ADVANCED FUNCTIONAL MATERIALS, Issue 4 2010
Yi Chen
Abstract Versatile strategies are currently being discovered for the fabrication of synthetic polypeptide-based hybrid hydrogels, which have potential applications in polymer therapeutics and regenerative medicine. Herein, a new concept,the reverse micellar hydrogel,is introduced, and a versatile strategy is provided for fabricating supramolecular polypeptide-based normal micellar hydrogel and reverse micellar hydrogels from the same polypeptide-based copolymer via the cooperation of host,guest chemistry and hydrogen-bonding interactions. The supramolecular hydrogels are thoroughly characterized, and a mechanism for their self-assembly is proposed. These hydrogels can respond to dual stimuli,temperature and pH,and their mechanical and controlled drug-release properties can be tuned by the copolymer topology and the polypeptide composition. The reverse micellar hydrogel can load 10% of the anticancer drug doxorubicin hydrochloride (DOX) and sustain DOX release for 45 days, indicating that it could be useful as an injectable drug delivery system. [source]

Changes in Basal Hypothalamic Chicken Gonadotropin-Releasing Hormone-I and Vasoactive Intestinal Polypeptide Associated with a Photo-Induced Cycle in Gonadal Maturation and Prolactin Secretion in Intact and Thyroidectomized Starlings (Sturnus vulgaris)

JOURNAL OF NEUROENDOCRINOLOGY, Issue 7 2002
A. Dawson
Abstract Chicken gonadotropin-releasing hormone-I (GnRH-I) and the avian prolactin-releasing hormone, vasoactive intestinal polypeptide (VIP), were measured in the basal hypothalamus in male starlings during photo-induced gonadal growth and the subsequent development and maintenance of reproductive photorefractoriness. Comparisons were made with thyroidectomized birds, which maintain breeding condition irrespective of changes in photoperiod. In intact birds, basal hypothalamic GnRH-I increased four-fold after photostimulation and then decreased 115-fold over 12 weeks to values characteristic of long-term photorefractoriness. Pituitary and plasma prolactin increased after photostimulation, reaching peak values when the testes were regressing, and returned to low values in long-term photorefractory birds. Basal hypothalamic VIP did not change after photostimulation in intact birds. In photostimulated thyroidectomized birds, values for basal hypothalamic GnRH-I and VIP, and for pituitary and plasma prolactin, remained no different to those of nonphotostimulated intact birds. These observations confirm that reproductive photorefractoriness is related to a decrease in hypothalamic GnRH-I. However, photorefractoriness in terms of prolactin secretion is not similarly related to a decrease in basal hypothalamic VIP. The mechanisms responsible for the decrease in prolactin in long-term photorefractory birds and for the total lack of photoperiodic responses in thyroidectomized birds remain unresolved. [source]

Efficient Synthesis of Protein-Drug Conjugates Using a Functionalizable Recombinant Elastin-Mimetic Polypeptide

MACROMOLECULAR BIOSCIENCE, Issue 11 2006
Doris Kaufmann
Abstract Summary: This report describes the efficient conjugation of doxorubicin-glycine-phenylalanine-leucine-glycine (1a) and rhodamine-glycine-phenylalanine-leucine-glycine (1b) units to a monodisperse elastin-mimetic polypeptide (EMM)7 bearing eight primary amine groups for chemical attachment. The synthetic approach is based on the solid-phase synthesis of 1a and 1b followed by chemical conjugation to the elastin-mimetic polypeptide in the presence of HOBt/PyBob as activating agents to form the polypeptide conjugates 2a and 2b. Conjugation efficiency was 61.2% (4.9 doxorubicin units per polypeptide chain) for 2a and 53.7% (4.3 rhodamine units per polypeptide chain) for 2b, demonstrating the feasibility of using these tailor-made, recombinant polypeptides as potential drug carriers for cancer therapy. Schematic structure of the elastin-mimetic polypeptide (EMM)7. [source]

Advances in the Synthesis and Characterization of Polypeptide-Based Hybrid Block Copolymers

MACROMOLECULAR SYMPOSIA, Issue 1 2004
Ivaylo Dimitrov
Abstract Linear polystyrene- block -poly(Z-L-lysine) copolymers with a very narrow molecular weight distribution (polydispersity index < 1.03) could be obtained via the ring-opening polymerization of Z-L-lysine- N -carboxyanhydride using ,-(primary amino hydrochloride)-polystyrenes as macroinitiators in N,N -dimethylformamide as the solvent at 40-80 °C. The block copolymer samples were analyzed by means of NMR, size exclusion chromatography, and analytical ultracentrifugation. [source]

Calcium Phosphate Crystallization on Electrospun Cellulose Non-Woven Fabrics Containing Synthetic Phosphorylated Polypeptides

