1.
Identification of a specific region of Plasmodium falciparum EBL-1 that binds to host receptor glycophorin B and inhibits merozoite invasion in human red blood cells.
Source
Department of Molecular Physiology and Pharmacology, Tufts University School of Medicine, Boston, MA 02111, USA; Sackler School Programs in Physiology, Pharmacology, and Microbiology, USA; Department of Pharmacology, University of Illinois College of Medicine, Chicago, IL 60612, USA.
Abstract
The malaria parasite Plasmodium falciparum invades human erythrocytes through multiple pathways utilizing several ligand-receptor interactions. These interactions are broadly classified in two groups according to their dependency on sialic acid residues. Here, we focus on the sialic acid-dependent pathway by using purified glycophorins and red blood cells (RBCs) to screen a cDNA phage display library derived from P. falciparum FCR3 strain, a sialic acid-dependent strain. This screen identified several parasite proteins including the erythrocyte-binding ligand-1, EBL-1. The phage cDNA insert encoded the 69-amino acid peptide, termed F2i, which is located within the F2 region of the DBL domain, designated here as D2, of EBL-1. Recombinant D2 and F2i polypeptides bound to purified glycophorins and RBCs, and the F2i peptide was found to interfere with binding of D2 domain to its receptor. Both D2 and F2i polypeptides bound to trypsin-treated but not neuraminidase or chymotrypsin-treated erythrocytes, consistent with known glycophorin B resistance to trypsin, and neither the D2 nor F2i polypeptide bound to glycophorin B-deficient erythrocytes. Importantly, purified D2 and F2i polypeptides partially inhibited merozoite reinvasion in human erythrocytes. Our results show that the host erythrocyte receptor glycophorin B directly interacts with the DBL domain of parasite EBL-1, and the core binding site is contained within the 69 amino acid F2i region (residues 601-669) of the DBL domain. Together, these findings suggest that a recombinant F2i peptide with stabilized structure could provide a protective function at blood stage infection and represents a valuable addition to a multi-subunit vaccine against malaria.
Copyright © 2012. Published by Elsevier B.V.
- PMID:
- 22273481
- [PubMed - as supplied by publisher]
Simultaneous Assessment of Asp Isomerization and Asn Deamidation inRecombinant Antibodies by LC-MS following Incubation at Elevated Temperatures.
Source
Pharma Research and Development Penzberg, Roche Diagnostics GmbH, Penzberg, Germany.
Abstract
The degradation of proteins by asparagine deamidation and aspartate isomerization is one of several chemical degradation pathways for recombinant antibodies. In this study, we have identified two solvent accessible degradation sites (light chain aspartate-56 and heavy chain aspartate-99/101) in the complementary-determining regions of arecombinant IgG1 antibody susceptible to isomerization under elevated temperature conditions. For both hot-spots, the degree of isomerization was found to be significantly higher than the deamidation of asparagine-(387, 392, 393) in the conserved CH3 region, which has been identified as being solvent accessible and sensitive to chemical degradation in previous studies. In order to reduce the time for simultaneous identification and functional evaluation of potential asparagine deamidation and aspartate isomerization sites, a test system employing accelerated temperature conditions and proteolytic peptide mapping combined with quantitative UPLC-MS was developed. This method occupies the formulation buffer system histidine/HCl (20 mM; pH 6.0) for denaturation/reduction/digestion and eliminates the alkylation step. The achieved degree of asparagine deamidation and aspartate isomerization was adequate to identify the functional consequence by binding studies. In summary, the here presented approach greatly facilitates the evaluation of fermentation, purification, formulation, and storage conditions on antibody asparagine deamidation and aspartate isomerization by monitoring susceptible marker peptides located in the complementary-determining regions ofrecombinant antibodies.
Bacterial cellulose modified using recombinant proteins to improve neuronal and mesenchymal cell adhesion.
Source
Centre of Biological Engineering, Universidade do Minho, Braga, Portugal.
