Assessment of estrogenic exposure in brown trout (Salmo trutta) in a Swiss midland river: integrated analysis of passive samplers, wild and caged fish, and vitellogenin mRNA and protein

This field study examined the vitellogenin (VTG) biomarker response under conditions of low and fluctuating activities of environmental estrogenicity. The present study was performed on immature brown trout (Salmo trutta) exposed to the small river Luetzelmurg, which is located in the prealpine Swiss midland region and receives effluents from a single sewage treatment plant (STP). To understand better factors influencing the relationship between estrogenic exposure and VTG induction, we compared VTG levels in caged (stationary) and feral (free-ranging) fish, VTG levels in fish from up- and downstream of the STP, and two different methods for quantifying VTG (enzyme-linked immunosorbent assay vs real-time reverse transcription-polymerase chain reaction), and we used passive samplers (polar organic chemical integrative sampler [POCIS]) to integrate the variable, bioaccumulative estrogenic load in the river water over time. The POCIS from the downstream site contained approximately 20-fold higher levels of bioassay-derived estrogen equivalents than the POCIS from the upstream site. In feral fish, this site difference in estrogenic exposure was reflected in VTG protein levels but not in VTG mRNA. In contrast, in caged fish, the site difference was evident only for VTG mRNA but not for VTG protein. Thus, the outcome of VTG biomarker measurements varied with the analytical detection method (protein vs mRNA) and with the exposure modus (caged vs feral). Our findings suggest that for environmental situations with low and variable estrogenic contamination, a multiple-assessment approach may be necessary for the assessment of estrogenic exposure in fish.

Cloning and expression of the gene encoding the major surface protein 5 (MSP5) of Anaplasma phagocytophilum and potential application for serodiagnosis

BACKGROUND: Anaplasma phagocytophilum (formerly known as the human granulocytic ehrlichia, Ehrlichia equi and Ehrlichia phagocytophila) is an obligate intracellular organism causing clinical disease in humans and various species of domestic animals. OBJECTIVES: The objectives of this investigation were to sequence and clone the major surface protein 5 (MSP5) of A phagocytophilum and to evaluate the suitability of this antigen in the serologic diagnosis of anaplasmosis in humans and dogs. METHODS: The msp5 gene of A phagocytophilum was sequenced, cloned, and expressed in Escherichia coli. The predicted amino acid sequence homology of the various MSP5/major antigenic protein 2 orthologs was compared among various Anaplasma and Ehrlichia species. Recombinant MSP5 of A phagocytophilum was used in an ELISA to detect antibodies in serum samples from humans and dogs infected with the organism. RESULTS: Serum samples from 104 individuals previously diagnosed with A phagocytophilum infection, as well as samples from clinically healthy humans, were tested. In addition, multiple samples from 4 dogs experimentally infected with 2 different geographic isolates of A phagocytophilum and 5 dogs naturally infected with a Swiss isolate were tested using ELISA. Using this group of immunofluorescent antibody test-positive and immunofluorescent antibody test-negative samples, we found the overall agreement between assays to be >90%. CONCLUSIONS: These results indicate that recombinant MSP5 has potential for use as a diagnostic test antigen to detect infection with A phagocytophilum in both dogs and humans. However, sequence similarities among orthologs of MSP5 in related species of anaplasma and ehrlichia suggest that cross-reactivity among these pathogens is likely if the entire peptide is used as a test antigen.

Assessing The Role Of Multi-protein Complexes In Determining Phenotype

Understanding regulatory mechanisms in complex biological systems is an important challenge, in particular to understand disease mechanisms, and to discover new therapies and drugs. In this paper, we consider the important question of cellular regulation of phenotype. Using single gene deletion data, we address the problem of linking a phenotype to underlying functional roles in the organism and provide a sound computational and statistical paradigm that can be extended to address more complex experimental settings such as multiple deletions. We apply the proposed approaches to publicly available data sets to demonstrate strong evidence for the involvement of multi-protein complexes in the phenotypes studied.

