1.
Differential expression and processing of secretogranin II in relation to the status of pheochromocytoma : implications for the production of the tumoral marker EM66.
Source
J Guillemot, Institut National de la Santé et de la Recherche Médicale (INSERM) Unité 982, Laboratory of Neuronal and Neuroendocrine Differentiation and Communication, European Institute for Peptide Research (IFRMP 23), University of Rouen, Mont-Saint-Aignan, France.
Abstract
We have previously demonstrated that measurement of tissue concentrations of the secretogranin II (SgII)-derived peptide EM66, may help to discriminate between benign and malignant pheochromocytomas and that EM66 represents a sensitive plasma marker of pheochromocytomas. Here, we investigated the gene expression and protein production of SgII in 13 normal adrenal glands, and 35 benign and 16 malignant pheochromocytomas with the goal to examine the molecular mechanisms leading to the marked variations of expression of EM66 in tumoral chromaffin tissue. EM66 levels were 16-fold higher in benign than in malignant pheochromocytomas and had an area under the ROC curve of 0.95 for distinction of benign and malignant tumors. Q-PCR experiments indicated that the SgII gene was significantly underexpressed in malignant compared to benign tumors. Western blot analysis using antisera directed against SgII and SgII-derived fragments revealed lower SgII protein and SgII-processing products in malignant tumors. Western blot also showed that low p-CREB concentrations seemed to be associated with the malignant status. In addition, the prohormone convertase PC1 and PC2 genes and proteins were overexpressed in benign compared to malignant pheochromocytomas. Low concentrations of EM66 found in malignant tumors are associated with reduced expression and production of SgII and SgII-derived peptides that could be ascribed to a decrease in SgII gene transcription, probably linked to p-CREB down-regulation, and to lower PC levels. These findings highlight the mechanisms leading to lower concentrations of EM66 in malignant pheochromocytoma and strengthen the notion that this peptide is a suitable marker of this neuroendocrine tumor.
- PMID:
- 22217803
- [PubMed - as supplied by publisher]
Recognition of Epidermal Transglutaminase by IgA and Tissue Transglutaminase 2 Antibodies in a Rare Case of Rhesus Dermatitis.
Source
Division of Microbiology, Tulane National Primate Research Center.
Abstract
Tissue transglutaminase 2 (tTG2) is an intestinal digestive enzyme which deamidates already partially digested dietary gluten e.g. gliadin peptides. In genetically predisposed individuals, tTG2 triggers autoimmune responses that are characterized by the production of tTG2 antibodies and their direct deposition into small intestinal wall (1,2). The presence of such antibodies constitutes one of the major hallmarks of the celiac disease (CD). Epidermal transglutaminase (eTG) is another member of the transglutaminase family that can also function as an autoantigen in a small minority of CD patients. In these relatively rare cases, eTG triggers an autoimmune reaction (a skin rash) clinically known as dermatitis herpetiformis (DH). Although the exact mechanism of CD and DH pathogenesis is not well understood, it is known that tTG2 and eTG share antigenic epitopes that can be recognized by serum antibodies from both CD and DH patients (3,4). In this study, the confocal microscopy examination of biopsy samples from skin lesions of two rhesus macaques (Macaca mulatta) with dermatitis (Table 1, Fig. 1 and 2) was used to study the affected tissues. In one animal (EM96) a spectral overlap of IgA and tTG2 antibodies (Fig. 3) was demonstrated. The presence of double-positive tTG2+IgA+ cells was focused in the deep epidermis, around the dermal papillae. This is consistent with lesions described in DH patients (3). When EM96 was placed on a gluten-free diet, the dermatitis, as well as tTG2+IgA+ deposits disappeared and were no longer detectable (Figs. 1-3). Dermatitis reappeared however, based on re-introduction of dietary gluten in EM96 (not shown). In other macaques including animal with unrelated dermatitis, the tTG2+IgA+ deposits were not detected. Gluten-free diet-dependent remission of dermatitis in EM96 together with presence of tTG2+IgA+ cells in its skin suggest an autoimmune, DH-like mechanism for the development of this condition. This is the first report of DH-like dermatitis in any non-human primate.
- PMID:
- 22214930
- [PubMed - in process]
Myofibroblasts in the Infarct Area: Concepts and Challenges.
