1.
Glucose-induced increase in circulating progenitor cells is blunted in polycystic amenorrhoeic subjects.
Source
Laboratory of Vascular Biology, Department of Biotechnology, Indian Institute of Technology Madras (IIT Madras), BT 415, Chennai 600036, India.
Abstract
BACKGROUNDGlucose-induced kinetics of bone marrow-derived stem cells in healthy females is presently unknown. The objectives of this study were to determine whether circulating levels of CD133(+), CD34(+) and CD133(+)CD34(+) cells increase in response to glucose load in healthy females and whether the kinetics is altered in amenorrhoeic women. The other objective of the work was to compare the endothelial differentiation potential of peripheral blood-derived endothelial progenitor cells (EPCs) from healthy versus amenorrhoeic women.METHODSIn this case-control study, 44 amenorrhoeic subjects and 36 age-matched females with no menstrual disturbance were recruited at Apollo Hospitals, a Tertiary health care center in Chennai, India. Circulating bone marrow-derived stem cells were measured by two color direct flow cytometry. Cultured progenitor cells were characterized at Day 7 and 14 for expression of endothelial markers and production of nitric oxide (NO) via immunofluoroscence.RESULTSThe amenorrhoeic subjects were insulin resistant with homeostatic model of assessment of insulin resistance values of 3.33 ± 0.3 versus 1.75 ± 0.148 observed for controls (P< 0.0001). Among the amenorrhoeic subjects, 38 subjects had polycystic ovaries with no signs of hyperandrogenism. Fasting levels of CD133(+), CD34(+) and CD133(+)CD34(+) cells were reduced in amenorrhoeic subjects (P< 0.001). There was a 1.5 to 2-fold increase in the circulating levels of these cells in response to 75 g oral glucose challenge at 1 and 2 h post-load conditions in controls, which was significantly blunted for CD133(+) (P< 0.001) and CD133(+)CD34(+) (P< 0.001) cells in amenorrhoeic subjects. A positive correlation was observed between estrogen and fasting CD133(+) (r= 0.205, P= 0.070), CD34(+) (r= 0.249, P= 0.027) and CD133(+)CD34(+) (r= 0.217, P= 0.055) cell counts. Additionally, fasting counts for CD34(+) and CD133(+)CD34(+) cells positively correlated with FSH and inversely correlated with LH and C-peptide in the polycystic group. Cultured cells from polycystic subjects exhibited reduced adherence to fibronectin and expressed lower levels of endothelial nitric-oxide synthase and NO.CONCLUSIONSOral glucose-induced increase in circulating numbers of CD133(+) and CD133(+)CD34(+) cells and endothelial differentiation potential of peripheral blood-derived EPCs is attenuated in insulin resistant amenorrhoeic subjects.
- PMID:
- 22252083
- [PubMed - as supplied by publisher]
Small surfactant-like peptides can drive soluble proteins into active aggregates.
Abstract
ABSTRACT:
BACKGROUND:
Inactive protein inclusion bodies occur commonly in Escherichia coli (E. coli) cells expressing heterologous proteins. Previously several independent groups have found that active protein aggregates or pseudo inclusion bodies can be induced by a fusion partner such as a cellulose binding domain from Clostridium cellulovorans (CBDclos) when expressed in E. coli. More recently we further showed that a short amphipathic helical octadecapeptide 18A (EWLKAFYEKVLEKLKELF) and a short beta structure peptide ELK16 (LELELKLKLELELKLK) have a similar property.
RESULTS:
In this work, we explored a third type of peptides, surfactant-like peptides, for performing such a "pulling-down" function. One or more of three such peptides (L6KD, L6K2, DKL6) were fused to the carboxyl termini of model proteins including Aspergillus fumigatus amadoriase II (AMA, all three peptides were used), Bacillus subtilis lipase A (LipA, only L6KD was used, hereinafter the same), Bacillus pumilus xylosidase (XynB), and green fluorescent protein (GFP), and expressed in E. coli. All fusions were found to predominantly accumulate in the insoluble fractions, with specific activities ranging from 25% to 92% of the native counterparts. Transmission electron microscopic (TEM) and confocal fluorescence microscopic analyses confirmed the formation of protein aggregates in the cell. Furthermore, binding assays with amyloid-specific dyes (thioflavin T and Cong red) to the AMA-L6KD aggregate and the TEM analysis of the aggregate following digestion with protease K suggested that the AMA-L6KD aggregate may contain structures reminiscent of amyloids, including a fibril-like structure core.
