1.
PET Imaging of Integrin Positive Tumors Using F Labeled Knottin Peptides.
Source
1. Molecular Imaging Program at Stanford (MIPS), Bio-X Program, Department of Radiology, Stanford University, California, 94305-5344, USA.
Abstract
Purpose: Cystine knot (knottin) peptides, engineered to bind with high affinity to integrin receptors, have shown promise as molecular imaging agents in living subjects. The aim of the current study was to evaluate tumor uptake and in vivo biodistribution of (18)F-labeled knottins in a U87MG glioblastoma model.Procedures: Engineered knottin mutants 2.5D and 2.5F were synthesized using solid phase peptide synthesis and were folded in vitro, followed by radiolabeling with 4-nitrophenyl 2-(18)F-fluoropropionate ((18)F-NFP). The resulting probes, (18)F-FP-2.5D and (18)F-FP-2.5F, were evaluated in nude mice bearing U87MG tumor xenografts using microPET and biodistribution studies.Results: MicroPET imaging studies with (18)F-FP-2.5D and (18)F-FP-2.5F demonstrated high tumor uptake in U87MG xenograft mouse models. The probes exhibited rapid clearance from the blood and kidneys, thus leading to excellent tumor-to-normal tissue contrast. Specificity studies confirmed that (18)F-FP-2.5D and (18)F-FP-2.5F had reduced tumor uptake when co-injected with a large excess of the peptidomimetic c(RGDyK) as a blocking agent.Conclusions: (18)F-FP-2.5D and (18)F-FP-2.5F showed reduced gallbladder uptake compared with previously published (18)F-FB-2.5D. (18)F-FP-2.5D and (18)F-FP-2.5F enabled integrin-specific PET imaging of U87MG tumors with good imaging contrasts. (18)F-FP-2.5D demonstrated more desirable pharmacokinetics compared to (18)F-FP-2.5F, and thus has greater potential for clinical translation.
- PMID:
- 22211146
- [PubMed - as supplied by publisher]
Improved Synthesis Strategy for Peptide Nucleic Acids (PNA) appropriate for Cell-specific Fluorescence Imaging.
Source
1. DKFZ, Central Peptide Synthesis Unit, INF 580, D-69120 Heidelberg, Germany.
Abstract
Progress in genomics and proteomics attended to the door for better understanding the recent rapid expanding complex research field of metabolomics. This trend in biomedical research increasingly focuses to the development of patient-specific therapeutic approaches with higher efficiency and sustainability. Simultaneously undesired adverse reactions are avoided. In parallel, the development of molecules for molecular imaging is required not only for the imaging of morphological structures but also for the imaging of metabolic processes like the aberrant expression of the cysteine protease cathepsin B (CtsB) gene and the activity of the resulting product associated with metastasis and invasiveness of malign tumors. Finally the objective is to merge imaging and therapy at the same level. The design of molecules which fulfil these responsibilities is pivotal and requires proper chemical methodologies. In this context our modified solid phase peptide chemistry using temperature shifts during synthesis is considered as an appropriate technology. We generated highly variable conjugates which consist of molecules useful as diagnostically and therapeutically active molecules. As an example the modular PNA products with the complementary sequence to the CtsB mRNA and additionally with a cathepsin B cleavage site had been prepared as functional modules for distinction of cell lines with different CtsB gene expression. After ligation to the modular peptide-based BioShuttle carrier, which was utilized to facilitate the delivery of the functional modules into the cells' cytoplasm, the modules were scrutinized.
- PMID:
- 22211082
- [PubMed - in process]
Regulation of Escherichia coli Biotin Biosynthesis: Isolation of BirA Super-Repressor Mutant Strains.
Source
Departments of Microbiology.
