Myoblast transplantation and adenoviral VEGF-C transfer in porcine model of coronary artery disease

Heart failure is a common and highly challenging medical disorder. The progressive increase of elderly population is expected to further reflect in heart failure incidence. Recent progress in cell transplantation therapy has provided a conceptual alternative for treatment of heart failure. Despite improved medical treatment and operative possibilities, end-stage coronary artery disease present a great medical challenge. It has been estimated that therapeutic angiogenesis would be the next major advance in the treatment of ischaemic heart disease. Gene transfer to augment neovascularization could be beneficial for such patients. We employed a porcine model to evaluate the angiogenic effect of vascular endothelial growth factor (VEGF)-C gene transfer. Ameroid-generated myocardial ischemia was produced and adenovirus encoding (ad)VEGF-C or β-galactosidase (LacZ) gene therapy was given intramyocardially during progressive coronary stenosis. Angiography, positron emission tomography (PET), single photon emission computed tomography (SPECT) and histology evidenced beneficial affects of the adVEGF-C gene transfer compared to adLacZ. The myocardial deterioration during progressive coronary stenosis seen in the control group was restrained in the treatment group. We observed an uneven occlusion rate of the coronary vessels with Ameroid constrictor. We developed a simple methodological improvement of Ameroid model by ligating of the Ameroid–stenosed coronary vessel. Improvement of the model was seen by a more reliable occlusion rate of the vessel concerned and a formation of a rather constant myocardial infarction. We assessed the spontaneous healing of the left ventricle (LV) in this new model by SPECT, PET, MRI, and angiography. Significant spontaneous improvement of myocardial perfusion and function was seen as well as diminishment of scar volume. Histologically more microvessels were seen in the border area of the lesion. Double staining of the myocytes in mitosis indicated more cardiomyocyte regeneration at the remote area of the lesion. The potential of autologous myoblast transplantation after ischaemia and infarction of porcine heart was evaluated. After ligation of stenosed coronary artery, autologous myoblast transplantation or control medium was directly injected into the myocardium at the lesion area. Assessed by MRI, improvement of diastolic function was seen in the myoblast-transplanted animals, but not in the control animals. Systolic function remained unchanged in both groups.

Inferring molecular function: contributions from functional linkages

In the current era of high-throughput sequencing and structure determination, functional annotation has become a bottleneck in biomedical science. Here, we show that automated inference of molecular function using functional linkages among genes increases the accuracy of functional assignments by >= 8% and enriches functional descriptions in >= 34% of top assignments. Furthermore, biochemical literature supports >80% of automated inferences for previously unannotated proteins. These results emphasize the benefit of incorporating functional linkages in protein annotation.

Ischemia-reperfusion injury in human liver transplantation : Mechanisms and effects on graft function

Liver transplantation is an established therapy for both acute and chronic liver failure. Despite excellent long-term outcome, graft dysfunction remains a problem affecting up to 15-30% of the recipients. The etiology of dysfunction is multifactorial, with ischemia-reperfusion injury regarded as one of the most important contributors. This thesis focuses on the inflammatory response during graft procurement and reperfusion in liver transplantation in adults. Activation of protein C was examined as a potential endogenous anti-inflammatory mechanism. The effects of inflammatory responses on graft function and outcome were investigated. Seventy adult patients undergoing liver transplantation in Helsinki University Central Hospital, and 50 multiorgan donors, were studied. Blood samples from the portal and the hepatic veins were drawn before graft procurement and at several time points during graft reperfusion to assess changes within the liver. Liver biopsies were taken before graft preservation and after reperfusion. Neutrophil and monocyte CD11b and L-selectin expression were analysed by flow cytometry. Plasma TNF-α, IL-6, IL-8, sICAM-1, and HMGB1 were determined by ELISA and Western-blotting. HMGB1 immunohistochemistry was performed on liver tissue specimens. Plasma protein C and activated protein C were determined by an enzyme-capture assay. Hepatic IL-8 release during graft procurement was associated with subsequent graft dysfunction, biliary in particular, in the recipient. Biliary marker levels increased only 5 7 days after transplantation. Thus, donor inflammatory response appears to influence recipient liver function with relatively long-lasting effects. Hepatic phagocyte activation and sequestration, with concomitant HMGB1 release, occurred during reperfusion. Neither phagocyte activation nor plasma cytokines correlated with postoperative graft function. Thus, activation of the inflammatory responses within the liver during reperfusion may be of minor clinical significance. However, HMGB1 was released from hepatocytes and were also correlated with postoperative transaminase levels. Accordingly, HMGB1 appears to be a marker of hepatocellular injury.

