Induction of apoptotic cell death by putrescine

The polyamines are essential for cellular growth and differentiation. Ornithine decarboxylase (ODC), which catalyses the first step in the biosynthesis of the polyamines, has a very fast turnover and is subject to a strong feedback control by the polyamines. In the present study, we show that overexpression of a metabolically stable ODC in CHO cells induced a massive cell death unless the cells were grown in the presence of the ODC inhibitor alpha-difluoromethylornithine (DFMO). Cells overexpressing wild-type (unstable) ODC, on the other hand, were not dependent on the presence of DFMO for their growth. The induction of cell death was correlated with a dramatic increase in cellular putrescine levels. Analysis using flow cytometry revealed perturbed cell cycle kinetics, with a large accumulation of cells with sub-G1 amounts of DNA, which is a typical sign of apoptosis. Another strong indication of apoptosis was the finding that one of the key enzymes in the apoptotic process, caspase-3, was induced when DFMO was omitted from the growth medium. Furthermore, inhibition of the caspase activity significantly reduced the recruitment of cells to the sub-G1 fraction. In conclusion, deregulation of polyamine homeostasis may negatively affect cell proliferation and eventually lead to cell death by apoptosis if putrescine levels become too high.     

Crystal structures of yeast beta-alanine synthase complexes reveal the mode of substrate binding and large scale domain closure movements

Beta-alanine synthase is the final enzyme of the reductive pyrimidine catabolic pathway, which is responsible for the breakdown of uracil and thymine in higher organisms. The fold of the homodimeric enzyme from the yeast Saccharomyces kluyveri identifies it as a member of the AcyI/M20 family of metallopeptidases. Its subunit consists of a catalytic domain harboring a di-zinc center and a smaller dimerization domain. The present site-directed mutagenesis studies identify Glu(159) and Arg(322) as crucial for catalysis and His(262) and His(397) as functionally important but not essential. We determined the crystal structures of wild-type beta-alanine synthase in complex with the reaction product beta-alanine, and of the mutant E159A with the substrate N-carbamyl-beta-alanine, revealing the closed state of a dimeric AcyI/M20 metallopeptidase-like enzyme. Subunit closure is achieved by a approximately 30 degrees rigid body domain rotation, which completes the active site by integration of substrate binding residues that belong to the dimerization domain of the same or the partner subunit. Substrate binding is achieved via a salt bridge, a number of hydrogen bonds, and coordination to one of the zinc ions of the di-metal center.     

A hybridisation barrier between two evolutionary lineages of <Emphasis Type="Italic">Barbarea vulgaris</Emphasis> (Brassicaceae) that differ in biotic resistances

Hybridisation barriers are likely to evolve during allopatric separation of populations, in parallel with divergent adaptation to different conditions in the two ranges. If the populations secondarily come into contact, limited interbreeding between them may affect the subsequent spread of the two lineages and their adaptations in the region of sympatry. Barbarea vulgaris include two genetically divergent lineages that differ in secondary metabolites and resistances to insects and a pathogen. The two plant types grow in different Eurasian ranges but co-occur in Denmark and neighbouring countries, posing the question why they have not merged and the resistances spread from one type into the range of the other. Here, we tested whether a hybridisation barrier contribute to this. Different proportions of plants of the two types were placed in net tents and pollinated by flies, and paternity of the resulting seeds determined with genetic markers. Lower proportions of fruits and seeds developed successfully in mixtures with higher proportions of heterotypic plants (i.e. of the other plant type). When combined with results on offspring paternity in a statistical analysis, we found that heterotypic pollen was much less successful in fertilizing embryos and that heterotypic seeds survived less frequently than the contypic. Mature F₁ hybrids in addition produced lower proportions of mature pollen. The two B. vulgaris plant types are thus separated by substantial prezygotic and postzygotic barriers, as strong as reported for crosses between closely related but taxonomically recognised plant species. This may explain why the two B. vulgaris types have not merged in sympatry and why genes for insect-resistance have not introgressed to any extent from one to the other.     

The occurrence of Chlamydia spp. in pigs with and without clinical disease

Background

Disk-associated cervical spondylomyelopathy (DA-CSM) is a multifactorial neurological disorder in which progressive caudal cervical spinal cord compression is mainly caused by one or more intervertebral disk protrusions. The Doberman pinscher breed seems predisposed for this condition. The underlying cause and pathophysiology of DA-CSM are currently unknown. Recently, wider intervertebral disks have been put forward as a risk factor for development of clinically relevant DA-CSM. However, little is known about other factors affecting intervertebral disk width. Therefore the aim of this study was to assess the association between intervertebral disk width, measured on magnetic resonance imaging (MRI), and clinical status, age, gender and intervertebral disk location in dogs with and without clinical signs of DA-CSM.

Methods

Doberman pinschers with clinical signs of DA-CSM (N=17),clinically normal Doberman pinschers (N=20), and clinically normal English Foxhounds (N=17), underwent MRI of the cervical vertebral column. On sagittal T2-weighted images, intervertebral disk width was measured from C2-C3 to C6-C7. Intra ?Cand interobserver agreement were assessed on a subset of 20 of the 54 imaging studies.

