1.
A fluorescent amino acid probe to monitor efficiency of peptide conjugation to glass surfaces for high density microarrays.
Source
Department of Bioengineering and Center for Bioengineering Research, Bourns College of Engineering, University of California at Riverside, 900 University Avenue, Riverside, CA 92521, USA. jiayu.liao@ucr.edu.
Abstract
Using a fluorescent NBD amino acid, new protease substrates were developed that are attractive because of the excellent chemical stability and long wavelength of excitation (480 nm) of the NBD fluorophore. The fluorescent peptides are synthesized by Fmoc solid-phase peptide synthesis. An example peptide was efficiently immobilized onto a microarray surface using click chemistry, and its proteolysis was monitored by fluorescence imaging. Excellent site specificity was achieved for the protease. Fluorescent peptides are also used to monitor the conjugation efficiency onto a surface using a standard microarray scanner.
- PMID:
- 22241083
- [PubMed - as supplied by publisher]
The Antibiotic Thermorubin Inhibits Protein Synthesis by Binding to Inter-Subunit Bridge B2a of the Ribosome.
Source
Department of Chemistry, Yale University, 225 Prospect Street, PO Box 208107, New Haven, CT 06520-8107, USA.
Abstract
Thermorubin is a small-molecule inhibitor of bacterial protein synthesis, but relatively little is known about the molecular mechanism by which it blocks translation. The structure of the complex between thermorubin and the 70S ribosome from Thermus thermophilus reported here shows that thermorubin interacts with the ribosome in a way that is distinct from any other known class of ribosome inhibitor. Though it is structurally similar to tetracycline, it binds to the ribosome at an entirely different location-the interface between the small and large subunits that is formed by inter-subunit bridge B2a. This region of the ribosome is known to play a role in the initiation of translation, and thus, the binding site we observe is consistent with evidence suggesting that thermorubin inhibits the initiation stage of proteinsynthesis. The binding of thermorubin induces a rearrangement of two bases on helix 69 of the 23S rRNA, and presumably, this rearrangement blocks the binding of an A-site tRNA, thereby inhibiting peptide bond formation. Due in part to its low solubility in aqueous media, thermorubin has not been used clinically, although it is a potent antibacterial agent with low toxicity (Therapeutic Index>200). The interactions between thermorubin and the ribosome, as well as its adjacency to the observed binding sites of three other antibiotic classes, may enable the design of novel derivatives that share thermorubin's mode of action but possess improved pharmacodynamic properties.
Copyright © 2011. Published by Elsevier Ltd.
- PMID:
- 22240456
- [PubMed - as supplied by publisher]
Bacteriocin formation by dominant aerobic sporeformers isolated from traditional maari.
Source
Food Technology Department (DTA / IRSAT / CNRST), Ouagadougou 03 BP 7047, Burkina Faso.
Abstract
The antimicrobial activity of 8 Bacillus spp. and 2 Lysinibacillus spp. representing the predominant aerobic sporeformers during traditional maari fermentations, a traditional fermented baobab seeds product from Burkina Faso, was investigated. The antimicrobial activity was assessed against a total of 31 indicator organisms representing various Gram-negative and positive pathogens. The screening showed that 3 Bacillus subtilis strains (B3, B122 and B222) in particular had antimicrobial activity against some Gram-positive organisms and were selected for further studies. It was found that the antimicrobial substances produced were heat stable, in-sensitive to catalase, sensitive to protease and trypsin but resistant to the proteolytic action of papain and proteinase K and equally active at pH values ranging from 3 to 11. Bacteriocin secretion started in late exponential growth phase and maximum activity was detected during the stationary growth phase. The production of bacteriocin by B. subtilis B3, B122 and B222 was dependent on the aeration conditions. Maximum production of bacteriocin was observed under reduced aeration. Specific primers were used to screen isolates B3, B122 and B222 for genes involved in the synthesis of the bacteriocins subtilosin A, subtilin, sublancin and ericin. Amplicons of the expected sizes were detected for iywB, sboA, sboX, albA and spaS involved in the biosynthesis of subtilosin and subtilin, respectively. The translated nucleotide sequences had 100% identity to the YiwB, SboX and SboA amino acid sequences of the subtilosin A producing B. subtilis subsp. subtilis strain 168. Interestingly there was a 3 amino acid deletion at the N-terminal part of AlbA in B3, B122 and B222 that probably alters the activity of this enzyme. Analysis of the spaS gene sequences of B3, B122 and B222, encoding a subtilin precursorpeptide, showed that the translated nucleotide sequence had 98% identity with the corresponding SpaS amino acid sequence of subtilin producing B. subtilis subsp. spizizenii strain ATCC6633.
Copyright © 2011 Elsevier B.V. All rights reserved.
- PMID:
- 22240061
- [PubMed - as supplied by publisher]
Fluorescent Peptide-PNA Chimeras for Imaging Monoamine Oxidase A mRNA in Neuronal Cells.
