1.
Ordered and disordered proteins as nanomaterial building blocks.
Source
Department of Bioengineering, University of California, Berkeley, CA, USA.
Abstract
Proteins possess a number of attractive properties that have contributed to their recent emergence as nanoscale building blocks for biomaterials and bioinspired materials. For instance, the amino acid sequence of a protein can be precisely controlled and manipulated via recombinant DNA technology, and proteins can be biosynthesized with very high purity and virtually perfect monodispersity. Most importantly, protein-based biomaterials offer the possibility of technologically harnessing the vast array of functions that these biopolymers serve in nature. In this review, we discuss recent progress in the field of protein-based biomaterials, with an overall theme of relating protein structure to material properties. We begin by discussing materials based on proteins that have well-defined three-dimensional structures, focusing specifically on elastin- and silk-like peptides. We then explore the newer field of materials based on intrinsically disordered proteins, using nucleoporin and neurofilament proteins as case studies. A key theme throughout the review is that specific environmental stimuli can trigger protein conformational changes, which in turn can alter macroscopic material properties and function. WIREs Nanomed Nanobiotechnol 2012 doi: 10.1002/wnan.1160 For further resources related to this article, please visit the WIREs website.
Copyright © 2012 Wiley Periodicals, Inc.
T cell receptor engineering.
Abstract
T lymphocytes express on their surface a heterodimeric αβ receptor, called the T cell receptor (TCR), which recognizes foreign antigens. Unlike antibodies, the recognition requires both an antigenic peptide epitope and a protein encoded by the major histocompatibility complex (MHC). In contrast to conventional antibody-directed target antigens, antigens recognized by the TCR can include the entire array of potential intracellular proteins, which are processed and delivered to the cell surface as a peptide/MHC complex. In the past 10 years, there have been significant efforts to engineer TCRs in various formats, which would allow improved recognition and destruction of virus-infected cells or cancer. The proposed therapeutic approaches involve either the use of engineered, high-affinity TCRs in soluble forms, analogous to antibody-directed therapies, or the use of engineered TCRs whose genes are reintroduced into autologous T cells and transferred back into patients (T cell adoptive therapies). This chapter describes three methods associated with the engineering of TCRs for these therapeutic purposes: (1) use of a yeast display system to engineer higher affinity single-chain VαVβ TCRs, called scTv; (2) use of a T cell display system to engineer higher affinity full-length TCRs; and (3) expression, purification, and characterization of soluble TCRs in an Escherichia coli system.
Copyright © 2012 Elsevier Inc. All rights reserved.
- PMID:
- 22230570
- [PubMed - in process]
Association of ferritin autoantibodies with giant cell arteritis/polymyalgia rheumatica.
Source
1Department of Immunology and Rheumatology, Medical University, Hannover, Germany.
Abstract
OBJECTIVES:
Polymyalgia rheumatica (PMR) and giant cell arteritis (GCA) are relatively common inflammatory disorders. Establishing the diagnosis however may be difficult, since so far no specific biomarkers of the disorders are available.
METHODS:
As a screening procedure, the authors used protein arrays for the detection of new autoantigens in GCA and PMR. The results of the protein array were confirmed by different ELISAs detecting IgG antibodies against the human ferritin heavy chain, N-terminal 27 amino acids of the human ferritin heavy chain or the homologous peptide of Staphylococcus epidermidis. Sera of patients with only GCA (n=64), only PMR (n=47) and both PMR and GCA (n=31) were used.
RESULTS:
In the ELISA using the human ferritin peptide, the sensitivity of IgG antibodies against ferritin was 92% in 36 GCA and/or PMR patients before initiation of treatment, 22/32 (69%) in patients with disease flares and 64/117 (55%) in the total cohort including treated and inactive patients. In controls, the false positive rate was 11/38 (29%) in systemic lupus erythematosus, 1/36 (3%) in rheumatoid arthritis, 0/31 (0%) in late onset rheumatoid arthritis, 3/46 (6.5%) in B-non-Hodgkin's lymphoma and 1/100 (1%) in blood donors. In the ELISA using the ferritin peptide of S epidermidis, 89% of 27 patients with untreated GCA and PMR were positive.
