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PubMed2005

Arsenic-contaminated water and extent of acute childhood malnutrition (wasting) in rural Bangladesh.

To investigate whether children's nutritional status, especially the extent of wasting [acute malnutrition as measured by weight-for-height Z (WHZ) score] is associated with arsenic contamination of drinking water, a survey was carried out in two discrete rural areas located less than 30 km southeast and northeast of Dhaka, the capital of Bangladesh. 761 children between 7 and 14 years of age were randomly selected, one child per household. The arsenic concentration in the tube well water used by each child and the prevalence and intensity of infection of three geo-helminths [Ascaris lumbricoides, Trichuris trichiura and hookworm (Ancylostoma duodenale or Necator americanus)] were determined. Each child had their height and weight measured and WHZ score determined using the World Health Organization (WHO) reference values. Basic socio-demographic data and knowledge of arsenic poisoning were collected using a questionnaire. 62.2% of the households studied were using well water with arsenic concentrations above the 0.01 mg/L WHO guideline (i.e. arsenic-contaminated households). The mean WHZ score of all the 761 children was -0.843 (sd 1.227) but children living in contaminated households were significantly more wasted than children living in noncontaminated households (mean difference = -0.361, p 0.001). When the effects of the differences in socio-economic status and prevalence of geo-helminths were also taken into account, the difference in means remained highly significant (-0.330, p = 0.006). This study suggests that arsenic contamination has a negative impact on children's acute nutritional status.

Acute Disease↗

PubMed2006

Preparation of germanium apatite and its cytotoxicity.

Germanium apatite was synthesized via the solid-state reaction between GeO(2) and (NH(4))(3)PO(4). The synthesized materials were characterized using XRD, and thermal analysis was carried out using TG-DTA. Ge(2)P(2)O(7) was preferentially produced at temperatures between 300-900 degrees C, and at temperatures above 1000 degrees C, germanium apatite (Ge(5)O(PO(4))(6), GeAp) was synthesized. In solubility tests, 0.36% and 0.65% of Ge ions were liberated from GeAp powder in distilled water at 37 and 80 degrees C after four weeks, respectively. A GeAp aqueous solution maintained at 37 degrees C was strongly acidic with a pH=1.67 after four weeks. The growth rate of human adult gingival fibroblast cells in a medium that included GeAp, HA, and GeO(2) was investigated. The growth rate of the cells in a 0.1 mg/ml GeAp medium was almost the same as that in the control. The cell growth was restricted in a 1.0 mg/ml GeAp medium, whereas the cell growth in a pH-adjusted 1.0 mg/ml GeAp medium at pH=7.60 was higher than that in non-adjusted medium at pH = 7.06.

Apatites↗

PubMedDate not supplied

[Space-temporal distribution of Boophilus microplus (Acari: Ixodidae) ticks analyzed by geoprocessing in the municipality of Seropédica, state of Rio de Janeiro, Brazil].

The Geo-Environmental System of the Federal University of Rio de Janeiro was used with the objective of analyzing the space-temporal distribution of Boophilus microplus ticks in the municipality of Seropédica in different types of pasture, breeding and animal density. Geo-environmental variables, climatic parameters, type of pasture, breeding and animal densities were evaluated and the results were signed. The pasture areas were categorized as very favorable, favorable, little favorable and unfavorable to the development of B. microplus. The lowest favorable rates occurred in the summer and the highest favorable rates occurred in the winter. In the summer, 100% of the areas were unfavorable with animal densities from 1.1 to 1.4 animal unit per hectare (AUH), native pastures and resistant animals. On the other hand, with susceptible animals, keeping the same conditions, the areas were classified as little favorable. Favorability was similar for animal densities from 0.5 to 1.4 AUH and was higher for animal densities from 1.5 to 1.9 AUH. In the winter, the areas were unfavorable with similar rates when resistant animals, with densities < 0.5AUH, native and mixed pastures were used. However, the area of occurrence of ticks was enlarged when cultivated pastures were used. Keeping the same conditions, but using susceptible animals, the favorability rate changed to little favorable for native and mixed pastures, expanding to favorable in cultivated pastures. The methodology used allowed the identification and the quantification of areas of occurrence of B. microplus, as well as its temporal distribution and the effects of animal density, breeding and pasture variations.