Abstract
Transcription of the Escherichia coli biotin (bio) operon is directly regulated by the biotin-protein ligase, BirA, the enzyme that covalently attaches biotin to its cognate acceptor proteins. Binding of BirA to the bio operator requires dimerization of the protein, which is triggered by BirA-catalyzed synthesis of biotinoyl-adenylate (biotinoyl-5' -AMP), the obligatory intermediate of the ligation reaction. Although several aspects of this regulatory system are well understood, no BirA super-repressor mutant strains had been isolated. Such super-repressor BirA proteins would repress the biotin operon transcription in vivo at biotin concentrations well below those needed for repression by wild type BirA. We isolated mutant strains having this phenotype by a combined selection-screening approach and resolved multiple mutations to give several birA super-repressor alleles each having a single mutation all of which showed repression dominant over the wild type allele. All of these mutant strains repressed bio operon transcription in vivo at biotin concentrations that gave derepression of the wild type strain and retained sufficient ligation activity for growth when overexpressed. All of the strains except that encoding G154D BirA showed derepression of bio operon transcription upon overproduction of a biotin accepting protein. The G154D BirA was a lethal mutation in single copy and the purified protein was unable to transfer biotin from enzyme bound biotinoyl-adenylate either to the natural acceptor protein or to a biotin accepting peptide sequence. Consistent with the transcriptional repression data, each of the purified mutant proteins showed increased affinity for the biotin operator DNA in electromobility shift assays. Surprisingly although most of the mutations were located in the catalytic domain all those tested excepting G154D BirA had normal ligase activity. Most of the mutations that gave super-repressor phenotypes altered residues located close to the dimerization interface of BirA. However, two mutations were located at sites well removed from the interface. The properties of the super-repressor mutants strengthen and extend other data indicating that BirA function entails extensive interactions among the three domains of the protein and shows that normal ligase activity does not ensure normal DNA binding.
- PMID:
- 22210766
- [PubMed - as supplied by publisher]
Colicin M hydrolyses branched lipids II from Gram-positive bacteria.
Source
Univ Paris-Sud, Laboratoire des Enveloppes Bactériennes et Antibiotiques, UMR 8619, Orsay, F-91405, France; CNRS, Orsay, F-91405, France.
Abstract
Lipids II found in some Gram-positive bacteria were prepared in radioactive form from l-lysine-containing UDP-MurNAc-pentapeptide. The specific lateral chains of Enterococcus faecalis, Enterococcus faecium and Staphylococcus aureus (di-l-alanine, d-isoasparagine, and pentaglycine, respectively) were introduced by chemical peptide synthesis using the Fmoc chemistry. The branched nucleotides obtained were converted into the corresponding lipids II by enzymaticsynthesis using the MraY and MurG enzymes. All of the lipids were hydrolysed by Escherichia coli colicin M at approximately the same rate as the meso-diaminopimelate-containing lipid II found in Gram-negative bacteria, thereby opening the way to the use of this enzyme as a broad spectrum antibacterial agent.
Copyright © 2011. Published by Elsevier Masson SAS.
- PMID:
- 22210388
- [PubMed - as supplied by publisher]
Altered regulation of nitric oxide and natriuretic peptide system in cisplatin-induced nephropathy.
Source
Department of Internal Medicine, Chonnam National University Medical School, Gwangju, Korea.
