1.
Characterization of autoreactive and bystander IL-17+ T cells induced in immunized C57BL/6 mice.
Source
Doheny Eye Institute, Keck School of Medicine of the University of Southern California, Los Angeles, CA90033, United States.
Abstract
Purpose.To characterize antigen-specific and bystander IL-17(+) T cells induced in immunized mice.Methods.C57BL/6 (B6) mice were immunized with the uveitogenic peptide IRBP(1-20) in either IFA or CFA. In vivo primed T cells were stimulated with syngeneic APCs with or without the immunizing peptide, under polarizing conditions. Activated T cells were analyzed for expression and production of IL-17.Results.B6 mice immunized with the uveitogenic peptide IRBP(1-20) generate two types of IL-17(+) T cell, one specific for the immunizing autoantigen (IRBP-Th17) and a much more abundant type (bystander-Th17) that is not reactive with the immunizing antigen. The bystander-Th17 can be demonstrated when in vivo primed T cells are cultured in Th17-polarizing conditions in the absence of antigen stimulation. Increased expansion of both types of Th17 cells is seen in mice immunized with IRBP(1-20)/CFA, but not with IRBP(1-20)/IFA. Both T cell types produce IL-17, IL-22, and IFN-?, but only bystander Th17 cells produce IL-10. Addition to culture medium with IL-6 and TGF-β1 caused more activation of bystander-Th17 T cells than IRBP-Th17 cells. When adoptively transferred into syngeneic naïve mice, the bystander-Th17 cells neutralize the pathogenic activity of the IRBP-Th17 cells.Conclusions.A procedure commonly used to induce autoimmune disease promotes two functionally antagonistic types of IL-17(+) T cells and that the pathogenic type is restricted to the population that specifically responds to the immunizing autoantigen. Molecular components of the CFA, rather than the immunizingpeptide, promote the generation of both types of IL-17(+) T cells.
- PMID:
- 22247477
- [PubMed - as supplied by publisher]
Statistical optimization of microalgae Pavlova lutheri cultivation conditions and its fermentation conditions by yeast, Candida rugopelliculosa.
Source
Marine Bioprocess Research Center, Pukyong National University, Busan 608-737, Republic of Korea.
Abstract
In this study, sequential strategy based design was applied to optimize the microalgae, Pavlova lutheri mass cultureconditions and fermentation conditions of the cultured algae by proteolytic yeast Candidia rugopelliculosa to obtain small peptide chains. This optimization of culture and fermentation conditions by response surface methodology (RSM) finally leads to effective purification of a bioactive peptide MPGPLSPL (793.01Da) with hydroxyl radical scavenging activity. Collectively, these results indicated that microalgae P. lutheri can enhance the hydroxyl radical inhibiting effect through protein hydrolysis process under RSM optimal condition.
Copyright © 2011 Elsevier Ltd. All rights reserved.
- PMID:
- 22244956
- [PubMed - as supplied by publisher]
Urotensin II and urocortin trigger the expression of myostatin, a negative regulator of cardiac growth, in cardiomyocytes.
Source
Pôle de recherche en Endocrinologie, Diabète et Nutrition, Institut de Recherche Expérimentale et Clinique, Cliniques Universitaires St-Luc and Université Catholique de Louvain, Brussels, Belgium.
Abstract
Urotensin II (UII) and urocortin (UCN) are potent contributors to the physiopathology of heart failure. Our study investigated the effects of UII and UCN on the expression of myostatin (Mstn) in primary culture of adult cardiomyocytes. Adult rat cardiomyocytes were stimulated for 48h with UII and UCN. Cell size and protein content were determined. Mstn gene expression was determined by real time quantitative polymerase chain reaction. Treatment with UII and UCN stimulates hypertrophy of adult cardiomyocytes. This effect was associated with a twofold increase of Mstn gene expression. We have established for the first time that the two hypertrophic peptides UII and UCN stimulate the expression of Mstn.
Copyright © 2012. Published by Elsevier Inc.
- PMID:
- 22244812
- [PubMed - as supplied by publisher]
Nanomolar cellular antisense activity of peptide nucleic acid (PNA) cholic acid ("umbrella") and cholesterol conjugates delivered by cationic lipids.