Animals↗

PubMed1991

Role of the urokinase receptor in facilitating extracellular matrix invasion by cultured colon cancer.

There is increasing evidence that urokinase secreted by tumor cells can be bound to a cell surface receptor retaining its full potential to activate plasminogen and subsequently cleave basement membrane constituents. This study was undertaken to discriminate between soluble and cell surface bound urokinase as a potential mediator of in vitro invasion by cultured colon cancer. Extracellular matrix invasion by a colon cancer cell line GEO, characterized as being a poor secretor of urokinase and having few receptors (less than 10(4) receptors/cell) was not augmented when these cells were made to secrete up to 8 times as much urokinase, in response to an exogenous urokinase gene driven by the Rous sarcoma virus long terminal repeat promoter. The majority of the plasminogen activator (greater than 95%) appeared in the culture medium, this reflecting the low numbers of binding sites displayed by GEO cells. In contrast, the cell line HCT 116 equipped with 10 times as many binding sites, (greater than 10(5)/cell), the majority of which are occupied with endogenous ligand, was an efficient invader of the extracellular matrix. Inhibition of urokinase binding to the cell surface receptors using an antibody to the A chain of the plasminogen activator reduced invasion by 65%. The cell line RKO is equipped with 3 x 10(5) receptors/cell, 15% of which are tagged with endogenous urokinase. Pretreatment of these cells with a concentration range of urokinase known to result in the majority of these binding sites being charged with the plasminogen activator led to a dose dependent increase in extracellular matrix invasion. Together, these data suggest that for cultured colon cancer, at least, invasion is a function of the amount of cell surface receptor bound urokinase.

Colonic Neoplasms↗

PubMed2006

Tuberculosis risks and socio-economic level: a case study of a city in the Brazilian south-east, 1998-2004.

OBJECTIVES: To explore tuberculosis (TB) risks in relation to potential determinants in the city of São José do Rio Preto, São Paulo State, Brazil; to analyse morbidity and mortality indicators in São José do Rio Preto, and to determine the relationship between the risk of TB and socio-economic level (SEL) using a geo-referenced information system (GIS) and the national census for 2000. METHOD: Standardised incidence rates and TB incidence and mortality rates were calculated. Socio-economic variables were determined using the statistical technique of principal component analysis. Data sources were the São Paulo State Data Analysis System (SEADE), the TB Notification Database (EPI-TB), the Information Department of the Brazilian Health Ministry (DATASUS), and the Brazilian Institute of Geography and Statistics (IBGE). New cases reported in 1998-1999 and 2003-2004 in the urban area of the city were geo-referenced and analysed. RESULTS: TB risk in the city is twice as high in areas of lower SEL than in areas with higher SEL. CONCLUSION: The identification of areas with different levels of risk enables the Municipal Health Department to propose innovative interventions to minimise the risk of disease at both individual and population level.

Brazil↗

PubMed2006

[Aboveground biomass of Tamarix on piedmont plain of Tianshan Mountains south slope].

Based on the geo-morphological and hydro-geological characteristics, the piedmont plain of Tianshan Mountains south slope was classified into 4 geo-morphological belts, i.e., flood erosion belt, groundwater spill belt, delta belt, and the joining belt of piedmont plain and Tarim floodplain. A field investigation on the Tamarix shrub in this region showed that there was a significant difference in its aboveground biomass among the four belts, ranged from 1428.53 kg x hm(-2) at groundwater spill belt to 111.18 kg x hm(-2) at the joining belt of piedmont plain and Tarim floodplain. The main reason for such a big difference might be the different density of Tamarix shrub on different belts. Both the Tamarix aboveground biomass and the topsoil's salinity were decreased with increasing groundwater level. Groundwater level was the main factor limiting Tamarix growth, while soil salinity was not.

Biomass↗

PubMed1988

Determination of the levels of urokinase and its receptor in human colon carcinoma cell lines.