MACROMOLECULAR MATERIALS & ENGINEERING, Issue 5 2009
Shinya Hayashi
Abstract The preparation of electrospun non-woven fabrics composed of cellulose and synthetic phosphorylated polypeptides, copoly[Ser(PO3H2)XAspY]s (X:Y,=,100:0, 75:25, 50:50, 25:75), is described. The non-wovens were subjected to an alternate immersion in CaCl2 and Na2HPO4 solutions to induce crystallization of calcium phosphate. The deposited calcium phosphate crystals were analyzed by means of EDX analysis and WXRD. The amounts of calcium phosphate deposition are greater for the cellulose non-woven fabrics containing copoly[Ser(PO3H2)XAspY] than those of cellulose-only non-woven fabrics. These results indicate that copoly[Ser(PO3H2)XAspY] can entrap Ca2+ ions around the fine fiber matrix to accelerate crystallization of the calcium phosphate. [source]

Post-translational modifications, but not transcriptional regulation, of major chloroplast RNA-binding proteins are related to Arabidopsis seedling development

PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 8 2006
Bai-Chen Wang
Abstract Chloroplast RNA-binding proteins are involved in stabilizing stored chloroplast mRNAs and in recruiting site-specific factors that mediate RNA metabolism. In the present study, we characterized two major chloroplast RNA-binding proteins, cp29A and cp29B, by MALDI-TOF MS, N-terminal sequencing, and ESI-MS/MS following 2D-PAGE separation. Polypeptides derived from cp29A were recovered with free N-terminus or with N-terminal acetylation. In addition to the two isoforms found for cp29A, an isoform derived from cp29B was also observed to have five amino acids cleaved from its N-terminus. Results of quantitative real-time RT-PCR indicate that both genes reached maximal rates of transcription 96,h after commencement of germination and maintained relatively high levels throughout the whole life cycle. Transcription of cp29A and cp29B did not vary significantly under light or dark conditions, although production of the acetylated and N-terminally cleaved protein isoforms exhibited light dependence. Exposure of etiolated Arabidopsis seedlings to light conditions for as short as 9,h restored the modified isoforms to levels similar to those found in green plants. Identification of post-translational modifications in major chloroplast RNA-binding proteins may help elucidate their roles in seedling development and in plant RNA stabilization during the greening process. [source]

Ancient Weapons for Attack and Defense: the Pore-forming Polypeptides of Pathogenic Enteric and Free-living Amoeboid Protozoa,

THE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 5 2004
MATTHIAS LEIPPE
ABSTRACT Pore-forming polypeptides have been purified from several amoeboid protozoans that are well-known human pathogens. Obligate enteric parasites, such as Entamoeba histolytica, and free-living but potentially highly pathogenic species, such as Naegleria fowleri, contain these cytolytic molecules inside cytoplasmic granules. Comprehensive functional and structural studies have been conducted that include isolation of the proteins from their natural sources, monitoring of their biological activity towards different targets, and molecular cloning of the genes of their precursors. In the case of the most prominent member of the protein family, with respect to protozoans, the three-dimensional structure of amoebapore A was solved recently. The amoebic pore-forming polypeptides can rapidly perforate human cells. The antibacterial activity of amoebapores and of related polypetides from free-living protozoa points to a more vital function of these molecules: inside the digestive vacuoles they combat growth of phagocytosed bacteria which are killed when their cytoplasmic membranes are permeabilized. The concommitant activity of these proteins towards host cells may be due to a coincidental selection for an efficient effector molecule. Nonetheless, several lines of evidence indicate that these factors are involved in pathogenesis of fatal diseases induced by amoeboid protozoa. [source]

Strongly Binding Cell-Adhesive Polypeptides of Programmable Valencies,

ANGEWANDTE CHEMIE, Issue 11 2010
Benjamin
Länger und stärker: Mit maßgeschneiderten multivalenten Polypeptiden , monodispersen Polypeptiden mit einer Zelladhäsionssequenz mit programmierbarer Valenz , konnte die Stärke der Adhäsion von Zellen an Oberflächen verstärkt und eingestellt werden. Sie enthielten bis zu 80 Wiederholungseinheiten der RGD-Sequenz (siehe Bild) und zeigten einen starken Widerstand gegen das Ablösen der Zellen unter Schubbeanspruchung. [source]

ChemInform Abstract: Molecular Assembly of Zinc,Nickel Hybrid Porphyrin Dimer Using Synthetic 4,-Helix Polypeptides.

CHEMINFORM, Issue 49 2002
Mamoru Nango
Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a "Full Text" option. The original article is trackable via the "References" option. [source]

Effects of short-term food deprivation on orexin-A-induced intestinal bicarbonate secretion in comparison with related secretagogues