Source
Department of Pharmacology, Cardiovascular Research Institute Maastricht, Maastricht University, 50 Universiteitssingel, 6229ER Maastricht, P.O. Box 616, 6200MD Maastricht, The Netherlands.
Abstract
Myofibroblasts are differentiated fibroblasts that hold a key role in wound healing and remodeling following myocardial infarction (MI). A large repertoire of stimuli, such as mechanical stretch, growth factors, cytokines, and vasoactivepeptides, induces myofibroblast differentiation. Myofibroblasts are responsible for the production and deposition of collagen, leading to the establishment of a dense extracellular matrix that strengthens the infarcted tissue and minimizes dilatation of the infarct area. In addition, cells contributing to fibrosis act on sites distal from the infarct area and promote collagen deposition in noninfarcted tissue, thus contributing to adverse remodeling and consequently to the development of congestive heart failure (CHF). Current drugs that are used to treat post-MI CHF do influence fibroblasts and myofibroblasts; however, their therapeutic efficacy is far from being regarded as ideal. Novel therapeutic agents targeting (myo)fibroblasts are being developed to successfully prevent the cardiac remodeling of sites remote from the infarct area and therefore hinder the establishment of CHF. The purpose of this review article is to discuss the basic concepts of the myofibroblasts' actions in cardiac wound healing processes, factors that influence them, currently available pharmacological agents, and future challenges in this area.
- PMID:
- 22214878
- [PubMed - as supplied by publisher]
Exposure of the lysine in the gamma chain dodecapeptide of human fibrinogen is not enhanced by adsorption to poly(ethylene terephthalate) as measured by biotinylation and mass spectrometry.
Source
Department of Biomedical Engineering and Center for Materials Innovation, Washington University, St. Louis, Missouri; Department of Neurology, Hope Center for Neurological Disorders and Alzheimer's Disease Research Center, Washington University School of Medicine, St. Louis, Missouri.
Abstract
Conformational changes in adsorbed fibrinogen may enhance the exposure of platelet adhesive sites that are inaccessible in solution. To test this hypothesis, mass spectrometric methods were developed to quantify chemical modification of lysine residues following adsorption of fibrinogen to biomaterials. The quantitative method used an internal standard consisting of isotope-labeled fibrinogen secreted by human HepG2 cells in culture. Lysine residues in the internal standard were partially reacted with NHS-biotin. For the experimental samples, normal human fibrinogen was adsorbed to poly(ethylene terephthalate) (PET) particles. The adsorbed fibrinogen was reacted with NHS-biotin and then eluted from the particles. Constant amounts of internal standard were added to sample fibrinogen and analyzed by liquid chromatography/tandem mass spectrometry. Biotinylation of the lysine residue in the platelet-adhesive gamma chain dodecapeptide (GCDP) was quantified by comparison with the internal standard. Approximately 80% of the GCDPpeptides were biotinylated when fibrinogen was reacted with NHS-biotin in solution or adsorbed onto PET. These results are generally consistent with previous antibody binding studies and suggest that other regions of fibrinogen may be crucial in promoting platelet adhesion to materials. The results do not directly address but are consistent with the hypothesis that only activated platelets adhere to adsorbed fibrinogen. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2011.
Copyright © 2011 Wiley Periodicals, Inc.
Dietary nitrogen and fish welfare.
Source
CCMAR-CIMAR L.A., Centro de Ciências do Mar do Algarve, Universidade do Algarve, Campus de Gambelas, 8005-139, Faro, Portugal, lconcei@ualg.pt.
Abstract
Little research has been done in optimizing the nitrogenous fraction of the fish diets in order to minimize welfare problems. The purpose of this review is to give an overview on how amino acid (AA) metabolism may be affected when fish are under stress and the possible effects on fish welfare when sub-optimal dietary nitrogen formulations are used to feed fish. In addition, it intends to evaluate the current possibilities, and future prospects, of using improved dietary nitrogen formulations to help fish coping with predictable stressful periods. Both metabolomic and genomic evidence show that stressful husbandry conditions affect AA metabolism in fish and may bring an increase in the requirement of indispensable AA. Supplementation in arginine and leucine, but also eventually in lysine, methionine, threonine and glutamine, may have an important role in enhancing the innate immune system. Tryptophan, as precursor for serotonin, modulates aggressive behaviour and feed intake in fish. Bioactive peptides may bring important advances in immunocompetence, disease control and other aspects of welfare of cultured fish. Fishmeal replacement may reduce immune competence, and the full nutritional potential of plant-protein ingredients is attained only after the removal or inactivation of some antinutritional factors. This review shows that AA metabolism is affected when fish are under stress, and this together with sub-optimal dietary nitrogen formulations may affect fish welfare. Furthermore, improved dietary nitrogen formulations may help fish coping with predictable stressful events.
