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1.
Soc Sci Med ; 301: 114787, 2022 05.
Article in English | MEDLINE | ID: mdl-35366460

ABSTRACT

With over 80 million people forcibly displaced worldwide, providing safe, healthy, and supportive places for refugees has become an imperative for national governments, aid organizations, and host communities. While much has been written about the needs of these displaced people, organizations and practitioners tend to focus on essential material needs, medical care, and food and water provisioning. Yet a growing body of evidence points to the potential role of social capital - the bonding, bridging, and linking social ties that connect us to one another - as a critical resource for these refugees. We have little data about social capital interventions at individual and community levels to assist with mental health for this vulnerable population, and even less methodical evidence about such interventions' impact. This systematic review analyzes nearly 400 articles to find patterns in the literature on how social-capital-based interventions can improve the mental health of refugees. Within the studies of interventions that met our filtering criteria, the reinforcement or creation of social capital, especially bridging and linking types, serves as a crucial resource to help this vulnerable group. Specifically, our review showed that community and multilevel social capital interventions are key to curbing mental health symptoms among refugees. Given this scanty evidence base among a group so vulnerable to mental health problems, this review serves as an explicit invitation for researchers to further examine social capital interventions among refugees.


Subject(s)
Mental Disorders , Refugees , Social Capital , Health Status , Humans , Mental Disorders/therapy , Mental Health , Refugees/psychology
2.
Soc Sci Med ; 212: 203-218, 2018 09.
Article in English | MEDLINE | ID: mdl-30048843

ABSTRACT

Despite two decades of research on social capital and health, intervention studies remain scarce. We performed a systematic review on social capital interventions in public health and searched the Pubmed and PsychInfo databases. The majority of interventions we identified focused on individual level change (e.g. encouraging social participation), as opposed to community level change. We included 17 manuscripts in the systematic review. We categorized studies according to the role of social capital in the interventions (as the direct target of intervention, as a channel/mediator, or as a segmenting variable) as well as the levels of interventions (individual, community levels vs. multilevel ). We conclude that the majority of interventions sought to directly strengthen social capital to influence health outcomes. Our review reveals (i) a lack of studies that incorporate a multilevel perspective and (ii) an absence of consideration of specific groups that might selectively benefit from social capital interventions (segmentation). Future research is needed on both questions to provide a more nuanced picture of how social capital can be manipulated to affect health outcomes.


Subject(s)
Health Promotion/methods , Public Health , Social Capital , Humans , Randomized Controlled Trials as Topic
3.
Front Immunol ; 9: 315, 2018.
Article in English | MEDLINE | ID: mdl-29520282

ABSTRACT

Age-associated changes in the immune system including alterations in surface protein expression are thought to contribute to an increased susceptibility for autoimmune diseases. The balance between the expression of coinhibitory and costimulatory surface protein molecules, also known as immune checkpoint molecules, is crucial in fine-tuning the immune response and preventing autoimmunity. The activation of specific inhibitory signaling pathways allows cancer cells to evade recognition and destruction by the host immune system. The use of immune checkpoint inhibitors (ICIs) to treat cancer has proven to be effective producing durable antitumor responses in multiple cancer types. However, one of the disadvantages derived from the use of these agents is the appearance of inflammatory manifestations termed immune-related adverse events (irAEs). These irAEs are often relatively mild, but more severe irAEs have been reported as well including several forms of vasculitis. In this article, we argue that age-related changes in expression and function of immune checkpoint molecules lead to an unstable immune system, which is prone to tolerance failure and autoimmune vasculitis development. The topic is introduced by a case report from our hospital describing a melanoma patient treated with ICIs and who subsequently developed biopsy-proven giant cell arteritis. Following this case report, we present an in-depth review on the role of immune checkpoint pathways in the development and progression of autoimmune vasculitis and its relation with an aging immune system.


