Charged Polypeptides Research Articles

Cloning and Analyses of a Dual Specific Serine/Thronine Protein Kinase Gene with High Conservative and Constitutive Expression in Oryza (OsSTK)

A cytoplasmic serine/thronine protein kinase gene (OsSTK), had been cloned from Oryza genus. It was found high conservative and constitutive expression in Oryza. OsSTK gene had two exons, separated by 114 bp short intron. The open reading frame of OsSTK gene that predicted encoded a 419 amino acids protein. The amino acid sequence of OsSTK had low identities (less than 53%) with any other known protein kinase. The phylogenetic tree based on the partial DNA sequences of OsSTK from different species and types of wild rice and cultivated rice, was close to the taxation system of rice. Interestingly, OsSTK had a serine, including basic amino acids and charged amino acids abundant polypeptide with a "GDGDGDGDG" sequence at N-terminal that had not been found in any other genes. OsSTK may play dual specificity that phosphorylates both serine/thronine and tyrosone, because the amino acids module of VIb, VIII and XI catalytic domain have both the serine/thronine and tyrosine kinase characters.

Mixtures of oppositely charged polypeptides as high-performance dispersing agents for single-wall carbon nanotubes

A new and simple strategy for the dispersion of single-wall carbon nanotubes in aqueous media is presented which does not rely on hydrophobic interactions between the polypeptidic dispersing agent and the nanotubes, and allows the surface charge of the resulting conjugate materials to be controlled.

Toxicity and Dose Response of Intra-Abdominally Administered Poly-L-α-Lysine and Poly-L-Glutamate for Postoperative Adhesion Protection

Background/Aims: Two differently charged polypeptides, poly-L-lysine (PL) and poly-L-glutamate (PG), have previously been shown to reduce postoperative intra-abdominal adhesions. This study aims to investigate the possible toxic effects and to establish a lowest effective antiadhesive dose. Methods: 152 mice were investigated with a well-known adhesion model and given different concentrations of the two differently charged polypeptides as well as only the cationic PL. Results: For the first time, a probable toxic level of PL given intraperitoneally (40 mg/kg) and the lowest significant concentration of PL and PG for antiadhesive purposes (1.6 mg/kg) could be established. Conclusion: The gap between the possible toxicity level of PL and the lowest efficient antiadhesive dose is probably too narrow, and the shape and charge of PL warrant continuous research for another polycation in the concept of differently charged polypeptides used as antiadhesive agents.

Protamine sulfate down-regulates thrombin generation by inhibiting factor V activation

AbstractProtamine sulfate is a positively charged polypeptide widely used to reverse heparin-induced anticoagulation. Paradoxically, prospective randomized trials have shown that protamine administration for heparin neutralization is associated with increased bleeding, particularly after cardiothoracic surgery with cardiopulmonary bypass. The molecular mechanism(s) through which protamine mediates this anticoagulant effect has not been defined. In vivo administration of pharmacologic doses of protamine to BALB/c mice significantly reduced plasma thrombin generation and prolonged tail-bleeding time (from 120 to 199 seconds). Similarly, in pooled normal human plasma, protamine caused significant dose-dependent prolongations of both prothrombin time and activated partial thromboplastin time. Protamine also markedly attenuated tissue factor-initiated thrombin generation in human plasma, causing a significant decrease in endogenous thrombin potential (41% ± 7%). As expected, low-dose protamine effectively reversed the anticoagulant activity of unfractionated heparin in plasma. However, elevated protamine concentrations were associated with progressive dose-dependent reduction in thrombin generation. To assess the mechanism by which protamine mediates down-regulation of thrombin generation, the effect of protamine on factor V activation was assessed. Protamine was found to significantly reduce the rate of factor V activation by both thrombin and factor Xa. Protamine mediates its anticoagulant activity in plasma by down-regulation of thrombin generation via a novel mechanism, specifically inhibition of factor V activation.

