Elm leaves ‘warned’ by insect egg deposition reduce survival of hatching larvae by a shift in their quantitative leaf metabolite pattern

Plants may take insect eggs on their leaves as a warning of future herbivory and intensify their defence against feeding larvae. Responsible agents are, however, largely unknown, and little knowledge is available on this phenomenon in perennial plants. We investigated how egg deposition affects the anti‐herbivore defence of elm against the multivoltine elm leaf beetle. Prior egg deposition caused changes in the quality of feeding‐damaged leaves that resulted in increased larval mortality and reduced reproductive capacity of the herbivore by harming especially female larvae. Chemical analyses of primary and secondary leaf metabolites in feeding‐damaged, egg‐free (F) and feeding‐damaged, egg‐deposited (EF)‐leaves revealed only small differences in concentrations when comparing metabolites singly. However, a pattern‐focused analysis showed clearly separable patterns of (F) and (EF)‐leaves because of concentration differences in especially nitrogen and phenolics, of which robinin was consumed in greater amounts by larvae on (EF) than on (F)‐leaves. Our study shows that insect egg deposition mediates a shift in the quantitative nutritional pattern of feeding‐damaged leaves, and thus might limit the herbivore's population growth by reducing the number of especially female herbivores. This may be a strategy that pays off in a long run particularly in perennial plants against multivoltine herbivores.     

Gamma irradiation preserves immunosuppressive potential and inhibits clonogenic capacity of human bone marrow‐derived mesenchymal stromal cells

Mesenchymal stromal cells (MSCs) are promising candidates for the treatment of graft‐versus‐host and autoimmune diseases. Here, by virtue of their immunosuppressive effects, they are discussed to exhibit inhibitory actions on various immune effector cells, including T lymphocytes that promote the underlying pathology. While it becomes apparent that MSCs exhibit their therapeutic effect in a transient manner, they are usually transplanted from third party donors into heavily immunocompromised patients. However, little is known about potential late complications of persisting third party MSCs in these patients. We therefore analysed the effect of gamma irradiation on the potency and proliferation of MSCs to elucidate an irradiation dose, which would allow inhibition of MSC proliferation while at the same time preserving their immunosuppressive function. Bone marrow‐derived MSCs (BM‐MSCs) were gamma‐irradiated at increasing doses of 5, 10 and 30 Gy and subsequently assessed by colony formation unit (CFU)‐assay, Annexin V‐staining and in a mixed lymphocyte reaction, to assess colony growth, apoptosis and the immunosuppressive capacity, respectively. Complete loss of proliferative capacity measured by colony formation was observed after irradiation with a dose equal to or greater than 10 Gy. No significant decrease of viable cells was detected, as compared to non‐irradiated BM‐MSCs. Notably, irradiated BM‐MSCs remained highly immunosuppressive in vitro for at least 5 days after irradiation. Gamma irradiation does not impair the immunosuppressive capacity of BM‐MSCs in vitro and thus might increase the safety of MSC‐based cell products in clinical applications.     

Two different lantibiotic-like peptides originate from the ericin gene cluster of Bacillus subtilis A1/3

A lantibiotic gene cluster was identified in Bacillus subtilis A1/3 showing a high degree of homology to the subtilin gene cluster and occupying the same genetic locus as the spa genes in B. subtilis ATCC 6633. The gene cluster exhibits diversity with respect to duplication of two subtilin-like genes which are separated by a sequence similar to a portion of a lanC gene. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analyses of B. subtilis A1/3 culture extracts confirmed the presence of two lantibiotic-like peptides, ericin S (3,442 Da) and ericin A (2,986 Da). Disruption of the lanB-homologous gene eriB resulted in loss of production of both peptides, demonstrating that they are processed in an eriB-dependent manner. Although precursors of ericins S and A show only 75% of identity, the matured lantibiotic-like peptides reveal highly similar physical properties; separation was only achieved after multistep, reversed-phase high-performance liquid chromatography. Based on Edman and peptidase degradation in combination with MALDI-TOF MS, for ericin S a subtilin-like, lanthionine-bridging pattern is supposed. For ericin A two C-terminal rings are different from the lanthionine pattern of subtilin. Due to only four amino acid exchanges, ericin S and subtilin revealed similar antibiotic activities as well as similar properties in response to heat and protease treatment. For ericin A only minor antibiotic activity was found.     