Subject(s)
Aging , Antineoplastic Agents , Autoimmune Diseases , Melanoma , Vasculitis , Aged , Aging/immunology , Aging/pathology , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/adverse effects , Autoimmune Diseases/chemically induced , Autoimmune Diseases/immunology , Autoimmune Diseases/pathology , Humans , Male , Melanoma/drug therapy , Melanoma/immunology , Melanoma/pathology , Vasculitis/chemically induced , Vasculitis/immunology , Vasculitis/pathology
4.
Oncol Rep ; 30(4): 1575-80, 2013 Oct.
Article in English | MEDLINE | ID: mdl-23934016

ABSTRACT

An effective circulating tumour marker is needed for melanoma especially with the advent of targeted therapies. Gene expression studies examining primary melanomas have shown that increased expression of osteopontin (SPP1) is associated with poor prognosis. Studies subsequently reported higher blood levels in melanoma patients with metastatic disease than those without. This study was designed to determine whether osteopontin plasma concentrations in disease-free patients after initial treatment predict survival. An enzyme-linked immunosorbent assay (ELISA) was used to measure osteopontin levels in stored plasma samples (N=215) from participants in the Leeds Melanoma Cohort. AJCC stage at sampling was statistically significant associated with osteopontin levels (p=0.03). Participants with untreated stage IV disease at sampling (n=10) had higher median osteopontin levels compared to those with treated stage I-III disease (n=158) (p<0.001) confirming previous findings. There was a trend for increased risk of death with increasing osteopontin levels but this was not statistically significant. If a level of 103.14 ng/ml (95th centile of healthy controls) was taken as the upper end of the normal range then 2.5% of patients with treated stage I-III (4/110), 17.6% of patients with untreated stage III (3/17) and 30% of patients with untreated stage IV disease (3/10) had higher levels. These findings suggest that plasma osteopontin levels warrant investigation as a tumour marker in a larger study in which the significance of change in levels over time should be studied in relation to detectable disease recurrence.


Subject(s)
Biomarkers, Tumor/blood , Melanoma/blood , Melanoma/mortality , Osteopontin/blood , Disease-Free Survival , Female , Humans , Male , Neoplasm Recurrence, Local , Neoplasm Staging , Osteopontin/biosynthesis , Osteopontin/genetics , Skin Neoplasms , Melanoma, Cutaneous Malignant
5.
Br J Cancer ; 107(7): 1131-7, 2012 Sep 25.
Article in English | MEDLINE | ID: mdl-22918393

ABSTRACT

BACKGROUND: Objectively measured circulating biomarkers of prognosis complementing existing clinicopathological models are needed in renal cell carcinoma (RCC). METHODS: Blood samples collected from 216 RCC patients in Leeds before nephrectomy (median follow-up 7 years) were analysed for C-reactive protein (CRP), osteopontin (OPN) and carbonic anhydrase IX (CA9) and prognostic significance determined. RESULTS: CA9, OPN and CRP were univariately prognostic for overall survival (OS), cancer-specific survival (CSS) and disease-free survival (DFS) with CRP and CA9 being independently prognostic for OS/CSS and OS, respectively. Including CA9, OPN and CRP with other conventional prognostic factors gave a superior predictive capacity when compared with a previously published pre-operative clinical nomogram (Karakiewicz et al, 2009). Osteopontin outperformed this nomogram and the post-operative SSIGN score for OS but not for CSS, being significantly predictive for non-cancer deaths. Osteopontin, CRP and CA9 outperformed stage (c-index 76% compared with 70% for stage) and OPN or CA9 identified several subsets of poor prognosis patients including in T1 patients, who may benefit from adjuvant therapy and increased surveillance. CONCLUSION: Circulating CA9, OPN and CRP add value to existing clinicopathological prognostic factors/models and support further studies to investigate their potential use in improving the clinical management of RCC.


Subject(s)
Biomarkers, Tumor/blood , C-Reactive Protein/metabolism , Carbonic Anhydrase IV/blood , Carcinoma, Renal Cell/blood , Kidney Neoplasms/blood , Osteopontin/blood , Adult , Aged , Aged, 80 and over , Carcinoma, Renal Cell/enzymology , Disease-Free Survival , Female , Follow-Up Studies , Humans , Kidney Neoplasms/enzymology , Male , Middle Aged , Nephrectomy/methods , Prognosis
6.
Child Care Health Dev ; 34(4): 530-41, 2008 Jul.
Article in English | MEDLINE | ID: mdl-19154554

