Infection pressure of human alveolar echinococcosis due to village and small town foxes (<Emphasis Type="Italic">Vuples vulpes</Emphasis>) living in close proximity to residents

This study investigated the epidemiological and ecological factors to assess the infection pressure of alveolar echinococcosis to human which are living in villages and small towns. Foxes and fox faeces were examined for Echinococcus multilocularis and foxes were observed by radio telemetry in Upper Bavaria, Germany. Forty-three percent of the village foxes (n = 65) had been infected with E. multilocularis. This prevalence rate did not differ significantly from the prevalence among rural foxes, which was 39% (n = 33; χ ² = 0.12, df = 1, p = 0.727) determined by the intestinal scraping technique. PCR analyses of fox faeces showed a higher infection rate of 35% (n = 26) among rural foxes than among foxes in villages and small towns (26%, n = 69; χ ² = 0.68, df = 1, p = 0.411). One quarter of the fox faecal samples come from private gardens of residents. The radio-tracking study on 17 foxes showed that foxes preferred the built-up area and grassland outside the villages. Village foxes concentrated their activity within a range of 500 m around the settlement. Sixty-four percent of all bearings for radio-tracked foxes showed positions in areas outside the town, and 36% of bearings were within the settlement. Village foxes, which are infected with E. multilocularis, are able to carry the parasite continuously into settlements and fox faeces present an immediate source of infection to humans, especially within their gardens. Therefore, foxes are responsible for environmental E. multilocularis egg contamination in the vicinity of humans, leading to an infection risk to inhabitants of villages and small towns.     

Pituitary adenylate cyclase-activating polypeptide is up-regulated in cortical pyramidal cells after focal ischemia and protects neurons from mild hypoxic/ischemic damage

The protective effect of pituitary adenylate cyclase-activating polypeptide (PACAP) in stroke models is poorly understood. We studied patterns of PACAP, vasoactive intestinal peptide, and the PACAP-selective receptor PAC1 after middle cerebral artery occlusion and neuroprotection by PACAP in cortical cultures exposed to oxygen/glucose deprivation (OGD). Within hours, focal ischemia caused a massive, NMDA receptor (NMDAR)-dependent up-regulation of PACAP in cortical pyramidal cells. PACAP expression dropped below the control level after 2 days and was normalized after 4 days. Vasoactive intestinal peptide expression was regulated oppositely to that of PACAP. PAC1 mRNA showed ubiquitous expression in neurons and astrocytes with minor changes after ischemia. In cultured cortical neurons PACAP27 strongly activated Erk1/2 at low and p38 MAP kinase at higher nanomolar concentrations via PAC1. In astrocyte cultures, effects of PACAP27 on Erk1/2 and p38 were weak. During OGD, neurons showed severely reduced Erk1/2 activity and dephosphorylation of Erk1/2-regulated Ser112 of pro-apoptotic Bad. PACAP27 stimulation counteracted Erk1/2 inactivation and Bad dephosphorylation during short-term OGD but was ineffective after expanded OGD. Consistently, PACAP27 caused MEK-dependent neuroprotection during mild but not severe hypoxic/ischemic stress. While PACAP27 protected neurons at 1–5 nmol/L, full PAC1 activation by 100 nmol/L PACAP exaggerated hypoxic/ischemic damage. PACAP27 stimulation of astrocytes increased the production of Akt-activating factors and conferred ischemic tolerance to neurons. Thus, ischemia-induced PACAP may act via neuronal and astroglial PAC1. PACAP confers protection to ischemic neurons by maintaining Erk1/2 signaling via neuronal PAC1 and by increasing neuroprotective factor production via astroglial PAC1.     

