively, after a median follow-up of 33.6 (95% CI 17.4-38.8) months. Lymphopenia (74.4% ) , neutropenia (52.6% ) , and leukopenia (39.7% ) , thrombocytopenia (29.5% ) and anemia (15.4% ) were the most common grade 3-4 hematologic adverse events (AE) . The most frequent non-hematologic AEs included nausea (43.6% ) , vomiting (33.3% ) , and anorexia (29.5% ) . Univariate and multivariate analysis showed that <4 cycles of bendamustine treatment was a poor prognostic factor for PFS (P=0.003) , and failure to accept fludarabine containing regimen was a poor prognostic factor for OS (P=0.009) . Conclusion: Bendamustine monotherapy has good efficacy and safety in the treatment of patient with R/R B-NHL.

[Expression of CD200 and INSM1 in gastrointestinal and pancreatic neuroendocrine neoplasms and its diagnostic values].

Objective: To investigate the expression and diagnostic values of CD200 and insulinoma associated protein 1 (INSM1) in gastrointestinal and pancreatic neuroendocrine neoplasm (GIP-NEN). Methods: The expression of CD200, INSM1, Syn and CgA was detected in 69 cases of GIP-NEN, 66 cases of gastrointestinal and pancreatic non-neuroendocrine neoplasm (GIP-nonNEN) and 16 cases of metastatic neuroendocrine neoplasm by immunohistochemistry, to compare the values of CD200, INSM1, Syn, CgA and their combinations in diagnosing GIP-NEN. Receiver operating characteristics (ROC) curve was used. Results: The immunoreactivity of CD200 was present in the cytoplasma and/or membrane of the neoplasms cells, the positive expression rates in GIP-NEN and GIP-nonNEN were significantly different (P<0.01). The sensitivity and specificity of CD200 for diagnosing GIP-NEN were 95.7% and 78.8%, respectively. There was significant difference of the positive rates of CD200 between neuroendocrine tumor and neuroendocrine carcinoma (P=0.05). The immunoreactivity of INSM1 was present in the nuclei of neoplasms cells. The positive expression rates in GIP-NEN and GIP-nonNEN were significantly different (P<0.01). The sensitivity and specificity of INSM1 for diagnosis of GIP-NEN were 85.5% and 95.5%, respectively. There were also significantly different positive rates of INSM1 between neuroendocrine tumor and neuroendocrine carcinoma, as well as between G1 and G3 neuroendocrine tumors (P<0.05). There was no difference in the area under ROC curve (AUC) of single stain of CD200, INSM1, Syn or CgA (0.857, 0.907, 0.890 and 0.833, respectively, P>0.05). The sensitivity of combined CD200+INSM1 stains for diagnosing GIP-NEN was significantly higher than that of Syn+CgA (85.5% vs. 63.8%, P<0.05). The AUC of two combinations were 0.962 and 0.925, respectively, which were not statistically different (P>0.05). Conclusions: CD200 and INSM1 are two novel markers of neuroendocrine neoplasm, which aid to diagnosis for GIP-NEN and exclude its mimickers. They are associated with tumor grades. Combining both as an immunohistochemical panel shows high sensitivity and specificity. Thus, the combined panel can be utilized as useful supplement for Syn and CgA.

[Regulatory relationship between lncRNA KCNQ1OT1 and miR-146a-3p in preeclampsia].

