1.
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 isoprenoidbiosynthesis 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.
[RNA interference of proteasome subunit of PSMbeta7 gene restricts proteasome subunit PSMbeta1 and PSMbeta5 mRNA expression and peptidyl-glutamyl peptide-hydrolyzing proteasome activity in neonatal cardiomyocytes].
Abstract
Using small interfering RNA (siRNA) transfection of neonatal cardiomyocytes to inhibit expression of nonproteolytic proteasome beta7 subunit, we observed a significant decrease in beta1 proteolytic subunit mRNA expression. Proteasome peptidyl-glutamyl peptide-hydrolyzing activity decreased to 28% (0.48 +/- 0.2 nM AMC/min) compared to control (1.7 +/- 0.5 nM AMC/min) (P < 0.05). Beta5 Subunit mRNA expression decreased 21 times (P < 0.05) with no changes in its chymotrypsin-like activity. Proteasome trypsin-like activity and activity of another proteolytic enzyme tripeptidyl-peptidase II remained unchanged.
- PMID:
- 22145409
- [PubMed - indexed for MEDLINE]
Thrombin and thrombin-derived peptides promote proliferation of cardiac progenitor cells in the form of cardiospheres without affecting their differentiation potential.
Source
Dept. of Anatomy, Histology, Forensic Medicine and Orthopedics, Sapienza University, Rome, Italy.
Abstract
Many studies demonstrated that human adult cardiac progenitor cells in the form of cardiospheres (CSps) could represent a powerful candidate for cardiac cell therapy. To achieve the clinical translation of this biotechnological product, the development of well-defined culture conditions is required to optimize their proliferation and differentiation. Thrombin, a serine protease acting through the protease-activated receptor 1 (PAR-1) signalling to modulate many cellular functions such as proliferation and differentiation in several cell types, is one of the factors included in the CSps medium. Therefore, the assessment of the effective dependence of the thrombin related cellular effects from PAR-signalling is strategic both for understanding the biological potential of these cells and for the GMP translation of the medium formulation, using synthesised analogs. In this study the effects of thrombin on human CSps and their potential relationship with the specific proteolytic activation of PAR-1 have been investigated in different culture conditions, including thrombin inhibitor hirudin and PAR-1 agonist/ antagonist peptides TFLLR and MUMB2. In this study we show that, in the presence of thrombin and TFLLR, CSps, in which PAR-1 expression was evidenced by immunofluorescence and western blot analysis, increase their proliferation activity (BrdU assay). Such increased proliferative rate was consistently associated with a higher phosphorylation level of the cell cycle inhibitor GSK3. Concerning the assessment of the potential effects of thrombin and its agonist on differentiation, both western blot and real-time PCR analysis for stemness, cardiac and vascular markers (such as cKit, cx43 and KDR) showed that CSps commitment was substantially unaffected, except for GATA4 mRNA, whose transcription was down-regulated in the presence of the natural protease, but not after treatment with TFLLR. In conclusion, activation of PAR-1-dependent signalling is important to support CSps proliferative potential, keeping unaltered or at best stable their differentiation properties. The availability of thrombin agonists, such as TFLLR, able to guarantee the required growth effect without affecting CSps lineage commitment, could represent a technological improvement for cost-effective, easy-to-handle and GMPtranslatable synthetic media.
- PMID:
- 22051170
- [PubMed - indexed for MEDLINE]
Increased matrix synthesis by fibroblasts with decreased proliferation on synthetic chitosan-gelatin porous structures.
Source
School of Chemical Engineering, Oklahoma State University, 423 Engineering North, Stillwater, Oklahoma 74078; telephone: 405-744-9115; F: 405-744-6338.