CONCLUSIONS:
This study shows that the surfactant-like peptides L6KD and it derivatives can act as a pull-down handler for converting soluble proteins into active aggregates, much like 18A and ELK16. These peptide-mediated protein aggregations might have important implications for protein aggregation in vivo, and can be explored for productionof functional biopolymers with detergent or other interfacial activities.
- PMID:
- 22251949
- [PubMed - as supplied by publisher]
Characterisation of the mgo operon in Pseudomonas syringae pv. syringae UMAF0158 that is required for mangotoxin production.
Abstract
ABSTRACT:
BACKGROUND:
Mangotoxin is an antimetabolite toxin that is produced by strains of Pseudomonas syringae pv. syringae; mangotoxin-producing strains are primarily isolated from mango tissues with symptoms of bacterial apical necrosis. The toxin is an oligopeptide that inhibits ornithine N-acetyl transferase (OAT), a key enzyme in the biosynthetic pathway of the essential amino acids ornithine and arginine. The involvement of a putative nonribosomalpeptide synthetase gene (mgoA) in mangotoxin production and virulence has been reported.
RESULTS:
In the present study, we performed a RT-PCR analysis, insertional inactivation mutagenesis, a promoter expression analysis and terminator localisation to study the gene cluster containing the mgoA gene. Additionally, we evaluated the importance of mgoC, mgoA and mgoD in mangotoxin production. A sequence analysis revealed an operon-like organisation. A promoter sequence was located upstream of the mgoB gene and was found to drive lacZ transcription. Two terminators were located downstream of the mgoD gene. RT-PCR experiments indicated that the four genes (mgoBCAD) constitute a transcriptional unit. This operon is similar in genetic organisation to those in the three other P. syringae pathovars for which complete genomes are available (P. syringae pv. syringae B728a, P. syringae pv. tomato DC3000 and P. syringae pv. phaseolicola 1448A). Interestingly, none of these three reference strains is capable of producing mangotoxin. Additionally, extract complementation resulted in a recovery of mangotoxin production when the defective mutant was complemented with wild-type extracts.
CONCLUSIONS:
The results of this study confirm that mgoB, mgoC, mgoA and mgoD function as a transcriptional unit and operon. While this operon is composed of four genes, only the last three are directly involved in mangotoxinproduction.
- PMID:
- 22251433
- [PubMed - as supplied by publisher]
B Lymphocytes Treated In Vitro with Antigen Coupled to Cholera Toxin B Subunit Induce Antigen-Specific Foxp3+ Regulatory T Cells and Protect against Experimental Autoimmune Encephalomyelitis.
Source
University of Gothenburg Vaccine Research Institute, Sahlgrenska Academy at the University of Gothenburg, SE-405 30, Göteborg, Sweden.
Abstract
The ability of activated B cells to protect against various experimental autoimmune or allergic diseases makes them attractive for use in cell-based therapies. We describe an efficient way to generate B cells with strong suppressive functions by incubating naive B cells with a relevant Ag conjugated to cholera toxin B subunit (CTB). This allows most B cells, irrespective of BCR, to take up and present Ag and induces their expression of latency-associated polypeptide (LAP)/TGF-β and after adoptive transfer also their production of IL-10. With OVA as model Ag, when naive T cells were cocultured in vitro with B cells pretreated with OVA conjugated to CTB (OVA/CTB) Ag-specific CD4(+) Foxp3 regulatory T (Treg) cells increased >50-fold. These cells effectively suppressed CD25(-)CD4(+) effector T (Teff) cells in secondary cultures. Adoptive transfer of OVA/CTB-treated B cells to mice subsequently immunized with OVA in CFA induced increase in Foxp3 Treg cells together with suppression and depletion of Teff cells. Likewise, adoptive transfer of B cells pulsed with myelin oligodendrocyte glycoprotein peptide(35-55) (MOGp) conjugated to CTB increased the number of Treg cells, suppressed MOGp-specific T cell proliferation and IL-17 and IFN-γ production, and prevented the development of experimental autoimmune encephalomyelitis. Similar effects were seen when B cells were given "therapeutically" to mice with early-stage experimental autoimmune encephalomyelitis. Our results suggest that B cells pulsed in vitro with relevant Ag/CTB conjugates may be used in cell therapy to induce Ag-specific suppression of autoimmune disease.