Objective: To observe the changes of the expression level of long non-coding RNA (lncRNA) KCNQ1OT1 and microRNA (miR)-146a-3p in placenta tissues of preeclampsia (PE) patients, as well as their effect and mechanism on the biological functions of trophoblast cells. Methods: A total of 45 cases of hospitalized PE patients in Hainan General Hospital from July 2017 to July 2018 were selected as the PE group, 55 normal pregnant women during the same period were chosed as the control group. The expression level of KCNQ1OT1 mRNA and miR-146a-3p in the placenta tissues between two groups were detected by using quantitative real time (qRT)-PCR. Pearson's test was furtherly analyzed the correlation between them. Human trophoblast cell line (HTR8/SVneo) were randomly divided into control and lipopolysaccharide (LPS) groups, and then LPS group were divide into four sub-groups,included LPS group, short hairpin RNA (sh)-KCNQ1OT1 (after silencing the expression of KCNQ1OT1), miR-146a-3p inhibitor and sh-KCNQ1OT1+miR-146a-3p inhibitor. The targeting relationship between KCNQ1OT1 and miR-146a-3p were predicted by bioinformatics software and confirmed by luciferase assay. The cell proliferation and invasion capacities were respectively detected by cell counting kit-8 (CCK-8) and transwell assay. The expression level of KCNQ1OT1 mRNA and miR-146a-3p were detected by qRT-PCR and the protein expression level of CXC chemokine ligand 12 （CXCL12） and CXC chemokine receptor type 4 (CXCR4) were tested by western blot. Results: (1) The mRNA expression level of KCNQ1OT1 in the placenta of PE group was lower than that of control group (0.23±0.03 vs 0.51±0.04, P<0.05), and the miR-146a-3p expression level was higher than that of the control group (0.49±0.03 vs 0.31±0.03, P<0.05), there were statistical significant differences between the two groups. (2) Luciferase assay showed that there was a targeting relationship between KCNQ1OT1 and mir-146a-3p. Compared with the control group, the mRNA expression level of KCNQ1OT1 in the LPS group were significantly decreased (0.91±0.03 vs 0.35±0.03, P<0.05), and the expression level of miR-146a-3p were significantly increased (0.22±0.03 vs 0.63±0.04, P<0.05). The cell proliferation, invasion and migration capacities and the protein expression of CXCL12 and CXCR4 significantly reduced in the LPS group compared with control group (all P<0.05). The mRNA expression level of KCNQ1OT1 (0.23±0.03) in the sh-KCNQ1OT1 group were further decreased, the expression of miR-146a-3p (0.85±0.03) were further increased, and the cell proliferation, invasion and migration capacities and the protein expression of CXCL12 and CXCR4 were all further reduced compared with control group，there were significant difference between two groups (all P<0.05). Comparing the miR-146a-3p inhibitor group, and sh-KCNQ1OT1+miR-146a-3p inhibitor group with the sh-KCNQ1OT1 group, respectively, the expression level of KCNQ1OT1 mRNA (0.78±0.04 vs 0.50±0.03) increased, and the expression level of miR-146a-3p (0.42±0.03 vs 0.46±0.03) decreased, the cell proliferation, invasion and migration capacities and the protein expression of CXCL12 and CXCR4 were all increased ,there were statistically significant differences (all P<0.05). Conclusion: KCNQ1OT1 could target the regulation of miR-146a-3p through CXCL12/CXCR4 pathway in the proliferation, invasion an migration of HTR8/SVneo cells, which may be involved in the pathogenesis of PE.

Relationship between the IL-4 gene promoter -590C/T (rs2243250) polymorphism and susceptibility to autoimmune diseases: a meta-analysis.

OBJECTIVE: Studies investigating the association between interleukin (IL)-4 gene promoter -590C/T (rs2243250) polymorphism and autoimmune diseases report conflicting results. To derive a more precise estimation of the relationship, a meta-analysis was performed. METHODS: A systematic literature search was conducted to identify relevant studies. Pooled odds ratio (OR) with 95% confidence interval (CI) was used to estimate the strength of association. RESULTS: A total of 6001 cases and 6788 controls from 24 studies were analysed. Significant association of the C allele of IL-4 rs2243250 polymorphism with rheumatoid arthritis (RA) was detected (odds ratio (OR) = 0.696, 95% confidence interval (CI) = 0.601-0.807). Stratification by ethnicity indicated an association between the IL-4 rs2243250 polymorphism and RA in Caucasians. Furthermore, the overall ORs of the associations between the C allele and multiple scleorosis (MS) were 1.340 (95% CI = 1.102-1.630). However, we failed to reveal any association between IL-4 rs2243250 polymorphism and systemic lupus erythematosus (SLE), type 1 diabetes (T1D) or Graves' disease (GD). CONCLUSIONS: The present study suggests that the IL-4 rs2243250 polymorphism might be associated with genetic susceptibility to autoimmune diseases, including RA and MS.

