Parvovirus Induced Alterations in Nuclear Architecture and Dynamics

The nucleus of interphase eukaryotic cell is a highly compartmentalized structure containing the three-dimensional network of chromatin and numerous proteinaceous subcompartments. DNA viruses induce profound changes in the intranuclear structures of their host cells. We are applying a combination of confocal imaging including photobleaching microscopy and computational methods to analyze the modifications of nuclear architecture and dynamics in parvovirus infected cells. Upon canine parvovirus infection, expansion of the viral replication compartment is accompanied by chromatin marginalization to the vicinity of the nuclear membrane. Dextran microinjection and fluorescence recovery after photobleaching (FRAP) studies revealed the homogeneity of this compartment. Markedly, in spite of increase in viral DNA content of the nucleus, a significant increase in the protein mobility was observed in infected compared to non-infected cells. Moreover, analyzis of the dynamics of photoactivable capsid protein demonstrated rapid intranuclear dynamics of viral capsids. Finally, quantitative FRAP and cellular modelling were used to determine the duration of viral genome replication. Altogether, our findings indicate that parvoviruses modify the nuclear structure and dynamics extensively. Intranuclear crowding of viral components leads to enlargement of the interchromosomal domain and to chromatin marginalization via depletion attraction. In conclusion, parvoviruses provide a useful model system for understanding the mechanisms of virus-induced intranuclear modifications.     

Transforming growth factor β2 is negatively regulated by endogenous retinoic acid during early heart morphogenesis

Vitamin A‐deficient (VAD) quail embryos lack the vitamin A‐active form, retinoic acid (RA) and are characterized by a phenotype that includes a grossly abnormal cardiovascular system that can be rescued by RA. Here we report that the transforming growth factor, TGFβ2 is involved in RA‐regulated cardiovascular development. In VAD embryos TGFβ2 mRNA and protein expression are greatly elevated. The expression of TGFβ receptor II is also elevated in VAD embryos but is normalized by treatment with TGFβ2‐specific antisense oligonucleotides (AS). Administration of this AS or an antibody specific for TGFβ2 to VAD embryos normalizes posterior heart development and vascularization, while the administration of exogenous active TGFβ2 protein to normal quail embryos mimics the excessive TGFβ2 status of VAD embryos and induces VAD cardiovascular phenotype. In VAD embryos pSmad2/3 and pErk1 are not activated, while pErk2 and pcRaf are elevated and pSmad1/5/8 is diminished. We conclude that in the early avian embryo TGFβ2 has a major role in the retinoic acid‐regulated posterior heart morphogenesis for which it does not use Smad2/3 pathways, but may use other signaling pathways. Importantly, we conclude that retinoic acid is a critical negative physiological regulator of the magnitude of TGFβ2 signals during vertebrate heart formation.     

Forest floor vegetation response to nitrogen deposition in Europe

Chronic nitrogen (N) deposition is a threat to biodiversity that results from the eutrophication of ecosystems. We studied long‐term monitoring data from 28 forest sites with a total of 1,335 permanent forest floor vegetation plots from northern Fennoscandia to southern Italy to analyse temporal trends in vascular plant species cover and diversity. We found that the cover of plant species which prefer nutrient‐poor soils (oligotrophic species) decreased the more the measured N deposition exceeded the empirical critical load (CL) for eutrophication effects (P = 0.002). Although species preferring nutrient‐rich sites (eutrophic species) did not experience a significantly increase in cover (P = 0.440), in comparison to oligotrophic species they had a marginally higher proportion among new occurring species (P = 0.091). The observed gradual replacement of oligotrophic species by eutrophic species as a response to N deposition seems to be a general pattern, as it was consistent on the European scale. Contrary to species cover changes, neither the decrease in species richness nor of homogeneity correlated with nitrogen CL exceedance (ExCL_empN). We assume that the lack of diversity changes resulted from the restricted time period of our observations. Although existing habitat‐specific empirical CL still hold some uncertainty, we exemplify that they are useful indicators for the sensitivity of forest floor vegetation to N deposition.     