ABSTRACT

BACKGROUND: In a substantial minority of children with a hemiparesis, motor impairments are accompanied by behavioural problems. This combination confronts parents with several persistent, frequently intense, sources of stress. At the same time, it is likely to reduce the effectiveness of psychosocial resources, such as feelings of competence, which would normally buffer the impact of the stressors. Aim To investigate the association between motor and behavioural problems in children with a hemiparesis and symptoms of stress in their parents, with particular attention to psychosocial factors which may mediate between the child's problems and parents' symptoms of stress. METHOD: Questionnaires assessing the medical, functional and behaviour problems of the child, and the parents' experience of stress were completed by the mothers and fathers of 108 children with a hemiparesis who were members of the Association for the Motor Handicapped in the Netherlands. RESULTS: Both parents reported (extremely) high levels of long-term stress significantly more frequently than parents in a normative sample. Indices of long-term stress were associated with the child's behavioural problems and, less strongly, with dysfunctionality in daily life. However, behavioural problems and dysfunctionality also reduced parents' feelings of competence and social support. A mediation analysis showed that feelings of incompetence and social isolation mediated between the child's problems and the parents' symptoms of stress. Fathers and mothers did not differ in level of reported stress, or in the associations between the child's problems and degree of experienced stress. CONCLUSION: Both parents of a child with a hemiparesis experience high levels of stress, which are strongly associated with feelings of incompetence and social isolation. This suggests that one focus of intervention should be the alleviation of parenting stress with particular attention to increasing perceived competence in the parenting role and reducing feelings of social isolation.


Subject(s)
Adaptation, Psychological , Fathers/psychology , Mothers/psychology , Parenting/psychology , Paresis/psychology , Stress, Psychological/psychology , Activities of Daily Living , Adolescent , Adult , Child , Child Behavior/physiology , Child Behavior/psychology , Child, Preschool , Female , Humans , Infant , Male , Middle Aged , Netherlands , Parent-Child Relations , Severity of Illness Index , Social Support , Surveys and Questionnaires , Time Factors , Young Adult
8.
Ecotoxicol Environ Saf ; 64(1): 85-99, 2006 May.
Article in English | MEDLINE | ID: mdl-16439022

ABSTRACT

An environmental risk assessment for alcohol ethoxylates (AE) is presented that integrates wastewater treatment plant monitoring, fate, and ecotoxicity research with a new application of mixture toxicity theory based on simple similar concentration addition of AE homologs in a species-sensitivity distribution (SSD) context. AEs are nonionic surfactants composed of a homologous series of molecules that range in alkyl chain length from 12 to 18 carbons and ethoxylates from 0 to 18 units. Chronic ecotoxicity of AE is summarized for 17 species in 60 tests and then normalized to monitoring data for AE mixtures. To do so, chronic aquatic toxicity was first expressed as EC10 per species (the concentration predicted to cause a 10% reduction in an important ecological endpoint). Normalization integrated several new quantitative structure-activity relationships for algae, daphnids, fish, and mesocosms and provided an interpretation of toxicity test data as a function of individual homologs in an AE mixture. SSDs were constructed for each homolog and the HC5 (hazardous concentration protective of 95% of species based on a small biological effect [the chronic EC10]) was predicted. Total mass of AE in monitored effluents from 29 sites in Europe, Canada, and the United States averaged 6.8, 2.8, and 3.55 microg/L, respectively. For risk assessment purposes, correction of exposure to account for fatty alcohol derived from sources other than AE and for sorbed components based on experimental evidence was used to determine AE concentrations in undiluted (100%) effluents from North America and Europe. Exposure and effect findings were integrated in a toxic unit (TU)-based model that considers the measured distribution of individual AE homologs in effluent with their corresponding SSDs. Use of environmentally relevant exposure corrections (bioavailability and accounting for AE-derived alcohol) resulted in TUs ranging from 0.015 to 0.212. Low levels of risk are concluded for AE in the aquatic environments of Europe and North America.


Subject(s)
Alcohols/toxicity , Water Pollutants, Chemical/toxicity , Adsorption , Alcohols/analysis , Algorithms , Animals , Biodegradation, Environmental , Daphnia , Eukaryota , Europe , Fishes , North America , Quantitative Structure-Activity Relationship , Reference Values , Risk Assessment , Species Specificity , Terminology as Topic , Water Microbiology , Water Pollutants, Chemical/analysis
9.
Ecotoxicol Environ Saf ; 64(1): 42-60, 2006 May.
Article in English | MEDLINE | ID: mdl-16005515