Abstract
Influence of mechanical characteristics and matrix architecture of substrates used in cell culture is an important issue to tissue engineering. Chitosan-based materials have been processed into porous structures, injectable gels and membranes, and are investigated to regenerate various tissues. However, the effect of these structures on cell growth and matrix production in accordance with each of the differing scaffolds has not been examined. We investigated the influence of porous structures, hydrogels, and membranes on the growth of normal human fibroblasts and their matrix production in a serum-free system. We used chitosan alone and in combination with gelatin. Injectable hydrogels were prepared using 2-glycerol phosphate. From the same solution, porous scaffolds and membranes were formed using controlled rate freezing and lyophilization, and air-drying, respectively. Fibroblast growth was evaluated on the 4th and 10th days using flow cytometry and CFDA-SE pre-staining. Cell morphology was assessed using actin and nucleus staining. Total protein content, collagen, tropoelastin, and MMP2/MMP-9 activity in the media supernatant were assessed by BCA, Sircol™, Fastin Elastin, and fluorogeneic peptide assays. Collagen accumulated in the matrix was assessed by Sircol™ assay after pepsin/acetic acid digestion and by Masson's Trichrome staining. These results showed increased viability of fibroblasts on chitosan-gelatin porous scaffold with decreased proliferation relative to tissueculture plastic (TCP) surface despite the cells showing spindle shape. The total protein, collagen, and tropoelastin contents were higher in the spent media from chitosan-gelatin porous scaffolds compared to other conditions. MMP2/MMP9 activity was comparable to TCP. An increase in collagen content was also observed in the matrix, suggesting increased matrix deposition. In summary, matrix production is influenced by the form of chitosan structures, which significantly affects the regenerative process. Biotechnol. Bioeng. © 2011 Wiley Periodicals, Inc.
Copyright © 2011 Wiley Periodicals, Inc.
The antifibrosis effect of adrenomedullin in human lung fibroblasts.
Source
Department of Respiratory Disease, Military General Hospital of Beijing PLA, Beijing, China.
Abstract
Adrenomedullin (AM) is a regulatory peptide involved in cellular proliferation and protein synthesis. The authors investigated AM and the AM receptor system in the human fetal lung fibroblasts (HFLFs), and assessed whether AM can inhibit proliferation and collagen synthesis in HFLFs under hypoxia. Fibroblasts were exposed to hypoxia (2% O(2)) after the addition of AM. The effects of AM and transforming growth factor β1 (TGF-β1) on the proliferation of fibroblasts were determined by the methanethiosulfonate (MTS) assay. Total collagen synthesis was determined by [(3)H]proline incorporation. TGF-β1 levels in the culture supernatant were measured by enzyme-linked immunosorbent assay (ELISA). The concentration of intracellular calciumion ([Ca(2+)](i)) in fibroblasts was detected with a laser scanning confocal microscope. AM, adrenomedullin receptor (ADMR), calcitonin receptor-like receptor (CRLR), AM receptor chaperone receptor activity-modifying protein-1 (RAMP1),RAMP2, and RAMP3 were detected in the HFLFs. The hypoxia-induced increases in cell proliferation, collagen synthesis, and TGF-β1 production were inhibited by AM. AM also inhibited proliferation and collagen synthesis in fibroblasts induced by TGF-β1. AM caused a decrease of the hypoxia-induced [Ca(2+)](i) in fibroblasts. This study suggests that AM is produced by HFLFs and AM may function as an antifibrosis factor that protects cells from hypoxic pulmonary damage through its receptors.
Characterizing the Escherichia coli O157:H7 proteome including protein associations with higher order assemblies.
Source
J. Craig Venter Institute, Rockville, Maryland, United States of America. rpieper@jcvi.org
Abstract
BACKGROUND:
The recent outbreak of severe infections with Shiga toxin (Stx) producing Escherichia coli (STEC) serotype O104:H4 highlights the need to understand horizontal gene transfer among E. coli strains, identify novel virulence factors and elucidate their pathogenesis. Quantitative shotgun proteomics can contribute to such objectives, allowing insights into the part of the genome translated into proteins and the connectivity of biochemical pathways and higher order assemblies of proteins at the subcellular level.
