VEGF-PLCgamma1 pathway controls cardiac contractility in the embryonic heart

The strength of the heart beat can accommodate in seconds to changes in blood pressure or flow. The mechanism for such homeostatic adaptation is unknown. We sought the cause of poor contractility in the heart of the embryonic zebrafish with the mutation dead beat. We find through cloning that this is due to a mutation in the phospholipase C gamma1 (plcgamma1) gene. In mutant embryos, contractile function can be restored by PLCgamma1 expression directed selectively to cardiac myocytes. In other situations, PLCgamma1 is known to transduce the signal from vascular endothelial growth factor (VEGF), and we show here that abrogation of VEGF also interferes with cardiac contractility. Somewhat unexpectedly, FLT-1 is the responsible VEGF receptor. We show that the same system functions in the rat. Blockage of VEGF-PLCgamma1 signaling decreases calcium transients in rat ventricular cardiomyocytes, whereas VEGF imposes a positive inotropic effect on cardiomyocytes by increasing calcium transients. Thus, the muscle of the heart uses the VEGF-PLCgamma1 cascade to control the strength of the heart beat. We speculate that this paracrine system may contribute to normal and pathological regulation of cardiac contractility.     

Influence of transferable genetic determinants on the outcome of typing methods commonly used for Enterococcus faecium

A variety of methods is used for a molecular typing of Enterococcus spp. and related gram-positive bacteria including macrorestriction analysis using pulsed-field gel electrophoresis (PFGE), ribotyping, rapid amplification of polymorphic DNA (RAPD), and amplified fragment length polymorphism (AFLP). To test the influence of transferable determinants on the outcome of different typing methods commonly used for enterococci, we established a homogenous strain collection of 24 transconjugants resulting from filter matings with antibiotic-resistant Enterococcus faecium. As expected, AFLP, RAPD, and PFGE all identified our model bacteria as strongly related. However, distinct differences in the resolving and discriminatory power of the tested methods could be clearly addressed. In PFGE, 22 of 24 transconjugants possessed less than a three-band difference to the recipient pattern and would be regarded as strongly related. Three different RAPD PCRs were tested; in two reactions, identical patterns for all transconjugants and the recipient were produced. One RAPD PCR produced an identical pattern for 18 transconjugants and the recipient and a clearly different pattern for the remaining 6 transconjugants due to a newly appearing fragment resulting from acquisition of the tetL gene. AFLP clusters all transconjugants into a group of major relatedness. Percent similarities were highly dependent on the method used for calculating the similarity coefficient (curve-based versus band-based similarity coefficient). Fragment patterns of digested plasmids showed the possession of nonidentical plasmids in most transconjugants. PFGE still could be recommended as the method of choice. Nevertheless, the more-modern AFLP approach produces patterns of comparable discriminatory power while possessing some advantages over PFGE (less-time-consuming internal standards). Plasmid fingerprints can be included to subdifferentiate enterococcal isolates possessing identical macrorestriction and PCR typing patterns.     

Histopathological studies on the local reactions induced by complete Freund's adjuvant (CFA), bacterial lipopolysaccharide (LPS), and synthetic lipopeptide (P3C) conjugates

The inflammatory reactions following subcutaneous application of adjuvants revealed characteristic pathological patterns. The injection of complete Freund's adjuvant (CFA) resulted in the formation of large lipid deposits encircled by an inflammatory reaction and concentrically arranged collagen bundles. Bacterial lipopolysaccharide (LPS) caused granulomatous aggregations of mononuclear cells with thrombotic vessel occlusions. Inoculation of the lipopeptide adjuvants induced accumulation of mononuclear cells with only minimal fibrotic changes which were resolved after day 28. Lipopeptide conjugates based on the head group tripalmitoyl-S-glyceryl-cysteinyl-serin (P3CS) can thus be used as effective immunogens and adjuvants without long-term tissue damage.     

Transforming growth factor-beta1 increases CXCR4 expression, stromal-derived factor-1alpha-stimulated signalling and human immunodeficiency virus-1 entry in human monocyte-derived macrophages

Stromal-derived factor-1 (SDF-1/CXCL12) and its receptor CXCR4 play crucial roles in leukocyte migration and activation, as well as embryogenesis, angiogenesis, cancer and viral pathogenesis. CXCR4 is one of the major human immunodeficiency virus-1 (HIV-1) coreceptors on macrophages. In many tissues macrophages are one of the predominant cell types infected by HIV-1 and act as a reservoir for persistent infection and viral dissemination. In patients infected by HIV-1, blood and tissue levels of transforming growth factor-beta1 (TGF-beta1) are increased. The purpose of this study was to evaluate the effects of TGF-beta1 on CXCR4 expression and function in primary human monocyte-derived macrophages (MDMs) and rat microglia. TGF-beta1 up-regulated CXCR4 and enhanced SDF-1alpha-stimulated ERK1,2 phosphorylation in these cells. The increased CXCR4 expression in human MDMs resulted in increased susceptibility of the cells to entry by dual-tropic CXCR4-using HIV-1 (D-X4). In contrast, TGF-beta1 failed to increase CCR5 expression or infection by a CCR5-using virus in MDMs. Our data demonstrate that TGF-beta1 enhances macrophage responsiveness to SDF-1alpha stimulation and susceptibility to HIV-1 by selectively increasing expression of CXCR4. The results suggest that increased expression of CXCR4 on macrophages may contribute to the emergence of dual-tropic X4 viral variants at later stages of HIV-1 infection.     

