Control of focal adhesion dynamics by material surface characteristics

The mechanisms of cell adhesion to the extracellular matrix (ECM) which are of fundamental importance for function, survival, and growth of cells involve the formation of focal adhesions to facilitate integrin signaling. Recently, it became evident that focal adhesions are not stable but move to enable cell migration and ECM formation. We examined the number, size, and dynamic behavior of focal adhesions in living MG-63 osteoblastic cells, which were cultured on titanium surfaces with different roughnesses and on stainless steel (SS). As a marker for focal adhesions we used GFP-tagged vinculin, a cytoskeletal protein. Focal adhesions were smaller on titanium and on SS than on collagen-coated glass coverslips. The corundum-blasted rough surface of titanium induced the smallest adhesions. On all the surfaces that we have tested, we observed a mobility of focal adhesions. On collagen-coated coverslips focal adhesions moved with a speed of 60 nm/min. The speed was reduced on titanium and still more restricted on SS. The topography did not affect the mobility of focal adhesions. We conclude that on the material surfaces that we have studied a reduced mobility of focal adhesions may strengthen the linkages between cell and ECM but impair the ability to dynamically organize and remodel the ECM. The results may have a great impact in the functional evaluation of tailored biomaterial surfaces for the application in tissue engineering.     

Reliable detection of macrolide-resistant Helicobacter pylori via fluorescence in situ hybridization in formalin-fixed tissue.

Macrolide-resistant Helicobacter (H.) pylori represent an increasing therapeutic problem. Macrolide resistance is usually determined phenotypically in vitro with methods such as E-test or agar dilution test. A prerequisite for those tests, however, is bacterial culture that is not routinely set up in the course of gastroscopy. In contrast, formalin-fixed, paraffin-embedded biopsies are regularly available from patients who have undergone gastroscopy. In such biopsies macrolide-resistant H. pylori can be detected by the genotype-based technique of fluorescence in situ hybridization (FISH). Experience gained by this new method, however, is still extremely limited, especially in formalin-fixed tissue. Therefore, we retrospectively investigated formalin-fixed, paraffin-embedded biopsy specimens by FISH in 104 patients suffering from therapy-resistant H. pylori gastritis. To test the accuracy of FISH, we initially examined specimens from 53 patients for whom results of the E-test were available. Next we analyzed biopsies from another 51 patients that had been selected since phenotypical resistance testing had failed despite documented culturing attempts. In all 104 patients, H. pylori was detected by FISH and could thus be investigated for macrolide resistance. Overall, macrolide-resistant bacteria were found in 71 patients (68.3%). In 49 of 53 patients (92.4%), FISH and E-test returned identical results (no significant discordance according to McNemar's chi(2)-test). Taken together, our study demonstrates that FISH is a highly sensitive and reliable method for detecting macrolide-resistant H. pylori in formalin-fixed, paraffin-embedded biopsy specimens, which represents the routine method of processing tissue obtained upon gastroscopy.     

Analysis of gene expression patterns in small amounts of human ventricular myocardium by a multiplex RNase protection assay

