Identification of a PAI-1-binding site within an intrinsically disordered region of vitronectin

© 2019 The Protein Society The serine protease inhibitor, plasminogen activator inhibitor Type-1 (PAI-1) is a metastable protein that undergoes an unusual transition to an inactive conformation with a short half-life of only 1–2 hr. Circulating PAI-1 is bound to a cofactor vitronectin, which stabilizes PAI-1 by slowing this latency conversion. A well-characterized PAI-1-binding site on vitronectin is located within the somatomedin B (SMB) domain, corresponding to the first 44 residues of the protein. Another PAI-1 recognition site has been identified with an engineered form of vitronectin lacking the SMB domain, yet retaining PAI-1 binding capacity (Schar, Blouse, Minor, Peterson. J Biol Chem. 2008;283:28487–28496). This additional binding site is hypothesized to lie within an intrinsically disordered domain (IDD) of vitronectin. To localize the putative binding site, we constructed a truncated form of vitronectin containing 71 amino acids from the N-terminus, including the SMB domain and an additional 24 amino acids from the IDD region. This portion of the IDD is rich in acidic amino acids, which are hypothesized to be complementary to several basic residues identified within an extensive vitronectin-binding site mapped on PAI-1 (Schar, Jensen, Christensen, Blouse, Andreasen, Peterson. J Biol Chem. 2008;283:10297–10309). Steady-state and stopped-flow fluorescence measurements demonstrate that the truncated form of vitronectin exhibits the same rapid biphasic association as full-length vitronectin and that the IDD hosts the elusive second PAI-1 binding site that lies external to the SMB domain of vitronectin

A Novel System for the Efficient Generation of Antibodies Following Immunization of Unique Knockout Mouse Strains

International audienceBACKGROUND: We wished to develop alternate production strategies to generate antibodies against traditionally problematic antigens. As a model we chose butyrylcholinesterase (BChE), involved in termination of cholinergic signaling, and widely considered as a poor immunogen. METHODOLOGY/PRINCIPAL FINDINGS: Jettisoning traditional laborious in silico searching methods to define putative epitopes, we simply immunized available BChE knock-out mice with full-length recombinant BChE protein (having been produced for crystallographic analysis). Immunization with BChE, in practically any form (recombinant human or mouse BChE, BChE purified from human serum, native or denatured), resulted in strong immune responses. Native BChE produced antibodies that favored ELISA and immunostaining detection. Denatured and reduced BChE were more selective for antibodies specific in Western blots. Two especially sensitive monoclonal antibodies were found capable of detecting 0.25 ng of BChE within one min by ELISA. One is specific for human BChE; the other cross-reacts with mouse and rat BChE. Immunization of wild-type mice served as negative controls. CONCLUSIONS/SIGNIFICANCE: We examined a simple, fast, and highly efficient strategy to produce antibodies by mining two expanding databases: namely those of knock-out mice and 3D crystallographic protein-structure analysis. We conclude that the immunization of knock-out mice should be a strategy of choice for antibody production

Pharmacological levels of withaferin A (Withania somnifera) trigger clinically relevant anticancer effects specific to triple negative breast cancer cells

Withaferin A (WA) isolated from Withania somnifera (Ashwagandha) has recently become an attractive phytochemical under investigation in various preclinical studies for treatment of different cancer types. In the present study, a comparative pathway-based transcriptome analysis was applied in epithelial-like MCF-7 and triple negative mesenchymal MDA-MB-231 breast cancer cells exposed to different concentrations of WA which can be detected systemically in in vivo experiments. Whereas WA treatment demonstrated attenuation of multiple cancer hallmarks, the withanolide analogue Withanone (WN) did not exert any of the described effects at comparable concentrations. Pathway enrichment analysis revealed that WA targets specific cancer processes related to cell death, cell cycle and proliferation, which could be functionally validated by flow cytometry and real-time cell proliferation assays. WA also strongly decreased MDA-MB-231 invasion as determined by single-cell collagen invasion assay. This was further supported by decreased gene expression of extracellular matrix-degrading proteases (uPA, PLAT, ADAM8), cell adhesion molecules (integrins, laminins), pro-inflammatory mediators of the metastasis-promoting tumor microenvironment (TNFSF12, IL6, ANGPTL2, CSF1R) and concomitant increased expression of the validated breast cancer metastasis suppressor gene (BRMS1). In line with the transcriptional changes, nanomolar concentrations of WA significantly decreased protein levels and corresponding activity of uPA in MDA-MB-231 cell supernatant, further supporting its anti-metastatic properties. Finally, hierarchical clustering analysis of 84 chromatin writer-reader-eraser enzymes revealed that WA treatment of invasive mesenchymal MDA-MB-231 cells reprogrammed their transcription levels more similarly towards the pattern observed in non-invasive MCF-7 cells. In conclusion, taking into account that sub-cytotoxic concentrations of WA target multiple metastatic effectors in therapy-resistant triple negative breast cancer, WA-based therapeutic strategies targeting the uPA pathway hold promise for further (pre)clinical development to defeat aggressive metastatic breast cancer

