Development of experimental genetic tools for Campylobacter fetus

Molecular analysis of the virulence mechanisms of the emerging pathogen Campylobacter fetus has been hampered by the lack of genetic tools. We report the development and functional analysis of Escherichia coli-Campylobacter shuttle vectors that are appropriate for C. fetus. Some vectors were constructed based on the known Campylobacter coli plasmid pIP1455 replicon, which confers a wide host range in Campylobacter spp. Versatility in directing gene expression was achieved by introducing a strong C. fetus promoter. The constructions carry features necessary and sufficient to detect the expression of phenotypic markers, including molecular reporter genes in both subspecies of C. fetus, while retaining function in C. jejuni. The capacity to express several gene products from different vectors in a single host can be advantageous but requires distinct plasmid replicons. To this end, replication features derived from a cryptic plasmid of C. fetus subsp. venerealis strain 4111/108, designated pCFV108, were adapted for a compatible series of constructions. The substitution of the C. coli replication elements reduced vector size while apparently limiting the host range to C. fetus. The complementation of a ciprofloxacin-resistant mutant phenotype via vector-driven gyrA expression was verified. Cocultivation demonstrated that shuttle vectors based on the pCFV108 replicon were compatible with pIP1455 replication functions, and the stable maintenance of two plasmids in a C. fetus subsp. venerealis host over several months was observed. The application of both vector types will facilitate the investigation of the genetics and cellular interactions of the emerging pathogen C. fetus.     

Biological Flora of Central Europe: Baldellia ranunculoides (Alismataceae)

Baldellia ranunculoides (L.) Parl. (Alismataceae) is a taxonomically problematic aquatic plant with an historically ill-defined distribution and global conservation status. This paper finds morphological, ecological and molecular evidence for two distinct taxa, probably best described as subspecies: (1) B. ranunculoides subsp. ranunculoides and (2) B. ranunculoides subsp. repens and provides detailed distribution data on their overlapping range, in different habitats, across the cool, high rainfall areas of western Europe and west Mediterranean. The two subspecies are amongst the relatively large number of threatened European and north Africa aquatic plants and this paper provides a systematic review of their relative conservation pressures and management needs, with particular emphasis on the status of both taxa in central Europe. Other observations indicate probable evolutionary relationships within B. ranunculoides s.l. and its associated taxa and the review points out where these and other research topics could potentially be pursued.     

Store-operated Ca2+ entry in astrocytes: different spatial arrangement of endoplasmic reticulum explains functional diversity in vitro and in situ

Ca(2+) signaling is the astrocyte form of excitability and the endoplasmic reticulum (ER) plays an important role as an intracellular Ca(2+) store. Since the subcellular distribution of the ER influences Ca(2+) signaling, we compared the arrangement of ER in astrocytes of hippocampus tissue and astrocytes in cell culture by electron microscopy. While the ER was usually located in close apposition to the plasma membrane in astrocytes in situ, the ER in cultured astrocytes was close to the nuclear membrane. Activation of metabotropic receptors linked to release of Ca(2+) from ER stores triggered distinct responses in cultured and in situ astrocytes. In culture, Ca(2+) signals were commonly first recorded close to the nucleus and with a delay at peripheral regions of the cells. Store-operated Ca(2+) entry (SOC) as a route to refill the Ca(2+) stores could be easily identified in cultured astrocytes as the Zn(2+)-sensitive component of the Ca(2+) signal. In contrast, such a Zn(2+)-sensitive component was not recorded in astrocytes from hippocampal slices despite of evidence for SOC. Our data indicate that both, astrocytes in situ and in vitro express SOC necessary to refill stores, but that a SOC-related signal is not recorded in the cytoplasm of astrocytes in situ since the stores are close to the plasma membrane and the refill does not affect cytoplasmic Ca(2+) levels.     

Anti-inflammatory effects of antibacterials on human bronchial epithelial cells

Background

Human Bronchial epithelial cells (hu-BEC) have been claimed to play a significant role in the pathogenesis of chronic inflammatory airway diseases like COPD. In this context IL-8 and GM-CSF have been shown to be key cytokines. Some antibiotics which are routinely used to treat lower respiratory tract infections have been shown to exert additional immunomodulatory or anti-inflammatory effects. We investigated whether these effects can also be detected in hu-BEC.

Methods

Hu-BEC obtained from patients undergoing lung resections were transferred to air-liquid-interface (ALI) culture. These cultures were incubated with cefuroxime (CXM, 10-62.5 mg/l), azithromycin (AZM, 0.1-1.5 mg/l), levofloxacin (LVX, 1-8 mg/l) and moxifloxacin (MXF, 1-16 mg/l). The spontaneous and TNF-α (10 ng/ml) induced expression and release of IL-8 and GM-CSF were measured using PCR and ELISA in the absence or presence of these antibiotics.