Abstract
A wide variety of biomaterials and bioactive molecules have been applied as scaffolds in neuronal tissue engineering. However, creating devices that enhance the regeneration of nervous system injuries is still a challenge, due the difficulty in providing an appropriate environment for cell growth and differentiation and active stimulation of nerve regeneration. In recent years, bacterial cellulose (BC) has emerged as a promising biomaterial for biomedical applications because of its properties such as high crystallinity, an ultrafine fiber network, high tensile strength, and biocompatibility. The small signaling peptides found in the proteins of extracellular matrix are described in the literature as promoters of adhesion and proliferation for several cell lineages on different surfaces. In this work, the peptide IKVAV was fused to a carbohydrate-binding module (CBM3) and used to modify BC surfaces, with the goal of promoting neuronal and mesenchymal stem cell (MSC) adhesion. The recombinant proteins IKVAV-CBM3 and (19)IKVAV-CBM3 were successfully expressed in E. coli, purified through affinity chromatography, and stably adsorbed to the BC membranes. The effect of these recombinant proteins, as well as RGD-CBM3, on cell adhesion was evaluated by MTS colorimetric assay. The results showed that the (19)IKVAV-CBM3 was able to significantly improve the adhesion of both neuronal and mesenchymal cells and had no effect on the other cell lineages tested. The MSC neurotrophin expression in cells grown on BC membranes modified with the recombinant proteins was also analyzed. © 2012 American Institute of Chemical Engineers Biotechnol. Prog.,, 2012.
Copyright © 2012 American Institute of Chemical Engineers (AIChE).
AMP-18 facilitates assembly and stabilization of tight junctions to protect the colonic mucosal barrier.
Source
Department of Medicine, University of Chicago, Chicago, Illinois.
Abstract
BACKGROUND:
Inflammatory bowel disease (IBD) is characterized by an injured epithelium. Development of agents that could enhance mucosal healing is a major goal in IBD therapeutics. The 18-kDa antrum mucosal protein (AMP-18) and a 21-mer peptide derived from AMP-18 stimulate accumulation of tight junction (TJ) proteins in cultured epithelial cells and mouse colonic mucosa to protect the mucosal barrier, suggesting it might be a useful agent to treat IBD.
METHODS:
We searched for molecular mechanisms by which AMP peptide or recombinant AMP-18 act on TJs in intact cell monolayers, or those disrupted by low-calcium medium. Roles of the p38 mitogen-activated protein kinase (MAPK) / heat shock protein (hsp)25 pathway and PKCζ were investigated by immunoblotting and confocal microscopy.
RESULTS:
AMP peptide activated p38 MAPK, which subsequently phosphorylated hsp25. Accumulated phospho-hsp25 was associated with perijunctional actin. AMP-18 also induced rapid phosphorylation of PKCζ and its colocalization with perijunctional actin in Caco2/bbe cells, which was accompanied by translocation and formation of complexes of "polarity proteins" in the TJ-containing detergent-insoluble fraction. Treatment with AMP-18 also stimulated accumulation of ZO-1, ZO-2, and JAM-A in nascent TJs known to associate with the multimeric p-PKCζ/Par6/ Cdc42/ECT2·GTP/Par3 polarity protein complex.
CONCLUSIONS:
AMP-18 facilitates translocation and assembly of multiple proteins into TJs and their association with and subsequent stabilization of the actin filament network. We speculate that improved barrier function induced by AMP-18 is mediated by enhanced TJ assembly. Thus, AMP-18 may provide a promising lead to develop agents effective in healing injured colonic epithelium in IBD. (Inflamm Bowel Dis 2012;).
Copyright © 2012 Crohn's & Colitis Foundation of America, Inc.
Neutralization of IL-17 ameliorates uveitis but damages photoreceptors in a murine model of spondyloarthritis.
Abstract
ABSTRACT:
INTRODUCTION:
Uveitis, or intraocular inflammatory disease, is a frequent extra-articular manifestation of several forms of arthritis. Despite the frequent co-occurrence of uveitis and arthritis, little is understood of the eye's predisposition to this disease. We recently described a previously unreported uveitis in a murine model of spondyloarthropathy triggered by autoimmunity to aggrecan, a prominent proteoglycan (PG) macromolecule in cartilage. In contrast to the joint and spine wherein interferon-gamma (IFNgamma) deficiency reduced disease, IFNgamma deficiency worsened uveitis. Given the regulatory role of IFNgamma on the Th17 response and the current focus of anti-interleukin-17 therapeutics in patients with uveitis and spondyloarthritis, we sought to determine the extent to which interleukin (IL)-17 mediates uveitis in the absence of IFNgamma.