ABSTRACT

Environmental monitoring indicates that the distribution of alcohol ethoxylate (AE) homologues in wastewater treatment plant (WWTP) effluents differs from the distribution in commercial AE products, with a relative higher proportion of fatty alcohol (AOH, which is AE with zero ethoxylation). To determine the contribution of AE-derived AOH to the total concentration of AE and AOH in WWTP effluents, we conducted a laboratory continuous activated-sludge study (CAS). This consisted of a test unit fed with AE-amended synthetic sewage and a control unit fed with only synthetic sewage to avoid AE contamination from the feed. The removal efficiencies of some 114 AE homologues were determined by the application of a specific and sensitive analytical method. The extent of the removal of AE ranged from 99.70% for C18 compounds to > 99.98% for C12-16. Relatively high-AOH concentrations were observed in the effluents from blank and test units. By building the concentration difference from the test minus the control unit, the AE in the CAS effluent originating from AE in the influent was determined. Thus, it could be shown that AOH represented only 19% of the total AE (EO0-18) in the CAS, while monitoring in 29 WWTP effluents (European, Canadian, and US) revealed in total a mean AOH fraction of 55% (5-82%) of the total AE (EO0-18). This shows that only a small fraction of AOH in WWTP effluents originates from AE entering the WWTP.


Subject(s)
Sewage/analysis , Surface-Active Agents/analysis , Water Pollutants, Chemical/analysis , Aerobiosis , Alcohols/analysis , Biodegradation, Environmental , Chromatography, High Pressure Liquid , Environmental Exposure/analysis , Environmental Exposure/statistics & numerical data , Environmental Monitoring , Indicators and Reagents , Mass Spectrometry , Reference Standards , Waste Disposal, Fluid
10.
Ecotoxicol Environ Saf ; 64(1): 61-74, 2006 May.
Article in English | MEDLINE | ID: mdl-16338005

ABSTRACT

Alcohol ethoxylates (AEs) are an important group of nonionic surfactants. Commercial AEs consist of a mixture of several homologues of varying carbon chain length (Cx) and degree of ethoxylation (EOy). The major disposal route of AE is down the drain to municipal wastewater treatment plants that discharge into receiving surface waters. Sorption of AE homologues onto activated sludge and river water solids is an important factor in assessing exposure of AE in the environment. This study presents the experimental determination of sorption coefficients for a wide array of AE homologues including five alcohols under environmentally relevant conditions and combines these data with literature data to generate a predictive model for the sorption of AEs in the environment. These results demonstrate that sorption can be effectively modeled using a log Kd vs. Cx and EOy predictive equation having the form log Kd = 0.331C - 0.00897EO - 1.126(R2 = 0.64).


Subject(s)
Alcohols/analysis , Fatty Alcohols/analysis , Sewage/analysis , Water Pollutants, Chemical/analysis , Adsorption , Biodegradation, Environmental , Chemical Phenomena , Chemistry, Physical , Environmental Monitoring , Forecasting , Fresh Water/analysis , Linear Models , Models, Statistical , Refuse Disposal , Risk Assessment , Thermodynamics , Waste Disposal, Fluid
11.
Ecotoxicol Environ Saf ; 64(1): 75-84, 2006 May.
Article in English | MEDLINE | ID: mdl-16256196

ABSTRACT

Traditionally, ecotoxicity quantitative structure-activity relationships (QSARs) for alcohol ethoxylate (AE) surfactants have been developed by assigning the measured ecotoxicity for commercial products to the average structures (alkyl chain length and ethoxylate chain length) of these materials. Acute Daphnia magna toxicity tests for binary mixtures indicate that mixtures are more toxic than the individual AE substances corresponding with their average structures (due to the nonlinear relation of toxicity with structure). Consequently, the ecotoxicity value (expressed as effects concentration) attributed to the average structures that are used to develop the existing QSARs is expected to be too low. A new QSAR technique for complex substances, which interprets the mixture toxicity with regard to the "ethoxymers" distribution (i.e., the individual AE components) rather than the average structure, was developed. This new technique was then applied to develop new AE ecotoxicity QSARs for invertebrates, fish, and mesocosms. Despite the higher complexity, the fit and accuracy of the new QSARs are at least as good as those for the existing QSARs based on the same data set. As expected from typical ethoxymer distributions of commercial AEs, the new QSAR generally predicts less toxicity than the QSARs based on average structure.


