Chitinase 3-like 1 secreted from cancer-associated fibroblasts promotes tumor angiogenesis via interleukin-8 secretion in colorectal cancer.

The cancer­stromal interaction has been demonstrated to promote tumor progression, and cancer-associated fibroblasts (CAFs), which are the main components of stromal cells, have attracted attention as novel treatment targets. Chitinase 3-like 1 (CHI3L1) is a chitinase-like protein, which affects cell proliferation and angiogenesis. However, the mechanisms through which cells secrete CHI3L1 and through which CHI3L1 mediates tumor progression in the cancer microenvironment are still unclear. Accordingly, the present study assessed the secretion of CHI3L1 in the microenvironment of colorectal cancer and evaluated how CHI3L1 affects tumor angiogenesis. CAFs and normal fibroblasts (NFs) established from colorectal cancer tissue, and human colon cancer cell lines were evaluated using immunostaining, cytokine antibody array, RNA interference, reverse transcription-quantitative PCR (RT-qPCR), ELISA, western blotting and angiogenesis assays. The expression and secretion of CHI3L1 in CAFs were stronger than those in NFs and colorectal cancer cell lines. In addition, interleukin-13 receptor α2 (IL-13Rα2), a receptor for CHI3L1, was not expressed in colorectal cancer cell lines, but was expressed in fibroblasts, particularly CAFs. Furthermore, the expression and secretion of IL-8 in CAFs was stronger than that in NFs and cancer cell lines, and recombinant CHI3L1 addition increased IL-8 expression in CAFs, whereas knockdown of CHI3L1 suppressed IL-8 expression. Furthermore, IL-13Rα2 knockdown suppressed the enhancement of IL-8 expression induced by CHI3L1 treatment in CAFs. For vascular endothelial growth factor-A (VEGFA), similar results to IL-8 were observed in an ELISA for comparison of secretion between CAFs and NFs and for changes in secretion after CHI3L1 treatment in CAFs; however, no significant differences were observed for changes in expression after CHI3L1 treatment or IL-13Rα2 knockdown in CAFs assessed using RT-qPCR assays. Angiogenesis assays revealed that tube formation in vascular endothelial cells was suppressed by conditioned medium from CAFs with the addition of human CHI3L1 neutralizing antibodies compared with control IgG, and also suppressed by conditioned medium from CAFs transfected with CHI3L1, IL-8 or VEGFA small interfering RNA compared with negative control small interfering RNA. Overall, the present findings indicated that CHI3L1 secreted from CAFs acted on CAFs to increase the secretion of IL-8, thereby affecting tumor angiogenesis in colorectal cancer.

Effect of a confirmatory testing algorithm on early acute HIV diagnosis in Korea.

ABSTRACT: The 17 Provincial Institutes of Health and Environment (PIHEs) in Korea use HIV antibody, antigen, and Western blot assays for confirmatory testing of HIV infection. The Korea Disease Control and Prevention Agency (KDCA) has further included p24 antigen neutralization and nucleic acid tests (NATs) since 2015. Our study aimed to investigate the effect of this new testing algorithm on the confirmation rate of HIV infection.Annual changes, from 2012 through 2017, in positive or indeterminate HIV confirmatory results were compared for the two algorithms between the PIHEs and the KDCA. Fiebig stages and Western blot p31 band were used to identify the diagnostic proportions of acute or early chronic HIV for the two algorithms.The number of positive cases in the samples requested from PIHEs for reconfirmation by the KDCA has steadily increased from 10.3% in 2014 to 33.3% in 2017. However, the number of indeterminate cases dropped sharply, from 71.9% in 2014 to 14.0% in 2017. The results for the p31 reactive band were 27.4% and 88.4% for the KDCA and PIHEs, respectively. Of positive cases reported by the KDCA, 22.9% were in the early acute stage and Fiebig stages I to II.The new testing algorithm has improved the diagnosis of HIV infections in the early acute stage. Early confirmatory diagnosis can prevent secondary transmission of HIV and provide early treatment opportunities for people living with HIV infection.

Multiclass cancer classification in fresh frozen and formalin-fixed paraffin-embedded tissue by DigiWest multiplex protein analysis.