Abstract
Cisplatin is a chemotherapeutic agent used for treating solid tumors. However, nephrotoxicity is the dose-limiting factor in its clinical use. The present study was aimed to determine whether altered regulation of the local nitric oxide (NO) and natriuretic peptide (NP) systems is involved in the pathogenesis of cisplatin-induced nephropathy. Cisplatin (6mg/kg) was injected intraperitoneally into male Sprague-Dawley rats. The control group was not treated with cisplatin. Expression levels of nitric oxide synthase (NOS), nitrotyrosine, soluble guanylyl cyclase and neutral endopeptidase (NEP) in the kidneys were determined 4days after treatment by semiquantitative immunoblotting. mRNA expression of NPs and natriuretic peptide receptors (NPRs) was determined by real-time polymerase chain reaction. The activities of soluble and particulate guanylyl cyclase were determined by measuring the amount of cyclic 3',5'-guanosine monophosphate (cGMP) generated in responses to sodium nitroprusside and atrial natriuretic peptide (ANP), respectively. In the test rats, creatinine clearance was decreased, while sodium and water excretion were increased. The expression of inducible NOS (iNOS) and nitrotyrosine was increased in the cortex/outer stripe of outer medullar and inner medullar, while that of endothelial and neuronal NOS was decreased in the inner medullar. Excretion of NO metabolites was increased in these rats. The catalytic activity of soluble guanyly cyclase was blunted in the papilla after cisplatin was administered. The mRNA expression of ANP, brain natriuretic peptide, and C-type natriuretic peptide was increased, while that of NPR-A and NPR-C were decreased in the test rats. The catalytic activity of soluble and particulate guanylyl cyclase in the papilla was blunted after cisplatin was administered. In conclusion, increased production of NO by iNOS may contribute to cytotoxic injury, resulting in cisplatin-induced nephropathy, while the up-regulation of renal natriuretic peptide synthesis together with the down-regulation of NEP and NPR-C may contribute to the natriuresis and diuresis seen in cisplatin-induced nephropathy.
Copyright © 2011. Published by Elsevier B.V.
- PMID:
- 22209992
- [PubMed - as supplied by publisher]
Apelin-12 stimulates acid secretion through an increase of histamine release in rat stomachs.
Source
Department of Gastroenterology and Hepatology, Saitama Medical Center, Saitama Medical University, 1981 Kamoda, Kawagoe City, Saitama 350-8550, Japan.
Abstract
BACKGROUND:
Apelin is a peptide that was originally isolated from bovine stomach extract and has been demonstrated to be an endogenous ligand for orphan receptor APJ. Both apelin and the APJ receptor are widely distributed in the whole body. Apelin is supposed to have important regulatory roles in the function of many organs such as in the cardiovascular system; however, the mechanism of apelin function has not been elucidated. In this study, we studied the action of apelin in acid secretion and demonstrated its mechanism of action.
METHODS:
Gastric lumen-perfused rats were prepared and their stomachs were perfused with a saline solution using a peristaltic pump. Apelin-12, 36 or Pyr(1)-apelin-13, were intravenously injected to examine their effects on acid secretion in rats. In some experiments, rats were pretreated with famotidine (0.33mg/kg) or atropine sulfate (0.1mg/kg) intravenously injected 5 or 15min before apelin injection. Furthermore, isolated vascularly perfused rat stomachs were prepared to examine the effect of apelin on histamine release, which was assayed in the effluent by radioimmunoassay. Messenger RNA of histidine decarboxylase (HDC) in gastric mucosa of isolated stomach was measured by real-time RT-PCR.
RESULTS:
Apelin-12 (20-100μg/kg) dose-dependently increased gastric acid secretion, with a maximum of 203% at 100μg/kg (n=5). Neither Pyr(1)-apelin-13 nor apelin-36 caused a significant increase in acid secretion. Famotidine completely blocked the stimulatory action of apelin on acid secretion. Apelin-12 (100μg/20ml/10min) markedly increased histamine release from isolated vascularly perfused rat stomachs by 278%, and also increased the mRNA of HDC by 480% of the control. Atropine sulfate did not abolish the effect of apelin on the secretion of gastric acid. Apelin-12 amplified an increase of acid secretion stimulated by gastrin injection.
CONCLUSION:
These results indicate that apelin-12 stimulates gastric acid secretion through an increase in histamine release and synthesis from gastric mucosa, suggesting that apelin might play a role in the secretion of gastric acid or serve as a regulating factor of the secretion of gastric acid.
Copyright © 2011. Published by Elsevier B.V.