Abstract
Monoamine oxidases (MAO) catalyze the oxidative deamination of many biogenic amines and are integral proteins found in the mitochondrial outer membrane. Changes in MAO-A levels are associated with depression, trait aggression and addiction. Here we report the synthesis, characterization and in vitro evaluation of novel fluorescent peptide-peptidenucleic acid (PNA) chimeras for MAOA mRNA imaging in live neuronal cells. The probes were designed to include MAOA-specific PNA dodecamers, separated by an N-terminal spacer to a m-opioid receptor targeting peptide(DAMGO), with a spacer and a fluorophore on the C-terminus. The probe was successfully delivered into human SH-SY5Y neuroblastoma cells through mu-opioid receptor-mediated endocytosis. The Kd by flow cytometry was 11.6 ± 0.8 nM. Uptake studies by fluorescence microscopy showed ~5-fold higher signal in human SH-SY5Y neuroblastoma cells than in negative control CHO-K1 cells that lack mu-opioid receptors. Moreover, a peptide-mismatch control sequence showed no significant uptake in SH-SY5Y cells. Such fluorescent mRNA imaging agents might enable real time imaging and quantitation of neuronal mRNAs in live animal models.
- PMID:
- 22239616
- [PubMed - as supplied by publisher]
Synthesis and Alignment of Discrete Polydiacetylene-Peptide Nanostructures.
Abstract
Oligopeptides bearing internal diacetylene units are shown to self-assemble in water into 1-D nanostructures and aligned macroscopic hydrogels. The diacetylene units can be photopolymerized into polydiacetylenes that run coincident to the nanostructure and noodle long axes, and the resulting nanostructures show evidence for ambipolar charge transport. This self-assembly, alignment and polymerization technique provides a rapid way to produce globally aligned collections of conjugated polymer chains.
- PMID:
- 22239459
- [PubMed - as supplied by publisher]
18F-Labeled N-(4-fluorobenzylidene)oxime-dimeric (ZHER2:477)2 .
Authors
Source
Molecular Imaging and Contrast Agent Database (MICAD) [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2004-2011.
2011 Nov 02 [updated 2012 Jan 04].
Excerpt
The 18F-labeled N-(4-fluorobenzylidene)oxime (FBO)-dimeric (ZHER2:477)2 conjugate, abbreviated as 18F-FBO-(ZHER2:477)2, is an affibody derivative synthesized by Cheng et al. for positron emission tomography (PET) of HER2-expressing tumors (1). Affibody molecules are a group of nonimmunogenic scaffold proteins that derive from the B-domain of staphylococcal surface protein A (2, 3). In the past several years, affibodies have drawn significant attention for developing imaging and therapeutic agents because of their unique features (3, 4). First, affibodies are small, with only 58 amino acid residues (~7 kDa) (3, 5). The small size allows affibodies to be generated with solid-phase peptide synthesis and to be cleared quickly from kidneys. Second, affibodies have a high binding affinity and specificity to their targets. Their binding affinity can be further improved by generating multimeric constructs through the solvent-exposed termini of affibody Z-domain. The anti-HER2 monomeric affibody ZHER2:4 is an example that has a binding affinity of ~50 nM, but its dimeric form, (ZHER2:4)2, exhibits an improved binding affinity of up to ~3 nM in vitro (6). Third, affibodies lack cysteine residues and disulfide bridges in structure, and they fold rapidly. These features make it possible to chemically synthesize fully functional molecules and to introduce unique cysteine residues or chemical groups into affibodies for site-specific labeling. Several anti-HER2 affibody derivatives have been synthesized in this way. The imaging agent HPEM-His6-(ZHER2:4)2-Cys was generated by radiobrominating the dimeric (ZHER2:4)2 through the cysteine residues that were introduced to the C-terminus of (ZHER2:4)2 (7). Several affibody derivatives (e.g., 68Ga-DOTA-ZHER2:342-pep2, 111In-DOTA-ZHER2:342-pep2, 111In-benzyl-DOTA-ZHER2:342, and 111In-benzyl-DTPA-ZHER2:342) were synthesized by coupling a chelating agent with a specifically protected site group of the ZHER2:342peptide chain (3). Furthermore, affibody proteins can be selected and optimized with a strategy of sequence mutation and affinity maturation, and an example selected with this strategy is the anti-HER2 affibody ZHER2:342, which has an increased affinity of 50 nM (ZHER2:4, the first generation) to 22 pM (8). The investigators at Stanford University first tested the feasibility of the monomeric and dimeric forms of anti-HER2 affibody ZHER2:477 for molecular imaging. Both forms of the ZHER2:477 molecule were radiofluorinated with an 18F-labeled prosthetic group of 4-18F-fluorobenzaldehyde (18F-FBO-ZHER2:477 and 18F-FBO-(ZHER2:477)2, respectively) (1). The investigators have also coupled 64Cu to the affibody through DOTA, leading to the development of imaging agents 64Cu-DOTA- ZHER2:477 and 64Cu-DOTA-(ZHER2:477)2 (9). Interestingly, these studies showed that smaller affibody constructs performed better in vivo in terms of tumor uptake and clearance in spite of the lower affinity in vitro. The investigators then generated a class of small proteins consisting of two α-helix bundles of the 3-helix affibody by deleting the helix 3 because the binding domain localizes in the α-helices 1 and 2 bundles (5). One of these 2-helix proteins is MUT-DS, which has α-helices 1 and 2 bundles, with a disulfide bridge being formed between the two inserted homocysteines (10-12). MUT-DS showed a binding affinity to HER2 in the low-nM range. The radiolabeled MUT-DS derivatives exhibited favorable pharmacokinetics for both imaging and therapy of HER2-expressing tumors (refer to MUT-DS derived agents in MICAD). This series of chapters summarizes the data obtained with the ZHER2:477 derivatives, and this chapter presents the data obtained with 18F-FBO-(ZHER2:477)2 (1).