ACTA PHYSIOLOGICA, Issue 3 2010
G. Flemström
Abstract Studies of gastrointestinal physiology in humans and intact animals are usually conducted after overnight fast. We compared the effects of orexin-A, vasoactive intestinal polypeptide (VIP), melatonin, serotonin, uroguanylin, ghrelin and prostaglandin E2 (PGE2) on duodenal bicarbonate secretion in fed and overnight fasted animals. This review is a summary of our findings. Secretagogues were administered by intra-arterial infusion or luminally (PGE2). Enterocyte intracellular calcium ([Ca2+]i) signalling was studied by fluorescence imaging. Total RNA was extracted, reverse transcripted to cDNA and expression of orexin receptors measured by quantitative real-time PCR. Orexin-A stimulates the duodenal secretion in continuously fed animals but not in food-deprived animals. Similarly, short-term fasting causes a 100-fold decrease in the amount of the muscarinic agonist bethanechol required for stimulation of secretion. In contrast, fasting does not affect secretory responses to intra-arterial VIP, melatonin, serotonin, uroguanylin and ghrelin, or that to luminal PGE2. Orexin-A induces [Ca2+]i signalling in enterocytes from fed rats but no significant [Ca2+]i responses occur in enterocytes from fasted animals. In addition, overnight fasting decreases the expression of mucosal orexin receptors. Short-term food deprivation thus decreases duodenal expression of orexin receptors and abolishes the secretory response to orexin-A as well as orexin-A-induced [Ca2+]i signalling. Fasting, furthermore, decreases mucosal sensitivity to bethanechol. The absence of declines in secretory responses to other secretagogues tested strongly suggests that short-term fasting does not affect the secretory capacity of the duodenal mucosa in general. Studies of intestinal secretion require particular evaluation with respect to feeding status. [source]

Possible common central pathway for resistin and insulin in regulating food intake

ACTA PHYSIOLOGICA, Issue 4 2009
C. Cifani
Abstract Aim:, Adipose tissue has been the object of intense research in the field of obesity and diabetes diseases in the last decade. Examination of adipocyte-secreted peptides led to the identification of a unique polypeptide, resistin (RSTN), which has been suggested as a link between obesity and diabetes. RSTN plays a clearly documented role in blocking insulin (INS)-induced hypoglycaemia. As brain injection of INS affects feeding behaviour, we studied the possible interaction between INS and RSTN in food-deprived rats, measuring effects on food intake. In addition, we examined how RSTN might affect neuropeptide Y (NPY)-induced feeding, as studies have shown that rat RSTN can interfere with the NPY system. Methods:, Overnight food-deprived rats were injected into the third brain ventricle (3V) with either INS (10 or 20 mUI), RSTN (0.1,0.4 nmol/rat), or saline before access to food. Another group of rats was injected into the 3V with RSTN alone, NPY alone or RSTN plus NPY. Their food intake and body weight were measured. Results:, Our results confirm the hypophagic effect of RSTN on food deprivation-induced food intake, and more importantly, show that RSTN neither potentiates nor blocks the effects of INS on food intake, but does reduce the hyperphagic effect of NPY. Conclusion:, The observation that RSTN does not modify feeding INS-induced hypophagia, but does influence NPY-induced feeding, points to the possibility that RSTN may be involved in control of food intake through an NPY-ergic mechanism as INS. [source]

Actin filament binding by a monomeric IQGAP1 fragment with a single calponin homology domain

CYTOSKELETON, Issue 4 2004
Scott C. Mateer
Abstract IQGAP1 is a homodimeric protein that reversibly associates with F-actin, calmodulin, activated Cdc42 and Rac1, CLIP-170, ,-catenin, and E-cadherin. Its F-actin binding site includes a calponin homology domain (CHD) located near the N-terminal of each subunit. Prior studies have implied that medium- to high-affinity F-actin binding (5,50 ,M Kd) requires multiple CHDs located either on an individual polypeptide or on distinct subunits of a multimeric protein. For IQGAP1, a series of six tandem IQGAP coiled-coil repeats (IRs) located past the C-terminal of the CHD of each subunit support protein dimerization and, by extension, the IRs or an undefined subset of them were thought to be essential for F-actin binding mediated by its CHDs. Here we describe efforts to determine the minimal region of IQGAP1 capable of binding F-actin. Several truncation mutants of IQGAP1, which contain progressive deletions of the IRs and CHD, were assayed for F-actin binding in vitro. Fragments that contain both the CHD and at least one IR could bind F-actin and, as expected, removal of all six IRs and the CHD abolished binding. Unexpectedly, a fragment called IQGAP12-210, which contains the CHD, but lacks IRs, could bind actin filaments. IQGAP12-210 was found to be monomeric, to bind F-actin with a Kd of ,47 ,M, to saturate F-actin at a molar ratio of one IQGAP12-210 per actin monomer, and to co-localize with cortical actin filaments when expressed by transfection in cultured cells. These collective results identify the first known example of high-affinity actin filament binding mediated by a single CHD. Cell Motil. Cytoskeleton 58:231,241, 2004. © 2004 Wiley-Liss, Inc. [source]

Cardiac expression patterns of endothelin-converting enzyme (ECE): Implications for conduction system development