CONCLUSION:
Antibodies against the ferritin peptide were present in up to 92% of untreated, active GCA and PMR patients. They can be useful as a diagnostic marker of PMR and GCA.
Anti-diuretic factors in insects: The role of CAPA peptides.
Source
Department of Biology, McMaster University, 1280 Main Street West, Hamilton, ON, Canada L8S 4K1.
Abstract
Insects have adapted to live in a wide variety of habitats and utilize an array of feeding strategies that present challenges to their ability to maintain osmotic balance. Regardless of the feeding strategy, water and ion levels within the haemolymph (insect blood) are maintained within a narrow range. This homeostasis involves the action of a variety of tissues, but is often chiefly regulated by the excretory system. Until recently, most research on the hormonal control of the excretory tissues has focused on factors known to have diuretic activities. In this mini-review, the current state of knowledge on anti-diuretic factors in insects will be discussed with a particular emphasis on the CAPA peptides in the blood-feeding Chagas' disease vector, Rhodnius prolixus.
Copyright © 2011. Published by Elsevier Inc.
Isotopic labelling of peptides in tissues enhances mass spectrometric profiling.
Source
REQUIMTE-CQFB, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Campus de Caparica, 2829-516, Caparica, Portugal; Turku Centre for Biotechnology, University of Turku & Åbo Akademi University, Turku, Finland.
Abstract
RATIONALE:
There is a need in imaging mass spectrometry to use the acquired isotope distribution to unequivocally determine the identity of a peptide ion. A way of achieving unambiguous differentiation of ions from protonated peptidesfrom other [M + H](+) ions in a tissue would be via the direct on-tissue incorporation of (18) O into peptides.
METHODS:
Tissues were first digested with trypsin for 3 h at 37 °C in a humidified chamber. For the (18) O-labelling of digested peptides 1 μL of H(2) (18) O/50 mM ammonium acetate (at pH 6.75) was added to the array of tryptic spots and incubated at room temperature for 20 min. α-Cyano-4-hydroxycinnamic acid was used as a matrix modifier. The mass spectral analysis of tissue sections was carried out using a matrix-assisted laser desorption/ionisation tandem time-of-flight (MALDI-TOF-TOF) instrument.
RESULTS:
On-tissue incorporation of (18) O into peptides cannot be carried out during the digestion step inside a humidified chamber. After tissue digestion for 3 h at 37 °C in an humidified chamber, (18) O labelling was carried out for 20 min at room temperature (no humidified chamber). No trypsin was needed to enhance the labelling.
CONCLUSIONS:
For first time the feasibility of (18) O-labelling of peptides in situ for tissues has been demonstrated. The method decouples protein digestion from peptide labelling and is performed in sequential steps. Furthermore, we observed that (18) O incorporation produces characteristic isotopic peptide distributions, thus making facile distinguishing peptides from other tissue molecular components that ionise in the MALDI ion source. Copyright © 2012 John Wiley & Sons, Ltd.
Copyright © 2012 John Wiley & Sons, Ltd.
Pharmacological Synergy: The Next Frontier on Therapeutic Advancement for Migraine.
Source
From The Headache Center of Southern CA-Headache Center, Encinitas, CA, USA (A. Blumenfeld); Virginia Commonwealth University-Department of Biostatistics, Richmond, VA, USA (C. Gennings); Headache Care Center-Medicine, Springfield, MO, USA (R. Cady).