Sections
18F-Labeled N-(4-fluorobenzylidene)oxime-monomeric ZHER2:477 .
Authors
Source
Molecular Imaging and Contrast Agent Database (MICAD) [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2004-2011.
2011 Nov 02 [updated 2012 Jan 04].
Excerpt
The 18F-labeled N-(4-fluorobenzylidene)oxime (FBO)-monomeric ZHER2:477 conjugate, abbreviated as 18F-FBO-ZHER2:477, is an affibody derivative synthesized by Cheng et al. for positron emission tomography (PET) of HER2-expressing tumors (1). Affibody molecules are a group of nonimmunogenic scaffold proteins that derive from the B-domain of staphylococcal surface protein A (2, 3). In the past several years, affibodies have drawn significant attention for developing imaging and therapeutic agents because of their unique features (3, 4). First, affibodies are small, with only 58 amino acid residues (~7 kDa) (3, 5). The small size allows affibodies to be generated with solid-phase peptide synthesis and to be cleared quickly by the kidneys. Second, affibodies have a high binding affinity and specificity to their targets. Their binding affinity can be further improved by generating multimeric constructs through the solvent-exposed termini of affibody Z-domain. The anti-HER2 monomeric affibody ZHER2:4 is an example that has a binding affinity of ~50 nM, but its dimeric form, (ZHER2:4)2, exhibits an improved binding affinity of up to ~3 nM (6). Third, affibodies lack cysteine residues and disulfide bridges in structure, and they fold rapidly. These features make it possible to chemically synthesize fully functional molecules and to introduce unique cysteine residues or chemical groups into affibodies for site-specific labeling. Several anti-HER2 affibody derivatives have been synthesized in this way. The imaging agent HPEM-His6-(ZHER2:4)2-Cys was generated by radiobrominating the dimeric (ZHER2:4)2 through the cysteine residues that were introduced to the C-terminus of (ZHER2:4)2 (7). Several affibody derivatives (e.g., 68Ga-DOTA-ZHER2:342-pep2, 111In-DOTA-ZHER2:342-pep2, 111In-benzyl-DOTA-ZHER2:342, and 111In-benzyl-DTPA-ZHER2:342) were synthesized by coupling a chelating agent with a specifically protected site group of the ZHER2:342peptide chain (3). Furthermore, these small affibody proteins can be selected and optimized with a strategy of sequence mutation and affinity maturation, and an example selected with this strategy is the anti-HER2 affibody ZHER2:342, which has an increased affinity of 50 nM (ZHER2:4, the first generation) to 22 pM (8). The investigators at Stanford University first tested the feasibility of the monomeric and dimeric forms of anti-HER2 affibody ZHER2:477 for molecular imaging. Both forms of the ZHER2:477 molecule were radiofluorinated with an 18F-labeled prosthetic group of 4-18F-fluorobenzaldehyde (18F-FBO-ZHER2:477 and 18F-FBO-(ZHER2:477)2, respectively) (1). The investigators have also coupled 64Cu to the affibody through DOTA, leading to the development of imaging agents 64Cu-DOTA- ZHER2:477 and 64Cu-DOTA-(ZHER2:477)2 (9). Interestingly, these studies showed that smaller affibody constructs performed better in vivo in terms of tumor uptake and clearance in spite of the lower affinity in vitro. The investigators then generated a class of small proteins consisting of two α-helix bundles of the 3-helix affibody by deleting the helix 3 because the binding domain localizes in the α-helices 1 and 2 bundles (5). One of these 2-helix proteins is MUT-DS, which has α-helices 1 and 2 bundles, with a disulfide bridge being formed between the two inserted homocysteines (10-12). MUT-DS showed a binding affinity to HER2 in the low-nM range. The radiolabeled MUT-DS derivatives exhibited favorable pharmacokinetics for both imaging and therapy of HER2-expressing tumors (refer to MUT-DS derived agents in MICAD). This series of chapters summarizes the data obtained with the ZHER2:477 derivatives, and this chapter presents the data obtained with 18F-FBO-ZHER2:477 (1).