Simultaneous detection of IFN-gamma and IL-4 mRNAs using RT-PCR and time-resolved fluorometry

Time-resolved fluorometry was applied in the detection of RT-PCR amplified mRNAs for the Th1 and Th2 cell-derived cytokines interferon gamma (IFN-gamma) and interleukin (IL-)4, respectively. RNA stimulated cells was reverse transcribed and the cDNAs for the cytokine mRNAs and the constantly expressed beta-actin (beta-ACT) mRNA were simultaneously amplified in one multiplex PCR reaction. The PCR conditions were optimized to minimize mutual inhibition of individual amplifications. One of the PCR primers in each primer pair was biotinylated, and the PCR products were captured onto streptavidin-coated microtitre plates. The three PCR products were detected with three different lanthanide labelled target-specific probes in solution hybridization. IFN-gamma, IL-4 and beta-ACT were detected with europium (Eu), terbium (Tb) and samarium (Sm) labelled probes, respectively, using time-resolved fluorometry. Small cell numbers used in microtitre plate cultures were sufficient to detect cytokine messages after mitogen stimulation. This sequence-based method provides a sensitive, specific, fast and nonisotopic alternative to conventional blotting and hybridisation with radioactive probes. In addition, the multiplex fluorogenic dye detection facilitates relative quantification of target mRNAs.     

Carrier-mediated transport of riboflavin in Ashbya gossypii

The filamentous hemiascomycete Ashbya gossypii is used for industrial riboflavin production. We examined riboflavin uptake and excretion at the plasma membrane using riboflavin auxotrophic and overproducing mutants. The riboflavin uptake system had low activity [Vmax = 20 +/- 4 nmol min(-1) g(-1) mycelial dry weight (dw)] and high affinity (KM = 40 +/- 12 microM). Inhibitor studies with the analogs FMN and FAD revealed high specificity of the uptake system. Excretion of riboflavin was not the consequence of non-specific permeability of the plasma membrane. Excretion rates in the mid-production phase were determined to be 2.5 nmol min(-1) g(-1) dw for wild-type cells and 66.7 nmol min(-1) g(-1) dw for an overproducing mutant, respectively. Inhibition of the reverse reaction, riboflavin uptake, led to an increase in apparent riboflavin efflux in the early production phase, indicating the presence of a separate excretion carrier. Riboflavin accumulation in A. gossypii vacuoles leading to product retention was found to be a secondary transport process. To address the question of whether a flux from the vacuoles back into the cytoplasm is present, we characterized efflux in hyphae in which the plasma membrane was permeabilized with digitonin. Efflux kinetics across the vacuolar membrane were unaffected by the lack of vacuolar H+ATPase activity and ATP, suggesting a passive mechanism. Based on the characterization of riboflavin transport processes in this study, the design of new production strains with improved riboflavin excretion may be possible.     

Frankia KB5 Possesses a Hydrogenase Immunologically Related to Membrane-Bound [NiFe]-Hydrogenases

The immunological relationship of the hydrogenase in Frankia KB5 to hydrogenases in other microorganisms was investigated using antisera raised against holo-[NiFe]-hydrogenases isolated from Alcaligenes latus, Azotobacter vinelandii, Ralstonia eutropha, and the small and large hydrogenase subunits from Bradyrhizobium japonicum. The antisera raised against the A. latus, R. eutropha, and B. japonicum (large subunit) polypeptides were found to recognize two polypeptides, corresponding to the unprocessed and processed forms of the hydrogenase subunit in Frankia KB5. None of the antisera, including the antibodies produced against the small hydrogenase subunit isolated from B. japonicum, recognized any polypeptide related to the small hydrogenase subunit in Frankia KB5. An immunogold localization study of the intracellular distribution of hydrogenase in Frankia KB5, with the cryo-section technique, showed that labeling in the membrane of both hyphae and vesicles was positively correlated with hydrogenase activity. Received: 6 November 2000 / Accepted: 18 December 2000     

Tropical forest fragmentation and nest predation – an experimental study in an Eastern Arc montane forest, Tanzania

When a habitat becomes fragmented and surrounded by another habitat this generally causes an increase in predation pressure at habitat transitions, often referred to as an edge effect. Edge effect in the form of enhanced nest predation intensities is one of the most cited explanations for bird population declines in fragmented landscapes. Here, we report results from a nest predation experiment conducted in a tropical montane forest landscape in the Uzungwa Mts., Tanzania. Using artificial nests with chicken eggs, we determined predation rates across a fragmentation gradient. The proportion of indigenous forest in four landscapes used in the study were 0.29, 0.58, 0.75 to 1.0. Nest predation intensities on artificial nests were about 19% higher inside intact forest than at edges in fragmented forest landscapes. Furthermore, predation intensities were relatively constant across a forest fragmentation gradient. Our results thus challenge the applicability and generality of the edge effect, derived from studies almost exclusively conducted in temperate regions rather than tropical forest ecosystems. Nest predation levels differences between tropical montane forest and that reported in other forest ecosystems are discussed.     