Abstract
The burden of migraine significantly impacts the individual sufferer, their families, the workplace, and society. The World Health Organization has identified migraine as an urgent public health priority and has initiated a global initiative to reduce the burden of migraine. Underlying the World Health Organization initiative is the need to discover means of optimizing migraine treatments and make them accessible to the broader portion of the world population. Development of acute migraine medications over the past several decades has largely centered on engineering highly specific receptor molecules that alter migraine pathophysiological mechanisms to abort or reverse the acute attack of migraine. The first product of this line of discovery was sumatriptan and heralded as a landmark therapeutic breakthrough. Sumatriptan is a 5-HT-1B/D receptor agonist considered to activate receptors involved in the pathophysiology specific to migraine. Large-scale regulatory/clinical studies demonstrated statistical superiority for sumatriptan over placebo in reduction or elimination of headache, nausea, photophobia, and phonophobia. Since the introduction of sumatriptan, 6 other triptan products have been released in the United States as acute treatments for migraine, all having the same mechanism of action and similar efficacy. Despite their utility as migraine abortive medications, the triptans do not successfully treat all attacks of migraine or necessarily treat all migraine associated symptoms. In fact, in less than 25% of attacks do subjects obtain and maintain a migraine-free response to treatment for at least beyond 24 hours. A wide range of non-triptan medications also have demonstrated efficacy in acute migraine. These include non-steroidal anti-inflammatory drugs (NSAIDs), opioids, phenothiazines, and valproic acid to name a few. Given the distinctly different mechanisms of actions of these various medications, it is likely that several unique pathophysiological mechanisms are involved in terminating acute episodes of migraine. Clinicians now capitalize on this observation and use migraine medication in combination with another to improve patient outcomes, for example, using an antiemetic with an opioid or a triptan and NSAIDs. More recently, the Food and Drug Adminstration has approved a combination product containing 85 mg of sumatriptan plus 500 mg of naproxen sodium for acute treatment of migraine. Clinical trials conducted prior to approval demonstrated that the combination of sumatriptan and naproxen was more effective as a migraine abortive than either of its components but that each component and the combination were more effective than placebo. Exactly how sumatriptan and naproxen interact to create therapeutic synergism is unknown though its mere occurrence suggests that models assisting medical understanding and prediction of pharmacological synergism may improve clinical outcome over products acting through a single receptor mechanism. Migraine is a syndrome, meaning it is defined by observed symptoms rather than known pathophysiology. Multiple pathogenic mechanisms are likely involved in generating this diverse array of symptoms understood as the migraine symptom complex. Sumatriptan and naproxen have independent mechanisms of action and target distinct aspects of the vascular and inflammatory processes hypothesized to underlie migraine. Sumatriptan acts on the 5-HT(1B) and 5-HT(1D) receptors, whereas naproxen inhibits the COX-1 and COX-2 enzymes. Sumatriptan has vasoconstricting effects as well as effects on neurogenic inflammation by decreasing the release of substance P and calcitonin gene-related peptide. In contrast, naproxen affects prostaglandins and other inflammatory mediators. Because sumatriptan and naproxen both relieve migraine yet interact with different cellular targets within the migraine pathway, it is reasonable to assume there is a unique synergy between these medications that improves treatment outcomes. Clinical trials supported this contention by demonstrating the combination of sumatriptan/naproxen alleviated migraine pain quickly (primarily based on the sumatriptan mechanism of action), and sustained the response longer (primarily based on the naproxen mechanism of action) than is possible when either drug is given alone. The working hypothesis is that when sumatriptan and naproxen are given at the same time, they affect different mechanisms of the migraine pathway and produce an enhanced therapeutic effect. The purpose of this article is to apply statistical analyses to data from phase II and phase III studies of the combination of sumatriptan and naproxen to determine if this enhanced therapeutic effect is synergistic. This methodology of accessing synergy can be used in the development of future combination migraine treatments to improve treatment outcomes.
© 2011 American Headache Society.
Pluripotency factor-mediated expression of the leptin receptor (OB-R) links obesity to oncogenesis through tumor-initiating stem cells.
Source
Department of Molecular Microbiology and Immunology, Bioinformatics Core, Norris Comprehensive Cancer Center at University of Southern California Epigenome Center and Division of Hematology, and Department of Pathology, University of Southern California Keck School of Medicine, Los Angeles, CA 90033.