MALDI based identification of soybean protein markers - Possible analytical targets for allergen detection in processed foods.
Source
Department of Food Safety and Food Quality, Research group Food Chemistry and Human Nutrition, Ghent University, Coupure Links 653, B-9000 Gent, Belgium; Laboratory for Protein Biochemistry and Biomolecular Engineering, Ghent University, KL Ledeganckstraat 35, B-9000 Gent, Belgium.
Abstract
Soybean (Glycine max) is extensively used all over the world due to its nutritional qualities. However, soybean is included in the "big eight" list of food allergens. According to the EU directive 2007/68/EC, food products containing soybeans have to be labeled in order to protect the allergic consumers. Nevertheless, soybeans can still inadvertently be present in food products. The development of analytical methods for the detection of traces of allergens is important for the protection of allergic consumers. Mass spectrometry of marker proteolytical fragments of protein allergens is growingly recognized as a detection method in food control. However, quantification of soybean at the peptide level is hindered due to limited information regarding specific stable markers derived after proteolytic digestion. The aim of this study was to use MALDI-TOF/MS and MS/MS as a fast screening tool for the identification of stable soybean derived tryptic markers which were still identifiable even if the proteins were subjected to various changes at the molecular level through a number of reactions typically occurring during food processing (denaturation, the Maillard reaction and oxidation). The peptides (401)Val-Arg(410) from the G1 glycinin (Gly m 6) and the (518)Gln-Arg(528) from the α' chain of the β-conglycinin (Gly m 5) proved to be the most stable. These peptides hold potential to be used as targets for the development of new analytical methods for the detection of soybean protein traces in processed foods.
Copyright © 2011. Published by Elsevier Inc.
Immunodistribution of amyloid beta protein (Aβ) and advanced glycation end-product receptors (RAGE) in choroid plexus and ependyma of resuscitated patients.
Source
Prof. Danuta Maślińska, Department of Experimental and Clinical Neuropathology, Mossakowski Medical Research Centre, Polish Academy of Sciences, 5 Pawińskiego St, 02-106 Warsaw, Poland, phone +48 22 608 65 02, fax +48 22 608 65 02, e-mail: maslinskad@cmdik.pan.pl.
Abstract
RAGE (receptor for advanced glycation end-products) participates in the influx transport of glycated Aβ (amyloid beta) from the blood to the brain. Because little is known of the RAGE operating in brain barriers such as those in the choroid plexus and ependyma, the aim of the present study was to examine the immunodistributions of RAGE and Aβ peptidesin the choroid plexus where the blood-cerebrospinal fluid barrier (B-CSF) is located, and in ependyma of the brain ventricles associated with functions of the cerebrospinal fluid-brain barrier (CSF-B). The study was performed on patients over 65 years successfully resuscitated after cardiac arrest with survival a few weeks. The control group consisted of age-matched individuals who were not resuscitated and died immediately after cardiac arrest. In resuscitated patients, but not in controls, RAGE receptors were localized in choroid plexus (CP) epithelial cells and in ependymal cells bordering the brain ventricles. These cells form the B-CSF and CSF-B barriers. The presence of Aβ was detected within the CP blood vessels and in the basement membrane of the CP epithelium. In numerous cytoplasmic vacuoles of CP epithelial and ependymal cells Aβ protein was found and our observations suggest that the contents of those vacuoles were undergoing progressive digestion. The results demonstrated that CP epithelium and ependymal cells, equipped with RAGE receptors, not only play an important role in the creation of amyloid deposits in the brain but are also places where Aβ may be utilized. The RAGE transportation system should be a main target in the therapy of brain amyloidosis, a well-known risk factor of Alzheimer disease.
