The properties of phosphodiesterase 11A4 GAF domains are regulated by modifications in its N-terminal domain

The tandem GAF domain of human phosphodiesterase 11A4 (hPDE11A4) requires 72 microm cGMP for half-maximal effective concentration (EC(50)) of a cyanobacterial adenylyl cyclase used as a reporter enzyme. Here we examine whether modifications in the N-terminus of PDE11A4 affect cGMP signalling. The N-terminus has two phosphorylation sites for cyclic nucleotide monophosphate-dependent protein kinases (Ser117, Ser168). Phosphorylation of both by cAMP-dependent protein kinase decreased the EC(50) value for cGMP from 72 to 23 microm. Phosphomimetic point mutations (S117D/S167D), which project complete phosphorylation, lowered the EC(50) value to 16 microm. Structural and sequence data indicate that 196 amino acids precede the start of the GAF domain in hPDE11A4. Removal of 197 amino acids yielded unregulated cyclase activity, whereas truncation by 196 amino acids resulted in a cGMP-regulated protein with a cGMP EC(50) value of 7.6 microm. Truncation by 176 amino acids was required for cGMP EC(50) values to decrease to below 10 microm; a construct truncated by 168 amino acids had an EC(50) value of 224 microm. The decrease in EC(50) values was accompanied by a sixfold increase in basal activity; the extent of cGMP stimulation remained unaffected, however. We conclude that N-terminal modifications strongly affect cGMP regulation of hPDE11A4.     

Dream imagery,rapid eye movement sleep behavior disorder,and hallucinations

Rapid eye movement (REM) sleep behavior disorder (RBD) and hypnagogic hallucinations are salient symptoms of abnormal and dissociated REM sleep that are frequently associated in serious neurological diseases. RBD is a strong, independent risk factor for hallucinations in narcolepsy (odds ratio: 4.3) and in Parkinson’s disease (odds ratio: 2.7). In Parkinson’s disease, RBD also predicts incident hallucinations and psychosis in prospective cohorts. Status dissociatus (a mixture of hallucinations, RBD, and dissociated sleep-wake states) is observed in patients with Guillain-Barré when hallucinating, but also in Lewy bodies dementia, delirium tremens, fatal familial insomnia, and Morvan’s chorea. This co-occurrence of RBD and visual hallucinations suggests a common, extensive lesion within REM sleep executive systems.

     

Cellular immune responses in mice infected with the intestinal nematode Trichuris muris.

CBA and B10.BR mice show variation in immune response to the intestinal nematode Trichuris muris. CBA mice develop strong resistance, eliminating worms from the intestine; B10BR mice are permissive and develop chronic infections. It is already known that resistance and permissiveness reflect differential T helper responses. The data reported here show that resistant CBA mice express good antigen-specific lymphocyte proliferative responses to infection, whereas cells from B10.BR mice are relatively anergic, although still responsive to Concanavalin A (ConA). The possibility that the altered proliferative responsiveness seen in infected B10.BR mice reflected quantitative or qualitative changes in T helper cells was examined by comparing cytokine production and expression of cell surface markers (CD4, CD8, and CD28) in mesenteric lymph node cells and spleen cells from both strains and comparing these with the characteristics of cells from resistant CBA mice and from CBA mice that had been rendered permissive to infection by a combination of irradiation and corticosteroid treatment. As expected, cells from B10.BR mice produced high levels of interferon-gamma (IFN-gamma), whereas those from CBA mice released high levels of IL-5, whether stimulated with adult worm somatic antigens, excretory/secretory antigens, or ConA. Immunosuppressed CBA mice produced high levels of both IFN-gamma and IL-5 throughout the experiment. FACS analysis revealed a decrease of CD4+ and an initial increase in CD8+ cells in all infected mice. No major changes occurred in the relative proportion of CD28(+) cells. Further evaluation of the CD28 costimulatory molecule, measured as mean fluorescence intensity, displayed down-regulation in permissive and immunosuppressed mice. The data obtained suggest that lymphocyte unresponsiveness and permissiveness to T. muris infection may be associated with a down-regulation or an initially reduced expression of costimulatory CD28 molecules.     