Structure-activity relationship of oxadiazoles and allylic structures in the Ames test: an industry screening approach

In recent years genotoxicity testing has become more and more important in the process of early screening for potential development compounds. In the case that a pharmacologically interesting structure is found to be positive in an in vitro mutagenicity test a straightforward approach starts by sorting out what substructure is responsible for the activity observed in the test. The Ames test is a rapid, convenient test system which has been effectively used in structure-activity relationship studies for mutagenicity, since it can rapidly establish differences in the mutagenic action of isomers and chemical analogs. The lead compound with a benzodiazepine-like structure and close analogs exhibited weak, but unequivocal positive effects in the Ames test (strains TA1535 and TA 100) after metabolic activation by rat liver homogenate fraction (S9). To identify substances within this class of compounds devoid of mutagenic liability an extensive structure-activity investigation was undertaken. More than 50 compounds were tested in the two critical bacterial strains, using a standard plate incorporation and a preincubation modification. It quickly became evident that the benzodiazepine structure was not involved. First hints that the allyl side chain were responsible for the Ames activity had to be refined in a more complex, but clear-cut structure-activity relationship during the course of the experiments. It was shown that all compounds with an allyl side chain, independent of the heterocycle, but surprisingly also all compounds with a specific arrangement of the heteroatoms in the oxadiazole ring, showed positive effects in at least one strain. Based on these investigations it was possible to select pharmacologically active structures without mutagenic liability.     

Multicontrast MRI of remyelination in the central nervous system

Although magnetic resonance imaging (MRI) represents the most sensitive tool for the detection of white matter abnormalities in patients with multiple sclerosis (MS), the heterogeneity of MS placques severely hampers the elucidation of specific pathophysiological processes. In order to identify putative MRI markers for de- and remyelination, we employed the cuprizone mouse model which leads to a selective and reversible demyelination of the corpus callosum with little or no axonal damage. Apart from histopathology, animals were studied with high-resolution three-dimensional MRI in vivo using multiple contrasts. While individual MRI findings significantly correlated with electron microscopy, the differentiation of regions with normal, demyelinated or remyelinated white matter by one contrast alone was less specific than by histology or electron microscopy. However, an accurate MRI prediction of the in vivo myelin status was achieved by a discriminant function analysis using a combination of T1, T2 and magnetization transfer contrast. With a correct assignment of 95% of all animals examined, the procedure will allow for the survey of new therapeutic approaches aiming at improved remyelination.     

Glycoprotein B of bovine herpesvirus type 4: Its phylogenetic relationship to gB equivalents of the herpesviruses

In order to estimate the phylogenetic relationship of BHV-4 among the herpesviruses, we have cloned and sequenced its glycoprotein B (gB). The 2.6 kb open reading frame codes for a 874 amino acid long protein. The comparison of its deduced amino acid sequence with those of its counterparts in 19 distinct herpesviruses groups BHV-4 into the -herpesvirinae. The calculation of an evolutionary tree emphasized that BHV-4 is more closely related to herpesvirus saimiri (HVS) than to Epstein-Barr virus (EBV). However, in contrast to EBV and HVS, the gB of BHV-4 contains a putative protease cleavage site and 20 potential N-glycosylation sites. The alignment of the amino acid sequences revealed that 10 cysteine and 7 proline residues, as well as the motifs SPF and GQLG, were completely conserved among the 20 investigated gBs.     

Prevalence of hepatitis-C virus RNA in serum and throat washings of children with chronic hepatitis

Serum samples from 46 children with chronic and probably transfusion acquired hepatitis were tested for the presence of hepatitis C virus (HCV) RNA by a “nested” polymerase chain reaction (PCR) assay, to judge a possible risk of HCV transmission from these patients. In 73% of the samples, viral RNA was detected, indicating a high virus prevalence in this patient group. High titers of HCV-RNA were observed in some sera as shown by the detection of virus in some samples even at dilutions of 10^?3. Comparison of simultaneously obtained PCR results and ALT values revealed no significant correlation between virus presence in serum and higher ALT levels. It was, however, shown that unusually high ALT values may reflect a high titer of viral RNA in serum. To investigate the prevalence of viral RNA in saliva, which could be a vehicle of virus transmission, 35 throat washing samples from the HCV-infected children were screened by PCR. Using three different sample preparation procedures, 20% of the throat washings were found to be positive for HCV-RNA. This indicates a prevalence of virus in this fluid lower than that reported previously. © 1994 Wiley-Liss, Inc.     

Heinzkörperchen und Blutfarbstoff

Zusammenfassung Heinzsche Körperchen, Methämoglobin und Sulfhämoglobin sind drei verschiedene, voneinander unabhängige und nebeneinander herlaufende Folgen gewisser Vergiftungen.Statt Methämoglobin empfiehlt sich als besserer Ausdruck: Häimglobin, statt Sulfhämoglobin Verdoglobin. Der Ausdruck hämoglobinämische Innenkörperchen entspricht in keiner Hinsicht dem tatsächlichen Befunde und ist daher auszumerzen.     

Antibiotic susceptibility of Helicobacter pylori in Germany: stable primary resistance from 1995 to 2000

The issue of antibiotic resistance in Helicobacter pylori is of particular concern and has become an important factor leading to eradication failure. This paper reports the prevalence of primary resistance to clarithromycin, amoxicillin, metronidazole and tetracycline among H. pylori isolates in the north-eastern part of Germany. A total of 1644 clinical H. pylori isolates was investigated over a period of 6 years from 1995 to 2000. The MICs were determined by the Etest. The overall rate of primary resistance was 26.2% for metronidazole and 2.2% for clarithromycin. No significant changes in the resistance rates during the period of investigation were observed. No isolate was resistant to amoxicillin or tetracycline. PCR-RFLP analysis for the detection of point mutations associated with clarithromycin resistance was performed with 36 H. pylori isolates. The A --> G transition mutation at position 2143 was detected in 19 H. pylori isolates (52.8%), whereas the mutation at position 2142 was found in 13 isolates (36.1%).