Assessment of Optimal Selected Prognostic Factors

The identification and assessment of prognostic factors is one of the major tasks in clinical research. The assessment of one single prognostic factor can be done by recently established methods for using optimal cutpoints. Here, we suggest a method to consider an optimal selected prognostic factor from a set of prognostic factors of interest. This can be viewed as a variable selection method and is the underlying decision problem at each node of various tree building algorithms. We propose to use maximally selected statistics where the selection is defined over the set of prognostic factors and over all cutpoints in each prognostic factor. We demonstrate that it is feasible to compute the approximate null distribution. We illustrate the new variable selection test with data of the German Breast Cancer Study Group and of a small study on patients with diffuse large B‐cell lymphoma. Using the null distribution for a p‐value adjusted regression trees algorithm, we adjust for the number of variables analysed at each node as well. (© 2004 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Intergeneric Transfer of Conjugative and Mobilizable Plasmids Harbored by Escherichia coli in the Gut of the Soil Microarthropod Folsomia candida (Collembola)

The gut of the soil microarthropod Folsomia candida provides a habitat for a high density of bacterial cells (T. Thimm, A. Hoffmann, H. Borkott, J. C. Munch, and C. C. Tebbe, Appl. Environ. Microbiol. 64:2660–2669, 1998). We investigated whether these gut bacteria act as recipients for plasmids from Escherichia coli. Filter mating with E. coli donor cells and collected feces of F. candida revealed that the broad-host-range conjugative plasmid pRP4-luc (pRP4 with a luciferase marker gene) transferred to fecal bacteria at estimated frequencies of 5.4 × 10⁻¹ transconjugants per donor. The mobilizable plasmid pSUP104-luc was transferred from the IncQ mobilizing strain E. coli S17-1 and less efficiently from the IncF1 mobilizing strain NM522 but not from the nonmobilizing strain HB101. When S17-1 donor strains were fed to F. candida, transconjugants of pRP4-luc and pSUP104-luc were isolated from feces. Additionally, the narrow-host-range plasmid pSUP202-luc was transferred to indigenous bacteria, which, however, could not maintain this plasmid. Inhibition experiments with nalidixic acid indicated that pRP4-luc plasmid transfer took place in the gut rather than in the feces. A remarkable diversity of transconjugants was isolated in this study: from a total of 264 transconjugants, 15 strains belonging to the alpha, beta, or gamma subclass of the class Proteobacteria were identified by DNA sequencing of the PCR-amplified 16S rRNA genes and substrate utilization assays (Biolog). Except for Alcaligenes faecalis, which was identified by the Biolog assay, none of the isolates was identical to reference strains from data banks. This study indicates the importance of the microarthropod gut for enhanced conjugative gene transfer in soil microbial communities.Gene transfer is a process by which bacterial populations substantially increase their rates of evolution and adaptation (12, 59). Particularly, plasmid-located genes, which are transferred by conjugation from donor to recipient cells, can disseminate rapidly between even phylogenetically different bacterial groups (17, 36, 41) and microbial communities in different spatial habitats (34, 71). Such microbial genetic networks should be considered in risk assessments of releases of genetically engineered microorganisms into the environment (22, 37, 43). The probability and rate of plasmid transfer from a donor to indigenous microorganisms in a given habitat are influenced by plasmid-borne genes which determine the type of transfer mechanism (self-transmissible or mobilizable) and the host range of autonomous plasmid replication. Additionally, specific physicochemical conditions, such as temperature, water potential, and the availability of energy (substrates) for donor and recipient cells, are important factors influencing gene transfer rates in terrestrial and aquatic environments (23, 53, 64).The spread of plasmid-borne genes is still extremely difficult to predict for terrestrial habitats, since a large variety of microhabitat conditions which are not well characterized exists. In bulk soil under laboratory conditions, conjugative gene transfer from recombinant bacterial donor strains to indigenous soil bacteria has been found only under specific selective conditions or on rare occasions (11, 20, 24, 27, 50, 61). Several studies failed to detect such transfer events, and it was concluded that heterogeneity and low densities of recipient cells, as well as a lack of substrates for microbial metabolism, prevented efficient plasmid transfer in bulk soil (19, 49, 54, 75). Plant exudates increased rates of gene transfer in soil (33, 48), and higher rates of gene transfer were found in rhizospheres than in bulk soil (50, 61). It was assumed that other microsites which favor gene transfer in terrestrial habitats are associated with soil invertebrates (74). However, to date little experimental evidence to prove this assumption is available.Intraspecies transconjugants of added Enterobacter cloacae donor and recipient cells could be isolated from microcosm experiments with the variegated cutworm, Peridroma saucia, and plant material (2). The investigators in that study concluded that gene transfer events happened, most likely, in the digestive tracts or in the feces of the insects. Another recent report demonstrated that a conjugative plasmid was transferred between fed Escherichia coli strains in the guts of Rhabditis nematodes (1). Earthworms mediated transport and enhanced plasmid transfer from added donor cells to added recipients and to indigenous bacteria in soil (14, 15). High rates of intraspecies plasmid transfer, comparable to those obtained in pure broth cultures, were detected with Bacillus thuringiensis in infected lepidopterous larvae (31).Microarthropods (collembolans and mites) are the most abundant invertebrate group in the majority of soils (5) but have not been recognized, so far, for their impact on microbial gene transfer. There are some indications that microarthropods harbor a large variety of microorganisms in their guts and thereby contribute to microbial biodiversity in terrestrial environments (7, 9, 57). In the accompanying paper, we have described the gut of Folsomia candida (Collembola) as a habitat and species-specific vector for microorganisms (67). The gut of this soil-dwelling insect, which has a volume of only several nanoliters, was found to be densely colonized, predominantly by rod-shaped bacterial cells. We were interested to know whether such bacterial cells act as recipients for plasmids and thereby promote gene transfer in microbial communities. F. candida feeds, under natural conditions, on bacteria (3), fungal mycelia (6, 66), and nematodes (35). Here, we report on the results of experiments in which plasmid-bearing E. coli strains were fed to F. candida in microcosms. Self-transferable plasmids, as well as mobilizable plasmids with different host ranges, and a nonmobilizable plasmid were included in this study in order to determine the specific capacities of these different classes of plasmids to spread into indigenous bacterial populations. For detection purposes, all plasmids were engineered by the insertion of the luciferase-encoding marker gene luc or lux (30, 47).     