The alternative sigma factor sigma B of Staphylococcus aureus modulates virulence in experimental central venous catheter-related infections

The impact of the alternative sigma factor sigma B (SigB) on pathogenesis of Staphylococcus aureus is not conclusively clarified. In this study, a central venous catheter (CVC) related model of multiorgan infection was used to investigate the role of SigB for the pathogenesis of S. aureus infections and biofilm formation in vivo. Analysis of two SigB-positive wild-type strains and their isogenic mutants revealed uniformly that the wild-type was significantly more virulent than the SigB-deficient mutant. The observed difference in virulence was apparently not linked to the capability of the strains to form biofilms in vivo since wild-type and mutant strains were able to produce biofilm layers inside of the catheter. The data strongly indicate that the alternative sigma factor SigB plays a role in CVC-associated infections caused by S. aureus.     

The cytochrome ba complex from the thermoacidophilic crenarchaeote Acidianus ambivalens is an analog of bc(1) complexes

A novel cytochrome ba complex was isolated from aerobically grown cells of the thermoacidophilic archaeon Acidianus ambivalens. The complex was purified with two subunits, which are encoded by the cbsA and soxN genes. These genes are part of the pentacistronic cbsAB-soxLN-odsN locus. The spectroscopic characterization revealed the presence of three low-spin hemes, two of the b and one of the a(s)-type with reduction potentials of +200, +400 and +160 mV, respectively. The SoxN protein is proposed to harbor the heme b of lower reduction potential and the heme a(s), and CbsA the other heme b. The soxL gene encodes a Rieske protein, which was expressed in E. coli; its reduction potential was determined to be +320 mV. Topology predictions showed that SoxN, CbsB and CbsA should contain 12, 9 and one transmembrane alpha-helices, respectively, with SoxN having a predicted fold very similar to those of the cytochromes b in bc(1) complexes. The presence of two quinol binding motifs was also predicted in SoxN. Based on these findings, we propose that the A. ambivalens cytochrome ba complex is analogous to the bc(1) complexes of bacteria and mitochondria, however with distinct subunits and heme types.     

Cyanamide mode of action during inhibition of onion (Allium cepa L.) root growth involves disturbances in cell division and cytoskeleton formation

Cyanamide is an allelochemical produced by hairy vetch (Vicia villosa Roth.). Its phyotoxic effect on plant growth was examined on roots of onion (Allium cepa L.) bulbs. Water solution of cyanamide (2-10 mM) restricted growth of onion roots in a dose-dependent manner. Treatment of onion roots with cyanamide resulted in a decrease in root growth rate accompanied by a decrease in accumulation of fresh and dry weight. The inhibitory effect of cyanamide was reversed by its removal from the environment, but full recovery was observed only for tissue treated with this chemical at low concentration (2-6 mM). Cytological observations of root tip cells suggest that disturbances in cell division may explain the strong cyanamide allelopathic activity. Moreover, in cyanamide-treated onion the following changes were detected: reduction of mitotic cells, inhibition of proliferation of meristematic cells and cell cycle, and modifications of cytoskeleton arrangement.     

Proton Pump Inhibitor Intake neither Predisposes to Spontaneous Bacterial Peritonitis or Other Infections nor Increases Mortality in Patients with Cirrhosis and Ascites

Background and Aim

The aim of this study was to assess the impact of proton pump inhibitor (PPI) intake on the development of spontaneous bacterial peritonitis (SBP) or other infections, as well as on mortality, in a thoroughly documented cohort of patients with cirrhosis and ascites.

Patients and Methods

We performed a retrospective analysis of follow-up data from 607 consecutive patients with cirrhosis undergoing their first paracentesis at a tertiary center. A binary logistic regression model investigating the association between PPI intake and SBP at the first paracentesis was calculated. Competing risk analyses and Cox models were used to investigate the effect of PPIs on the cumulative incidence of SBP or other infections and transplant-free survival, respectively. Adjustments were made for age, hepatocellular carcinoma, history of variceal bleeding, varices and model of end-stage liver disease score.