At present, there is a lack of availability of differentiation markers for colon carcinoma. This may, in part, be a consequence of the diversified function of the normal human colon. This study addresses the possibility that the expression of urokinase and its receptor is inversely related to differentiation in colon carcinoma. Six colon carcinoma cell lines including three well-differentiated (CBS, GEO, FET) and three poorly differentiated ones (HCT116, HCT116b, RKO) were screened for urokinase receptor display and secretion of the plasminogen activator. A radioreceptor assay was used to determine receptor levels. Binding of radioactive urokinase to colon cells was saturable, specific, and time dependent. Cell-bound 125I-labeled protease was unaffected by the presence of epidermal growth factor, low-molecular-weight urokinase, plasminogen, or transferrin. Time course studies revealed that maximum amounts of radioactive tracer were bound in a 30-min period with no change occurring over the course of a 90-min incubation. Scatchard analysis of ligand binding indicated that the well-and poorly differentiated cells could be separated on the basis of receptor display; the aggressive RKO, HCT116, and HCT116b expressed in excess of 10(5) sites per cell, while the more indolent CBS, GEO, and FET possessed less than 1.5 X 10(4) receptors per cell. The colon carcinoma cells were also analyzed for urokinase in the conditioned medium. Low levels of the plasminogen activator (0.8 to 1.3 ng/ml/10(6) cells/72 h) were associated with the more "mature" cells. This was in contrast to the elevated levels of the protease (3.9 to 11.4 ng/ml/10(6) cells/72 h) present in the medium derived from the more aggressive cells (HCT 116, HCT116b, RKO). Thus, secreted urokinase and/or the expression of cellular receptor for the plasminogen activator may provide useful measurements of the degree of undifferentiation of in vitro colon carcinoma.

Binding, Competitive↗

PubMed1997

Role of alpha 5 beta 1 integrin in determining malignant properties of colon carcinoma cells.

We characterized the expression of alpha 5 beta 1 integrin in two distinct phenotypes of colon carcinoma cell lines. Highly invasive colon cell lines (designated Group I cell lines) expressed higher levels of integrin alpha 5 beta 1 mRNA and protein than did poorly invasive colon cell lines (designated Group III cell lines). The relatively high expression of integrin alpha 5 beta 1 in Group I cell lines resulted in strong enhancement of cell adhesion to fibronectin (FN) tissue culture plates, whereas Group III cell lines showed little or no enhancement of cell adhesion by coating. There was no significant difference between Group I and Group III cell lines with respect to cell adhesion to laminin and collagen IV. Cell adhesion to FN in Group I cells was mainly mediated by integrin alpha 5 beta 1 because a monoclonal anti-alpha 5 subunit antibody could block cell adhesion to FN, whereas anti-alpha 2 and anti-alpha 3 antibodies had no effect on cell adhesion to FN. The divergence of alpha 5 beta 1 expression in these two distinct colon carcinoma phenotypes suggested that high expression of alpha 5 beta 1 might contribute to malignant progression in this model system. To test this hypothesis, GEO cells, a Group III cell line that did not express alpha 5 integrin, were transfected with the alpha 5 subunit. Stable transfection of alpha 5 sense cDNA into a typical GEO-limiting dilution clone led to the expression of alpha 5 subunit mRNA and cell surface alpha 5 beta 1 protein. The alpha 5 sense transfectants showed enhanced attachment to FN-coated plates and were more tumorigenic when the cells were injected into athymic nude mice. These results indicate that inappropriately high alpha 5 beta 1 integrin expression contributes to malignant progression in colon carcinoma.

Animals↗

PubMed1997

8-Chloro-cyclic AMP inhibits autocrine and angiogenic growth factor production in human colorectal and breast cancer.

8-Chloro-cyclic AMP (8-Cl-cAMP) is a cAMP analogue that specifically down-regulates type I protein kinase A, a signaling protein directly involved in cell proliferation and neoplastic transformation, and that causes growth inhibition in a variety of human cancer cell types. In this report, we have investigated the effects of 8-Cl-cAMP on the expression of several growth factors in human colon (GEO and LS174T) and breast (MDA-MB468) cancer cell lines. 8-Cl-cAMP treatment caused in the three cancer cell lines a significant dose- and time-dependent inhibition in the expression of various endogenous autocrine growth factors, such as transforming growth factor alpha, amphiregulin, and CRIPTO, and of two angiogenic factors, such as vascular endothelial growth factor and basic fibroblast growth factor, at both the mRNA and protein levels. Furthermore, 8-Cl-cAMP treatment markedly inhibited the ability of all three cell lines to invade a basement membrane matrix in a chemoinvasion assay. Finally, 8-Cl-cAMP-induced inhibition of GEO tumor growth in nude mice was accompanied by a significant suppression of transforming growth factor alpha, amphiregulin, CRIPTO, basic fibroblast growth factor, and vascular endothelial growth factor production by the tumor cells, and of neoangiogenesis, as detected by factor VIII staining of host blood cells. These results demonstrate that 8-Cl-cAMP is a novel anticancer drug that inhibits the production of various autocrine and paracrine tumor growth factors that are important in sustaining autonomous local growth and facilitate invasion and metastasis.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed2026

Integrative analysis and experiment validation of SLC12A8 as a biomarker for the malignant transition from endometriosis to endometriosis associated ovarian cancer.