One-Step Expression and Tyrosine O-Sulfonation of Ax21 in Escherichia coli.
Source
School of Municipal and Environmental Engineering, Shandong Jianzhu University, Jinan, China, biohou@sdjzu.edu.cn.
Abstract
Ax21 (activator of Xa21-mediated immunity), a pathogen-associated molecular pattern secreted by Xanthomonas oryzae pv. oryzae, can be perceived by a membrane-located pattern recognition receptor Xa21 and triggered immune responses in rice. An Ax21-derived peptide (17-amino acid) containing a sulfated tyrosine-22 (axY(S)22) is sufficient for Ax21 activity. Here, we expressed Ax21 and O-sulfated its tyrosine-22 through coexpressing a putative tyrosine sulfotransferase, raxST, and two other genes involved in the synthesis of 3'-phosphoadenosine 5'-phosphosulfate in Escherichia coli BL21 (DE3). The sulfated Ax21 fused with a histidine tag in its N-terminus was extracted and bound onto a Ni-NTA agarose and then cleaved with Factor Xa and CNBr in turn. Δax21Y(S)22, a 36-amino acid peptidecovering axY(S)22 in the lysate supernatant, was finally yielded after ultrafiltration. The purified peptide was further verified by Tricine-SDS-PAGE and isoelectrofocusing electrophoresis. Lesion length analysis, reactive oxygen speciesproduction, and mitogen-activated protein kinase (MAPK) activation of rice leaves inoculated with Δax21Y(S)22 confirmed the activity of the sulfated peptide. Overall, this study successfully established an efficient system for expression and purification of a sulfated peptide. In addition, the sulfotransferase activity of RaxST was confirmed for the first time.
[68Ga-labeled peptides for clinical trials - Production according to the German Drug Act: The Göttingen experience.]
Source
Priv.-Doz. Dr. rer. nat. Birgit Meller, Abteilung für Nuklearmedizin, Universitätsmedizin Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, Deutschland, Tel. 05 51/39 49 84, Fax 05 51/39 85 26, E-Mail: birgit.meller@medizin.uni-halle.de.
Abstract
The AMG implies far-reaching implications for the synthesis of new radiopharmaceuticals for clinical trials. Aim, methods: As a part of the DFG-funded Clinical Research Group (KFO 179) a project designated "Immuno-PET for assessment of early response to radiochemotherapy of advanced rectal cancer" was initiated. This trial is focused on a trivalent bispecific humanized monoclonal antibody, and a 68Ga-labeled peptide. Following the new regulatory framework we established a GMP-compliant cleanroom laboratory and applied for a manufacturing permission. Results: During the project constructural, personnel and organizational conditions for a successful application were established, including a quality management system. A GMP-conform cleanroom laboratory class C was constructed, equipped with a two-chamber lock. The actual manufacturing is performed in a closed system with subsequent sterile filtration. The manufacturing processes have been automatised and validated as well as the necessary quality controls. The manufacturing permission was granted after an official inspection. Conclusions: The new German Drug Act is considered as a break in the production practice of nuclear medicine. The early involvement and communication with the authorities avoids time-consuming and costly planning errors. It is much to be hoped that the new legal situation in Germany will not cause serious impairments in the realization of clinical trials in German nuclear medicine.
[Glucagon and glucagon-like peptides the role in control glucose homeostasis. Part I].