Histomorphology and immunohistochemistry are the most common ways of cancer classification in routine cancer diagnostics, but often reach their limits in determining the organ origin in metastasis. These cancers of unknown primary, which are mostly adenocarcinomas or squamous cell carcinomas, therefore require more sophisticated methodologies of classification. Here, we report a multiplex protein profiling-based approach for the classification of fresh frozen and formalin-fixed paraffin-embedded (FFPE) cancer tissue samples using the digital western blot technique DigiWest. A DigiWest-compatible FFPE extraction protocol was developed, and a total of 634 antibodies were tested in an initial set of 16 FFPE samples covering tumors from different origins. Of the 303 detected antibodies, 102 yielded significant correlation of signals in 25 pairs of fresh frozen and FFPE primary tumor samples, including head and neck squamous cell carcinomas (HNSC), lung squamous cell carcinomas (LUSC), lung adenocarcinomas (LUAD), colorectal adenocarcinomas (COAD), and pancreatic adenocarcinomas (PAAD). For this signature of 102 analytes (covering 88 total proteins and 14 phosphoproteins), a support vector machine (SVM) algorithm was developed. This allowed for the classification of the tissue of origin for all five tumor types studied here with high overall accuracies in both fresh frozen (90.4%) and FFPE (77.6%) samples. In addition, the SVM classifier reached an overall accuracy of 88% in an independent validation cohort of 25 FFPE tumor samples. Our results indicate that DigiWest-based protein profiling represents a valuable method for cancer classification, yielding conclusive and decisive data not only from fresh frozen specimens but also FFPE samples, thus making this approach attractive for routine clinical applications.

Sensitive detection of fluorescence in western blotting by merging images.

The western blotting technique is widely used to analyze protein expression levels and protein molecular weight. The chemiluminescence method is mainly used for detection due to its high sensitivity and ease of manipulation, but it is unsuitable for detailed analyses because it cannot be used to detect multiple proteins simultaneously. Recently, more attention has been paid to the fluorescence detection method because it is more quantitative and is suitable for the detection of multiple proteins simultaneously. However, fluorescence detection can be limited by poor image resolution and low detection sensitivity. Here, we describe a method to detect fluorescence in western blots using fluorescence microscopy to obtain high-resolution images. In this method, filters and fluorescent dyes are optimized to enhance detection sensitivity to a level similar to that of the chemiluminescence method.

CDCA2 promotes lung adenocarcinoma cell proliferation and predicts poor survival in lung adenocarcinoma patients.

Cell division cycle associated 2(CDCA2) is overexpressed in neuroblastoma and oral squamous cell carcinoma, and its overexpression positively correlates to tumor progression. However, the biological and clinical significance of CDCA2 in lung adenocarcinoma(LAC) has never been investigated. We determined the expression profile and clinical significance of CDCA2 using The Cancer Genome Atlas(TCGA) and tissue microarray(TMA). Furthermore, we explored the biological function of CDCA2 both in vitro and in vivo. A great upregulation of CDCA2 was observed in LAC tissues compared with adjacent normal tissues. Importantly, Cox regression analysis indicated that high level of CDCA2 was an independent risk factor for overall survival(OS) in LAC patients (TCGA: HR = 1.720, p = 0.004; TMA: HR = 1.971, p = 0.023). Inhibition of CDCA2 suppressed the proliferation of LAC cells via G1 phase arrest by downregulating cyclin E1(CCNE1), while overexpression of CDCA2 promoted LAC cells proliferation by upregulating CCNE1. Moreover, the oncogenic activity of CDCA2 was also confirmed in vivo. In conclusion, CDCA2 promotes proliferation of LAC cells and predicts poor prognosis in LAC patients. CDCA2 might play a significant role in LAC progression.

Axl Is a Potential Cancer Prognostic Marker for the Migration and Invasion of Nasopharyngeal Carcinoma.