Abstract
Misregulation of a pluripotency-associated transcription factor network in adult tissues is associated with the expansion of rare, highly malignant tumor-initiating stem cells (TISCs) through poorly understood mechanisms. We demonstrate that robust and selective expression of the receptor for the adipocyte-derived peptide hormone leptin (OB-R) is a characteristic feature of TISCs and of a broad array of embryonic and induced pluripotent stem cells and is mediated directly by the core pluripotency-associated transcription factors OCT4 and SOX2. TISCs exhibit sensitized responses to leptin, including the phosphorylation and activation of the pluripotency-associated oncogene STAT3 and induction of Oct4 and Sox2, thereby establishing a self-reinforcing signaling module. Exposure of cultured mouse embryonic stem cells to leptin sustains pluripotency in the absence of leukemia inhibitory factor. By implanting TISCs into leptin-deficient ob/ob mice or into comparably overweight Lepr(db/db) mice that produce leptin, we provide evidence of a central role for the leptin-TISC-signaling axis in promoting obesity-induced tumor growth. Differential responses to extrinsic, adipocyte-derived cues may promote the expansion of tumor cell subpopulations and contribute to oncogenesis.
The internal region leucine-rich repeat 6 of decorin interacts with LRP-1, modulates TGF-β dependent signaling and inhibits TGF-β dependent fibrotic response in skeletal muscles.
Source
Catholic University of Chile, Chile;
Abstract
Decorin is a small proteoglycan, composed of 12 leucine-rich repeats (LRRs) that modulates the activity of transforming growth factor type β(TGF-β) and other growth factors, and thereby influences proliferation and differentiation in a widearray of physiological and pathological processes, such as fibrosis, in several tissues and organs. Previously we described two novel modulators of this TGF-β dependent signaling pathway: LDL receptor related protein (LRP-1) and decorin. Here we have identified the regions in decorin that are responsible for interaction with LRP-1 and that are involved in TGF-β dependent binding and signaling. Specifically, we used decorin deletion mutants, as well as peptidesderived from internal LRR regions, to determine the LRRs responsible for these decorin functions. Our results indicate that LRR6 and LRR5 participate in the interaction with LRP-1 and TGF-β as well as in its dependent signaling. Furthermore, the internal region (LRR6i), composed of 11 amino-acids, is responsible for decorin binding to LRP-1 and subsequent TGF-β dependent signaling. Finally, using an in vivo approach, we also demonstrate that the LRR6 region of decorin can inhibit TGF-β mediated action in response to skeletal muscle injury.
Challenges and Strategies in Developing Microneedle Patches For Transdermal Delivery of Protein and Peptide Therapeutics.
Source
School of Pharmacy, Shanghai Jiao Tong University 800 Dongchuan Road, Shanghai, 200240, P. R. CHINA. tjin@sjtu.edu.cn.
Abstract
The birth of microneedles, an array of needles sufficiently long to penetrate epidermis but small enough to do not cause skin injury and pain feeling, has offered a highly promising solution for non-invasive delivery of protein and peptide drugs, a long-cherished desire over eighty years. However, the attempts to develop clinically feasible microneedle transdermal delivery methods encountered series of difficulties, for which a decade research efforts have yet to result in a single product. Microneedles may be incorporated into devices as skin pre-treatment tools, skin microinjectors as well as transdermal patches by their functions in drug delivery. They may also be categorized to insoluble solid microneedles, hollow microneedles, soluble/degradable solid microneedles and phase-transition microneedles by their structure and forming materials. This review article is aimed to update the progress and discuss the technical challenges raised in developing protein/peptide loaded microneedle patches.
- PMID:
- 22201589
- [PubMed - as supplied by publisher]
Functional peptidomics of amphibian skin secretion: A novel Kunitz-type chymotrypsin inhibitor from the African hyperoliid frog, Kassina senegalensis.
Source
Natural Drug Discovery group, School of Pharmacy, Queen's University, Belfast BT9 7BL, Northern Ireland, UK.