Antibacterial activity of six novel peptides from Tityus discrepans scorpion venom. A fluorescent probe study of microbial membrane Na + permeability changes

Six novel peptides (named bactridines) were isolated from Tityus discrepans scorpion venom. From mass spectrometry molecular masses were 6916, 7362, 7226, 7011, 7101 and 7173 Da (bactridines 1–6). Bactridines 1 and 2 were sequenced by Edman degradation. The sequences and in silico analysis, indicated that they are positively charged polypeptides comprised of 61 and 64 amino acids (AA), respectively, bactridine 1 and bactridine 2 containing 4 disulfide bridges. Bactridine 1 was only toxic to cockroaches and crabs, and bactridine 2–6 were only toxic to mice. Bactridine 1 has a 78% sequence identity with ardiscretin. Ardisctretin is an insect specific sodium toxin which also produces a small depolarization and induces repetitive firing in squid axons resembling those of DDT [1,10(pchlorobenzyl) 2-trichloretane] in its ability to slow down action potential, to induce repetitive firing. Measured as the minimal inhibitory concentration, bactridines had high antibacterial activity against a wide range of Gram positive and Gram negative bacteria. Complete bacterial growth inhibition occurred at concentrations from 20 to 80 μM depending on the bacteria and peptide tested. Effects on membrane Na + permeability induced by bactridines were observed on Yersinia enterocolitica loaded with 1 μM CoroNa™ Red. CoroNa™ Red fluorescence leakage from bacteria was observed after exposure to 0.3 μM of any bactridine tested, indicating that they modified Na + membrane permeability. This effect was blocked by 10 μM amiloride and by 25 μM mibefradil drugs that affect Na + and Ca 2+ channels respectively. We found no evidence of changes of K + or Ca 2+ concentrations neither inside nor outside the bacteria in experiments using the fluorescent dyes Fluo 4AM (10 μM) and PBFI (20 μM).

Positioning and guidance of neurons on gold surfaces by directed assembly of proteins using Atomic Force Microscopy

We demonstrate that Atomic Force Microscopy nanolithography can be used to control effectively the adhesion, growth and interconnectivity of cortical neurons on Au surfaces. We demonstrate immobilization of neurons at well-defined locations on Au surfaces using two different types of patterned proteins: 1) poly- d-lysine (PDL), a positively charged polypeptide used extensively in tissue culture and 2) laminin, a component of the extracellular matrix. Our results show that both PDL and laminin patterns can be used to confine neuronal cells and to control their growth and interconnectivity on Au surfaces, a significant step towards the engineering of artificial neuronal assemblies with well-controlled neuron position and connections.

Calcite Crystal Growth Kinetics in the Presence of Charged Synthetic Polypeptides

Poly-l-glutamic (pGlu) and poly-l-aspartic (pAsp) acids, as analogues of naturally occurring soluble acidic proteins involved in biomineralization processes, and poly-l-lysine (pLys), were used to investigate calcite growth kinetics as a function of the interaction between the charged polypeptides and the calcite surface. The kinetics of calcite crystal growth was determined in a simplified precipitation model system by inoculating well-defined calcite seed crystals into a moderately supersaturated solution containing one of the polypeptides. The parabolic rate law was found to be valid for the calcite crystal growth, the integration of ions into the spiral steps at the calcite crystal surface being the rate-determining mechanism. Small amounts of pGlu or pAsp caused an inhibition of calcite crystal growth, the effect being pAsp > pGlu, and the exponential dependence of the growth rate on supersaturation confirmed that surface nucleation was the growth controlling mechanism in the presence of the two acidic polypeptides. The pLys nonselective, weak, electrostatic adsorption at the crystal surface was probably responsible for increasing the calcite growth rate at low concentrations and for inhibiting it at higher concentrations. The strongest interactions between the crystal surfaces and the polypeptides were observed for the calcite/pAsp systems. They could account for coordinative interactions between the side chain carboxylic groups of the predominantly planar arrangement of the pAsp structure (β-pleated sheet) and Ca2+ ions from the calcite surface.

Biocompatible polypeptide microcapsules via templating mesoporous silica spheres

We reported the stepwise formation of biocompatible poly( l-lysine)/poly( l-glutamic acid) (PLL/PGA) multilayer films on mesoporous silica (MS) spheres via layer-by-layer (LbL) self-assembly technique. In-situ QCM revealed the nonlinear (exponential) growth of PLL/PGA multilayer films at both pH 5.5 and pH 7.0 conditions. ξ-Potential measurements of the multilayer coated particles indicated that the multilayer surface was being charge-overcompensated in each adsorption step, thereby facilitating adsorption of the next oppositely charged polypeptide onto the MS spheres. Hollow polypeptide capsules could be obtained by subsequently removing silica cores in HF solution. By using enzyme-preloaded MS spheres as capsule templates, a general approach was developed for encapsulating enzymes in biocompatible microcapsules with high loading and retained bioactivity. The loading amount for several enzymes with different sizes and their bioactivity after encapsulation were also reported.