Abstract
Glucose homeostasis is controlled primarily by the opposing actions of insulin and glucagon, hormones that are secreted by the islets of Langerhans from β-cells and α-cells and Δ-cells, their role in glucose homeostasis still needs identifying. Insulin secretion is increased in response to elevated blood glucose to maintain normoglycemia by stimulating glucose transport in muscles and adipocytes and reducing glucose production by inhibiting gluconeogenesis in the liver. Whereas glucagon secretion is suppressed by hyperglycemia, it is stimulated during hypoglycemia, promoting hepatic glucose production and ultimately raising blood glucose levels. Glucagon secretion from pancreatic α-cells is regulated by various mechanisms including glycemia, neural input, and secretion from neighboring β-cells. Glucagon primarily acts on liver to initiate glycogenolysis and gluconeogenesis, resulting in a rapid increase in endogenous production of glucose. With longer stimulation, glucagon action on the liver results in a glucose-sparing activation of free fatty acid oxidation and production of ketones.
- PMID:
- 22248782
- [PubMed - in process]
Overexpression of a modified protein from amaranth seed in Escherichia coli and effect of environmental conditions on the protein expression.
Source
Centro de Investigación para el Desarrollo Integral Regional, CIDIIR-IPN, Sinaloa, Mexico.
Abstract
Amaranth seeds are considered as an excellent complementary source of food protein due to their balanced amino acid composition. Amarantin acidic subunit has the potential as a functional and nutraceutical protein, and it is structurally a good candidate for modification. The aim of this work was to improve its functionality, then the primary structure was modified into the third variable region of 11S globulins, by inserting antihypertensive peptides: four Val-Tyr in tandem and Arg-Ile-Pro-Pro in the C-terminal region. Modified protein was expressed in Escherichia coli Origami (DE3) and was purified. The culture conditions, including the culture media, temperature, agitation speed and air flow were tested in order to obtain an increased expression levels of the modified protein. A 2(3) factorial design was used for evaluate the effect of environmental conditions on modified protein production. The results indicated that the yield of modified protein could be increased by up 3-fold in bioreactor as compared with flask. In addition, the temperature, the agitation speed and the oxygen were significant factors on the expression of the antihypertensive protein. The maximum production was 99mg protein-L(-1). The hydrolyzed protein showed a high inhibitory activity of the angiotensin converting enzyme (IC(50)=0.047mgmL(-1)).
Copyright © 2012 Elsevier B.V. All rights reserved.
Synthesis and characterization of an aggrecan mimic.
Source
Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN 47907, USA.
Abstract
Aggrecan (AGG) is a large, aggregating proteoglycan present throughout the body, but predominantly found in articular cartilage. The principle features of AGG, its hyaluronan (HA) binding domain and its abundance of covalently attached glycosaminoglycans (GAGs), make it an essential component of the functional ability of articular cartilage. Current tissue engineering constructs have attempted to stimulate AGG production, but have been unable to produce adequate amounts of mature AGG, and hence have suffered a mismatch in mechanical properties. To address these deficiencies, an AGG mimic was synthesized to match AGG functional properties and provide greater control within tissue engineering constructs. Chondroitin sulfate was functionalized with HA-specific binding peptides to replicate both the GAG presence and HA-binding ability of AGG, respectively. Upon characterization and testing, the mimic was able to effectively bind to HA, increase the compressive strength of cartilage extracellular matrix-based constructs, and protect the other extracellular matrix (ECM) components from degradation, replicating the important functions of AGG. In particular, the mimic produced a 78% increase in compressive strength of the ECM-based constructs, and was able to significantly reduce the degradation of both HA and collagen. The initial characterization of the newly synthesized AGG mimic demonstrates its potential in tissue engineering constructs, and provides an essential basis for more explorative studies of the AGG mimic's abilities as an AGG substitute and beyond.
Copyright © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
Analysis of YM-216391 Biosynthetic Gene Cluster and Improvement of the Cyclopeptide Production in Heterologous Host.