Abstract
Amphibian skin secretions are, for the most part, complex peptidomes. While many peptide components have been biologically- and structurally-characterised into discrete "families", some of which are analogues of endogenous vertebrate regulatory peptides, a substantial number are of unique structure and unknown function. Among the components of these secretory peptidomes is an array of protease inhibitors. Inhibitors of trypsin are of widespread occurrence in different taxa and are representative of many established structural classes, including Kunitz, Kazal and Bowman-Birk. However, few protease inhibitors with activity against other specific proteases have been described from this source. Here we report for the first time, the isolation and structural characterisation of an inhibitor of chymotrypsin of Kunitz-type from the skin secretion of the African hyperoliid frog, Kassina senegalensis. To this end, we employed a functional peptidomic approach. This scheme involves fractionation of the peptidome, functional end-point screening, structural characterisation of resultant actives followed by molecular cloning of biosynthetic precursor-encoding cDNA(s). The novel mature and active polypeptide identified consisted of 62 amino acid residues (average molecular mass 6776.24 Da), of which 6 were positionally-conserved cysteines. The P(1) position within the active site was occupied by a phenylalanyl residue. Bioinformatic analysis of the sequence using BLAST, revealed a structural similarity to Kunitz-type chymotrypsin inhibitors from other organisms, ranging from silkworms to snakes.
Copyright © 2011 Elsevier Masson SAS. All rights reserved.
A calmodulin-dependent translocation pathway for small secretory proteins.
Source
Cell Biology and Metabolism Program, National Institute of Child Health and Human Development, 18 Library Drive, Building 18, Room 101, National Institutes of Health, Bethesda, MD 20892, USA; Department of Biology, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA.
Abstract
Metazoans secrete an extensive array of small proteins essential for intercellular communication, defense, and physiologic regulation. Their synthesis takes mere seconds, leaving minimal time for recognition by the machinery for cotranslational protein translocation into the ER. The pathway taken by these substrates to enter the ER is not known. Here, we show that both in vivo and in vitro, small secretory proteins can enter the ER posttranslationally via a transient cytosolic intermediate. This intermediate contained calmodulin selectively bound to the signal peptides of small secretory proteins. Calmodulin maintained the translocation competence of small-protein precursors, precluded their aggregation and degradation, and minimized their inappropriate interactions with other cytosolic polypeptide-binding proteins. Acute inhibition of calmodulin specifically impaired small-protein translocation in vitro and in cells. These findings establish a mammalian posttranslational pathway for small-protein secretion and identify an unexpected role for calmodulin in chaperoning these precursors safely through the cytosol.
Copyright © 2011 Elsevier Inc. All rights reserved.
The type 1 diabetes associated HLA-DQ8-trans dimer accomodates a uniquepeptide repertoire.
Source
Leiden University Medical Center, Netherlands;
Abstract
HLA-DQ2 and HLA-DQ8 are strongly predisposing haplotypes for type 1 diabetes (T1D). Yet, HLA-DQ2/8 heterozygous individuals have a synergistically increased risk compared to HLA-DQ2 or HLA-DQ8 homozygote subjects that may result from the presence of a transdimer formed between the α-chain of HLA-DQ2 (DQA1*0501) and the β-chain of HLA-DQ8 (DQB1*0302). We generated cells exclusively expressing this transdimer (HLA-DQ8trans), characterized itspeptide binding repertoire and defined a unique transdimer specific peptide-binding motif that was found to be distinct from those of HLA-DQ2 and HLA-DQ8. This motif predicts an array of peptides of islet autoantigens as candidate T cell epitopes, many of which selectively bind to the HLA-transdimer, whereas others bind to both HLA-DQ8 and transdimer with similar affinity. Our findings provide a molecular basis for the association between HLA-DQ transdimers and T1D and set the stage for rational testing of potential diabetogenic peptide epitopes.
Structural and Antioxidant Modification of Wheat Peptides Modified by the Heat and Lipid Peroxidation Product Malondialdehyde.
Source
Authors are with State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan Univ., Wuxi, 214122, Jiangsu Province, P. R. China. Direct inquiries to author Shi (E-mail: yhshi@jiangnan.edu.cn).
