Semicarbazide-sensitive amine oxidase in annulo-aortic ectasia disease: relation to elastic lamellae-associated proteins.

Lysyl oxidases (Lox), which are members of the amine oxidase family, are involved in the maturation of elastic lamellae and collagen fibers. Modifications of amine oxidases in idiopathic annulo-aortic ectasia disease (IAAED) have never been investigated. Our aim was to examine the expression of several proteins that might interfere with elastic fiber organization in control (n=10) and IAAED (n=18) aortic tissues obtained at surgery. Expression of amine oxidases and semicarbazide-sensitive amine oxidase (SSAO), and cellular phenotypic markers were examined by immunohistopathology and confocal microscopy. The expression of these proteins was assessed in relation to clinical and histomorphological features of the arterial wall. In control aorta, SSAO staining was expressed along elastic lamellae, whereas in aneurysmal areas of IAAED, SSAO was markedly decreased, in association with severe disorganization of elastic lamellae. Smooth muscle myosin heavy chain was also decreased in IAAED compared with controls, indicating smooth muscle cell dedifferentiation. Multiple regression analysis showed that elastic lamellar thickness (ELT) was correlated positively with the SSAO:elastin ratio and negatively with the Lox:elastin ratio, and that the clinical features of IAAED (aneurysm, thoracic aorta diameter, and aortic insufficiency) were positively correlated with ELT but not with SSAO. The relationship between SSAO expression and ELT suggests that this amine oxidase may be involved in elastic fiber organization. However, in advanced IAAED, the deficit in SSAO expression could be secondary to the decrease and fragmentation of elastic fibers and/or to vascular smooth muscle cell dedifferentiation.     

Modélisation mathématique du traitement par laser endoveineux (LEV)

Background and ObjectivesEndovenous laser treatment (ELT) has been recently proposed as an alternative in the treatment of reflux of the great saphenous vein (GSV) and small saphenous vein (SSV). Successful ELT depends on the selection of optimal parameters required to achieve an optimal vein damage while avoiding side effects. Mathematical modeling of ELT could provide a better understanding of the ELT process and could determine the optimal dosage as a function of vein diameter.Study design–materials and methodsThe model is based on calculations describing the light distribution using the diffusion approximation of the transport theory, the temperature rise using the bioheat equation and the laser-induced injury using the Arrhenius damage model. The geometry to simulate ELT was based on a 2D model consisting of a cylindrically symmetric blood vessel including a vessel wall and surrounded by an infinite homogenous tissue. The mathematical model was implemented using the Macsyma-Pdease2D software (Macsyma Inc., Arlington, MA, USA). Damage to the vein wall for CW and single shot energy was calculated for one vein diameter (3 mm). In pulsed mode, the pullback distance (3, 5 and 7 mm) was considered. For CW mode simulation, the pullback speed (1, 2, 3 mm/s) was the variable. The total dose was expressed as joules per centimeter in order to perform comparison to results already reported in clinical studies.ResultsIn pulsed mode, for a 3 mm vein diameter, irrespective of the pullback distance (2, 5 or 7 mm), a minimum fluence of 15 J/cm is required to obtain a permanent damage of the intima. In continuous mode, for a 3 mm vein diameter, 65 J/cm are required to obtain a permanent damage of the vessel wall. Finally, the use of different wavelengths (810 or 980 nm) played only a minor influence on these results.Discussion and conclusionThe parameters determined by mathematical modeling are in keeping with those used in clinical practice. They confirm that thermal damage of the inner vein wall is required to obtain a permanent occlusion. However, in order to obtain a high rate of success, without adverse events, the knowledge of the vein diameter after tumescent anesthesia is required. When performing ELT, the pulsed mode should be preferred for 2 reasons: 1) it requires less energy since the vein is only damaged sequentially and not continuously, 2) it reduces the risk of too high an energy inside the vein when pullback speed is not controlled. So, the pulsed mode requires a very precise positioning of the fiber after each pullback and the time of treatment is longer but it has been prove that this technique is reproducible, safe and efficient [29].This model should be a very useful tool to simulate ELT.     