DEVELOPMENTAL DYNAMICS, Issue 6 2008
David Sedmera
Abstract The spatiotemporal distribution of the endothelin-converting enzyme (ECE) protein in the embryonic chick heart and the association of this polypeptide with the developing cardiac conduction system is described here for the first time. Further, we show how cardiac hemodynamic load directly affects ECE level and distribution. Endothelin (ET) is a cytokine involved in the inductive recruitment of Purkinje fibers. ET is produced by proteolytic cleavage of Big-ET by ECE. We generated an antibody against chick ECE recognizing a single band at ,70 kD to correlate the cardiac expression of this protein with that reported previously for its mRNA. ECE protein expression was more widespread compared to its mRNA, being present in endothelial cells, mesenchymal cells, and myocytes, and particularly enriched in the trabeculae and nascent ventricular conduction system. The myocardial expression was significantly modified under experimentally altered hemodynamic loading. In vivo, ET receptor blockade with bosentan delayed activation sequence maturation. These data support a role for ECE in avian cardiac conduction system differentiation and maturation. Developmental Dynamics 237:1746,1753, 2008. © 2008 Wiley-Liss, Inc. [source]

ghrelin is a novel target of Pax4 in endocrine progenitors of the pancreas and duodenum

DEVELOPMENTAL DYNAMICS, Issue 1 2008
Qian Wang
Abstract Pax4 -deficient mice have a severe gastrointestinal endocrine deficiency: they lack most pancreatic cells that produce insulin or somatostatin and various duodenal endocrine cell types. Remarkably, Pax4 -deficient mice also have an overabundance of ghrelin-expressing cells in the pancreas and duodenum. Detailed analysis of the Pax4 nullizygous pancreas determined that the mutant islets are largely composed of a distinctive endocrine cell type that expresses ghrelin, glucagon, islet amyloid polypeptide (IAPP), and low levels of Pdx1. Lineage-tracing analysis revealed that most of these unique endocrine cells directly arose from Pax4 -deficient progenitors. Previous in vitro work reported that Pax4 is a transcriptional repressor of islet amyloid polypeptide (IAPP) and glucagon. In this study, we expanded those results by showing that Pax4 is also a repressor of gherlin. Together, our data further support the notion that Pax4 activity is necessary to establish appropriate patterns of gene expression in endocrine progenitors of the digestive tract. Developmental Dynamics 237:51,61, 2008. © 2007 Wiley-Liss, Inc. [source]

Prep2: Cloning and expression of a new prep family member

DEVELOPMENTAL DYNAMICS, Issue 3 2002
Klaus Haller
Abstract We describe Prep2, a new murine homeobox-containing gene closely related to Prep1. The PREP2 protein belongs to the three amino acid loop extension (TALE) superclass of homeodomain-containing proteins and encodes a polypeptide of 462 residues. As for PREP1, PREP2 binds an appropriate site on DNA as a heterodimer with PBX1A. Northern analysis, immunoblotting, immunohistochemistry, and in situ hybridization show widespread Prep2 expression during organogenesis and in the adult. The data suggest that Prep2 functions to varying degrees in a broad array of tissues and developmental processes. © 2002 Wiley-Liss, Inc. [source]

Two Na,K-ATPase ,2 subunit isoforms are differentially expressed within the central nervous system and sensory organs during zebrafish embryogenesis

DEVELOPMENTAL DYNAMICS, Issue 2 2002
Johannes R. Rajarao
Abstract We have identified cDNAs encoding a second zebrafish ortholog of the human Na,K-ATPase ,2 subunit. The ,2b cDNA encodes a 292 amino acid-long polypeptide with 74% identity to the previously characterized zebrafish ,2a subunit. By using a zebrafish meiotic mapping panel, we determined that the ,2b gene (atp1b2b) was tightly linked to markers on linkage group 5, whereas the ,2a gene was located on linkage group 23. In situ hybridization analysis shows that in developing zebrafish embryos, atp1b2a and atp1b2b are predominantly expressed in the nervous system. ,2a transcripts were abundantly expressed throughout brain as well as spinal cord neurons and lateral line ganglia. In contrast, ,2b mRNA expression was primarily detected in sensory organs, including retina, otic vesicles, and lateral line neuromast cells. These results suggest that the ,2a and ,2b genes play distinct roles in developing brain and sensory organs, and raise the possibility that the functions encoded by the single mammalian ,2 gene may be partitioned between the two zebrafish ,2 orthologs. © 2002 Wiley-Liss, Inc. [source]

Active immunization against (Pro3)GIP improves metabolic status in high-fat-fed mice

DIABETES OBESITY & METABOLISM, Issue 9 2010
I. A. Montgomery
Aim: Ablation of gastric inhibitory polypeptide (GIP) receptor signalling can prevent many of the metabolic abnormalities associated with dietary-induced obesity-diabetes. The present study was designed to assess the ability of active immunization against (Pro3)GIP to counter metabolic dysfunction associated with diet-induced obesity in high-fat-fed mice. Methods: Normal male Swiss NIH mice were injected (s.c.) once every 14 days for 98 days with complexed (Pro3)GIP peptide, with transfer to a high-fat diet on day 21. Results: Active immunization against (Pro3)GIP resulted in circulating GIP antibody production and significantly (p < 0.05 p < 0.01) reduced circulating blood glucose concentrations compared to high-fat control mice from day 84 onwards. Glucose levels were not significantly different from lean controls. The glycaemic response to i.p. glucose was correspondingly improved (p < 0.01) in (Pro3)GIP-immunized mice. Furthermore, circulating and glucose-stimulated plasma insulin levels were significantly (p < 0.01 to p < 0.001) depressed compared to high-fat control mice. Liver triglyceride, pancreatic insulin and circulating LDL-cholesterol levels were also significantly reduced in (Pro3)GIP-immunized mice. These changes were independent of any effects on food intake or body weight. The glucose-lowering effect of native GIP was annulled in (Pro3)GIP-immunized mice consistent with the induction of biologically effective GIP-specific neutralizing antibodies. Conclusion: These results suggest that immunoneutralization of GIP represents an effective means of countering the disruption of metabolic processes induced by high-fat feeding. [source]