BACKGROUND: The Axl receptor tyrosine kinase has been demonstrated to be elevated and activated in many human cancers including liver, lung, breast, and pancreatic cancer. Its high expression has been considered as a cancer biomarker for predicting poor prognosis and increased invasiveness/metastasis. OBJECTIVES: The aim of the study was to investigate the clinical significance of Axl in nasopharyngeal carcinoma (NPC) and its role in cell migration and invasion. MATERIAL AND METHODS: We detected Axl expression in 86 collected NPC tissues and 20 collected normal nasopharyngeal epithelial tissues using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemical staining. Axl was knocked down by a specific shRNA in NPC cell lines, 5-8F and 6-10B. Transwell assays were used to determine NPC cell migration and invasion. RESULTS: The expressions of Axl mRNA and protein in NPC tissues were significantly higher than those in normal nasopharyngeal epithelial tissues (p < 0.05, respectively). The positive expression of Axl was significantly correlated with distant metastasis and high TNM stage in NPC (p < 0.05, respectively). Furthermore, Axl positive expression was correlated with a worse overall survival of NPC patients (p < 0.05). Multivariate Cox repression analysis indicated that Axl was an independent factor for predicting overall survival of NPC patients (p < 0.05). In vitro studies found that Axl knockdown significantly reduced the number of migrated and invaded 5-8F and 6-10B cells (p < 0.05, respectively). CONCLUSIONS: The positive expression of Axl is correlated with the poor clinicopathological features in NPC. Furthermore, Axl is an independent prognostic marker for predicting overall survival of NPC patients. Functionally, Axl may facilitate tumour progression by promoting NPC cell migration and invasion.

HIV testing patterns for United States Air Force personnel, 2008-2012.

OBJECTIVE: This study evaluated 3rd generation human immunodeficiency virus (HIV) test patterns and HIV infection rates in the United States Air Force (USAF). STUDY DESIGN: Retrospective database study. METHODS: HIV enzyme-linked immunoassay (ELISA) and Western blot tests were analysed for all USAF personnel from 2008 to 2012. For new HIV cases, unadjusted and adjusted annual rates were calculated per 100,000 persons. RESULTS: In total, 1,608,665 tests were performed in 626,298 individuals, with a reactive ELISA observed in 809 (0.001%) persons. Western blot (n = 1949) results included 378 (19.4%) positive, 1283 (65.8%) negative, and 288 (15.0%) indeterminate (WBi). Unadjusted annual HIV rates were between 16.7 and 20.6 per 100,000 persons during the study period. The overall age-adjusted rate was 14.8 cases per 100,000 persons tested. Blacks/African Americans had the highest risk of HIV (risk ratio 7.9 [95% confidence interval 5.78, 9.95] compared to Whites). CONCLUSIONS: WBi results, which can cause delays in determining HIV status, were relatively common with the 3rd generation assay. However, this will be mitigated by a planned transition to a 4th generation assay. Although the overall rate of HIV in the USAF is lower than US civilian adults, HIV prevention efforts targeting young Blacks/African Americans may help to reduce HIV incidence in the USAF.

MiR-215, an activator of the CTNNBIP1/ß-catenin pathway, is a marker of poor prognosis in human glioma.

MicroRNA-215 (miR-215) promotes tumor growth in various human malignancies. However, its role has not yet been determined in human glioma. Here, we found that levels of miR-215 were higher in glioma tissues than in corresponding non-neoplastic brain tissue. High miR-215 expression was correlated with higher World Health Organization (WHO) grades and shorter overall survival. Multivariate and univariate analysis indicated that miR-215 expression was an independent prognostic factor. We also found that TGF-beta1, phosphorylated beta-catenin, alpha-SMA, and fibronectin were increased in glioma tissues. Additionally, CTNNBIP1, a direct target of miR-215, was decreased in glioma compared to adjacent normal tissue. These data indicate that miR-215 activates Wnt/ß-catenin signaling by increasing ß-catenin phosphorylation, α-SMA expression, and fibronectin expression. It promotes TGF-ß1-induced oncogenesis by suppressing CTNNBIP1 in glioma. In summary, miR-215 is overexpressed in human glioma, is involved in TGF-ß1-induced oncogenesis, and can be used as a marker of poor prognosis in glioma patients.