Role of lysophosphatidic acid in the regulation of uterine leiomyoma cell proliferation by phospholipase D and autotaxin

Phospholipase D (PLD) hydrolyzes phosphatidylcholine into phosphatidic acid (PA), a lipidic mediator that may act directly on cellular proteins or may be metabolized into lysophosphatidic acid (LPA). We previously showed that PLD contributed to the mitogenic effect of endothelin-1 (ET-1) in a leiomyoma cell line (ELT3 cells). In this work, we tested the ability of exogenous PA and PLD from Streptomyces chromofuscus (scPLD) to reproduce the effect of endogenous PLD in ELT3 cells and the possibility that these agents acted through LPA formation. We found that PA, scPLD, and LPA stimulated thymidine incorporation. LPA and scPLD induced extracellular signal-regulated kinase (ERK(1/2)) mitogen-activated protein kinase activation. Using Ki16425, an LPA(1)/LPA(3) receptor antagonist and small interfering RNA targeting LPA(1) receptor, we demonstrated that scPLD acted through LPA production and LPA(1) receptor activation. We found that scPLD induced LPA production by hydrolyzing lysophosphatidylcholine through its lysophospholipase D (lysoPLD) activity. Autotaxin (ATX), a naturally occurring lysoPLD, reproduced the effects of scPLD. By contrast, endogenous PLD stimulated by ET-1 failed to produce LPA. These results demonstrate that scPLD stimulated ELT3 cell proliferation by an LPA-dependent mechanism, different from that triggered by endogenous PLD. These data suggest that in vivo, an extracellular lysoPLD such as ATX may participate in leiomyoma growth through local LPA formation.     

Phosphatidylinositol 3-kinase but not tuberin is required for PDGF-induced cell migration

The loss of function of the tumor suppressor gene TSC2 and its protein product tuberin promotes the development of benign lesions by stimulating cell growth, although the role of tuberin in regulating cell migration and metastasis has not been characterized. In addition, the role of phosphatidylinositol 3-kinase (PI 3-kinase), an important signaling event regulating cell migration, in modulating tuberin-deficient cell motility remains unknown. Using a tuberin-deficient rat smooth muscle cell line, ELT3, we demonstrate that platelet-derived growth factor (PDGF) stimulates cell migration by 3.2-fold, whereas vascular endothelial growth factor (VEGF), transforming growth factor (TGF)-alpha, and basic fibroblast growth factor (bFGF) increase migration by 2.1-, 2.1-, and 2.6-fold, respectively. Basal and PDGF-induced migration in tuberin-deficient ELT3, ELT4, and ERC15 cells was not significantly different from that of tuberin-positive transformed rat kidney epithelial 2, airway smooth muscle, and pulmonary arterial vascular smooth muscle cells. Expression of tuberin in tuberin-deficient ELT3 cells also had little effect on cell migration. In parallel experiments, the role of PI 3-kinase activation in ELT3 cell migration was investigated. LY-294002, a PI 3-kinase inhibitor, decreased PDGF-induced migration in a concentration-dependent manner with an IC(50) of approximately 5 microM. LY-294002 also abrogated ELT3 cell migration stimulated by bFGF and TGF-alpha but not by VEGF and phorbol 12-myristate 13-acetate. Furthermore, transient expression of constitutively active PI 3-kinase (p110*) was sufficient to induce ELT3 cell migration. However, the migration induced by p110* was less than that induced by growth factors, suggesting other signaling pathways are also critically important in modulating growth factor-induced cell migration. These data suggest that PI 3-kinase is required for growth factor-induced cell migration and loss of tuberin appears to have little effect on cell migration.     

藕节止血作用研究

本实验通过测定毛细管凝血时间(CT)、剪尾法出血时间(BT)、活化部分凝血酶时间(APTT)、凝血酶原时间(PT)、凝血酶时间(TT)、优球蛋白溶解时间(ELT)等指标研究藕节醇提物及其不同极性段的体内止血作用.结果表明,藕节醇提物显著缩短小鼠CT、BT,显示很好的止血作用,可能是主要通过缩短APTT、PT,延长ELT...     

Mathematical modeling of endovenous laser treatment (ELT)

Background and objectives  

Endovenous laser treatment (ELT) has been recently proposed as an alternative in the treatment of reflux of the Great Saphenous Vein (GSV) and Small Saphenous Vein (SSV). Successful ELT depends on the selection of optimal parameters required to achieve an optimal vein damage while avoiding side effects. Mathematical modeling of ELT could provide a better understanding of the ELT process and could determine the optimal dosage as a function of vein diameter.     

Determination of the specific radloactivity of proteins separated by two-dimensional gel electrophoresis

Tritlum-labeled proteins, separated by two-dimensional gel electrophoresis, can be quantitatively extracted using sodium dodecyl sulfate (SDS)-urea buffer and subsequently acid-precipitated in the presence of serum albumin as carrier at low temperature (0–4°C). Their radioactivity can be counted efficiently under these conditions. Besides a better efficiency of counting, this method has some other advantages over the classical procedures using H₂O₂. The amounts of the eluted proteins can be easily measured in the SDS solution, using the Lowry method, and therefore specific radioactivity can be calculated. Also SDS can be removed easily, and the proteins can be used for further experiments.     