Inhibition of Escherichia coli precursor-16S rRNA processing by mouse intestinal contents

The correlation between ribosome content and growth rate found in many bacterial species has proved useful for estimating the growth activity of individual cells by quantitative in situ rRNA hybridization. However, in dynamic environments, the stability of mature ribosomal RNA causes problems in using cellular rRNA contents for direct monitoring of bacterial growth activity in situ . In a recent paper, Cangelosi and Brabant suggested monitoring the content of precursors in rRNA synthesis (pre-rRNAs) as an alternative approach. These are rapidly broken down after the cessation of bacterial growth. We have applied fluorescence in situ hybridization of pre-16S rRNA to Escherichia coli cells growing in vitro in extracts from two different compartments of the mouse intestine: the caecal mucus layer, where E. coli grew rapidly, and the contents of the caecum, which supported much slower bacterial growth. The amounts of 23S rRNA and pre-16S rRNA measured for E. coli growing in intestinal mucus corresponded to that expected for bacteria with the observed growth rate. In contrast, the slow-growing E. coli cells present in intestinal contents turned out to have an approximately ninefold higher content of pre-16S rRNA than cultures of the same strain growing rapidly in rich media. We present results suggesting that the mouse intestinal contents contain an agent that inhibits the growth of E. coli by disturbing its ability to process pre-16S rRNA.     

Harmful singlet oxygen can be helpful

Highly reactive harmful singlet oxygen O2(1delta(g)) can be helpful while relaxing to its triplet ground state O2(3sigma(g)-). The energy emitted during this relaxation from the excited energy state is discernable at 634 nm. We report here on the effect of this energy as photon illumination and as energy transfer in air on the production of reactive oxygen species (ROS) by human monocytes, measured as isoluminol-enhanced chemiluminescence. We demonstrate up to 60% decrease in the secretion of ROS after 2-min illumination of the monocytes stimulated with phorbol myristate acetate (PMA). The results provide in vitro documentation of the utility of singlet oxygen energy in modifying cellular behaviour.     

Caspase multiplexing: simultaneous homogeneous time-resolved quenching assay (TruPoint) for caspases 1, 3, and 6

Caspases are a group of cysteine proteases involved in apoptosis and inflammation. A multiparametric homogeneous assay capable of measuring activity of three different caspases in a single well of a microtiter plate is described. Different fluorescent europium, samarium, terbium, and dysprosium chelates were coupled to a caspase substrate peptide, their luminescence properties, were analyzed, and their function in a time-resolved fluorescence quenching-based caspase 3 assay was studied. Substrates for caspases 1, 2, 3, 6, and 8 and granzyme B were also synthesized and their specificities for different caspases were determined. By selecting suitable lanthanide chelates and substrates we developed a multiparametric homogeneous time-resolved fluorescence quenching-based assay for caspases 1, 3, and 6. The assay was capable of measuring the activity of both single caspases and a mixture of three caspases mixed in the same well.     

Isolation and characterization of O-acetylated glucomannans from aspen and birch wood

O-acetylated glucomannans were isolated from aspen and birch wood employing two different procedures and thereafter subjected to carbohydrate analysis by NMR spectroscopy and MALDI mass spectrometry. In one of the isolation procedures, acetone-extracted aspen or birch wood meal was extracted with dimethyl sulfoxide and then with hot water. Fractionation of the hemicellulose-containing extracts by size-exclusion chromatography was subsequently performed. In the other procedure, fractional precipitation with ethanol was used to isolate glucomannans from lyophilized process water produced by mechanical pulping of aspen. The aspen and birch glucomannans are O-acetylated at the C-2 or C-3 position of some of the mannose residues (random distribution), with a degree of acetylation of approx 0.3. In both cases the degree of polymerization was approx 16, indicating that low-molecular mass fractions of the glucomannans in hardwood have been isolated here.     