Abstract
YM-216391, an antitumor natural product, represents a new class of cyclic peptides containing a polyoxazole-thiazole moiety. Herein we describe its gene cluster encoding the biosynthetic paradigm featuring a ribosomally synthesizing precursor peptide followed by a series of novel posttranslational modifications, which include i) cleavage of both N-terminal leader peptide and C-terminal extension peptide and cyclization in a head-to-tail fashion, ii) conversion of an L-Ile to D-allo-Ile, and iii) -hydroxylation of Phe by a P450 monooxygenase followed by further heterocyclization and oxidation to form a phenyloxazole moiety. The cluster was heterologously expressed in Streptomyces lividans to bypass difficult genetic manipulation. Deletion of the ymR3 gene, encoding a putative transcriptional regulator, increased the YM-216391 yield about 20-fold higher than the original yields for the heterologous expression of wild-type cluster, which set the stage for further combinatorial biosynthesis.
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.
The Apoplastic Oxidative Burst Peroxidase in Arabidopsis Is a Major Component of Pattern-Triggered Immunity.
Source
School of Biological Sciences, Royal Holloway, University of London, Egham, Surrey TW20 0EX, United Kingdom.
Abstract
In plants, reactive oxygen species (ROS) associated with the response to pathogen attack are generated by NADPH oxidases or apoplastic peroxidases. Antisense expression of a heterologous French bean (Phaseolus vulgaris) peroxidase (FBP1) cDNA in Arabidopsis thaliana was previously shown to diminish the expression of two Arabidopsis peroxidases (peroxidase 33 [PRX33] and PRX34), block the oxidative burst in response to a fungal elicitor, and cause enhanced susceptibility to a broad range of fungal and bacterial pathogens. Here we show that mature leaves of T-DNA insertion lines with diminished expression of PRX33 and PRX34 exhibit reduced ROS and callose deposition in response to microbe-associated molecular patterns (MAMPs), including the synthetic peptides Flg22 and Elf26 corresponding to bacterial flagellin and elongation factor Tu, respectively. PRX33 and PRX34 knockdown lines also exhibited diminished activation of Flg22-activated genes after Flg22 treatment. These MAMP-activated genes were also downregulated in unchallenged leaves of the peroxidase knockdown lines, suggesting that a low level of apoplastic ROS production may be required to preprime basal resistance. Finally, the PRX33 knockdown line is more susceptible to Pseudomonas syringae than wild-type plants. In aggregate, these data demonstrate that the peroxidase-dependent oxidative burst plays an important role in Arabidopsis basal resistance mediated by the recognition of MAMPs.
The role of the Src family kinase Lyn in the immunomodulatory activities of cathelicidin peptide LL-37 on monocytic cells.
Source
Department of Microbiology and Immunology, University of British Columbia, Vancouver, Canada.
Abstract
Cathelicidin LL-37 is a multifunctional, immunomodulatory and antimicrobial host-defense peptide of the human immune system. Here, we identified the role of SFKs in mediating the chemokine induction activity of LL-37 in monocytic cells. LL-37 induced SFK phosphorylation; and chemical inhibitors of SFKs suppressed chemokine production in response to LL-37 stimulation. SFKs were required for the downstream activation of AKT, but Ca(2+)-flux and MAPK induction were SFK-independent. Through systematic siRNA knockdown of SFK members, a requirement for Lyn in mediating LL-37 activity was identified. The involvement of Lyn in cathelicidin activities was further confirmed using Lyn-knockout mouse BMDMs. The role of SFKs and Lyn was also demonstrated in the activities of the synthetic cationic IDR peptides, developed as novel, immunomodulatory therapeutics. These findings elucidate the common molecular mechanisms mediating the chemokine induction activity of natural and synthetic cationic peptides in monocytic cells and identify SFKs as a potential target for modulating peptide responses.
Immunomodulatory effects and improved prognosis of experimental autoimmune encephalomyelitis after O-tetradecanoyl-genistein treatment.
Source
Department of Parasitology, Microbiology and Immunology, Institute of Biological Sciences, Federal University of Juiz de Fora, Juiz de Fora, Brazil.