Abstract
Wheat peptides, the biological active peptides derived from foods, has an array of biological actions, including antiobesity, antimicrobial, and angiotensin I-converting enzyme inhibitory effects in mammalian species. Recent studies showed that some wheat peptides may show the noteworthy antioxidant potency against the peroxidation of lipids or fatty acids, but the effect of oxidation on its antioxidant activities is unclear. In the present study, we demonstrate that heat and malandialdehyde (MDA)-oxidized wheat peptides lose its surface hydrophobicity and reducing power, and show a relatively lower free radical-scavenging activitiy in vitro. Those modifications also lead to gradual formation of aggregates in wheat peptides and induce more reactive oxygen species (ROS) production in vivo. These findings indicate that oxidation may influence the functional properties and directly alter the structure of wheat peptides, and lead to the loss of its antioxidant potency both in vitro and in vivo, thereby providing a novel explanation for some of the potential health risks proposed for oxidized food in human.
© 2011 Institute of Food Technologists®
- PMID:
- 22181681
- [PubMed - as supplied by publisher]
Expression of genes belonging to the IGF-system in glial tumors.
Source
Institute of Molecular Biology and Genetics, Kyiv, Ukraine. dmitrenko@imbg.org.ua
Abstract
Increased expression of the insulin-like growth factor (IGF) family members, IGF1, IGF2, their receptors and binding proteins, or combinations thereof has been documented in various malignancies including gliomas. The results of multiple investigations suggest that the IGFs can play a paracrine and/or autocrine role in promoting tumor growth in situ during tumor progression but that these roles may vary depending on the tissue of origin. Enhanced IGF1 expression was not found in glioblastomas and it was supposed that IGF1 participation in the development of glial tumors may be substituted by protein products of highly expressed other genes, also participating in PI3K and MAPK pathways. Increased expression of IGF-binding protein genes in brain tumors makes the picture even more complicated. As other binding proteins, IGFBPs regulate the activity of their ligands by prolonging their half-life. The discrepancies arising from conflicting evidence on the results obtained by different laboratories in human gliomas are discussed. Our data highlight the importance of viewing the IGF-related proteins as a complex multifactorial system and show that changes in the expression levels of any one component of the system, in a given malignancy, should be interpreted with caution. As IGF targeting for anticancer therapy is rapidly becoming clinical reality, an understanding of this complexity is very timely.
- PMID:
- 22168049
- [PubMed - indexed for MEDLINE]
A genome-wide study reveals rare CNVs exclusive to extreme phenotypes of Alzheimer disease.
Source
1] Inserm U614, Faculté de Médecine, Rouen, France [2] CNR-MAJ, CHU Rouen, CHU Lille, CHU Pitié Salpétrière, Paris, France.
Abstract
Studying rare extreme forms of Alzheimer disease (AD) may prove to be a useful strategy in identifying new genes involved in monogenic determinism of AD. Amyloid precursor protein (APP), PSEN1, and PSEN2 mutations account for only 85% of autosomal dominant early-onset AD (ADEOAD) families. We hypothesised that rare copy number variants (CNVs) could be involved in ADEOAD families without mutations in known genes, as well as in rare sporadic young-onset AD cases. Using high-resolution array comparative genomic hybridisation, we assessed the presence of rare CNVs in 21 unrelated ADEOAD cases, having no alteration on known genes, and 12 sporadic AD cases, with an age of onset younger than 55 years. The analysis revealed the presence of 7 singleton CNVs (4 in ADEOAD and 3 in sporadic cases) absent in 1078 controls and 912 late-onset AD cases. Strikingly, 4 out of 7 rearrangements target genes (KLK6, SLC30A3, MEOX2, and FPR2) encoding proteins that are tightly related to amyloid-β peptide metabolism or signalling. Although these variants are individually rare and restricted to particular subgroups of patients, these findings support the causal role, in human pathology, of a set of genes coding for molecules suspected for a long time to modify Aβ metabolism or signalling, and for which animal or cellular models have already been developed.European Journal of Human Genetics advance online publication, 14 December 2011; doi:10.1038/ejhg.2011.225.