Results

Eighty-six percent of patients were receiving PPIs. After adjusting for potential confounding factors, PPI intake was neither associated with increased SBP prevalence at the first paracentesis (odds ratio (OR):1.11,95% confidence interval (95%CI):0.6–2.06; P = 0.731) nor cumulative incidence of SBP (subdistribution hazard ratio (SHR): 1.38; 95%CI:0.63–3.01; P = 0.42) and SBP or other infections (SHR:1.71; 95%CI:0.85–3.44; P = 0.13) during follow-up. Moreover, PPI intake had no impact on transplant-free survival in both the overall cohort (hazard ratio (HR):0.973,95%CI:0.719–1.317; P = 0.859) as well as in the subgroups of patients without SBP (HR:1.01,95%CI:0.72–1.42; P = 0.971) and without SBP or other infections at the first paracentesis (HR:0.944,95%CI:0.668–1.334; P = 0.742).

Conclusions

The proportion of cirrhotic patients with PPI intake was higher than in previous reports, suggesting that PPI indications were interpreted liberally. In our cohort with a particularly high prevalence of PPI intake, we observed no association between PPIs and SBP or other infections, as well as mortality. Thus, the severity of liver disease and other factors, rather than PPI treatment per se may predispose for infectious complications.     

The Reference Index Method for the Macrophyte‐BasedAssessment of Rivers – a Contribution to the Implementationof the European Water Framework Directive in Germany

The European Water Framework Directive requires ecological status classification and monitoring of surface and ground waters using biological indicators. To act as a component of the “Macrophytes and Phytobenthos” biological quality element, as demanded by the Directive, a macrophyte‐based assessment system was developed for application in river site types in Germany. Macrophyte abundance data were collected from 262 sites in 202 rivers. Seven biocoenotic river site types were established using differences in characteristic macrophyte communities reflecting ecoregion, channel width, water depth, current velocity, water hardness, and ground water influence. For four of these river site types, a macrophyte assessment system was developed, for the remaining three river site types data were insufficient for developing an assessment system. Ecological status classification of river sites is based on the calculation of a Reference Index value, in some cases supplemented by additional vegetation criteria. The Reference Index quantifies the deviation of species composition and abundance from reference conditions and classifies sites as one of the five possible ecological quality classes specified in the Directive. The assessment of long river stretches with changing river site types along its course is discussed based on an example from the Forstinninger Sempt River, southeast Germany. (© 2005 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Cloning and Some Novel Characteristics of Mitochondrial Hsp70 from Chinese Hamster Cells

The cDNA for Chinese hamster mitochondrial Hsp70 (mHsp70) was cloned and sequenced using a polymerase chain reaction probe based on conserved regions in the Hsp70 family of proteins. The encoded protein consists of 679 amino acids which includes a N-terminal mitochondrial targeting sequence of 46 amino acids. The mHsp70 protein contains several sequence signatures that are characteristics of prokaryotic and eukaryotic organellar Hsp70 homologs. In a phylogenetic tree based on Hsp70 sequences, it branches with the gram-negative proteobacteria, supporting the endosymbiotic origin of mitochondria from this group of prokaryotes. The mHsp70 cDNA was transcribed and translatedin vitroand its import into isolated rat heart mitochondria was examined. The precursor mHsp70 was converted into a mature form of lower molecular mass (≈71 kDa) which became resistant to trypsin digestion. The import of mHsp70 into mitochondria was not observed in the presence of an uncoupler of energy metabolism or when the N-terminal presequence was lacking. The cDNA for mHsp70 was expressed inEscherichia coliand a polyclonal antibody to the purified recombinant protein was raised. The antibody shows no cross-reactivity to recombinant cytosolic Hsp70 protein and in 2-D gel blots it reacted specifically with the mHsp70 protein only. In immunofluorescence experiments, the antibody predominantly labeled mitochondria, and the observed labeling pattern was identical to that seen with a monoclonal antibody to the mitochondrial Hsp60 chaperonin. The affinity-purified antibody to mHsp70 was also employed to examine the subcellular distribution of the protein by cryoelectron microscopy and the immunogold-labeling technique. In these experiments, in addition to mitochondria, labeling with mitochondrial Hsp70 antibody was also observed on the plasma membrane and in unidentified cytoplasmic vesicles and granules. These studies raise the possibility that similar to the Hsp60 chaperonin and a number of other mitochondrial proteins, mHsp70 may have an extramitochondrial role.