 End-stage human heart failure is associated with changes in expression of steady-state messenger RNA (mRNA) levels. These changes correspond to alterations in protein levels and myocardial function and may have clinical implications regarding etiology, clinical state, or prognosis. However, analysis of mRNA levels in endomyocardial biopsies can be accomplished only by the quantitative polymerase chain reaction, which is difficult to standardize. The aim of the study was to evaluate whether the RNase protection assay is applicable to measure mRNAs of multiple genes simultaneously in small amounts of ventricular myocardium comparable to myocardial biopsies. Total RNA was prepared from left ventricular myocardium from terminally failing hearts with idiopathic (n=9) or ischemic cardiomyopathy (n=7) and from nonfailing control hearts (n=10). mRNA was measured by an optimized RNase protection assay for the β₁-adrenoceptor, the stimulatory G protein α-subunit (G_sα), phospholamban, the calcium ATPase of the sarcoplasmic reticulum (SERCA), β-myosin heavy chain (β-MHC), and the atrial natriuretic peptide (ANP). We extracted 10.7±2.1 μg total RNA from three myocardial biopsies taken in vitro. All of the six genes were measurable in duplicate in a total of 7 μg RNA. mRNAs of β₁-adrenoceptor, phospholamban, and SERCA were lower in failing than in nonfailing myocardium by 50%, 33%, and 42% respectively, whereas β-MHC and G_sα mRNAs were unchanged. mRNA of ANP was expressed at high levels only in the failing myocardium, providing a highly specific and sensitive marker for discriminating nonfailing and failing hearts. A direct comparison with ANP and G_sα levels obtained by Northern blot analysis with 7.5 μg total RNA showed a good correlation between the two methods. The RNase protection assay is thus a suitable method for simultaneous measurements of multiple mRNA levels in human myocardial biopsies. Changes in mRNA levels closely reflected those identified by other methods using larger amounts of RNA. Increased myocardial ANP mRNA levels determined by the RNase protection assay may serve as a molecular marker of heart failure. Received: 12 May 1997 / Accepted: 8 September 1997     

The effect of tourniquet release timing on perioperative blood loss in simultaneous bilateral cemented total knee arthroplasty: a prospective randomized study.

Today the use of pneumatic tourniquet is commonly accepted in total knee arthroplasty (TKA) to reduce perioperative blood loss. There are a few prospective randomised and nonrandomised studies that compare the effect of tourniquet release timing in cementless or cemented unilateral TKA. However, many of these studies show an inadequate reporting and methodology. This randomized prospective study was designed to investigate the efficiency of tourniquet release timing in preventing perioperative blood loss in a simultaneous bilateral TKA study design. To our knowledge, this is the first study of its kind, in which the effect of tourniquet release timing on perioperative blood loss was investigated in simultaneous bilateral cemented TKA to compare both techniques intraindividually. In 20 patients (40 knees) one knee was operated with tourniquet release and hemostasis before wound closure, and the other knee with tourniquet release after wound closure and pressure dressing. We found no significant difference in total blood loss between both techniques (p=0.930), but a significant difference in operating time (p=0.035). There were no postoperative complications at a follow-up of 6 month. Other studies report an increase the blood loss in early tourniquet release and an increase the risk of early postoperative complications in deflation of tourniquet after wound closure. In this study we found no significant difference in perioperative blood loss and no increase of postoperative complications. Therefore, we recommend a tourniquet release after wound closure to reduce the duration of TKA procedure and to avoid possible risks of extended anaesthesia.     

Functional characterization of the human atrial essential myosin light chain (hALC-1) in a transgenic rat model

Most patients with hypertrophic cardiomyopathy and congenital heart diseases express the atrial essential myosin light chains (ALC-1) in their ventricles, partially replacing the ventricular essential light chains (VLC-1). This VLC-1/ALC-1 isoform shift is correlated with an increase in cross-bridge cycling kinetics as measured using skinned fibers from the hypertrophied ventricles of human hearts.To study the functional importance of hALC-1 in the intact perfused heart, we generated a transgenic rat model (TGR) overexpressing hALC-1 in the heart. Twelve-week-old TGR rats expressed 17±4 g hALC-1 per mg of whole SDS-soluble protein. Their perfused heart contractility parameters were evaluated using the Langendorff preparation. Expression of hALC-1 was accompanied by statistically significant improvements (P<0.001) in the contractile parameters of the hearts of the TGR compared to the age matched control (WKY) animals, represented by increases from 20.8±2.3 to 45.1±3.6 mmHg/g heart weight in the developed left ventricular pressure, 1,035.7±89.8 to 2,181±135.4 mmHg/s in the contraction rate, and 713±60.2 to 1,364±137.4 mmHg/s in the relaxation rate in the WKY and the TGR groups respectively. Characterizing the functional effects of hALC-1 at the whole organ level represents a step towards gene therapy of heart failure.     