Abstract
Limited cellular uptake and low bioavailability of peptide nucleic acids (PNAs) have restricted widespread use of PNAs as antisense/antigene agents for cells in culture and not least for in vivo applications. We now report the synthesis and cellular antisense activity in cultured HeLa pLuc705 cells of cholesterol and cholic acid ("umbrella") derivatives of splice correction antisense PNA oligomers. While the conjugates alone were practically inactive up to 1 μM, their activity was dramatically improved when delivered by a cationic lipid transfection agent (LipofectAMINE2000). In particular, PNAs, conjugated to cholesterol through an ester hemisuccinate linker or to cholic acid exhibited low nanomolar activity (EC50 ~ 25 nM). Excellent sequence specificity was retained as mismatch PNA conjugates did not show any significant antisense activity. Furthermore, we show that increasing the transfection volume improved transfection efficiency suggesting that accumulation (condensation) of the PNA/lipid complex on the cellular surface is part of the uptake mechanism. These results provide a novel, simple method for very efficient cellular delivery of PNA oligomers, especially using PNA-cholic acid conjugates which, in contrast to PNA-cholesterol conjugates exhibit sufficient water solubility. The results also question the generality of using cholic acid "umbrella" derivatives as a delivery modality for antisense oligomers.
- PMID:
- 22243634
- [PubMed - as supplied by publisher]
Extracellular Overexpression of Recombinant Thermobifida fusca cutinase by Alpha-hemolysin Secretion System in E. coli BL21(DE3).
Abstract
ABSTRACT:
BACKGROUND:
Extracellular expression of proteins has an absolute advantage in a large-scale industrial production. In our previous study, Thermobifida fusca cutinase, an enzyme mainly utilized in textile industry, was expressed via type II secretory system in Escherichia coli BL21(DE3), and it was found that parts of the expressed protein was accumulated in the periplasmic space. Due to the fact that alpha-hemolysin secretion system can export target proteins directly from cytoplasm across both cell membrane of E. coli to the culture medium, thus in the present study we investigated the expression of cutinase using this alpha-hemolysin secretion system.
RESULTS:
T. fusca cutinase was fused with the specific signal peptide of alpha-hemolysin scretion system and expressed in E. coli BL21(DE3). In addition, HlyB and HlyD, strain-specific translocation components of alpha-hemolysin secretion system, were coexpressed to facilitate the enzyme expression. The cultivation of this engineered cell showed that cutinase activity in the culture medium reached 334 U/ml, which is 2.5 times that from type II secretion pathway under the same culture condition. The recombinant cutinase was further purified. Biochemical characterization of purified enzyme, which had an alpha-hemolysin secretion pathway signal peptide attached, had substrate specificity, pH and temperature profile, as well as application capability in bioscouring similar to that of wild-type cutinase.
CONCLUSIONS:
In the present study, T. fusca cutinase was successfully secreted to the culture media by alpha-hemolysin secretion system. This is the first report of cutinase being efficiently secreted by this pathway. Due to the limited cases of successful expression of industrial enzyme by E. coli alpha-hemolysin secretion system, our study further explored the utilization of this pathway in industrial enzymes.
Co-culture of osteocytes and neurons on a unique patterned surface.
Source
University of Delaware, Department of Biological Science, 118 Wolf Hall, and Department of Chemistry and Biochemistry, 104 Brown Lab, Newark, Delaware 19716University of Delaware, Department of Physical Therapy, 301 McKinly Lab and Program in Biomechanics and Movement Science, Newark, Delaware 19716Rice University, Department of Biochemistry and Cell Biology, W100 George R. Brown Hall, Houston, Texas 77251University of Delaware, Department of Biological Science, 118 Wolf Hall, Newark, Delaware 19716.