Upregulation of spondin-2 predicts poor survival of colorectal carcinoma patients.

Colorectal cancer (CRC) is the third and second most common cancer in males and females worldwide, respectively. Spondin-2 is a conserved secreted extracellular matrix protein and a candidate cancer biomarker. Here we found that Spondin-2 mRNA was upregulated in CRC tissues using quantitative RT-PCR and data-mining of public Oncomine microarray datasets. Spondin-2 protein was increased in CRC tissues, as revealed by immunohistochemistry analyses of two tissue microarrays containing 180 cases. Spondin-2 gene expression was significantly associated with CRC stage, T stage, M stage and Dukes stage, while its protein was associated with age and M stage. Kaplan-Meier analysis revealed that the upregulated Spondin-2 mRNA and protein predicted poor prognosis of CRC patients. Univariate and multivariate Cox regression analyses indicated that grade, recurrence, N stage and high Spondin-2 were independent predictors of overall survival of CRC patients. ELISA revealed that plasma Spondin-2 was upregulated in CRC and dropped after surgery. Receiver operating characteristic curve analysis demonstrated that plasma Spondin-2 has superior predictive performance for CRC with an area under the curve of 0.959 and the best sensitivity/specificity of 100%/90%. Furthermore, ectopic expression of Spondin-2 enhanced colon cancer cell proliferation. Spondin-2 could be an independent diagnostic and prognostic biomarker of colon cancer.

MicroRNA-21 plays an oncogenic role by targeting FOXO1 and activating the PI3K/AKT pathway in diffuse large B-cell lymphoma.

The prognostic implications of miR-21, miR-17-92 and miR-155 were evaluated in diffuse large B-cell lymphoma (DLBCL) patients, and novel mechanism by which miR-21 contributes to the oncogenesis of DLBCL by regulating FOXO1 and PI3K/AKT/mTOR pathway was investigated. The expressions of miR-21, miR-17-92 and miR-155 measured by quantitative reverse-transcription-PCR were significantly up-regulated in DLBCL tissues (n=200) compared to control tonsils (P=0.012, P=0.001 and P<0.0001). Overexpression of miR-21 and miR-17-92 was significantly associated with shorter progression-free survival (P=0.003 and P=0.014) and overall survival (P=0.004 and P=0.012). High miR-21 was an independent prognostic factor in DLBCL patients treated with rituximab-combined chemotherapy. MiR-21 level was inversely correlated with the levels of FOXO1 and PTEN in DLBCL cell lines. Reporter-gene assay showed that miR-21 directly targeted and suppressed the FOXO1 expression, and subsequently inhibited Bim transcription in DLBCL cells. MiR-21 also down-regulated PTEN expression and consequently activated the PI3K/AKT/mTOR pathway, which further decreased FOXO1 expression. Moreover, miR-21 inhibitor suppressed the expression and activity of MDR1, thereby sensitizing DLBCL cells to doxorubicin. These data demonstrated that miR-21 plays an important oncogenic role in DLBCL by modulating the PI3K/AKT/mTOR/FOXO1 pathway at multiple levels resulting in strong prognostic implication. Therefore, targeting miR-21 may have therapeutic relevance in DLBCL.

Overexpression of AKIP1 promotes angiogenesis and lymphangiogenesis in human esophageal squamous cell carcinoma.

A-kinase-interacting protein 1 (AKIP1) is found to be overexpressed in breast and prostate cancers, suggesting that AKIP1 might act as a potent oncogenic protein. However, the clinical significance and biological role of AKIP1 in cancer progression remain largely unknown. Herein, we report that AKIP1 is markedly overexpressed in esophageal squamous cell carcinoma (ESCC) cell lines and clinical ESCC samples. AKIP1 expression significantly correlates with ESCC progression and patients' shorter survival time. Furthermore, we find that overexpressing AKIP1 induces, whereas silencing AKIP1 reduces, ESCC angiogenesis and lymphangiogenesis both in vitro and in vivo. Moreover, we demonstrate that AKIP1 transcriptionally upregulates vascular endothelial growth factor-C (VEGF-C) via interaction with its promoter through cooperation with multiple transcriptional factors, including SP1, AP2 and nuclear factor-κB (NF-κB). Importantly, significant correlation between levels of AKIP1 and VEGF-C is observed in a cohort of human ESCC, as well as in non-small cell lung cancer, hepatocellular carcinoma and ovarian cancer. Hence, these findings indicate an important role for AKIP1 in ESCC angiogenesis and lymphangiogenesis, and uncover a novel mechanism for the upregulation of VEGF-C in cancers.

