Case studies on the use of biotechnologies and on biosafety provisions in four African countries

This review is based on a study commissioned by the European Commission on the evaluation of scientific, technical and institutional challenges, priorities and bottlenecks for biotechnologies and regional harmonisation of biosafety in Africa. Biotechnology was considered within four domains: agricultural biotechnologies (‘Green’), industrial biotechnologies and biotechnologies for environmental remediation (‘White’), biotechnologies in aquaculture (‘Blue’) and biotechnologies for healthcare (‘Red’). An important consideration was the decline in partnerships between the EU and developing countries because of the original public antipathy to some green biotechnologies, particularly genetically modified organisms (GMOs) and food from GM crops in Europe. The study focus reported here was West Africa (Ghana, Senegal, Mali and Burkina Faso).The overall conclusion was that whereas high-quality research was proceeding in the countries visited, funding is not sustained and there is little evidence of practical application of biotechnology and benefit to farmers and the wider community. Research and development that was being carried out on genetically modified crop varieties was concentrating on improving food security and therefore unlikely to have significant impact on EU markets and consumers. However, there is much non-controversial green biotechnology such as molecular diagnostics for plant and animal disease and marker-assisted selection for breeding that has great potential application. Regarding white biotechnology, it is currently occupying only a very small industrial niche in West Africa, basically in the sole sector of the production of liquid biofuels (i.e., bio-ethanol) from indigenous and locally planted biomass (very often non-food crops). The presence of diffused small-scale fish production is the basis to develop and apply new (Blue) aquaculture technologies and, where the research conditions and the production sector can permit, to increase this type of production and the economy of this depressed areas. However, the problems bound to environmental protection must not be forgotten; priority should be given to monitor the risks of introduction of foreign species. Red biotechnologies potentially bring a vast domain of powerful tools and processes to achieve better human health, most notably improved diagnostics by molecular techniques, better targeting of pathogens and a better knowledge of their sensitivities to drugs to permit better treatment.Biosafety regulatory frameworks had been initiated in several countries, starting with primary biosafety law. However, disparate attitudes to the purpose of biosafety regulation (e.g., fostering informed decision-making versus ‘giving the green-light for a flood of GMOs’) currently prevent a needed consensus for sub-regional harmonisation. To date, most R&D funding has come from North America with some commercial interests from Asia, but African biotechnology workers expressed strong desire for (re-)engagement with interested parties from the European Union. Although in some of the visited countries there are very well qualified personnel in molecular biology and biosafety/regulation, the main message received is that human resources and capacity building in-house are still needed. This could be achieved through home-based courses and capacity-building including funds for post-degree research to motivate and retain trained staff.     

Acetylation and phosphorylation of SRSF2 control cell fate decision in response to cisplatin

SRSF2 is a serine/arginine-rich protein belonging to the family of SR proteins that are crucial regulators of constitutive and alternative pre-mRNA splicing. Although it is well known that phosphorylation inside RS domain controls activity of SR proteins, other post-translational modifications regulating SRSF2 functions have not been described to date. In this study, we provide the first evidence that the acetyltransferase Tip60 acetylates SRSF2 on its lysine 52 residue inside the RNA recognition motif, and promotes its proteasomal degradation. We also demonstrate that the deacetylase HDAC6 counters this acetylation and acts as a positive regulator of SRSF2 protein level. In addition, we show that Tip60 downregulates SRSF2 phosphorylation by inhibiting the nuclear translocation of both SRPK1 and SRPK2 kinases. Finally, we demonstrate that this acetylation/phosphorylation signalling network controls SRSF2 accumulation as well as caspase-8 pre-mRNA splicing in response to cisplatin and determines whether cells undergo apoptosis or G(2)/M cell cycle arrest. Taken together, these results unravel lysine acetylation as a crucial post-translational modification regulating SRSF2 protein level and activity in response to genotoxic stress.     

Renovascular hypertension using a modified two-kidney, one-clip approach in mice is not dependent on the α1 or α2 Na-K-ATPase ouabain-binding site

Endogenous cardiotonic steroids, through their interaction with the ouabain-binding site of the Na-K-ATPase α-subunit, have been implicated in a variety of cardiovascular disease states including hypertension. We have previously shown that ACTH-induced hypertension is abolished in mutant mice expressing ouabain-resistant α1- and α2-subunits. To further evaluate hypertension resistance in these mutant mice, we examined blood pressure changes in a modified model of 2-kidney, 1-clip (2K1C) renovascular hypertension. To reliably generate 2K1C hypertension, we used polyvinyl tubing (inner diameter: ～0.27 mm) to accurately gauge the degree of renal artery stenosis. Using this method, virtually all of the clipped mice became hypertensive and there was no incidence of apparent renal ischemia. By telemetry, in response to renal artery clipping, blood pressure in wild-type mice (α1 ouabain-resistant, α2 ouabain-sensitive) increased from 97 ± 3 to 136 ± 7 mmHg. In α1-resistant, α2-resistant mice, pressure increased from 93 ± 2 to 123 ± 4 mmHg, and in α1-sensitive, α2-resistant mice, blood pressure increased from 95 ± 2 to 139 ± 5 mmHg. Blood pressure changes were equivalent in all three groups. In sham mice, blood pressure did not change (96 ± 1 to 95 ± 2 mmHg). Renin mRNA expression was dramatically elevated in the left vs. the right kidney, and plasma renin concentration was elevated similarly in all genotypes. These data indicate that sensitivity of the α1- or α2-Na-K-ATPase binding site to cardiotonic steroids is not a prerequisite for the development of 2K1C renovascular hypertension. In addition, use of a polyurethane cuff to constrict the renal artery provides a reliable method for producing 2K1C hypertension in mice.     

