Dynamics and memory of heterochromatin in living cells

Posttranslational histone modifications are important for gene regulation, yet the mode of propagation and the contribution to heritable gene expression states remains controversial. To address these questions, we developed a chromatin in vivo assay (CiA) system employing chemically induced proximity to initiate and terminate chromatin modifications in living cells. We selectively recruited HP1α to induce H3K9me3-dependent gene silencing and describe the kinetics and extent of chromatin modifications at the Oct4 locus in fibroblasts and pluripotent cells. H3K9me3 propagated symmetrically and continuously at average rates of ~0.18 nucleosomes/hr to produce domains of up to 10 kb. After removal of the HP1α stimulus, heterochromatic domains were heritably transmitted, undiminished through multiple cell generations. Our data enabled quantitative modeling of reaction kinetics, which revealed that dynamic competition between histone marking and turnover, determines the boundaries and stability of H3K9me3 domains. This framework predicts the steady-state dynamics and spatial features of the majority of euchromatic H3K9me3 domains over the genome.     

Ethylene Glycol Metabolism by Pseudomonas putida

In this study, we investigated the metabolism of ethylene glycol in the Pseudomonas putida strains KT2440 and JM37 by employing growth and bioconversion experiments, directed mutagenesis, and proteome analysis. We found that strain JM37 grew rapidly with ethylene glycol as a sole source of carbon and energy, while strain KT2440 did not grow within 2 days of incubation under the same conditions. However, bioconversion experiments revealed metabolism of ethylene glycol by both strains, with the temporal accumulation of glycolic acid and glyoxylic acid for strain KT2440. This accumulation was further increased by targeted mutagenesis. The key enzymes and specific differences between the two strains were identified by comparative proteomics. In P. putida JM37, tartronate semialdehyde synthase (Gcl), malate synthase (GlcB), and isocitrate lyase (AceA) were found to be induced in the presence of ethylene glycol or glyoxylic acid. Under the same conditions, strain KT2440 showed induction of AceA only. Despite this difference, the two strains were found to use similar periplasmic dehydrogenases for the initial oxidation step of ethylene glycol, namely, the two redundant pyrroloquinoline quinone (PQQ)-dependent enzymes PedE and PedH. From these results we constructed a new pathway for the metabolism of ethylene glycol in P. putida. Furthermore, we conclude that Pseudomonas putida might serve as a useful platform from which to establish a whole-cell biocatalyst for the production of glyoxylic acid from ethylene glycol.     

Short-term changes in-stream macroinvertebrate communities following a severe fire in the Lake Tahoe basin,California

Rivers, lakes and wetlands are good examples of ecosystems that provide multiple, concurrent, services to mankind. Human society has often exploited these systems by enhancing one ecosystem service at the expense of another. Loch Leven, Scotland, UK, is a good example of this. Over the past 150 years, the lake has been subjected to hydrological modification, fish stocking and pollution control to improve the delivery of key goods and services. This study uses historical records to explore the results of these interventions on the ecosystem services that were targeted for improvement and the knock-on effects on other services provided by the lake. The results suggest that, when management changes are being considered to enhance particular ecosystem services, the potentially damaging effects on other ecosystem services should be taken into account. This requires a better understanding of the role of ecosystem function in delivering ecosystem services, and of the links between multiple ecosystem services, than is currently available. While further research is clearly needed, the value of long-term datasets in providing knowledge and understanding through ‘hindsight’ should not be underestimated. The study concludes that successful management actions are likely to be those that incorporate lessons learned from previous decisions.     

Exploratory study on the influence of climatological parameters on <Emphasis Type="BoldItalic">Mycoplasma hyopneumoniae</Emphasis> infection dynamics

The objective of the present work was to elucidate the potential relationship between Mycoplasma hyopneumoniae infection and seroconversion dynamics and climatological conditions in four groups of pigs from the same farm born in different seasons of the year. Nasal swabs and blood samples were taken from 184 pigs at 1, 3, 6, 9, 12, 15, 18, 22 and 25 (slaughter age) weeks of age. Outside climatologic parameters, including temperature (°C), relative humidity (%), precipitation (l/m²) and wind speed (m/s) were recorded weekly from January 2003 to June 2004. Percentage of nPCR detection of M. hyopneumoniae in nasal swabs was associated significantly with the weekly precipitation rate [P = 0.0018, OR = 1.31 (IC = 1.11–1.55)]; the higher the precipitation rate, the higher the probability of being M. hyopneumoniae nPCR-positive. On the other hand, the percentage of seropositive pigs had a significant association with mean weekly temperature rate [P = 0.0012, OR = 0.89 [IC = 0.84–0.95]); the lower the temperature, the higher the probability of being M. hyopneumoniae seropositive. Animals born in autumn (when higher precipitations rates were recorded), entering finishing units in winter (when lower temperatures were recorded), and reaching slaughter in spring, had the highest probability of being infected by M. hyopneumoniae and the highest probability of being M. hyopneumoniae seropositive.     

Behavioural resistance against a protozoan parasite in the monarch butterfly

1. As parasites can dramatically reduce the fitness of their hosts, there should be strong selection for hosts to evolve and maintain defence mechanisms against their parasites. One way in which hosts may protect themselves against parasitism is through altered behaviours, but such defences have been much less studied than other forms of parasite resistance. 2. We studied whether monarch butterflies (Danaus plexippus L.) use altered behaviours to protect themselves and their offspring against the protozoan parasite Ophryocystis elektroscirrha (McLaughlin & Myers (1970), Journal of Protozoology, 17, p. 300). In particular, we studied whether (i) monarch larvae can avoid contact with infectious parasite spores; (ii) infected larvae preferentially consume therapeutic food plants when given a choice or increase the intake of such plants in the absence of choice; and (iii) infected female butterflies preferentially lay their eggs on medicinal plants that make their offspring less sick. 3. We found that monarch larvae were unable to avoid infectious parasite spores. Larvae were also not able to preferentially feed on therapeutic food plants or increase the ingestion of such plants. However, infected female butterflies preferentially laid their eggs on food plants that reduce parasite growth in their offspring. 4. Our results suggest that animals may use altered behaviours as a protection against parasites and that such behaviours may be limited to a single stage in the host-parasite life cycle. Our results also suggest that animals may use altered behaviours to protect their offspring instead of themselves. Thus, our study indicates that an inclusive fitness approach should be adopted to study behavioural defences against parasites.     