Transgenic Mice Over-expressing Endothelin-1 in Testis Transactivated by a Cre/loxP System Showed Decreased Testicular Capillary Blood Flow

It is generally believed that too high or low levels of endothelin-1 (ET-1), a strong vasoconstrictor, may be detrimental to animals. Therefore, in order to understand the in vivo function of ET-1, we used a conditional transgenic approach, Cre/loxP recombination system, to generate transgenic mice that over-express ET-1 in a tissue-specific manner. In such a strategy a single transgenic mouse line, ELSE, was initially generated where a general promoter, human elongation factor 1alpha (hEF1alpha) promoter, was used to drive the expression of a loxP-flanked sequence containing the lacZ reporter gene and a STOP cassette before the ET-1 cDNA, the recombinational competency of which was confirmed in an Escherichia coli test system. In ELSE mice, expression of the reporter lacZ was limited to spermatozoa and spermatogonia as well as Sertoli, Leydig and endothelial cells in the testis, thus confirming the suitability of these mice for the generation of testes-limited ET-1 expression. To generate transgenic progeny with ET-1 over-expression in the testis (successful recombination, ELSE/ELT), ELSE mice were mated with EIIa-cre mice expressing Cre recombinase in pre-implantation mouse embryos. These ELSE/ELT mice exhibiting testis-specific ET-1 over-expression had normal reproductive function and showed no obvious alterations in gross testicular morphology. Although over-expression of ET-1 leads to reduction of testicular blood flow, young adult ELSE/ELT mice showed no obvious signs of inflammation, fibrosis or abnormal proliferation of cells in the testes of young ELSE/ELT mice by histochemical analyses.     

Sedimentation equilibrium in macromolecular solutions of arbitrary concentration. II. Two protein components

Relations describing sedimentation equilibrium in solutions containing two macromolecular solute components are derived for the following cases: (1) two nonassociating proteins at arbitrary concentration, (2) one dilute self-associating protein in the presence of a second inert protein at arbitrary concentration, and (3) two proteins at arbitrary concentration that can associate to form a single heterocomplex of arbitrary composition. As in earlier work (R. C. Chatelier and A. P. Minton (1987) Biopolymers, 26, 507–524), the relations are obtained by using scaled particle theory to calculate the thermodynamic activity of each species present at a given radial distance in the centrifuge. The results of numerical simulations of sedimentation equilibrium are presented as the dependence of apparent molecular weights, or apparent weight-average molecular weights, upon solution composition. Semiempirical methods are presented, by means of which the weight-average molecular weights of self- and heteroassociating proteins in highly nonideal solutions may be estimated from experimental data. It is found that the semiempirical methods yield reasonably accurate estimates of the true weight-average molecular weight over a broad range of experimental conditions, providing that the partial specific volumes of two components in a heteroassociating system do not differ by more than about 0.05 mL/g.     

Protein structure determination from pseudocontact shifts using ROSETTA

Paramagnetic metal ions generate pseudocontact shifts (PCSs) in nuclear magnetic resonance spectra that are manifested as easily measurable changes in chemical shifts. Metals can be incorporated into proteins through metal binding tags, and PCS data constitute powerful long-range restraints on the positions of nuclear spins relative to the coordinate system of the magnetic susceptibility anisotropy tensor (Δχ-tensor) of the metal ion. We show that three-dimensional structures of proteins can reliably be determined using PCS data from a single metal binding site combined with backbone chemical shifts. The program PCS-ROSETTA automatically determines the Δχ-tensor and metal position from the PCS data during the structure calculations, without any prior knowledge of the protein structure. The program can determine structures accurately for proteins of up to 150 residues, offering a powerful new approach to protein structure determination that relies exclusively on readily measurable backbone chemical shifts and easily discriminates between correctly and incorrectly folded conformations.     

Protein sequence analysis using Hewlett-Packard biphasic sequencing cartridges in an applied biosystems 473A protein sequencer

Protein sequence analysis using an adsorptive biphasic sequencing cartridge, a set of two coupled columns introduced by Hewlett-Packard for protein sequencing by Edman degradation, in an Applied Biosystems 473A protein sequencer has been demonstrated. Samples containing salts, detergents, excipients, etc. (e.g., formulated protein drugs) can be easily analyzed using the ABI sequencer. Simple modifications to the ABI sequencer to accommodate the cartridge extend its utility in the analysis of difficult samples. The ABI sequencer solvents and reagents were compatible with the HP cartridge for sequencing. Sequence information up to ten residues can be easily generated by this nonoptimized procedure, and it is sufficient for identifying proteins by database search and for preparing a DNA probe for cloning novel proteins.     