Tocopherol-associated protein-1 accelerates apoptosis induced by alpha-tocopheryl succinate in mesothelioma cells

Alpha-tocopheryl succinate (alpha-TOS), a redox-silent analogue of vitamin E, induces apoptosis in multiple cell lines in a selective manner, by activating the intrinsic pathway. Since it is a highly hydrophobic compound, it may require a carrier protein for its trafficking to intracellular targets like mitochondria. We studied the role of the ubiquitous tocopherol-associated protein-1 (TAP1 or sec14-like 2) in apoptosis induction by alpha-TOS in malignant mesothelioma (MM) cells. Over-expression of TAP1 in MM cells sensitised them to apoptosis by low doses of alpha-TOS which were sub-apoptotic for the parental cells. Apoptosis induced in TAP1-over-expressing cells was mitochondria- and caspase-dependent, as suggested by dissipation of mitochondrial trans-membrane potential and inhibition by zVAD-fmk, respectively. Binding assays showed affinity of alpha-TOS for TAP1. Finally, TAP1 over-expressing cells accumulated alpha-TOS at higher levels compared to their normal counterparts. We suggest that TAP1 may act as an intracellular shuttle for alpha-TOS, promoting apoptosis initiated by this vitamin E analogue, as shown here for MM cells.

E- and P-selectin are not required for the development of experimental autoimmune encephalomyelitis in C57BL/6 and SJL mice

In multiple sclerosis and in its animal model experimental autoimmune encephalomyelitis (EAE), inflammatory cells migrate across the endothelial blood-brain barrier (BBB) and gain access to the CNS. It is well-established that alpha4 integrins are actively involved in leukocyte recruitment across the BBB during EAE. In contrast, the role of endothelial E- and P-selectin in this process has been a controversial issue. In this study, we demonstrate that P-selectin protein can be detected in meningeal blood vessel endothelial cells in healthy SJL and C57BL/6 mice and on rare parenchymal CNS blood vessels in C57BL/6, but not SJL, mice. During EAE, expression of P-selectin but not E-selectin was found up-regulated on inflamed CNS microvessels surrounded by inflammatory infiltrates irrespective of their meningeal or parenchymal localization with a more prominent immunostaining detected in C57BL/6 as compared with SJL mice. P-selectin immunostaining could be localized to CNS endothelial cells and to CD41-positive platelets adhering to the vessel wall. Despite the presence of P-selectin in wild-type mice, E/P-selectin-deficient SJL and C57BL/6 mice developed clinical EAE indistinguishable from wild-type mice. Absence of E- and P-selectin did neither influence the activation of myelin-specific T cells nor the composition of the cellular infiltrates in the CNS during EAE. Finally, endothelial-specific tetracycline-inducible expression of E-selectin at the BBB in transgenic C57BL/6 mice did not alter the development of EAE. Thus, E- and P-selectin are not required for leukocyte recruitment across the BBB and the development of EAE in C57BL/6 and in SJL mice.

Signal peptide and helical bundle domains of virulent canine distemper virus fusion protein restrict fusogenicity

Persistence in canine distemper virus (CDV) infection is correlated with very limited cell-cell fusion and lack of cytolysis induced by the neurovirulent A75/17-CDV compared to that of the cytolytic Onderstepoort vaccine strain. We have previously shown that this difference was at least in part due to the amino acid sequence of the fusion (F) protein (P. Plattet, J. P. Rivals, B. Zuber, J. M. Brunner, A. Zurbriggen, and R. Wittek, Virology 337:312-326, 2005). Here, we investigated the molecular mechanisms of the neurovirulent CDV F protein underlying limited membrane fusion activity. By exchanging the signal peptide between both F CDV strains or replacing it with an exogenous signal peptide, we demonstrated that this domain controlled intracellular and consequently cell surface protein expression, thus indirectly modulating fusogenicity. In addition, by serially passaging a poorly fusogenic virus and selecting a syncytium-forming variant, we identified the mutation L372W as being responsible for this change of phenotype. Intriguingly, residue L372 potentially is located in the helical bundle domain of the F(1) subunit. We showed that this mutation drastically increased fusion activity of F proteins of both CDV strains in a signal peptide-independent manner. Due to its unique structure even among morbilliviruses, our findings with respect to the signal peptide are likely to be specifically relevant to CDV, whereas the results related to the helical bundle add new insights to our growing understanding of this class of F proteins. We conclude that different mechanisms involving multiple domains of the neurovirulent A75/17-CDV F protein act in concert to limit fusion activity, preventing lysis of infected cells, which ultimately may favor viral persistence.