Trogocytosis Is a Gateway to Characterize Functional Diversity in Melanoma-Specific CD8+ T Cell Clones.
Source
Sharett Institute of Oncology, Hadassah Medical Organization, Jerusalem 91120, Israel;
Abstract
Trogocytosis, the transfer of membrane patches from target to immune effector cells, is a signature of tumor-T cell interaction. In this study, we used the trogocytosis phenomenon to study functional diversity within tumor-specific T cell clones with identical TCR specificity. MART-1(26-35)-specific CD8 T cell clones, which differed in their trogocytosis capacity (low [2D11], intermediate [2G1], high [2E2]), were generated from melanoma patients. Functional evaluation of the clones showed that the percentage of trogocytosis-capable T cells closely paralleled each clone's IFN-γ and TNF-α production, lysosome degranulation, and lysis of peptide-pulsed targets and unmodified melanoma. The highly cytotoxic 2E2 clone displayed the highest TCR peptide binding affinity, whereas the low-activity 2D11 clone showed TCR binding to peptide-MHC in a CD8-dependent manner. TCR analysis revealed Vβ16 for clones 2E2 and 2G1 and Vβ14 for 2D11. When peptide-affinity differences were bypassed by nonspecific TCR stimulation, clones 2E2 and 2D11 still manifested distinctive signaling patterns. The high-activity 2E2 clone displayed prolonged phosphorylation of ribosomal protein S6, an integrator of MAPK and AKT activation, whereas the low-activity 2D11 clone generated shorter and weaker phosphorylation. Screening the two clones with identical TCR Vβ by immunoreceptor array showed higher phosphorylation of NK, T, and B cell Ag (NTB-A), a SLAM family homophilic receptor, in clone 2E2 compared with 2G1. Specific blocking of NTB-A on APCs markedly reduced cytokine production by CD8 lymphocytes, pointing to a possible contribution of NTB-A costimulation to T cell functional diversity. This finding identifies NTB-A as a potential target for improving anti-cancer immunotherapy.
Natriuresis and diuretic hormone synergism in R. prolixus upper Malpighian tubules is inhibited by the anti-diuretic hormone, RhoprCAPA-α2.
Source
Department of Biology, McMaster University, Hamilton, Ontario, Canada L8S 4K1.
Abstract
Insects contain an array of hormones that coordinate the actions of the excretory system to achieve osmotic and ionic balance. In the hematophagous insect, Rhodnius prolixus, two diuretic hormones have been identified, serotonin (5HT) and a corticotropin releasing factor-related peptide (RhoprDH), and both lead to an increase in fluid secretion by Malpighian tubules (MTs). However, only 5HT activates reabsorption by the lower MTs to recover K(+) and Cl(-). An anti-diuretic hormone (RhoprCAPA-α2) is believed to coordinate the cessation of the rapid diuresis following blood meal engorgement. However, the role of RhoprCAPA-α2 on fluid secretion by MTs stimulated by RhoprDH was previously unknown. Here we demonstrate that, unlike the inhibitory effect on 5HT-stimulated secretion by MTs, RhoprCAPA-α2 does not inhibit secretion stimulated by RhoprDH although it does abolish the synergism that occurs between the two diuretic hormones. In addition, we show that the natriuresis elicited by either diuretic hormone is reduced by RhoprCAPA-α2. Using electrophysiological tools, we investigate the possible mechanism by which this complex regulatory pathway is achieved. Analysis of the pH of secreted fluid as well as the triphasic response in transepithelial potential in MTs treated with diuretic hormones, suggests that RhoprCAPA-α2 does not inhibit the V-type H(+) ATPase. Taken together, these results indicate that RhoprCAPA-α2 functions to reduce the rapid diuresis following blood feeding, and in addition, it inhibits the natriuresis associated with diuretic hormone stimulated MTs. This may reflect an important regulatory mechanism related to the slow diuresis that occurs as the K(+)-rich blood cells are digested.
