RSSLow expression and secretion of circulating soluble CTLA-4 in peripheral blood mononuclear cells and sera from type 1 diabetic children.
Source
Division of Paediatrics & Diabetes Research Centre, Department of Molecular & Clinical Medicine, Faculty of Health Sciences, Linköping University, Linköping, Sweden. anna.ryden@liu.se.
Abstract
BACKGROUND:
High levels of soluble cytotoxic T-lymphocyte antigen 4 (soluble CTLA-4), an alternative splice form of the regulatory T-cell (Treg) associated CTLA-4 gene, have been associated with type 1 diabetes (T1D) and other autoimmune diseases, such as Grave's disease and myasthenia gravis. At the same time, studies have shown soluble CTLA-4 to inhibit T-cell activation through B7 binding. This study aimed to investigate the role of soluble CTLA-4 in relation to full-length CTLA-4 and other Treg-associated markers in T1D children and in individuals with high or low risk of developing the disease.
METHODS:
T1D children were studied at 4 days, 1 and 2 years after diagnosis in comparison to individuals with high or low risk of developing the disease. Isolated peripheral blood mononuclear cells were stimulated with the T1D-associated glutamic acid decarboxylase 65 and phytohaemagglutinin. Subsequently, soluble CTLA-4, full-length CTLA-4, FOXP3 and TGF-β mRNA transcription were quantified and protein concentrations of soluble CTLA-4 were measured in culturesupernatant and sera.
RESULTS AND CONCLUSIONS:
Low protein concentrations of circulating soluble CTLA-4 and a positive correlation between soluble CTLA-4 mRNA and protein were seen in T1D, in parallel with a negative correlation in healthy subjects. Further, low levels of mitogen-induced soluble CTLA-4 were accompanied by low C-peptide levels. Interestingly, low mitogen-induced soluble CTLA-4 mRNA and low TGF-β mRNA expression were seen in high risk individuals, suggesting an alteration in activation and down-regulating immune mechanisms during the pre-diabetic phase. Copyright © 2011 John Wiley & Sons, Ltd.
Copyright © 2011 John Wiley & Sons, Ltd.
- PMID:
- 22218756
- [PubMed - in process]
Temporary transvenous VDD pacing as a bridge to permanent pacemaker implantation in patients with sepsis and haemodynamically significant atrioventricular block.
Source
Service de Cardiologie, Hôpital Européen Georges Pompidou, 20 rue Leblanc 75015, Paris, France.
Abstract
AimsPermanent pacemaker (PM) implantation is temporarily contraindicated in patients (pts) with sepsis. In patients with symptomatic atrioventricular (AV) block and infection, prolonged VVI pacing is therefore usually ensured by a ventricular pacing lead (PL) connected to an external PM generator. In patients with normal sinus function and heart failure, the VVI mode can exacerbate haemodynamic dysfunction. A single AV PL can be attractive to achieve physiological pacing. This study was designed to assess the efficacy and safety of temporary VDD pacing as a bridge to permanent PM implantation in patients with complete AV block until control of infection.Methods and resultsThis study included eight patients with complete AV block and sepsis with negative blood culture. Due to the presence of congestive heart failure, a single bipolar AV PL connected to an external VDD PM generator. At VDD implantation, P-wave amplitude was 1.9 ± 1.6 mV and R-wave was 11.3 ± 5.2 mV. The ventricular pacing threshold was 0.53 ± 0.1 V for a 0.5 ms pulse. Antibiotic therapy was instituted in all patients. A permanent VDD or DDD PM was implanted after 8 ± 2.5 days of temporary VDD pacing. At permanent PM implantation, the mean brain natriuretic peptide level had decreased and sepsis was controlled in all patients. No recurrence of sepsis was observed with a mean follow-up of 15.8 ± 5.3 months.ConclusionTemporary VDD pacing is a safe and effective method to achieve prolonged AV physiological pacing in patients with AV block until infection has been controlled.
The effects of media on pharmaceutically relevant transporters in the human HT-29 adenocarcinoma cell line: Does culture media need to be controlled?