Quantum dot / optical protein bio-nano hybrid system biosensing

The integration of novel nanomaterials with highly-functional biological molecules has advanced multiple fields including electronics, sensing, imaging, and energy harvesting. This work focuses on the creation of a new type of bio-nano hybrid substrate for military biosensing applications. Specifically it is shown that the nano-scale interactions of the optical protein bacteriorhodopsin and colloidal semiconductor quantum dots can be utilized as a generic sensing substrate. This work spans from the basic creation of the protein to its application in a novel biosensing system. The functionality of this sensor design originates from the unique interactions between the quantum dot and bacteriorhodopsin molecule when in nanoscale proximity. A direct energy transfer relationship has been established between coreshell quantum dots and the optical protein bacteriorhodopsin that substantially enhances the protein’s native photovoltaic capabilities. This energy transfer phenomena is largely distance dependent, in the sub-10nm realm, and is characterized experimentally at multiple separation distances. Experimental results on the energy transfer efficiency in this hybrid system correlate closely to theoretical predictions. Deposition of the hybrid system with nano-scale control has allowed for the utilization of this energy transfer phenomena as a modulation point for a functional biosensor prototype. This work reveals that quantum dots have the ability to activate the bacteriorhodopsin photocycle through both photonic and non-photonic energy transfer mechanisms. By altering the energy transferred to the bacteriorhodopsin molecule from the quantum dot, the electrical output of the protein can be modulated. A biosensing prototype was created in which the energy transfer relationship is altered upon target binding, demonstrating the applicability of a quantum dot/bacteriorhodopsin hybrid system for sensor applications. The electrical nature of this sensing substrate will allow for its efficient integration into a nanoelectronics array form, potentially leading to a small-low power sensing platform for remote toxin detection applications.

Alternative splicing of pre-mRNA in cancer: focus on G protein-coupled peptide hormone receptors

Through alternative splicing, multiple different transcripts can be generated from a single gene. Alternative splicing represents an important molecular mechanism of gene regulation in physiological processes such as developmental programming as well as in disease. In cancer, splicing is significantly altered. Tumors express a different collection of alternative spliceoforms than normal tissues. Many tumor-associated splice variants arise from genes with an established role in carcinogenesis or tumor progression, and their functions can be oncogenic. This raises the possibility that products of alternative splicing play a pathogenic role in cancer. Moreover, cancer-associated spliceoforms represent potential diagnostic biomarkers and therapeutic targets. G protein-coupled peptide hormone receptors provide a good illustration of alternative splicing in cancer. The wild-type forms of these receptors have long been known to be expressed in cancer and to modulate tumor cell functions. They are also recognized as attractive clinical targets. Recently, splice variants of these receptors have been increasingly identified in various types of cancer. In particular, alternative cholecystokinin type 2, secretin, and growth hormone-releasing hormone receptor spliceoforms are expressed in tumors. Peptide hormone receptor splice variants can fundamentally differ from their wild-type receptor counterparts in pharmacological and functional characteristics, in their distribution in normal and malignant tissues, and in their potential use for clinical applications.

Can RapidTEG accelerate the search for coagulopathies in the patient with multiple injuries?

Hypothesis: Early recognition of coagulopathy may improve the care of patients with multiple injuries. Rapid thrombelastography (RapidTEG) is a new variant of thrombelastography (TEG), in which coagulation is initiated by the addition of protein tissue factor. The kinetics of coagulation and the times of measurement were compared for two variants of TEG--RapidTEG and conventional TEG, in which coagulation was initiated with kaolin. The measurements were performed on blood samples from 20 patients with multiple injuries. The RapidTEG results were also compared with conventional measurements of blood coagulation. The mean time for the RapidTEG test was 19.2 +/- 3.1 minutes (mean +/- SD), in comparison with 29.9 +/- 4.3 minutes for kaolin TEG and 34.1 +/- 14.5 minutes for conventional coagulation tests. The mean time for the RapidTEG test was 30.8 +/- 5.72 minutes, in comparison with 41.5 +/- 5.66 minutes for kaolin TEG and 64.9 +/- 18.8 for conventional coagulation tests---measured from admission of the patients to the resuscitation bay until the results were available. There were significant correlations between the RapidTEG results and those from kaolin TEG and conventional coagulation tests. RapidTEG is the most rapid available test for providing reliable information on coagulopathy in patients with multiple injuries. This has implications for improving patient care.