Sections
Unveiling the biosynthetic puzzle of destruxins in Metarhizium species.
Source
Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, People's Republic of China.
Abstract
Insect pathogenic fungi produce a plethora of insecticidally and pharmaceutically active compounds, including 39 cyclohexadepsipeptide destruxins (dtxs). Even though dtxs were first discovered more than 50 y ago, the genes responsible for their biosynthesis were unknown until this study. Based on our comparative genomic information and targeted gene disruptions, we report the gene cluster for dtx biosynthesis in the insect pathogen Metarhizium robertsii. The nonribosomal peptide synthetase DtxS1 has six adenylation domains, two of which are capable of selecting different amino acids to synthesize dtx B and its analogs. The cytochrome P450 enzyme DtxS2 converts dtx B into other dtxs by a chain of reactions, each producing a new derivative. The aldo-keto reductase DtxS3 and aspartic acid decarboxylase DtxS4 are responsible for the conversion and provision of the first and last substrates for the dtx assembly line, respectively. Insect bioassays showed that dtxs could suppress both cellular and humoral immune responses thereby assisting fungal propagation in insects. The differing abilities of Metarhizium species to produce toxins is dependent on the presence of the dtxS1 gene. The toxigenic species are capable of killing multiple orders of insects, whereas the nontoxigenic Metarhizium spp. have narrow host ranges. Thus, the acquisition or retention of the dtx biosynthesis gene cluster in Metarhizium lineages has been coordinated with the evolution of fungal host specificity. The data from this study will facilitate the development of dtxs as bioinsecticides or pharmaceuticals.
Clinical efficacy and safety of once-weekly glucagon-like Peptide-1 agonists in development for treatment of type 2 diabetes mellitus in adults.
Source
Cabarrus Family Medicine, Harrisburg, NC.
Abstract
OBJECTIVE:
To review pharmacologic, pharmacokinetic, efficacy, and safety data of once-weekly glucagon-likepeptide-1 (GLP-1) agonists exenatide long-acting release (LAR), albiglutide, and taspoglutide in treatment of type 2 diabetes mellitus (T2DM).
DATA SOURCES:
A MEDLINE search (1966-August 2011) was conducted using the following key words: type 2 diabetes mellitus, glucagon-like peptide-1 agonists once weekly, glucagon-like peptide-1 agonists, exenatide LAR, albiglutide, and taspoglutide.
STUDY SELECTION AND DATA EXTRACTION:
All articles published in English identified from the data sources were evaluated, prioritizing randomized controlled trials with human data. The references of published articles identified were examined for additional studies appropriate for the review.
DATA SYNTHESIS:
Native GLP-1 increases glucose-dependent insulin secretion, decreases glucagon secretion, and slows gastric emptying in healthy individuals, but these effects may be blunted in patients with T2DM. Because native GLP-1 is rapidly degraded by dipeptidyl peptidase-IV, it is not a practical treatment option. Currently, 2 GLP-1 receptor agonists have been approved by the Food and Drug Administration: exenatide twice daily and liraglutide once daily. Several additional GLP-1 agonists, including exenatide LAR, albiglutide, and taspoglutide, are in various stages of clinical trials and have been modified to increase their half-lives. These agents have shown significant improvements in hemoglobin A(1c), fasting plasma glucose, and postprandial plasma glucose, as well as improvements in body weight, blood pressure, and lipid parameters. These agents allow for less-frequent dosing schedules, improved glycemic control throughout the day, and improved treatment satisfaction compared to some available agents. GLP-1 agonists have been well tolerated, with the most common adverse effects being gastrointestinal related, which occurred early in therapy but typically resolved after 4-8 weeks. The incidence of hypoglycemia was infrequent and mild during therapy.
CONCLUSIONS:
Once-weekly GLP-1 agonists provide similar glycemic control with weight reduction, as well as overall higher treatment satisfaction for patients because of their ease of use and need for less-frequent dosing compared to some available agents.
Bio-inspired Silicification of Silica-binding Peptide-Silk Protein Chimeras: Comparison of Chemically and Genetically Produced Proteins.