Subject(s)
Alcohols/toxicity , Environmental Pollution/analysis , Surface-Active Agents/toxicity , Water Pollutants, Chemical/toxicity , Algorithms , Animals , Chromatography, High Pressure Liquid , Daphnia , Fishes , Models, Statistical , Quantitative Structure-Activity Relationship
12.
Chemosphere ; 54(8): 1145-53, 2004 Feb.
Article in English | MEDLINE | ID: mdl-14664842

ABSTRACT

The aim of this study was the comparison between predicted environmental concentrations (PEC) derived using a generic aspacial model, European Union System for the Evaluation of Substances (EUSES), and a geo-referenced model, the Geo-referenced Regional Environmental Assessment Tool for European Rivers (GREAT-ER). The PECs of some consumer-product ingredients (boron, LAS) and professional uses (EDTA, NTA and Triclosan) were calculated for the river catchment of the Itter, a small tributary to the river Rhine. The PEClocal and PECregional for the water compartment generated by EUSES (default scenario) were subsequently refined with data that realistically reflects the region of North Rhine-Westphalia (NRW scenario) and the Itter catchment (Itter scenario). The results of the three scenarios were then compared with the PECinitial and PECcatchment calculated by GREAT-ER, that was designed as a higher-tiered exposure assessment tool, and with concrete concentrations in the Itter, measured as 24-h composite samples. While the PECregional of all scenarios was close to the lower end of the measured concentrations, the geo-referenced PECs described equally well the real spacial situation. The measured environmental concentrations confirmed the built-in conservatism of the PEClocal calculations by EUSES showing for all investigated chemicals an unrealistically high PEClocal (default). The refinement in the more realistic scenarios could not provide a straight forward general improvement of the PEClocal. In conclusion, when the EUSES prognosis is refined using more detailed substance and regional specific data, it may provide a fairly accurate modelling especially of substances that are not eliminated in the environment. However, in the case of eliminable substances, it does not match the accuracy of higher-tiered geo-referenced exposure models like GREAT-ER.


Subject(s)
Environmental Monitoring/methods , Household Products/analysis , Models, Theoretical , Rivers , Water Pollutants, Chemical/analysis , Arylsulfonates/analysis , Arylsulfonates/chemistry , Boron/analysis , Computer Simulation , Databases, Factual , Edetic Acid/analysis , Environmental Exposure/analysis , European Union , Nitrilotriacetic Acid/analogs & derivatives , Nitrilotriacetic Acid/analysis , Triclosan/analysis
13.
Chemosphere ; 54(8): 1135-44, 2004 Feb.
Article in English | MEDLINE | ID: mdl-14664843

ABSTRACT

A computer simulation of the environmental concentrations of some typical consumer-product ingredients was performed using the geo-referenced exposure model GREAT-ER (Geo-referenced Regional Environmental Assessment Tool for European Rivers) in the river Itter. Boron and LAS were chosen as typical detergent ingredients along with EDTA, NTA and Triclosan as examples of household and cosmetic product ingredients. The simulations were based on consumption figures of the respective chemical in consumer products in the year 2000. For EDTA, the consumption figure used for the calculation had to be extended to commercial products since the EDTA-use in domestic products could not account for the measured concentrations alone. The resulting PEC (Predicted Environmental Concentration) for all investigated compounds showed very good accordance to the measured concentrations in the Itter which were monitored in the same year. The concentrations did not deviate more than by a factor of 3. GREAT-ER's calculated 90th-percentile was never exceeded by the monitoring result thus reflecting a reasonable accuracy.


Subject(s)
Environmental Monitoring/methods , Household Products/analysis , Rivers , Water Pollutants, Chemical/analysis , Arylsulfonates/analysis , Arylsulfonates/chemistry , Boron/analysis , Computer Simulation , Databases, Factual , Edetic Acid/analysis , Germany , Models, Theoretical , Nitrilotriacetic Acid/analogs & derivatives , Nitrilotriacetic Acid/analysis , Sensitivity and Specificity , Triclosan/analysis
14.
Eur J Biochem ; 268(12): 3407-15, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11422370