THE BERGMAN KERNEL FUNCTION

In this note, we point out that a large family of n x n matrix valued kernel functions defined on the unit disc D subset of C, which were constructed recently in [9], behave like the familiar Bergman kernel function on ID in several different ways. We show that a number of questions involving the multiplication operator on the corresponding Hilbert space of holomorphic functions on D can be answered using this likeness.

Structural Studies on the Second Mycobacterium smegmatis Dps: Invariant and Variable Features of Structure, Assembly and Function

A second DNA binding protein from stationary-phase cells of Mycobacterium smegmatis (MsDps2) has been identified from the bacterial genome. It was cloned, expressed and characterised and its crystal structure was determined. The core dodecameric structure of MsDps2 is the same as that of the Dps from the organism described earlier (MsDps1). However, MsDps2 possesses a long N-terminal tail instead of the C-terminal tail in MsDps1. This tail appears to be involved in DNA binding. It is also intimately involved in stabilizing the dodecamer. Partly on account of this factor, MsDps2 assembles straightway into the dodecamer, while MsDps1 does so on incubation after going through an intermediate trimeric stage. The ferroxidation centre is similar in the two proteins, while the pores leading to it exhibit some difference. The mode of sequestration of DNA in the crystalline array of molecules, as evidenced by the crystal structures, appears to be different in MsDps1 and MsDps2, highlighting the variability in the mode of Dps–DNA complexation. A sequence search led to the identification of 300 Dps molecules in bacteria with known genome sequences. Fifty bacteria contain two or more types of Dps molecules each, while 195 contain only one type. Some bacteria, notably some pathogenic ones, do not contain Dps. A sequence signature for Dps could also be derived from the analysis.

The mixed black hole partition function for the STU model

We evaluate the mixed partition function for dyonic BPS black holes using the recently proposed degeneracy formula for the STU model. The result factorizes into the OSV mixed partition function times a proportionality factor. The latter is in agreement with the measure factor that was recently conjectured for a class of N = 2 black holes that contains the STU model.

Lung structure and function studied by synchrotron radiation

A novel method for functional lung imaging was introduced by adapting the K-edge subtraction method (KES) to in vivo studies of small animals. In this method two synchrotron radiation energies, which bracket the K-edge of the contrast agent, are used for simultaneous recording of absorption-contrast images. Stable xenon gas is used as the contrast agent, and imaging is performed in projection or computed tomography (CT) mode. Subtraction of the two images yields the distribution of xenon, while removing practically all features due to other structures, and the xenon density can be calculated quantitatively. Because the images are recorded simultaneously, there are no movement artifacts in the subtraction image. Time resolution for a series of CT images is one image/s, which allows functional studies. Voxel size is 0.1mm3, which is an order better than in traditional lung imaging methods. KES imaging technique was used in studies of ventilation distribution and the effects of histamine-induced airway narrowing in healthy, mechanically ventilated, and anaesthetized rabbits. First, the effect of tidal volume on ventilation was studied, and the results show that an increase in tidal volume without an increase in minute ventilation results a proportional increase in regional ventilation. Second, spiral CT was used to quantify the airspace volumes in lungs in normal conditions and after histamine aerosol inhalation, and the results showed large patchy filling defects in peripheral lungs following histamine provocation. Third, the kinetics of proximal and distal airway response to histamine aerosol were examined, and the findings show that the distal airways react immediately to histamine and start to recover, while the reaction and the recovery in proximal airways is slower. Fourth, the fractal dimensions of lungs was studied, and it was found that the fractal dimension is higher at the apical part of the lungs compared to the basal part, indicating structural differences between apical and basal lung level. These results provide new insights to lung function and the effects of drug challenge studies. Nowadays the technique is available at synchrotron radiation facilities, but the compact synchrotron radiation sources are being developed, and in relatively near future the method may be used at hospitals.

Rotorcraft parameter estimation using radial basis function neural network

Increased emphasis on rotorcraft performance and perational capabilities has resulted in accurate computation of aerodynamic stability and control parameters. System identification is one such tool in which the model structure and parameters such as aerodynamic stability and control derivatives are derived. In the present work, the rotorcraft aerodynamic parameters are computed using radial basis function neural networks (RBFN) in the presence of both state and measurement noise. The effect of presence of outliers in the data is also considered. RBFN is found to give superior results compared to finite difference derivatives for noisy data. (C) 2010 Elsevier Inc. All rights reserved.

Asymmetric cross-coupled differential pair configuration to realize neuron activation function and its derivative

In this brief, we present a new circuit technique to generate the sigmoid neuron activation function (NAF) and its derivative (DNAF). The circuit makes use of transistor asymmetry in cross-coupled differential pair to obtain the derivative. The asymmetry is introduced through external control signal, as and when required. This results in the efficient utilization of the hard-ware by realizing NAF and DNAF using the same building blocks. The operation of the circuit is presented in the subthreshold region for ultra low-power applications. The proposed circuit has been experimentally prototyped and characterized as a proof of concept on the 1.5-mum AMI technology.