Abstract
Neural and skeletal communication is essential for the maintenance of bone mass and transmission of pain, yet the mechanism(s) of signal transduction between these tissues is unknown. The authors established a novel system to co-culture murine long bone osteocyte-like cells (MLO-Y4) and primary murine dorsal root ganglia (DRG) neurons. Assessment of morphology and maturation marker expression on perlecan domain IV peptide (PlnDIV) and collagen type-1 (Col1) demonstrated that PlnDIV was an optimal matrix for MLO-Y4 culture. A novel matrix-specificity competition assay was developed to expose these cells to several extracellular matrix proteins such as PlnDIV, Col1, and laminin (Ln). The competition assay showed that approximately 70% of MLO-Y4 cells preferred either PlnDIV or Col1 to Ln. To co-culture MLO-Y4 and DRG, we developed patterned surfaces using micro-contact printing to create 40 μm × 1 cm alternating stripes of PlnDIV and Ln or PlnDIV and Col1. Co-culture on PlnDIV/Ln surfaces demonstrated that these matrix molecules provided unique cues for each cell type, with MLO-Y4 preferentially attaching to the PlnDIV lanes and DRG neurons to the Ln lanes. Approximately 80% of DRG were localized to Ln. Cellular processes from MLO-Y4 were closely associated with axonal extensions of DRG neurons. Approximately 57% of neuronal processes were in close proximity to nearby MLO-Y4 cells at the PlnDIV-Ln interface. The surfaces in this new assay provided a unique model system with which to study the communication between osteocyte-like cells and neurons in an in vitro environment.
- PMID:
- 22239813
- [PubMed - as supplied by publisher]
Minimal essential length of Clostridium botulinum C3 peptides to enhance neuronal regenerative growth and connectivity in a non-enzymatic mode.
Source
Center for Anatomy, Functional Cell Biology, Charité-Universitätsmedizin Berlin, Germany Department of Morphology & BIOMED Institute, Hasselt University, Belgium Université Pierre et Marie Curie, Institut de la Vision, Paris, France) Institute of Toxicology, Hannover Medical School (MHH), Germany.
Abstract
C3 ADP-ribosyltransferase is a valuable tool to study Rho-dependent cellular processes. In the current study we investigated the impact of enzyme-deficient peptides derived from Clostridium botulinum C3 transferase in the context of neuronal process elongation and branching, synaptic connectivity, and putative beneficial effects on functional outcome following traumatic injury to the CNS. By screening a range of peptidic fragments we identified three short peptides from C3bot that promoted axon and dendrite outgrowth in cultivated hippocampal neurons. Furthermore, one of these fragments, a 26-amino acid peptide covering the residues 156-181 enhanced synaptic connectivity in primary hippocampal culture. This peptide was also effective to foster axon outgrowth and re-innervation in organotypical brain slice culture. To evaluate the potential of the 26mer to foster repair mechanisms after CNS injury we applied this peptideto mice subjected to spinal cord injury by either compression impact or hemisection. A single local administration at the site of the lesion improved locomotor recovery. In addition, histological analysis revealed an increased serotonergic input to lumbar motoneurons in treated compared to control mice. Pull-down assays showed that lesion-induced up-regulation of RhoA activity within the spinal cord was largely blocked by C3bot peptides despite the lack of enzymatic activity. © 2012 The Authors Journal of Neurochemistry© 2012 International Society for Neurochemistry.
© 2012 The Authors. Journal of Neurochemistry © 2012 International Society for Neurochemistry.
Peptidergic Regulation of Thymocyte Differentiation, Proliferation, and Apoptosis during Aging of the Thymus.
Source
St. Petersburg Institute of Bioregulation and Gerontology, North Western Division of the Russian Academy of Medical Sciences, Russia. miayy@yandex.ru.
Abstract
The effects of T-31, AB-17, and AB-9 peptides on old (passage 8) thymocyte culture were studied. Only AB-9 peptideexhibited a complex geroprotective effect on thymocytes during their aging. Peptide AB-9 stimulated proliferative activity and differentiation of thymocytes and inhibited their apoptosis.
- PMID:
- 22238759
- [PubMed - in process]
In Vitro Study of Neuroprotective Properties of GK-2, a New Original Nerve Growth Factor Mimetic.
Source
V. V. Zakusov Institute of Pharmacology, Russian Academy of Medical Sciences, Moscow, Russia. niipharm@mail.ru.
Abstract
New nerve growth factor (NGF) mimetic GK-2, a substituted dimeric dipeptide, in a concentration of up to 10(-9)M produced a protective effect on the culture of immortalized mouse hippocampal neurons (line HT-22) after addition of H(2)O(2)and glutamate. GK-2 in a concentration of 10(-8)M protected rat PC-12 pheochromocytoma cells from the neurotoxin MPTP. The neuroprotective effect of this peptide on the model of oxidative stress was also observed in the primary culture of embryonic rat hippocampal neurons.