- PMID:
- 22209991
- [PubMed - as supplied by publisher]
N-Succinimidyl 4-[(18)F]-fluoromethylbenzoate-labeled dimeric RGD peptide for imaging tumor integrin expression.
Source
Department of Medical Imaging and Nuclear Medicine, Fourth Affiliated Hospital, Harbin Medical University, Harbin, 150001, China.
Abstract
RGD peptides, radiolabeled with (18)F, have been used in the clinic for PET imaging of tumor angiogenesis in cancer patients. RGD peptides are typically labeled using a prosthetic group such as N-succinimidyl 4-[(18)F]-fluorobenzoate ([(18)F]SFB) or 4-nitrophenyl 2-[(18)F]-fluoropropionate ([(18)F]NPFP). However, the complex radiosynthetic procedures have impeded their broad application in clinical studies. We previously radiolabeled proteins and peptides with the prosthetic group, N-succinimidyl 4-[(18)F]-fluoromethylbenzoate ([(18)F]SFMB), which was prepared in a simple one-step procedure. In this study, we labeled a PEGylated cyclic RGD peptide dimer, PEG(3)-E[c(RGDyK)](2) (PRGD2), using [(18)F]SFMB and evaluated for imaging tumor αvβ3 integrin expression with positron emission tomography (PET). [(18)F]SFMB was prepared in one step using [(18)F]fluoride displacement of a nitrobenzenesulfonate leaving group under mild reaction conditions followed by HPLC purification. The (18)F-labeled peptide, [(18)F]FMBPRGD2 was prepared by coupling PRGD2 with [(18)F]SFMB in pH 8.6 borate buffer and purified with HPLC. The direct labeling on BMBPRGD2 was also attempted. A Siemens Inveon PET was used to image the uptake of the [(18)F]FMBPRGD2 into a U87MG xenograft mouse model. [(18)F]FMBPRGD2, was prepared with a 15% overall radiochemical yield (uncorrected) in a total synthesis time of 90 min, which was considerably shorter than the preparation of [(18)F]SFB- and [(18)F]NPFP-labeled RGD peptides. The direct labeling, however, was not successful. High quality microPET images using [(18)F]FMBPRGD2 clearly visualized tumors by 15 min with good target to background ratio. Early tracer accumulation in the bladder suggests fast renal clearance. No obvious bone uptake can be detected even at 4-h time point indicating that fluorine attachment is stable in mice. In conclusion, N-succinimidyl 4-[(18)F]-fluoromethylbenzoate ([(18)F]SFMB) prosthetic group can be a good alternative for labeling RGD peptides to image αvβ3 integrin expression and for labeling other peptides.
- PMID:
- 22209865
- [PubMed - as supplied by publisher]
Synthesis of uriedo and thiouriedo derivatives of peptide conjugated heterocycles - A new class of promising antimicrobials.
Source
Department of Studies in Chemistry, Manasagangotri, University of Mysore, Mysore 570 006, India.
Abstract
Forty five new derivatives of ureas and thioureas were synthesized by the reaction of peptide conjugated heterocycles with isocyanates and isothiocyanates respectively. All the compounds have been characterized by IR, (1)H NMR, mass and elemental analysis. The compounds were evaluated for their ability to inhibit the growth of a panel of microorganisms and all the synthesized compounds displayed an excellent antimicrobial activity. From structure-activity relationship studies, it was apparent that thioureas infact is slightly more active than ureas. Also, substituents on the phenyl ring of the title compounds play a key role in the activity. Further, compound 40 is nearly twenty times more potent than the standard used. These results present a platform for the further studies in this line.
Copyright © 2011 Elsevier Masson SAS. All rights reserved.
- PMID:
- 22209564
- [PubMed - as supplied by publisher]
Vasoactive Intestinal Peptide Enhances TNF-α-Induced IL-6 and IL-8 Synthesisin Human Proximal Renal Tubular Epithelial Cells by NF-κB-Dependent Mechanism.
