Transmission of Megatrypanum Trypanosomes to Cervus dama by Tabanidae1

Four fallow deer, Cervus dama, became infected with Trypanosoma (Megatrypanum) sp. by oral application of triturated guts from tabanids collected in an area with deer but without any cattle; four control calves remained negative. Upon challenge with triturated guts from tabanids from an area with pastured cattle, the four calves became infected with Trypanosoma (M.) theileri. The prepatent period in deer was five days or less. Haematopota spp. and Tabanus spp. were identified as vectors of the deer trypanosomes. It is concluded that the trypanosomes of C. dama belong to a Megatrypanum species that is not identical with T. theileri.     

Thermodynamic analysis of trinitrotoluene biodegradation and mineralization pathways

Biodegradation of 2,4,6-trinitrotoluene (TNT) proceeds through several different metabolic pathways. However, the reaction steps which are considered rate-controlling have not been fully determined. Glycolysis and other biological pathways contain biochemical reactions which are acutely rate-limiting due to enzyme control. These rate-limiting steps also have large negative Gibbs free energy changes. Because xenobiotic compounds such as TNT can be used by biological systems as nitrogen, carbon, and energy sources, it is likely that their degradation pathways also contain acutely rate-limiting steps. Identification of these rate-controlling reactions will enhance and better direct genetic engineering techniques to increase specific enzyme levels.This article identifies likely rate-controlling steps (or sets of steps) in reported TNT biodegradation pathways by estimating the Gibbs free energy change for each step and for the overall pathways. The biological standard Gibbs free energy change of reaction was calculated for each pathway step using a group contribution method specifically tailored for biomolecules. The method was also applied to hypothetical "pathways" constructed to mineralize TNT using several different microorganisms. Pathways steps that have large negative Gibbs free energy changes are postulated to be potentially rate-controlling. The microorganisms which utilize degradation pathways with the largest overall (from TNT to citrate) negatiave Gibbs free energy changes were also determined. Such microorganisms can extract more energy from the starting substrate and are thus assumed to have a competitive advantage over other microorganisms. Results from this modeling-based research are consistent with much experimental work available in the literature. (c) 1996 John Wiley & Sons, Inc.     

Physical mapping of three fruit ripening genes：Endopolygalacturonase,ACC oxidase and ACC synthase from apple（Malus x domestica） in an apple rootstock A106（Malus sieboldii

The apple rootstock,A106(Malus sieboldii),had 17 bivalents in pollen mother cells at meiotic metaphase 1,and 17 chromosomes in a haploid pollen cell.Karyotypes were prepared from root-tip cells with 2n=34 chromosomes,Seven out of 82 karyotypes(8.5%) showed one pari of satellites at the end of the short arm of chromosome 3.C-bands were shown on 6 pairs of chromosomes 2,4,6,8,14,and 16 near the telomeric regions of short arms.Probes for three ripening-related genes from Malus x domestica:endopolygalacturonase(EPG,0.6kb),ACC oxidase(1.2kb),and ACC synthase(2kb)were hybridized in situ to metaphase chromosomes of A106.Hybridization sites for the EPG gene were observed on the long arm of chromosome 14 in 15 out of 16 replicate spreads and proximal to the centromere of chromosomes 6 and 11.For the ACC oxidase gene,hylridization sites were observed in the telomeric region of the short arm of chromosomes 5 and 11 in 87% and 81% of 16 spreads respectively,proxiaml to the centromere of chromosome 1 in 81% of the spreads,and on the long arm of chromosome 13 in 50% of the spreads. Physical mapping of three fruit ripening genes in an apple rootstock A106.Twenty five spreads were studied for the ACC synthase gene and hybridization sites were observed in the telomeric region of the short arm of chromosome 12 in 96% of the spreads.chromosomes 9 and 10 in 76% of the spreads,and chromosome 17 in 56% of the spreads.     