METHODOLOGY/PRINCIPAL FINDINGS:
We examined protein profiles in cell lysate fractions of STEC strain 86-24 (serotype O157:H7), following growth in cell culture or bacterial isolation from intestines of infected piglets, in the context of functionally and structurally characterized biochemical pathways of E. coli. Protein solubilization in the presence of Triton X-100, EDTA and high salt was followed by size exclusion chromatography into the approximate M(r) ranges greater than 280 kDa, 280-80 kDa and 80-10 kDa. Peptide mixtures resulting from these and the insoluble fraction were analyzed by quantitative 2D-LC-nESI-MS/MS. Of the 2521 proteins identified at a 1% false discovery rate, representing 47% of all predicted E. coli O157:H7 gene products, the majority of integral membrane proteins were enriched in the high M(r) fraction. Hundreds of proteins were enriched in a M(r) range higher than that predicted for a monomer supporting their participation in protein complexes. The insoluble STEC fraction revealed enrichment of aggregation-prone proteins, including many that are part of large structure/function entities such as the ribosome, cytoskeleton and O-antigen biosynthesis cluster.
SIGNIFICANCE:
Nearly all E. coli O157:H7 proteins encoded by prophage regions were expressed at low abundance levels or not detected. Comparative quantitative analyses of proteins from distinct cell lysate fractions allowed us to associate uncharacterized proteins with membrane attachment, potential participation in stable protein complexes, and susceptibility to aggregation as part of larger structural assemblies.
A late phase of LTD in cultured cerebellar Purkinje cells requires persistent dynamin-mediated endocytosis.
Source
The Solomon H. Snyder Dept. of Neuroscience, The Johns Hopkins Univ. School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205. dlinden@jhmi.edu.
Abstract
Long-term synaptic depression (LTD) of cerebellar parallel fiber-Purkinje cell synapses is a form of use-dependent synaptic plasticity that may be studied in cell culture. One form of LTD is induced postsynaptically through an mGlu1/Ca influx/protein kinase Cα (PKCα) cascade, and its initial expression requires phosphorylation of ser-880 in the COOH-terminal PDZ-ligand region of GluA2 and consequent binding of PICK1. This triggers postsynaptic clathrin/dynamin-mediated endocytosis of GluA2-containing surface AMPA receptors. Cerebellar LTD also has a late phase beginning 45-60 min after induction that is blocked by transcription or translation inhibitors. Here, I have sought to determine the expression mechanism of this late phase of LTD by applying various drugs and peptides after the late phase has been established. Neither bath application of mGluR1 antagonists (JNJ-16259685, LY-456236) nor the PKC inhibitor GF-109203X starting 60-70 min after LTD induction attenuated the late phase. Similarly, achieving the whole cell configuration with a second pipette loaded with the peptide PKC inhibitor PKC(19-36) starting 60 min postinduction also failed to alter the late phase. Late internal perfusion with peptides designed to disrupt PICK1-GLUA2 interaction or PICK1 dimerization failed to impact late phase LTD expression. However, late internal perfusion with two different blockers of dynamin, the drug dynasore and a dynamin inhibitory peptide (QVPSRPNRAP), produced rapid and complete reversal of cerebellar LTD expression. These findings suggest that the protein synthesis-dependent late phase of LTD requires persistent dynamin-mediated endocytosis, but not persistent PICK1-GluA2 binding nor persistent activation of the upstream mGluR1/PKCα signaling cascade.
[Screening of optimal signal peptide for heterologous xylanase secretion by Bacillus subtilis].
Source
Department of Animal Science, Northwest A&F University, Yangling 712100, China.
Abstract
OBJECTIVE:
We searched optimal signal peptide for heterologous and exogenous secretion of xylanase in Bacillus subtilis.
METHODS:
We constructed a screening vector for signal peptides from B. subtilis. The Alkali resistance xylanase gene (xynA) from Bacillus pumilus was chosen as reporter gene and cloned into E. coli and B. subtilis shuttle vector pGJ148 which has maltose-inducible promoter Pglv and spectinomycin resistant gene. 24 Sec-type signal peptides (SPs) was amplified from B. Subtilis 1A747 and cloned into the screening vector for the expression of xynA in B. Subtilis WB700. The xylanase activity of the culture supernatant were detected after 24h incubation.