A robust,cost‐effective method for DNA,RNA and protein co‐extraction from soil,other complex microbiomes and pure cultures

The soil microbiome is inherently complex with high biological diversity, and spatial heterogeneity typically occurring on the submillimetre scale. To study the microbial ecology of soils, and other microbiomes, biomolecules, that is, nucleic acids and proteins, must be efficiently and reliably co‐recovered from the same biological samples. Commercial kits are currently available for the co‐extraction of DNA, RNA and proteins but none has been developed for soil samples. We present a new protocol drawing on existing phenol–chloroform‐based methods for nucleic acids co‐extraction but incorporating targeted precipitation of proteins from the phenol phase. The protocol is cost‐effective and robust, and easily implemented using reagents commonly available in laboratories. The method is estimated to be eight times cheaper than using disparate commercial kits for the isolation of DNA and/or RNA, and proteins, from soil. The method is effective, providing good quality biomolecules from a diverse range of soil types, with clay contents varying from 9.5% to 35.1%, which we successfully used for downstream, high‐throughput gene sequencing and metaproteomics. Additionally, we demonstrate that the protocol can also be easily implemented for biomolecule co‐extraction from other complex microbiome samples, including cattle slurry and microbial communities recovered from anaerobic bioreactors, as well as from Gram‐positive and Gram‐negative pure cultures.     

The determination of the activity of immobilised amidases using an ammonia-sensitive glass electrode.

Ribosomal proteins, separated from rRNA by the LiCl-urea method, are then precipitated by adding one volume of 20% trichloroacetic acid. We show here that this precipitate can be dissolved directly into unbuffered Tris-OH for electrophoresis on acrylamide gels without further washing of the pellet or dialyzing of the solution.Ribosomal proteins can also be separated from rRNA by using 66% acetic acid in presence of 0.1 m Mg²⁺. We show here that the ribosomal proteins can be precipitated directly from the acetic acid by adding ten volumes of 10% trichloroacetic acid. This precipitate is then easily dissolved in unbuffered Tris-OH to prepare the proteins for acrylamide-gel electrophoresis without further treatment.     

Proportional means regression for censored medical costs

The semiparametric proportional means model specifies that the mean function for the cumulative medical cost over time conditional on a set of covariates is equal to an arbitrary baseline mean function multiplied by an exponential regression function. We demonstrate how to estimate the vector-valued regression parameter using possibly censored lifetime costs. The estimator is consistent and asymptotically normal with an easily estimable covariance matrix. Simulation studies show that the proposed methodology is appropriate for practical use. An application to AIDS is provided.     

Discrete breathers in protein structures

Recently, using a numerical surface cooling approach, we have shown that highly energetic discrete breathers (DBs) can form in the stiffest parts of nonlinear network models of large protein structures. In the present study, using an analytical approach, we extend our previous results to low-energy discrete breathers as well as to smaller proteins. We confirm and further scrutinize the striking site selectiveness of energy localization in the presence of spatial disorder. In particular, we find that, as a sheer consequence of disorder, a non-zero energy gap for exciting a DB at a given site either exists or not. Remarkably, in the former case, the gaps arise as a result of the impossibility of exciting small-amplitude modes in the first place. In contrast, in the latter case, a small subset of linear edge modes acts as accumulation points, whereby DBs can be continued to arbitrary small energies, while unavoidably approaching one of such normal modes. In particular, the case of the edge mode seems peculiar, its dispersion relation being simple and little system dependent. Concerning the structure-dynamics relationship, we find that the regions of protein structures where DBs form easily (zero or small gaps) are unfailingly the most highly connected ones, also characterized by weak local clustering. Remarkably, a systematic analysis on a large database of enzyme structures reveals that amino-acid residues involved in catalysis tend to be located in such regions. This finding reinforces the idea that localized modes of nonlinear origin may play an important biological role, e.g., by providing a ready channel for energy storage and/or contributing to lower energy barriers of chemical reactions.     