Source
Department of Industrial and Physical Pharmacy, College of Pharmacy, Purdue University, West Lafayette, Indiana 47907.
Abstract
The HT-29 cell line forms a confluent monolayer with tight junctions, but displays different phenotypes when cultured for 21 days in galactose-supplemented media (differentiated) versus glucose-supplemented media (dedifferentiated). This study is aimed at elucidating how media differences might affect selected drug transporter expression and peptide-based substrate transport toward reducing this variability. A vial of HT-29 cells was amplified and cultured over several passages in four different mediums (American Type Culture Collection recommended McCoy's 5A versus Dulbecco's modified Eagle's media containing glucose, galactose, or neither carbohydrate) with normal supplementation. Transporter mRNA expression was characterized at days 5 and 21 postseeding utilizing SABiosciences quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) drug transporter arrays. Transport studies using [H]histidine, [(3) H]glycylsarcosine, [(3) H]valacyclovir, and [(3) H]carnosine were performed to assess the functional effects of oligopeptide transporter expression changes in HT-29 cells grown in each media. qRT-PCR arrays illustrated variable, media-dependent transporter expression between both the initial and differentiated time points. Permeability studies illustrated considerable media-dependent differences in both paracellular and transcellular substrate fluxes. The results demonstrate that these cells exhibit differing monolayer characteristics and genotypic/phenotypic profile properties when cultured under different media. The results suggest a need for standardization of culture methodologies for reducing inter- and intralaboratory variability. © 2011 Wiley Periodicals, Inc.
Copyright © 2011 Wiley Periodicals, Inc.
Functionalized self-assembling peptide nanofiber hydrogel as a scaffold for rabbit nucleus pulposus cells.
Source
Department of Orthopedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, People's Republic of China.
Abstract
In this study, a new functionalized peptide RLN was designed containing the bioactive motif link N, the amino terminalpeptide of link protein. A link N nanofiber scaffold (LN-NS) was self-assembled by mixing peptide solution of RLN and RADA16. The characterization of LN-NS was tested using atomic force microscopy (AFM). The biocompatibility and bioactivity of this nanofiber scaffold for rabbit nucleus pulposus cells (NPCs) were also evaluated. This designer functionalized nanofiber scaffold exhibited little cytotoxicity and promoted NPCs adhesion obviously. In three-dimensional cell culture experiments, confocal reconstructed images testified that the functionalized LN-NS-guided NPCs migration from the surface into the hydrogel considerably, in which the RADA16 scaffold did not. Moreover, the functionalized LN-NS significantly stimulated the biosynthesis of extracelluar matrices (ECM) by NPCs. Our findings demonstrate that the functionalized nanofiber scaffold containing link N had excellent biocompatibility and bioactivity with rabbit NPCs and could be useful in the nucleus pulposus regeneration. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2011.
Copyright © 2011 Wiley Periodicals, Inc.
Exposure of the lysine in the gamma chain dodecapeptide of human fibrinogen is not enhanced by adsorption to poly(ethylene terephthalate) as measured by biotinylation and mass spectrometry.
Source
Department of Biomedical Engineering and Center for Materials Innovation, Washington University, St. Louis, Missouri; Department of Neurology, Hope Center for Neurological Disorders and Alzheimer's Disease Research Center, Washington University School of Medicine, St. Louis, Missouri.
Abstract
Conformational changes in adsorbed fibrinogen may enhance the exposure of platelet adhesive sites that are inaccessible in solution. To test this hypothesis, mass spectrometric methods were developed to quantify chemical modification of lysine residues following adsorption of fibrinogen to biomaterials. The quantitative method used an internal standard consisting of isotope-labeled fibrinogen secreted by human HepG2 cells in culture. Lysine residues in the internal standard were partially reacted with NHS-biotin. For the experimental samples, normal human fibrinogen was adsorbed to poly(ethylene terephthalate) (PET) particles. The adsorbed fibrinogen was reacted with NHS-biotin and then eluted from the particles. Constant amounts of internal standard were added to sample fibrinogen and analyzed by liquid chromatography/tandem mass spectrometry. Biotinylation of the lysine residue in the platelet-adhesive gamma chain dodecapeptide (GCDP) was quantified by comparison with the internal standard. Approximately 80% of the GCDPpeptides were biotinylated when fibrinogen was reacted with NHS-biotin in solution or adsorbed onto PET. These results are generally consistent with previous antibody binding studies and suggest that other regions of fibrinogen may be crucial in promoting platelet adhesion to materials. The results do not directly address but are consistent with the hypothesis that only activated platelets adhere to adsorbed fibrinogen. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2011.