Autoantibodies directed against bactericidal/permeability-increasing protein in patients with cystic fibrosis: association with microbial respiratory tract colonization

BACKGROUND: Cystic fibrosis (CF) is associated with the appearance of serum autoantibodies directed against bactericidal/permeability-increasing protein (BPI). OBJECTIVES: To determine the age-specific seroprevalence rates of anti-BPI-IgG and IgA in a population of patients with CF and to correlate anti-BPI antibody concentrations with microbial respiratory tract colonization and pulmonary function variables at the time of serum sampling and 6 years thereafter. METHODS: Determination of BPI antibodies of the IgG and IgA isotypes using a commercial enzyme-linked immunosorbent assay in sera of a CF serum bank of 1992; correlation of anti-BPI antibody concentrations with age, clinical score, pulmonary function variables in 1992 and 1998, total serum immunoglobulin isotype concentrations and respiratory tract colonization with Pseudomonas aeruginosa and Aspergillus spp. RESULTS: Seventy-one patients (age in 1992, 14.1 +/- 7.5 years) were studied. Reactivities for anti-BPI-IgG and IgA were found in 28 (39%) and 26 (37%) patients, respectively. The seroprevalence of anti-BPI-IgA, but not IgG, increased significantly with age. P. aeruginosa colonization was associated with elevated concentrations of anti-BPI-IgG (P = 0.003) and IgA (P = 0.037). There were significant negative correlations between pulmonary function variables (vital capacity, forced expiratory volume in 1 s) in 1992 and 1998, respectively, and concentrations of anti-BPI-IgG or IgA in a multiple regression analysis. Anti-BPI-IgG, but not IgA, remained significantly associated with P. aeruginosa colonization (P = 0.006) and with reduced vital capacity (P = 0.01) in 1998 after correction for total serum isotype concentration. CONCLUSIONS: Anti-BPI-IgG are strongly associated with concurrent P. aeruginosa colonization and with long term restrictive pulmonary function abnormalities.

Analysis of protein expression in zebrafish during gonad differentiation by targeted proteomics

The molecular mechanisms governing sex determination and differentiation in the zebrafish (Danio rerio) are not fully understood. To gain more insights into the function of specific genes in these complex processes, the expression of multiple candidates needs to be assessed, preferably on the protein level. Here, we developed a targeted proteomics method based on selected reaction monitoring (SRM) to study the candidate sex-related proteins in zebrafish which were selected based on a global proteomics analysis of adult gonads and representational difference analysis of male and female DNA, as well as on published information on zebrafish and other vertebrates. We employed the developed SRM protocols to acquire time-resolved protein expression profiles during the gonad differentiation period in vas::EGFP transgenic zebrafish. Evidence on protein expression was obtained for the first time for several candidate genes previously studied only on the mRNA level or suggested by bioinformatic predictions. Tuba1b (tubulin alpha 1b), initially included in the study as one of the potential housekeeping proteins, was found to be preferentially expressed in the adult testis with nearly absent expression in the ovary. The revealed changes in protein expression patterns associated with gonad differentiation suggest that several of the examined proteins, especially Ilf2 and Ilf3 (interleukin enhancer-binding factors 2 and 3), Raldh3 (retinaldehyde dehydrogenase type 3), Zgc:195027 (low density lipoprotein-related receptor protein 3) and Sept5a (septin 5a), may play a specific role in the sexual differentiation in zebrafish.

14-3-3 proteins interact with the beta-thymosin repeat protein Csp24.

Conditioned stimulus pathway protein 24 (Csp24) is a beta-thymosin-like protein that is homologous to other members of the family of beta-thymosin repeat proteins that contain multiple actin binding domains. Actin co-precipitates with Csp24 and co-localizes with it in the cytosol of type-B photoreceptor cell bodies. Several signal transduction pathways have been shown to regulate the phosphorylation of Csp24 and contribute to cellular plasticity. Here, we report the identification of the adapter protein 14-3-3 in lysates of the Hermissenda circumesophageal nervous system and its interaction with Csp24. Immunoprecipitation experiments using an antibody that is broadly reactive with several isoforms of the 14-3-3 family of proteins showed that Csp24 co-precipitates with 14-3-3 protein, and nervous systems stimulated with 5-HT exhibited a significant increase in co-precipitated Csp24 probed with a phosphospecific antibody as compared with controls. These results indicate that post-translational modifications of Csp24 regulate its interaction with 14-3-3 protein, and suggest that this mechanism may contribute to the control of intrinsic enhanced excitability.

RNAi reduces expression and intracellular retention of mutant cartilage oligomeric matrix protein.

