Inactivation of a gene that is highly conserved in Gram-positive bacteria stimulates degradation of non-native proteins and concomitantly increases stress tolerance in Lactococcus lactis

Exposure of cells to elevated temperatures triggers the synthesis of chaperones and proteases including components of the conserved Clp protease complex. We demonstrated previously that the proteolytic subunit, ClpP, plays a major role in stress tolerance and in the degradation of non-native proteins in the Gram-positive bacterium Lactococcus lactis. Here, we used transposon mutagenesis to generate mutants in which the temperature- and puromycin-sensitive phenotype of a lactococcal clpP null mutant was partly alleviated. In all mutants obtained, the transposon was inserted in the L. lactis trmA gene. When analysing a clpP, trmA double mutant, we found that the expression normally induced from the clpP and dnaK promoters in the clpP mutant was reduced to wild-type level upon introduction of the trmA disruption. Additionally, the degradation of puromycyl-containing polypeptides was increased, suggesting that inactivation of trmA compensates for the absence of ClpP by stimulating an as yet unidentified protease that degrades misfolded proteins. When trmA was disrupted in wild-type cells, both stress tolerance and proteolysis of puromycyl peptides was enhanced above wild-type level. Based on our results, we propose that TrmA, which is well conserved in several Gram-positive bacteria, affects the degradation of non-native proteins and thereby controls stress tolerance.     

The Agrobacterium rhizogenes pRi TL-DNA segment as a gene vector system for transformation of plants

Summary A plant gene transfer system was developed from the Agrobacterium rhizogenes pRi15834 TL-DNA region. Intermediate integration vectors constructed from ColE1-derived plasmids served as cloning vectors in Escherichia coli and formed cointegrates into the TL-DNA after transfer to A. rhizogenes. An A. rhizogenes strain with pBR322 plasmid sequences replacing part of the TL-DNA was also constructed. Plasmids unable to replicate in Agrobacterium can integrate into this TL-DNA by homologous recombination through pBR322 sequences. No loss of pathogenicity was observed with the strains formed after integration of intermediate vectors or strains carrying pBR322 in the TL-DNA segment. Up to 15 kb of DNA have been transferred to plant cells with these systems. The T-DNA from a binary vector was cotransformed into hairy roots which developed after transfer of the wild-type pRi T-DNA. Tested on Lotus corniculatus the TL-derived vector system transformed 90% of the developed roots and the T-DNA from the binary vector was cotransformed into 60% of the roots. Minimum copy numbers of one to five were found. Both constitutive and organ-specific plant genes were faithfully expressed after transfer to the legume L. corniculatus.     

Characterization of <Emphasis Type="Italic">Campylobacter</Emphasis> phages including analysis of host range by selected <Emphasis Type="Italic">Campylobacter</Emphasis> Penner serotypes

Background  

The predominant food borne pathogen in the western world today is Campylobacter. Campylobacter specific bacteriophages (phages) have been proposed as an alternative agent for reducing the burden of Campylobacter in broilers. One concern in relation to phage biocontrol is the narrow host range often displayed by phages. To identify the potential of phages as a Campylobacter reducing agent we needed to determine their infectivity on a panel of isolates representing the Campylobacter strains found in broilers as well as humans.     

The impact of tail tip amputation and ink tattoo on C57BL/6JBomTac mice

Genetic material for polymerase chain reaction (PCR) and Southern blot analysis on transgenic mice is normally obtained by tail biopsy. Additionally, it may be necessary to tattoo the mice, as it is essential to have a good and permanent identification. The aim of this study was to evaluate the effects of amputating the tip of the tail to obtain a biopsy for genetic analysis and of ink tattooing on welfare in C57BL/6J mice, a strain often used as genetic background for transgenes. The behaviour of the animals, fluctuating asymmetry (FA, a measure of developmental instability) and the level of restitution in the remaining part of the tail were evaluated and used for an assessment of the impact of these procedures on the welfare of the animals. One group of mice was marked by tail tattooing at various ages. Another group of mice were tail amputated at 12 or 20 days of age. Body weight and FA were followed, and at the end of the experiment, the level of fear/anxiety was assessed using a light-dark box. In the group of tail-amputated animals observation of climbing behaviour and a beam walking test for balance was performed. Seven weeks after tail amputation, the animals were euthanized. The remaining part of the tail was evaluated histopathologically. Body weight, behaviour in the light-dark box and balance test results were not influenced by tail amputation or tattooing. FA was only transiently increased by tattooing. Climbing behaviour was reduced just after tail amputation at 20 days of age. No signs of neuromas were found in the amputated tails, but seven weeks after amputation a significant number of mice did not have fully regenerated glandular tissue and hair follicles in the tail. It is concluded that both tail amputation and tail tattooing seem to have minor short-term negative effects on welfare and that the tissues on the tail probably do not regenerate fully after amputation.     