Lung, liver, prostate, bladder malignancies risk in systemic lupus erythematosus: evidence from a meta-analysis.

OBJECTIVE: The objective of this paper is to examine some solid tumors incidence in patients with systemic lupus erythematosus (SLE) derived from population-based cohort studies by means of meta-analysis. METHODS: Relevant electronic databases were searched for studies characterizing the associated risk of overall malignancy and four site-specific malignancies (lung, liver, prostate, bladder cancer) in patients with SLE. The meta-analysis procedure was used to pool standardized incidence rates (SIRs) with 95% confidence intervals (CIs) to evaluate the association. RESULTS: A total of seven cohort studies were identified, of which six provided the SIR for overall malignancy, seven reported the SIR for lung cancer, five for liver cancer, four for prostate cancer and six for bladder cancer. Overall, lung and liver cancers were more frequently observed in patients with SLE with SIR of 1.16 (95% CI = 1.12-1.21), 1.68 (95% CI = 1.33-2.13) and 2.44 (95% CI = 1.46-4.05), respectively. However, the risk of prostate cancer appeared to be somewhat reduced in male patients with SLE (SIR = 0.71, 95% CI = 0.57-0.89). CONCLUSIONS: This meta-analysis shows that SLE patients are at increased risk of developing cancer, particularly of the lung, bladder and liver. However, males with SLE have a decreased risk of prostate cancer.

Genetic polymorphisms of VKORC1, CYP2C9, CYP4F2 in Bai, Tibetan Chinese.

BACKGROUND: VKORC1, CYP2C9 and CYP4F2 are three critical genes associated with inter-individual variation of warfarin dose. Many dosing algorithms containing these gene polymorphisms and demographic characteristics have been set up for better use of warfarin. However, with distinct gene mutation frequencies among different ethnics, dosing algorithms differ greatly. For Chinese, related research just concentrate on Han Chinese, ignoring other Chinese ethnicities. This study aims to detect the popular polymorphisms in these three critical genes in Bai, Tibetan Chinese, to start the exploration of better use of warfarin in Chinese minorities. METHODS: PCR-based methods were used to analyze VKORC1 3673G > A, CYP2C9*3, CYP4F2 rs2108622 C > T in Han, Bai and Tibetan Chinese. RESULTS: The differences among the mutation frequencies of the studied genes in three ethnicities were not statistically significant. The frequency of A-allele of VKORC1 3673G > A was 92.8%, 90.2%, 90.8% in Bai, Tibetan, Han Chinese, respectively. The frequency of *3-allele in CYP2C9*3 was low in Bai (4.5%), Tibetan (2.8%) and Han Chinese (4.6%). Approximately one fourth of each ethnic had the mutant T-allele of CYP4F2 rs108622. However, Bai Chinese got statistically higher A-allele frequency of VKORC1 3673G > A than previously studied Han Chinese did. CONCLUSIONS: Bai Chinese got significant higher A-allele frequency of VKORC1 3673G > A.

Prevention of murine acute graft-versus-host disease by recipient-derived TGFbeta1-treated dendritic cells.