Recent progress in research on small post-translationally modified peptide signals in plants.
Source
National Institute for Basic Biology, Nishigonaka 38, Myodaiji, Okazaki, Aichi 444-8585, Japan.
Abstract
Peptide signaling plays a major role in various aspects of plant growth and development, as has been shown in recent biochemical, genetic and bioinformatic studies. There are over a dozen secreted peptides recognized in plants known to regulate cellular functions. To become functional, these secreted peptide signals often undergo post-translational modifications, such as tyrosine sulfation, proline hydroxylation, and hydroxyproline arabinosylation, and successive proteolytic processing. These types of 'small post-translationally modified peptide signals' are one of the major groups of peptide signals found in plants. In parallel with the discovery of peptide signals, specific receptors for such peptide signals were identified as being membrane-localized leucine-rich repeat receptor kinases. This short review highlights the recent progress in research on small post-translationally modified peptide signals, including our own research.
© 2011 The Author. Journal compilation © 2011 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.
Inhibition of the STAT3 signaling pathway is involved in the antitumor activity of cepharanthine in SaOS2 cells.
Source
Institute of Vascular Medicine, Medical Research Center, Peking University Third Hospital, and Key Laboratory of Cardiovascular Molecular Biology and Regulatory Peptides, Ministry of Health, Beijing 100191, China.
Abstract
Aim:To investigate the molecular mechanisms underlying the antitumor activity of cepharanthine (CEP), an alkaloid extracted from Stephania cepharantha Hayata.Methods:Human osteosarcoma cell line SaOS2 was used. MTT assay, Hoechst 33342 nuclear staining, flow cytometry, Western blotting and nude mouse xenografts of SaOS2 cells were applied to examine the antitumor activity of CEP in vitro and in vivo. The expression levels of STAT3 and its downstream signaling molecules were measured with Western blotting and immunochemistry analysis. The activity of STAT3 was detected based on the phosphorylation level of STAT3, luciferase gene reporter assay and translocation of STAT3 to the nucleus.Results:Treatment of SaOS2 cells with CEP (2.5-20 μmol/L) inhibited the cell growth in a concentration- and time-dependent manner. CEP (10 μmol/L) caused cell cycle arrest at G(1) phase and induced apoptosis of SaOS2 cells. CEP (10 and 15 μmol/L) significantly decreased the expression of STAT3 in SaOS2 cells. Furthermore, CEP (5 and 10 μmol/L) significantly inhibited the expression of target genes of STAT3, including the anti-apoptotic gene Bcl-xL and the cell cycle regulators c-Myc and cyclin D1. In nude mouse xenografts of SaOS2 cells, CEP (20 mg·kg(-1)·d(-1), ip for 19 d) significantly reduced the volume and weight of the tumor.Conclusion:Our findings suggest that inhibition of STAT3 signaling pathway is involved in the anti-tumor activity of CEP.
Resveratrol, a neuroprotective supplement for Alzheimer's disease.
Source
School of Traditional Chinese Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China. feili@northwestern.edu.
Abstract
The polyphenolic compound resveratrol (3,4',5-trihydroxystilbene) is a naturally occurring phytochemical which has been found in more than 70 plant species, including herbs and human food products such as grapes, berries, and peanuts. Resveratrol was first isolated in 1940; however, little attention was paid to it until its benefits in coronary heart disease were studied in 1992. Since then, increasing evidence has indicated that resveratrol may be useful in treating cardiovascular diseases, cancers, pain, inflammation, tissue injury, and in reducing the risk of neurodegenerative disorders, especially Alzheimer's disease (AD). AD is characterized by a progressive dementia, and is one of the most common neurodegenerative disorders in the elderly. It has been reported that resveratrol exhibits neuroprotective benefits in animal models of AD. Resveratrol promotes the non-amyloidogenic cleavage of the amyloid precursor protein, enhances clearance of amyloid beta-peptides, and reduces neuronal damage. Despite the effort spent trying to understand the mechanisms by which resveratrol functions, the research work in this field is still incomplete. Many concerns such as bioavailability, biotransformation, synergism with other dietary factors, and risks inherent to its possible pro-oxidant activities still need to be addressed. This review summarizes and discusses the neuroprotective effects of resveratrol on AD, and their potential mechanisms.