Abstract
Experimental autoimmune encephalomyelitis (EAE) is a murine autoimmune disease used to study multiple sclerosis (MS), a human inflammatory demyelinating disease of the central nervous system. Genistein, an isoflavonoid phytoestrogenic compound found in soy, is known to reverse clinical signs of EAE. Although genistein has some potential in clinical application, it has some disadvantages related to its chemical structure, such as rapid in vivo metabolism and a fast decline in serum after oral administration. The present work investigates the treatment of EAE by using 7-O-tetradecanoyl-genistein (TDG), a more lipophilic analog of genistein obtained by esterification. The clinical course of EAE was investigated in C57Bl/6 mice immunized with myelin oligodendrocyte glycoprotein peptide (MOG)(35-55) in complete Freund's adjuvant supplemented with Mycobacterium tuberculosis H37RA. After 14days of MOG immunization, mice were treated with TDG for seven days. Numbers of IL-17-producing cells and Foxp3 by CD4(+) T cells and CTLA-4 expression by CD3(+) T cells from brain were determined by flow cytometry. Levels of IL-6, IFN-γ and IL-10 were evaluated by ELISA. Brain sections were stained by hematoxylin and eosin method. The data obtained indicate that TDG treatment ameliorates the clinical signs of EAE, which correlates with a decrease of IL-17-producing cells and an increase in Foxp3(+)CD4(+) cells in the brain. TDG is also shown to enhance IL-10 production and CTLA-4 expression and to reduce IFN-γ and IL-6. Altogether, these findings suggest an immunomodulatory therapeutic role for TDG in EAE and multiple sclerosis.
Copyright © 2011. Published by Elsevier B.V.
Mitochondria-targeted antioxidant attenuates high glucose-induced P38 MAPK pathway activation in human neuroblastoma cells.
Source
Department of Endocrinology Provincial Hospital Affiliated to Shandong University, Jinan 250021, P.R. China.
Abstract
Excessive mitochondrial free radical production and the related mitogen-activated protein kinase P38 (P38 MAPK) activation are key regulators in the pathogenesis of high glucose-induced cell stress. Increasing evidence has emphasized the impact of hyperglycemia on neurons and the consequent neuronal stresses eventually resulting in neurodegeneration and neuronal death. In this study, we employed a novel mitochondria-targeted antioxidant, SS31peptide, on high glucose-insulted neuroblastoma cells (SH-SY5Y). Our results showed that high glucose promoted significantly increased P38 phosphorylation which was efficiently suppressed by the application of the SS31 peptideunder the experimental conditions. The inhibition of high glucose-induced P38 activation by the SS31 peptide was associated with the impact of the SS31 peptide on attenuating high glucose-induced mitochondrial ROS (reactive oxygen species) elevation and mitochondrial membrane potential collapse. The addition of SS31 peptide significantly attenuated high-gluose-induced apoptosis. Therefore, our study suggests that elimination of high glucose-induced mitochondrial oxidative stress helps to rescue SH-SY5Y cells from high glucose-related P38 MAPK pathway disturbances, and the SS31 peptide has the potential to serve as a new treatment strategy against hyperglycemia-instigated neuronal perturbations.
Turkish scorpion Buthacus macrocentrus: General characterization of the venom and description of Bu1, a potent mammalian Na(+)-channel α-toxin.
Source
Department of Biology, Faculty of Science and Art, Eskisehir Osmangazi University, 26480 Eskisehir, Turkey; Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, UNAM, Av. Universidad, 2001, Col. Chamilpa, Apartado Postal 510-3, Cuernavaca, Morelos 62210, Mexico.