RESULTS:
The screening of these signal peptides revealed differences in xylanase activity of the culture supernatants, The recombinant strain containing YnfF signal peptide showed the highest xylanase acitivity (37.2 IU/mL).
CONCLUSION:
Experiment proved screening of signal peptides is effective way for optimization of the export of heterologous protein in B. subtilis.
- PMID:
- 22043800
- [PubMed - indexed for MEDLINE]
Production of N-acetylgalactosaminyl-transferase 2 (GalNAc-T2) fused with secretory signal Igκ in insect cells.
Source
Department of Immunology, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic; Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL, USA.
Abstract
The human UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyl-transferase 2 (GalNAc-T2) is one of the key enzymes that initiate synthesis of hinge-region O-linked glycans of human immunoglobulin A1 (IgA1). We designed secreted soluble form of human GalNAc-T2 as a fusion protein containing mouse immunoglobulin light chain kappa secretory signal and expressed it using baculovirus and mammalian expression vectors. The recombinant protein was secreted by insect cells Sf9 and human HEK 293T cells in the culture medium. The protein was purified from the media using affinity Ni-NTA chromatography followed by stabilization of purified protein in 50mM Tris-HCl buffer at pH 7.4. Although the purity of recombinant GalNAc-T2 was comparable in both expression systems, the yield was higher in Sf9 insect expression system (2.5mg of GalNAc-T2 protein per 1L culture medium). The purified soluble recombinant GalNAc-T2 had an estimated molecular mass of 65.8kDa and its amino-acid sequence was confirmed by mass-spectrometric analysis. The enzymatic activity of Sf9-produced recombinant GalNAc-T2 was determined by the quantification of enzyme-mediated attachment of GalNAc to synthetic IgA1 hinge-region peptide as the acceptor and UDP-GalNAc as the donor. In conclusion, murine immunoglobulin kappa secretory signal was used for production of secreted enzymatically active GalNAc-T2 in insect baculovirus expression system.
Copyright © 2011 Elsevier Inc. All rights reserved.
TRAIL promotes a pro-survival signal in erythropoietin-deprived human erythroblasts through the activation of an NF-kB/IkBalpha pathway.
Source
Dipartimento di Medicina e Scienze dell 'Invecchiamento, Università G. d 'Annunzio Chieti-Pescara, Chieti, Italy.
Abstract
The biological activity of TNF-related apoptosis inducing ligand (TRAIL) was analyzed in primary human erythroblasts derived from mononuclear cells of blood donors, kept in culture in the presence of 20 percent foetal calf serum, growth factors (EPO, SCF, IL-3) and glucocorticoids (10-6 M dexamethasone, 10-6 M oestradiol) or under growth factor and serum starvation. In the presence of growth factors and serum, primary erythroblasts showed a differential expression of TRAIL-Receptors (Rs) at various degrees of maturation and responded to TRAIL treatment with a mild cytotoxicity. On the other hand, in the absence of serum and growth factors, TRAIL treatment unexpectedly up-regulated TRAIL-R4 decoy receptor and promoted erythroblast survival. The concomitant activation of NF-kB/IkB survival pathway was detected with Western blotting and immunofluorescence procedures and confirmed by experiments performed with SN50, a pharmacological inhibitor of the NF-kB/IkB pathway. Our study indicates that TRAIL has a twofold activity on erythroid lineages: it induces a mild erythroid cell cytotoxicity in the presence of serum and growth factors, while it promotes erythroid cell survival through the activation of the NF-kB/IkB pathway under starvation conditions.
- PMID:
- 22023762
- [PubMed - indexed for MEDLINE]
Biomimetic self-templating supramolecular structures.
Source
Department of Bioengineering, University of California, Berkeley, California 94720, USA.
