Reduced baroreflex sensitivity in alcoholic cirrhosis: relations to hemodynamics and humoral systems

In cirrhosis, arterial vasodilatation leads to central hypovolemia and activation of the sympathetic nervous and renin-angiotensin-aldosterone systems. As the liver disease and circulatory dysfunction may affect baroreflex sensitivity (BRS), we assessed BRS in a large group of patients with cirrhosis and in controls who were all supine and some after 60 degrees passive head-up and 30 degrees head-down tilting in relation to central hemodynamics and activity of the sympathetic nervous and renin-angiotensin-aldosterone systems. One-hundred and five patients (Child classes A/B/C: 21/55/29) and 25 (n=11 + 14) controls underwent a full hemodynamic investigation. BRS was assessed by cross-spectral analysis of variabilities between blood pressure and heart rate time series. The median BRS was significantly lower in the supine cirrhotic patients, 3.7 (range 0.3-30.7) ms/mmHg than in matched controls (n=11): 14.3 (6.1-23.6) ms/mmHg, P<0.001. A stepwise multiple-regression analysis revealed that serum sodium (P=0.044), heart rate (P=0.027), and central circulation time (P=0.034) independently correlated with BRS. Head-down tilting had no effects on BRS, but, after head-up tilting, BRS was similar in the patients (n=23) and controls (n=14). In conclusion, BRS is reduced in cirrhosis in the supine position and relates to various aspects of cardiovascular dysfunction, but no further reduction was observed in parallel with the amelioration of the hyperdynamic circulation after head-up tilting. The results indicate that liver dysfunction and compensatory mechanisms to vasodilatation may be involved in the low BRS, which may contribute to poor cardiovascular adaptation in cirrhosis.     

MEK inhibition impairs influenza B virus propagation without emergence of resistant variants

Influenza A and B viruses are still a major worldwide threat. We demonstrate that influenza B virus infection induces signaling via the Raf/MEK/ERK cascade, a process required for efficient virus production. Expression of dominant-negative Raf and ERK mutants or treatment with a MEK inhibitor (U0126) strongly impaired viral propagation, while selective activation of the pathway resulted in increased virus titers. MEK inhibition appears to interfere with a distinct viral nuclear export process. Most importantly, no resistant virus variants emerged in the presence of U0126 demonstrating that influenza viruses cannot easily adapt to the missing cellular function.     

Critical role of Ca2+ ions in the reaction mechanism of Euphorbia characias peroxidase

A cationic peroxidase was isolated and characterized from the latex of the perennial Mediterranean plant Euphorbia characias. The purified enzyme contained one heme prosthetic group identified as ferric iron-protoporphyrin IX. In addition, the purified peroxidase contained 1 mol of endogenous calcium per mol of enzyme; removal of this calcium ion resulted in almost complete loss of the enzyme activity. However, when excess Ca(2+) was added to the native enzyme the catalytic efficiency was enhanced by 3 orders of magnitude. The mechanism of activation was studied using a wide range of spectroscopic and analytic techniques. Analysis of the steady state by stopped-flow measurements suggests that the main effect of calcium ions is to favor the oxidation of the ferric enzyme by hydrogen peroxide to form compound I, whereas the other steps of the catalytic cycle seem to be affected to a lesser extent. UV/vis absorption spectra and CD measurements show that the heme iron is pentacoordinated high-spin in native enzyme and remains so after the binding of Ca(2+). Only minor changes in the secondary or tertiary structure of the protein could be detected by fluorescence or CD measurements in the presence of Ca(2+) ions, except for a significant perturbation of the Fe(3+) inner sphere geometry, as detected by EPR measurements. We propose that Ca(2+) binding to a low affinity site induces a reorientation of the distal histidine changing the almost inactive form of Euphorbia peroxidase to a high activity form. This is the first example of a peroxidase that responds as an on/off switch to variations in the external Ca(2+) level.     

Parasitoids on the loose – experimental lack of support of the parasitoid movement hypothesis

Forest fragmentation can disrupt important ecological processes both within and among species. Of particular interest is the extent to which fragmentation decouples economically important pest species from their parasites. In an influential paper, Roland and Taylor (1997) advanced the hypothesis that forest fragmentation at a relatively fine spatial scale would disrupt the ability of parasitoids to aggregate in response to host density, thereby facilitating the build-up of host populations at isolated sites. Our study provides the first experimental test of this notion. We augmented existing densities of forest tent caterpillar individuals at two types of sites: isolated forest fragments and continuous tracts of forest. Of the four parasitoid species studied by Roland and Taylor (1997), two were encountered in our experiment. For the sarcophagid fly Arachnidomyia aldrichi and the tachinid Carcelia malacosomae, we found no difference in parasitism rates among forest fragments and continuous forest tracts. A third tachinid species unique to this study, Lespesia frenchii , is known to be a broad generalist with respect to host choice. In this species, we observed significantly elevated parasitism in forest fragments, and higher parasitism at the forest edge than within the forest. All of these findings suggest complex effects of forest fragmentation on parasitoid movement, and warn against broad generalizations from observational studies. Forest fragmentation may affect movement patterns in a multitude of ways, significantly obscuring or modifying the simplest mechanisms proposed by Roland and Taylor (1997).     