- PMID:
- 22211686
- [PubMed - as supplied by publisher]
[What's new in dermatological research?].
Source
Université de Franche Comté, EA3181, IFR133 et Service de Dermatologie, CHU de Besançon, 2 place Saint-Jacques 25030 Besançon cedex, France.
Abstract
Dermatological research has been very active this year. Most of the numerous fields investigated involve the mechanisms of cutaneous regeneration and barrier function. A novel target of early ultraviolet-induced skin photodamage, the Syk kinase, has been recently identified. Synergistic relationship between telomere damage and cutaneous progerin production during cell senescence may also participate in the natural skin aging process. Interestingly, ultraviolet radiation induces an inhibitory effect on subcutaneous lipogenesis. Androgenetic alopecia or common baldness is not characterized by loss of hair follicle stem cells but by a defect in the conversion of hair follicle stem cells into active progenitor cells. It has been shown that the cornified envelope functions not only as a physicomechanical barrier, but also as both a biochemical line of antoxidant defense and an immunological line of defense. Like human papillomaviruses, Merckel cell polyomavirus belongs to the skin microbiome and different studies have demonstrated the protective role of epidermal resident microflora through the activation of innate immunity. Production of antimicrobial peptides and the activation of inflammasome and plasmacytoid dendritic cells are involved in the modulation of the cutaneous barrier function. Results from different studies suggest that IL-22 and IL-36 may be common mediators of both innate and adaptive immune responses. All these pathways interact not only to maintain cutaneous homeostasis and integrity (wound healing) but also to regulate autoinflammatory and autoimmune dermatoses (psoriasis, lupus, rosacea, atopic dermatitis, etc…). In addition, molecular mechanisms that regulate T helper type 2 differentiation and the retention at the site of inflammation of Th2 cells have been identified. New promising therapeutic targets for different chronic dermatosis are thus suggested. Mechanobiology and mechanotransduction are also emerging fields that investigate mechanical interactions between living cells and their environment and the conversion of mechanical cues into biochemical signals. Electronic second skin is now a current concept through bio-integrated epidermal electronics platforms used for different monitoring and stimulations of body functions.
Copyright © 2011 Elsevier Masson SAS. All rights reserved.
Trackable and Targeted Phage as Positron Emission Tomography (PET) Agent for Cancer Imaging.
Source
1. Molecular Imaging Center, Department of Radiology, University of Southern California, Los Angeles 90033, USA.
Abstract
The recent advancement of nanotechnology has provided unprecedented opportunities for the development of nanoparticle enabled technologies for detecting and treating cancer. Here, we reported the construction of a PET trackable organic nanoplatform based on phage particle for targeted tumor imaging. Method: The integrin α(v)β(3) targeted phage nanoparticle was constructed by expressing RGD peptides on its surface. The target binding affinity of this engineered phage particle was evaluated in vitro. A bifunctional chelator (BFC) 1,4,7,10-tetraazadodecane-N,N',N",N"'-tetraacetic acid (DOTA) or 4-((8-amino-3,6,10,13,16,19-hexaazabicyclo [6.6.6] icosane-1-ylamino) methyl) benzoic acid (AmBaSar) was then conjugated to the phage surface for (64)Cu(2+) chelation. After (64)Cu radiolabeling, microPET imaging was performed in U87MG tumor model and the receptor specificity was confirmed by blocking experiments. Results: The phage-RGD demonstrated target specificity based on ELISA experiment. According to the TEM images, the morphology of the phage was unchanged after the modification with BFCs. The labeling yield was 25 ± 4% for (64)Cu-DOTA-phage-RGD and 46 ± 5% for (64)Cu-AmBaSar-phage-RGD, respectively. At 1 h time point, (64)Cu-DOTA-phage-RGD and (64)Cu-AmBaSar-phage-RGD have comparable tumor uptake (~ 8%ID/g). However, (64)Cu-AmBaSar-phage-RGD showed significantly higher tumor uptake (13.2 ± 1.5 %ID/g, P<0.05) at late time points compared with (64)Cu-DOTA-phage-RGD (10 ± 1.2 %ID/g). (64)Cu-AmBaSar-phage-RGD also demonstrated significantly lower liver uptake, which could be attributed to the stability difference between these chelators. There is no significant difference between two tracers regarding the uptake in kidney and muscle at all time points tested. In order to confirm the receptor specificity, blocking experiment was performed. In the RGD blocking experiment, the cold RGD peptide was injected 2 min before the administration of (64)Cu-AmBaSar-phage-RGD. Tumor uptake was partially blocked at 1 h time point. Phage-RGD particle was also used as the competitive ligand. In this case, the tumor uptake was significantly reduced and the value was kept at low level consistently. Conclusion: In this report, we constructed a PET trackable nanoplatform based on phage particle and demonstrated the imaging capability of these targeted agents. We also demonstrated that the choice of chelator could have significant impact on imaging results of nano-agents. The method established in this research may be applicable to other receptor/ligand systems for theranostic agent construction, which could have an immediate and profound impact on the field of imaging/therapy and lay the foundation for the construction of next generation cancer specific theranostic agents.