Abstract
The venom of the scorpion Buthacus macrocentrus of Turkey was fractionated by high performance liquid chromatography (HPLC) and its mass finger print analysis was obtained by spectrometry. More than 70 different fractions were obtained, allowing the determination of the molecular masses of at least 60 peptides ranging between 648 and 44,336 Da. The venom is enriched with peptides containing molecular masses between 3200-4500 Da, and 6000-7500 Da. They very likely correspond to K(+)-channel and Na(+)-channel specific peptides, respectively, as expected from venoms of scorpions of the family Buthidae, already determined for other species. The major component obtained from HPLC was shown to be lethal to mice and was further purified and characterized. It contains 65 amino acid residues maintained closely packed by 4 disulfide bridges, and shows a molecular weight of 7263 Da. Additionally, a cDNA from the venomous glands of this scorpion was used in conjunction with sequence data from Edman degradation and mass spectrometry for cloning the gene that codes for Bu1 as we named this toxin. This gene codes for a 67 amino acid residues peptide, where the two last are eliminated post-translationally for production of an amidated C-terminal arginine. Its sequence is closely related to toxins from the species Leiurus quinquestriatus, as revealed by a phylogenetic tree analysis. Electrophysiological results conducted with Bu1 using patch-clamp techniques indicate that it modifies the Na(+) currents, in a similar way as other well known α-scorpion toxins. These results support the conclusion that this species of scorpions is dangerous to humans, having an epidemiological interest for the country.
Copyright © 2012 Elsevier Ltd. All rights reserved.
F-spondin regulates the differentiation of human cementoblast-like (HCEM) cells via BMP7 expression.
Source
Center of Oral Clinical Examination, Hiroshima University Hospital, Hiroshima University, Hiroshima 734-8553, Japan.
Abstract
Cementum plays an important role in the attachment of connective tissue to the root surface. However, the detailed mechanism of cementum formation has not yet been clarified. We previously established human cementoblast-like cell lines (HCEM) and human periodontal ligament cell lines (HPL) by infection of hTERT gene. Using those cell lines, we compared the gene expression of them and identified F-spondin as a cementoblast specific gene. In this study, to clarify the role of F-spondin in the differentiation of periodontal ligament cells to cementoblasts, we compared the gene expression of F-spondin-overexpressed HPL (HPL-spondin) cells with HPL parent cells. We found that several genes expressed higher level in HPL-spondin cells than HPL cells, such as heparin sulfate 6-sulfotranferase, calcitonin-related polypeptide beta, bone morphogenetic proteins 7 (BMP7), BMP2 and BMP8B. Among those genes, we focused on BMP7 and examined the interaction between F-spondin and BMP7, because BMP7 was reported to enhance cementoblast function. Moreover, we further examined the effect of BMP7 peptide on the expression of mineralization-associated genes in HCEM cells. RT-PCR and real-time PCR analyses showed that HPL-spondin expressed BMP7, but not HPL cells. And BMP7 and phospho-Smad1/5/8 protein production were detected in HPL-spondin by Western blot. siSPON1 inhibited expression of type I collagen, runt-related transcription factor 2 (RUNX2) and bone sialoprotein (BSP) mRNA in HCEM cells. And the mineralization tended to be decreased in siSPON1 treated cells by ALZ staining and the quantification analysis. Moreover, we examined the effect of BMP7 peptide on the gene expressions of HCEM cells by RT-PCR. Increase of the osteopontin and BSP mRNA was observed in BMP7 treated HCEM cells. These findings indicate that F-spondin regulates the differentiation of HCEM cells via BMP7 expression.
Copyright © 2012. Published by Elsevier Inc.
Extracellular signal-regulated kinase 1/2 activation is involved in intermedin1-53 attenuating myocardial oxidative stress injury induced by ischemia/reperfusion.
Source
Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Peking University, Beijing 100191, China.