METHODS:
Antigen specific T cell cytokine production was measured in splenocyte cultures using multiplex-ELISA. Transgenic (Tg) mice expressing the T cell receptor (TCR) recognizing the dominant arthritogenic epitope in the G1 domain of PG (TCR-Tg), also lacking IFNgamma, were immunized with PG. Mice were then systemically administered an anti-IL-17 neutralizing antibody. The onset and severity of peripheral arthritis was evaluated by clinical scoring criteria and histology. Uveitis was assessed using intravital videomicroscopy, which visualizes leukocyte trafficking within the vasculature and tissue of the iris, and by histology.
RESULTS:
TCR-Tg splenocytes stimulated in vitro with recombinant G1 peptide demonstrated exacerbated production of cytokines such as macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, IL-1beta, and most notably IL-17A as a consequence of IFNgamma deficiency. In vivo, IL-17 inhibition prevented the component of PG-induced arthritis that occurs independently of IFNgamma. 4 Blockade of IL-17 ameliorated the ongoing leukocyte trafficking responses within the iris vasculature and tissue, which coincided with reduced infiltration of leukocytes within the anterior and posterior eye segments. However, the anti-IL-17 treatment resulted in unanticipated photoreceptor toxicity.
CONCLUSIONS:
These data support a protective, regulatory role for IFNgamma in suppression of IL-17-mediated intraocular disease and to a lesser extent, joint disease. The unanticipated photoreceptor toxicity raises some caution regarding the use of anti-IL-17 therapeutics until the mechanism of this potential effect is determined.
The function of platelet-derived growth factor in the differentiation of mouse tongue striated muscle.
Source
Erika Suzuki, Kayoko Aoyama, Tadayoshi Fukui, Yoshiki Nakamura, Department of Orthodontics, Tsurumi University School of Dental Medicine, Tsurumi-ku, Yokohama, Japan Akira Yamane, Department of Biophysics, Tsurumi University School of Dental Medicine, Tsurumi-ku, Yokohama, Japan.
Abstract
Suzuki E., Aoyama K., Fukui T., Nakamura Y., Yamane A. The function of platelet-derived growth factor in the differentiation of mouse tongue striated muscle. Orthod Craniofac Res 2012;15:39-51. © John Wiley & Sons A/S STRUCTURED ABSTRACT: Objective - to determine the function of platelet-derived growth factor (PDGF) in the final differentiation phase of tongue striated muscle cells. Materials and Methods - We analyzed the expressions of PDGF-A, -B, platelet-derived growth factor receptor (PDGFR)-α, and PDGFR-β in mouse tongues between embryonic days (E) 11 and 15. Furthermore, we examined the effects of human recombinant PDGF-AB and the peptide antagonist for PDGFRs using an organ culture system of mouse embryonic tongue. Mouse tongues at E12 were cultured in BGJb medium containing human recombinant PDGF-AB for 4 days or the peptide antagonist for PDGF receptors for 8 days. Results - PDGF-A, -B, PDGFR-α, and -β were expressed in the differentiating muscle cells between E11 and 15. The human recombinant PDGF-AB induced increases in the mRNA expressions of myogenin and muscle creatine kinase (MCK) and the number of fast myosin heavy chain (fMHC)-positive cells, markers for the differentiation of muscle cells. On the other hand, the peptide antagonist for PDGFRs induced suppressions in the mRNA expressions of myogenin and MCK, and the number of fMHC-positive cells. Both the PDGF-AB and the antagonist failed to affect the expressions of cell proliferation markers. Conclusion - These results suggest that PDGF functions as a positive regulator in the final differentiation phase of tongue muscle cells in mouse embryos.
© 2012 John Wiley & Sons A/S.
Recombinant protein scaffolds for tissue engineering.
Source
CSIRO Materials Science and Engineering, Bayview Avenue, Clayton 3169, Australia.
Abstract
New biological materials for tissue engineering are now being developed using common genetic engineering capabilities to clone and express a variety of genetic elements that allow cost-effective purification and scaffold fabrication from these recombinant proteins, peptides or from chimeric combinations of these. The field is limitless as long as the gene sequences are known. The utility is dependent on the ease, product yield and adaptability of these protein products to the biomedical field. The development of recombinant proteins as scaffolds, while still an emerging technology with respect to commercial products, is scientifically superior to current use of natural materials or synthetic polymer scaffolds, in terms of designing specific structures with desired degrees of biological complexities and motifs. In the field of tissue engineering, next generation scaffolds will be the key to directing appropriate tissue regeneration. The initial period of biodegradable synthetic scaffolds that provided shape and mechanical integrity, but no biological information, is phasing out. The era of protein scaffolds offers distinct advantages, particularly with the combination of powerful tools of molecular biology. These include, for example, the production of human proteins of uniform quality that are free of infectious agents and the ability to make suitable quantities of proteins that are found in low quantity or are hard to isolate from tissue. For the particular needs of tissue engineering scaffolds, fibrous proteins like collagens, elastin, silks and combinations of these offer further advantages of natural well-defined structural scaffolds as well as endless possibilities of controlling functionality by genetic manipulation.