Absence of p300 induces cellular phenotypic changes characteristic of epithelial to mesenchyme transition

p300 is a transcriptional cofactor and prototype histone acetyltransferase involved in regulating multiple cellular processes. We generated p300 deficient (p300−) cells from the colon carcinoma cell line HCT116 by gene targeting. Comparison of epithelial and mesenchymal proteins in p300− with parental HCT116 cells showed that a number of genes involved in cell and extracellular matrix interactions, typical of ‘epithelial to mesenchyme transition' were differentially regulated at both the RNA and protein level. p300− cells were found to have aggressive ‘cancer' phenotypes, with loss of cell–cell adhesion, defects in cell–matrix adhesion and increased migration through collagen and matrigel. Although migration was shown to be metalloproteinase mediated, these cells actually showed a downregulation or no change in the level of key metalloproteinases, indicating that changes in cellular adhesion properties can be critical for cellular mobility

Laboratory-based evaluation of legionellosis epidemiology in Ontario, Canada, 1978 to 2006

BACKGROUND: Legionellosis is a common cause of severe community acquired pneumonia and respiratory disease outbreaks. The Ontario Public Health Laboratory (OPHL) has conducted most testing for Legionella species in the Canadian province of Ontario since 1978, and represents a multi-decade repository of population-based data on legionellosis epidemiology. We sought to provide a laboratory-based review of the epidemiology of legionellosis in Ontario over the past 3 decades, with a focus on changing rates of disease and species associated with legionellosis during that time period. METHODS: We analyzed cases that were submitted and tested positive for legionellosis from 1978 to 2006 using Poisson regression models incorporating temporal, spatial, and demographic covariates. Predictors of infection with culture-confirmed L. pneumophila serogroup 1 (LP1) were evaluated with logistic regression models. Results: 1,401 cases of legionellosis tested positive from 1978 to 2006. As in other studies, we found a late summer to early autumn seasonality in disease occurrence with disease risk increasing with age and in males. In contrast to other studies, we found a decreasing trend in cases in the recent decade (IRR 0.93, 95% CI 0.91 to 0.95, P-value = 0.001); only 66% of culture-confirmed isolates were found to be LP1. CONCLUSION: Despite similarities with disease epidemiology in other regions, legionellosis appears to have declined in the past decade in Ontario, in contrast to trends observed in the United States and parts of Europe. Furthermore, a different range of Legionella species is responsible for illness, suggesting a distinctive legionellosis epidemiology in this North American region

Absence of Host Plasminogen Activator Inhibitor 1 Prevents Cancer Invasion and Vascularization

Acquisition of invasive/metastatic potential through protease expression is an essential event in tumor progression. High levels of components of the plasminogen activation system, including urokinase, but paradoxically also its inhibitor, plasminogen activator inhibitor 1 (PAI1), have been correlated with a poor prognosis for some cancers. We report here that deficient PAI1 expression in host mice prevented local invasion and tumor vascularization of transplanted malignant keratinocytes. When this PAI1 deficiency was circumvented by intravenous injection of a replication-defective adenoviral vector expressing human PAI1, invasion and associated angiogenesis were restored. This experimental evidence demonstrates that host-produced PAI is essential for cancer cell invasion and angiogenesis

When is the Best Time to Sample Aquatic Macroinvertebrates in Ponds for Biodiversity Assessment?

Ponds are sites of high biodiversity and conservation value, yet there is little or no statutory monitoring of them across most of Europe. There are clear and standardized protocols for sampling aquatic macroinvertebrate communities in ponds but the most suitable time(s) to undertake the survey(s) remains poorly specified. This paper examined the aquatic macroinvertebrate communities from 95 ponds within different landuse types over three seasons (spring, summer and autumn) to determine the most appropriate time to undertake sampling to characterise biodiversity. The combined samples from all three seasons provided the most comprehensive record of the aquatic macroinvertebrate taxa recorded within ponds (alpha and gamma diversity). Samples collected during the autumn survey yielded significantly greater macroinvertebrate richness (76% of the total diversity) than either spring or summer surveys. Macroinvertebrate diversity was greatest during autumn in meadow and agricultural ponds but taxon richness among forest and urban ponds did not differ significantly temporally. The autumn survey provided the highest measures of richness for Coleoptera, Hemiptera and Odonata. However, richness of the aquatic insect order Trichoptera was highest in spring and lowest in autumn. The results illustrate that multiple surveys, covering more than one season, provide the most comprehensive representation of macroinvertebrate biodiversity. When sampling can only be undertaken on one occasion, the most appropriate time to undertake surveys to characterise the macroinvertebrate community biodiversity is during the autumn; although this may need to be modified if other floral and faunal groups need to be incorporated in to the sampling programme