- PMID:
- 22235396
- [PubMed - in process]
Amyloid-β oligomers induce differential gene expression in adult human brain slices.
Source
Federal University of Rio de Janeiro, Brazil;
Abstract
Cognitive decline in Alzheimers disease (AD) is increasingly attributed to the neuronal impact of soluble oligomers of the amyloid-β peptide (AβOs). Current knowledge on the molecular and cellular mechanisms underlying the toxicity of AβOs stems largely from rodent-derived cell/tissue culture experiments or from transgenic models of AD, which do not necessarily recapitulate the complexity of the human disease. Here, we used DNA microarray and RT-PCR to investigate changes in transcription in adult human cortical slices exposed to sub-lethal doses of AβOs. Results revealed a set of 27 genes that showed consistent differential expression upon exposure of slices from three different donors to AβOs. Functional classification of differentially expressed genes revealed that AβOs impact pathways important for neuronal physiology and known to be dysregulated in AD, including vesicle trafficking, cell adhesion, actin cytoskeleton dynamics and insulin signaling. Most genes (70%) were down-regulated by AβO treatment, suggesting a predominantly inhibitory effect on the corresponding pathways. Significantly, AβOs induced downregulation of synaptophysin, a presynaptic vesicle membrane protein, suggesting a mechanism by which oligomers cause synapse failure. Results provide insight into early mechanisms of pathogenesis of AD, and suggest that the neuronal pathways affected by AβOs may be targets for development of novel diagnostic or therapeutic approaches.
Potential interaction of brain natriuretic peptide with hyperadiponectinemia in preeclampsia.
Source
1Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences.
Abstract
Adiponectin was recently reported to have roles in the pathophysiology of preeclampsia. Moreover, elevation of adiponectin and brain natriuretic peptide (BNP) has been observed in preeclampsia. We examined the possible links between adiponectin and BNP in the pathophysiology of preeclampsia. We performed a cross-sectional study in 56 preeclampsia patients and 56 controls matched for gestational age and body mass index. The BNP, leptin and adiponectin levels were measured by ELISA, and their mRNA expressions were evaluated in omental adipose tissue by real-time PCR. The effects of BNP on adiponectin and leptin mRNA expression and secretion were investigated in primary cultures of adipocytes from obese and normal weight women. The BNP, adiponectin and leptin levels were significantly higher in preeclampsia patients compared with controls. The adiponectin level was significantly increased in normal weight preeclampsia patients compared with overweight preeclampsia patients. Adiponectin mRNA expression was significantly increased in adipose tissues of preeclampsia patients compared with controls, and was also significantly increased in normal weight preeclampsia patients compared with overweight preeclampsia patients, while leptin was not. BNP and adiponectin showed significant positive correlations in both normal weight and overweight preeclampsia patients. BNP had a significantly weaker effect on adiponectin in overweight preeclampsia patients compared with normal weight preeclampsia patients. Moreover, BNP had a weaker effect on adiponectin production in adipocytes from overweight women compared with adipocytes from normal weight women using primary culture of human adipocytes. These data suggested that BNP may play a role in hyperadiponectinemia of preeclampsia patients. The weaker effect of BNP on adiponectin production may participate in the pathophysiology of overweight preeclampsia patients.
Evaluation of the peptide nucleic acid fluorescence in situ hybridisation technology for yeast identification directly from positive blood cultures: an Italian experience.
Source
Microbiology Institute, AO "Ospedale San Carlo Borromeo", Milano, Italy Microbiology Laboratory, AOU "Maggiore della Carità", Novara, Italy Microbiology Laboratory, AO "Ospedale Domenico Cotugno", Napoli, Italy Biochemical and Microbiology Laboratory, PO "Ospedale Santo Spirito", Pescara, Italy Microbiology and Virology Laboratory, AO "Ospedale Niguarda - Ca' Granda", Milano, Italy Microbiology and Virology Laboratory, AO "Ospedali Riuniti di Ancona", Ancona, Italy Microbiology Institute, University of Sassari, Sassari, Italy.