Endometriosis (EM) is a chronic inflammatory, estrogen‑dependent benign gynecological disorder. A subset of patients with EM may subsequently develop endometriosis‑associated ovarian cancer (EAOC), implying a biological continuum between these two conditions. Nevertheless, the molecular events underlying the progression from benign endometriotic lesions toward EAOC remain incompletely characterized. In this study, transcriptomic datasets retrieved from the GEO database were interrogated through differentially expressed gene screening, functional enrichment analysis, and weighted gene co‑expression network analysis (WGCNA) to identify key genes and pathways relevant to EM and EAOC. Candidate genes were further prioritized by integrating survival analysis via the Kaplan‑Meier Plotter, LASSO regression, random‑forest modeling, and CIBERSORT immune‑infiltration profiling. Loss and gain‑of‑function cellular models were established using siRNA and overexpression plasmids, and in‑vitro functional assays were performed to characterize the phenotypic effects of target genes.We identified several candidate genes associated with EM and EAOC and evaluated their discriminatory performance. Among them, SLC12A8 elevated expression across EM and EAOC tissues and exhibited moderate diagnostic capacity. Higher SLC12A8 expression was also associated with poorer prognosis in EAOC patients. In‑vitro experiments further demonstrated that SLC12A8 modulates proliferation, invasion, and migration in both EM and EAOC cell lines. Collectively, our exploratory research findings support SLC12A8 as a candidate functional mediator and potential biomarker linked to EM‑EAOC pathological progression, thereby extending the mechanistic understanding of these disorders.

Female↗

PubMed2025

Discovery of novel diagnostic biomarkers of hepatocellular carcinoma associated with immune infiltration.

OBJECTIVE: Diagnosis of hepatocellular carcinoma (HCC) remains challenging for clinicians. Machine learning approaches and big data analyses are viable strategies for identifying HCC diagnostic markers. MATERIALS AND METHODS: In this study, we downloaded mRNA expression profiles of HCC from the GEO database and used random forest and machine learning algorithms, such as least absolute shrinkage and selection operator, to screen for reliable diagnostic genes. Disease Ontology, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Set Enrichment Analysis enrichment analyses were performed to explore differential gene functions and disease pathways. CIBERSORT was performed to calculate the immune cell infiltration of HCC and the correlation between diagnostic genes and immune cells. Cell experiments were performed to evaluate the function of R-spondin 3 (RSPO3) in HCC cells. Immunohistochemical staining was used to evaluate the protein expression of CD138, CD206 and iNOS. RESULTS: The results indicated that extracellular matrix protein 1 (ECM1), Niemann-Pick C1-Like 1 (NPC1L1) and RSPO3 were down-regulated in HCC compared with the normal group (p < 0.05), which was validated in clinical tissue samples. Moreover, ECM1, NPC1L1 and RSPO3 had high diagnostic values (AUC > 0.75) for HCC in both training and test groups. Immuno-infiltration analysis revealed that ECM1 and RSPO3 were highly positively correlated with neutrophil and macrophage M2 levels, whereas they were negatively correlated with Tregs. RSPO3-si affected cell proliferation and apoptosis in HCC. Furthermore, RSPO3 exhibited a positive correlation with tumour progression, the proportion of plasma cells and M2 macrophages in mice, while showing a negative association with M1 macrophages. CONCLUSION: The present study identified ECM1, NPC1L1 and RSPO3 as new diagnostic biomarkers for HCC based on normal and diseased samples from HCC, meanwhile the pro-oncogenic function of RSPO3 and its regulation on immune infiltration have been confirmed.

Carcinoma, Hepatocellular↗

PubMed2025

Anoikis classification of lung squamous cell carcinoma reveals correlation with clinical prognosis and immune characteristics.