Overexpression of CPE-ΔN predicts poor prognosis in colorectal cancer patients.

Carboxypeptidase E (CPE) is one of the most important carboxypeptidases involved in biosynthesis of numerous peptide hormones and neurotransmitters and has an important role in endocrine regulation. A splice variant of CPE (CPE-ΔN) has been detected and the mechanism of CPE-ΔN action in tumorigenesis has been studied in many different cancers. The aim of this study was to examine CPE-ΔN expression in human colorectal cancer (CRC) and to evaluate its possible use as a potential prognostic marker. Two hundred nineteen primary colorectal tumors and corresponding normal tissues were included in the study. We have analyzed CPE-ΔN isoform expression by qRT-PCR and Western blot in 219 CRC patients. Correlations between CPE-ΔN mRNA expression and clinicopathological variables were determined with chi-square tests. Survival probabilities were determined using Kaplan-Meier analysis, and univariate and multivariate analyses of the prognostic factors were performed with a Cox regression model. Our results show that CPE-ΔN is overexpressed in colorectal tumor tissue and that high CPE-ΔN mRNA expression is closely correlated with tumor differentiation, pT classification, pN classification, tumor recurrence, and lymph node metastasis (P = 0.042, 0.036, 0.031, 0.006, and 0.008, respectively). However, no correlation was observed between CPE-ΔN expression and age, gender, tumor localization, gross features, and the tumor size. In addition, patients with high CPE-ΔN expression had a significantly shorter survival (P < 0.001, logrank test). Tumor differentiation, gross feature, pT classification, pN classification, tumor recurrence, lymph node metastasis, and CPE-ΔN status were significantly associated with poor prognosis after performing a univariate Cox survival analysis. High CPE-ΔN expression was also identified as an independent prognostic factor using a multivariate analysis (P = 0.011). Based on these results, we can conclude that CPE-ΔN expression might be a potential prognostic marker for colorectal cancer patients.

Snail is an independent prognostic predictor for progression and patient survival of gastric cancer.

The present study investigated the clinical significance of Snail, a zinc-finger transcription factor, in the development and progression of gastric cancer. To elucidate the relationship between Snail expression and dedifferentiation status with cancer stem cell phenotype in gastric cancer cells, we used western blot analysis, RT-PCR, quantitative real-time PCR and flow cytometry. Immunohistochemistry staining and evaluation of Snail expression in 10 human normal gastric samples versus 103 clinicopathologically characterized gastric cancer tissues followed by statistical analyses were applied to evaluate the prognostic value of Snail expression for progression and patient survival of gastric carcinomas. The results showed that functional Snail expression interlinks dedifferentiation status with cancer stem cell phenotype in gastric cancer cells. In addition, expression levels of Snail in gastric cancer tissues were significantly associated with tumor cell differentiation, local tumor growth, lymph node status, distant metastasis and tumor stage. The overall survival rate of gastric cancer patients with high Snail expression was significantly lower than for those patients with low Snail expression. Multivariate Cox regression analyses showed that Snail expression is an independent prognostic predictor for patient survival of gastric carcinomas. Thus, our data suggest that Snail expression could be a reliable independent prognostic factor to predict gastric carcinoma progression, which might open a new avenue for potential clinical intervention with functional Snail expression in gastric cancer patients.

High sensitivity of an ELISA kit for detection of the gamma-isoform of 14-3-3 proteins: usefulness in laboratory diagnosis of human prion disease.