Interactions between Homopolypeptides and Lightly Cross-Linked Microgels

The relative importance of electrostatic and nonelectrostatic interactions in peptide-microgel systems was evaluated by micromanipulator-assisted light microscopy, confocal microscopy, and circular dichroism. For this purpose, the interaction of various homopolypeptides with lightly cross-linked polyelectrolyte gel particles ( approximately 70 microm in diameter) was studied with focus on peptide-induced microgel deswelling and its relation to peptide distribution within the microgel particles. Negatively charged poly-l-glutamic acid (pGlu) and poly-l-aspartic acid (pAsp), as well as uncharged poly-l-proline (pPro) and poly-l-threonine (pThr), were found to not bind to negatively charged poly(acrylic acid) microgels under the conditions investigated, but were instead depleted from the microgel particles. Positively charged poly-l-arginine (pArg), poly-l-histidine (pHis), and poly-l-lysine (pLys), on the other hand, interacted strongly with the oppositely charged microgel particles and caused significant deswelling of these. In parallel, cationic acrylamidopropyltriethylammoniumchloride (APTAC) microgels bound negatively charged polypeptides to a much higher extent than positively charged and uncharged ones. These findings suggest that electrostatic interactions dominate peptide binding and resulting microgel deswelling in these systems. Nevertheless, although the amount of cationic peptide bound to the anionic microgel particles was similar for cationic pLys, pArg, and pHis, peptide-induced gel deswelling differed significantly, as did the change in peptide conformation after microgel binding and the peptide distribution within the microgels. These effects, as well as pH dependent binding and release of titrable pHis, are discussed in terms of the effects of the charge density of, and structural differences between, the cationic homopolypeptides on the interaction with the oppositely charged microgel particles.

Intermediate vimentin filaments and their role in intracellular organelle distribution

Intermediate filaments (IF) represent one of three main cytoskeletal structures in most animal cells. The human IF protein family includes about 70 members divided into five main groups. The characteristic feature of IF is that in various cells and tissues they are formed by proteins of different groups. Structures of all IF proteins follow a unique scheme: a central alpha-helical part is flanked at the N and C ends by positively charged polypeptide chains devoid of a clear secondary structure. The central part is highly conserved for all proteins in all animals, whereas the N and C termini strongly differ both in size and amino acid composition. This review covers the broad spectrum of recent investigations of IF structure and diverse functions. Special attention is paid to the regulatory mechanisms of IF functions, mainly to phosphorylation by different protein kinases whose role is well studied. The review gives examples of hereditary diseases associated with mutations of some IF proteins, which point to an important physiological role of these cytoskeletal structures.

Morphology-Controlled Assembly of ZnO Nanostructures: A Bioinspired Method and Visible Luminescence

In a bioinspired methodology, positively charged polypeptides and polyamines directly catalyse ZnO mineralization under "green" conditions of room temperature and neutral pH. The polyamines not only act as mineralizing agents for the formation of ZnO nanoparticles, but also self-assemble the nanoparticles to form spindle-like morphologies at these very ambient conditions. Both the directional growth of ZnO and its luminescent property have a pH dependency. At higher pH, the ZnO shape changes to a rodlike morphology that exhibits green photoluminescence with different intensity than that for ZnO spindles.

Electron Capture/Transfer versus Collisionally Activated/Induced Dissociations: Solo or Duet?

New ion fragmentation technologies--electron capture dissociation (ECD) and electron-transfer dissociation (ETD)--are based on interaction of multiply charged polypeptides with either free electrons (ECD) or anionic species (ETD). After initial difficulties, these ECD/ETD (ExD) technologies are now being increasingly implemented in high-throughput proteomics work. This critical analysis presents arguments for the combined use of ExD with the conventional low-energy collisional excitation CID/CAD (CxD). It is argued that the database search, a key technology in MS/MS-based proteomics, is vulnerable with respect to the incomplete sequence information obtainable with either of the techniques, peptide MS/MS homology being a major complicating factor. De novo sequencing is viewed as the only adequate answer to this challenge and it can be achieved only with combined use of ExD and CxD. The payoff in the form of additional sequence information is projected to exceed the costs of such implementation. The greatest impact of combining ExD and CxD is expected in high-resolution instruments.

