Effect of transgenesis on reproductive traits of rabbit males

The influence of foreign transgene integration on the reproductive capabilities of rabbit males is not known. Therefore, we analyzed their ejaculate characteristics, reproductive capabilities, occurrence of pathological spermatozoa and histological structure of the testis. We have generated transgenic rabbits by microinjection of WAP-hFVIII gene into pronucleus of fertilized egg. We observed that the libido, volume and pH value of the ejaculate did not differ significantly between transgenic and non-transgenic male lines. The motility, concentration, osmolarity, thermoresistant test of spermatozoa (at 1 or 6 h) and the percentage of alive spermatozoa were significantly different (p < 0.001) among transgenic and non-transgenic males. No significant differences were found between transgenic and non-transgenic male lines in the occurrence of pathological spermatozoa and histology of the testis. The ability of spermatozoa from transgenic and non-transgenic males to fertilize eggs was ranged within 96 and 100%; while the yield of transgenic embryos ranged from 43 to 57%. Our results show that mammary gland specific over-expression mWAP-hFVIII gene construct does not affect reproductive traits of transgenic rabbit males.     

Recombinant expression systems: the obstacle to helminth vaccines?

The need for alternative ways to control helminth parasites has in recent years led to a boost in vaccination experiments with recombinant antigens. Despite the use of different expression systems, only a few recombinants induced high levels of protection against helminths. This is often attributed to the limitations of the current expression systems. Therefore, the need for new systems that can modify and glycosylate the expressed antigens has been advocated. However, analysis of over 100 published vaccine trials with recombinant helminth antigens indicates that it is often not known whether the native parasite antigen itself can induce protection or, if it does, which epitopes are important. This information is vital for a well-thought-out strategy for recombinant production. So, in addition to testing more expression systems, it should be considered that prior evaluation and characterization of the native antigens might help the development of recombinant vaccines against helminths in the long term.     

Recombinant production of <Emphasis Type="Italic">Streptococcus equisimilis</Emphasis> streptokinase by <Emphasis Type="Italic">Streptomyces lividans</Emphasis>

Background  

Streptokinase (SK) is a potent plasminogen activator with widespread clinical use as a thrombolytic agent. It is naturally secreted by several strains of beta-haemolytic streptococci. The low yields obtained in SK production, lack of developed gene transfer methodology and the pathogenesis of its natural host have been the principal reasons to search for a recombinant source for this important therapeutic protein. We report here the expression and secretion of SK by the Gram-positive bacterium Streptomyces lividans. The structural gene encoding SK was fused to the Streptomyces venezuelae CBS762.70 subtilisin inhibitor (vsi) signal sequence or to the Streptomyces lividans xylanase C (xlnC) signal sequence. The native Vsi protein is translocated via the Sec pathway while the native XlnC protein uses the twin-arginine translocation (Tat) pathway.     

Tuning of Pectin Methylesterification: PECTIN METHYLESTERASE INHIBITOR 7 MODULATES THE PROCESSIVE ACTIVITY OF CO-EXPRESSED PECTIN METHYLESTERASE 3 IN A pH-DEPENDENT MANNER*

Pectin methylesterases (PMEs) catalyze the demethylesterification of homogalacturonan domains of pectin in plant cell walls and are regulated by endogenous pectin methylesterase inhibitors (PMEIs). In Arabidopsis dark-grown hypocotyls, one PME (AtPME3) and one PMEI (AtPMEI7) were identified as potential interacting proteins. Using RT-quantitative PCR analysis and gene promoter::GUS fusions, we first showed that AtPME3 and AtPMEI7 genes had overlapping patterns of expression in etiolated hypocotyls. The two proteins were identified in hypocotyl cell wall extracts by proteomics. To investigate the potential interaction between AtPME3 and AtPMEI7, both proteins were expressed in a heterologous system and purified by affinity chromatography. The activity of recombinant AtPME3 was characterized on homogalacturonans (HGs) with distinct degrees/patterns of methylesterification. AtPME3 showed the highest activity at pH 7.5 on HG substrates with a degree of methylesterification between 60 and 80% and a random distribution of methyl esters. On the best HG substrate, AtPME3 generates long non-methylesterified stretches and leaves short highly methylesterified zones, indicating that it acts as a processive enzyme. The recombinant AtPMEI7 and AtPME3 interaction reduces the level of demethylesterification of the HG substrate but does not inhibit the processivity of the enzyme. These data suggest that the AtPME3·AtPMEI7 complex is not covalently linked and could, depending on the pH, be alternately formed and dissociated. Docking analysis indicated that the inhibition of AtPME3 could occur via the interaction of AtPMEI7 with a PME ligand-binding cleft structure. All of these data indicate that AtPME3 and AtPMEI7 could be partners involved in the fine tuning of HG methylesterification during plant development.     

Drivers of temporal changes in temperate forest plant diversity vary across spatial scales

Global biodiversity is affected by numerous environmental drivers. Yet, the extent to which global environmental changes contribute to changes in local diversity is poorly understood. We investigated biodiversity changes in a meta‐analysis of 39 resurvey studies in European temperate forests (3988 vegetation records in total, 17–75 years between the two surveys) by assessing the importance of (i) coarse‐resolution (i.e., among sites) vs. fine‐resolution (i.e., within sites) environmental differences and (ii) changing environmental conditions between surveys. Our results clarify the mechanisms underlying the direction and magnitude of local‐scale biodiversity changes. While not detecting any net local diversity loss, we observed considerable among‐site variation, partly explained by temporal changes in light availability (a local driver) and density of large herbivores (a regional driver). Furthermore, strong evidence was found that presurvey levels of nitrogen deposition determined subsequent diversity changes. We conclude that models forecasting future biodiversity changes should consider coarse‐resolution environmental changes, account for differences in baseline environmental conditions and for local changes in fine‐resolution environmental conditions.     

Synthesis of novel 5-amino-thiazolo[4,5-d]pyrimidines as E. coli and S. aureus SecA inhibitors

An efficient synthesis of a library of 5-amino-thiazolo[4,5-d]pyrimidines is reported. Regioselective displacements of chlorines, as well as regioselective diazotation reactions are described, which allow the introduction of structural diversity on the scaffold by consecutive reactions. Screening of this focused library led to the discovery of SecA inhibitors from Escherichia coli and Staphylococcus aureus.     

Mild detritylation of nucleic acid hydroxyl groups by warming up

It is challenging to effectively deprotect hydroxyl groups of acid-or-base sensitive bio-macromolecules without causing even minor defects and compromising high quality of final products. We report here a mild detritylation strategy in mildly acidic buffers to remove the DMTr protection from the 5'-hydroxyl groups of synthetic nucleic acids. The DMTr-groups can be easily and effectively removed at pH 4.5 or 5.0 with slight warming up (40 °C), offering virtually quantitative deprotection. This warming-up strategy is particularly useful for deprotection of the modified nucleic acids that are sensitive to the conventional acid deprotection. As a first step towards our long-term goal of synthesizing defect-free nucleic acids, our novel and simple strategy further increases the quality of synthetic nucleic acids.