Activity, Expression and Genetic Variation of Canine β-Defensin 103: A Multifunctional Antimicrobial Peptide in the Skin of Domestic Dogs.
Source
Department of Microbiology and Immunology, UC Davis School of Medicine, Davis, Calif., USA.
Abstract
The skin functions as more than a physical barrier to infection. Epithelial cells of the skin can synthesize antimicrobialpeptides, including defensins, which exhibit direct antimicrobial activity. Here we characterize the expression pattern, genetic variation and activity of the major β-defensin expressed in canine skin, canine β-defensin 103 (CBD103). The gene encoding CBD103 exhibits two forms of polymorphism: a common 3-basepair deletion allele and a gene copy-number variation. Golden retrievers and Labrador retrievers were the only breeds that encoded the variant allele of CBD103, termed CBD103ΔG23. Both these breeds also exhibited a CBD103 gene copy-number polymorphism that ranged from 2 to 4 gene-copies per diploid genome. Recombinant CBD103 and CBD103ΔG23, as well as the human ortholog human β-defensin 3 (hBD3) and hBD3ΔG23, showed potent and comparable antimicrobial killing against both methicillin-susceptible and methicillin-resistant Staphylococcus pseudintermedius. Skin biopsy specimens from dogs with atopic dermatitis revealed CBD103 expression levels similar to those in healthy controls and comparable at lesional and nonlesional sites. This expression pattern in dogs differs from the previously reported reduced expression of the human ortholog in atopic dermatitis. Overall, the similarities of CBD103 and its human ortholog reported here support the notion that the domestic dog may serve as a valuable model for studying β-defensin biology in the skin.
Copyright © 2012 S. Karger AG, Basel.
Mapping of B cell epitopes on desmoglein 3 in pemphigus vulgaris patients by the use of overlapping peptides.
Source
Department of Dermatology, University of Lübeck, Lübeck, Germany.
Abstract
BACKGROUND:
Pemphigus vulgaris (PV) is a severe autoimmune blistering disease associated with autoantibodies to desmoglein 3 (Dsg 3), a transmembrane glycoprotein of the cadherin family. Previous studies mainly focused on the mapping of conformational epitopes of Dsg 3 using recombinant fragments of Dsg 3 and competition ELISA.
OBJECTIVE:
Here, we performed a mapping of linear B cell epitopes on Dsg 3 in PV patients by the use of overlapping synthetic peptides.
METHODS:
A set of 254 overlapping synthetic peptides of 14 amino acids length covering the entire Dsg 3 extracellular domain was generated. Sera of patients with active PV (n=10) and healthy volunteers (n=10) were tested for IgG reactivity with the 254 peptides by ELISA. Testing each peptide separately, 7 major antigenic sites were identified. In order to validate these reactivities, 7 corresponding peptides of 13-33 amino acids in length were generated and employed by ELISA. Additional sera of active PV patients (n=17) and healthy volunteers (n=20) were tested and the most reactive peptide was used to specifically purify anti-Dsg 3 antibodies from PV sera (n=3).
RESULTS:
The major autoantibody reactivity in PV sera was mapped to amino acids 333-356 within the EC3 domain. Purifying patients IgG using the identified peptide, however, failed to induce acantholysis in keratinocyte dissociation assay.
CONCLUSION:
We conclude that linear epitopes do not play a major pathogenic role in human PV.
Copyright © 2011 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.
Comparative analysis of recombinant Human Papillomavirus 8 L1 production in plants by a variety of expression systems and purification methods.
Source
Istituto di Virologia Vegetale, CNR, Strada delle Cacce, Torino, Italy.