Abstract
In nature, helical macromolecules such as collagen, chitin and cellulose are critical to the morphogenesis and functionality of various hierarchically structured materials. During tissue formation, these chiral macromolecules are secreted and undergo self-templating assembly, a process whereby multiple kinetic factors influence the assembly of the incoming building blocks to produce non-equilibrium structures. A single macromolecule can form diverse functional structures when self-templated under different conditions. Collagen type I, for instance, forms transparent corneal tissues from orthogonally aligned nematic fibres, distinctively coloured skin tissues from cholesteric phase fibre bundles, and mineralized tissues from hierarchically organized fibres. Nature's self-templated materials surpass the functional and structural complexity achievable by current top-down and bottom-up fabrication methods. However, self-templating has not been thoroughly explored for engineering synthetic materials. Here we demonstrate the biomimetic, self-templating assembly of chiral colloidal particles (M13 phage) into functional materials. A single-step process produces long-range-ordered, supramolecular films showing multiple levels of hierarchical organization and helical twist. Three distinct supramolecular structures are created by this approach: nematic orthogonal twists, cholesteric helical ribbons and smectic helicolidal nanofilaments. Both chiral liquid crystalline phase transitions and competing interfacial forces at the interface are found to be critical factors in determining the morphology of the templated structures during assembly. The resulting materials show distinctive optical and photonic properties, functioning as chiral reflector/filters and structural colour matrices. In addition, M13 phages with genetically incorporated bioactive peptide ligands direct both soft and hard tissue growth in a hierarchically organized manner. Our assembly approach provides insight into the complexities of hierarchical assembly in nature and could be expanded to other chiral molecules to engineer sophisticated functional helical-twisted structures.
Comment in
Cloning and expression of hydroxynitrile lyase gene from Eriobotrya japonica in Pichia pastoris.
Source
Institute of Pharmaceutical Biotechnology, Fuzhou University, Fuzhou, Fujian 350108, China.
Abstract
Hydroxynitrile lyase gene (hnl) from Eriobotrya japonica was successfully amplified using the method of SEFA PCR (Self-Formed Adaptor PCR). The complete sequence was 5.5 kbp in length, including 3100 bp of the upstream promoter region, 1659 bp of the coding sequence, three introns and 315 bp of the downstream transcription terminator. The phylogenetic analysis illustrated that the obtained hnl exhibited 66-70% identity to the reported isozymes from almond, black cherry and Japanese apricot. The EjHNL had 552 amino acids including a 25 amino acid-long signal peptide. The conserved characteristic structures of HNLs, such as FAD-binding motif, N-glycosylation sites and active sites were observed. The coding sequence of the hnl was inserted into pPIC9K vector for heterologous expression in Pichia pastoris. The HNL activity of the culture supernatant reached 15 U/ml after 96 h of induction by methanol. The specific activity of the recombinant HNL was about 197 U/mg. The enantiomeric excess value of the product R-mandelonitrile attained 98.6% and the value of K(m) of the recombinant HNL was determined to be 0.47 mM based on the kinetic data. The optimum temperature and pH of the recombinant HNL were 40°C and 6.0 respectively. The experimental data indicated that the obtained recombinant HNL showed similar catalytic characteristics with the natural EjHNL. The expression of the recombinant HNL in P. pastoris could present another available biocatalyst for the synthesis of R-selective cyanohydrins.
Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.
Hypoxia potentiates microRNA-mediated gene silencing through posttranslational modification of Argonaute2.
Source
Graduate Program in Biochemistry, Sackler School of Graduate Biomedical Sciences, Boston, Massachusetts 02111, USA.