Source
Division of Nephrology, Xiangya Hospital, Central South University, Changsha, Hunan, 410008, China.
Abstract
Vasoactive intestinal polypeptide (VIP) is a 28-amino acid neuropeptide with vasodilator, bronchodilator, and anti-inflammatory effects. But little is known about its pro-inflammatory effects. We investigated the effect of VIP on the secretion of interleukin-6 (IL-6) and interleukin-8 (IL-8), two pro-inflammatory cytokines, in TNF-α-activated proximal renal tubular epithelial cell line (HK-2 cells). Cultured HK-2 cells were treated with TNF-α in the presence or absence of VIP with a dose range from 1 to 100 nM, followed by analysis of pro-inflammatory cytokines (IL-6 and IL-8) induction and their signal events including activation of the NF-κB pathway. We report here that tumor necrosis factor-α (TNF-α) increased IL-6 and IL-8 production, and that these effects were potentiated by VIP at 10 nM in HK-2 cells. However, VIP at 1 and 100 nM did not display this function. Consistent with these observations, we were able to show that VIP at 10 nM upregulated TNF-α-induced phosphorylation of IκB-α, leading to IκB-α degradation and the subsequent nuclear translocation of NF-κB. Furthermore, VIP-enhanced activation of NF-κB transcription activity was demonstrated using a NF-κB reporter construct upon transient transfection into HK-2 cells. These results strongly suggest that VIP synergistically enhances TNF-α-stimulated IL-6 and IL-8 synthesis via activating the NF-κB pathway in HK-2 cells.
Molecular Structure and Peptidoglycan Recognition of Mycobacterium tuberculosis ArfA (Rv0899).
Source
Sanford Burnham Medical Research Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA.
Abstract
Mycobacterium tuberculosis ArfA (Rv0899) is a membrane protein encoded by an operon that is required for supporting bacterial growth in acidic environments. Its C-terminal domain (C domain) shares significant sequence homology with the OmpA-like family of peptidoglycan-binding domains, suggesting that its physiological function in acid stress protection may be related to its interaction with the mycobacterial cell wall. Previously, we showed that ArfA forms three independently structured modules, and we reported the structure of its central domain (B domain). Here, we describe the high-resolution structure and dynamics of the C domain, and we identify ArfA as a peptidoglycan-binding protein and elucidate the molecular basis for its specific recognition of diaminopimelate-type peptidoglycan. The C domain of ArfA adopts the characteristic fold of the OmpA-like family. It exhibits pH-dependent conformational dynamics (with significant heterogeneity at neutral pH and a more ordered structure at acidic pH), which could be related to its acid stress response. The C domain associates tightly with polymeric peptidoglycan isolated from M. tuberculosis and also associates with a soluble peptide intermediate of peptidoglycan biosynthesis. This enabled us to characterize the peptidoglycan binding site where five highly conserved ArfA residues, including two key arginines, establish the specificity for diaminopimelate- but not Lys-type peptidoglycan. ArfA is the first peptidoglycan-binding protein to be identified in M. tuberculosis. Its functions in acid stress protection and peptidoglycan binding suggest a link between the acid stress response and the physicochemical properties of the mycobacterial cell wall.
Copyright © 2011. Published by Elsevier Ltd.
Evaluation of Amoebicidal Potential of Paneth Cell Cryptdin-2 against Entamoeba histolytica.
Source
Department of Microbiology, Basic Medical Sciences Block, Panjab University, Chandigarh, India.
Abstract
BACKGROUND:
Amoebiasis is a major public health problem in tropical and subtropical countries. Currently, metronidazole is the gold choice medication for the treatment of this disease. However, reports have indicated towards the possibility of development of metronidazole-resistance in Entamoeba strains in near future. In view of the emergence of this possibility, in addition to the associated side effects and mutagenic ability of the currently available anti-amoebic drugs, there is a need to explore newer therapeutics against this disease. In this context, the present study evaluated the amoebicidal potential of cryptdin-2 against E. histolytica.