Th2- and to a lesser extent Th1-type cytokines upregulate the production of both CXC (IL-8 and gro-alpha) and CC (RANTES,eotaxin, eotaxin-2, MCP-3 and MCP-4) chemokines in human airway epithelial cells

BACKGROUND: Both CXC and CC chemokines play an important role in leukocyte recruitment. However, a systematic examination of their production by human airway epithelial cells (HAECs) has not been carried out. The objective of this study was to investigate whether Th1- and Th2-type cytokines regulate chemokine production in HAECs. METHODS: HAECs were grown from both nasal and bronchial tissue and subsequently stimulated with either Th1- or Th2-type cytokines. RESULTS: Constitutive mRNA expression for gro-alpha, IL-8 and RANTES was seen in both human nasal and human bronchial epithelial cells. IL-4 was the strongest stimulus for both gene expression and protein production of the chemokines RANTES, IL-8 and gro-alpha, while both IL-13 and IFN-gamma were weaker inducers of these chemokines, with the exception of gro-alpha (IL-13 was a strong stimulus for gro-alpha production). TNF-alpha synergized with IL-4, and to a lesser extent with IFN-gamma and IL-13, to release RANTES, IL-8 and gro-alpha. IL-4 and to a lesser extent IL-13 and IFN-gamma stimulated the production of MCP-3 and -4, eotaxin and eotaxin-2 immunoreactivities. However, no induction of the mRNAs encoding these chemokines was observed, suggesting that they may be released from a preformed pool within the HAECs. CONCLUSION: These findings suggest that when released into the airways, Th2- and to a lesser extent Th1-type cytokines may stimulate recruitment of eosinophils and neutrophils through the release of CC (RANTES, MCP-3 and -4, eotaxin and eotaxin-2) and CXC chemokines (gro-alpha and IL-8).     

Identification, isolation, and characterization of a species-specific 30-kDa antigen of Oesophagostomum dentatum

In the search for a serology tool for the diagnosis of nonpatent as well as patent infections with Oesophagostomum dentatum in pigs a water-soluble, unglycosilated antigen of about 30 kDa specific for the third-stage larvae of the parasite was purified by ion-exchange chromatography. In Western blots, the antigen was first detected by antibodies at day 7 postinfection. Cross-reactivity with O. quadrispinulatum, Ascaris suum, or Trichuris suis was not detected. It is suggested that this protein is a suitable tool for the species-specific serodiagnosis of O. dentatum infection in pigs. Received: 15 June 1998 / Accepted: 28 September 1998     

Inter- and intramolecular interactions in polyelectrolyte complex formation with polyampholytes

Employing polyampholytes (inclusively polybetaines) of different chemical structure containing carboxylic groups and various basic nitrogen functions, homosymplex formation, as well as the competition between homo- and heterosymplex formation on addition of an appropriate polyelectrolyte, was investigated in dependence of pH and ionic strength by means of viscometry and turbidimetry. With most, but not with all, of the polyampholytes, the expected viscosity minimum at the isoelectric point, with its steepness depending on polyampholyte structure, was observed. Competition of homo- and heterosymplex formation at and near the isoelectric point is mainly governed by the pK values of the species involved, the level of zwitter-ion formation of the polyampholyte and the effect of non-Coulombic interactions, for example, via hydrogen bonds.     

13C NMR spectroscopic studies on polyanion-polycation complexes and their component polyelectrolytes

Position and intensities of the ¹³C NMR signals and relaxation times T₁ of several anionic and cationic polyelectrolytes in the solid state were compared with those of the appropriate polyanion-polycation complexes. At a high charge density of the components, the most significant changes of the parameters in question due to complex formation are observed for the C atoms adjacent to the charge centers, indicating a strong Coulombic interaction. At lower charge density, conformational changes of the polymer chains have also to be taken into consideration.