Cell-Specific siRNA Delivery by Peptides and Antibodies.
Source
Department of Bioengineering and Institute for Bioengineering and Biopharmaceutical Research, Hanyang University, Seoul, South Korea; Department of Internal Medicine/Section for Infectious Diseases, Yale School of Medicine, New Haven, Connecticut, USA.
Abstract
Cellular targeting and intracellular delivery of small interfering RNA (siRNA) remain a critical barrier to the clinical application of RNA interference. This chapter provides an overview of various delivery agents employing protein ligands mediating cell-specific delivery of siRNA. Specifically, the chapter details methodologies for the conjugation of antibody or peptide ligands to i) the cationic peptide-oligo-9-arginine (ii) the polymer poly(lactic-co-glycolic acid) (PLGA) and (iii) a lipid-vesicle (liposome).
Copyright © 2012 Elsevier Inc. All rights reserved.
- PMID:
- 22208983
- [PubMed - in process]
Efficient Intracellular Delivery of Nucleic Acid Pharmaceuticals Using Cell-Penetrating Peptides.
Source
Institute for Chemical Research, Kyoto University , Uji, Kyoto 611-0011, Japan.
Abstract
Over the last 20 years, researchers have designed or discovered peptides that can permeate membranes and deliver exogenous molecules inside a cell. These peptides, known as cell-penetrating peptides (CPPs), typically consist of 6-30 residues, including HIV TAT peptide, penetratin, oligoarginine, transportan, and TP10. Through chemical conjugation or noncovalent complex formation, these structures successfully deliver bioactive and membrane-impermeable molecules into cells. CPPs have also gained attention as an attractive vehicle for the delivery of nucleic acid pharmaceuticals (NAPs), including genes/plasmids, short oligonucleotides, and small interference RNAs and their analogues, due to their high internalization efficacy, low cytotoxicity, and flexible structural design. In this Account, we survey the potential of CPPs for the design and optimization of NAP delivery systems. First, we describe the impact of the N-terminal stearylation of CPPs. Endocytic pathways make a major contribution to the cellular uptake of NAPs. Stearylation at the N-terminus of CPPs with stearyl-octaarginine (R8), stearyl-(RxR)(4), and stearyl-TP10 prompts the formation of a self-assembled core-shell nanoparticle with NAPs, a compact structure that promotes cellular uptake. Researchers have designed modifications such as the addition of trifluoromethylquinoline moieties to lysine residues to destabilize endosomes, as exemplified by PepFect 6, and these changes further improve biological responsiveness. Alternatively, stearylation also allows implantation of CPPs onto the surface of liposomes. This feature facilitates "programmed packaging" to establish multifunctional envelope-type nanodevices (MEND). The R8-MEND showed high transfection efficiency comparable to that of adenovirus in non-dividing cells. Understanding the cellular uptake mechanisms of CPPs will further improve CPP-mediated NAP delivery. The cellular uptake of CPPs and their NAP complex involves various types of endocytosis. Macropinocytosis, a mechanism which is also activated in response to stimuli such as growth factors or viruses, is a primary pathway for arginine-rich CPPs because high cationic charge density promotes this endocytic pathway. The use of larger endosomes (known as macropinosomes) rather than clathrin- or caveolae-mediated endocytosis has been reported in macropinocytosis which would also facilitate the endocytosis of NAP nanoparticles into cells.