Copyright © 2011 Wiley Periodicals, Inc.
A new paradigm for stem cell therapy: Substance-P as a stem cell-stimulating agent.
Source
Department of Genetic Engineering, Kyung Hee University, Yongin, 146-701, Korea.
Abstract
Bone marrow is a reservoir for hematopoietic stem cells, endothelial precursor cells, and bone marrow stromal cells (also generally called mesenchymal stem cells), whose positive role in tissue repair is highly anticipated. In this report, we introduce a novel function of substance-P (SP), an 11-amino-acid peptide, as an injury-inducible messenger to mobilize bone marrow stem cells to the blood and finally to engage in tissue repair. This new drug may substitute for ex vivo cell culture of therapeutic cells by stimulating cell proliferation in the bone marrow in vivo and mobilizing those therapeutic cells to the patient's own blood stream. Again, the additional role of SP in mitigating inflammation-mediated tissue damage can further rationalize the clinical development of SPpeptide as a stem cell stimulant.
Identification of a novel Brain Derived Neurotrophic Factor (BDNF)-inhibitory factor: Regulation of BDNF by Teneurin C-terminal Associated Peptide(TCAP)-1 in immortalized embryonic mouse hypothalamic cells.
Source
Department of Cell and Systems Biology, University of Toronto, Toronto Ontario, Canada.
Abstract
The teneurins are a family of four large transmembrane proteins that are highly expressed in the central nervous system (CNS) where they have been implicated in development and CNS function. At the tip of the carboxyl terminus of each teneurin lies a 43-amino acid sequence, that when processed, could liberate an amidated 41-residue peptide. We have called this region the teneurin C-terminal associated peptide (TCAP). Picomolar concentrations of the synthetic version of TCAP-1 inhibit stress-induced cocaine reinstatement in rats. Because cocaine-seeking is associated with increased brain derived neurotrophic factor (BDNF) in the brain, we examined whether synthetic mouse TCAP-1 has the potential to regulate BDNF expression in immortalized mouse neurons. Immortalized mouse neurons (N38; mHypoE38) show strong FITC-labeled [K(8)]-TCAP-1 uptake and BDNF labeling in the cytosol. Moreover, FITC-labeled [K(8)]-TCAP-1 bound competitively to membrane fractions. In culture, the labeled TCAP-1 peptide could be detected on cell membranes within 15min and subsequently became internalized in the cytosol and trafficked toward the nucleus. Administration of 10(-8)M unlabeled TCAP-1 to cultures of the N38 cells resulted in a significant decrease of total cell BDNF immunoreactivity over 4h as determined by western blot and ELISA analyses. Real-time PCR, utilizing primers to the various BDNF transcripts showed a significant decline of promoter IIB- and VI-driven transcripts. Taken together, these studies indicated that in vitro, TCAP-1 induces a significant decline in BDNF transcription and protein labeling in embyronic mouse immortalized hypothalamic neurons. Thus, TCAP-1 may act as a novel BDNF inhibitory factor.
Copyright © 2011. Published by Elsevier B.V.
Extinction of Tumor Antigen Expression by SF2/ASF in JCV-Transformed Cells.
Source
Department of Neuroscience, Center for Neurovirology, Temple University School of Medicine, Philadelphia, PA, USA.