BACKGROUND: The gamma-isoform of the 14-3-3 protein (14-3-3 gamma) is expressed in neurons, and could be a specific marker for neuronal damage. This protein has been reported as a detectable biomarker, especially in the cerebrospinal fluid (CSF) of Creutzfeldt-Jakob disease (CJD) patients by Western blotting (WB) or enzyme-linked immunosorbent assays (ELISAs). Western blotting for 14-3-3 gamma is not sensitive, and the reported data are conflicting among publications. An ELISA specific for 14-3-3 gamma is not available. METHODS: CJD patients (n=114 sporadic CJD patients, 7 genetic CJD, and 3 iatrogenic CJD) and 99 patients with other neurodegenerative diseases were examined in this study. The CSF samples obtained were analyzed by Western blotting for 14-3-3 gamma, and by ELISA for total tau protein. We evaluated the sensitivity and specificity of the newly developed sandwich ELISA for 14-3-3 gamma. RESULTS: The cut-off value of the 14-3-3 gamma ELISA was >1, 683 AU/ml; and sensitivity was 95.2%, with 72.7% specificity. This specificity was the same for the total tau protein ELISA. Seven CJD cases were negative by WB but positive using the 14-3-3 gamma ELISA, indicating that the ELISA is more sensitive. All 21 cases of early stage CJD could be diagnosed using a combination of the 14-3-3γ ELISA and diffusion weighted MR imaging (DWI-MRI). CONCLUSION: The 14-3-3 gamma ELISA was more sensitive than conventional WB, and was useful for laboratory diagnosis of CJD, similar to the ELISA for the tau protein. Using DWI-MRI and these ELISA tests on CSF, diagnosis of CJD will be possible even at early stages of the disease.

Evidence for disease and antipsychotic medication effects in post-mortem brain from schizophrenia patients.

Extensive research has been conducted on post-mortem brain tissue in schizophrenia (SCZ), particularly the dorsolateral prefrontal cortex (DLPFC). However, to what extent the reported changes are due to the disorder itself, and which are the cumulative effects of lifetime medication remains to be determined. In this study, we employed label-free liquid chromatography-mass spectrometry-based proteomic and proton nuclear magnetic resonance-based metabonomic profiling approaches to investigate DLPFC tissue from two cohorts of SCZ patients grouped according to their lifetime antipsychotic dose, together with tissue from bipolar disorder (BPD) subjects, and normal controls (n=10 per group). Both techniques showed profound changes in tissue from low-cumulative-medication SCZ subjects, but few changes in tissue from medium-cumulative-medication subjects. Protein expression changes were validated by Western blot and investigated further in a third group of subjects who were subjected to high-cumulative-medication over the course of their lifetime. Furthermore, key protein expression and metabolite level changes correlated significantly with lifetime antipsychotic dose. This suggests that the detected changes are present before antipsychotic therapy and, moreover, may be normalized with treatment. Overall, our analyses revealed novel protein and metabolite changes in low-cumulative-medication subjects associated with synaptogenesis, neuritic dynamics, presynaptic vesicle cycling, amino acid and glutamine metabolism, and energy buffering systems. Most of these markers were altered specifically in SCZ as determined by analysis of the same brain region from BPD patients.

Most positive HIV western blot tests do not diagnose new cases in New York City: implications for HIV testing programs.

OBJECTIVE: To evaluate HIV testing efforts based on surveillance data. METHODS: We determined the contribution of new diagnoses to all positive confidential HIV-1 Western blotting conducted in New York City between 2004 and 2006 based on clinical history recorded in the HIV Surveillance Registry, by testing site type. RESULTS: Of 31,504 positive Western blots reported and linked to Registry cases, 36.8% were new diagnoses and 63.2% were repeat positive tests. City health department clinics and private physicians' offices reported greater proportions of new diagnoses than other testing sites (64.4% and 58.3% vs. 31.1%). The percentage of positive tests at health department clinics that were new diagnoses increased from 59.8% in 2004 to 69.0% in 2006 (P = 0.001), coinciding with efforts to expand HIV testing. Repeat positive testers were significantly older, more likely to have an injection drug use history or AIDS, and less likely to be foreign-born. CONCLUSIONS: Repeat testing of known HIV-infected persons is common and an inefficient use of HIV prevention resources when the purpose of testing is to diagnose previously unidentified infections. Initiatives to increase HIV testing should be evaluated routinely using surveillance data to determine the proportion of infected persons identified who are newly diagnosed.

