Functionalized quinolizinium-based fluorescent reagents for modification of cysteine-containing peptides and proteins

A series of quinolizinium-based fluorescent reagents were prepared by visible light-mediated gold-catalyzed cis-difunctionalization between quinolinium diazonium salts and electron-deficient alkyne-linked phenylethynyl trimethylsilanes. The electron-deficient alkynyl group of the quinolizinium-based fluorescent reagents underwent nucleophilic addition reaction with the sulfhydryl group on cysteine-containing peptides and proteins. The quinolizinium-based fluorescent reagents were found to function as highly selective reagents for the modification of cysteine-containing peptides and proteins with good to excellent conversions (up to 99%). Moreover, the modified BCArg mutants bearing cationic quinolizinium compounds 1b, 1d, 1e and 1h exhibit comparable activity in enzymatic and cytotoxicity assays to the unmodified one.

New quinolizinium-based fluorescent reagents were made by visible light-mediated gold-catalyzed cis-difunctionalization of quinolinium diazonium salts and trimethylsilyl alkyne derivatives.     

水蛭中的抗凝血多肽与蛋白质

中医认为水蛭有破血，逐淤，通经等功效。１８８４年Ｈａｙｃｒａｆｔ发现医用水蛭的提取物含有抗凝血的物质。１９５５年这种抗凝血物质被Ｍａｒｋｗａｒｄｔ确定为蛋白质，并命名为水蛭素（Ｈｉｒｕｄｉｎ）。由此开始了对吸血水蛭中抗凝血物质的多方面研究，相继发现了...     

Micropatterned immobilization of membrane-mimicking polymer and peptides for regulation of cell behaviors in vitro

To regulate the behaviors and functions of endothelial cells (ECs) on the biomaterials on titanium (Ti), a biomimetic micropattern (ridge/groove: 25/25 μm) of polymer of 2-methacryloyloxyethyl phosphorylcholine (polyMPC) and Gly-Arg-Glu-Asp-Val-Tyr (GREDVY) was fabricated. PMMPC (monomer contain MPC and methacrylic acid (MA)) containing carboxyl groups was chosen, and PMMPC was cross-linked with hexamethylene diamine through condensation reaction of amino and carboxyl. Simultaneously, the carboxyl groups of cross-linked PMMPC (PMMPC-HD) can react with amino groups of polydopamine which can adhered on many materials firmly. GREDVY was immobilized on polydopamine but not on PMMPC-HD because amino and carboxyl groups can react with catechol and amino groups of polydopamine. IR and ¹H NMR demonstrated that PMMPC-HD was successfully synthesized. And the QCM-D (quartz crystal microbalance with dissipation) and IR approved that PMMPC-HD and GREDVY can be immobilized on polydopamine (PDA). Platelet adhesion and whole blood adhesion on micropattern modificated with PMMPC and GREDVY (Ti-PDA-M/R(P)) showed better hemocompatibility than other samples. Endothelial cells were regulated in the direction of micropattern showing elongated ECs were closer to a healthy, athero-protective phenotype than ECs cultured in vitro without micropattern. NO and PGI₂ release were upregulated. Simultaneously the number of SMCs on Ti-PDA-M/R(P) was the smaller that of other samples, which demonstrated that the Ti-PDA-M/R(P) had property of inhibiting SMCs proliferation to a certain extent.

The Ti-PDA-M/R(P) biomimetic micropattern was successfully fabricated with PMMPC-HD and GREDVY. The Ti-PDA-M/R(P) micropattern can regulate EC morphology, orientation and functions, and inhibit platelet adhesion and proliferation of SMCs.     

Synthesis and application of caged peptides and proteins.

Caged compounds have covalently attached groups that are rapidly cleaved upon exposure to UV light. Attachment of photolabile groups makes the molecule inert until photolysis releases it in its bioactive form. When caged compounds are applied to the experimental system in advance, the concentration jump of biologically active substances can be brought about immediately in a limited area upon irradiation with pulsed and focused UV light. Therefore, caged compounds of low molecular weight, which are commercially available, have been used effectively to study the mechanisms of temporal biological phenomena, such as muscle contraction, intracellular signaling, and neurotransmission. Because many proteins and peptides play important roles in these phenomena, their caged derivatives should serve as powerful tools to clarify complex biological systems. To prepare caged proteins and peptides, several groups have improved upon a chemical modification method, as well as developed two new methods: (1) nonsense codon suppression and (2) solid-phase peptide synthesis. In this review, we summarize recent advances made in the design, preparation, and application of caged peptides and proteins.     