Oxytocin and Vasopressin Are Dysregulated in Williams Syndrome, a Genetic Disorder Affecting Social Behavior

The molecular and neural mechanisms regulating human social-emotional behaviors are fundamentally important but largely unknown; unraveling these requires a genetic systems neuroscience analysis of human models. Williams Syndrome (WS), a condition caused by deletion of ∼28 genes, is associated with a gregarious personality, strong drive to approach strangers, difficult peer interactions, and attraction to music. WS provides a unique opportunity to identify endogenous human gene-behavior mechanisms. Social neuropeptides including oxytocin (OT) and arginine vasopressin (AVP) regulate reproductive and social behaviors in mammals, and we reasoned that these might mediate the features of WS. Here we established blood levels of OT and AVP in WS and controls at baseline, and at multiple timepoints following a positive emotional intervention (music), and a negative physical stressor (cold). We also related these levels to standardized indices of social behavior. Results revealed significantly higher median levels of OT in WS versus controls at baseline, with a less marked increase in AVP. Further, in WS, OT and AVP increased in response to music and to cold, with greater variability and an amplified peak release compared to controls. In WS, baseline OT but not AVP, was correlated positively with approach, but negatively with adaptive social behaviors. These results indicate that WS deleted genes perturb hypothalamic-pituitary release not only of OT but also of AVP, implicating more complex neuropeptide circuitry for WS features and providing evidence for their roles in endogenous regulation of human social behavior. The data suggest a possible biological basis for amygdalar involvement, for increased anxiety, and for the paradox of increased approach but poor social relationships in WS. They also offer insight for translating genetic and neuroendocrine knowledge into treatments for disorders of social behavior

The multiplex bead array approach to identifying serum biomarkers associated with breast cancer