METHODS/PRINCIPAL FINDINGS:
In the present study, cryptdin-2 exhibited potent in-vitro amoebicidal activity against E. histolytica in a concentration dependent manner at a minimum amoebicidal concentration (MAC) of 4 mg/L. Scanning electron microscopy as well as phase contrast microscopic investigations of cryptdin-2 treated trophozoites revealed that the peptide was able to induce significant morphological alterations in terms of membrane wrinkling, leakage of the cytoplasmic contents and damaged plasma membrane suggesting a possible membrane dependent amoebicidal activity. N-phenyl napthylamine (NPN) uptake assay in presence of sulethal, lethal as well as twice the lethal concentrations further confirmed the membrane-dependent mode of action of cryptdin-2 and suggested that the peptidecould permeabilize the plasma membrane of E. histolytica. It was also found that cryptdin-2 interfered with DNA, RNA as well as protein synthesis of E. histolytica exerting the highest effect against DNA synthesis. Thus, the macromolecular synthesis studies correlated well with the observations of membrane permeabilization studies. SIGNIFICANCE/
CONCLUSIONS:
The amoebicidal efficacy of cryptdin-2 suggests that it may be exploited as a promising option to combat amoebiasis or, at least, may act as an adjunct to metronidazole and/or other available anti-amoebic drugs.
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. Cell culture 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.
Structure-activity studies on alpha-conotoxins.
Source
Institute for Molecular Bioscience, The University of Queensland, St Lucia Queensland 4072, Australia. p.alewood@imb.uq.edu.au.
Abstract
Conotoxins are small bioactive highly structured peptides from the venom of marine cone snails (genus Conus). Over the past 50 million years these molluscs have developed a complex venom cocktail for each species that is comprised of 100-2000 distinct cysteine- rich peptides for prey capture and defence. This review focuses on an important and well-studied class of conotoxins, the α- conotoxins. These α-conotoxins are potent and selective antagonists of various subtypes of the nicotinic acetylcholine receptors (nAChRs). Key structure-activity relationship studies are presented to illustrate the common motifs, structural features and pharmacophores that define this interesting peptide class. Additionally, their synthesis, chemical modifications, the development of more selective and stable analogues and their therapeutic potential are discussed.
- PMID:
- 22204424
- [PubMed - in process]
Ribosomal biosynthesis of α-amanitin in Galerina marginata.
Source
Department of Energy Plant Research Laboratory, Michigan State University, E. Lansing, MI 48824, United States.
Abstract
Amatoxins, including α-amanitin, are bicyclic octapeptides found in mushrooms (Agaricomycetes, Agaricales) of certain species in the genera Amanita, Galerina, Lepiota, and Conocybe. Amatoxins and the chemically similar phallotoxins are synthesized on ribosomes in Amanita bisporigera, Amanita phalloides, and Amanita ocreata. In order to determine if amatoxins are synthesized by a similar mechanism in another, distantly related mushroom, we obtained genome survey sequence data from a monokaryotic isolate of Galerinamarginata, which produces α-amanitin. The genome of G. marginata contains two copies of the α-amanitin gene (GmAMA1-1 and GmAMA1-2). The α-amanitin proprotein sequences of G. marginata (35 amino acids) are highly divergent from AMA1 of A. bisporigera except for the toxin region itself (IWGIGCNP in single-letter amino acid code) and the amino acids immediately upstream (N[A/S]TRLP). G. marginata does not contain any related toxin-encoding sequences besides GmAMA1-1 and GmAMA1-2. DNA from two other α-amanitin-producing isolates of Galerina (G. badipes and G. venenata) hybridized to GmAMA1, whereas DNA from the toxin non-producing species Galerinahybrida did not. Expression of the GmAMA1 genes was induced by growth on low carbon. RNASeq evidence indicates that both copies of GmAMA1 are expressed approximately equally. A prolyl oligopeptidase (POP) is strongly implicated in processing of the cyclic peptide toxins of A. bisporigera and Conocybe apala. G. marginata has two predicted POP genes; one, like AbPOPB of A. bisporigera, is present only in the toxin-producing isolates of Galerina and the other, like AbPOPA of A. bisporigera, is present in all species. Our results indicate that G.marginata biosynthesizes amatoxins on ribosomes by a pathway similar to Amanita species, involving a genetically encoded proprotein of 35 amino acids that is post-translationally processed by a POP. However, due to the high degree of divergence, the evolutionary relationship between AMA1 in the genera Amanita and Galerina is unclear.