Abstract
Novel protein chimeras constituted of 'silk' and a silica-binding peptide (KSLSRHDHIHHH) were synthesized by genetic or chemical approaches and their influence on silica-silk based chimera composite formation evaluated. Genetic chimeras were constructed from 6 or 15 repeats of the 32 amino acid consensus sequence of Nephila clavipes spider silk ([SGRGGLGGQG AGAAAAAGGA GQGGYGGLGSQG]n) to which one silica binding peptide was fused at the N terminus. For the chemical chimera, 25 equivalents of the silica binding peptide were chemically coupled to natural Bombyx mori silk after modification of tyrosine groups by diazonium coupling and EDC/NHS activation of all acid groups. After silica formation under mild, biomaterial compatible conditions the effect of peptide addition on the properties of the silk and chimeric silk-silica composite materials was explored. The composite biomaterial properties could be related to the extent of silica condensation and to the higher number of silica binding sites in the chemical chimera as compared to the genetically derived variants. In all cases, the structure of the protein / chimera in solution dictated the type of composite structure that formed with the silica deposition process having little effect on the secondary structural composition of the silk based materials. Similarly to our study of genetic silk based chimeras containing the R5 peptide (SSKKSGSYSGSKGSKRRIL), the role of the chimeras (genetic and chemical) used in the present study resided more in aggregation and scaffolding than in the catalysis of condensation. The variables of peptideidentity, silk construct (number of consensus repeats or silk source) and approach to synthesis (genetic or chemical) can be used to 'tune' the properties of the composite materials formed and is a general approach which can be used to prepare a range of materials for biomedical and sensor based applications.
A defect in the synthesis of Interferon-γ by the T cells of Complement-C5 deficient mice leads to enhanced susceptibility for tuberculosis.
Source
Department of Pathology and Laboratory Medicine, University of Texas Health Sciences Center, Houston, TX 77030, USA.
Abstract
Interferon-γ (IFNγ) plays a major role during host defense against Mycobacterium tuberculosis (Mtb). T cells produce IFNγ in response to IL-12 and IL-18 secreted from Mtb infected macrophages. IFNγ in turn, induces nitric oxide secretion in macrophages that kills Mtb. IFNγ knockout mice are thus hyper-susceptible to tuberculosis. We reported earlier that Complement-C5 deficient (C5(-/-)) congenic mice are more susceptible to tuberculosis and showed reduced IL-12 synthesis in their macrophages. Using C5(-/-) congenic mice that carry a deletion in the C5 gene and the wild type C5(+/+) mice, we demonstrate here that, the C5(-/-) derived CD3(+) T cells, have an additional defect in the synthesis of IFNγ. C5(-/-) T cells produced lower levels of IFNγ upon stimulation by antigen presenting cells (APCs) infected with Mtb or when stimulated directly with a combination of IL-12 and IL-18. The latter was in part due to a reduced phosphorylation of STAT4 following IL-12/IL-18 stimulation. Addition of C5a peptide to IL-12/IL-18 partially restored STAT4 phosphorylation and IFNγ synthesis in C5(-/-) T cells indicating that IL-12/IL-18 mediated signaling within CD3(+) T cells involves C5a peptide. Finally, C5(-/-) T cells derived from M. bovis BCG or Mtb infected mice showed a reduced expression of T-bet (T-box expressed in T cells) transcription factor, which correlated well with a reduced T cell secretion of IFNγ. Since T-bet mediated IFNγ synthesis facilitates Th1 expansion, C5(-/-) mouse derived T cells appear to have an intrinsic defect in the production of IFNγ, which is related to C5 deficiency and this may explain their increased susceptibility to infection with Mtb and BCG.
Copyright © 2011 Elsevier Ltd. All rights reserved.
[The role of apelin in pathogenesis of cardiovascular diseases and metabolic disorders].
Source
Katedra i Zakład Fizjologii Doświadczalnej i Klinicznej, Warszawski Uniwersytet Medyczny, Warszawa. agnieszka.cudnoch@wum.edu.pl
Abstract
Apelin is a recently discovered biologically active peptide present in several isoforms that are agonists for orphan receptor APJ. Apelin and APJ receptor were found in the central nervous system and in different peripheral tissues. In the cardiovascular system the peptide is present both in the heart and in the endothelium and smooth muscles cells of the vascular wall. Acting on cardiomyocytes apelin exerts positive inotropic effect, in the endothelium it releases nitric oxide, which mediates its vasodilatory action, while acting directly on smooth muscles cells it causes vasoconstriction. Apelin interacts with other compounds regulating blood pressure; for instance with angiotensin II, vasopressin, and with the sympathetic nervous system. Special attention is focused on the possibility of positive role of apelin in hypertension, initial stages of heart failure and ischaemic heart disease. Synthesis of apelin in adipocytes permits to include thispeptide among adipokines. In the adipose tissue its production is increased in obesity and by insulin. It appears that apelin may play essential role in pathogenesis of insulin-resistant obesity. In patients with type 2 diabetes apelin improves glucose tolerance in initial stages of the illness. However, further experimental and clinical studies are required for full evaluation of significance of positive and negative aspects of the role of apelin in the cardiovascular and metabolic diseases.