Abstract
Human papillomavirus 8 (HPV-8), one of the high-risk cutaneous papillomaviruses (cHPVs), is associated with epidermodysplasia verruciformis and nonmelanoma skin cancer in immuno-compromised individuals. Currently, no vaccines against cHPVs have been reported; however, recent studies on cross-neutralizing properties of their capsid proteins (CP) have fostered an interest in vaccine production against these viruses. We examined the potential of producing HPV-8 major CP L1 in Nicotiana benthamiana by agroinfiltration of different transient expression vectors: (i) the binary vector pBIN19 with or without silencing suppressor constructs, (ii) the nonreplicating Cowpea mosaic virus-derived expression vector pEAQ-HT and (iii) a replicating Tobacco mosaic virus (TMV)-based vector alone or with signalpeptides. Although HPV-8 L1 was successfully expressed using pEAQ-HT and TMV, a 15-fold increase was obtained with pEAQ-HT. In contrast, no L1 protein could be immune detected using pBIN19 irrespective of whether silencing suppressors were coexpressed, although such constructs were required for identifying L1-specific transcripts. A fourfold yield increase in L1 expression was obtained when 22 C-terminal amino acids were deleted (L1ΔC22), possibly eliminating a nuclear localization signal. Electron microscopy showed that plant-made HPV-8 L1 proteins assembled in appropriate virus-like particles (VLPs) of T = 1 or T = 7 symmetry. Ultrathin sections of L1ΔC22-expressing cells revealed their accumulation in the cytoplasm in the form of VLPs or paracrystalline arrays. These results show for the first time the production and localization of HPV-8 L1 protein in planta and its assembly into VLPs representing promising candidate for potential vaccine production.
© 2012 The Authors. Plant Biotechnology Journal © 2012 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.
A molecular carrier to transport and deliver cisplatin into endometrial cancer cells.
Source
NCI of Naples Pascale Foundation, Molecular Biology and Viral Oncogenesis C.N.R, Institute of Protein Biochemistry INBIOS srl, Naples, Italy University of Udine, Dept. of Statistics University of Naples Federico II, Dept. of Biological Sciences Second University of Naples, Dept. of Gynecology, Obstetrics and Reproduction.
Abstract
The leader peptide of a recombinant manganese superoxide dismutase (rMnSOD-Lp) acts as a molecular carrier. Clonogenic tests on normal (MRC-5) and endometrial adeno-carcinoma cells (HTB-112) were carried out in the presence of rMnSOD-Lp, cisplatin alone (CC) or cisplatin conjugated to the rMnSOD-Lp (rMnSOD-Lp-CC). The platinum delivered into the cells was measured by atomic spectrophotometric absorbance. The treatments on tumor and normal cells were finally evaluated by LM and TM microscopy. Tumor cell death in the case of 0.5 μM cisplatin on its own was minimal, while in the presence of 0.5 μM rMnSOD-Lp-CC, no tumor cells survived. Atomic absorbance analysis showed that rMnSOD-Lp-CC delivered approximately 4 times more cisplatin into HTB-112 cells than the amount delivered using cisplatin alone. By LM observation, the cells treated with rMnSOD-Lp-CC showed signs of nuclear and cytoplasmic fragmentation, i.e. apoptosis induced by the treatment. The therapeutic effect of rMnSOD-Lp-CC on endometrial cancer cells was significant, while on the normal cells it showed only a minimal toxicity. We believe that rMnSOD-Lp deserves to be considered as a molecular carrier to deliver cisplatin directly into tumor cells, thus transforming its antireplicative activity into a specific and selective antitumor agent. © 2012 John Wiley & Sons A/S.
© 2012 John Wiley & Sons A/S.
[Construction and toxicity the recombinant SpltMNPV expressing the scorpion toxin gene].
Source
School of Biology and Basic Medical Science, Medical College of Soochow University, Suzhou 215123, China. alicetangsz@yahoo.com.cn
Abstract
OBJECTIVE:
To develop a high toxic recombinant Spodoptera litura multicapsid nucleopolyhedroviruse (SpltMNPV) insecticide.
METHODS:
We constructed a recombinant transfer vector that was characterized by disrupting of ecdysterioid UDP-glucosyltransferase (egt) gene and expressing the mature peptide of the Chinese scorpion, B. martensi Karsch (BmK ITal) gene at the control of ie-1 promoter. The transfer vector and the SpltMNPV II DNA cotransfected the SpLi cells.Recombinant viruses were purified by the end point dilution and fluorescent spot purification.
RESULTS:
We successfully screened the recombinant SpltMNPV-deltaegt-Pph-egfp-ie-1-BmK ITal of which the egt gene was knocked out and expressed the mature peptide of the BmK ITal gene at the control of ie-1 promoter. Bioassays showed that, compared to the wide-type SpltMNPV, the speed of the recombinant virus killing the S. litura (LT50) increased by 0.7-0.8 days.