Abstract
Summary Fungaemia is an increasing nosocomial pathology. The 'gold standard' for detection of fungaemia is bloodculture, but it is time-consuming and its sensitivity for early detection is low. On the other hand, yeasts present different antifungal sensitivity patterns to be quickly detected to allow an effective treatment. The aim of this study was to evaluate the diagnostic performances of PNA-FISH to directly identify yeasts from blood cultures and to compare results with those obtained by culture. A total of 176 blood cultures positive for yeasts at direct Gram stain and 24 negative blood cultures as control collected from 15 Italian hospitals, included in a network coordinated by the Medical Mycology Committee, Italian Society of Clinical Microbiology (AMCLI), were examined both by culture and PNA-FISH technology. Sensitivity of the PNA-FISH technique evaluated for five Candida species was 99.3% and specificity, 100%. Distinguishing which yeast is implicated in fungaemia and whether the infection is caused by multiple species are important for the selection of antifungal therapy. The PNA-FISH technique is a very useful approach because the test discriminates between groups of Candida species with different susceptibility pattern, particularly against azoles and echinocandins, with only a 90-minute turn-around time after the Gram-stain reading.
© 2012 Blackwell Verlag GmbH.
- PMID:
- 22233292
- [PubMed - as supplied by publisher]
Two types of assays for detecting frog sperm chemoattraction.
Source
Department of Animal Sciences, University of Illinois, Urbana-Champaign.
Abstract
Sperm chemoattraction in invertebrates can be sufficiently robust that one can place a pipette containing the attractivepeptide into a sperm suspension and microscopically visualize sperm accumulation around the pipette(1). Sperm chemoattraction in vertebrates such as frogs, rodents and humans is more difficult to detect and requires quantitative assays. Such assays are of two major types - assays that quantitate sperm movement to a source of chemoattractant, so-called sperm accumulation assays, and assays that actually track the swimming trajectories of individual sperm. Sperm accumulation assays are relatively rapid allowing tens or hundreds of assays to be done in a single day, thereby allowing dose response curves and time courses to be carried out relatively rapidly. These types of assays have been used extensively to characterize many well established chemoattraction systems - for example, neutrophil chemotaxis to bacterial peptides and sperm chemotaxis to follicular fluid. Sperm tracking assays can be more labor intensive but offer additional data on how chemoattractancts actually alter the swimming paths that sperm take. This type of assay is needed to demonstrate the orientation of sperm movement relative to the chemoattrractant gradient axis and to visualize characteristic turns or changes in orientation that bring the sperm closer to the egg. Here we describe methods used for each of these two types of assays. The sperm accumulation assay utilized is called a "two-chamber" assay. Amphibian sperm are placed in a tissue culture plate insert with a polycarbonate filter floor having 12 μm diameter pores. Inserts with sperm are placed into tissue culture plate wells containing buffer and a chemoatttractant carefully pipetted into the bottom well where the floor meets the wall (see Fig. 1). After incubation, the top insert containing the sperm reservoir is carefully removed, and sperm in the bottom chamber that have passed through the membrane are removed, pelleted and then counted by hemocytometer or flow cytometer. The sperm tracking assay utilizes a Zigmond chamber originally developed for observing neutrophil chemotaxis and modified for observation of sperm by Giojalas and coworkers(2,3). The chamber consists of a thick glass slide into which two vertical troughs have been machined. These are separated by a 1 mm wide observation platform. After application of a cover glass, sperm are loaded into one trough, the chemoattractant agent into the other and movement of individual sperm visualized by video microscopy. Video footage is then analyzed using software to identify two-dimensional cell movements in the x-y plane as a function of time (xyt data sets) that form the trajectory of each sperm.
The ghrelin gene products and exendin-4 promote survival of human pancreatic islet endothelial cells in hyperglycaemic conditions, through phosphoinositide 3-kinase/Akt, extracellular signal-related kinase (ERK)1/2 and cAMP/protein kinase A (PKA) signalling pathways.
Source
Department of Internal Medicine, University of Turin, Corso Dogliotti 14, 10126, Turin, Italy.
Abstract
AIMS/HYPOTHESIS:
Pancreatic islet microendothelium exhibits unique features in interdependent relationship with beta cells. Gastrointestinal products of the ghrelin gene, acylated ghrelin (AG), unacylated ghrelin (UAG) and obestatin (Ob), and the incretin, glucagon-like peptide-1 (GLP-1), prevent apoptosis of pancreatic beta cells. We investigated whether the ghrelin gene products and the GLP-1 receptor agonist exendin-4 (Ex-4) display survival effects in human pancreatic islet microendothelial cells (MECs) exposed to chronic hyperglycaemia.