Abstract
Intermedin (IMD)(1-53) is a novel member of the calcitonin gene-related peptide superfamily and has potent cardioprotective effects against myocardial injury induced by ischemia-reperfusion (I/R). To explore the mechanism of the IMD(1-53) cardioprotective effect, we studied the anti-oxidant effects of IMD(1-53) on myocardial injury induced by I/R in vivo in rat and H(2)O(2) treatment in vitro in rat cardiomyocytes. Compared with sham treatment, I/R treatment induced severe lipid peroxidation injury in rat myocardium: plasma malondialdehyde (MDA) content and myocardial LDH activity was increased by 34% and 85% (all P<0.01); Mn-superoxide dismutase (Mn-SOD) and catalase (CAT) activity was reduced 80% and 86% (all P<0.01), respectively, and the protein levels of the NADPH oxidase complex subunits gp91(phox) and p47(phox) were markedly increased, by 86% (P<0.05) and 95% (P<0.01), respectively; IMD(1-53) treatment ameliorated lipid peroxidation injury: plasma MDA content and myocardial LDH activity was decreased by 30% (P<0.05) and 36% (P<0.01); Mn-SOD and CAT activity was elevated 1.0- and 4.3-fold (all P<0.01), respectively; and the protein levels of gp91(phox) and p47(phox) were reduced, by 28% and 36% (both P<0.05), respectively. Concurrently, IMD(1-53) treatment markedly promoted cell viability and inhibited apoptosis in cardiomyocytes as compared with H(2)O(2) treatment alone. Furthermore, IMD(1-53) increased the ratio of p-ERK to ERK by 66% (P<0.05) as compared with I/R alone, and the protective effect of IMD(1-53) on H(2)O(2)-induced apoptosis was abolished by preincubation with PD98059, a MEK inhibitor. IMD(1-53) may improve the oxidative stress injury induced by I/R via inhibiting the production of reactive oxygen species and enhancing ERK phosphorylation.
Copyright © 2012. Published by Elsevier Inc.
Bacteriocin-producing oral streptococci and inhibition of respiratory pathogens.
Source
Department of Bio-Medical Sciences sect. Microbiology, University of Catania, Via Androne 81, 95124, Catania (I).
Abstract
The use of bacteria as probiotics is in continuous development thanks to their capacity to maintain or restore a host's natural microbiome by interference with and/or inhibition of other microrganisms, mediated by antimicrobial peptideproduction such as bacteriocins. In the oral cavity, Streptococcus salivarius, a non-pathogenic and predominant oral species, is one of the major bacteriocin producers that is able to coexist in this environment and reduce the frequency of colonization of the main pathogens involved in upper respiratory tract infections. The aim of this study was to screen oral bacteria colonizing healthy children for their use as potential oral probiotics. Eighty-one α-haemolytic streptococci isolated from nasal and/or pharyngeal swabs of thirty-one healthy children aged between two and twelve years were isolated. Among them, 13 α-haemolytic streptococci were selected for their bacteriocin-like inhibitory activity against potential pathogens. These strains were tested for bacteriocin production and assayed for their capacity to adhere to HEp-2 cell lines. Our data showed that 13 bacteriocin producer strains were able to inhibit different gram-positive pathogens. Among them one strain, S. salivarius 24SMB, deposited as DSM 23307, was selected as a potential oral probiotic thanks to its safety assessment, ability to inhibit S. pneumoniae and the absence of virulence and antibiotic resistance genes.
© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.
β-Amyloid peptide is internalized into chick retinal neurons and alters the distribution of myosin Vb.
Source
Instituto de Bioquímica Médica - Universidade Federal do Rio de Janeiro; Departamento de Biociências da Atividade Física - Escola da Educação Física e Desportos - Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, 21941-902, Brazil.
Abstract
The most common neurodegenerative disorder afflicting the aging human population is Alzheimer's disease. A major hallmark of Alzheimer's disease is dementia from a loss of neuronal function, attributed to the presence and accumulation of β-amyloid peptide into senile plaques. Preceding senile plaque formation, abnormalities in axons can be observed as changes in morphologies and intracellular trafficking. Recently, it has been recognized that β-amyloid also accumulates within neurons and this intraneuronal β-amyloid accumulation has been reported to be critical in the disruption of synapses and cognitive function. Here we report on the internalization of a fluorescently labeled β-amyloidpeptide into cultured chick retinal neurons. The pattern of β-amyloid distribution during the time course of incubation is reminiscent of the endocytic pathway. Furthermore, the distribution of the internalized β-amyloid peptide converges with that of myosin Vb and both relocalize from the axon to cell body. These observations are consistent with the hypothesis that Alzheimer's disease proceeds as a result of an imbalance between β-amyloid production and β-amyloid clearance, suggesting a role for myosin Vb in this process. © 2012 Wiley Periodicals, Inc.
No comments:
Post a Comment