Expression of an anti apoptotic recombinant short peptide in mammalian cells

Understanding the mechanisms of the apoptotic and anti apoptotic processes may lead to a better way to control these cascades. Here we demonstrated for the first time the feasibility to express a short functional peptide in mammalian cells that abrogates the apoptosis cascade through interference with the proteolytic activity of the initiator caspase 9 and the executing caspase 3 enzymes. The expression of a short peptide that includes the pseudo-substrate motif of the apoptosis inhibitor protein P35 (Asp-Gln-Met-Asp) leads to the abrogation of cell death induced through either the mitochondrial or the death receptors pathways.Short open reading frames have been detected in several mammalian mRNAs, primarily upstream of the main long reading frame (uORFs), however, direct evidence for de-novo peptides translation has not been provided. Utilizing biochemical and imaging techniques we demonstrate here that the functional recombinant peptide was localized to the cytpoplasmic fraction of the cell.In conclusion, this work demonstrates that ribosomes recognize short ORFs to translate stable short recombinant peptides in mammalian cells. Expression of these intracellular peptides results in the knock down of apoptotic processes to generate apoptosis resistant stable cells.     

Quinidine and procainamide inhibit murine macrophage uptake of apoptotic and necrotic cells: A novel contributing mechanism of drug-induced-lupus

A number of mechanisms have been proposed to explain the etiology of drug-induced lupus (DIL) but the effect of apoptotic and necrotic cell handling has not been previously examined.Objective. To evaluate the effect of quinidine and procainamide at therapeutic range concentrations, on the uptake of apoptotic and necrotic thymocytes by murine peritoneal macrophages and on macrophage survival, as a novel mechanism for DIL.Methods. Thymocytes were stained and induced to undergo apoptosis by serum withdrawal. Apoptosis was evaluated using annexin V and propidum iodide (PI) and PI staining. Necrosis was induced by heating. Peritoneal macrophages were treated with quinidine or procainamide at a range of therapeutic concentrations and incubated with stained apoptotic and necrotic thymocytes. Apoptotic and necrotic cell uptake was evaluated by flow cytometry using double staining of thymocytes and macrophages and by confocal microscopy. Green fluorescent latex beads were used as controls for phagocytosis.Results. Significantly decreased uptake of apoptotic and necrotic cells was seen in the presence of quinidine and procainamide. The documented effect was mainly on the number of apoptotic/necrotic cells per macrophage. Uptake of fluorescent latex beads offered to resident macrophages was not significantly affected by quinidine or procainamide. No pro-apoptotic effect of quinidine or procainamide on macrophages was seen.Conclusion. Quinidine and procainamide at therapeutic range concentrations specifically inhibit clearance of apoptotic and necrotic cells by peritoneal macrophages. Altered handling of apoptotic and necrotic cells may represent a contributing mechanism for DIL.     

Diversity in fertility potential and organo-sulphur compounds among garlics from Central Asia

Extending the collection of garlic (Allium sativum L.) accessions is an important means that is available for broadening the genetic variability of this cultivated plant, with regard to yield, quality, and tolerance to biotic and abiotic traits; it is also an important means for restoring fertility and flowering. In the framework of the EU project Garlic and Health, 120 garlic accessions were collected in Central Asia – the main centre of garlic diversity. Plants were documented and thereafter maintained in field collections in both Israel and The Netherlands. The collection was evaluated for biological and economic traits. Garlic clones vary in most vegetative characteristics (leaf number, bulb size and structure), as well as in floral scape elongation and inflorescence development. A clear distinction was made between incomplete bolting and bolting populations; most of the accessions in the latter populations produced flowers with fertile pollen and receptive stigma. Wide variations were recorded with regard to differentiation of topsets, their size, number and rapidity of development. Furthermore, significant variation in organo-sulphur compounds (alliin, isoalliin, allicin and related dipeptides) was found within garlic collections and between plants grown under differing environmental conditions. Genetic fingerprinting by means of AFLP markers revealed three distinct groups within this collection, differing also in flowering ability and organo-S content.     