Abstract
Hypoxia contributes to the pathogenesis of various human diseases, including pulmonary artery hypertension (PAH), stroke, myocardial or cerebral infarction, and cancer. For example, acute hypoxia causes selective pulmonary artery (PA) constriction and elevation of pulmonary artery pressure. Chronic hypoxia induces structural and functional changes to the pulmonary vasculature, which resembles the phenotype of human PAH and is commonly used as an animal model of this disease. The mechanisms that lead to hypoxia-induced phenotypic changes have not been fully elucidated. Here, we show that hypoxia increases type I collagen prolyl-4-hydroxylase [C-P4H(I)], which leads to prolyl-hydroxylation and accumulation of Argonaute2 (Ago2), a critical component of the RNA-induced silencing complex (RISC). Hydroxylation of Ago2 is required for the association of Ago2 with heat shock protein 90 (Hsp90), which is necessary for the loading of microRNAs (miRNAs) into the RISC, and translocation to stress granules (SGs). We demonstrate that hydroxylation of Ago2 increases the level of miRNAs and increases the endonuclease activity of Ago2. In summary, this study identifies hypoxia as a mediator of the miRNA-dependent gene silencing pathway through posttranslational modification of Ago2, which might be responsible for cell survival or pathological responses under low oxygen stress.
- PMID:
- 21969601
- [PubMed - indexed for MEDLINE]
- PMCID: PMC3232924
- [Available on 2012/6/1]
[The regulated effect of the coded amino acids on the basic cellular processes in young and old animals].
Abstract
The aspects of the regulatory effect of 20 coded amino acids on the basic cellular processes--proliferation and apoptosis--are discussed. This effect is performed due to the regulation by the amino acids of the specific genes at the levels of the transcription and translation. This leads to the triggering of the regulation of any cellular processes. The investigations in organotypic culture of the tissues of the different genesis have demonstrated that the different amino acids perform the cell proliferation or apoptosis. The group of low molecular mass hydrophile amino acids with the charge chains stimulated the cell proliferation in the tissues of mesodermal genesis. The other group of high molecular mass hydrophobe amino acids stimulated the cell proliferation in the tissues of ectodermal genesis. Thus, the coded amino acids are not only the structural elements of the proteins, but can participate in the regulation of the specific genes, controlling the cellular cycle. The number of the active amino acids is decreased in 2.7 times in the explants from the old rats, as compared to the young, reflecting the disturbances in the amino acid transport and gene expression by the aging.
- PMID:
- 21957573
- [PubMed - indexed for MEDLINE]
A new daunomycin-peptide conjugate: synthesis, characterization and the effect on the protein expression profile of HL-60 cells in vitro.
Source
Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Eötvös Loránd University, Budapest, Hungary.
Abstract
Daunomycin (Dau) is a DNA-binding antineoplastic agent in the treatment of various types of cancer, such as osteosarcomas and acute myeloid leukemia. One approach to improve its selectivity and to decrease the side effects is the conjugation of Dau with oligopeptide carriers, which might alter the drug uptake and intracellular fate. Here, we report on the synthesis, characterization, and in vitro biological properties of a novel conjugate in which Dau is attached, via an oxime bond, to one of the cancer specific small peptides (LTVSPWY) selected from a random phage peptide library. The in vitro cytostatic effect and cellular uptake of Dau═Aoa-LTVSPWY-NH(2) conjugate were studied on various human cancer cell lines expressing different levels of ErbB2 receptor which could be targeted by the peptide. We found that the new daunomycin-peptide conjugate is highly cytostatic and could be taken up efficiently by the human cancer cells studied. However, the conjugate was less effective than the free drug itself. RP-HPLC data indicate that the conjugate is stable at least for 24 h in the pH 2.5-7.0 range of buffers, as well as in cell culture medium. The conjugate in the presence of rat liver lysosomal homogenate, as indicated by LC-MS analysis, could be degraded. The smallest, Dau-containing metabolite (Dau═Aoa-Leu-OH) identified and prepared expresses DNA-binding ability. In order to get insight on the potential mechanism of action, we compared the protein expression profile of HL-60 human leukemia cells after treatment with the free and peptide conjugated daunomycin. Proteomic analysis suggests that the expression of several proteins has been altered. This includes three proteins, whose expression was lower (tubulin β chain) or markedly higher (proliferating cell nuclear antigen and protein kinase C inhibitor protein 1) after administration of cells with Dau-conjugate vs free drug.
[Self-synchronization of the protein synthesis rhythm in HaCaT cultures of human keratinocytes].