Bioactive peptides and proteins as alternative antiplatelet drugs

Antiplatelet drugs reduce the risks associated with atherothrombotic events and show various applications in diverse cardiovascular diseases including myocardial infarctions. Efficacy of the current antiplatelet medicines including aspirin, clopidogrel, prasugrel and ticagrelor, and the glycoprotein IIb/IIIa antagonists, are limited due to their increased risks of bleeding, and antiplatelet drug resistance. Hence, it is important to develop new effective antiplatelet drugs, with fewer side-effects. The vast repertoire of natural peptides can be explored towards this goal. Proteins and peptides derived from snake venoms and plants represent exciting candidates for the development of novel and potent antiplatelet agents. Consequently, this review discusses multiple peptides that have displayed antiplatelet aggregation activity in preclinical drug development stages. This review also describes the antiplatelet mechanisms of the peptides, emphasizing the signaling pathways intervened by them. Also, the hurdles encountered during the development of peptides into antiplatelet drugs have been listed. Finally, hitherto unexplored peptides with the potential to prevent platelet aggregation are explored.     

Binding sites on tau proteins as components for antimicrobial peptides

To design new antimicrobial peptides, we have focused on various proteins which are not essential for self-defense but carry important responsibilities for biosystems. Previously, we reported that highly efficient antimicrobial properties or antiviral properties are inherent in the nuclear translocation signals and binding sites on laminin receptors. Here we introduce microtubule binding sites on tau proteins as new components for antimicrobial peptides. Strong antimicrobial activities against Staphylococcus aureus and Escherichia coli were found in tandem sequences of the binding sites on tau proteins. Moreover, the binding sites obtained significantly strong antimicrobial activities against bacteria and fungi when combined with a nuclear localization signal (NLS) and/or a peptide derived from a binding site of a laminin receptor. The antimicrobial activities of some of the tau-derived peptides were not affected by salt, cations, or serum that simulate the natural environment present in blood. Tau proteins so far have only been known as one of the microtubule-associated proteins (MAPs) which are especially abundant in the central nervous system within the brain. Our finding demonstrates that the binding sites on tau proteins possess high potential for becoming components in antimicrobial peptides. Designs based on binding sites of various proteins could become a useful method in peptide antibiotic research.     

Activation of mitochondrial ATP-dependent protease by peptides and proteins

We examined the effect of peptides or protein on the proteolytic and ATPase activities of mitochondrial ATP-dependent LON protease purified from bovine adrenal cortex. Peptides/proteins including angiotensin I which stimulated ATPase activity without hydrolysis of any peptide bonds stimulated proteolysis of 125I-labeled substrates at low concentrations; whereas at high concentrations they competitively inhibited proteolysis, thus displaying a biphasic activity profile. All peptides and proteins thus examined stimulated degradation of 125I-labeled substrates. When an ATP analog was substituted for ATP, only inhibition; i.e., no stimulation, of proteolysis by unlabeled peptides was observed. Without activator peptides, degradation of [125I] peptides was higher in the presence of an ATP analog than that in the presence of ATP. ADP, a product of the ATPase reaction, inhibited the proteolytic activity in the absence of an activator peptide but not in its presence. From analogy to E. coli ATP-dependent protease La (LON), we suggest that the activator peptides stimulated the proteolysis by releasing enzyme-bound ADP.     

Rational design of composition and activity correlations for pH-responsive and glutathione-reactive polymer therapeutics.

Limited cytoplasmic delivery of enzyme-susceptible drugs remains a significant challenge facing the development of protein and nucleic acid therapies that act in intracellular compartments. "Smart" pH-responsive, membrane-destabilizing polymers present a new approach to shuttling therapeutic molecules past the endosomal membrane and into the cytoplasm of targeted cells. This report describes the use of a functionalized monomer, pyridyl disulfide acrylate (PDSA), to develop pH-responsive, membrane-destabilizing, and glutathione-reactive polymers by copolymerization with several pH-responsive and hydrophobic monomers. The activity of the carriers is described as a function of (a) increasing the length of the hydrophobic alkyl group substituted onto the pH-responsive monomer and (b) the incorporation of a hydrophobic monomer such as butyl acrylate (BA) on the pH sensitivity and membrane-destabilizing activity of new polymer compositions. The membrane-destabilizing activity of different polymer compositions was evaluated as a function of pH and polymer concentration using the red blood cell (RBC) hemolysis assay. Hemolysis results show that the increase in the hydrophobic character of the polymer backbone results in a shift in the pH sensitivities and an increase in the membrane-destabilizing activity. Results show that the observed hemolytic activities and pH sensitivity profiles could be designed across a range that matches the properties needed for enhancing the cytoplasmic delivery of macromolecular therapeutic.     