Lysine synthesis control in Corynebacterium glutamicum RC 115 in mixed substrate (glucose-acetate) medium

The effect of acetate as a glucose co-substrate on growth, lysine synthesis and experimental lysine yield from carbon substrates by Corynebacterium glutamicum RC 115 was investigated. It was found that low amounts of acetate, injected with a glucose-acetate pulse into the steady-state continuous culture in bioreactor, caused a slight decrease in the specific rates of glucose uptake and bacterial growth, but a significant increase in the cell specific rate of lysine synthesis and an increase in lysine yield. In contrast, acetate injected in high amounts was followed by a drastic decrease in the values of these parameters. A strong increase in experimental lysine yield under the latter conditions was reached in the response to pyruvate addition. Therefore it was shown that acetate in low concentrations can be used as a glucose co-substrate to increase the cell specific rate of lysine synthesis and lysine yield by C. glutamicum RC 115. Pyruvate supplementation was found as a promising method to enhance lysine synthesis by bacterial cells grown in glucose-acetate media with an increased concentration of acetate.     

A novel role for retinoids in patterning the avian forebrain during presomite stages

Retinoids, and in particular retinoic acid (RA), are known to induce posterior fates in neural tissue. However, alterations in retinoid signalling dramatically affect anterior development. Previous reports have demonstrated a late role for retinoids in patterning craniofacial and forebrain structures, but an earlier role in anterior patterning is not well understood. We show that enzymes involved in synthesizing retinoids are expressed in the avian hypoblast and in tissues directly involved in head patterning, such as anterior definitive endoderm and prechordal mesendoderm. We found that in the vitamin A-deficient (VAD) quail model, which lacks biologically active RA from the first stages of development, anterior endodermal markers such as Bmp2, Bmp7, Hex and the Wnt antagonist crescent are affected during early gastrulation. Furthermore, prechordal mesendodermal and prospective ventral telencephalic markers are expanded posteriorly, Shh expression in the axial mesoderm is reduced, and Bmp2 and Bmp7 are abnormally expressed in the ventral midline of the neural tube. At early somite stages, VAD embryos have increased cell death in ventral neuroectoderm and foregut endoderm, but normal cranial neural crest production, whereas at later stages extensive apoptosis occurs in head mesenchyme and ventral neuroectoderm. As a result, VAD embryos end up with a single and reduced telencephalic vesicle and an abnormally patterned diencephalon. Therefore, we propose that retinoids have a dual role in patterning the anterior forebrain during development. During early gastrulation, RA acts in anterior endodermal cells to modulate the anteroposterior (AP) positional identity of prechordal mesendodermal inductive signals to the overlying neuroectoderm. Later on, at neural pore closure, RA is required for patterning of the mesenchyme of the frontonasal process and the forebrain by modulating signalling molecules involved in craniofacial morphogenesis.     

Accumulation of genistein and lipophilic genistein derivatives in lipoproteins during incubation with human plasma in vitro

Atherosclerosis is initiated by the uptake and retention of oxidized low-density lipoprotein (LDL) into the arterial intima. We have previously shown that dietary isoflavone phytoestrogens inhibit LDL oxidation in vitro. The inhibition could have been caused by undetected isoflavone metabolites associated with lipoproteins. In the present study, we incubated human plasma with [3H]genistein, both with and without the lecithin:cholesterol acyltransferase (LCAT) inhibitor dithionitrobenzoic acid (DTNB). Our results indicated that the 3H-label was attached to both high-density lipoprotein (HDL) and LDL, and that it represented both underivatized genistein and lipophilic derivatives of genistein, part of which were identified as fatty acid monoesters. The latter was demonstrated by the findings that DTNB decreased the HDL and LDL associated radioactivity in the lipophilic fraction isolated by hydrophobic chromatography and that saponification hydrolysis liberated a corresponding part of the 3H-label. Two-dimensional reversed-phase thin-layer chromatography (TLC) demonstrated that a corresponding part of the radioactivity comigrated with genistein monoester standards in the absence of DTNB but was abolished if DTNB had been present in the incubation. In summary, incubation of plasma with [3H]genistein resulted in accumulation of underivatized genistein as well as lipophilic genistein derivatives in lipoproteins. A smaller part of the latter were genistein monoesters, while part remained unidentified. Our results suggest an explanation for the increased oxidation resistance of isolated LDL during intake of soybean isoflavones.