Nitric oxide synthase inhibitor and IL-18 enhance the anti-tumor immune response of rats carrying an intrahepatic colon carcinoma

The role of nitric oxide (NO) produced by adherent spleen cells in the systemic immunosuppression developing in tumor-bearing hosts was investigated. After therapeutic immunization of rats carrying an intrahepatic colon carcinoma, H1D2, the spleen cell antitumor immune responsiveness was analyzed. Compared to parallel immunized tumor-free rats, tumor-bearing rats (TB rats) had a greatly reduced proliferative T-cell response to wild-type tumor stimulator cells. The TB rats had a depressed proliferative response to anti-CD3 and to the superantigen SEA. TB rats with small tumors had a stronger response to IL-18-producing H1D2 stimulator cells than to wild type H1D2 cells. This was not the case with TB rats carrying larger tumors. Also the IFN-gamma production and cytotoxicity against the wild-type tumor cells and the NK sensitive YAC cells were depressed in spleen cells of TB rats after 5-day restimulation with wild-type tumor cells. A part of this immunosuppression was mediated by adherent spleen cells, mostly consisting of macrophages. An important mode of action appears to involve their production of an enhanced level of nitric oxide, since the competitive nitric oxide synthase (NOS) inhibitor L-NAME could partially counteract the suppression in vitro. We conclude that NOS inhibitors in combination with immunostimulatory cytokines, such as IL-18, could be useful tools to enhance anti-tumor immune responses in TB rats and therefore to increase the efficiency of immunotherapies.     

Nontoxic and toxic oligopeptides with D-amino acids and unusual residues in Microcystis aeruginosa PCC 7806

Toxic and nontoxic peptides were isolated from the cyanobacterium Microcystis aeruginosa PCC 7806 by a procedure including extraction of cells with water-saturated 1-butanol, chromatography of the extract on silica gel plates and high performance liquid chromatography (HPLC) on Partisil-5. The toxin was shown to be only a minor constituent, being negatively charged and thus separable by electrophoresis, within the HPLC-purified fraction. It contained erythro-β-methyl-D-Asp, D-Glu, D-Ala, L-Leu, and L-Arg known to be part of the Microcystis peptide-toxin with M_r 994. The major part of the HPLC-purified fraction was assigned, however, to a nontoxic peptide with a M_r of 956. Partial hydrolysis studies of the nontoxic peptide(s) revealed amino acid sequences composed of D-Glu, N-methyl-Phe, and 3,4-dehydro-Pro, aside from the common L-amino acids. Cyclic linkage in the nontoxic peptide(s) appears likely.     

Chromosomal instability and delayed apoptosis in long-term T-lymphocyte cultures irradiated with carbon ions and x rays

In this study, we examined genomic instability induced by 250 kV X rays and 100 MeV/nucleon carbon ions in long-term lymphocyte cultures from two healthy donors. Two biological end points, delayed apoptosis and chromosomal instability, were studied in descendants of cells irradiated with three different doses of the particular radiation up to 22 population doublings. The delayed apoptosis showed no clear dependence on radiation dose, culture time or radiation quality. A persistent significant increase in the rate of apoptosis up to 36 days after X irradiation was observed for a dose of 4 Gy in donor 1 only. For both donors and radiations, de novo aberration yields were significantly increased in comparison to control values up to day 36. For both radiations, chromosome-type aberrations were seen more frequently than chromatid-type aberrations in both donors up to 22 days postirradiation. In both donors, carbon ions were more effective than X rays with respect to the induction of chromosome instability. A dose of 0.25 Gy of carbon ions corresponding to 1.4 ion traversals per cell nucleus was effective in the induction of instability in our cell system.