Mutations in cartilage oligomeric matrix protein (COMP), a large extracellular glycoprotein expressed in musculoskeletal tissues, cause two skeletal dysplasias, pseudoachondroplasia and multiple epiphyseal dysplasia. These mutations lead to massive intracellular retention of COMP, chondrocyte death and loss of growth plate chondrocytes that are necessary for linear growth. In contrast, COMP null mice have only minor growth plate abnormalities, normal growth and longevity. This suggests that reducing mutant and wild-type COMP expression in chondrocytes may prevent the toxic cellular phenotype causing the skeletal dysplasias. We tested this hypothesis using RNA interference to reduce steady state levels of COMP mRNA. A panel of shRNAs directed against COMP was tested. One shRNA (3B) reduced endogenous and recombinant COMP mRNA dramatically, regardless of expression levels. The activity of the shRNA against COMP mRNA was maintained for up to 10 weeks. We also demonstrate that this treatment reduced ER stress. Moreover, we show that reducing steady state levels of COMP mRNA alleviates intracellular retention of other extracellular matrix proteins associated with the pseudoachondroplasia cellular pathology. These findings are a proof of principle and the foundation for the development of a therapeutic intervention based on reduction of COMP expression.

Ribozyme-mediated reduction of wild-type and mutant cartilage oligomeric matrix protein (COMP) mRNA and protein.

Dominant-negative mutations in the homopentameric extracellular matrix glycoprotein cartilage oligomeric matrix protein (COMP) result in inappropriate intracellular retention of misfolded COMP in the rough endoplasmic reticulum of chondrocytes, causing chondrocyte cell death, which leads to two skeletal dysplasias: pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (EDM1). COMP null mice show no adverse effects on normal bone development and growth, suggesting a possible therapy involving removal of COMP mRNA. The goal of this study was to assess the ability of a hammerhead ribozyme (Ribo56, designed against the D469del mutation) to reduce COMP mRNA expression. In COS7 cells transfected with plasmids that overexpress wild-type or mutant COMP mRNA and Ribo56, the ribozyme reduced overexpressed normal COMP mRNA by 46% and mutant COMP mRNA by 56% in a dose-dependent manner. Surprisingly, the use of recombinant adenoviruses to deliver wild-type or mutant COMP mRNA and Ribo56 simultaneously into COS7 cells proved problematic for the activity of the ribozyme to reduce COMP expression. However, in normal human costochondral cells (hCCCs) infected only with adenoviruses expressing Ribo56, expression of endogenous wild-type COMP mRNA was reduced in a dose-dependent manner by 50%. In chondrocytes that contain heterozygous COMP mutations (D469del, G427E and D511Y) that cause PSACH, Ribo56 was more effective at reducing COMP mRNA (up to 70%). These results indicate that Ribo56 is effective at reducing mutant and wild-type COMP levels in cells and suggests a possible mode of therapy to reduce the mutant protein load.

Gene by BMI Interactions Influencing C-Reactive Protein Levels in European-Americans

C-Reactive Protein (CRP) is a biomarker indicating tissue damage, inflammation, and infection. High-sensitivity CRP (hsCRP) is an emerging biomarker often used to estimate an individual’s risk for future coronary heart disease (CHD). hsCRP levels falling below 1.00 mg/l indicate a low risk for developing CHD, levels ranging between 1.00 mg/l and 3.00 mg/l indicate an elevated risk, and levels exceeding 3.00 mg/l indicate high risk. Multiple Genome-Wide Association Studies (GWAS) have identified a number of genetic polymorphisms which influence CRP levels. SNPs implicated in such studies have been found in or near genes of interest including: CRP, APOE, APOC, IL-6, HNF1A, LEPR, and GCKR. A strong positive correlation has also been found to exist between CRP levels and BMI, a known risk factor for CHD and a state of chronic inflammation. We conducted a series of analyses designed to identify loci which interact with BMI to influence CRP levels in a subsample of European-Americans in the ARIC cohort. In a stratified GWA analysis, 15 genetic regions were identified as having significantly (p-value < 2.00*10-3) distinct effects on hsCRP levels between the two obesity strata: lean (18.50 kg/m2 < BMI < 24.99 kg/m2) and obese (BMI ≥ 30.00 kg/m2). A GWA analysis performed on all individuals combined (i.e. not a priori stratified for obesity status) with the inclusion of an additional parameter for BMI by gene interaction, identified 11 regions which interact with BMI to influence hsCRP levels. Two regions containing the genes GJA5 and GJA8 (on chromosome 1) and FBXO11 (on chromosome 2) were identified in both methods of analysis suggesting that these genes possibly interact with BMI to influence hsCRP levels. We speculate that atrial fibrillation (AF), age-related cataracts and the TGF-β pathway may be the biological processes influenced by the interaction of GJA5, GJA8 and FBXO11, respectively, with BMI to cause changes in hsCRP levels. Future studies should focus on the influence of gene x bmi interaction on AF, age-related cataracts and TGF-β.