METHODS:
Islet MECs were cultured in high glucose concentration and treated with AG, UAG, Ob or Ex-4. Apoptosis was assessed by DNA fragmentation, Hoechst staining of the nuclei and caspase-3 activity. Western blot analyses and pharmacological inhibition of protein kinase B (Akt) and extracellular signal-related kinase (ERK)1/2 pathways, detection of intracellular cAMP levels and blockade of adenylyl cyclase (AC)/cAMP/protein kinase A (PKA) signalling were performed. Levels of NO, IL-1β and vascular endothelial growth factor (VEGF)-A in cell culture supernatant fractions were measured.
RESULTS:
Islet MECs express the ghrelin receptor GHS-R1A as well as GLP-1R. Treatment with AG, UAG, Ob and Ex-4 promoted cell survival and significantly inhibited glucose-induced apoptosis, through activation of PI3K/Akt, ERK1/2 phosphorylation and intracellular cAMP increase. Moreover, peptides upregulated B cell lymphoma 2 (BCL-2) and downregulated BCL-2-associated X protein (BAX) and CD40 ligand (CD40L) production, and significantly reduced the secretion of NO, IL-1β and VEGF-A.
CONCLUSIONS/INTERPRETATION:
The ghrelin gene-derived peptides and Ex-4 exert cytoprotective effects in islet MECs. The anti-apoptotic effects involve phosphoinositide 3-kinase (PI3K)/Akt, ERK1/2 and cAMP/PKA pathways. These peptides could therefore represent a potential tool to improve islet vascularisation and, indirectly, islet cell function.
Human and rat hepatocyte toxicity and protein phosphatase 1 and 2A inhibitory activity of naturally occurring desmethyl-microcystins and nodularins.
Source
Food Chemistry and Toxicology, University of Kaiserslautern, Kaiserslautern 67659, Germany.
Abstract
Contamination of water, foods and food supplements by various genera of cyanobacteria is a serious health problem worldwide for humans and animals, largely due to the toxic effects of microcystins (MCs) and nodularin (NOD), a group of hepatotoxic cyclic peptides. The toxins occur in variable structures resulting in more than 90 different MCs and 8 different NODs, many of them not having been investigated for their toxic potency. Potent MCs such as MC-LR have been shown to elicit their hepatotoxic potency via inhibition of hepatic protein phosphatases (PP) 1 and 2A leading to over-phosphorylation of vital cellular proteins. This mechanism of action is also thought to be responsible for the long term tumor promoting action of certain MCs and NOD in the liver. Here, we report on the isolation of certain MCs and NOD as well as a number of their desmethylated derivatives from algae bloom. Subsequently, we determined the cytotoxicity of these compounds in isolated primary human and rat hepatocytes in culture. In parallel experiments, we analyzed the inhibitory potency of these congeners on PP1 and 2A using commercially available enzymes. We found in primary rat hepatocyts that MC-LR, -YR and NOD were more cytotoxic, namely in the 10 to >50nM range, while MC-RR was not. The desmethylated congeners of MC-LR, -YR, and NOD were equally or more-toxic as/than their fully methylated counterparts. In primary human hepatocytes we could show that MC-LR, NOD and the desmethylated variants [(3)Asp]MC-LR, [(7)Dha]MC-LR and [(1)Asp]NOD were cytotoxic in the 20 to >600nM range. Inhibition data with human, bovine and rabbit protein phosphatases 1 and 2A were roughly in accordance with the cytotoxicity findings in human and rat hepatocytes, i.e. desmethylation had no pronounced effects on the inhibitory potencies. Thus, a variety of naturally occurring desmethylated MC and NOD congeners have to be considered as being at least as toxic as the corresponding fully methylated derivatives.
Copyright © 2011. Published by Elsevier Ireland Ltd.
Protective action of NDP-MSH in experimental subarachnoid hemorrhage.
Source
Center for Surgical Research, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy.