The cytokineplast: purified, stable, and functional motile machinery from human blood polymorphonuclear leukocytes

We examined the formation of motile, chemotactically active, anucleate fragments from human blood polymorphonuclear leukocytes (PMN, granulocytes), induced by the brief application of heat. These granule-poor fragments are former protopods (leading fronts, lamellipodia) that become uncoupled from the main body of the cell and leave it, at first with a connecting filament that breaks and seals itself. The usual random orientation of such filaments can be controlled by preorientation of cells in a gradient of the chemotactic peptide, N-formylmethionylleucylphenylalanine (F-Met-Leu-Phe) (2x10(-9) M- 1x10(-8)). Cytochalsin B, 2.5-5 μg/ml, prevents fragment formation; colchicine, 10(-5) M, does not. In scanning electron micrographs, fragments are ruffled and the cell body rounded up and rather smooth. In transmission electron micrographs, fragments contain microfilaments but lack centrioles and microtubules. Like intact cells, both bound and free fragments can respond chemotactically to an erythrocyte destroyed by laser microirradiation (necrotaxis); the free, anucleate fragments may do so repeatedly, even after having been held overnight at ambient temperatures. We propse the name cytokineplast for the result of this self-purification of motile apparatus. The exodus of the motile machinery from the granulocyte requires anchoring of the bulk of the cell to glass and uncoupling, which may involve heat-induced dysfunction of the centrosome. In ultrastructural studies of the centrosomal region after heat, centriolar structure remains intact, but pericentriolar osmiophilic material appears condensed, and microtubules are sparse. These changes are found in all three blood cell types examined: PMN, eosinophil, and monocyte. Of these, the first two make fragments under our conditions; the more sluggish monocyte does not. Uncoupling is further linked to centrosomal dysfunction by the observation that colchicines-treated granulocytes (10(-5)M, to destroy the centrosome’s efferent arm) make fragments after less heat than controls. If motive force and orientation are specified mainly from the organelle-excluding leading front, then endoplasmic streaming in PMN is a catch-up phenomenon, and microtubules do not provide the vector of locomotion but rather stabilize and orient the “baggage” (nucleus, granuloplasm)—i.e., they prevent fishtailing. Moreover, constraints emanating from the centrosome may now be extended to include, maintenance of the motile machinery as an integral part of the cell.     

Severe diarrhoea caused by Aeromonas veronii biovar sobria in a patient with metastasised GIST.

This report describes the isolation of Aeromonas veronii biovar sobria as the causative enteropathogen of diarrhoea in an oncological patient after failure of detection of other infectious agents. The case points out the severe and long course of the infection, the diagnostic dilemma, and the prompt recovery after antibiotic treatment.     

In vitro evaluation of native entomopathogenic fungi and neem (Azadiractha indica) extracts on Spodoptera frugiperda

The control of Spodoptera frugiperda is based on synthetic insecticides, so some alternatives are the use of entomopathogenic fungi (EF) and neem extract. The objective of the study was to evaluate in vitro effectiveness of native EF and neem extracts on S. frugiperda larvae. Six EF were identified by DNA sequencing of ITS regions from three EF (Fusarium solani, Metarrhizium robertsii, Nigrospora spherica and Penicillium citrinum). They were evaluated in concentrations of 1 × 10⁸ spores/ mL. In addition, a second bioassay was carried out evaluating only F. solani, M. robertsii and N. sphaerica and the addition of vegetable oil. On the other hand, extraction of secondary metabolites from neem seed (Azadirachta indica) was carried out by performing, mass (g) and solvent volume (mL ethanol and water) combinations, which were subjected to microwaves and ultrasound. Subsequently, these extracts were evaluated in concentrations of 3%, 4% and 5%. A survival analysis was performed for each of the bioassays. With respect to the results of the first bioassay, F. solani obtained a probability of survival of 0.476 on the seventh day, while in the second bioassay, M. robertsii obtained 0.488 survival probability. This suggests that the expected percentage of larvae that stay alive on the sixth day is 48.8%. However, in the evaluation of the neem extract the combination 1:12/70% to 4% caused 84% mortality of larvae. The use of native HE and neem extracts has potential for the control of S. frugiperda.     

Evidence of a leading role for VEGF in Bartonella henselae-induced endothelial cell proliferations

Bartonella henselae causes the vasculoproliferative disorders bacillary angiomatosis (BA) and bacillary peliosis (BP). The pathomechanisms of these tumorous proliferations are unknown. Our results suggest a novel bacterial two-step pathogenicity strategy, in which the pathogen triggers growth factor production for subsequent proliferation of its own host cells. In fact, B. henselae induces host cell production of the angiogenic factor vascular endothelial growth factor (VEGF), leading to proliferation of endothelial cells. The presence of B. henselae pili was associated with host cell VEGF production, as a Pil- mutant of B. henselae was unable to induce VEGF production. In turn, VEGF-stimulated endothelial cells promoted the growth of B. henselae. Immunohistochemistry for VEGF in specimens from patients with BA or BP revealed increased VEGF expression in vivo. These findings suggest a novel bacteria-dependent mechanism of tumour growth.     