CONCLUSION:
The insecticidal effect of SpltNPV could be increased by inserting the foreign gene, which provided a further opportunity to develop the SpltNPV into commercially viable products to control the S. litura.
- PMID:
- 22260048
- [PubMed - in process]
Production, purification, and characterization of the cecropin from Plutella xylostella, pxCECA1, using an intein-induced self-cleavable system in Escherichia coli.
Source
State Key Laboratory of Virology, College of Life Science, Wuhan University, Wuhan, 430072, People's Republic of China.
Abstract
Antimicrobial peptides (AMPs) are widely expressed and play an important role in innate immune defense against infectious agents such as bacteria, viruses, fungi, and parasites. Cecropins are a family of AMPs synthesized in the fat body of insects that have proven effective at killing specific pathogens. In order to fulfill their clinical potential as antimicrobial drugs, a simple, cost-effective method to express AMPs is sorely needed. In this study, we expressed and characterized the cecropin from Plutella xylostella (pxCECA1) using an intein-dependent expression system in Escherichia coli. We cloned the pxCECA1 gene from larva by RT-PCR and fused the encoding sequence of mature pxCECA1 with an intein gene and a chitin-binding domain gene (CBD) in pTWIN1 plasmid. The fusion protein CBD-intein-pxCECA1 was expressed in E. coli BL21 (DE3) and separated by flowing cell extracts through a chitin column. Subsequently, self-cleavage of the intein at its C-terminus was induced in a temperature- and pH-dependent manner, resulting in the release of mature pxCECA1. The optimal conditions for self-cleavage were determined to be pH 6.0 for 48 h at 4°C, under which 12.3 mg of recombinant pxCECA1 could be recovered from 1 l of E. coli culture. The purified pxCECA1 displayed antimicrobial activity against a broad variety of gram-positive and gram-negative bacteria. This preparation was especially effective against Staphylococcus aureus, including methicillin-resistant strains. Catalase release assays demonstrated that pxCECA1 acts as a microbicidal agent. These results show for the first time that the IMPACT-TWIN expression system is an efficient, cost-effective way to produce fully functional AMPs and that the AMP pxCECA1 is a novel microbicidal agent with promising therapeutic applications.
Zinc-finger nuclease-mediated gene correction using single AAV vector transduction and enhancement by Food and Drug Administration-approved drugs.
Source
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX, USA.
Abstract
An emerging strategy for the treatment of monogenic diseases uses genetic engineering to precisely correct the mutation(s) at the genome level. Recent advancements in this technology have demonstrated therapeutic levels of gene correction using a zinc-finger nuclease (ZFN)-induced DNA double-strand break in conjunction with an exogenous DNA donor substrate. This strategy requires efficient nucleic acid delivery and among viral vectors, recombinant adeno-associated virus (rAAV) has demonstrated clinical success without pathology. However, a major limitation of rAAV is the small DNA packaging capacity and to date, the use of rAAV for ZFN gene delivery has yet to be reported. Theoretically, an ideal situation is to deliver both ZFNs and the repair substrate in a single vector to avoid inefficient gene targeting and unwanted mutagenesis, both complications of a rAAV co-transduction strategy. Therefore, a rAAV format was generated in which a single polypeptide encodes the ZFN monomers connected by a ribosome skipping 2A peptideand furin cleavage sequence. On the basis of this arrangement, a DNA repair substrate of 750 nucleotides was also included in this vector. Efficient polypeptide processing to discrete ZFNs is demonstrated, as well as the ability of this single vector format to stimulate efficient gene targeting in a human cell line and mouse model derived fibroblasts. Additionally, we increased rAAV-mediated gene correction up to sixfold using a combination of Food and Drug Administration-approved drugs, which act at the level of AAV vector transduction. Collectively, these experiments demonstrate the ability to deliver ZFNs and a repair substrate by a single AAV vector and offer insights for the optimization of rAAV-mediated gene correction using drug therapy.Gene Therapy advance online publication, 19 January 2012; doi:10.1038/gt.2011.211.
A whole cell assay to measure caspase-6 activity by detecting cleavage of lamin a/c.
Source
Department of Biochemical Pharmacology, Genentech, Inc., South San Francisco, California, United States of America.