Acute graft-versus-host disease (GVHD) remains the major barrier to allogeneic bone marrow transplantation (allo-BMT). Evidence has accumulated that transforming growth factor beta1-treated dendritic cells (TGFbeta-DC), deficient in surface costimulatory molecules, inhibit alloantigen-specific T-cell responses and induce graft hyporeactivity. To analyze the effect of TGFbeta-DC on GVHD after allo-BMT, 5.0 x 10(6) recipient-derived TGFbeta-DC were injected into C57BL/6 (H-2b) with bone marrow-splenocyte grafts from major histocompatibility complex (MHC) disparate BALB/c mice (H-2d). Survival analysis showed TGFbeta-DC cotransplantation resulted in significant prolongation of allograft survival, namely a mean survival time (MST) of 44.3 +/- 4.5 days, versus the untreated MST of 9.5 +/- 0.6 days (P < .01). However, mature DC aggravated the GVHD with an MST of 6.6 +/- 0.6 days (P < .01). In addition, the third-party C3H-derived TGFbeta-DC did not enhance the survival rate (MST = 9.7 +/- 0.5 days). Furthermore, serum IFN-gamma, IL-12, and IL-18 levels in TGFbeta-DC cotransplanted mice were reduced compared with untreated BMT hosts, while serum IL-10 levels were not changed. These results suggest that TGFbeta-DC cotransplantation may attenuate the severity of GVHD after BMT.

High-throughput screening for ligand-induced c-fos mRNA expression by branched DNA assay in Chinese hamster ovary cells.

We have developed a generally useful screening assay for receptor agonists and antagonists in Chinese Hamster Ovary (CHO) cells. Three key features of the assay make it applicable to a broad range of receptors: (1) the use of CHO cells as host cells to overexpress receptors, (2) measurement of endogenous c-fos mRNA, which responds to a wide spectrum of stimuli, and (3) the use of branched chain DNA assay which is highly sensitive, quantifiable, amenable to high-throughput analysis, and easy to execute. The combination of these features provides a powerful means to screen rapidly for peptide and small molecule ligands for a variety of receptors. CHO cells overexpressing insulin receptor were used as a test system to compare conventional signaling assays with the high-throughput c-fos branched DNA assay.

The effects of cryotherapy on muscle damage in rats subjected to endurance training.

Sixty 60-day-old Sprague-Dawley male rats were subjected to exhaustive and non-exhaustive exercise treatment over a period of 3, 5 and 7 weeks. Moderate exercise consisted of running on the treadmill at 10% grade at 10 m/min for 20 min for 5 days per week, while exhaustive running consisted of running at 10% grade at 15 m/min with an increase of 5 m/min every 5 min for 5 days per week. Half of the exercised rats were subjected to immersion in 4 degrees C water for 5 min after each exercise bout. Histological analyses of the gastrocnemius showed that ultrastructural damage of myofibrils occurred after 5 weeks of exhaustive running and 7 weeks of moderate running. With post-exercise immersion in 4 degrees C water for 5 min, the occurrence of ultrastructural damage advanced by 2 weeks in both treatments. It was concluded that the application of cryotherapy to the exercise groups was deleterious. Muscle damage occurred earlier in the treatment group and the degree of damage was also more serious over the same time period. This study supports the recommendation that the use of cryotherapy to reduce pain and haematoma formation should be coupled with rest for at least 48 hours.

[Primary epididymal tumors: report of 24 cases].

24 cases of primary epididymal tumors including 23 benign and 1 malignant tumors were treated. In these cases, 66.7% situated at epididymal tail, and 12.5% involved both tail and body. Smooth muscle tumor of one case was bilateral, and adenomatoid tumor of another one was multiple. The symptoms of primary epididymal tumors were mild even absent, so the tumor was easily confused with non-tumorous mass of epididymis. Benign epididymal tumor should be differentiated from tuberculosis, chronic inflammation or granuloma. Besides signs of malignant mass, malignant epididymal tumor usually showed thickened spermatic cord, especially enlarged ductus deferens. The removal of the tumor mass or whole epididymis of the same side could cure benign epididymal tumor without recurrence. Malignant epididymal tumor should be treated as malignancy of testis or spermatic cord, with adjuvant chemotherapy or radiotherapy as indicated by the pathological type.