Characterization of the interaction of lipid A and lipopolysaccharide with human serum albumin: implications for an endotoxin carrier function for albumin

The interactions of lipid A and lipopolysaccharide (LPS) with human serum albumin (HSA) were examined using fluorescence methods. Lipid A binds HSA with a stoichiometry of 2:1 with dissociation constants of 1.0 µM and 6.0 µM for the high- and low-affinity interactions, respectively. Lipid A displaces HSA-bound dansylsarcosine competitively, but not HSA-bound warfarin, suggesting that domain III-A, and not domain 11-A, is a lipid A binding site. Domain I does not contribute a site for lipid A. Based on these data, and the structural similarity between subdomains III-A and III-B, it is proposed that these two regions of HSA represent the high- and low-affinity sites of interaction of lipid A. Whole LPS also binds HSA, displacing dansylsarcosine, and its lipid A moiety appears to be the interaction site. However, there are differences between LPS and free lipid A. Polymyxin B forms ternary complexes with LPS bound to HSA, suggesting that the regions on LPS recognized by HSA and polymyxin B are different. The observed affinity of lipid A for HSA and mass action effects due to its abundance in the circulation would imply a major LPS carrier function for HSA.

Structure determination and biochemical studies on Bacillus stearothermophilus E53Q serine hydroxymethyltransferase and its complexes provide insights on function and enzyme memory

Serine hydroxymethyltransferase (SHMT) belongs to the alpha-family of pyridoxal 5'-phosphate-dependent enzymes and catalyzes the reversible conversion of L-Ser and etrahydrofolate to Gly and 5,10-methylene tetrahydrofolate. 5,10-Methylene tetrahydrofolate serves as a source of one-carbon fragment in many biological processes. SHMT also catalyzes the tetrahydrofolate-independent conversion of L-allo-Thr to Gly and acetaldehyde. The crystal structure of Bacillus stearothermophilus SHMT (bsSHMT) suggested that E53 interacts with the substrate, L-Ser and etrahydrofolate. To elucidate the role of E53, it was mutated to Q and structural and biochemical studies were carried out with the mutant enzyme. The internal aldimine structure of E53QbsSHMT was similar to that of the except for significant changes at Q53, Y60 and Y61. The wild-type enzyme, carboxyl of Gly and side chain of L-Ser were in two conformations in the respective external aldimine structures. The mutant enzyme was completely inactive for tetrahydrofolate-depen dent cleavage of L-Ser, whereas there was a 1.5-fold increase in the rate of tetrahydrofolate-independent reaction with L-allo-Thr. The results obtained from these studies suggest that E53 plays an essential role in tetrahydrofolate/5-formyl tetrahydrofolate binding and in the proper positioning of C beta of L-Ser for direct attack by N5 of tetrahydrofolate. Most interestingly, the structure of the complex obtained by cocrystallization of E53QbsSHMT with Gly and 5-formyl tetrahydrofolate revealed the gem-diamine form of pyridoxal 5'-phosphate bound to Gly and active site Lys. However, density for 5-formyl tetrahydrofolate was not observed. Gly carboxylate was in a single conformation, whereas pyridoxal 5'-phosphate had two distinct conformations. The differences between the structures of this complex and Gly external aldimine suggest that the changes induced by initial binding of 5-formyl tetrahydrofolate are retained even though 5-formyl tetrahydrofolate is absent in the final structure. Spectral studies carried out with this mutant enzyme also suggest that 5-formyl tetrahydrofolate binds to the E53QbsSHMT-Gly complex forming a quinonoid intermediate and falls off within 4 h of dialysis, leaving behind the mutant enzyme in the gemdiamine form. This is the first report to provide direct evidence for enzyme memory based on the crystal structure of enzyme complexes.

Microcalorimetric indications for ligand binding as a function of the protein for galactoside-specific plant and avian lectins

The process cascade leading to the final accommodation of the carbohydrate ligand in the lectin’s binding site comprises enthalpic and entropic contributions of the binding partners and solvent molecules. With emphasis on lactose, N-acetyllactosamine, and thiodigalactoside as potent inhibitors of binding of galactoside-specific lectins, the question was addressed to what extent these parameters are affected as a function of the protein. The microcalorimetric study of carbohydrate association to the galectin from chicken liver (CG-16) and the agglutinin from Viscum album (VAA) revealed enthalpy–entropy compensation with evident protein type-dependent changes for N-acetyllactosamine. Reduction of the entropic penalty by differential flexibility of loops or side chains and/or solvation properties of the protein will have to be reckoned with to assign a molecular cause to protein type-dependent changes in thermodynamic parameters for lectins sharing the same monosaccharide specificity.