Abstract
Caspase-6 is a cysteinyl protease implicated in neurodegenerative conditions including Alzheimer's and Huntington's disease making it an attractive target for therapeutic intervention. A greater understanding of the role of caspase-6 in disease has been hampered by a lack of suitable cellular assays capable of specifically detecting caspase-6 activity in an intact cell environment. This is mainly due to the use of commercially available peptide substrates and inhibitors which lack the required specificity to facilitate development of this type of assay. We report here a 384-well whole-cell chemiluminescent ELISA assay that monitors the proteolytic degradation of endogenously expressed lamin A/C during the early stages of caspase-dependent apoptosis. The specificity of lamin A/C proteolysis by caspase-6 was demonstrated against recombinant caspase family members and further confirmed in genetic deletion studies. In the assay, plasma membrane integrity remained intact as assessed by release of lactate dehydrogenase from the intracellular environment and the exclusion of cell impermeable peptide inhibitors, despite the induction of an apoptotic state. The method described here is a robust tool to support drug discovery efforts targeting caspase-6 and is the first reported to specifically monitor endogenous caspase-6 activity in a cellular context.
Conformational Differences Unfold a Wide Range of Enterotoxigenic Abilities Exhibited by rNSP4 Peptides from Different Rotavirus Strains.
Source
Department of Microbiology & Cell Biology.
Abstract
NSP4 has been recognized as the rotavirus-encoded enterotoxin. However, a few studies failed to support its diarrheagenic activity. As recombinant NSP4 (rNSP4) peptides of different lengths were used in the limited number of studies, a comparison of relative diarrheagenic potential of NSP4 from different strains could not be possible. To better understand the diarrheagenic potential of NSP4 from different strains, in this report we have evaluated the enterotoxigenic activity of the deletion mutant ΔN72 that lacks the N-terminal 72 residues and the biologically relevant ΔN112 peptide which when derived from SA11 rotavirus strain were previously shown to be highly diarrheagenic in newborn mice. Detailed comparative analysis of biochemical and biophysical properties and diarrheagenic activity of therecombinant ΔN72 peptides from seventeen different strains under identical conditions revealed wide differences among themselves in their resistance to trypsin cleavage, thioflavin T (ThT) binding, multimerization and conformation without any correlation with their diarrhea inducing abilities. These results support our previously proposed concept for the requirement of a unique conformation for optimal biological functions conferred by cooperation between the N- and C-terminal regions of the cytoplasmic tail.
Functional importance of GLP-1 receptor species and expression levels in cell lines.
Source
Novo Nordisk, Diabetes Research Unit, Novo Nordisk Park, 2760 Maaloev, Denmark.
Abstract
Of the mammalian species, only the GLP-1 receptors of rat and human origin have been described and characterized. Here, we report the cloning of the homologous GLP-1 receptors from mouse, rabbit, pig, cynomolgus monkey and chimp. The GLP-1 receptor is highly conserved across species, thus underlining the physiological importance of thepeptide hormone and its receptor across a wide range of mammals. We expressed the receptors by stable transfection of BHK cells, both in cell lines with high expression levels of the cloned receptors, as well as in cell lines with lower expression levels, more comparable to endogenous expression of these receptors. High expression levels of cloned GLP-1 receptors markedly increased the potency of GLP-1 and other high affinity ligands, whereas the K(d) values were not affected. For a low affinity ligand like the ago-allosteric modulator Compound 2, expression levels of the human GLP-1 receptor were important for maximal efficacy as well as potency. The two natural metabolites of GLP-1, GLP-1(9-37) and GLP-1(9-36)amide were agonists when tested on a cell line with high expression of the recombinant human GLP-1 receptor, whereas they behaved as (low potent) antagonists on a cell line that expressed the receptor endogenously, as well as cells expressing a moderate level of the recombinant human GLP-1 receptor. The amide form was a more potent agonist than the free acid from. In conclusion, receptor expression level is an important parametre for selecting cell lines with cloned GLP-1 receptors for functional characterization of physiological and pharmaceutical ligands.
Copyright © 2011 Elsevier B.V. All rights reserved.
Solution structure of native and recombinant expressed toxin CssII from the venom of the scorpion centruroides suffusus suffusus, and their effects on Nav1.5 Sodium channels.
Source
Instituto de Química, Departamento de Química de Biomacromoléculas, Universidad Nacional Autónoma de México, UNAM, Ciudad Universitaria, D.F., 04510, Mexico.