Mechanisms by which pesticides affect insect immunity.
Source
USDA-ARS, Pollinating Insects Research Unit, Dept. Biology UMC 5310, Utah State University, Logan, UT 84322-5310, USA.
Abstract
The current state of knowledge regarding the effect of pesticides on insect immunity is reviewed here. A basic understanding of these interactions is needed for several reasons, including to improve methods for controlling pest insects in agricultural settings, for controlling insect vectors of human diseases, and for reducing mortality in beneficial insects. Bees are particularly vulnerable to sublethal pesticide exposures because they gather nectar and pollen, concentrating environmental toxins in their nests in the process. Pesticides do have effects on immunity. Organophosphates and some botanicals have been found to impact hemocyte number, differentiation, and thus affect phagocytosis. The phenoloxidase cascade and malanization have also been shown to be affected by several insecticides. Many synthetic insecticides increase oxidative stress, and this could have severe impacts on the production of some antimicrobial peptides in insects, but research is needed to determine the actual effects. Pesticides can also affect grooming behaviors, rendering insects more susceptible to disease. Despite laboratory data documenting pesticide/pathogen interactions, little field data is available at the population level.
Copyright © 2011. Published by Elsevier Inc.
Oocyte specific oolemmal SAS1B involved in sperm binding through intra-acrosomal SLLP1 during fertilization.
Source
Department of Cell Biology, Center for Research in Contraceptive and Reproductive Health, University of Virginia, Charlottesville, VA 22908, USA.
Abstract
Molecular mechanisms by which fertilization competent acrosome-reacted sperm bind to the oolemma remain uncharacterized. To identify oolemmal binding partner(s) for sperm acrosomal ligands, affinity panning was performed with mouse oocyte lysates using sperm acrosomal protein, SLLP1 as a target. An oocyte specific membrane metalloproteinase, SAS1B (Sperm Acrosomal SLLP1 Binding), was identified as a SLLP1 binding partner. cDNA cloning revealed six SAS1B splice variants, each containing a zinc binding active site and a putative transmembrane domain, with signal peptides in three variants. SAS1B transcripts were ovary specific. SAS1B protein was first detected in early secondary follicles in day 3 ovaries. Immunofluorescence localized SAS1B to the microvillar oolemma of M2 oocytes. After fertilization, SAS1B decreased on the oolemma and became virtually undetectable in blastocysts. In transfected CHO-K1 cells SAS1B localized to the surface of unpermeabilized cells. Recombinant and native SLLP1 co-localized with SAS1B to the microvillar domain of ovulated M2 oocytes. Molecular interactions between mouse SLLP1 and SAS1B were demonstrated by surface plasmon resonance, far-western, yeast two-hybrid, recombinant- and native-co-IP analyses. SAS1B bound to SLLP1 with high affinity. SAS1B had protease activity, and SAS1B protein or antibody significantly inhibited fertilization. SAS1B knockout female mice showed a 34% reduction in fertility. The study identified SAS1B-SLLP1 as a pair of novel sperm-egg binding partners involving the oolemma and intra-acrosomal compartment during fertilization.
Copyright © 2011. Published by Elsevier Inc.
Proteasome subtypes and the processing of tumor antigens: increasing antigenic diversity.
Source
Ludwig Institute for Cancer Research, Brussels Branch and de Duve Institute, Université catholique de Louvain, Brussels, Belgium.
Abstract
Protein degradation by the proteasome releases peptides that can be loaded on MHC class I molecules and presented to cytolytic T lymphocytes. Several mechanisms were recently found to increase the diversity of antigenic peptidesdisplayed at the cell surface, thereby maximizing the efficacy of immune responses. The proteasome was shown to produce spliced antigenic peptides, which are made of two fragments initially not contiguous in the parental protein. Different proteasome subtypes also produce distinct sets of antigenic peptides: the standard proteasome and the immunoproteasome, containing different catalytic subunits, have different cleavage specificities and produce different sets of peptides. Moreover, recent work confirmed the existence of two additional proteasome subtypes that are intermediate between the standard and the immunoproteasome, and each produce a unique peptide repertoire.
Copyright © 2011 Elsevier Ltd. All rights reserved.