Copyright © 2011. Published by Elsevier Inc.
Cloning, expression, purification, and characterization of a glutamate-specific endopeptidase from Bacillus licheniformis.
Source
School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, PR China.
Abstract
A gene encoding a glutamate-specific endopeptidase (GSE) from Bacillus licheniformis (BL) has been cloned in Escherichia coli cells. The recombinant protein was expressed as cytoplasmic insoluble inclusion bodies. Immobilized metal affinity chromatography was employed to purify the protein, and then a 27-kDa GSE intermediate was obtained by gradient urea dialysis. The remaining pro-peptide was completely removed by treatment with trypsin to obtain mature GSE-BL with a molecular weight of 26kD at a final yield of up to 140.9mg/L. With Z (benzyloxycarbomyl)-Phe-Leu-Glu-pNA (p-nitroanilide) as the substrate, the optimal temperature and pH conditions for the enzyme were 37°C and 8.5, respectively, K(m) was 1.495±0.034mM, and V(max) was 50.237μmol/mgmin. The presence of calcium and ferrous ions enhanced the catalytic activity of GSE-BL. These results suggest that the recombinant protein is a relatively stable specific proteinase that could be effectively utilized in protein structure analysis, peptide synthesis, and the food industry.
Copyright © 2011 Elsevier Inc. All rights reserved.
Exogenous Glucagon-Like Peptide-1 for Hyperglycemia in Critically Ill Patients (January).
Source
< Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, Detroit, MI.
Abstract
OBJECTIVE:
To review literature evaluating the safety and efficacy of exogenous glucagon-like peptide-1 (GLP-1) for hyperglycemia in critically ill patients.
DATA SOURCES:
PubMed was queried (inception to September 3, 2011), using the search term glucagon-likepeptide-1. The search was limited to studies published in English and conducted in humans. Regular and late-breaking abstracts from the American Diabetes Association Scientific Sessions in 2009 and 2010 were also searched using the same search term.
STUDY SELECTION AND DATA EXTRACTION:
All abstracts were screened for eligibility, which consisted of studies reporting the effects of intravenous GLP-1 administration on glycemic control in critically ill patients. Data extracted from eligible trials included study and population characteristics, measures of glycemic efficacy, and safety.
DATA SYNTHESIS:
Our search resulted in the identification of 2105 potentially relevant articles; of those, 7 were reviewed. All included publications evaluated the use of intravenous GLP-1 (1.2-3.6 pmol/kg/min) compared with insulin or placebo infused for 4.5-72 hours in critically ill patients. The majority (n = 4) of studies included only patients from a surgical intensive care setting, and 71% (n = 5) of trials included those with a history of diabetes. Relative to insulin or placebo, GLP-1 therapy effectively lowered blood glucose concentrations in all trials. Out of 81 total study participants receiving GLP-1, only 4 had documentedhypoglycemia (<60 mg/dL), 4 reported nausea, and 2 experienced vomiting. No other serious adverse events were reported.
CONCLUSIONS:
All trials reviewed suggest that GLP-1 may be a promising agent for the management of hyperglycemia in critically ill patients, regardless of diabetes status. Additional studies in more heterogeneous intensive care settings comparing GLP-1 with insulin, the current standard of care, are necessary. These studies should evaluate long-term safety and effectiveness of GLP-1 therapy on morbidity and mortality outcomes in critically ill populations.