Abstract
The three-dimensional structures of the long-chain mammalian scorpion β-toxin CssII from Centruroides suffusus suffusus and of its recombinant form, HisrCssII, were determined by NMR. The neurotoxin CssII (nCssII) is a 66 amino acid long peptide with four disulfide bridges; it is the most abundant and deadly toxin from the venom of this scorpion. Both native and recombinant CssII structures were determined by nuclear magnetic resonance using a total of 828 sequential distance constraints derived from the volume integration of the cross peaks observed in 2D NOESY spectra. Both nCssII and HisrCssII structures display a mixed α/β fold stabilized by four disulfide bridges formed between pairs of cysteines: C1-C8, C2-C5, C3-C6, and C4-C7 (the numbers indicate the relative positions of the cysteine residues in the primary structure), with a distortion induced by two cis-prolines in its C-terminal part. The native CssII electrostatic surface was compared to both the recombinant one and to the Cn2 toxin, from the scorpion Centruroides noxius, which is also toxic to mammals. Structural features such N- and C-terminal differences could influence toxin specificity and affinity towards isoforms of different sub-types of Na(v) channels.
Copyright © 2012. Published by Elsevier B.V.
Leptin receptors.
Source
Centre for Cardiovascular Science, Queen's Medical Research Institute, University of Edinburgh, Edinburgh, Scotland, EH16 4TH, UK.
Abstract
The hormone leptin, secreted predominantly from adipose tissue, plays a crucial role in the regulation of numerous neuroendocrine functions, from energy homeostasis to reproduction. Genetic deficiency as a consequence of leptin or leptin receptor mutations, although rare in humans, leads to early onset of chronic hyperphagia and massive obesity. In most human obesity, however, leptin levels are chronically elevated. Under these conditions of persistent hyperleptinaemia, and particularly when obesity is associated with a high-fat diet, leptin resistance develops, and signalling through the leptin receptor is curtailed, fuelling further weight gain. Here, we review the role of leptin receptors in the regulation of feeding and obesity development. Leptin receptors are found in each of the major components of the CNS "feeding" circuitry-the brainstem, hypothalamus and distributed reward centres. Through these receptors, leptin exerts influences on signalling and integration within these circuits to alter feeding behaviours. Although some progress is now being made with peptide analogues, the leptin receptor has not proved to be amenable to small molecule pharmacological intervention to date. Where clinical benefit from recombinant leptin administration has been achieved, this has been under circumstances of complete endogenous leptin deficiency or relative hypoleptinaemia such as in lipodystrophy.
One-step immunopurification and lectinochemical characterization of the Duffy atypical chemokine receptor from human erythrocytes.
Source
Department of Immunochemistry, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, R. Weigla 12, 53-114, Wrocław, Poland.
Abstract
Duffy antigen/receptor for chemokines (DARC) is a glycosylated seven-transmembrane protein acting as a blood group antigen, a chemokine binding protein and a receptor for Plasmodium vivax malaria parasite. It is present on erythrocytes and endothelial cells of postcapillary venules. The N-terminal extracellular domain of the Duffy glycoprotein carries Fy(a)/Fy(b) blood group antigens and Fy6 linear epitope recognized by monoclonal antibodies. Previously, we have shown that recombinant Duffy protein expressed in K562 cells has three N-linked oligosaccharide chains, which are mainly of complex-type. Here we report a one-step purification method of Duffy protein from human erythrocytes. DARC was extracted from erythrocyte membranes in the presence of 1% n-dodecyl-β-D-maltoside (DDM) and 0.05% cholesteryl hemisuccinate (CHS) and purified by affinity chromatography using immobilized anti-Fy6 2C3 mouse monoclonal antibody. Duffy glycoprotein was eluted from the column with synthetic DFEDVWN peptide containing epitope for 2C3 monoclonal antibody. In this single-step immunoaffinity purification method we obtained highly purified DARC, which migrates in SDS-polyacrylamide gel as a major diffuse band corresponding to a molecular mass of 40-47 kDa. In ELISA purified Duffy glycoprotein binds anti-Duffy antibodies recognizing epitopes located on distinct regions of the molecule. Results of circular dichroism measurement indicate that purified DARC has a high content of α-helical secondary structure typical for chemokine receptors. Analysis of DARC glycans performed by means of lectin blotting and glycosidase digestion suggests that native Duffy N-glycans are mostly triantennary complex-type, terminated with α2-3- and α2-6-linked sialic acid residues with bisecting GlcNAc and α1-6-linked fucose at the core.
- PMID:
- 22246380
- [PubMed - as supplied by publisher]
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