ABSTRACT

To generate specific tools for, in particular, localization studies of the eukaryotic elongation factor 1A (eEF1A), we have applied phage display in various formats to affinity-improve and map epitopes of two previously isolated, low-affinity single-chain Fv (scFv) G3 and D1. The scFv differ in their reactivity toward the eEF1A isoforms, eEF1A-1 and eEF1A-2. By PCR-based randomization of six residues within the variable light chain CDR3 (LCDR3), and subsequent phage-based affinity-selection, two 'families' of affinity-improved scFv were obtained. The scFv of highest affinity, A8, has a Kd of 9 nM to eEF1A-1. Interestingly, two affinity-improved scFvs have abnormally short LCDR3 consisting of two and four residues compared to 11 in the parental scFv. Hence, the LCDR3 of the parental clones may play a modulating rather than a direct role in antigen-binding. Despite different preferences for the eEF1A isoforms, both families of scFv recognize antigenic determinant(s), which was mapped to residues 413-450 of eEF1A-1/2 by Western blot analysis of recombinant human eEF1A (hEF1A) fragments. Prior to the Western blotting analysis, the epitope location had been suggested using a novel approach where phage-antibody repertoire derived scFv were used to select phage-displayed peptides. Hereby, peptides containing a SFXD motif, matching the SFSD(414-418) sequence found in hEF1A-1 were isolated. The structure of eukaryotic EF1A from yeast indicates a discontinuous nature of the epitope with distal functional elements juxtaposed by the protein fold. Finally, the scFv A8 was applied for immunofluorescence studies of transformed human amnion cells and MCF-7 fibroblasts. In both cases a perinuclear localization of hEF1A was observed. No evidence for the reported nuclear localization of hEF1A was obtained.


Subject(s)
Bacteriophages/genetics , Epitope Mapping , Eukaryotic Initiation Factor-1 , Immunoglobulin Variable Region/chemistry , Peptide Initiation Factors/genetics , Amino Acid Sequence , Base Sequence , Cell Line, Transformed , DNA Primers , Fluorescent Antibody Technique , Humans , Molecular Sequence Data , Peptide Initiation Factors/chemistry , Protein Conformation
15.
Eur J Biochem ; 268(4): 1095-106, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11179976

ABSTRACT

The inhibitory mechanism of serine proteinase inhibitors of the serpin family is based on their unique conformational flexibility. The formation of a stable proteinase-serpin complex implies insertion of the reactive centre loop of the serpin into the large central beta-sheet A and a shift in the relative positions of two groups of secondary structure elements, the smaller one including alpha-helix F. In order to elucidate this mechanism, we have used phage-display and alanine scanning mutagenesis to map the epitopes for four monoclonal antibodies against alpha-helix F and its flanking region in the serpin plasminogen activator inhibitor-1 (PAI-1). One of these is known to inhibit the reaction between PAI-1 and its target proteinases, an effect that is potentiated by vitronectin, a physiological carrier protein for PAI-1. When combined with the effects these antibodies have on PAI-1 activity, our epitope mapping points to the mobility of amino-acid residues in alpha-helix F and the loop connecting alpha-helix F and beta-strand 3A as being important for the inhibitory function of PAI-1. Although all antibodies reduced the affinity of PAI-1 for vitronectin, the potentiating effect of vitronectin on antibody-induced PAI-1 neutralization is based on formation of a ternary complex between antibody, PAI-1 and vitronectin, in which PAI-1 is maintained in a state behaving as a substrate for plasminogen activators. These results thus provide new details about serpin conformational changes and the regulation of PAI-1 by vitronectin and contribute to the necessary basis for rational design of drugs neutralizing PAI-1 in cancer and cardiovascular diseases.


Subject(s)
Antibodies, Monoclonal/immunology , Epitope Mapping , Plasminogen Activator Inhibitor 1/immunology , Alanine/genetics , Antibodies, Monoclonal/pharmacology , Binding Sites, Antibody , Epitopes/immunology , Epitopes/isolation & purification , Humans , Models, Molecular , Mutagenesis, Site-Directed , Peptide Library , Plasminogen Activator Inhibitor 1/genetics , Plasminogen Activator Inhibitor 1/metabolism , Protein Conformation , Species Specificity , Vitronectin/metabolism
16.
Electrophoresis ; 21(3): 509-16, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10726750

ABSTRACT

A procedure was established for selecting phage antibodies (phage-abs) from phage-displayed antibody repertoires by panning against proteins, separated by sodium dodecyl phosphate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto nitrocellulose membranes (Western blots). This immobilization strategy is applicable for secondary rounds of panning in selections against semipurified proteins, and directs the selection toward antibodies suitable as immunochemical reagents in Western blots. In model experiments, enrichment factors as high as 1.9x10(5) were obtained in a single round of panning. Furthermore, we demonstrate the application of this approach by selection of phage-abs recognizing the human Werner protein, which is defective in a premature aging syndrome.