- PMID:
- 22125210
- [PubMed - in process]
Are amyloid-degrading enzymes viable therapeutic targets in Alzheimer's disease?
Source
Institute of Molecular & Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK Sechenov Institute of Evolutionary Physiology and Biochemistry of Russian Academy of Sciences, St. Petersburg, Russia.
Abstract
J. Neurochem. (2012) 120 (Suppl. 1), 167-185. ABSTRACT: The amyloid cascade hypothesis of Alzheimer's disease envisages that the initial elevation of amyloid β-peptide (Aβ) levels, especially of Aβ(1-42) , is the primary trigger for the neuronal cell death specific to onset of Alzheimer's disease. There is now substantial evidence that brain amyloid levels are manipulable because of a dynamic equilibrium between their synthesis from the amyloid precursor protein and their removal by amyloid-degrading enzymes (ADEs) providing a potential therapeutic strategy. Since the initial reports over a decade ago that two zinc metallopeptidases, insulin-degrading enzyme and neprilysin (NEP), contributed to amyloid degradation in the brain, there is now an embarras de richesses in relation to this category of enzymes, which currently number almost 20. These now include serine and cysteine proteinases, as well as numerous zinc peptidases. The experimental validation for each of these enzymes, and which to target, varies enormously but up-regulation of several of them individually in mouse models of Alzheimer's disease has proved effective in amyloid and plaque clearance, as well as cognitive enhancement. The relative status of each of these enzymes will be critically evaluated. NEP and its homologues, as well as insulin-degrading enzyme, remain as principal ADEs and recently discovered mechanisms of epigenetic regulation of NEP expression potentially open new avenues in manipulation of AD-related genes, including ADEs.
© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.
Activation of α-secretase cleavage.
Source
Institute of Pharmacy and Biochemistry, Johannes Gutenberg University Mainz, Johann-Joachim-Becherweg 30, Mainz, Germany.
Abstract
J. Neurochem. (2012) 120 (Suppl. 1), 46-54. ABSTRACT: Alpha-secretase-mediated cleavage of the amyloid precursor protein (APP) releases the neuroprotective APP fragment sαAPP and prevents amyloid β peptide (Aβ) generation. Moreover, α-secretase-like cleavage of the Aβ transporter 'receptor for advanced glycation end products' counteracts the import of blood Aβ into the brain. Assuming that Aβ is responsible for the development of Alzheimer's disease (AD), activation of α-secretase should be preventive. α-Secretase-mediated APP cleavage can be activated via several G protein-coupled receptors and receptor tyrosine kinases. Protein kinase C, mitogen-activated protein kinases, phosphatidylinositol 3-kinase, cAMP and calcium are activators of receptor-induced α-secretase cleavage. Selective targeting of receptor subtypes expressed in brain regions affected by AD appears reasonable. Therefore, the PACAP receptor PAC1 and possibly the serotonin 5-HT(6) receptor subtype are promising targets. Activation of APP α-secretase cleavage also occurs upon blockade of cholesterol synthesis by statins or zaragozic acid A. Under physiological statin concentrations, the brain cholesterol content is not influenced. Statins likely inhibit Aβ production in the blood by α-secretase activation which is possibly sufficient to inhibit AD development. A disintegrin and metalloproteinase 10 (ADAM10) acts as α-secretase on APP. By targeting the nuclear retinoic acid receptor β, the expression of ADAM10 and non-amyloidogenic APP processing can be enhanced. Excessive activation of ADAM10 should be avoided because ADAM10 and also ADAM17 are not APP-specific. Both ADAM proteins cleave various substrates, and therefore have been associated with tumorigenesis and tumor progression.
© 2011 The Author. Journal of Neurochemistry © 2011 International Society for Neurochemistry.
Mechanism for release of alkaline phosphatase caused by glycosylphosphatidylinositol deficiency in patients with hyperphosphatasia-mental retardation syndrome.
Source
Research Institute for Microbial Diseases, Osaka University, Japan;
Abstract
Hyperphosphatasia Mental Retardation syndrome (HPMR), an autosomal recessive disease characterized by mental retardation and elevated serum alkaline phosphatase (ALP) levels, is caused by mutations in the coding region of the phosphatidylinositol glycan anchor biosynthesis, class V (PIGV) gene, the product of which is a mannosyltransferase essential for glycosylphosphatidylinositol (GPI) biosynthesis. Mutations found in four families caused amino acid substitutions A341E, A341V, Q256K and H385P, which drastically decreased expression of PIGV protein. Hyperphosphatasia resulted from secretion of ALP, a GPI-anchored protein normally expressed on the cell surface, into serum due to PIGV deficiency. In contrast, a previously reported PIGM deficiency, in which there is a defect in the transfer of the first mannose, does not result in hyperphosphatasia. To provide insights into the mechanism of ALP secretion in HPMR patients, we took advantage of CHO cell mutants that are defective in various steps of GPIbiosynthesis. Secretion of ALP requires GPI transamidase, which in normal cells, cleaves C-terminal GPI attachment signal peptide and replaces it with GPI. The GPI-anchored protein was secreted substantially into medium from PIGV-, PIGB- and PIGF-deficient CHO cells, in which incomplete GPI bearing mannose was accumulated. In contrast, ALP was degraded in PIGL-, DPM2- or PIGX-deficient CHO cells, in which incomplete shorter GPIs that lacked mannose were accumulated. Our results suggest that GPI transamidase recognizes incomplete GPI bearing mannose and cleaves a hydrophobic signal peptide, resulting in secretion of soluble ALP. These results explain the molecular mechanism of hyperphosphatasia in HPMR.