[Histopathology appearance of intrauterine residue after medical abortion by mifepristone and prostaglandin analogue].

Four hundred and fifty pregnant women were recruited for termination of early gestation by mifepristone combined with dl-15-methyl PGF2 alpha or misoprostol. Eight-four out of 450 subjects received curettage because of heavy or prolonged vaginal bleeding and slow decline of urinary hCG levels. Histopathology examinations of specimens obtained during curettage revealed denatured, necrotic and obscure villi and trophoblasts in 77 specimens, which accounted for 91.7%. Among them, 68 samples were mingled with inflammatory cell infiltration, and 15 with decidual cells, only 3 were villi and trophoblasts alone. The remaining 7 specimens were decidua in 6 and inflammatory infiltration in 1, which accounted for 7.1% and 1.2% respectively. This study suggested that the major cause resulting in heavy or prolonged vaginal bleeding after medical abortion by mifepristone and prostaglandin analogue was residual villi and trophoblasts with inflammatory cell infiltration.

EBV gene expression, EBNA antibody responses and EBV+ peripheral blood lymphocytes in post-transplant lymphoproliferative disease.

Epstein-Barr virus (EBV) is associated with the development of several B cell malignancies including Burkitt's lymphoma (BL), post-transplant lymphoproliferative disease (PTLD), and AIDS-related lymphomas. The latter two diseases result from EBV-driven B cell proliferation in the absence of normal immunosurveillance and as such, represent a heterogenous family of lymphoproliferative disorders. This article reviews studies on EBV gene expression and antibody development in PTLD and introduces recent information on the levels of EBV+ peripheral blood lymphocytes to discuss possible mechanisms of pathogenesis under varying conditions of immunosuppression.

EBV-immortalized isogenic human B-cell clones exhibit differences in DNA-protein complex formation on the BZLF1 and BRLF1 promoter regions among latent, lytic and TPA-activated cell lines.

The reactivation of Epstein-Barr virus from latency requires the transcriptional induction of the viral encoded lytic cycle initiator gene, BZLF1, and a concomitant switch from OriP to OriLyt directed viral DNA replication. To investigate the role of host cell factors in these events, a series of EBV-immortalized clonal lymphoblastoid cell lines (LCL) were derived from the spontaneous outgrowth of peripheral blood lymphocytes from a single EBV-seropositive individual. We show that the state of virus activation among this family of isogenic clonal LCL differs, suggesting that each B-cell clone expresses a different complement of cellular factors that influence the state of viral activation. As a first step in the identification of factors involved in EBV reactivation, nuclear extracts were prepared from tightly latent, spontaneously replicating and latent LCL treated with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and sodium butyrate. The extracts were used in gel mobility shift analyses to compare DNA-protein complex formation among a series of target DNA sequences, including OriLyt and promoter sequences from BZLF1 and BRLF1. The DNA-protein complex patterns were reproducible and indistinguishable among extracts obtained from the latent LCL, but differed from those observed in extracts obtained from the spontaneously replicating LCL, particularly in regard to the binding of a CREB protein to the BZLF1 promoter. Moreover, extracts prepared from LCL treated with TPA to induce virus reactivation resulted in the formation of complexes that differed from those prepared from the spontaneously replicating LCL. Taken together, these data suggest that B-cell factors govern the state of viral activation and that EBV may be reactivated by more than one pathway.

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.

Evidence for restricted Epstein-Barr virus latent gene expression and anti-EBNA antibody response in solid organ transplant recipients with posttransplant lymphoproliferative disorders.