Subject(s)
Antibodies, Viral/immunology , Bacteriophages/immunology , DNA Helicases/immunology , Base Sequence , Blotting, Western , DNA Fingerprinting , DNA Primers , Enzyme-Linked Immunosorbent Assay , Exodeoxyribonucleases , Humans , Polymerase Chain Reaction , RecQ Helicases , Werner Syndrome Helicase
17.
Biochimie ; 81(12): 1079-87, 1999 Dec.
Article in English | MEDLINE | ID: mdl-10607402

ABSTRACT

Phage display technology has been used in a variety of contexts to understand and manipulate biomolecular interactions between proteins and other biomolecules. In this paper we describe the establishment of a phage display system for elucidation of the interactions between the GTPase Ras and its panel of effectors. It is shown how technical problems associated with phage display of a protein with unpaired cysteines, likely to be caused by the oxidizing environment of the bacterial periplasm into which the protein is directed, can be overcome by cysteine replacement based on functional and structural studies. First, the catalytic domain (residues 1-166) of mammalian H-Ras (Ras) was observed to be displayed on phage in an incorrect conformation not detectable by antibodies recognizing conformational epitopes on Ras. Although truncation of the phage coat protein used as fusion partner (g3p) resulted in minor improvements in the display, Ras was tailored for phage display by cysteine replacement. By replacing the three cysteines at positions 51, 80 and 118 of Ras with the corresponding residues in Saccharomyces cerevisiae RAS1, the resulting fusion-phage is recognized by the conformation-dependent anti-Ras antibodies. Furthermore, display of cysteine-free Ras is demonstrated by GTP-analogue dependent binding to the Ras-binding domain of the Ras-effector Raf1. These data pave the way for analysis of Ras-effector interactions using phage display technology yet demonstrate that phage display of proteins with normally reduced cysteines should be approached with caution.


Subject(s)
Fungal Proteins , Saccharomyces cerevisiae Proteins , ras Proteins/chemistry , ras Proteins/genetics , Amino Acid Sequence , Amino Acid Substitution , Animals , Base Sequence , Binding Sites , Coliphages/genetics , Cysteine/genetics , DNA Primers/genetics , Drug Stability , Escherichia coli/genetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Structure, Tertiary , Proto-Oncogene Proteins c-raf/chemistry , Proto-Oncogene Proteins c-raf/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae/genetics , Two-Hybrid System Techniques , ras Proteins/metabolism
18.
Syst Appl Microbiol ; 22(2): 269-79, 1999 May.
Article in English | MEDLINE | ID: mdl-10390878

ABSTRACT

Rice plants that were grown in flooded rice soil microcosms were examined for their ability to exhibit sulfate reducing activity. Washed excised rice roots showed sulfate reduction potential when incubated in anaerobic medium indicating the presence of sulfate-reducing bacteria. Rice plants, that were incubated in a double-chamber (phylloshpere and rhizosphere separated), showed potential sulfate reduction rates in the anoxic rhizosphere compartment. These rates decreased when oxygen was allowed to penetrate through the aerenchyma system of the plants into the anoxic root compartment, indicating that sulfate reducers on the roots were partially inhibited by oxygen or that sulfate was regenerated by oxidation of reduced S-compounds. The potential activity of sulfate reducers on rice roots was consistent with MPN enumerations showing that H2-utilizing sulfate-reducing bacteria were present in high numbers on the rhizoplane (4.1 x 10(7) g-1 root fresh weight) and in the adjacent rhizosperic soil (2.5 x 10(7) g-1 soil dry weight). Acetate-oxidizing sulfate reducers, on the other hand, showed highest numbers in the unplanted bulk soil (1.9 x 10(6) g-1 soil dry weight). Two sulfate reducing bacteria were isolated from the highest dilutions of the MPN series and were characterized physiologically and phylogenetically. Strain F1-7b which was isolated from the rhizoplane with H2 as electron donor was related to subgroup II of the family Desulfovibrionaceae. Strain EZ-2C2, isolated from the rhizoplane on acetate, grouped together with Desulforhabdus sp. and Syntrophobacter wolinii. Other strains of sulfate-reducing bacteria originated from bulk soil of rice soil microcosms and were isolated using different electron donors. From these isolates, strains R-AcA1, R-IbutA1, R-PimA1 and R-AcetonA170 were Gram-positive bacteria which were affiliated with the genus Desulfotomaculum. The other isolates were members of subgroup II of the Desulfovibrionaceae (R-SucA1 and R-LacA1), were related to Desulforhabdus sp. (strain BKA11), Desulfobulbus (R-PropA1), or culstered between Desulfobotulus sapovorans and Desulfosarcina variabilis (R-ButA1 and R-CaprA1).