Ischemic preconditioning in rat heart: No correlation between glycogen content and return of function

We tested the hypothesis that glycogen levels at the beginning of ischemia affect lactate production during ischemia and postischemic contractile function.Isolated working rat hearts were perfused at physiological workload with bicarbonate buffer containing glucose (10 mmol/L). Hearts were subjected to four different preconditioning protocols, and cardiac function was assessed on reperfusion. Ischemic preconditioning was induced by either one cycle of 5 min ischemia followed by 5, 10, or 20 min of reperfusion (PC5/5, PC5/10, PC5/20), or three cycles of 5 min ischemia followed by 5 min of reperfusion (PC3 × 5/5). All hearts were subjected to 15 min total, global ischemia, followed by 30 min of reperfusion. We measured lactate release, timed the return of aortic flow, compared postischemic to preischemic power, and determined tissue metabolites at selected time points.Compared with preischemic function, cardiac power during reperfusion improved in groups PC5/10 and PC5/20, but was not different from control in groups PC5/5 and PC3 × 5/5. There was no correlation between preischemic glycogen levels and recovery of function during reperfusion. There was also no correlation between glycogen breakdown (or resynthesis) and recovery of function. Lactate accumulation during ischemia was lowest in group PC5/20 and highest in the group with three cycles of preconditioning (PC3 × 5/5). Lactate release during reperfusion was significantly higher in the groups with low recovery of power than in the groups with high recovery of power.In glucose-perfused rat heart recovery of function is independent from both pre- and postischemic myocardial glycogen content over a wide range of glycogen levels. The ability to utilize lactate during reperfusion is an indicator for postischemic return of contractile function.     