Complete genomic organization of the human JAK3 gene and mutation analysis in severe combined immunodeficiency by single-strand conformation polymorphism

JAK3 deficiency in humans results in autosomal recessive T-B+ severe combined immunodeficiency disease (SCID), a severe immunodeficiency that can only be cured by bone marrow transplantation. We unraveled the complete organization of the human JAK3 gene, which includes 23 exons. This information allowed us to set up a molecular screening test that enabled us to diagnose JAK3 deficiency in 14 patients from 12 unrelated families with T-B+ SCID. In order to define the mutations, we used a nonradioactive single-strand conformation polymorphism (SSCP)/heteroduplex (HD) assay based on exon-specific polymerase chain reaction (PCR). In this cohort of patients, 15 independent JAK3 gene mutations have been identified, including 7 that have not been described previously. Mutation analysis information was used for genetic counseling and prenatal diagnosis.     

Maternal Vitamin C Deficiency during Pregnancy Persistently Impairs Hippocampal Neurogenesis in Offspring of Guinea Pigs

While having the highest vitamin C (VitC) concentrations in the body, specific functions of VitC in the brain have only recently been acknowledged. We have shown that postnatal VitC deficiency in guinea pigs causes impairment of hippocampal memory function and leads to 30% less neurons. This study investigates how prenatal VitC deficiency affects postnatal hippocampal development and if any such effect can be reversed by postnatal VitC repletion. Eighty pregnant Dunkin Hartley guinea pig dams were randomized into weight stratified groups receiving High (900 mg) or Low (100 mg) VitC per kg diet. Newborn pups (n = 157) were randomized into a total of four postnatal feeding regimens: High/High (Control); High/Low (Depleted), Low/Low (Deficient); and Low/High (Repleted). Proliferation and migration of newborn cells in the dentate gyrus was assessed by BrdU labeling and hippocampal volumes were determined by stereology. Prenatal VitC deficiency resulted in a significant reduction in postnatal hippocampal volume (P<0.001) which was not reversed by postnatal repletion. There was no difference in postnatal cellular proliferation and survival rates in the hippocampus between dietary groups, however, migration of newborn cells into the granular layer of the hippocampus dentate gyrus was significantly reduced in prenatally deficient animals (P<0.01). We conclude that a prenatal VitC deficiency in guinea pigs leads to persistent impairment of postnatal hippocampal development which is not alleviated by postnatal repletion. Our findings place attention on a yet unrecognized consequence of marginal VitC deficiency during pregnancy.     

Activated protein C inhibits the release of macrophage inflammatory protein-1-alpha from THP-1 cells and from human monocytes

Several lines of evidence have implicated activated protein C (APC) to be an endogenous inhibitor of the inflammatory septic cascade. APC may exhibit direct anti-inflammatory properties, independent of its antithrombotic effects. Chemokines influence the interaction of monocytes at the endothelium during infection and sepsis and are involved in the molecular events leading to an adverse and lethal outcome of sepsis. Defining regulatory mechanisms on the monocytic release profile of the proinflammatory C-C chemokines macrophage inflammatory protein-1-alpha (MIP-1-alpha) and monocyte chemoattractant protein-1 (MCP-1) might have therapeutic implications for the treatment of sepsis. We established a monocytic cell model of inflammation by the addition of lipopolysaccharide (LPS) and examined the effect of human APC on LPS-stimulated chemokine release from the monocytic cell line THP-1. We found that human APC in supra-physiological concentrations of 2.5-10 microg/ml inhibited the LPS-induced release of the chemokines MIP-1-alpha and MCP-1, as measured by enzyme-linked immunosorbent assays (ELISA) at 6 up to 24 h. In addition to experiments on THP-1 cells, recombinant human APC in concentrations of 50 ng/ml was found to have an inhibiting effect on the release of MIP-1-alpha from freshly isolated mononuclear cells of septic patients. The ability of APC to decrease the release of the C-C chemokine MIP-1-alpha from the monocytic cell line THP-1 and from human monocytes may identify a novel immunomodulatory pathway by which APC exerts its anti-inflammatory action and may contribute to control the inflammatory response in sepsis.     

Characterization of legumain

The mammalian legumain, also called asparaginyl endopeptidase (AEP), is critically involved in the processing of bacterial antigens for MHC class II presentation. In order to investigate the substrate specificity of AEP in the P1' position, we created a peptide library and digested it with purified pig kidney AEP. Digestion was less efficient only when proline was in the P1' position. Maximum AEP activity was found in lysosomal fractions of different types of antigen presenting cells (APC). When the multiple sclerosis-associated autoantigen myelin basic protein (MBP) was digested with AEP, the immunodominant epitope 83-99 was destroyed. Myoglobin as an alternative substrate was AEP resistant. These results suggest an important, but not necessarily critical role for AEP in lysosomal antigen degradation.