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.
Copyright © 2012. Published by Elsevier Inc.
Potential of the gastric motility drug lorglumide in prostate cancer imaging.
Source
Department of Neuroradiology, University of Tübingen, Germany; Peptide Synthesis Laboratory, Interfaculty Institute of Biochemistry, University of Tübingen, Germany.
Abstract
The use of tissue-specific receptor ligands is a promising approach for cancer diagnostics and therapy. Lorglumide, a highly effective competitive ligand for the cholecystokinine-A receptor (CCKRA) was conjugated to a fluorescent dye and a magnetic resonance imaging (MRI) contrast agent to obtain a bifunctional marker for tissue with high CCKRA expression. An intermediate conjugate containing only lorglumide and a fluorescent dye was also produced. By performing CCKRA mRNA expression analysis on carcinoma cell lines we found that CCKRA is highly expressed in PC3 prostate carcinoma cells compared to U373 glioma and U2OS osteosarcoma cells. Uptake, specificity and detection sensitivity of both lorglumide conjugates was evaluated by confocal laser scanning microscopy, fluorescence activated cell sorting (FACS) and magnetic resonance relaxometry. While the conjugate containing only lorglumide and rhodamine isothiocyanate as fluorescent dye showed clearly higher uptake than the bifunctional conjugate in FACS analysis, both conjugates clearly showed preferential staining of the PC3 prostate carcinoma cells. Magnetic resonance relaxometry experiments with the bifunctional conjugate containing the MRI contrast agent gadolinium-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid confirmed the higher PC3-affinity of the lorglumide ligand. Confocal laser scanning microscopy images of PC3/U2OS mixed cell cultures incubated with the bifunctional conjugate also clearly showed PC3 preference and cytoplasmic dot-like staining concurring with uptake by receptor binding and subsequent receptor internalization. Considering these results, CCKRA ligands like lorglumide could play a role in the future design of prostate-cancer-specific markers.
Copyright © 2012. Published by Elsevier B.V.
Click Chemistry Facilitates Formation of Reporter Ions and SimplifiedSynthesis of Amine-Reactive Multiplexed Isobaric Tags for Protein Quantification.
Abstract
We report the development of novel reagents for cell-level protein quantification, referred to as Caltech Isobaric Tags (CITs), which offer several advantages in comparison with other isobaric tags (e.g., iTRAQ and TMT). Click chemistry, copper-catalyzed azide-alkyne cycloaddition (CuAAC), is applied to generate a gas-phase cleavable linker suitable for the formation of reporter ions. Upon collisional activation, the 1,2,3-triazole ring constructed by CuAAC participates in a nucleophilic displacement reaction forming a six-membered ring and releasing a stable cationic reporter ion. To investigate its utility in peptide mass spectrometry, the energetics of the observed fragmentation pathway are examined by density functional theory. When this functional group is covalently attached to a target peptide, it is found that the nucleophilic displacement occurs in competition with formation of b- and y-type backbone fragment ions regardless of the amino acid side-chains present in the parent bioconjugate, confirming that calculated reaction energetics of reporter ion formation are similar to those of backbone fragmentations. Based on these results, we apply this selective fragmentation pathway for the development of CIT reagents. For demonstration purposes, duplex CIT reagent is prepared using a single isotope-coded precursor, allyl-d5-bromide, with reporter ions appearing at m/z 164 and 169. Isotope-coded allyl azides for the construction of the reporter ion group can be prepared from halogenated alkyl groups which are also employed for the mass balance group via N-alkylation, reducing the cost and effort for synthesis of isobaric pairs. Owing to their modular designs, an unlimited number of isobaric combinations of CIT reagents are, in principle, possible. The reporter ion mass can be easily tuned to avoid overlapping with common peptide MS/MS fragments as well as the low mass cut-off problems inherent in ion trap mass spectrometers. The applicability of the CIT reagent is tested with several model systems involving protein mixtures and cellular systems.
Phytochelatin synthase: of a protease a peptide polymerase made.
Source
Plant Science Institute, Department of Biology, Carolyn Hoff Lynch Biology Laboratory, University of Pennsylvania, Philadelphia, PA 19104, USA.