[Expression of transcription factor Sp1 in human gastric cancer tissue and its correlation with prognosis].

OBJECTIVE: To investigate the expression of transcription factor Sp1 in gastric cancer tissue and its correlation with prognosis. METHODS: Sp1 expression patterns in specimens of 86 gastric cancers, 57 normal gastric tissues and 53 metastatic lymph nodes were detected by immunohistochemical method. The activity of Sp1 in the tumor and normal tissues was examined by electrophoretic mobility shift analysis (EMSA). The correlation between transcription factor Sp1 expression of tumors and patients' prognosis were statistically analyzed using Cox proportional hazard model. RESULTS: In normal gastric tissue, Sp1 protein was predominantly expressed in the nuclei of cell located in the mucous neck region, but neither in the gastric pit cells with foveolar differentiation, nor in cells of the glandular epithelium with glandular differentiation. Strong Sp1 expression was also detected in tumor cells, but very weak or even no Sp1 expression in stromal cells or normal glandular cells surrounding the tumor. The median survival time of patients with negative, weak, and strong Sp1 expression was 43, 37, and 8 months, respectively (P < 0.01). Spl expression (P < 0.01) and stage (P < 0.001) were demonstrated as independent prognostic factor by multivariate analysis. CONCLUSION: Normal and malignant gastric tissue are found to have its own unique Sp1 expression patterns. Sp1 expression may be used as an important survival predictor in human gastric cancer.

TRPV1 recapitulates native capsaicin receptor in sensory neurons in association with Fas-associated factor 1.

TRPV1, a cloned capsaicin receptor, is a molecular sensor for detecting adverse stimuli and a key element for inflammatory nociception and represents biophysical properties of native channel. However, there seems to be a marked difference between TRPV1 and native capsaicin receptors in the pharmacological response profiles to vanilloids or acid. One plausible explanation for this overt discrepancy is the presence of regulatory proteins associated with TRPV1. Here, we identify Fas-associated factor 1 (FAF1) as a regulatory factor, which is coexpressed with and binds to TRPV1 in sensory neurons. When expressed heterologously, FAF1 reduces the responses of TRPV1 to capsaicin, acid, and heat, to the pharmacological level of native capsaicin receptor in sensory neurons. Furthermore, silencing FAF1 by RNA interference augments capsaicin-sensitive current in native sensory neurons. We therefore conclude that FAF1 forms an integral component of the vanilloid receptor complex and that it constitutively modulates the sensitivity of TRPV1 to various noxious stimuli in sensory neurons.

Immunodetection of the bacteriocin lacticin RM: analysis of the influence of temperature and Tween 80 on its expression and activity.

Immunoassays with specific antibodies offer higher sensitivity than do bioassays with indicator strains in the detection and quantification of several bacteriocins. Here we present the purification of lacticin RM and the production of specific polyclonal antibodies to a synthetic peptide resembling an internal fragment of the mature bacteriocin. The specificity and sensitivity of the generated polyclonal antibodies were evaluated in various immunoassays. The detection limits of lacticin RM were found to be 1.9, 0.16, and 0.18 micro g ml(-1) for Western blot, immuno-dot blot, and noncompetitive indirect enzyme-linked immunosorbent assays, respectively. Immunoassay sensitivities were 12.5-fold higher than that of the agar diffusion test (ADT). The production of lacticin RM showed temperature dependency, with 3, 4.2, 12.7, 28.9, 37.8, and 12 micro g ml(-1) at 37, 30, 20, 15, 10, and 4 degrees C, respectively. Temperature-stability analysis demonstrated that lacticin RM is sensitive to mild temperature, but the loss of activity does not seem to result from protein degradation. Tween 80 increased the concentration of lacticin RM eightfold and probably affected the results of the ADT either by enhancing the activity of lacticin RM or by increasing the sensitivity of the indicator strain. The use of antibodies for the specific detection and quantification of lacticin RM can expand our knowledge of its production and stability, with important implications for further investigation and future application.