Abstract
Subarachnoid hemorrhage (SAH) is still a major cause of morbidity and mortality. α-Melanocyte stimulating hormone (α-MSH) and other melanocortin peptides exert potent neuroprotective action and they might modulate key molecules involved in SAH-induced vasospasm. The aim of this research was to determine whether treatment with the α-MSH analog Nle4,DPhe7-α-MSH (NDP-MSH) exerts protective effects in experimental SAH in the rat. Initial experiments examined effects of NDP-MSH on the basilar artery phenotype in the absence of injury. In these tests intrathecal injection of small concentrations (10ng) of the peptide induced a tolerant phenotype similar to that observed after ischemic preconditioning. Then the effect of systemic treatment with NDP-MSH (100μg i.v.) on experimental SAH was evaluated. SAH was induced by a single-blood injection into the cisterna magna. The basilar artery phenotype was examined at 4h and the artery caliber at 5days following SAH. Expression of 96 genes was analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR) using Custom Taqman Low-Density Arrays. Four hours after SAH, the transcriptional profile of the basilar artery was deeply disrupted. Transcript alteration included genes involved in inflammation, stress response, apoptosis, and vascular remodeling. Treatment with NDP-MSH prevented most of these transcription changes and decreased phosphorylation of extracellular-signal-regulated kinases (ERK1/2) and inhibitor protein IκBα. Vasospasm on day 5 was significantly reduced by NDP-MSH administration. These results combine with others on CNS inflammation to suggest that the melanocortins could be safe and effective therapeutic candidates to treat SAH-related complications.
Copyright © 2011. Published by Elsevier Inc.
Diminished levels of nasal S100A7 (psoriasin) in seasonal allergic rhinitis: an effect mediated by Th2 cytokines.
Abstract
ABSTRACT:
BACKGROUND:
S100A7 is an antimicrobial peptide involved in several inflammatory diseases. The aim of the present study was to explore the expression and regulation of S100A7 in seasonal allergic rhinitis (SAR).
METHODS:
Nasal lavage (NAL) fluid was obtained from healthy controls before and after lipopolysaccharide (LPS) provocation, from SAR patients before and after allergen challenge, and from SAR patients having completed allergen-specific immunotherapy (ASIT). Nasal biopsies, nasal epithelial cells and blood were acquired from healthy donors. The airway epithelial cell line FaDu was used for in vitro experiments. Real-time RT-PCR and immunohistochemistry were used to determine S100A7 expression in nasal tissue and cells. Release of S100A7 in NAL and culture supernatants was measured by ELISA. The function of recombinant S100A7 was explored in epithelial cells, neutrophils and peripheral blood mononuclear cells (PBMC).
RESULTS:
Nasal administration of LPS induced S100A7 release in healthy non-allergic subjects. The level of S100A7 was lower in NAL from SAR patients than from healthy controls, and it was further reduced in the SAR group 6 h post allergen provocation. In contrast, ASIT patients displayed higher levels after completed treatment. S100A7 was expressed in the nasal epithelium and in glands, and it was secreted by cultured epithelial cells. Stimulation with IL-4 and histamine repressed the epithelial S100A7 release. Further, recombinant S100A7 induced activation of neutrophils and PBMC.
CONCLUSIONS:
The present study shows an epithelial expression and excretion of S100A7 in the nose after microbial stimulation. The levels are diminished in rhinitis patients and in the presence of an allergic cytokine milieu, suggesting that the antimicrobial defense is compromised in patients with SAR.
[Calcitonin gene-related peptide promoting migration of rat bone marrow mesenchymal stem cells and stimulating expression of vascular endothelial growth factor].
Source
Department of Orthopaedics and Traumatology, Nanfang Hospital, Southern Medical University, Guangzhou Guangdong 510515, PR China.
Abstract
OBJECTIVE:
To explore the effects of calcitonin gene-related peptide (CGRP) on the migration of bone marrow mesenchymal stem cells (BMSCs) and vascular endothelial growth factor (VEGF) expression in vitro.
METHODS:
The BMSCs were isolated from Sprague Dawley rats using whole bone marrow adherence method. At 1, 2, and 3 weeks after culture, the expressions of CGRP receptor (CGRPR) was detected by Western blot. The BMSCs were treated with CGRP at concentration 1 x 10(-8) mol/L (experimental group) and did not treated (control group), and the efficacy of BMSCs migration was analyzed by Transwell chamber assay after 72 hours; at 1, 3, 5, and 7 days, the mRNA expressions of vascular cell adhesion molecule 1 (VCAM-1) were detected by real-time fluorescent quantitative PCR; the protein expressions of VEGF were examined using immunohistochemistry and Western blot.