Cloning and expression of genes encoding transferrin-binding protein A and B from Actinobacillus pleuropneumoniae serotype 5

Actinobacillus pleuropneumoniae is an important primary pathogen in pigs, which causes a highly contagious pleuropneumonia. As an adaptation to the iron-restricted environment of the host, A. pleuropneumoniae possesses iron acquisition pathways mediated by surface receptors that specifically bind transferrin from the host. The receptor is composed of two receptor proteins, transferrin-binding protein A and B (TbpA and B), which are both capable of binding to transferrin. An impairment of iron uptake mechanisms is likely to reduce virulence. For this reason, these two proteins can be useful as a candidate target for A. pleuropneumoniae vaccination. To do this, genes encoding the TbpA and B from a serotype 5 isolate of A. pleuropneumoniae were amplified from genomic DNA template by PCR and cloned into a pRSET prokaryotic expression vector, generating the pRSET-A.pp-TbpA and B. Escherichia coli BL21(DE3)pLysS competent cells were transformed with each construct followed by the induction of protein expression by the addition of IPTG. Bands corresponding to the predicted sizes (110 and 60 kDa) were seen on the SDS-PAGE. Polyclonal antibodies raised against recombinant TbpA and B from mice were reacted with bacterial proteins. This result indicates that the recombinant proteins can induce immunological responses and might be useful as candidate targets for A. pleuropneumoniae vaccination.     

Antibacterial essential oils in malodorous cancer patients: clinical observations in 30 patients.

Malodorous necrotic ulcers in cancer patients are of major concern as it leads to social isolation and poor quality of life. Current medications and topical therapies have proven inadequate in their ability to reduce foul smell to acceptable levels. We report the positive experience we have had in using antibacterial essential oils in patients with incurable head and neck cancer and associated malodorous necrotic ulcers. All patients received a standard course of therapy with oral or systemic antibiosis. In addition, we rinsed the ulcers with an antibacterial essential oil mix (mainly based on Eucalyptus oil) twice a day. All patients experienced complete resolution of the foul smell by only the third or fourth day of therapy. As a secondary effect we saw that besides smell reduction the oils had anti-inflammatory effects on neoplastic ulcers. In some patients ulcers started to heal and achieved complete re-epithiliazation. The patients experienced great personal relief upon resolution of their malodorous conditions. Quality of life improved significantly with the resulting reintroduction of social contact with friends and relatives.     

Postglacial-period and Recent Invasions Shape the Population Genetics of Botryllid Ascidians along European Atlantic Coasts

The colonial urochordate Botryllus schlosseri is a sedentary species of Mediterranean origin that became cosmopolitan, probably because of postglacial-period dispersal and human-mediated invasions of colonies attached to ship hulls. Here we studied microsatellite allele diversity of Atlantic coast populations from an area ranging from European regions south of the last glacial front to regions that had been permanently ice-covered. Gene diversity levels varied dramatically among populations residing in areas subject to different glacial conditions. Five populations from the Iberian Peninsula, in an area south of the last glacial front, as well as two populations from presumed refugia in Brittany, expressed high gene diversity values (expected heterozygosity [He]: 0.76–0.80; average number of alleles per locus [A]: 7.25–8.75). Two populations inhabiting areas that experienced permafrost conditions (Helgoland Island, Germany, and Plymouth, England) had intermediate values (He: 0.40–0.42; A: 3.0–4.0), whereas the Auchenmalg, Scotland, population, from an area previously covered by ice, showed a remarkably low value (He: 0.17; A: 1.75). Therefore, most European populations of B. schlosseri mirrored the movement of the ice front in the last ice age. A second population from the area that was covered by permanent ice (Lossiemouth, Scotland), however, had a high He of 0.61 and an intermediate A of 3.67. Results were compared with recent invasions (populations less than 200 years old) in the United States and New Zealand that had a higher degree of genetic variation than the European native populations established thousands of years ago. Given the overall dearth of studies on this subject, we suggest that in contemporary established Botryllus populations, gene diversity is affected by ecological factors, some of which can be traced directly to the last ice age. Other parameters of gene diversity are influenced by selection pressure, which might be more intense in northern regions.     

Might cardiac catheterization influence diurnal rhythm of the steroid stress hormones secretion?

Glucocorticoids (GCs) are steroid hormones produced by the adrenal cortex in reaction to stress stimuli. GCs production is not stable over a 24-hour period; the plasma concentration peaks in the morning (approximately upon awakening) and then the plasma levels decrease, reaching the nadir in the evening. In our experiments, the levels of cortisol, cortisone, DHEA and DHEAS were tested in young female pigs (n=23) during heart catheterization at two different day times (in the morning and in the afternoon). The non-parametric Mann-Whitney test for statistical analysis was used. We found only minimal statistical differences in studied markers between the morning and afternoon group (p>0.05). The absence of circadian variation in GCs levels could originate either at an early age of our experimental pigs, or in stressful conditions on the experiment day, or most likely the day before (e.g. social isolation, fasting, transport, and catheterization), respectively. We can conclude there is no difference in the stress load between morning and afternoon experiments, and therefore we can assume the stress load is not a limiting factor for the timing when catheterization should be preferably performed.