Abstract
The human neurotropic polyomavirus JC (JCV) induces a broad range of neural-origin tumors in experimental animals and has been repeatedly detected in several human cancers, most notably neural crest-origin tumors including medulloblastomas and glioblastomas. The oncogenic activity of JCV is attributed to the viral early gene products, large T and small t antigens, as evident by results from in vitro cell culture and in vivo animal studies. Recently, we have shown that alternative splicing factor, SF2/ASF, has the capacity to exert a negative effect on transcription and splicing of JCV genes in glial cells through direct association with a specific DNA motif within the viral promoter region. Here, we demonstrate that SF2/ASF suppresses large T antigen expression in JCV-transformed tumor cell lines, and the expression of SF2/ASF in such tumor cells thereby inhibits the transforming capacity of the viral tumor antigens. Moreover, down-regulation of SF2/ASF in viral-transformed tumor cell lines induces growth and proliferation of the tumor cells. Mapping analysis of the minimal peptide domain of SF2/ASF responsible for JCV promoter silencing and tumor suppressor activity suggests that amino acid residues 76 to 100 of SF2/ASF are functionally sufficient to suppress the growth of the tumor cells. These observations demonstrate a role for SF2/ASF in JCV-mediated cellular transformation and provide a new avenue of research to pathogenic mechanisms of JCV-induced tumors.
Evidence for a new post-translational modification in Staphylococcus aureus: Hydroxymethylation of asparagine and glutamine.
Source
Protein Analysis Facility, University of Lausanne, 1015 Lausanne, Switzerland.
Abstract
Staphylococcus aureus is an opportunistic pathogen whose infectious capacity depends on surface proteins, which enable bacteria to colonize and invade host tissues and cells. We analyzed "trypsin-shaved" surface proteins of S. aureus cultures by high resolution LC-MS/MS at different growth stages and culture conditions. Some modified peptideswere identified, with a mass shift corresponding to the addition of a CH(2)O group (+30.0106u). We present evidence that this shift corresponds to a hyxdroxymethylation of asparagine and glutamine residues. This known but poorly documented post-translational modification was only found in a few proteins of S. aureus grown under specific conditions. This specificity seemed to exclude the hypothesis of an artifact due to sample preparation. Altogether hydroxymethylation was observed in 35 peptides from 15 proteins in our dataset, which corresponded to 41 modified sites, 35 of them being univocally localized. While no function can currently be assigned to this post-translational modification, we hypothesize that it could be linked to modulation of virulence factors, since it was mostly found on some surface proteins of S. aureus.
Copyright © 2011. Published by Elsevier B.V.
Statins in Unconventional Secretion of Insulin-Degrading Enzyme and Degradation of the Amyloid-β Peptide.
Source
Department of Neurology, University of Bonn, Bonn, Germany.
Abstract
Population-based studies demonstrated that statins might decrease the risk of developing Alzheimer's disease (AD). Statins inhibit the 3-hydroxy-3-methyl-glutaryl-coenzyme-A reductase and thereby de novo synthesis of cholesterol. Cellculture and animal studies indicated that cholesterol affects the proteolytic processing of the amyloid precursor protein and the generation of amyloid-β (Aβ). Recently, we have demonstrated that statins can also stimulate the degradation of Aβ. The statin-induced clearance of Aβ could be attributed to increased release of the insulin-degrading enzyme (IDE) via an exosome-related unconventional secretory pathway. Interestingly, this statin-induced secretion of exosome-associated IDE was independent of cellular cholesterol concentrations, but rather caused by impairment of isoprenoid biosynthesis and protein prenylation. We further identified a new hexapeptide sequence in the C-terminal region of IDE, named the SlyX motif that is critically involved in IDE secretion. Taken these findings together, the increased clearance of Aβ by stimulated secretion of IDE might contribute to the protective effects of statins against AD.
Copyright © 2011 S. Karger AG, Basel.
Prolonged expression of MHC class I - peptide expression in bone marrow derived retrovirus transfected matured dendritic cells by continuous centrifugation in the presence of IL-4.
Source
Department of Pharmacology, Faculty of Medicine, University of Ruhuna, Sri Lanka.