Exendin-4 protects pancreatic beta cells from human islet amyloid polypeptide-induced cell damage: potential involvement of AKT and mitochondria biogenesis

DIABETES OBESITY & METABOLISM, Issue 9 2010
R. Fan
Aim: Glucagon-like peptide-1 (GLP-1) stimulates beta-cell proliferation and enhances beta-cell survival, whereas oligomerization of human islet amyloid polypeptide (hIAPP) may induce beta-cell apoptosis and reduce beta-cell mass. Type 2 diabetes is associated with increased expression of IAPP. As GLP-1-based therapy is currently developed as a novel antidiabetic therapy, we examined the potential protective action of the GLP-1 receptor agonist exendin-4 on hIAPP-induced beta-cell apoptosis. Methods: The study was performed in clonal insulinoma (INS-1E) cells. Both method of transcriptional and translational and sulphorhodamine B (SRB) assays were used to evaluate cell viability and cell mass. Western blot analysis was applied to detect protein expression. Transfection of constitutively active protein kinase B (PKB/AKT) was performed to examine the role of AKT. Mitochondrial biogenesis was quantified by mitogreen staining and RT-PCR. Results: First, we confirmed that hIAPP induced cell apoptosis and growth inhibition in INS-1E cells. These effects were partially protected by exendin-4 in association with partial recovery of the hIAPP-mediated AKT inhibition. Furthermore, AKT constitutive activation attenuated hIAPP-induced apoptosis, whereas PI3K/AKT inhibition abrogated exendin-4-mediated effects. These findings suggest that the antiapoptotic and proliferative effects of exendin-4 in hIAPP-treated INS-1E cells were partially mediated through AKT pathway. Moreover, hIAPP induced FOXO1 but inhibited pdx-1 nucleus translocation. These effects were restored by exendin-4. Finally, mitogreen staining and RT-PCR revealed enhanced mitochondrial biogenesis by exendin-4 treatment. Conclusions: Collectively, these results suggest that GLP-1 receptor agonist protects beta cells from hIAPP-induced cell death partially through the activation of AKT pathway and improved mitochondrial function. [source]

Changes in gene expression and morphology of mouse embryonic stem cells on differentiation into insulin-producing cells in vitro and in vivo

DIABETES/METABOLISM: RESEARCH AND REVIEWS, Issue 5 2009
Ortwin Naujok
Abstract Background Embryonic stem (ES) cells have the potential to produce unlimited numbers of surrogate insulin-producing cells for cell replacement therapy of type 1 diabetes mellitus. The impact of the in vivo environment on mouse ES cell differentiation towards insulin-producing cells was analysed morphologically after implantation. Methods ES cells differentiated in vitro into insulin-producing cells according to the Lumelsky protocol or a new four-stage differentiation protocol were analysed morphologically before and after implantation for gene expression by in situ reverse transcription polymerase chain reaction and protein expression by immunohistochemistry and ultrastructural analysis. Results In comparison with nestin positive ES cells developed according to the reference protocol, the number of ES cells differentiated with the four-stage protocol increased under in vivo conditions upon morphological analysis. The cells exhibited, in comparison to the in vitro situation, increased gene and protein expression of Pdx1, insulin, islet amyloid polypeptide (IAPP), the GLUT2 glucose transporter and glucokinase, which are functional markers for glucose-induced insulin secretion of pancreatic beta cells. Renal sub-capsular implantation of ES cells with a higher degree of differentiation achieved by in vitro differentiation with a four-stage protocol enabled further significant maturation for the beta-cell-specific markers, insulin and the co-stored IAPP as well as the glucose recognition structures. In contrast, further in vivo differentiation was not achieved with cells differentiated in vitro by the reference protocol. Conclusions A sufficient degree of in vitro differentiation is an essential prerequisite for further substantial maturation in a beta-cell-specific way in vivo, supported by cell-cell contacts and vascularisation. Copyright © 2009 John Wiley & Sons, Ltd. [source]

RD Lawrence Lecture 2008 Targeting GLP-1 release as a potential strategy for the therapy of Type 2 diabetes

DIABETIC MEDICINE, Issue 8 2008
F. M. Gribble
Abstract Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are gastrointestinal hormones that play an important role in stimulating postprandial insulin release from pancreatic ,-cells. Agents that either mimic GLP-1 or prevent its degradation are now available for the treatment of Type 2 diabetes, and strategies to enhance endogenous GLP-1 release are under assessment. As intestinal peptides have a range of actions, including appetite regulation and coordination of fat metabolism, harnessing the enteric endocrine system is a promising new field for drug development. [source]

Dorothy Hodgkin Lecture 2008 Gastric inhibitory polypeptide (GIP) revisited: a new therapeutic target for obesity,diabetes?