RESULTS:
CGRPR expressed stably in the cultured BMSCs and reached the peak at 2 weeks. CGRP had a significantly enhanced role in promoting cell migration. The number of cell migration was (3.20 +/- 1.77) cells/HP in experimental group and (1.11 +/- 0.49) cells/HP in control group, showing significant difference (t = 4.230, P = 0.001). In experimental group, the expressions of VCAM-1 mRNA increased with time and reached the peak at 7 days. There were significant differences in the expressions of VCAM-1 mRNA between control group and experimental group at 3, 5, and 7 days (P < 0.05). Immunocytochemistry results showed positive DAB staining for VEGF at 5 and 7 days in experimental group. Western blot results showed that the protein expressions of VEGF increased significantly at 5 and 7 days in experimental group when compared with control group (P < 0.05), which was signfiantly higher at 5 days than at 7 days in experimental group (P < 0.05).
CONCLUSION:
CGRP can promote the migration of BMSCs and stimulate the protein expression of VEGF, which may plays an important role in regulating bone metabolism by increasing angiogenesis.
- PMID:
- 22229198
- [PubMed - in process]
Expression and purification of E. coli BirA biotin ligase for in vitro biotinylation.
Source
Protein Production Core Facility, Department of Biochemistry, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA.
Abstract
The extremely tight binding between biotin and avidin or streptavidin makes labeling proteins with biotin a useful tool for many applications. BirA is the Escherichia coli biotin ligase that site-specifically biotinylates a lysine side chain within a 15-amino acid acceptor peptide (also known as Avi-tag). As a complementary approach to in vivo biotinylation of Avi-tag-bearing proteins, we developed a protocol for producing recombinant BirA ligase for in vitro biotinylation. The target protein was expressed as both thioredoxin and MBP fusions, and was released from the corresponding fusion by TEV protease. The liberated ligase was separated from its carrier using HisTrap HP column. We obtained 24.7 and 27.6mg BirA ligase per liter of culture from thioredoxin and MBP fusion constructs, respectively. The recombinant enzyme was shown to be highly active in catalyzing in vitro biotinylation. The described protocol provides an effective means for making BirA ligase that can be used for biotinylation of different Avi-tag-bearing substrates.
Copyright © 2012. Published by Elsevier Inc.
The veA gene of the pine needle pathogen Dothistroma septosporum regulates sporulation and secondary metabolism.
Source
Institute of Molecular BioSciences, Massey University, Palmerston North, New Zealand.
Abstract
Fungi possess genetic systems to regulate the expression of genes involved in complex processes such as development and secondary metabolite biosynthesis. The product of the velvet gene veA, first identified and characterized in Aspergillus nidulans, is a key player in the regulation of both of these processes. Since its discovery and characterization in many Aspergillus species, VeA has been found to have similar functions in other fungi, including the Dothideomycete Mycosphaerella graminicola. Another Dothideomycete, Dothistroma septosporum, is a pine needle pathogen that produces dothistromin, a polyketide toxin very closely related to aflatoxin (AF) and sterigmatocystin (ST) synthesized by Aspergillus spp. Dothistromin is unusual in that, unlike most other secondary metabolites, it is produced mainly during the early exponential growth phase in culture. It was therefore of interest to determine whether the regulation of dothistromin production in D. septosporum differs from the regulation of AF/ST in Aspergillus spp. To begin to address this question, a veA ortholog was identified and its function analyzed in D. septosporum. Inactivation of the veA gene resulted in reduced dothistromin production and a corresponding decrease in expression of dothistromin biosynthetic genes. Expression of other putative secondary metabolite genes in D. septosporum such as polyketide synthases and non-ribosomal peptide synthases showed a range of different responses to loss of Ds-veA. Asexual sporulation was also significantly reduced in the mutants, accompanied by a reduction in the expression of a putative stuA regulatory gene. The mutants were, however, able to infect Pinus radiata seedlings and complete their life cycle under laboratory conditions. Overall this work suggests that D. septosporum has a veA ortholog that is involved in the control of both developmental and secondary metabolite biosynthetic pathways.
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