Heterogeneity of Soluble Neural Cell Adhesion Molecule

Soluble neural cell adhesion molecule (NCAM) from rat brain neuronal cell culture media consists predominantly of a polypeptide of Mr approximately 115,000. Minor amounts of a polypeptide of Mr approximately 180,000 and two inconsistently appearing components of Mr 160,000 and 145,000 are also observed. The Mr 115,000 component is derived from the neuronal membrane NCAM components NCAM-A of Mr 190,000, NCAM-B of Mr 140,000, or both. Thus, as a part of the catabolism of membrane NCAM-A plus -B, a minor fraction is posttranslationally cleaved and recovered in the media as discernible soluble NCAM polypeptides. The half-life of membrane NCAM-A plus -B is less than 24 h. Astrocyte culture media contains a predominant soluble NCAM component of Mr 120,000 derived from membrane-associated NCAM-C. A close comparison of deglycosylated soluble NCAM from astrocyte and neuronal cultures showed a small but consistent difference in Mr, a result suggesting that different NCAM polypeptides are released from the membrane of neurons and astrocytes. In contrast to the Mr 115,000-120,000 NCAM polypeptides, the Mr 180,000 polypeptide from neuronal culture media does not seem to be derived from membrane-attached NCAM and may therefore represent a secreted NCAM isoform.     

No evidence for recognition errors in Acrocephalus warblers

Failure to recognise own eggs (recognition errors) may be an important selective force behind acceptance of parasitic eggs, leading to a balance between rejecters and acceptors in a host population (the equilibrium hypothesis). We predicted that recognition errors should occur frequently among host species with intermediate rejection rates, whose rejection behaviour shows many conditional responses. The reed warbler Acrocephalus scirpaceus and great reed warbler A. arundinaceus fulfil these requirements. These two species were therefore used in an experiment where host birds were exposed to a common cuckoo Cuculus canorus dummy, either <2 m or 5–10 m from the nest, at fishponds in southern Moravia (Czech Republic). The hosts responded to the cuckoo dummy, great reed warblers being much more aggressive than reed warblers, and both species being more aggressive towards the dummy when it was close to the nest than when it was farther away. We furthermore predicted that there should be more eggs rejected (ejected or nest abandoned) due to recognition errors among hosts exposed to a dummy close to the nest than among both those exposed to a dummy farther away from the nest and towards controls not exposed to cuckoo dummies. When comparing egg loss between groups of birds that were exposed to a cuckoo dummy with those that were not, we found no significant difference. However, partial egg loss was frequent among hosts in the studied population, most probably due to cuckoo depredation. We discuss why there were no detectable recognition errors in the studied population, when other researchers have claimed to have found such errors in host populations elsewhere.     

Tissue organization ofFarrea occa (Porifera,Hexactinellida)