DIABETIC MEDICINE, Issue 7 2008
P. R. Flatt
Abstract There is increasing realization that gastric inhibitory polypeptide (GIP) has actions outside of the pancreas and gastrointestinal tract. Most significant is the presence of functional GIP receptors on adipocytes and the appreciation that GIP, secreted strongly in response to fat ingestion, plays a role in the translation of excessive amounts of dietary fat into adipocyte tissue stores. Such effects open up the possibility of exploiting GIP receptor antagonism for the treatment of obesity and insulin resistance. This is borne out by studies in high-fat-fed mice or ob/ob mice with either genetic knockout of GIP receptor or chemical ablation of GIP action using the GIP receptor antagonist, (Pro3)GIP. By causing preferential oxidation of fat, blockade of GIP signalling clears triglyceride deposits from liver and muscle, thereby respectively restoring mechanisms for suppression of hepatic glucose output and cellular glucose uptake. Further studies are needed to determine the applicability of this research to human obesity,diabetes. However, proof of concept is provided by emerging evidence that rapid cure of diabetes in grossly obese subjects undergoing Roux-en-Y bypass surgery is mediated in part by surgical bypass of GIP-secreting K-cells in the upper small intestine. [source]

Incretins and other peptides in the treatment of diabetes

DIABETIC MEDICINE, Issue 3 2007
J. F. Todd
Abstract Glucagon-like peptide-1 (7-36) amide (GLP-1) is a gut hormone, released postprandially, which stimulates insulin secretion and insulin gene expression as well as pancreatic B-cell growth. Together with glucose-dependent insulinotropic polypeptide (GIP), it is responsible for the incretin effect which is the augmentation of insulin secretion following oral administration of glucose. Patients with Type 2 diabetes have greatly impaired or absent incretin-mediated insulin secretion which is mainly as a result of decreased secretion of GLP-1. However, the insulinotropic action of GLP-1 is preserved in patients with Type 2 diabetes, and this has encouraged attempts to treat Type 2 diabetic patients with GLP-1. GLP-1 also possesses a number of potential advantages over existing agents for the treatment of Type 2 diabetes. In addition to stimulating insulin secretion and promoting pancreatic B-cell mass, GLP-1 suppresses glucagon secretion, delays gastric emptying and inhibits food intake. Continuous intravenous and subcutaneous administration significantly improves glycaemic control and causes reductions in both HbA1c and body weight. However, GLP-1 is metabolized extremely rapidly in the circulation by the enzyme dipeptidyl peptidase IV (DPP-IV). This is the probable explanation for the short-lived effect of single doses of native GLP-1, making it an unlikely glucose-lowering agent. The DPP-IV resistant analogue, exenatide, has Food and Drug Administration (FDA) approval for the treatment of Type 2 diabetes and selective DPP-IV inhibitors are under development. Both approaches have demonstrated remarkable efficacy in animal models and human clinical studies. Both are well tolerated and appear to have advantages over current therapies for Type 2 diabetes, particularly in terms of the effects on pancreatic B-cell restoration and potential weight loss. [source]

Immobilization and Electrochemistry of Negatively Charged Proteins on Modified Nanocrystalline Metal Oxide Electrodes

ELECTROANALYSIS, Issue 12 2005
Emmanuel Topoglidis
Abstract The immobilization of two acidic, low isoelectric point proteins, green fluorescence protein and ferredoxin (FRD) is investigated on nanocrystalline, mesoporous TiO2 and SnO2 electrodes. Modification of these electrodes with a cationic polypeptide (poly- L -lysine) or an aminosilane prior to protein immobilization is found to enhance protein binding at least ten fold, attributed to more favorable protein/electrode electrostatic interactions. Cyclic voltammetry studies of FRD-modified SnO2 electrodes indicate reversible protein electrochemistry with a midpoint potential of ,0.59,V (vs. Ag/AgCl) and an interfacial electron transfer rate constant of 0.45,s,1. [source]

Comprehensive proteome analysis by chromatographic protein prefractionation

ELECTROPHORESIS, Issue 7-8 2004
Pierre Lescuyer
Abstract Protein copy number is distributed from 7 to 8 orders of magnitude in cells and probably up to 12 orders of magnitude in plasma. Classical silver-stained two-dimensional electrophoresis (2-DE) can only display up to four orders of magnitude. This is a major drawback since it is assumed that most of the regulatory proteins are low-abundance gene products. It is thus clear that the separation of low copy number proteins in amounts sufficient for postseparation analysis is an important issue in proteome studies to complete the comprehensive description of the proteome of any given cell type. The visualization of a polypeptide on a 2-DE gel will depend on the copy number, on the quantity loaded onto the gel and on the method of detection. As the amount of protein that can be loaded onto a gel is limited, one efficient solution is to fractionate the sample prior to 2-DE analysis. Several approaches exist including subcellular fractionation, affinity purification and chromatographic and electrophoretic protein prefractionation. The chromatographic step adds a new dimension in the protein separation using specific protein properties. It allows proteins to be adsorbed to a surface and eluted differentially under certain conditions. This review article presents studies combining chromatography-based methods to 2-DE analysis and draws general conclusions on this strategy. [source]