BACKGROUND: Anoikis is a new mode of cell death that has been shown to correlate significantly with tumors. However, the clinical prognostic significance of anoikis in lung squamous cell carcinoma (LUSC) remains poorly studied. METHODS: The differentially expressed ARGs and candidate genes were selected by the differential analysis to construct a predictive model. Independent prognostic gene was determined by Cox and LASSO analysis and we used the HCC95 and NCI H520 cell line to verify the gene function. We used the data from TCGA, GEO, GeneCards, and Harmonizome databases to analyze the immune microenvironment, functional enrichment, and drug sensitivity analysis. RESULTS: We identified 717 differentially expressed and selected 3 ARGs (FADD, SNAI1, and BAG4) to construct a predictive model. We found that SNAI1 is an independent prognostic gene and confirmed that knocking out the SNAI1 inhibited the HCC95/NCI H520 cell proliferation. We used single-sample gene-set enrichment analysis (ssGSEA) to evaluate the immune infiltration based on the 3 ARG expression levels. We constructed a risk score and provided a visual representation of the prophetic implications of the ARGs-based signature through a nomogram. We found 15 susceptible drugs in the high-risk group and 15 sensitive drugs in the low-risk group by the drug sensitivity analysis. CONCLUSION: We used ARGs to construct a prognosis model for LUSC that can accurately predict the prognosis of LUSC patients. ARGs, especially SNAI1, play an essential role in developing LUSC. These findings could provide individualized treatment plans and new research ideas for LUSC patients.

Humans↗

PubMed2026

Identification of NR4A2 as a Potential Predictive Biomarker for Atherosclerosis.

INTRODUCTION/OBJECTIVE: Atherosclerosis, a leading cause of death globally, is characterized by the buildup of immune cells and lipids in medium to large-sized arteries. However, its precise mechanism remains unclear. The purpose of this study is to explore innovative and reliable biomarkers as a viable approach for the identification and management of atherosclerosis. METHODS: The atherosclerosis-related datasets GSE100927 and GSE66360 were retrieved from the Gene Expression Omnibus (GEO) database. The Limma package in the R programming language was utilized, applying the criteria of |logFC| > 1 and P < 0.05. Subsequently, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed on the 127 identified DEGs using R. Machine learning techniques were then applied to these data to explore and pinpoint potential biomarkers. The diagnostic potential of these markers was assessed via Receiver Operating Characteristic (ROC) curve analysis. Finally, western blot, real-time quantitative PCR (qRT-PCR), and immunohistochemistry (IHC) were employed to confirm the key biomarkers. RESULTS: Our research indicated that a total of 127 DEGs linked to atherosclerosis were successfully identified. Through the application of machine learning methods, eight critical genes were highlighted. Among these, Nuclear Receptor Subfamily 4 Group A Member-2 (NR4A2) emerged as the most promising marker for further investigation. CIBERSORT analysis revealed that NR4A2 expression levels were significantly correlated with multiple immune cell types, including B cells, plasma cells, and macrophages. Additional validation experiments confirmed that NR4A2 expression was indeed elevated in atherosclerotic plaques, supporting its potential as a biomarker for atherosclerosis. CONCLUSION: Our study identified NR4A2 as a potential immune-related biomarker for the diagnosis and treatment of atherosclerosis.

Atherosclerosis↗

PubMed2025

CS Ratio is an immune-related prognostic biomarker for cervical cancer.