Increase of the pharmacological and pharmacokinetic efficacy of negatively charged polypeptide recombinant hirudin in rats via parenteral route by association with cationic liposomes

Two biodegradable cationic lipids, stearylamine and DC-Chol, were chosen to investigate the effect of cationic lipids on the in vitro and in vivo characteristics of hydrophilic proteins or peptides of low isoelectric point. Thrombin inhibitor recombinant hirudin variant-2 (rHV2) was selected as the model drug. The cationic lipids were found to achieve higher entrapment efficiency of rHV2 in liposomes than zwitterionic lipids. The positively charged liposomes became less positive and relatively stable in serum after loading rHV2. The cationic liposomes induced sustained release of rHV2 in the presence of plasma, significantly prolonged the antithrombotic efficacy and plasma level of rHV2 after intravenous injection in rats in comparison with neutral lipid liposomes, especially for stearylamine group. Both clotting times correlated well with plasma rHV2 levels. No serious adverse events were observed and physical state of rats was satisfactory for all the formulations. Electrostatic interaction between negative charge of rHV2 and cationic liposomes was confirmed and it might affect all the characteristics of rHV2 loaded cationic vehicles. The findings suggest that cationic liposomes may be a potential sustained-release delivery system for parenteral administration of hydrophilic proteins or peptides with low isoelectric point to prolong efficacy and improve bioavailability.

Porphyrin “Mille-Feuilles” photo-electrodes

A porphyrin “Mille-Feuilles” photo-electrode is fabricated layer-by-layer by deposition of anionic [zinc meso-tetrakis( p-sulfonatophenyl) porphyrin] 4− (ZnTPPS 4−) and positively charged polypeptides on a 11-mercaptoundecanoic acid-modified gold electrode to form a photoactive film. This work demonstrates that it is possible to form 3D structures having regularly spaced layers of redox molecules. UV–visible spectra of the multilayer films display the characteristic absorption bands of the porphyrins and the absorbance increases linearly with the number of bilayers. The cyclic voltammetric response of the films in contact with 1,2-dichloroethane (DCE) varies from that of a modified electrode to that of a supported liquid film. Under illumination, a large photocurrent response for oxygen reduction with a maximum for a 5-bilayer film is observed.

Biological Variation of the Platelet Proteome in the Elderly Population and Its Implication for Biomarker Research

Knowledge about the extent of total variation experienced between samples from different individuals is of great importance for the design of not only proteomics but every clinical study. This variation defines the smallest statistically significant detectable signal difference when comparing two groups of individuals. We isolated platelets from 20 healthy human volunteers aged 56-100 years because this age group is most commonly encountered in the clinics. We determined the technical and total variation experienced in a proteome analysis using two-dimensional DIGE with IPGs in the pI ranges 4-7 and 6-9. Only spots that were reproducibly detectable in at least 90% of all gels (n = 908) were included in the study. All spots had a similar technical variation with a median coefficient of variation (cv) of about 7%. In contrast, spots showed a more diverse total variation between individuals with a surprisingly low median cv of only 18%. Because most known biomarkers show an effect size in a 1-2-fold range of their cv, any future clinical proteomics study with platelets will require an analytical method that is able to detect such small quantitative differences. In addition, we calculated the minimal number of samples (sample size) needed to detect given protein expression differences with statistical significance.

Osteoformin Accelerates Fresh Fracture Healing in Rats

Negatively charged resins have been shown to stimulate bone repair. In previous studies, the negatively charged polypeptide polyaspartate, which has been named Osteoformin, has been shown to stimulate osteoblast differentiation in vitro. The objective of the current study was to investigate the potential effect of Osteoformin on experimental femoral fracture healing in vivo. Thirty-six male Sprague-Dawley rats were used. The femurs of anesthetized rats were stabilized with intramedullary pins and subjected to closed midshaft transverse facture by bending to failure. In experimental rats, fracture sites were injected with 100 microg of Osteoformin dissolved in 0.1 mL phosphate buffered saline (PBS) at day 1 and day 7 after surgery; in controls groups, fracture sites received 0.1 mL PBS at the intervals indicated above. Between 2 and 4 weeks after fracture, the animals were sacrificed and the healing femurs were removed for radiographic and histologic analysis. Osteoformin decreased the healing time of fresh fractures in rats, as indicated by histologic and radiographic assessments. The results of this study show that Osteoformin improves femoral fracture healing in rats.