The Pseudomonas aeruginosa patatin-like protein PlpD is the archetype of a novel Type V secretion system

ENVIRONMENTAL MICROBIOLOGY, Issue 6 2010
Richard Salacha
Summary We discovered a novel secreted protein by Pseudomonas aeruginosa, PlpD, as a member of the bacterial lipolytic enzyme family of patatin-like proteins (PLPs). PlpD is synthesized as a single molecule consisting of a secreted domain fused to a transporter domain. The N-terminus of PlpD includes a classical signal peptide followed by the four PLP conserved blocks that account for its lipase activity. The C-terminus consists of a POTRA (polypeptide transport-associated) motif preceding a putative 16-stranded ,-barrel similar to those of TpsB transporters of Type Vb secretion system. We showed that the C-terminus remains inserted into the outer membrane while the patatin moiety is secreted. The association between a TpsB component and a passenger protein is a unique hybrid organization that we propose to classify as Type Vd. More than 200 PlpD orthologues exist among pathogenic and environmental bacteria, which suggests that bacteria secrete numerous PLPs using this newly defined mechanism. [source]

Reduction of fumarate, mesaconate and crotonate by Mfr, a novel oxygen-regulated periplasmic reductase in Campylobacter jejuni

ENVIRONMENTAL MICROBIOLOGY, Issue 3 2010
Edward Guccione
Summary Methylmenaquinol : fumarate reductase (Mfr) is a newly recognized type of fumarate reductase present in some ,-proteobacteria, where the active site subunit (MfrA) is localized in the periplasm, but for which a physiological role has not been identified. We show that the Campylobacter jejuni mfrABE operon is transcribed from a single promoter, with the mfrA gene preceded by a small open reading-frame (mfrX) encoding a C. jejuni -specific polypeptide of unknown function. The growth characteristics and enzyme activities of mutants in the mfrA and menaquinol : fumarate reductase A (frdA) genes show that the cytoplasmic facing Frd enzyme is the major fumarate reductase under oxygen limitation. The Mfr enzyme is shown to be necessary for maximal rates of growth by fumarate respiration and rates of fumarate reduction in intact cells measured by both viologen assays and 1H-NMR were slower in an mfrA mutant. As periplasmic fumarate reduction does not require fumarate/succinate antiport, Mfr may allow more efficient adaptation to fumarate-dependent growth. However, a further rationale for the periplasmic location of Mfr is suggested by the observation that the enzyme also reduces the fumarate analogues mesaconate and crotonate; fermentation products of anaerobes with which C. jejuni shares its gut environment, that are unable to be transported into the cell. Both MfrA and MfrB subunits were localized in the periplasm by immunoblotting and 2D-gel electrophoresis, but an mfrE mutant accumulated unprocessed MfrA in the cytoplasm, suggesting a preassembled MfrABE holoenzyme has to be recognized by the TAT system for translocation to occur. Gene expression studies in chemostat cultures following an aerobic-anaerobic shift showed that mfrA is highly upregulated by oxygen limitation, as would be experienced in vivo. Our results indicate that in addition to a role in fumarate respiration, Mfr allows C. jejuni to reduce analogous substrates specifically present in the host gut environment. [source]

Potential multidrug resistance gene POHL: An ecologically relevant indicator in marine sponges

ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 1 2001
Anatoli Krasko
Abstract Sponges are sessile filter feeders found in all aquatic habitats from the tropics to the arctic. Against potential environmental hazards, they are provided with efficient defense systems, e.g., protecting chaperones and/or the P-170/multidrug resistance pump system. Here we report on a further multidrug resistance pathway that is related to the pad one homologue (POH1) mechanism recently identified in humans. It is suggested that proteolysis is involved in the inactivation of xenobiotics by the POH1 system. Two cDNAs were cloned, one from the demosponge Geodia cydoniumand a second from the hexactinellid sponge Aphrocallistes vastus. The cDNA from G. cydonium, termed GCPOHL, encodes a deduced polypeptide with a size of 34,591 Da and that from A. vastus, AVPOHL, a protein of a calculated Mr of 34,282. The two sponge cDNAs are highly similar to each other as well as to the known sequences from fungi (Schizosaccharomyces pombe and Saccharomyces cerevisiae) and other Metazoa (from Schistosoma mansoni to humans). Under controlled laboratory conditions, the expression of the potential multidrug resistance gene POHL is, in G. cydonium, strongly upregulated in response to the toxins staurosporin (20 ,M) or taxol (50 ,M); the first detectable transcripts appear after 1 d and reach a maximum after 3 to 5 d of incubation. The relevance of the expression pattern of the G. cydonium gene POHL for the assessment of pollution in the field was determined at differently polluted sites in the area around Rovinj (Croatia; Mediterranean Sea, Adriatic Sea). The load of the selected sites was assessed by measuring the potency of XAD-7 concentrates of water samples taken from those places to induce the level of benzo[a]pyrene monooxygenase (BaPMO) in fish and to impair the multidrug resistance (MDR)/P-170 extrusion pump in clams. These field experiments revealed that the levels of inducible BaPMO activity in fish and of the MDR potential by the water concentrates are highly correlated with the level of expression of the potential multidrug resistance gene POHL in G. cydonium. This report demonstrates that the detoxification POH pathway, here mediated by the G. cydonium GCPOHL gene, is an additional marker for the assessment of the environmental load in a given marine area. [source]

