A Highlights from MBoC Selection: More than just a ticket canceller: the mitochondrial processing peptidase tailors complex precursor proteins at internal cleavage sites

Most mitochondrial proteins are synthesized as precursors that carry N-terminal presequences. After they are imported into mitochondria, these targeting signals are cleaved off by the mitochondrial processing peptidase (MPP). Using the mitochondrial tandem protein Arg5,6 as a model substrate, we demonstrate that MPP has an additional role in preprotein maturation, beyond the removal of presequences. Arg5,6 is synthesized as a polyprotein precursor that is imported into mitochondria and subsequently separated into two distinct enzymes. This internal processing is performed by MPP, which cleaves the Arg5,6 precursor at its N-terminus and at an internal site. The peculiar organization of Arg5,6 is conserved across fungi and reflects the polycistronic arginine operon in prokaryotes. MPP cleavage sites are also present in other mitochondrial fusion proteins from fungi, plants, and animals. Hence, besides its role as a “ticket canceller” for removal of presequences, MPP exhibits a second conserved activity as an internal processing peptidase for complex mitochondrial precursor proteins.     

Short- and long-term effects of silver nanoparticles on human microvascular endothelial cells

AIM: To study the response to silver nanoparticles (Ag NP) of human microvascular endothelial cells, protagonists of angiogenesis.METHODS: We cultured human microvascular endothelial cells and endothelial colony-forming cells in their corresponding growth medium. Stock solutions of Ag NP were prepared in culture medium and sonicated before use. They were added at different concentrations and for different times to culture media. The toxicity of Ag NP was investigated by measuring the reduction of yellow tetrazolium salt to dark purple formazan (MTT assay) at 575 nm. After staining with trypan blue, cell proliferation was assessed by counting viable cells. The lactate dehydrogenase leakage assay was performed on culture media by following the oxidation of NADH to NAD+ and monitoring the reaction kinetically at 340 nm. Reactive oxygen species production was quantified using 2’-7’-dichlorofluorescein diacetate. The alkaline comet assay was performed after mixing the cells with low melting-point agarose. Electrophoresis was then conducted and the samples were stained with ethidium bromide and analyzed with a fluorescence microscope.RESULTS: Ag NP are cytotoxic in a dose and time dependent fashion for HMEC. At high concentrations, Ag NP determine loss of membrane integrity as demonstrated by the increased activity of lactate dehydrogenase in the culture medium. Ag NP rapidly stimulate the formation of free radicals. However, pre-incubation with Trolox, apocynin, or N-acetyl-L-cysteine, antioxidants which have different structure and act through different mechanisms, is not sufficient to prevent cytotoxicity. Ag NP also induce DNA damage dose-dependently, as shown by comet assay. When exposed to sublethal concentrations of Ag NP for long times, the cells remain viable but are growth retarded. Interestingly, removal of Ag NP partially rescues cell growth. Also genotoxicity is reversible upon removal of Ag NP from culture medium, suggesting that no permanent modifications occur. It is noteworthy that Ag NP are cytotoxic and genotoxic also for endothelial progenitors, in particular for endothelial colony-forming cells, which participate to angiogenesis.CONCLUSION: Silver nanoparticles are cytotoxic and genotoxic for human microvascular endothelial cells and might become a useful tool to control excessive angiogenesis.     

Characterization of two heparan sulphate-binding sites in the mycobacterial adhesin Hlp

Background  

The histone-like Hlp protein is emerging as a key component in mycobacterial pathogenesis, being involved in the initial events of host colonization by interacting with laminin and glycosaminoglycans (GAGs). In the present study, nuclear magnetic resonance (NMR) was used to map the binding site(s) of Hlp to heparan sulfate and identify the nature of the amino acid residues directly involved in this interaction.     

A DNA vaccine against tuberculosis based on the 65 kDa heat-shock protein differentially activates human macrophages and dendritic cells

Background

Application of plasmid DNA for immunization of food-producing animals established new standards of food safety. The addition of foreign products e.g. pDNA into the food chain should be carefully examined to ensure that neither livestock animals nor consumers develop unpredicted or undesirable side-effects.

Methods

A quantitative real-time PCR (QRTPCR) methodology was developed to study the biodistribution and persistence of plasmid DNA vaccine pDNAX (pVAX-Hsp60 TM814) in mice and beef cattle. The linear quantification range and the sensitivity of the method was found to be 10 – 10⁹ copies per reaction (500 ng/gDNA) and 3 copies per reaction, respectively.