Yersinia enterocolitica invasin triggers phagocytosis via beta1 integrins, CDC42Hs and WASp in macrophages

The Yersinia outer surface protein invasin binds to β1 integrins on target cells and has been shown to trigger phagocytic uptake by macrophages. Here, we investigated the role of the actin regulator Wiskott–Aldrich syndrome protein (WASp), its effector the Arp2/3 complex and the Rho-GTPases CDC42Hs, Rac and Rho in invasin/β1 integrin-triggered phagocytosis. During uptake of invasin-coated latex beads, the α5β1 integrin, WASp and the Arp2/3 complex were recruited to the developing actin-rich phagocytic cups in primary human macrophages. Blockage of β1 integrins by specific antibodies, inhibition of Arp2/3 function by microinjection of inhibitors or the use of WASp knockout macrophages inhibited phagocytic cup formation and uptake. Furthermore, microinjection of the dominant negative GTPase mutants N17CDC42Hs, N17Rac or the Rho-specific inhibitor C3-transferase into macrophages greatly attenuated invasin-induced formation of cups. These data suggest that during invasin-triggered phagocytosis β1 integrins activate actin polymerization via CDC42Hs, its effector WASp and the Arp2/3 complex. The contribution of Rac and Rho to phagocytic cup formation also suggests a complex interplay between different Rho GTPases during phagocytosis of pathogens.     

Large-conductance calcium-activated potassium channel activity is absent in human and mouse neutrophils and is not required for innate immunity

Large-conductance Ca²⁺-activated K⁺ (BK) channels are reported to be essential for NADPH oxidase-dependent microbial killing and innate immunity in leukocytes. Using human peripheral blood and mouse bone marrow neutrophils, pharmacological targeting, and BK channel gene-deficient (BK^–/–) mice, we stimulated NADPH oxidase activity with 12-O-tetradecanoylphorbol-13-acetate (PMA) and performed patch-clamp recordings on isolated neutrophils. Although PMA stimulated NADPH oxidase activity as assessed by O₂^– and H₂O₂ production, our patch-clamp experiments failed to show PMA-activated BK channel currents in neutrophils. In our studies, PMA induced slowly activating currents, which were insensitive to the BK channel inhibitor iberiotoxin. Instead, the currents were blocked by Zn²⁺, which indicates activation of proton channel currents. BK channels are gated by elevated intracellular Ca²⁺ and membrane depolarization. We did not observe BK channel currents, even during extreme depolarization to +140 mV and after elevation of intracellular Ca²⁺ by N-formyl-L-methionyl-L-leucyl-phenylalanine. As a control, we examined BK channel currents in cerebral and tibial artery smooth muscle cells, which showed characteristic BK channel current pharmacology. Iberiotoxin did not block killing of Staphylococcus aureus or Candida albicans. Moreover, we addressed the role of BK channels in a systemic S. aureus and Yersinia enterocolitica mouse infection model. After 3 and 5 days of infection, we found no differences in the number of bacteria in spleen and kidney between BK^–/– and BK^+/+ mice. In conclusion, our experiments failed to identify functional BK channels in neutrophils. We therefore conclude that BK channels are not essential for innate immunity. killing assay; reactive oxygen species; BK-deficient mice; mice infection     

Mitochondria can recognize and assemble fragments of a beta-barrel structure

β-barrel proteins are found in the outer membranes of eukaryotic organelles of endosymbiotic origin as well as in the outer membrane of Gram-negative bacteria. Precursors of mitochondrial β-barrel proteins are synthesized in the cytosol and have to be targeted to the organelle. Currently, the signal that assures their specific targeting to mitochondria is poorly defined. To characterize the structural features needed for specific mitochondrial targeting and to test whether a full β-barrel structure is required, we expressed in yeast cells the β-barrel domain of the trimeric autotransporter Yersinia adhesin A (YadA). Trimeric autotransporters are found only in prokaryotes, where they are anchored to the outer membrane by a single 12-stranded β-barrel structure to which each monomer is contributing four β-strands. Importantly, we found that YadA is solely localized to the mitochondrial outer membrane, where it exists in a native trimeric conformation. These findings demonstrate that, rather than a linear sequence or a complete β-barrel structure, four β-strands are sufficient for the mitochondria to recognize and assemble a β-barrel protein. Remarkably, the evolutionary origin of mitochondria from bacteria enables them to import and assemble even proteins belonging to a class that is absent in eukaryotes.