Copyright © 2011. Published by Elsevier Ltd.
Diverse Roles of G-Protein Coupled Receptors in Regulation of Neurohypophyseal Hormone Secretion.
Source
Department of Physiology and Biophysics University of Colorado School of Medicine Aurora, CO 80045.
Abstract
The magnocellular neurons in the supraoptic nucleus project to the neural lobe and release vasopressin and oxytocin into the peripheral circulation where they act on the kidney to promote fluid retention or stimulate smooth muscles in the vasculature, uterus and mammary glands to support blood pressure, promote parturition, or induce milk let-down respectively. Hormone release is regulated by complex afferent pathways carrying information about plasma osmolality, blood pressure and volume, cervical stretch, and suckling. These afferent pathways utilize a broad array of neurotransmitters and peptides that activate both ligand-gated ion channels and G-protein coupled receptors (GPCRs). The ligand-gated ion channels induce rapid changes in membrane potential resulting in generation of action potentials, initiation of exocytosis, and release of hormone into the periphery. In contrast, the GPCRs activate a host of diverse signaling cascades that modulate action potential firing and regulate other cellular functions required to support hormone release (e.g. hormone synthesis, processing, packaging and trafficking). The diversity of these actions is critical for integration of the distinct regulatory signals into a response appropriate for maintaining homeostasis. This review will describe several diverse roles of GPCRs in magnocellular neurons focusing primarily on adrenergic, purinergic, and peptidergic (neurokinin and angiotensin) receptors.
© 2011 The Authors. Journal of Neuroendocrinology © 2011 Blackwell Publishing Ltd.
Phage-Chips for Novel Optically Readable Tissue Engineering Assays.
Source
Department of Bioengineering, and Berkeley Nanoscience and Nanoengineering Institute, University of California, Berkeley , Berkeley, California 94720, United States.
Abstract
We report novel phage-based array chips that are optically readable for cell proliferation and morphology assays. Using M13 phages that were engineered to display RGD on its major coat proteins and/or immobilize FGFb on its minor coat proteins, we prepared arrays of phage spot matrices composed of self-assembled nanofibrous network structures. We cultured fibroblasts on the arrays and, using surface plasmon resonance (SPR) spectroscopy, monitored the effects of the biochemical cues displayed by the phage on cell proliferation and morphology. This study demonstrates the utility of engineered phages as promising coating materials for lab-on-a-chip (LOC) platforms, allowing sensitive monitoring of the effects of functional peptides on cell growth. Phage-chips have great potential for use as high-throughput screening systems for biochemical assays and biosensors and the discovery of novel drugs.
Insights into high affinity SUMO recognition by SUMO-interacting motifs (SIM) revealed by a combination of NMR and peptide array analysis.
Source
Beckman Research Institute of the City of Hope, United States;
Abstract
The small ubiquitin-like modifiers (SUMO) regulate many essential cellular functions. Only one type of SUMO-interacting motif (SIM) has been identified that can extend the β-sheet of SUMO as either a parallel or an anti-parallel strand. The molecular determinants of the bound orientation and paralogue-specificity of a SIM is unclear. To address this question, we have conducted structural studies of SUMO1 in complex with a SUMO1-specific SIM that binds to SUMO1 with high affinity without post-translational modifications using nuclear magnetic resonance methods. In addition, the SIM sequence requirements have been investigated by peptide arrays in comparison to another high affinity SIM that binds in the opposing orientation. We found that anti-parallel binding SIMs tolerate more diverse sequences, whereas the parallel binding SIMs prefer the more strict sequences consisting of I/V-D-L-T that have a preference in high affinity SUMO2 and 3 binding. Comparison of two high affinity SUMO1-binding SIMs that binds in opposing orientations has revealed common SUMO1-specific interactions needed for high affinity binding. This study has significantly advanced our understanding of the molecular determinants underlining SUMO-SIM recognition.
Peptide stapling is a strategy for constraining short peptides typically in an alpha-helical conformation. Stapling is carried out by covalently linking the side-chains of two amino acids, Stapled Peptide Design
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