Abstract
In cultures of human keratinocytes HaCaT contained in a serum-free medium on glass, a circahoralian rhythm of proteinsynthesis was found similar to the one in hepatocytes in vitro. The intensity of the synthesis was determined by the inclusion of 3H-leucine corrected for the pool of free marked leucine. Rhythm was studied in washed 1- or 2-day cultures after the change of the medium. The medium conditioned with keratinocytes HaCaT synchronized the rarefied hepatocyte cultures nonsynchronous in the control. Therefore, the keratinocytes liberate synchronizing factors into the medium. A BAPTA-AM chelator of calcium ions eliminates the protein synthesis rhythm both in dense hepatocyte cultures synchronous in the control and in the HaCaT keratinocyte cultures. The effect of the H7 inhibitor of protein kinases was analogous. Thus, both in keratinocytes and hepatocytes, self-synchronization of fluctuations of the intensity of protein synthesis takes place. The mechanism of self-synchronization is the calcium-depending phosphorylation of cell proteins.
- PMID:
- 21950057
- [PubMed - indexed for MEDLINE]
Basic peptide-morpholino oligomer conjugate that is very effective in killing bacteria by gene-specific and nonspecific modes.
Source
Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA.
Abstract
Basic peptides covalently linked to nucleic acids, or chemically modified nucleic acids, enable the insertion of such a conjugate into bacteria grown in liquid medium and mammalian cells in tissue culture. A unique peptide, derived from human T cells, has been employed in a chemical synthesis to make a conjugate with a morpholino oligonucleotide. This new conjugate is at least 10- to 100-fold more effective than previous peptides used in altering the phenotype of host bacteria if the external guide sequence methodology is employed in these experiments. Bacteria with target genes expressing chloramphenicol resistance, penicillin resistance, or gyrase A function can effectively be reduced in their expression and the host cells killed. Several bacteria are susceptible to this treatment, which has a broad range of potency. The loss in viability of bacteria is not due only to complementarity with a target RNA and the action of RNase P, but also to a non-gene-specific tight binding of the complexed nontargeted RNA to the basic polypeptide-morpholino oligonucleotide.
- PMID:
- 21949365
- [PubMed - indexed for MEDLINE]
- PMCID: PMC3189074
- [Available on 2012/4/4]
The unfolded protein response is a major mechanism by which LRP1 regulates Schwann cell survival after injury.
Source
Department of Anesthesiology, University of California, San Diego, La Jolla, California 92093-0629, USA.
Abstract
In peripheral nerve injury, Schwann cells (SCs) must survive to exert a continuing and essential role in successful nerve regeneration. Herein, we show that peripheral nerve injury is associated with activation of endoplasmic reticulum (ER) stress and the adaptive unfolded protein response (UPR). The UPR culminates in expression of C/EBP homology protein (CHOP), a proapoptotic transcription factor in SCs, unless counteracted by LDL receptor-related protein-1 (LRP1), which serves as a major activator of phosphatidylinositol 3-kinase (PI3K). Sciatic nerve crush injury in rats induced expression of the ER chaperone GRP78/BIP, reflecting an early, corrective phase of the UPR. However, when LRP1 signaling was inhibited with receptor-associated protein, PI3K activity was decreased and CHOP protein expression increased, particularly in myelinating SCs. In cultured SCs, the PKR-like ER kinase target eIF2α was phosphorylated and CHOP was induced by (1) inhibiting PI3K, (2) treating the cells with tumor necrosis factor-α (TNF-α), or (3) genetic silencing of LRP1. CHOP gene deletion in SCs decreased cell death in response to TNF-α. Furthermore, the effects of TNF-α on phosphorylated eIF2α, CHOP, and SC death were blocked by adding LRP1 ligands that augment LRP1-dependent cell signaling to PI3K. Collectively, our results support a model in which UPR-activated signaling pathways represent a major challenge to SC survival in nerve injury. LRP1 functions as a potent activator of PI3K in SCs and, by this mechanism, limits SC apoptosis resulting from increased CHOP expression in nerve injury.