BACKGROUND: The tumor microenvironment (TME) plays a crucial role in cancer progression but its complex structure significant variability among patients present considerable challenges for research. Recent studies have demonstrated that macrophage polarization states defined by the expression levels of CXCL9 SPP1 (CS Ratio) are more prognostically relevant than traditional M1/M2 markers. The CS polarization state reflects a highly coordinated network of pro-tumor anti-tumor variables offering a simplified yet effective immune response indicator for the complex TME. The CS Ratio has been shown to correlate with the abundance of anti-tumor immune cells the gene expression programs of tumor-infiltrating cells responses to immunotherapy. Cervical cancer, one of the most common gynecological malignancies, still faces limited therapeutic options. CXCL9, a member of the CXC chemokine family, plays a critical role in immune regulation, inflammation, tumor growth, angiogenesis, and metastasis. Similarly, SPP1, a cytokine, influences immune-related pathways by regulating molecules such as interferon-γ and interleukin-12. However, no studies have systematically investigated the role of the CS Ratio in cervical cancer or its relationship with immunotherapy characteristics. Research in this area could provide critical insights into the role and clinical potential of the CS Ratio in cervical cancer and related tumors. METHODS: The expression ratio of CXCL9 to SPP1 was analyzed in cervical cancer patients using data from the Gene Expression Omnibus (GEO) database, which revealed significant differences. Data for cervical cancer patients were obtained from The Cancer Genome Atlas (TCGA) database. The optimal cutoff value for the CS Ratio was determined using the maxstat package in R, and Kaplan-Meier (KM) survival curves were constructed. Patients were categorized into High and Low groups based on the median CS Ratio. Immune scores were analyzed, and immune cell infiltration was assessed using CIBERSORT. Differences in the CS Ratio were evaluated across patients with varying pathological T stages and FIGO stages. Additionally, receiver operating characteristic (ROC) analysis was performed using the pROC package in R to calculate the area under the curve (AUC). Univariate and multivariate Cox regression analyses were performed to evaluate the potential of the CS Ratio as an independent prognostic factor in cervical cancer. A Cox regression-based nomogram integrating four key features was subsequently developed for the TCGA-CESC cohort. Nomogram performance was assessed using calibration curves and ROC analysis. RESULTS: The CS Ratio was significantly lower in cervical cancer patients compared to normal controls (P < 0.05). KM survival curves indicated that patients in the CS High group exhibited better prognoses. Immune score analysis revealed significantly higher immune scores (P < 0.05) and lower tumor purity (P < 0.05)in the CS High group compared to the Low group. CIBERSORT analysis revealed significantly higher proportions of CD8+ T cells (P < 0.05) and M1 macrophages (P < 0.05), and a significantly lower proportion of M2 macrophages (P < 0.05), in the CS High group compared to the Low group. The CS Ratio significantly decreased with advancing FIGO stage (P < 0.05). Both univariate (P < 0.05) and multivariate Cox regression analyses (P < 0.05) confirmed the CS Ratio as an independent prognostic factor. ROC analysis demonstrated that the CS Ratio had higher AUC values for predicting 1-year (AUC=0.69), 3-year (AUC=0.66), and 5-year OS (AUC=0.68) than CXCL9 or SPP1 alone. The Cox regression-based nomogram integrating four key features demonstrated predictive capability for 1-, 3-, and 5-year OS in CESC patients (Concordance Index = 0.751; 95% CI: 0.678-0.824; p = 1.50Í10-11). Significant survival differences were observed between the high-risk and low-risk groups based on the nomogram score. ROC analysis yielded high AUC values for survival prediction: 0.85 (95% CI: 0.94-0.75) at 1-year, 0.74 (95% CI:0.84-0.64) at 3-year, and 0.72 (95% CI:0.84-0.61) at 5-year. CONCLUSION: The CS Ratio may serve as a more effective prognostic biomarker for cervical cancer patients.

CXCL9↗

PubMed1998

Gene trap expression and mutational analysis for genes involved in the development of the mammalian nervous system.

We have used a large-scale gene trap approach for the isolation and mutation of genes that might play roles in the developing nervous system. After in vitro integration of two different gene trap vectors (pGT1.8geo: Skarnes et al. [1995] Proc. Natl. Acad. Sci. USA 92:6592-6596; IRES beta geo: Chowdhury et al. [1997] Nucleic Acids Res. 25:1531-1536) in mouse embryonic stem (ES) cell lines, we created 64 transgenic mouse lines. The expression analysis of the reporter gene during embryogenesis of heterozygous embryos revealed 47 lines with a variety of patterns. Around one-third (36%) of these gene trap lines showed spatiotemporal expression that was either restricted predominantly in the developing nervous system (11 lines; 17%) or widespread but with very high levels of expression in the nervous tissue (12 lines; 19%). In most cases, a correlation was found between the in vitro and the in vivo patterns of the reporter gene expression. Thus far, preliminary mutant analysis of 16 gene trap lines with potentially interesting expression patterns in the developing nervous system showed that mice homozygous for eight (50%) insertions were lethal, whereas the homozygous mice from five gene trap lines (31%) showed a lower than expected Mendelian ratio of live homozygous animals. Analysis of beta-galactosidase reporter gene expression during embryogenesis has shown that four transgenic lines are useful lacZ in situ markers for specific regions of the developing nervous system. Here, we discuss some in vivo and in vitro selection criteria that may increase the number of the trapped genes potentially involved in the control of neural development and some future strategies to improve further the efficiency of the gene trap approach.