Abstract
Background & objectives: Dendritic cells (DCs) are potent antigen presenting cells which proceed from immature to a mature stage during their differentiation. There are several methods of obtaining long lasting mature antigen expressing DCs and different methods show different levels of antigen expressions. We investigated bone marrow derived DCs for the degree of maturation and genetically engineered antigen presentation in the presence of interleukin-4 (IL-4) as a maturity enhancer. Methods: DCs and transfected retrovirus were cultured together in the presence of granulocyte-macrophage colony stimulating factor (GMCSF)-IL4, GMCSF +IL4, lipopolysaccharide (LPS). B 7.1, B7.2 and CD11c were measured by the degree of immune fluorescence using enhanced green fluorescent protein (EGFP) shuttled retrovirus transfected antigen. Degree of MHC class I molecule with antigen presentation of antigen was also evaluated by fluorescence activated cell sorting. The antigen presenting capacity of transfected DCs was investigated. Bone marrow DCs were generated in the presence of GMCSF and IL-4 in vitro. Dividing bone marrow cells were infected with EGFP shuttled retrovirus expressing SSP2 by prolonged centrifugation for three consecutive days from day 5, 6 and 7 and continued to culture in the presence of GMSCF and IL-4 until day 8. Results: IL-4 as a cytokine increased the maturation of retrovirus transfected DCs by high expression of B 7-1 and B 7-2. Also, IL-4 induced DC enhanced by the prolonged centrifugation and it was shown by increased antigen presentation of these dendric cells as antigen presenting cell (APC). Cytolytic effects were significantly higher in cytotoxic T cell response (CTLs) mixed with transfected DCs than CTLs mixed with pulsed DCs. Interpretation & conclusions: There was an enhanced antigen presentation by prolonged expression of antigen loaded MHC class I receptors in DCs in the presence of IL-4 by prolonged centrifugation.
A single conformational transglutaminase 2 epitope contributed by three domains is critical for celiac antibody binding and effects.
Source
Department of Biochemistry and Molecular Biology, Medical and Health Science Center, University of Debrecen, Nagyerdei krt 98, Debrecen H-4032, Hungary.
Abstract
The multifunctional, protein cross-linking transglutaminase 2 (TG2) is the main autoantigen in celiac disease, an autoimmune disorder with defined etiology. Glutamine-rich gliadin peptides from ingested cereals, after their deamidation by TG2, induce T-lymphocyte activation accompanied by autoantibody production against TG2 in 1-2% of the population. The pathogenic role and exact binding properties of these antibodies to TG2 are still unclear. Here we show that antibodies from different celiac patients target the same conformational TG2 epitope formed by spatially close amino acids of adjacent domains. Glu153 and 154 on the first alpha-helix of the core domain and Arg19 on first alpha-helix of the N-terminal domain determine the celiac epitope that is accessible both in the closed and open conformation of TG2 and dependent on the relative position of these helices. Met659 on the C-terminal domain also can cooperate in antibody binding. This composite epitope is disease-specific, recognized by antibodies derived from celiac tissues and associated with biological effects when passively transferred from celiac mothers into their newborns. These findings suggest that celiac antibodies are produced in a surface-specific way for which certain homology of the central glutamic acid residues of the TG2 epitope with deamidated gliadin peptides could be a structural basis. Monoclonal mouse antibodies with partially overlapping epitope specificity released celiac antibodies from patient tissues and antagonized their harmful effects in cell culture experiments. Such antibodies or similar specific competitors will be useful in further functional studies and in exploring whether interference with celiac antibody actions leads to therapeutic benefits.
PPARα activation inhibits endothelin-1-induced cardiomyocyte hypertrophy by prevention of NFATc4 binding to GATA-4.
Source
Department of Pharmacology and Toxicology, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, China.