- PMID:
- 21940431
- [PubMed - indexed for MEDLINE]
- PMCID: PMC3188465
- [Available on 2012/3/21]
Increased phagocyte-like NADPH oxidase and ROS generation in type 2 diabetic ZDF rat and human islets: role of Rac1-JNK1/2 signaling pathway in mitochondrial dysregulation in the diabetic islet.
Source
Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Detroit, Michigan, USA.
Abstract
OBJECTIVE:
To determine the subunit expression and functional activation of phagocyte-like NADPH oxidase (Nox), reactive oxygen species (ROS) generation and caspase-3 activation in the Zucker diabetic fatty (ZDF) rat and diabetic human islets.
RESEARCH DESIGN AND METHODS:
Expression of core components of Nox was quantitated by Western blotting and densitometry. ROS levels were quantitated by the 2',7'-dichlorofluorescein diacetate method. Rac1 activation was quantitated using the gold-labeled immunosorbent assay kit.
RESULTS:
Levels of phosphorylated p47(phox), active Rac1, Nox activity, ROS generation, Jun NH(2)-terminal kinase (JNK) 1/2 phosphorylation, and caspase-3 activity were significantly higher in the ZDF islets than the lean control rat islets. Chronic exposure of INS 832/13 cells to glucolipotoxic conditions resulted in increased JNK1/2 phosphorylation and caspase-3 activity; such effects were largely reversed by SP600125, a selective inhibitor of JNK. Incubation of normal human islets with high glucose also increased the activation of Rac1 and Nox. Lastly, in a manner akin to the ZDF diabetic rat islets, Rac1 expression, JNK1/2, and caspase-3 activation were also significantly increased in diabetic human islets.
CONCLUSIONS:
We provide the first in vitro and in vivo evidence in support of an accelerated Rac1-Nox-ROS-JNK1/2 signaling pathway in the islet β-cell leading to the onset of mitochondrial dysregulation in diabetes.
- PMID:
- 21911753
- [PubMed - indexed for MEDLINE]
- PMCID: PMC3198065
- [Available on 2012/11/1]
Single-pot derivatisation strategy for enhanced gliotoxin detection by HPLC and MALDI-ToF mass spectrometry.
Source
Department of Biology, National University of Ireland Maynooth, Maynooth, Co. Kildare, Ireland.
Abstract
Gliotoxin is produced by non-ribosomal peptide synthesis and secreted from certain fungi, including Aspergillus fumigatus. It is an epipolythiodioxopiperazine that contains an intact disulphide bridge and is the focus of intense research as a consequence of its negative immunomodulatory properties. Gliotoxin detection is generally enabled by reversed-phase-high-performance liquid chromatography (RP-HPLC), with absorbance detection (220-280 nm), or liquid chromatography-mass spectrometry, yet detection is not readily achievable by matrix-assisted laser desorption ionisation-time-of-flight mass spectrometry (MALDI-ToF MS). We have developed a single-pot derivatisation strategy which uses sodium borohydride-mediated reduction of gliotoxin followed by immediate alkylation of exposed thiols by 5'-iodoacetamidofluorescein to yield a stable product, diacetamidofluorescein-gliotoxin (GT-(AF)(2)), of molecular mass 1103.931 Da ((M+H)+). This product is readily detectable by RP-HPLC and exhibits a 6.8-fold increase in molar absorptivity compared with gliotoxin, which results in a higher sensitivity of detection (40 ng; 125 pmoL). GT-(AF)(2) also fluoresces (excitation/emission, 492:518 nm). Unlike free gliotoxin, the product (>800 fmol) is detectable by MALDI-ToF MS. Sporidesmin A can also be detected by RP-HPLC and MALDI-ToF MS (>530 fmol) using this strategy. We also demonstrate that the strategy facilitates detection of gliotoxin (mean ± SD = 3.55 ± 0.07 μg 100 μL(-1); n = 2) produced by A. fumigatus, without the requirement for organic extraction of culture supernatants and associated solvent removal. GT-(AF)(2) is also detectable (150 ng; 460 pmol) by thin-layer chromatography.
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