The variable subdomain of Escherichia coli SecA functions to regulate SecA ATPase activity and ADP release

Bacterial SecA proteins can be categorized by the presence or absence of a variable subdomain (VAR) located within nucleotide-binding domain II of the SecA DEAD motor. Here we show that VAR is dispensable for SecA function, since the VAR deletion mutant secAΔ519-547 displayed a wild-type rate of cellular growth and protein export. Loss or gain of VAR is extremely rare in the history of bacterial evolution, indicating that it appears to contribute to secA function within the relevant species in their natural environments. VAR removal also results in additional secA phenotypes: azide resistance (Azi(r)) and suppression of signal sequence defects (PrlD). The SecAΔ(519-547) protein was found to be modestly hyperactive for SecA ATPase activities and displayed an accelerated rate of ADP release, consistent with the biochemical basis of azide resistance. Based on our findings, we discuss models whereby VAR allosterically regulates SecA DEAD motor function at SecYEG.     

Mosaiktrisomie 8p11.21q11.21 als Pr?disposition für myeloische Leuk?mien

Juvenile myelomonocytic leukemia (JMML) is a unique myeloproliferative disorder of early childhood in which mutations in NRAS, KRAS, PTPN11, NF1 and CBL are frequently found. Using high-resolution oligo array-based comparative genomic hybridization (aCGH), 20 JMML samples were investigated for submicroscopic genomic copy number alterations. Besides known cytogenetic aberrations, ten samples displayed additional submicroscopic alterations. Interestingly, an almost identical gain of chromosome 8 was identified in two patients. Subsequently, fluorescence in situ hybridization indicated a constitutional partial trisomy 8 mosaic (cT8M) in both patients. A survey on 27 cT8M patients with reported malignancies showed a predominance of myeloid malignancies including JMML. Our results dramatically reduce the critical region on chromosome 8 to 8p11.21q11.21. To determine how constitutional partial trisomy 8 mosaicisms may contribute to leukemogenesis in different mutational subtypes of JMML and other myeloid malignancies, further investigations are required.     

Comparative analysis of Neph gene expression in mouse and chicken development

Neph proteins are evolutionarily conserved members of the immunoglobulin superfamily of adhesion proteins and regulate morphogenesis and patterning of different tissues. They share a common protein structure consisting of extracellular immunoglobulin-like domains, a transmembrane region, and a carboxyl terminal cytoplasmic tail required for signaling. Neph orthologs have been widely characterized in invertebrates where they mediate such diverse processes as neural development, synaptogenesis, or myoblast fusion. Vertebrate Neph proteins have been described first at the glomerular filtration barrier of the kidney. Recently, there has been accumulating evidence suggesting a function of Neph proteins also outside the kidney. Here we demonstrate that Neph1, Neph2, and Neph3 are expressed differentially in various tissues during ontogenesis in mouse and chicken. Neph1 and Neph2 were found to be amply expressed in the central nervous system while Neph3 expression remained localized to the cerebellum anlage and the spinal cord. Outside the nervous system, Neph mRNAs were also differentially expressed in branchial arches, somites, heart, lung bud, and apical ectodermal ridge. Our findings support the concept that vertebrate Neph proteins, similarly to their Drosophila and C. elegans orthologs, provide guidance cues for cell recognition and tissue patterning in various organs which may open interesting perspectives for future research on Neph1-3 controlled morphogenesis.     

Immunofluorescent visualisation of focal adhesion kinase in human skeletal muscle and its associated microvasculature

Within animal skeletal muscle, focal adhesion kinase (FAK) has been associated with load-dependent molecular and metabolic adaptation including the regulation of insulin sensitivity. This study aimed to generate the first visual images of the localisation of FAK within human skeletal muscle fibres and its associated microvasculature using widefield and confocal immunofluorescence microscopy. Percutaneous muscle biopsies, taken from five lean, active males, were frozen and 5-μm cryosections were incubated with FAK antibodies for visualisation in muscle fibres and the microvasculature. Anti-myosin heavy chain type I was used for fibre-type differentiation. Muscle sections were also incubated with anti-dihydropyridine receptor (DHPR) to investigate co-localisation of FAK with the t-tubules. FITC-conjugated Ulex europaeus Agglutinin I stained the endothelium of the capillaries, whilst anti-smooth muscle actin stained the vascular smooth muscle of arterioles. Fibre-type differences in the intensity of FAK immunofluorescence were determined with image analysis software. In transversely and longitudinally orientated fibres, FAK was localised at the sarcolemmal regions. In longitudinally orientated fibres, FAK staining also showed uniform striations across the fibre and co-staining with DHPR suggests FAK associates with the t-tubules. There was no fibre-type difference in sarcoplasmic FAK content. Within the capillary endothelium and arteriolar smooth muscle, FAK was distributed heterogeneously as clusters. This is the first study to visualise FAK in human skeletal muscle microvasculature and within the (sub)sarcolemmal and t-tubule regions using immunofluorescence microscopy. This technique will be an important tool for investigating the role of FAK in the intracellular signalling of human skeletal muscle and the endothelium of its associated microvasculature.