Abstract
Peroxisome proliferator-activated receptor alpha (PPARα) has been implicated in the pathogenesis of cardiac hypertrophy, although its mechanism of action remains largely unknown. To determine the effect of PPARα activation on endothelin-1 (ET-1)-induced cardiomyocyte hypertrophy and explore its molecular mechanisms, we evaluated the interaction of PPARα with nuclear factor of activated T-cells c4 (NFATc4) in nuclei of cardiomyocytes from neonatal rats in primary culture. In ET-1-stimulated cardiomyocytes, data from electrophoretic mobility-shift assays (EMSA) and co-immunoprecipitation (co-IP) revealed that fenofibrate (Fen), a PPARα activator, in a concentration-dependent manner, enhanced the association of NFATc4 with PPARα and decreased its interaction with GATA-4, in promoter complexes involved in activation of the rat brain natriuretic peptide (rBNP) gene. Effects of PPARα overexpression were similar to those of its activation by Fen. PPARα depletion by small interfering RNA abolished inhibitory effects of Fen on NFATc4 binding to GATA-4 and the rBNP DNA. Quantitative RT-PCR and confocal microscopy confirmed inhibitory effects of PPARα activation on elevation of rBNP mRNA levels and ET-1-induced cardiomyocyte hypertrophy. Our results suggest that activated PPARα can compete with GATA-4 binding to NFATc4, thereby decreasing transactivation of NFATc4, and interfering with ET-1 induced cardiomyocyte hypertrophy.
Copyright © 2011. Published by Elsevier Inc.
Identification of extracellular siderophores and a related peptide from the endophytic fungus Epichloë festucae in culture and endophyte-infected Lolium perenne.
Source
Lipid Profiling and Signaling Group, MRC HNR, Elsie Widdowson Laboratory, Cambridge, UK.
Abstract
A number of genes encoding non-ribosomal peptide synthetases (NRPSs) have been identified in fungi of Epichloë/Neotyphodium species, endophytes of Pooid grasses, including sidN, putatively encoding a ferrichrome siderophore-synthesizing NRPS. Targeted gene replacement and complementation of sidN in Epichloë festucae has established that extracellular siderophore epichloënin A is the major product of the SidN enzyme complex (Johnson et al., 2007a). We report here high resolution mass spectrometric fragmentation experiments and NMR analysis of an isolated fraction establishing that epichloënin A is a siderophore of the ferrichrome family, comprising a cyclic sequence of four glycines, a glutamine and three N(δ)-trans-anhydromevalonyl-N(δ)-hydroxyornithine (AMHO) moieties. Epichloënin A is unusual among ferrichrome siderophores in comprising an octapeptide rather than hexapeptide sequence, and in incorporating a glutamine residue. During this investigation we have established that desferrichrome siderophores with pendant trans-AMHO groups can be distinguished from those with pendant cis-AMHO groups by the characteristic neutral loss of an hydroxyornithine moiety in the MS/MS spectrum. A minor component, epichloënin B, has been characterized as the triglycine variant by mass spectrometry. A peptide characterized by mass spectrometry as the putative deoxygenation product, epichloëamide has been detected together with ferriepichloënin A in guttation fluid from ryegrass (Lolium perenne) plants infected with wild-type E. festucae, but not in plants infected with the ΔsidN mutant strain, and also detected at trace levels in wild-type E. festucae fungal culture.
Copyright © 2011 Elsevier Ltd. All rights reserved.
Generation of glucose-responsive, insulin-producing cells from human umbilical cord blood-derived mesenchymal stem cells.
Abstract
We sought to assess the potential of human cord blood-derived mesenchymal stem cells (CB-MSCs) to derive insulin-producing, glucose-responsive cells. We show here that differentiation protocols based on step-wise culture conditions initially described for human embryonic stem cells (hESCs) lead to differentiation of cord blood-derived precursors towards a pancreatic endocrine phenotype, as assessed by marker expression and in vitro glucose-regulated insulin secretion. Transplantation of these cells in immune-deficient animals shows human c-peptide production in response to a glucose challenge. This data suggests that human cord blood may be a promising source for regenerative medicine approaches for the treatment of diabetes mellitus.
PEGylation Potentiates the Effectiveness of an Antagonistic Peptide That Targets the EphB4 Receptor with Nanomolar Affinity.