Animals↗

PubMed1998

Disruption of murine alpha-enolase by a retroviral gene trap results in early embryonic lethality.

Gene trapping with the retroviral ROSA beta geo vector was used to generate lines of mice carrying disrupted genes. Both cDNA and genomic flanks have been cloned from a number of these lines. One mutation has been shown to disrupt the alpha-enolase gene by insertion of the splice-trap vector into the first intron. In adult mice, lacZ expression was detected only in testes. Embryonic expression was detected from 10.5-day postcoitum embryos and was seen as a diffuse staining pattern over much of the embryo, consistent with the housekeeping gene function of alpha-enolase. This mutation results in an early recessive embryonic lethality. Mice heterozygous for the mutation have no obvious phenotype. Mutations of this gene in humans are reported to be associated with rare autosomal-dominant, non-spherocytic haemolytic anaemia. This phenotype is not reproduced in mice heterozygous for this mutation.

Animals↗

PubMed1999

Increased transforming growth factor-alpha levels in human colon carcinoma cell lines over-expressing protein kinase C.

Transforming growth factor-alpha (TGF-alpha) is synthesized as a membrane-bound precursor protein, pro-TGF-alpha, that is converted to a soluble form by 2 endoproteolytic cleavages. Several factors have been implicated in the regulation of the second rate-limiting step, including protein kinase C (PKC). Earlier results indicated a potential role for the conventional class of PKC isozymes in the observed increase in TGF-alpha in the conditioned media of 2 human colon carcinoma cell lines. The present study addresses the potential role of specific PKC isozymes in this process using sense and anti-sense expression vectors for PKC isozymes. Two human colon carcinoma cell lines, HCT 116 and GEO, were transfected with plasmids, leading to the over-expression of PKC-alpha, -betaI or -betaII; and the secretion of TGF-alpha into the conditioned medium was determined. Over-expression of either PKC-betaI or PKC-betaII in these cell lines enhanced the levels of TGF-alpha in the media 2- to 5-fold. Over-expression of PKC-alpha did not alter the amount of TGF-alpha in the media to a significant extent. Transfection of HCT 116 cells with the anti-sense PKC-betaI cDNA resulted in a reduction in PKC-betaI protein expression. This was accompanied by a decrease in the amount of TGF-alpha in the conditioned media. Our results indicate that modulation of PKC-beta protein levels alters the amount of TGF-alpha found in the conditioned media from these colon carcinoma cells.

Colonic Neoplasms↗

PubMed1998

Gene-trapping to identify and analyze genes expressed in the mouse hippocampus.

Mice harboring random gene-trap insertions of a lacZ (beta-galactosidase)-neomycin resistance fusion cassette (beta-geo) were analyzed for expression in the hippocampus. In 4 of 15 lines reporter gene activity was observed in the hippocampal formation. In the obn line, enzyme activity was detected in the CA1-3 hippocampal subfields, in hpk expression was restricted to CA1, but in both lines reporter activity was also present in other brain regions. In the third line, kin, reporter activity was robustly expressed throughout the stratum pyrimidale of CA1-3, with only low-level expression elsewhere. The final line (glnC) displayed ubiquitous expression of the reporter and was not analyzed further. Fusion transcripts for the first three lines were characterized; all encode polypeptides with features of membrane-associated signalling proteins. The obn fusion identified a human cDNA (B2-1) encoding a pleckstrin homology (PH) domain, while hpk sequences matched the Epstein-Barr Virus (EBV) inducible G-protein coupled receptor, EBI-1. kin identified an alternative form of the abl-related nonreceptor tyrosine kinase c-arg. Electrophysiological studies on mice homozygous for the insertions revealed normal synaptic transmission, paired pulse facilitation and paired-pulse depression at Schaffer collateral-commissural CA1 synapses, and normal long-term potentiation (LTP) in obn and kin. hpk mice displayed an increase in hippocampal CA1 long-term potentiation (LTP), suggesting a role for this receptor in synaptic plasticity.

Amino Acid Sequence↗