Summary The tissue organization ofFarrea occa has been examind by light microscopy, transmission electron microscopy (TEM), and scanning electron microscopy (SEM). It was found to agree closely with the hexactinellid model established forRhabdocalyptus dawsoni by Mackie and Singla (1983) in consisting of a thin general syncytium incorporating few discrete cellular components, several of which share membrane continuity with the general syncytium by distinctive plug junctions. The general syncytium, supported by a thin collagenous mesolamella, is specialized regionally as dermal membrane, gastral membrane, peripheral trabecular strands, and primary reticulum (R1) of flagellated chambers. Extensions of the syncytium, which lack mesolamella support, form the distinctive secondary reticulum (R2) inside chambers and a newly discovered structure, the inner membrane, which occupies the central region of flagellated chambers. The choanosyncytia are enucleate networks of collar bodies and stolons embedded in R1 and plugged to R1 and choanoblasts. The discrete cell population consists of choanoblasts and archeocytes located in the thin mesohyle space and plugged to syncytial elements, cystencytes and vacuolar cells also located in the mesohyle but lacking plug connections, and granular cells emergent on R1 and apparently not bearing plug connections. The status of scleroblast syncytia has not been resolved. Large populations of rod-shaped bacteria occupy the mesohyle space; intracellular ovoid bodies, possible symbiotic prokaryotes, are common in R1 and R2. The previously unknown inner membrane probably functions to control flagellar activity on a very localized scale and to accumulate and release egesta in packages.Abbreviations ac archeocyte congeries - ap apopyle - ar archeocyte - b bacterium - c collagen fibrils - cb collar body - cbl choanoblast - cbs collar body socket - ch choanosome - cm collar microvilli - co choanocyte collar - cr crystalloid - cs connecting strand - dm dermal membrane - dv debris vacuole - e exhalant opening - ex exhalant space - f flagellum - fn filamentous network - Gb Golgi body - gf glycocalyx filaments - gm gastral membrane - im inner membrane - is inhalant space - ml mesolamella - ms mesohyle space - mt mitochondrion - n1 nucleus of R1 - o ostium - ob ovoid body - os osmiophilic body - pm plasma membrane - pr prosopyle - pt peripheral trabeculae - R1 primary reticulum - R2 secondary reticulum - s spicule space - ser smooth endoplasmic reticulum - ts trabecular strand     

Cathepsin B mediates tumor necrosis factor-induced arachidonic acid release in tumor cells

Arachidonic acid (AA) generated by cytosolic phospholipase A2 (cPLA2) has been suggested to function as a second messenger in tumor necrosis factor (TNF)-induced death signaling. Here, we show that cathepsin B-like proteases are required for the TNF-induced AA release in transformed cells. Pharmaceutical inhibitors of cathepsin B blocked TNF-induced AA release in human breast (MCF-7S1) and cervix (ME-180as) carcinoma as well as murine fibrosarcoma (WEHI-S) cells. Furthermore, TNF-induced AA release was significantly reduced in cathepsin B-deficient immortalized murine embryonic fibroblasts. Employing cPLA2-deficient MCF-7S1 cells expressing ectopic cPLA2 or cPLA2-deficient immortalized murine embryonic fibroblasts, we showed that cPLA2 is dispensable for TNF-induced AA release and death in these cells. Furthermore, TNF-induced cathepsin B-dependent AA release could be dissociated from the cathepsin B-independent cell death in MCF-7S1 cells, whereas both events required cathepsin B activity in other cell lines tested. These data suggest that cathepsin B inhibitors may prove useful not only in the direct control of cell death but also in limiting the damage-associated inflammation.     

Seasonal sea ice cover as principal driver of spatial and temporal variation in depth extension and annual production of kelp in Greenland

We studied the depth distribution and production of kelp along the Greenland coast spanning Arctic to sub‐Arctic conditions from 78 ºN to 64 ºN. This covers a wide range of sea ice conditions and water temperatures, with those presently realized in the south likely to move northwards in a warmer future. Kelp forests occurred along the entire latitudinal range, and their depth extension and production increased southwards presumably in response to longer annual ice‐free periods and higher water temperature. The depth limit of 10% kelp cover was 9–14 m at the northernmost sites (77–78 ºN) with only 94–133 ice‐free days per year, but extended to depths of 21–33 m further south (73 ºN–64 ºN) where >160 days per year were ice‐free, and annual production of Saccharina longicruris and S. latissima, measured as the size of the annual blade, ranged up to sevenfold among sites. The duration of the open‐water period, which integrates light and temperature conditions on an annual basis, was the best predictor (relative to summer water temperature) of kelp production along the latitude gradient, explaining up to 92% of the variation in depth extension and 80% of the variation in kelp production. In a decadal time series from a high Arctic site (74 ºN), inter‐annual variation in sea ice cover also explained a major part (up to 47%) of the variation in kelp production. Both spatial and temporal data sets thereby support the prediction that northern kelps will play a larger role in the coastal marine ecosystem in a warmer future as the length of the open‐water period increases. As kelps increase carbon‐flow and habitat diversity, an expansion of kelp forests may exert cascading effects on the coastal Arctic ecosystem.