Impact of immunization technology and assay application on antibody performance - a systematic comparative evaluation.
Source
Research and Development, SDIX, Newark, Delaware, United States of America.
Abstract
Antibodies are quintessential affinity reagents for the investigation and determination of a protein's expression patterns, localization, quantitation, modifications, purification, and functional understanding. Antibodies are typically used in techniques such as Western blot, immunohistochemistry (IHC), and enzyme-linked immunosorbent assays (ELISA), among others. The methods employed to generate antibodies can have a profound impact on their success in any of these applications. We raised antibodies against 10 serum proteins using 3 immunization methods: peptide antigens (3 per protein), DNA prime/protein fragment-boost ("DNA immunization"; 3 per protein), and full length protein. Antibodies thus generated were systematically evaluated using several different assay technologies (ELISA, IHC, and Western blot). Antibodies raised against peptides worked predominantly in applications where the target protein was denatured (57% success in Western blot, 66% success in immunohistochemistry), although 37% of the antibodies thus generated did not work in any of these applications. In contrast, antibodies produced by DNA immunization performed well against both denatured and native targets with a high level of success: 93% success in Western blots, 100% success in immunohistochemistry, and 79% success in ELISA. Importantly, success in one assay method was not predictive of success in another. Immunization with full length protein consistently yielded the best results; however, this method is not typically available for new targets, due to the difficulty of generating full length protein. We conclude that DNA immunization strategies which are not encumbered by the limitations of efficacy (peptides) or requirements for full length proteins can be quite successful, particularly when multiple constructs for each protein are used.
Highly efficient method of preparing human catalytic antibody light chains and their biological characteristics.
Source
*Research Center for Applied Medical Engineering and.
Abstract
The ultimate goal of catalytic antibody research is to develop new patient therapies that use the advantages offered by human catalytic antibodies. The establishment of a high-throughput method for obtaining valuable candidate catalytic antibodies must be accelerated to achieve this objective. In this study, based on our concept that we can find antibody light chains with a high probability of success if they include a serine protease-like catalytic triad composed of Ser, His, and Asp on a variable region of the antibody structure, we amplified and cloned DNAs encoding human antibody light chains from germline genes of subgroup II by seminested PCR using two primer sets designed for this purpose. Seven DNA fragments encoding light chains in 17 clones were derived from germline gene A18b, 6 DNA fragments from A3/A19, 2 DNA fragments from A17, and a clone DNA fragment from A5 and O11/O1. All light chains expressed in Escherichia coli and highly purified under nondenaturing conditions exhibited amidolytic activity against syntheticpeptides. Some of the light chains exhibited unique features that suppressed the infectious activity of the rabies virus. Furthermore, the survival rate of mice in which a lethal level of the rabies virus was coinoculated directly into the brain with light chain 18 was significantly improved. In the case of humans, these results demonstrate that high-throughput selection of light chains possessing catalytic functions and specificity for a target molecule can be attained from a light-chain DNA library amplified from germline genes belonging to subgroup II.-Hifumi, E., Honjo, E., Fujimoto, N., Arakawa, M., Nishizono, A., Uda, T. Highly efficient method of preparing human catalytic antibody light chains and their biological characteristics.
Sample Preparation Techniques for the Untargeted LC-MS-Based Discovery ofPeptides in Complex Biological Matrices.
Source
Laboratory for Analytical Biotechnology and Innovative Peptide Biology, Department of Biotechnology, Delft University of Technology, Julianalaan 67, 3706 AV Zeist, The Netherlands.
Abstract
Although big progress has been made in sample pretreatment over the last years, there are still considerable limitations when it comes to overcoming complexity and dynamic range problems associated with peptide analyses from biological matrices. Being the little brother of proteomics, peptidomics is a relatively new field of research aiming at the direct analysis of the small proteins, called peptides, many of which are not amenable for typical trypsin-based analytics. In this paper, we present an overview of different techniques and methods currently used for reducing a sample's complexity and for concentrating low abundant compounds to enable successful peptidome analysis. We focus on techniques which can be employed prior to liquid chromatography coupled to mass spectrometry for peptide detection and identification and indicate their advantages as well as their shortcomings when it comes to the untargeted analysis of native peptides from complex biological matrices.
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