Source
Sanford-Burnham Medical Research Institute, La Jolla, California, United States of America.
Abstract
The EphB4 receptor tyrosine kinase together with its preferred ligand, ephrin-B2, regulates a variety of physiological and pathological processes, including tumor progression, pathological forms of angiogenesis, cardiomyocyte differentiation and bone remodeling. We previously reported the identification of TNYL-RAW, a 15 amino acid-long peptide that binds to the ephrin-binding pocked of EphB4 with low nanomolar affinity and inhibits ephrin-B2 binding. Although ephrin-B2 interacts promiscuously with all the EphB receptors, the TNYL-RAW peptide is remarkably selective and only binds to EphB4. Therefore, this peptide is a useful tool for studying the biological functions of EphB4 and for imaging EphB4-expressing tumors. Furthermore, TNYL-RAW could be useful for treating pathologies involving EphB4-ephrin-B2 interaction. However, the peptide has a very short half-life in cell culture and in the mouse blood circulation due to proteolytic degradation and clearance by the kidneys and reticuloendothelial system. To overcome these limitations, we have modified TNYL-RAW by fusion with the Fc portion of human IgG1, complexation with streptavidin or covalent coupling to a 40 KDa branched polyethylene glycol (PEG) polymer. These modified forms of TNYL-RAW all have greatly increased stability in cell culture, while retaining high binding affinity for EphB4. Furthermore, PEGylation most effectively increases peptide half-life in vivo. Consistent with increased stability, submicromolar concentrations of PEGylated TNYL-RAW effectively impair EphB4 activation by ephrin-B2 in cultured B16 melanoma cells as well as capillary-like tube formation and capillary sprouting in co-cultures of endothelial and epicardial mesothelial cells. Therefore, PEGylated TNYL-RAW may be useful for inhibiting pathological forms of angiogenesis through a novel mechanism involving disruption of EphB4-ephrin-B2 interactions between endothelial cells and supporting perivascular mesenchymal cells. Furthermore, the PEGylated peptide is suitable for other cell culture and in vivo applications requiring prolonged EphB4 receptor targeting.
Differentiation of mesenchymal stem cells derived from pancreatic islets and bone marrow into islet-like cell phenotype.
Source
Molecular Biotechnology Centre (MBC), University of Turin, Turin, Italy.
Abstract
BACKGROUND:
Regarding regenerative medicine for diabetes, accessible sources of Mesenchymal Stem Cells (MSCs) for induction of insular beta cell differentiation may be as important as mastering the differentiation process itself.
METHODOLOGY/PRINCIPAL FINDINGS:
In the present work, stem cells from pancreatic islets (human islet-mesenchymal stem cells, HI-MSCs) and from human bone marrow (bone marrow mesenchymal stem cells, BM-MSCs) were cultured in custom-made serum-free medium, using suitable conditions in order to induce differentiation into Islet-like Cells (ILCs). HI-MSCs and BM-MSCs were positive for the MSC markers CD105, CD73, CD90, CD29. Following this induction, HI-MSC and BM-MSC formed evident islet-like structures in the culture flasks. To investigate functional modifications after induction to ILCs, ultrastructural analysis and immunofluorescence were performed. PDX1 (pancreatic duodenal homeobox gene-1), insulin, C peptide and Glut-2 were detected in HI-ILCs whereas BM-ILCs only expressed Glut-2 and insulin. Insulin was also detected in the culture medium following glucose stimulation, confirming an initial differentiation that resulted in glucose-sensitive endocrine secretion. In order to identify proteins that were modified following differentiation from basal MSC (HI-MSCs and BM-MSCs) to their HI-ILCs and BM-ILCs counterparts, proteomic analysis was performed. Three new proteins (APOA1, ATL2 and SODM) were present in both ILC types, while other detected proteins were verified to be unique to the single individual differentiated cells lines. Hierarchical analysis underscored the limited similarities between HI-MSCs and BM-MSCs after induction of differentiation, and the persistence of relevant differences related to cells of different origin.