Subject(s)
Gram-Negative Anaerobic Straight, Curved, and Helical Rods/physiology , Plant Roots/microbiology , Soil Microbiology , Sulfur-Reducing Bacteria/physiology , Gram-Negative Anaerobic Straight, Curved, and Helical Rods/classification , Oryza/microbiology , Phylogeny , Sulfur-Reducing Bacteria/classification
19.
FEBS Lett ; 431(3): 448-52, 1998 Jul 24.
Article in English | MEDLINE | ID: mdl-9714561

ABSTRACT

Using a semi-synthetic phage displayed antibody repertoire, isoform-specific and cross-reactive phage-antibodies to eukaryotic elongation factor 1A (eEF1A) have been selected. Enrichment of specific antibodies was found to depend on the presence of glycerol. Further selections against lactate dehydrogenase (LDH) revealed that the dominance of a phage-antibody clone to LDH was inhibited by glycerol, a notable feature for selection strategies where a broad variety of binding clones is desired. The impact of glycerol in distinct steps of the selection protocol was examined and glycerol found to affect certain antibody-antigen interactions. Furthermore, the nonspecific phage binding was lowered by three orders of magnitude at a 20% (v/v) glycerol concentration.


Subject(s)
Antibodies/genetics , Coliphages/drug effects , Glycerol/pharmacology , Base Sequence , Blotting, Western , Coliphages/genetics , DNA Primers
20.
Syst Appl Microbiol ; 21(4): 569-78, 1998 Dec.
Article in English | MEDLINE | ID: mdl-9924825

ABSTRACT

In rice paddy fields the bulk soil is anoxic, but oxygenated zones occur in the surrounding of the rice roots to where oxygen is transported via the aerenchyma system of the rice plants. In the anaerobic soil compartments sulfate is consumed by sulfate-reducing bacteria. In the rhizosphere the reduced sulfur compounds can be reoxidized by sulfur-oxidizing bacteria. Measurements of the potential activity of thiosulfate-oxidizing bacteria in soil slurries derived from planted rice soil microcosms showed turnover rates of 2-6 mumol d-1 g-dw-1. Thiosulfate was oxidized to sulfate with tetrathionate as intermediate. Most probable number (MPN) enumeration with three aerobic media and one anaerobic nitrate-amended medium showed that thiosulfate-oxidizing bacteria were abundant in paddy soil and in rhizosphere soil at numbers of 10(5) to 10(6) per gram dry weight soil. Nine isolates of S-oxidizing bacteria were obtained from enrichment cultures or from the highest dilutions of the MPN series and were affiliated to four different phylogenetic groups. These isolates were characterized by physiological properties and by comparative 16S rDNA sequence analysis. Three isolates (TA1-AE1, TA1-A1 and TA12-21) were shown to be facultatively chemolithoautotrophic strains of Ancylobacter aquaticus. Three further isolates (Tv6-2b, Z2A-6A and Z4A-2A) were also facultatively chemolithoautotrophic and were affiliated with the Xanthobacter sp. group, probably representing new strains of X. flavus or X. tagetidis. Strain SZ-2111 was phylogenetically related to Bosea thiooxidans. However, the genus Bosea is described as obligately heterotrophic, whereas strain 5Z-2111 was able to grow autotrophically. The isolates 5Z-C1 and TBW3 were obligate chemolithoautotrophs and were closely affiliated with Thiobacillus thioparus. Our results showed that S-oxidizing bacteria were abundant and active in rice paddy soil and consisted of physiologically and phylogenetically diverse populations.


Subject(s)
Bacteria , Oryza/microbiology , Soil Microbiology , Thiosulfates/metabolism , Anaerobiosis , Bacteria/classification , Bacteria/isolation & purification , Bacteria/metabolism , DNA, Bacterial/genetics , Microscopy, Phase-Contrast , Molecular Sequence Data , Oxidation-Reduction , Phylogeny , Plant Roots/microbiology , Polymerase Chain Reaction , RNA, Ribosomal, 16S/analysis
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