Epstein-Barr virus (EBV) is associated with the development of two human B-cell malignancies, Burkitt's lymphoma and lymphomas that occur in the immunosuppressed host. The latter category of disease has become important recently as it is seen primarily in organ transplant recipients and individuals with acquired immunodeficiency syndrome. One possible mechanism for lymphoma development involves a reduction in or lack of EBV-specific cytotoxic T-cell recognition. In support of this model are previous observations that the expression of EBV nuclear antigen 2 (EBNA2) and latent membrane protein, two viral antigens associated with major histocompatibility complex class I-restricted T-cell killing, are downregulated in Burkitt's lymphoma and in early passage lymphoblastoid cell lines (LCL) derived from the malignant lesions. To determine whether a similar mechanism could occur in the development of posttransplant lymphoproliferative disorders (PTLD), we compared EBV gene expression among 23 PTLD tumor lesions obtained from 11 solid organ transplant recipients and among LCL derived from 3 of these lesions. In this report, we demonstrate, by Southern blot, Western blot, and immunofluorescence analysis, that (1) the tumor lesions exhibit varying patterns of restricted viral gene expression; (2) LCL derived from these lesions may represent the in vitro selection of cell subpopulations; and (3) immunosuppressed individuals have a markedly reduced antibody response to the latent cycle antigens, EBNA1, EBNA2, and EBNA-LP, but not to the lytic cycle viral capsid antigen when compared with normal immunocompetent controls.

Cell transformation by ras and regulation of its protein product.

We are studying the biological activity and regulation of mammalian Ras protein in tumours and in physiological signalling. We have shown that GAP (the GTPase-activating protein) is a potent negative regulator of normal Ras in cells. Reduction or loss of the NF1 gene product neurofibromin, in association with genetic abnormalities of the NF1 locus, has been identified in schwannoma cell lines from patients with neurofibromatosis and in melanoma and neuroblastoma lines from patients without neurofibromatosis. Although loss of neurofibromin in the schwannoma lines was associated with a high proportion of normal Ras protein in the active GTP-bound state, Ras-GTP appeared to be appropriately regulated in the melanoma and neuroblastoma lines, which contain normal levels of GAP. Therefore the GTPase-activating activity of neurofibromin is not essential for negative regulation of Ras in some cell types and the putative tumour suppressor function of neurofibromin in such cell types is independent of its GTPase-activating activity. Mitogen activation of Ras in fibroblasts is mediated primarily by exchange factors, which probably interact with a region on the Ras protein distinct from the region required for interaction with GAP. Multiple full-length cDNAs have identified a mouse gene whose products are related to yeast CDC25 guanine nucleotide exchange factor.

Isolation of multiple mouse cDNAs with coding homology to Saccharomyces cerevisiae CDC25: identification of a region related to Bcr, Vav, Dbl and CDC24.

In Saccharomyces cerevisiae, the product of the CDC25 gene is an essential Ras activator that appears to function by stimulating guanine nucleotide exchange on Ras. Using the ability of a mouse cDNA expression library to complement yeast cells lacking functional CDC25, Martegani et al. have identified a 1.7 kb partial cDNA from a gene, designated CDC25Mm, with homology to CDC25. We have now screened a mouse brain cDNA library to identify full-length clones of CDC25Mm. This cloning has led to the isolation of six distinct full-length cDNAs, each of which appear to be derived from the CDC25Mm gene, since their 3' 2 kb appear to be identical and to encode the same 661 C-terminal amino acids. Three cDNAs are predicted to encode protein products of 666 or 667 amino acids. The other three cDNAs encode products that are 836, 1120 and 1260 amino acids, respectively. A 241 amino acid region near the N-terminus of the two largest products was found to have homology to a domain shared by Bcr, Vav, Dbl and CDC24. Polyclonal antibodies raised to a peptide encoded by all the cDNAs have identified at least two protein products in NIH3T3 fibroblasts. Their apparent molecular weights are 75 and 95 kDa, which correspond closely to those predicted to be encoded, respectively, by the two shorter classes of cDNAs. In NIH3T3, the 95 kDa form is much more abundant than the 75 kDa form, while PC-12 pheochromocytoma cells contain relatively high levels of the 75 kDa form. We conclude that CDC25Mm is a complex gene whose protein products are regulated in a tissue-specific manner.