Abstract
Of the mechanisms known to protect vascular plants and some algae, fungi and invertebrates from the toxic effects of nonessential heavy metals such as As, Cd or Hg, one of the most sophisticated is the enzyme-catalyzed synthesis of phytochelatins (PCs). PCs, (γ-Glu-Cys)(n) Gly polymers, which serve as high-affinity, thiol-rich cellular chelators and contribute to the detoxification of heavy metal ions, are derived from glutathione (GSH; γ-Glu-Cys-Gly) and related thiols in a reaction catalyzed by phytochelatin synthases (PC synthases, EC 2.3.2.15). Using the enzyme from Arabidopsis thaliana (AtPCS1) as a model, the reasoning and experiments behind the conclusion that PC synthases are novel papain-like Cys protease superfamily members is presented. The status of S-substituted GSH derivatives as generic PC synthase substrates and the sufficiency of the N-terminal domain of the enzyme from eukaryotic and its half-size equivalents from prokaryotic sources, for net PC synthesis and deglycylation of GSH and its derivatives, respectively, are emphasized. The question of the common need or needs met by PC synthases and their homologs is discussed. Of the schemes proposed to account for the combined protease and peptide polymerase capabilities of the eukaryotic enzymes versus the limited protease capabilities of the prokaryotic enzymes, two that will be considered are the storage and homeostasis of essential heavy metals in eukaryotes and the metabolism of S-substituted GSH derivatives in both eukaryotes and prokaryotes.
Copyright © Physiologia Plantarum 2012.
- PMID:
- 22224506
- [PubMed - as supplied by publisher]
[Regulatory effect of glucagon-like peptide-1 on cell proliferation of skeletal myoblast strain L6 and its possible signal mechanism].
Source
Burns Institute, First Hospital Affiliated to the PLA General Hospital, Beijing 100048, China.
Abstract
OBJECTIVE:
To study the regulatory effect of glucagon-like peptide-1 (GLP-1) on cell proliferation of skeletal myoblast strain L6 and its possible signal mechanism.
METHODS:
L6 cells cultured in DMEM high glucose culture medium containing 10% FBS were divided into control group (C, without addition), GLP-1 group (G, added with 10 nmol/L GLP-1), PI3K inhibitor group (W, added with 50 nmol/L PI3K specific inhibitor wortmannin), and GLP-1 + PI3K inhibitor group (GW, added with 10 nmol/L GLP-1 and 50 nmol/L wortmannin) according to the random number table. Cell proliferation activity was detected with MTT assay at post culture hour (PCH) 24, 48, 72 (denoted as absorbance value). At PCH 24, the change in cell cycle was evaluated with flow cytometer, the expression level of proliferating cell nuclear antigen (PCNA) was determined with immunohistochemical staining, the protein levels of phosphorylated PI3K (p-PI3K) and p-Akt were determined with Western blotting. Data were processed with multi-group analysis of variance.
RESULTS:
(1) The cell proliferation activity at PCH 48, 72 in G group was respectively 0.660 +/- 0.120, 0.870 +/- 0.240, all significantly higher than those in C group (0.530 +/- 0.060, 0.700 +/- 0.100, with F value respectively 5.46, 5.90, P < 0.05 or P < 0.01). The cell proliferation activity in W group at each time point was lower than that in C group. The cell proliferation activity in GW group at PCH 48, 72 was respectively 0.510 +/- 0.080, 0.740 +/- 0.160, all lower than those in G group (with F value respectively 5.46, 5.90, P < 0.05 or P < 0.01). (2) The percentage of S phase cell in G group at PCH 24 [(15.7 +/- 0.4)%] was significantly higher than that in C group [(13.6 +/- 0.6)%] and GW group [(10.1 +/- 0.6)%], while that in W group [(6.8 +/- 1.2)%] was lower than that in C group (with F values all equal to 15.39, P values all below 0.01). (3) PCNA level in G group at PCH 24 [(51.24 +/- 1.18)%] was markedly higher than that in C group [(36.72 +/- 1.56)%] and GW group [(25.90 +/- 1.22)%], and while in W group [(21.70 +/- 0.09)%] was lower than that in C group (with F values equal to 783.80, P values all below 0.05). (4) The protein level of p-Akt in G group at PCH 24 was significantly higher than that in the other 3 groups, while that in W group was lower than that in C group (with F values equal to 94.43, P values all below 0.01). There was no obvious difference in protein level of p-PI3K at PCH 24 among G, GW, and C groups ( F = 20.94, P > 0.05). The protein level of p-PI3K at PCH 24 in W group was lower than that in C group (F = 20.94, P < 0.05).
CONCLUSIONS:
GLP-1 can promote cell proliferation of skeletal myoblast by accelerating the progression of cell cycle and increasing the synthesis of DNA, which can be attributed to PI3K/Akt signal pathway.
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