Results

Intervertebral disk width was not significantly different between Doberman pinschers with clinical signs of DA-CSM, clinically normal Doberman pinschers or clinically normal English Foxhounds (p=0.43). Intervertebral disk width was positively associated with increasing age (p=0.029). Each monthly increase in age resulted in an increase of disk width by 0.0057mm. Intervertebral disk width was not significantly affected by gender (p=0.056), but was significantly influenced by intervertebral disk location (p <0.0001). The assessed measurements were associated with a good intra ?Cand interobserver agreement.

Conclusions

The present study does not provide evidence that wider intervertebral disks are associated with clinical status in dogs with and without DA-CSM. Instead, it seems that cervical intervertebral disk width in dogs is positively associated with increase in age.     

The involvement of cytosolic lipases in converting phosphatidyl choline to substrate for galactolipid synthesis in the chloroplast envelope

Here we report that cytosolic phospholipases are involved in the utilization of phosphatidylcholine (PC) as substrate for chloroplast-localized synthesis of monogalactosyldiacylglycerol (MGDG). Isolated chloroplasts were pre-incubated with lysoPC and [14C]18:0-CoA to form [14C]PC. When soluble plant proteins (cytosol) and UDP-galactose were added, [14C] MGDG was formed. An inhibitor of phospholipase D markedly lowered the formation of [14C]MGDG, whereas thermolysin pretreatment of the chloroplasts was without effect. The cytosolic activity resided in the >100-kDa fraction. In a second approach, [14C]PC-containing lipid mixtures were incubated with cytosol. Degradation of [14C]PC to [14C]diacylglycerol was highest when the lipid composition of the mixture mimicked that of the outer chloroplast envelope. We also investigated whether PC of extraplastidic origin could function as substrate for MGDG synthesis. Isolated chloroplasts were incubated with enriched endoplasmic reticulum containing radiolabelled acyl lipids. In the presence of cytosol and UDP-galactose, there was a time-dependent transfer of [14C]PC from this fraction to chloroplasts, where [14C]MGDG was formed. We conclude that chloroplasts recruit cytosolic phospholipase D and phosphatidic acid phosphatase to convert PC to diacylglycerol. Apparently, these lipases do not interact with chloroplast surface proteins, but rather with outer membrane lipids, either for association to the envelope or for substrate presentation.     

Threonine 788 in integrin subunit beta1 regulates integrin activation

In the present study, the functional role of suggested phosphorylation of the conserved threonines in the cytoplasmic domain of integrin subunit beta1 was investigated. Mutants mimicking phosphorylated and unphosphorylated forms of beta1 were expressed in beta1 deficient GD25 cells. T788 in beta1 was identified as a site with major influence on integrin function. The mutation to A788 strongly reduced beta1-dependent cell attachment and exposure of the extracellular 9EG7 epitope, whereas replacement of T789 with alanine did not interfere with the ligand-binding ability. Talin has been shown to mediate integrin activation, but the talin head domain bound equally well to the wild-type beta1 and the mutants indicating that the T788A mutation caused defect integrin activation by another mechanism. The phosphorylation-mimicking mutation T788D was fully active in promoting cell adhesion. GD25 cells expressing beta1T788D accumulated increased number of focal contacts and migrated slowly compared to GD25 beta1 wild-type. An analogous phenotype is seen when focal adhesion kinase activation is abrogated. However, neither the beta1T788D nor the beta1T788A mutation failed to induce tyrosine phosphorylation of focal adhesion kinase. The results suggest that phosphorylation of T788 in integrin beta1 promotes inside-out receptor activation, as well as focal contact accumulation.     

Peroxisome proliferation in Norway spruce induced by ozone

Summary Catalase (EC 1.11.1.6) activity (both total and specific activity) of particulate fractions of needles of Norway spruce [Picea abies (L.) Karst.] was elevated approximately 2-fold following exposure of trees to 60–70 g/m³ of ozone during the growing season compared to trees receiving charcoal filtered air (about 15 g/m³ ozone). Measurements were from homogenates fractionated into particulate and soluble (supernatent) activities. In contrast, the catalase activity of the supernatant was unchanged in response to ozone treatment. Catalase activity declined as the needles aged comparing current, 1-, and 2-year needles but the ozone-induced increment remained constant. Electron microscope cytochemistry using peroxidatic coupling with 3,3-diaminobenzidine carried out in parallel, revealed catalase-containing peroxisomes both in situ and in the particulate fractions analyzed for catalase activity. The tissue volume occupied by peroxisomes in response to needle age and ozone appeared to vary approximately in proportion to the measured catalase activity. Overall cytochemical reactivity for catalase declined with needle age, but, for all years, was greater in needles of trees receiving air supplemented with ozone compared to those of trees receiving charcoal filtered air.Abbreviations DAB 3,3-diaminobenzidine tetrahydrochloride