Results

The spontaneous IL-8 and GM-CSF release was significantly reduced with MXF (8 mg/l) by 37 ± 20% and 45 ± 31%, respectively (both p < 0.01). IL-8 release in TNF-α stimulated hu-BEC decreased by 16 ± 8% (p < 0.05) with AZM (1.5 mg/l). With MXF a concentration dependent decrease of IL-8 release was noted up to 39 ± 7% (p < 0.05). GM-CSF release from TNF-α stimulated hu-BEC was maximally decreased by 35 ± 24% (p < 0.01) with MXF (4 mg/l).

Conclusion

Using ALI cultures of hu-BEC we observed differential effects of antibiotics on spontaneous and TNF-α induced cytokine release. Our data suggest that MXF and AZM, beyond bactericidal effects, may attenuate the inflammatory process mediated by hu-BEC.     

A fault detection service for wide area distributed computations

The potential for faults in distributed computing systems is a significant complicating factor for application developers. While a variety of techniques exist for detecting and correcting faults, the implementation of these techniques in a particular context can be difficult. Hence, we propose a fault detection service designed to be incorporated, in a modular fashion, into distributed computing systems, tools, or applications. This service uses well-known techniques based on unreliable fault detectors to detect and report component failure, while allowing the user to trade off timeliness of reporting against false positive rates. We describe the architecture of this service, report on experimental results that quantify its cost and accuracy, and describe its use in two applications, monitoring the status of system components of the GUSTO computational grid testbed and as part of the NetSolve network-enabled numerical solver. This revised version was published online in July 2006 with corrections to the Cover Date.     

Feeling by sight or seeing by touch?

We have addressed the role of occipital and somatosensory cortex in a tactile discrimination task. Sight-ed and congenitally blind subjects rated the roughness and distance spacing for a series of raised dot patterns. When judging roughness, intermediate dot spacings were perceived as being the most rough, while distance judgments generated a linear relation. Low-frequency rTMS applied to somatosensory cortex disrupted roughness without affecting distance judgments, while rTMS to occipital cortex disrupted distance but not roughness judgments. We also tested an early blind patient with bilateral occipital cortex damage. Her performance on the roughness determination task was normal; however, she was greatly impaired with distance judgments. The findings suggest a double-dissociation effect in which roughness and distance are primarily processed in somatosensory and occipital cortex, respectively. The differential effect of rTMS on task performance and corroborative clinical evidence suggest that occipital cortex is engaged in tactile tasks requiring fine spatial discrimination.     

Divergent segmentation mechanism in the short germ insect Tribolium revealed by giant expression and function

Segmentation is well understood in Drosophila, where all segments are determined at the blastoderm stage. In the flour beetle Tribolium castaneum, as in most insects, the posterior segments are added at later stages from a posteriorly located growth zone, suggesting that formation of these segments may rely on a different mechanism. Nevertheless, the expression and function of many segmentation genes seem conserved between Tribolium and Drosophila. We have cloned the Tribolium ortholog of the abdominal gap gene giant. As in Drosophila, Tribolium giant is expressed in two primary domains, one each in the head and trunk. Although the position of the anterior domain is conserved, the posterior domain is located at least four segments anterior to that of Drosophila. Knockdown phenotypes generated with morpholino oligonucleotides, as well as embryonic and parental RNA interference, indicate that giant is required for segment formation and identity also in Tribolium. In giant-depleted embryos, the maxillary and labial segment primordia are normally formed but assume thoracic identity. The segmentation process is disrupted only in postgnathal metamers. Unlike Drosophila, segmentation defects are not restricted to a limited domain but extend to all thoracic and abdominal segments, many of which are specified long after giant expression has ceased. These data show that giant in Tribolium does not function as in Drosophila, and suggest that posterior gap genes underwent major regulatory and functional changes during the evolution from short to long germ embryogenesis.     

Cell migration: mechanisms of rear detachment and the formation of migration tracks

Cell migration is central to many biological and pathological processes, including embryogenesis, tissue repair and regeneration as well as cancer and the inflammatory response. In general, cell migration can be usefully conceptualized as a cyclic process. The initial response of a cell to a migration-promoting agent is to polarize and extend protrusions in the direction of migration. These protrusions can be large, broad lamellipodia or spike-like filopodia, are usually driven by actin polymerization, and are stabilized by adhering to the extracellular matrix (ECM) via transmembrane receptors of the integrin family linked to the actin cytoskeleton. These adhesions serve as traction sites for migration as the cell moves forward over them, and they must be disassembled at the cell rear, allowing it to detach. The mechanisms of rear detachment and the regulatory processes involved are not well understood. The disassembly of adhesions that is required for detachment depends on a coordinated interaction of actin and actin-binding proteins, signaling molecules and effector enzymes including proteases, kinases and phosphatases. Originally, the biochemically regulated processes leading to rear detachment of migrating cells were thought not to be necessarily accompanied by any loss of cell material. However, it has been shown that during rear detachment long tubular extensions, the retracting fibers, are formed and that "membrane ripping" occurs at the cell rear. By this process, a major fraction of integrin-containing cellular material is left behind forming characteristic migration tracks that exactly mark the way a cell has taken.