Matrix effects in PIXE analysis of aerosols and ashes

A comparison of instrumental neutron activation analysis (INAA) and proton-induced X-ray emission (PIXE) results for sizefractionated atmospheric aerosols (“coarse” and “fine” fractions with an equivalent aerodynamic diameter of 2–10 Μm and < 2 Μm, respectively, or the PM10 fraction) showed that PIXE yielded significantly lower results for the PM10 and coarse fractions, especially for elements with a low Z resulting from a particle size effect. Somewhat lower PIXE results were also obtained for the fine fraction of atmospheric aerosols. A correction is also needed for irregularly shaped deposits of combustion aerosols collected by a cascade impactor in 11 size fractions ranging from 0.016 to 14.3 Μm, as well as for thick samples of fly and bottom ashes. An equivalent layer thickness (ELT) model is proposed to correct the matrix effects in PIXE. The approaches for the calculation of ELT using a comparison of PIXE and INAA results or by comparing PIXE results obtained using two different incident proton beam energies (1.31 and 2.35 MeV) are described. The correction for the ash pellets and irregular deposits are also discussed.     

Synthesis of 32P-labelled protein probes using a modified thioredoxin fusion protein expression system in Escherichia coli

The thioredoxin fusion protein expression system from invitrogen was modified so that 32P-labelled recombinant proteins can be easily obtained in large quantities for functional studies. Proteins that are prone to form the inclusion bodies can be functionally expressed as thioredoxin fusion proteins in Escherichia coli. After expression, the recombinant proteins can be easily phosphorylated with 32P-gamma ATP and the 32P-labelled protein can be obtained functionally via a mild proteolytic digestion to cleave off the thioredoxin moiety. A deletion construct of the Ah receptor nuclear translocator protein was used as an example to illustrate how this protein expression system works.     

A novel method for analyzing phosphoproteins using SELDI-TOF MS in combination with a series of recombinant proteins

A novel SELDI-TOF MS-based method for analyzing phosphoproteins was developed using a series of recombinant wild-type and mutant ribosomal P2 proteins. We demonstrated that the phosphorylation status of the overexpressed proteins in cells was easily and rapidly confirmed using this method. The ribosomal P2 protein contained two phosphorylation sites, which were sequentially phosphorylated in vivo. We also quantitatively detected the phosphoprotein by using SELDI-TOF MS.     

Towards a microarray of functional membrane proteins: Assembly of a surface-attachable,membrane-protein-anchored membrane structure using apolipoprotein A-1

Apolipoprotein A-1 (apoA-I) forms a discoidal membrane structure with phospholipids during the formation of high density lipoprotein and plays a role in the reverse cholesterol transport pathway. The discoidal membrane nanostructure, Nanodisc, can be easily assembled in vitro using apoA-I protein and phospholipids. In this study, the possibility of exploiting this unique membrane structure for the immobilization of membrane proteins on solid surfaces while the proteins are embedded in the membrane was investigated. By using His6-tagged full-length apoA-I, a surface-attachable, membrane-protein-anchored membrane (SAMPAM) structure, in which membrane proteins of interest are embedded into the membrane, was reconstituted. When the SAMPAM was immobilized on a Ni-NTA surface, the structure maintained its size and shape, indicating that the new proposed architecture may be useful for the display of membrane proteins on a solid surface in a membrane-associated form.     

Optimal docking area: a new method for predicting protein-protein interaction sites

Understanding energetics and mechanism of protein-protein association remains one of the biggest theoretical problems in structural biology. It is assumed that desolvation must play an essential role during the association process, and indeed protein-protein interfaces in obligate complexes have been found to be highly hydrophobic. However, the identification of protein interaction sites from surface analysis of proteins involved in non-obligate protein-protein complexes is more challenging. Here we present Optimal Docking Area (ODA), a new fast and accurate method of analyzing a protein surface in search of areas with favorable energy change when buried upon protein-protein association. The method identifies continuous surface patches with optimal docking desolvation energy based on atomic solvation parameters adjusted for protein-protein docking. The procedure has been validated on the unbound structures of a total of 66 non-homologous proteins involved in non-obligate protein-protein hetero-complexes of known structure. Optimal docking areas with significant low-docking surface energy were found in around half of the proteins. The 'ODA hot spots' detected in X-ray unbound structures were correctly located in the known protein-protein binding sites in 80% of the cases. The role of these low-surface-energy areas during complex formation is discussed. Burial of these regions during protein-protein association may favor the complexed configurations with near-native interfaces but otherwise arbitrary orientations, thus driving the formation of an encounter complex. The patch prediction procedure is freely accessible at http://www.molsoft.com/oda and can be easily scaled up for predictions in structural proteomics.