Production of biologically active equine interleukin 12 through expression of p35, p40 and single chain IL-12 in mammalian and baculovirus expression systems

EQUINE VETERINARY JOURNAL, Issue 7 2001
E. L. J. McMONAGLE
Summary Interleukin-12 (IL-12) is a key cytokine in the development of cell-mediated immune responses. Bioactive IL-12 is a heterodimeric cytokine composed of disulphide linked p35 and p40 subunits. The aim of this study was to verify biologically activity of the products expressed from equine interleukin-12 (IL-12) p35 and p40 cDNAs and to establish whether equine IL-12 could be expressed as a p35/p40 fusion polypeptide, as has been reported for IL-12a of several mammalian species. We report production of equine IL-12 through expression of p35 and p40 subunits in mammalian and insect cells and of a p35:p40 fusion polypeptide in mammalian cells. Conditioned medium recovered from cultures transiently transfected with constructs encoding equine p35 and p40 subunits or single chain IL-12 enhanced IFN-, production in cells derived from equine lymph nodes. Preincubation of IFN-, inducing preparations with anti-p40 monoclonal antibody resulted in a significant decrease in IFN-, induction capacity. Medium recovered from p35 and p40-expressing baculovirus infected cultures enhanced target cell IFN-, production and proliferation. Experimental studies in mice and other animals have revealed a therapeutic benefit of IL-12 in cancer, inflammatory and infectious disease and an adjuvant effect in prophylactic regimes. Production of a bioactive species-specific IL-12 is a first step towards an investigation of its potential application in equine species. [source]

Organic anion-transporting polypeptide (OATP) transporter family and drug disposition

EUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 2003
R. B. Kim
Abstract Drug transporters are increasingly recognized as a key determinant of drug disposition. Recent studies have revealed that targeted expression of drug uptake and efflux transporters to specific cell membrane domains allows for the efficient directional movement of many drugs in clinical use. While the role of certain efflux transporters such as MDR1 (P-glycoprotein) in drug disposition has been extensively studied, emerging evidence suggests that uptake transporters may also be important to the intestinal absorption and renal or hepatic elimination of drugs. Members of the organic anion-transporting polypeptide (OATP) family of drug uptake transporters have been found capable of transporting a large array of structurally divergent drugs. Moreover, expression of OATP isoforms in the gastrointestinal tract, liver and kidney, as well as at the level of the blood,brain barrier, has important implications for our understanding of the factors governing drug absorption, elimination and tissue penetration. [source]

Tauroursodeoxycholic acid mobilizes ,-PKC after uptake in human HepG2 hepatoma cells

EUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 6 2002
Helena Glasova
Abstract Background Tauroursodeoxycholic acid (TUDCA) may exert anticholestatic effects via Ca++ - and ,-protein kinase C (,-PKC)-dependent apical vesicular insertion of canalicular transporters in cholestatic hepatocytes (Hepatology 2001; 33: 1206,16). Tauroursodeoxycholic acid is mainly taken up into liver cells by Na+ -taurocholate cotransporting polypeptide (Ntcp). Tauroursodeoxycholic acid selectively translocates ,-PKC, a key mediator of regulated exocytosis, to hepatocellular membranes. It is unclear whether TUDCA exerts its effects on ,-PKC after carrier-mediated uptake into liver cells or by interaction with extracellular/membraneous structures. Materials and methods Human hepatoblastoma HepG2 cells lacking Ntcp were stably transfected with pcDNA3·1/Ntcp or sham-transfected with pcDNA3·1 [+]. Distribution of ,-PKC was studied using a Western blotting technique. Uptake of [3H]taurocholic acid (TCA) was determined radiochemically. Results [3H]taurocholic acid uptake was approximately 180-fold higher in Ntcp-transfected than in sham-transfected cells. Phorbol 12-myristate 13-acetate (1 µmol L,1; positive control) increased membrane binding of ,-PKC by 34% in Ntcp-transfected and by 37% in sham-transfected cells. Tauroursodeoxycholic acid (10 µmol L,1) increased membrane-associated ,-PKC by 19% in Ntcp-transfected, but not in sham-transfected cells (,13%). Taurocholic acid (10 µmol L,1) did not affect the distribution of ,-PKC. Conclusion Carrier-mediated uptake is a prerequisite for TUDCA-induced translocation of ,-PKC to hepatocellular membranes. [source]