Approximate methods for step function response of undamped non-linear spring mass systems with arbitrary hardening type spring characteristic

The possibility of applying two approximate methods for determining the salient features of response of undamped non-linear spring mass systems subjected to a step input, is examined. The results obtained on the basis of these approximate methods are compared with the exact results that are available for some particular types of spring characteristics. The extension of the approximate methods for non-linear systems with general polynomial restoring force characteristics is indicated.

Heme oxygenase-1 in cardiovascular diseases

Myocardial infarction (MI) and heart failure are major causes of morbidity and mortality worldwide. Treatment of MI involves early restoration of blood flow to limit infarct size and preserve cardiac function. MI leads to left ventricular remodeling, which may eventually progress to heart failure, despite the established pharmacological treatment of the disease. To improve outcome of MI, new strategies for protecting the myocardium against ischemic injury and enhancing the recovery and repair of the infarcted heart are needed. Heme oxygenase-1 (HO-1) is a stress-responsive and cytoprotective enzyme catalyzing the degradation of heme into the biologically active reaction products biliverdin/bilirubin, carbon monoxide (CO) and free iron. HO-1 plays a key role in maintaining cellular homeostasis by its antiapoptotic, anti-inflammatory, antioxidative and proangiogenic properties. The present study aimed, first, at evaluating the role of HO-1 as a cardioprotective and prohealing enzyme in experimental rat models and at investigating the potential mechanisms mediating the beneficial effects of HO-1 in the heart. The second aim was to evaluate the role of HO-1 in 231 critically ill intensive care unit (ICU) patients by investigating the association of HO-1 polymorphisms and HO-1 plasma concentrations with illness severity, organ dysfunction and mortality throughout the study population and in the subgroup of cardiac patients. We observed in an experimental rat MI model, that HO-1 expression was induced in the infarcted rat hearts, especially in the infarct and infarct border areas. In addition, pre-emptive HO-1 induction and CO donor pretreatment promoted recovery and repair of the infarcted hearts by differential mechanisms. CO promoted vasculogenesis and formation of new cardiomyocytes by activating c-kit+ stem/progenitor cells via hypoxia-inducible factor 1 alpha, stromal cell-derived factor 1 alpha (SDF-1a) and vascular endothelial growth factor B, whereas HO-1 promoted angiogenesis possibly via SDF-1a. Furthermore, HO-1 protected the heart in the early phase of infarct healing by increasing survival and proliferation of cardiomyocytes. The antiapoptotic effect of HO-1 persisted in the late phases of infarct healing. HO-1 also modulated the production of extracellular matrix components and reduced perivascular fibrosis. Some of these beneficial effects of HO-1 were mediated by CO, e.g. the antiapoptotic effect. However, CO may also have adverse effects on the heart, since it increased the expression of extracellular matrix components. In isolated perfused rat hearts, HO-1 induction improved the recovery of postischemic cardiac function and abrogated reperfusion-induced ventricular fibrillation, possibly in part via connexin 43. We found that HO-1 plasma levels were increased in all critically ill patients, including cardiac patients, and were associated with the degree of organ dysfunction and disease severity. HO-1 plasma concentrations were also higher in ICU and hospital nonsurvivors than in survivors, and the maximum HO-1 concentration was an independent predictor of hospital mortality. Patients with the HO-1 -413T/GT(L)/+99C haplotype had lower HO-1 plasma concentrations and lower incidence of multiple organ dysfunction. However, HO-1 polymorphisms were not associated with ICU or hospital mortality. The present study shows that HO-1 is induced in response to stress in both experimental animal models and severely ill patients. HO-1 played an important role in the recovery and repair of infarcted rat hearts. HO-1 induction and CO donor pretreatment enhanced cardiac regeneration after MI, and HO-1 may protect against pathological left ventricular remodeling. Furthermore, HO-1 induction potentially may protect against I/R injury and cardiac dysfunction in isolated rat hearts. In critically ill ICU patients, HO-1 plasma levels correlate with the degree of organ dysfunction, disease severity, and mortality, suggesting that HO-1 may be useful as a marker of disease severity and in the assessment of outcome of critically ill patients.

Ring loading of a long, thin, circular cylindrical shell enclosing a soft, solid core: A recalculation by the love function method of elasticity.

The problem is solved using the Love function and Flügge shell theory. Numerical work has been done with a computer for various values of shell geometry parameters and elastic constants.