- PMID:
- 22202493
- [PubMed - as supplied by publisher]
Studying protein-peptide interactions using benzophenone units: A case study of protein kinase B/Akt and its inhibitor PTR6154.
Source
Institute of Chemistry, Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel.
Abstract
Protein-protein interactions (PPIs) govern nearly all processes in living cells. Peptides play an important role in studying PPIs. Peptides carrying photoaffinity labels that covalently bind the interacting protein can be used to obtain more accurate information regarding PPIs. Benzophenone (BP) is a useful photoaffinity label that is widely used to study PPIs. We developed a one-pot two-step synthesis for the preparation of novel BP units. To map the binding site more thoroughly, linkers of various lengths were attached to the BP moiety. These units can be incorporated into peptidesequences using well-established solid phase peptide synthesis (SPPS) protocols. As a proof of concept, we studied the interaction between protein kinase B (PKB/Akt) and its synthetic peptide inhibitor, PTR6154. The methodology is general and can be implemented to study PPIs in a variety of biological systems.
Copyright © 2011. Published by Elsevier Inc.
A simple oxazolidine linker for solid-phase synthesis of peptide aldehydes.
Source
Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing 210008, China.
Abstract
A very simple and cheap linker has been used for solid-phase synthesis of peptide aldehydes. Protected amino acid aldehydes are immobilized on 2-Cl(trt) resin as oxazolidine formation via diethanolamine. After classical Fmoc SPPS, treatment of the resin with AcOH/DCM/H(2)O (8:1:1) affords peptide aldehydes in high yield and purity.
Copyright © 2011 Elsevier Ltd. All rights reserved.
Paenibacillus polymyxa PKB1 Produces Variants of Polymyxin B-Type Antibiotics.
Source
Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2E9, Canada.
Abstract
Polymyxins are cationic lipopeptide antibiotics active against many species of Gram-negative bacteria. We sequenced the gene cluster for polymyxin biosynthesis from Paenibacillus polymyxa PKB1. The 40.8 kb gene cluster comprises three nonribosomal peptide synthetase-encoding genes and two ABC transporter-like genes. Disruption of a peptidesynthetase gene abolished all antibiotic production, whereas deletion of one or both transporter genes only reduced antibiotic production. Computational analysis of the peptide synthetase modules suggested that the enzyme system produces variant forms of polymyxin B (1 and 2), with D-2,4-diaminobutyrate instead of L-2,4-diaminobutyrate in amino acid position 3. Two antibacterial metabolites were resolved by HPLC and identified by high-resolution mass spectrometry and MS/MS sequencing as the expected variants 3 and 4 of polymyxin B(1) (1) and B(2) (2). Stereochemical analysis confirmed the presence of both D-2,4-diaminobutyrate and L-2,4-diaminobutyrate residues.
Copyright © 2011 Elsevier Ltd. All rights reserved.
Cellular uptake of an α-AApeptide.
Source
Department of Chemistry, University of South Florida, 4202 E. Fowler Ave, Tampa, FL 33620, USA. Jianfengcai@usf.edu.
Abstract
Some short and cationic peptides such as the Tat peptide can cross the cell membrane and function as vectors for intracellular delivery. Here we show that an α-AApeptide is able to penetrate the membranes of living cells from an extracellular environment and enter the endosome and cytoplasm of cells. The efficiency of the cellular uptake is comparable to a Tat peptide (48-57) of the same length and is unexpectedly superior to an α-peptide with identical functional groups. The mechanism of uptake is similar to that of the Tat peptide and is through endocytosis by an energy-dependent pathway. Due to the easy synthesis of the α-AApeptides, their resistance to proteolytic hydrolysis, and their low cytotoxicity, α-AApeptides represent a new class of transporters for the delivery of drugs.
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