Rational design of composition and activity correlations for pH-sensitive and glutathione-reactive polymer therapeutics.

Limited cytoplasmic delivery of enzyme-susceptible drugs remains a significant challenge facing the development of protein and nucleic acid therapies that act in intracellular compartments. "Smart" pH-sensitive, membrane-destabilizing polymers present an attractive approach to shuttle therapeutic molecules past the endosomal membrane and into the cytoplasm of targeted cells. This report describes the use of a new functionalized monomer, pyridyl disulfide acrylate (PDSA), to develop pH-sensitive, membrane-destabilizing, and glutathione-reactive polymers by copolymerization with several pH-sensitive and hydrophobic monomers. The activity of the carriers is described as a function of (a) the influence of increasing the length of the hydrophobic alkyl group substituted onto the pH-sensitive monomer and (b) of the effect of incorporating a hydrophobic monomer such as butyl acrylate (BA) on the pH sensitivity and membrane-destabilizing activity of new polymer compositions. The membrane-destabilizing activity of different polymer compositions was evaluated as a function of pH and polymer concentration using the red blood cells (RBC) hemolysis assay. Hemolysis results show that the increase in the hydrophobic character of polymer backbone results in a shift in the pH sensitivity profile and an increase in the membrane-destabilizing activity. Results show that the observed hemolytic activities and pH sensitivity profiles could be designed across a range that matches the properties needed for drug carriers to enhance the cytoplasmic delivery of therapeutic cargos.     

Analysis of albumin-associated peptides and proteins from ovarian cancer patients

BACKGROUND: Albumin binds low-molecular-weight molecules, including proteins and peptides, which then acquire its longer half-life, thereby protecting the bound species from kidney clearance. We developed an experimental method to isolate albumin in its native state and to then identify [mass spectrometry (MS) sequencing] the corresponding bound low-molecular-weight molecules. We used this method to analyze pooled sera from a human disease study set (high-risk persons without cancer, n = 40; stage I ovarian cancer, n = 30; stage III ovarian cancer, n = 40) to demonstrate the feasibility of this approach as a discovery method. METHODS: Albumin was isolated by solid-phase affinity capture under native binding and washing conditions. Captured albumin-associated proteins and peptides were separated by gel electrophoresis and subjected to iterative MS sequencing by microcapillary reversed-phase tandem MS. Selected albumin-bound protein fragments were confirmed in human sera by Western blotting and immunocompetition. RESULTS: In total, 1208 individual protein sequences were predicted from all 3 pools. The predicted sequences were largely fragments derived from proteins with diverse biological functions. More than one third of these fragments were identified by multiple peptide sequences, and more than one half of the identified species were in vivo cleavage products of parent proteins. An estimated 700 serum peptides or proteins were predicted that had not been reported in previous serum databases. Several proteolytic fragments of larger molecules that may be cancer-related were confirmed immunologically in blood by Western blotting and peptide immunocompetition. BRCA2, a 390-kDa low-abundance nuclear protein linked to cancer susceptibility, was represented in sera as a series of specific fragments bound to albumin. CONCLUSION: Carrier-protein harvesting provides a rich source of candidate peptides and proteins with potential diverse tissue and cellular origins that may reflect important disease-related information.     

A synthetic polymer matrix for the delayed or pulsatile release of water-soluble peptides.

The design of a polymeric peptide release system with a controlled delay time and a burst-free pre-release phase is described. In general, the system consists of a blend of a tyrosine-derived polyarylate and a fast-degrading copolymer of lactic and glycolic acid (PLGA). Due to the peptide-like structure of the polyarylate backbone, peptide-polymer interactions prevented the release of peptide from neat polyarylate films. The addition of PLGA acts as a 'delayed' excipient: as PLGA degrades, it generates acidic degradation products that cause a drop in the internal pH of the polyarylate matrix. This drop in pH weakens the peptide-polymer interactions and causes the release of peptide to commence. The initial molecular weight of PLGA can be used to control the length of time before degradation occurs. Consequently, this parameter can also be used to control the duration of the delay period prior to peptide release. As a specific model system, blends of poly(DTH adipate) with three different copolymers of lactic and glycolic acid were prepared and used for the delayed release of Integrilin, a synthetic water-soluble heptapeptide (clinically used in antithrombic injections) that acts as a highly potent glycoprotein IIb/IIIa antagonist. Blends composed of a 1:1 weight ratio of poly(DTH adipate) and PLGA and containing Integrilin (15%, w/w) were prepared. In vitro release studies were conducted in phosphate buffered solution at 37 degrees C and the release of Integrilin was followed by HPLC. As the initial molecular weight of PLGA varied from 12000 to 62000, the duration of the delay period prior to release increased from 5 to 28 days.     