Introduction Breast cancer is the most common type of cancer seen in women in western countries. Thus, diagnostic modalities sensitive to early-stage breast cancer are needed. Antibody-based array platforms of a data-driven type, which are expected to facilitate more rapid and sensitive detection of novel biomarkers, have emerged as a direct, rapid means for profiling cancer-specific signatures using small samples. In line with this concept, our group constructed an antibody bead array panel for 35 analytes that were selected during the discovery step. This study was aimed at testing the performance of this 35-plex array panel in profiling signatures specific for primary non-metastatic breast cancer and validating its diagnostic utility in this independent population. Methods Thirty-five analytes were selected from more than 50 markers through screening steps using a serum bank consisting of 4,500 samples from various types of cancer. An antibody-bead array of 35 markers was constructed using the Luminex (TM) bead array platform. A study population consisting of 98 breast cancer patients and 96 normal subjects was analysed using this panel. Multivariate classification algorithms were used to find discriminating biomarkers and validated with another independent population of 90 breast cancer and 79 healthy controls. Results Serum concentrations of epidermal growth factor, soluble CD40-ligand and proapolipoprotein A1 were increased in breast cancer patients. High-molecular-weight-kininogen, apolipoprotein A1, soluble vascular cell adhesion molecule-1, plasminogen activator inhibitor-1, vitamin-D binding protein and vitronectin were decreased in the cancer group. Multivariate classification algorithms distinguished breast cancer patients from the normal population with high accuracy (91.8% with random forest, 91.5% with support vector machine, 87.6% with linear discriminant analysis). Combinatorial markers also detected breast cancer at an early stage with greater sensitivity. Conclusions The current study demonstrated the usefulness of the antibody-bead array approach in finding signatures specific for primary non-metastatic breast cancer and illustrated the potential for early, high sensitivity detection of breast cancer. Further validation is required before array-based technology is used routinely for early detection of breast cancer.Kenny HA, 2008, J CLIN INVEST, V118, P1367, DOI 10.1172/JCI33775Shah FD, 2008, INTEGR CANCER THER, V7, P33, DOI 10.1177/1534735407313883Carlsson A, 2008, EUR J CANCER, V44, P472, DOI 10.1016/j.ejca.2007.11.025Nolen BM, 2008, BREAST CANCER RES, V10, DOI 10.1186/bcr2096Brogren H, 2008, THROMB RES, V122, P271, DOI 10.1016/j.thromres.2008.04.008Varki A, 2007, BLOOD, V110, P1723, DOI 10.1182/blood-2006-10-053736Madsen CD, 2007, J CELL BIOL, V177, P927, DOI 10.1083/jcb.200612058Levenson VV, 2007, BBA-GEN SUBJECTS, V1770, P847, DOI 10.1016/j.bbagen.2007.01.017VAZQUEZMARTIN A, 2007, EUR J CANCER, V43, P1117GARCIA M, 2007, GLOBAL CANC FACTS FIMoore LE, 2006, CANCER EPIDEM BIOMAR, V15, P1641, DOI 10.1158/1055-9965.EPI-05-0980Borrebaeck CAK, 2006, EXPERT OPIN BIOL TH, V6, P833, DOI 10.1517/14712598.6.8.833Zannis VI, 2006, J MOL MED-JMM, V84, P276, DOI 10.1007/s00109-005-0030-4Jemal A, 2006, CA-CANCER J CLIN, V56, P106Silva HC, 2006, NEOPLASMA, V53, P538Chahed K, 2005, INT J ONCOL, V27, P1425Jain KK, 2005, EXPERT OPIN PHARMACO, V6, P1463, DOI 10.1517/14656566.6.9.1463Abe O, 2005, LANCET, V365, P1687Paradis V, 2005, HEPATOLOGY, V41, P40, DOI 10.1002/hep.20505Molina R, 2005, TUMOR BIOL, V26, P281, DOI 10.1159/000089260Furberg AS, 2005, CANCER EPIDEM BIOMAR, V14, P33Benoy IH, 2004, CLIN CANCER RES, V10, P7157Song JS, 2004, BLOOD, V104, P2065, DOI 10.1182/blood-2004-02-0449Schairer C, 2004, J NATL CANCER I, V96, P1311, DOI 10.1093/jnci/djh253Hellman K, 2004, BRIT J CANCER, V91, P319, DOI 10.1038/sj.bjc.6601944Roselli M, 2004, CLIN CANCER RES, V10, P610Zhou AW, 2003, NAT STRUCT BIOL, V10, P541, DOI 10.1038/nsb943Hapke S, 2003, BIOL CHEM, V384, P1073Miller JC, 2003, PROTEOMICS, V3, P56Amirkhosravi A, 2002, BLOOD COAGUL FIBRIN, V13, P505Bonello N, 2002, HUM REPROD, V17, P2272Li JN, 2002, CLIN CHEM, V48, P1296Louhimo J, 2002, ANTICANCER RES, V22, P1759Knezevic V, 2001, PROTEOMICS, V1, P1271Di Micco P, 2001, DIGEST LIVER DIS, V33, P546Ferrigno D, 2001, EUR RESPIR J, V17, P667Webb DJ, 2001, J CELL BIOL, V152, P741Gion M, 2001, EUR J CANCER, V37, P355Schonbeck U, 2001, CELL MOL LIFE SCI, V58, P4Blackwell K, 2000, J CLIN ONCOL, V18, P600Carriero MV, 1999, CANCER RES, V59, P5307Antman K, 1999, JAMA-J AM MED ASSOC, V281, P1470Loskutoff DJ, 1999, APMIS, V107, P54Molina R, 1998, BREAST CANCER RES TR, V51, P109Bajou K, 1998, NAT MED, V4, P923Chan DW, 1997, J CLIN ONCOL, V15, P2322Chu KC, 1996, J NATL CANCER I, V88, P1571vanDalen A, 1996, ANTICANCER RES, V16, P2345Yamamoto N, 1996, CANCER RES, V56, P2827KOCH AE, 1995, NATURE, V376, P517HADDAD JG, 1995, J STEROID BIOCHEM, V53, P579FOEKENS JA, 1994, J CLIN ONCOL, V12, P1648GEARING AJH, 1993, IMMUNOL TODAY, V14, P506HUTCHENS TW, 1993, RAPID COMMUN MASS SP, V7, P576DECLERCK PJ, 1992, J BIOL CHEM, V267, P11693GABRIJELCIC D, 1992, AGENTS ACTIONS S, V38, P350BIEGLMAYER C, 1991, TUMOR BIOL, V12, P138DNISTRIAN AM, 1991, TUMOR BIOL, V12, P82VANDALEN A, 1990, TUMOR BIOL, V11, P189KARAS M, 1988, ANAL CHEM, V60, P2299, DOI 10.1021/ac00171a028LERNER WA, 1983, INT J CANCER, V31, P463WESTGARD JO, 1981, CLIN CHEM, V27, P493TROUSSEAU A, 1865, CLIN MED HOTEL DIEU, V3, P654*R PROJ, R PROJ STAT COMP1