Annexin A8 Identifies a Subpopulation of Transiently Quiescent c-Kit Positive Luminal Progenitor Cells of the Ductal Mammary Epithelium

We have previously shown that Annexin A8 (ANXA8) is strongly associated with the basal-like subgroup of breast cancers, including BRCA1-associated breast cancers, and poor prognosis; while in the mouse mammary gland AnxA8 mRNA is expressed in low-proliferative isolated pubertal mouse mammary ductal epithelium and after enforced involution, but not in isolated highly proliferative terminal end buds (TEB) or during pregnancy. To better understand ANXA8’s association with this breast cancer subgroup we established ANXA8’s cellular distribution in the mammary gland and ANXA8’s effect on cell proliferation. We show that ANXA8 expression in the mouse mammary gland was strong during pre-puberty before the expansion of the rudimentary ductal network and was limited to a distinct subpopulation of ductal luminal epithelial cells but was not detected in TEB or in alveoli during pregnancy. Similarly, during late involution its expression was found in the surviving ductal epithelium, but not in the apoptotic alveoli. Double-immunofluorescence (IF) showed that ANXA8 positive (+ve) cells were ER-alpha negative (−ve) and mostly quiescent, as defined by lack of Ki67 expression during puberty and mid-pregnancy, but not terminally differentiated with ∼15% of ANXA8 +ve cells re-entering the cell cycle at the start of pregnancy (day 4.5). RT-PCR on RNA from FACS-sorted cells and double-IF showed that ANXA8+ve cells were a subpopulation of c-kit +ve luminal progenitor cells, which have recently been identified as the cells of origin of basal-like breast cancers. Over expression of ANXA8 in the mammary epithelial cell line Kim-2 led to a G₀/G₁ arrest and suppressed Ki67 expression, indicating cell cycle exit. Our data therefore identify ANXA8 as a potential mediator of quiescence in the normal mouse mammary ductal epithelium, while its expression in basal-like breast cancers may be linked to ANXA8’s association with their specific cells of origin.     

Natural killer cells promote early CD8 T cell responses against cytomegalovirus

Understanding the mechanisms that help promote protective immune responses to pathogens is a major challenge in biomedical research and an important goal for the design of innovative therapeutic or vaccination strategies. While natural killer (NK) cells can directly contribute to the control of viral replication, whether, and how, they may help orchestrate global antiviral defense is largely unknown. To address this question, we took advantage of the well-defined molecular interactions involved in the recognition of mouse cytomegalovirus (MCMV) by NK cells. By using congenic or mutant mice and wild-type versus genetically engineered viruses, we examined the consequences on antiviral CD8 T cell responses of specific defects in the ability of the NK cells to control MCMV. This system allowed us to demonstrate, to our knowledge for the first time, that NK cells accelerate CD8 T cell responses against a viral infection in vivo. Moreover, we identify the underlying mechanism as the ability of NK cells to limit IFN-alpha/beta production to levels not immunosuppressive to the host. This is achieved through the early control of cytomegalovirus, which dramatically reduces the activation of plasmacytoid dendritic cells (pDCs) for cytokine production, preserves the conventional dendritic cell (cDC) compartment, and accelerates antiviral CD8 T cell responses. Conversely, exogenous IFN-alpha administration in resistant animals ablates cDCs and delays CD8 T cell activation in the face of NK cell control of viral replication. Collectively, our data demonstrate that the ability of NK cells to respond very early to cytomegalovirus infection critically contributes to balance the intensity of other innate immune responses, which dampens early immunopathology and promotes optimal initiation of antiviral CD8 T cell responses. Thus, the extent to which NK cell responses benefit the host goes beyond their direct antiviral effects and extends to the prevention of innate cytokine shock and to the promotion of adaptive immunity.