Electrospray‐assisted laser desorption/ionization and tandem mass spectrometry of peptides and proteins

We have constructed an electrospray-assisted laser desorption/ionization (ELDI) source which utilizes a nitrogen laser pulse to desorb intact molecules from matrix-containing sample solution droplets, followed by electrospray ionization (ESI) post-ionization. The ELDI source is coupled to a quadrupole ion trap mass spectrometer and allows sampling under ambient conditions. Preliminary data showed that ELDI produces ESI-like multiply charged peptides and proteins up to 29 kDa carbonic anhydrase and 66 kDa bovine albumin from single-protein solutions, as well as from complex digest mixtures. The generated multiply charged polypeptides enable efficient tandem mass spectrometric (MS/MS)-based peptide sequencing. ELDI-MS/MS of protein digests and small intact proteins was performed both by collisionally activated dissociation (CAD) and by nozzle-skimmer dissociation (NSD). ELDI-MS/MS may be a useful tool for protein sequencing analysis and top-down proteomics study, and may complement matrix-assisted laser desorption/ionization (MALDI)-based measurements.

3D-ITIES supported on porous reticulated vitreous carbon

Abstract A thin aqueous film prepared by layer-by-layer assembly of oppositely charged polypeptides on 4-aminobenzoic acid modified porous reticulated vitreous carbon (RVC) electrodes was used with the aim of forming a 3D-ITIES. The film containing a redox mediator, namely ferri/ferrocyanide, was placed in contact with an organic solution 1,2-dichloroethane containing an electron acceptor, 7,7′,8,8′-tetracyanoquinodimethane (TCNQ). The reduction of TCNQ was studied to characterise the electrochemical properties of the supported ITIES. When the film is dye-sensitised, a reduction photocurrent was observed upon illumination. This photocurrent was an order of magnitude larger than that measured on a planar electrode.

Direct characterization of intact polypeptides by matrix‐assisted laser desorption electrospray ionization quadrupole Fourier transform ion cyclotron resonance mass spectrometry

We report the characterization of a recently introduced hybrid ionization source, matrix-assisted laser desorption electrospray ionization (MALDESI), coupled to a quadrupole Fourier transform ion cyclotron resonance mass spectrometry (QFT-ICR-MS) system. We first demonstrate the ability of MALDESI-QFT-ICR MS to directly analyze and provide high mass measurement accuracy (approximately 1 part-per-million) of a polypeptide using internal calibration. Second, we show the potential of MALDESI-QFT-ICR MS for the top-down characterization of multiply charged polypeptide cations. Finally, we demonstrate sub-femtomole detection limits in MALDESI-QFT-ICR MS using a combination of naturally occurring peptides and their respective stable isotope labeled forms. The results presented herein demonstrate the feasibility of several potential applications for MALDESI-QFT-ICR MS for the direct analysis of intact biological molecules.

Orientation of Polyelectrolyte Multilayers Containing Charged Polypeptides Determined by ATR-FTIR and AFM

We report on factors influencing the orientation of polyelectrolyte multilayers (PEM) containing charged polypeptides. As factors, the presence and absence of mechanical unidirectional texturization, the conformation, and the molecular weight will be outlined in four typical samples. For that, the charged cationic homopolypeptide poly(L-lysine) (PLL) was used, whose conformations can be changed from random coil to α-helix by salt type or pH value. PEM of PLL and poly(vinylsulfate) (PVS) were prepared by consecutive adsorption. Dichroic in situ attenuated total reflection infrared (ATR-IR) spectroscopy and atomic force microscopy (AFM) were used for orientation and surface topology characterization, respectively. Untexturized surfaces, the random coil conformation, and low molecular weight of PLL caused the formation of isotropically structured PEM films, and the α-helical conformation and the high molecular weight of PLL resulted in anisotropic PEM films. Polypeptide-containing PEM could be used as biomimetic coatings for specific interaction with α-helical proteins.