CONCLUSIONS/SIGNIFICANCE:
Proteomic analysis highlighted differences in the MSCs according to site of origin, reflecting spontaneous differentiation and commitment. A more detailed understanding of protein assets may provide insights required to master the differentiation process of HI-MSCs to functional beta cells based only upon cultureconditioning. These findings may open new strategies for the clinical use of BM-MSCs in diabetes.
A novel neurotrophic drug for cognitive enhancement and Alzheimer's disease.
Source
Cellular Neurobiology Laboratory, The Salk Institute for Biological Studies, La Jolla, California, United States of America.
Abstract
Currently, the major drug discovery paradigm for neurodegenerative diseases is based upon high affinity ligands for single disease-specific targets. For Alzheimer's disease (AD), the focus is the amyloid beta peptide (Aß) that mediates familial Alzheimer's disease pathology. However, given that age is the greatest risk factor for AD, we explored an alternative drug discovery scheme that is based upon efficacy in multiple cell culture models of age-associated pathologies rather than exclusively amyloid metabolism. Using this approach, we identified an exceptionally potent, orally active, neurotrophic molecule that facilitates memory in normal rodents, and prevents the loss of synaptic proteins and cognitive decline in a transgenic AD mouse model.
Short-Chain Fatty Acids Stimulate Glucagon-Like Peptide-1 Secretion via the G-Protein-Coupled Receptor FFAR2.
Source
Cambridge Institute for Medical Research, Wellcome Trust/Medical Research Council Building, Addenbrooke's Hospital, Cambridge, U.K.
Abstract
Interest in how the gut microbiome can influence the metabolic state of the host has recently heightened. One postulated link is bacterial fermentation of "indigestible" prebiotics to short-chain fatty acids (SCFAs), which in turn modulate the release of gut hormones controlling insulin release and appetite. We show here that SCFAs trigger secretion of the incretin hormone glucagon-like peptide (GLP)-1 from mixed colonic cultures in vitro. Quantitative PCR revealed enriched expression of the SCFA receptors ffar2 (grp43) and ffar3 (gpr41) in GLP-1-secreting L cells, and consistent with the reported coupling of GPR43 to Gq signaling pathways, SCFAs raised cytosolic Ca(2+) in L cells in primary culture. Mice lacking ffar2 or ffar3 exhibited reduced SCFA-triggered GLP-1 secretion in vitro and in vivo and a parallel impairment of glucose tolerance. These results highlight SCFAs and their receptors as potential targets for the treatment of diabetes.
Recombinant expression, purification, and antimicrobial activity of a novel hybrid antimicrobial peptide LFT33.
Source
College of Animal Science and Technology, Northeast Agricultural University, 59 Mucai Street, Xiangfang District, Harbin, 150030, China, fengxingjun2008@163.com.
Abstract
With great therapeutic potential against antibiotic-resistant bacteria, viruses, and even parasites, antimicrobial peptides(AMPs) have received increased interest as pharmaceutical agents in recent years. It is a worthy yet challenging work to carry out the implement and improvement of AMPs production using bioengineering techniques. In the present study, a novel hybrid peptide LFT33 was designed derived from LfcinB and thanatin. The cDNA fragment encoding LFT33 with preferred codons of Escherichia coli was chemically synthesized and ligated into the vector pET32a(+) to express the LFT33 fusion protein. The fusion protein was successfully expressed in soluble form in E. coli induced under optimized conditions. After purification by affinity chromatography, the fusion protein was cleaved successfully by enterokinase and released the peptide LFT33. About 0.5 mg of the recombinant LFT33 was obtained by reversed-phase high performance liquid chromatography from 1 l of culture medium. Mass spectrometry analysis of the purified recombinant LFT33 demonstrated that the molecular weight perfectly matched the calculated mass (4,195 Da). The recombinantpeptide LFT33 caused an increase in antimicrobial activity (IC(50) = 16-64 μg/ml) against given strains and did not show hemolytic activity for human erythrocytes. The results indicated that the hybrid peptide LFT33 could serve as a promising candidate for pharmaceutical agents.
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