The use of senior nurses in administering a standardised protocol for the pre-operative optimisation of high risk surgical patients.

The pre-optimisation study is outlined. The pre-operative optimisation of high risk elective surgical patients in a District General Hospital is discussed in relation to a research study. Issues related to the development of a protocol are examined. The use of standardised protocols to guide the pre-optimisation process are focused on, emphasising the role of the senior nurse in administering the protocol.     

Synthesis of a surface molecular imprinting polymer based on silica and its application in the identification of nitrocellulose

A surface molecular imprinting polymer (MIP) based on silica (SiO₂/MIP) with excellent selective identification properties towards nitrocellulose (NC) was synthesized with methylacrylic acid as a functional monomer and NC as a template molecule, through simple in situ polymerization. The functional groups of SiO₂/MIP were studied through Fourier transform infrared spectroscopy. The morphology, crystalline state and thermostability of SiO₂/MIP were investigated respectively by scanning electron microscopy, X-ray diffraction and thermogravimetric analysis. Binding capacity and selectivity studies of SiO₂/MIP for NC and its analogues were carried out through ultraviolet-visible spectrophotometry. The thermal analysis and study of crystalline states confirmed the successful imprinting of NC in the polymer networks. The optimized conditions were found to be a polymerization temperature of 45 °C and a functional monomer to cross-linking ratio of 1 : 3. The adsorption capacity of SiO₂/MIP was improved considerably compared with that of polymers prepared by traditional imprinting technology, with a maximum adsorption amount of 1.7 mg mg⁻¹ in 2 mg ml⁻¹ NC solution, compared with an adsorption capacity of about 0.5 mg mg⁻¹ for a traditional MIP. According to the selectivity study, more NC was adsorbed by SiO₂/MIP than its analogues; the best adsorption capacity of SiO₂/MIP for NC was approaching 5 times that for carboxymethyl cellulose (CMC). The results show that it would be possible to apply SiO₂/MIP for the detection of NC, to give improved sensitivity in security checking and improved contaminant adsorption.

A novel surface molecular imprinting polymer was prepared which displayed excellent specificity, selectivity and a large adsorption capacity for nitrocellulose.     

Identification of Leishmania genes encoding proteins containing tandemly repeating peptides

A genomic Leishmania major DNA expression library was screened using antibodies raised against L. major membranes. Two different clones were identified that encoded proteins containing regions of tandemly repeated peptides. Clone 20 encodes a repetitive peptide of 14 amino acids, while clone 39 encodes a repetitive peptide of 10 amino acids. DNA from clone 20 hybridized with two RNA species of 9,500 and 5,200 nucleotides in length, while DNA from clone 39 hybridized to a single RNA species of 7,500 nucleotides. Antibodies against clone 20 fusion protein recognized a series of L. major proteins of apparent mol wt 250,000. Regions of repetitive peptides is a characteristic shared by many malarial protein antigens and this feature has been implicated in immune evasion. Intracellular parasites such as Leishmania and Plasmodia, therefore, may have evolved similar mechanisms consisting of the expression of proteins containing tandemly repeating peptides that are involved in immune evasion.     

Development and optimisation of a radioimmunoassay for plasma captopril

A specific and sensitive radioimmunoassay has been developed to monitor plasma levels of captopril, the first orally active angiotensin-converting enzyme inhibitor. Because of the reactive nature of the captopril thiol group, captopril was measured as the captopril-N-ethylmaleimide complex (captopril-NEM). Accuracy studies, using samples with known added concentrations of captopril, were satisfactory (r = 0.95, p less than 0.001, y = 0.97x - 2.02), and the radioimmunoassay results compared well with those determined by a gas chromatography/mass spectrometric method (r = 0.98, p less than 0.0005, y = 1.2x - 19). The minimum detection limit of the assay was 2 micrograms captopril/litre plasma.