Results

Persistence of pDNAX in mice muscle tissue was restricted to injection site and the amount of pDNAX showed delivery formulation dependent (naked pDNA, electroporation, cationic liposome complexes) and mouse age-dependent clearance form injection site but pDNAX was still detectable even after 365 days. The QRTPCR analysis of various muscle tissue samples of vaccinated beef bulls performed 242–292 days after the last revaccination proved that residual pDNAX was found only in the injection site. The highest plasmid levels (up to 290 copies per reaction) were detected in the pDNAX:CDAN/DOPE group similarly to mice model. No pDNA was detected in the samples from distant muscles and draining lymph nodes.

Conclusion

Quantitative real-time PCR (QRTPCR) assay was developed to assess the residual pDNA vaccine pVAX-Hsp60 TM814 in mice and beef cattle. In beef cattle, ultra low residual level of pDNA vaccine was only found at the injection site. According to rough estimation, consumption of muscles from the injection site represents almost an undetectable intake of pDNA (400 fg/g muscle tissue) for consumers. Residual plasmid in native state will hardly be found at measurable level following further meat processing. This study brings supportive data for animal and food safety and hence for further approval of pDNA vaccine field trials.     

Quantification of daunorubicin and daunorubicinol in plasma by capillary electrophoresis

Capillary electrophoresis (CE) with laser-induced fluorescence detection was applied to quantify daunorubicin and daunorubicinol in plasma. Separation was carried out in a 47 cm×50 μm I.D. fused-silica capillary, with a running buffer, pH 5 containing 60 μM spermine and 70% acetonitrile. Sample preparation was done either by protein precipitation with acetonitrile or by liquid–liquid extraction. The assay can be applied in a concentration range from 40 mg/l down to 2 μg/l for daunorubicin and from 1 mg/l to 2 μg/l for daunorubicinol. Precision and accuracy were between 2.9 and 14.5% (n=6) on 1 day and between 1.0 and 14.7% from day to day (n=6) for both analytes. Thus, the CE method enables precise and accurate quantification of daunorubicin and daunorubicinol in small sample volumes over a wide concentration range.     

Conservation priorities for Ethiopian sheep breeds combining threat status,breed merits and contributions to genetic diversity

Prioritizing livestock breeds for conservation needs to incorporate both genetic and non-genetic aspects important for the survival of the breeds. Here, we apply a maximum-utility-strategy to prioritize 14 traditional Ethiopian sheep breeds based on their threat status, contributions to farmer livelihoods (current breed merits) and contributions to genetic diversity. Contributions of the breeds to genetic diversity were quantified using Eding''s marker-estimated kinship approaches. Non-genetic aspects included threats (e.g. low population size, low preferences by farmers) and current merits (economic, ecological and cultural merits). Threat analysis identified eight of the 14 breeds as threatened. Analysis of current merits showed that sub-alpine and arid-lowland breeds contribute most to farmer livelihoods in comparison to other breeds. The highest contribution to the genetic diversity conserved was from the Simien breed. Simien showed high between-breed (low between-breed kinship = 0.04) as well as high within-breed diversity (low within-breed kinship = 0.09 and high H_E = 0.73 and allelic richness = 6.83). We combined the results on threat status, current breed merits and contributions to genetic diversity to produce a ranking of the 14 breeds for conservation purposes. Our results balance the trade-offs between conserving breeds as insurance against future uncertainties and current sustainable utilization. The ranking of breeds provides a basis for conservation strategies for Ethiopian sheep and contributes to a regional or global conservation plan.     

Assessing the contribution of breeds to genetic diversity in conservation schemes

The quantitative assessment of genetic diversity within and between populations is important for decision making in genetic conservation plans. In this paper we define the genetic diversity of a set of populations, S, as the maximum genetic variance that can be obtained in a random mating population that is bred from the set of populations S. First we calculated the relative contribution of populations to a core set of populations in which the overlap of genetic diversity was minimised. This implies that the mean kinship in the core set should be minimal. The above definition of diversity differs from Weitzman diversity in that it attempts to conserve the founder population (and thus minimises the loss of alleles), whereas Weitzman diversity favours the conservation of many inbred lines. The former is preferred in species where inbred lines suffer from inbreeding depression. The application of the method is illustrated by an example involving 45 Dutch poultry breeds. The calculations used were easy to implement and not computer intensive. The method gave a ranking of breeds according to their contributions to genetic diversity. Losses in genetic diversity ranged from 2.1% to 4.5% for different subsets relative to the entire set of breeds, while the loss of founder genome equivalents ranged from 22.9% to 39.3%.