Acute stress disrupts performance of zebrafish in the cued and spatial memory tests: the utility of fish models to study stress-memory interplay

The zebrafish (Danio rerio) has emerged as a promising model organism for affective or cognitive neuroscience research, and may be useful to study the interplay between memory and anxiety-related states. To assess the effects of acute psychological stress on spatial and cued memory, adult zebrafish were trained in an aquatic plus-maze for 14 days using food bait as a reward. Two ecologically relevant stressors (alarm pheromone or Indian leaf fish exposure) were applied to acutely stress zebrafish immediately prior to the final (testing) trial. Overall, acute single inescapable stress markedly impaired spatial and cued memory in zebrafish plus-maze test, reducing the number of correct arm entries and time spent in the target arm. This observation parallels rodent and clinical literature on memory-impairing effects of acute stress, strongly supporting the utility of zebrafish in neurobehavioral research.     

The kinase VRK1 is required for normal meiotic progression in mammalian oogenesis

The kinase VRK1 has been implicated in mitotic and meiotic progression in invertebrate species, but whether it mediates these events during mammalian gametogenesis is not completely understood. Previous work has demonstrated a role for mammalian VRK1 in proliferation of male spermatogonia, yet whether VRK1 plays a role in meiotic progression, as seen in Drosophila, has not been determined. Here, we have established a mouse strain bearing a gene trap insertion in the VRK1 locus that disrupts Vrk1 expression. In addition to the male proliferation defects, we find that reduction of VRK1 activity causes a delay in meiotic progression during oogenesis, results in the presence of lagging chromosomes during formation of the metaphase plate, and ultimately leads to the failure of oocytes to be fertilized. The activity of at least one phosphorylation substrate of VRK1, p53, is not required for these defects. These results are consistent with previously defined functions of VRK1 in meiotic progression in Drosophila oogenesis, and indicate a conserved role for VRK1 in coordinating proper chromosomal configuration in female meiosis.     

Structural basis of ligand specificity in the human pentraxins, C-reactive protein and serum amyloid P component

The normal physiological roles of the phylogenetically conserved human plasma proteins C-reactive protein (CRP) and serum amyloid P component (SAP) are not known. Novel drugs targeting their ligand specificities are in clinical development as both proteins have significant pathophysiological effects, SAP in promoting amyloidosis and CRP in exacerbating ischemic injury. Both proteins bind to phosphoethanolamine and we show here that, under physiological conditions, phosphoethanolamine is bound with higher affinity by human SAP than by human CRP. An explanation is provided by X-ray crystal structures that show SAP residue Tyr74 allowing additional hydrophobic protein-ligand interactions compared with the equivalent Thr76 of CRP. Docking simulations show many more low energy positions for phosphoethanolamine bound by CRP than by SAP and are consistent with the crystallographic and functional binding results. These fundamental observations on structure-activity relationships will aid the design of improved pentraxin targeting drugs.     

Holocarboxylase synthetase is a chromatin protein and interacts directly with histone H3 to mediate biotinylation of K9 and K18

Holocarboxylase synthetase (HCS) mediates the binding of biotin to lysine (K) residues in histones H2A, H3 and H4; HCS knockdown disturbs gene regulation and decreases stress resistance and lifespan in eukaryotes. We tested the hypothesis that HCS interacts physically with histone H3 for subsequent biotinylation. Co-immunoprecipitation experiments were conducted and provided evidence that HCS co-localizes with histone H3 in human cells; physical interactions between HCS and H3 were confirmed using limited proteolysis assays. Yeast two-hybrid (Y2H) studies revealed that the N-terminal and C-terminal domains in HCS participate in H3 binding. Recombinant human HCS was produced and exhibited biological activity, as evidenced by biotinylation of its known substrate, recombinant p67. Recombinant histone H3.2 and synthetic H3-based peptides were also good targets for biotinylation by recombinant HCS (rHCS) in vitro, based on tracing histone-bound biotin with [³H]biotin, streptavidin and anti-biotin antibody. Biotinylation site-specific antibodies were generated and revealed that both K9 and K18 in H3 were biotinylated by HCS. Collectively, these studies provide conclusive evidence that HCS interacts directly with histone H3, causing biotinylation of K9 and K18. We speculate that the targeting of HCS to distinct regions in human chromatin is mediated by DNA sequence, biotin, RNA, epigenetic marks or chromatin proteins.     

Absence of PmrAB-mediated phosphoethanolamine modifications of Citrobacter rodentium lipopolysaccharide affects outer membrane integrity

The PmrAB two-component system of enterobacteria regulates a number of genes whose protein products modify lipopolysaccharide (LPS). The LPS is modified during transport to the bacterial outer membrane (OM). A subset of PmrAB-mediated LPS modifications consists of the addition of phosphoethanolamine (pEtN) to lipid A by PmrC and to the core by CptA. In Salmonella enterica, pEtN modifications have been associated with resistance to polymyxin B and to excess iron. To investigate putative functions of pEtN modifications in Citrobacter rodentium, ΔpmrAB, ΔpmrC, ΔcptA, and ΔpmrC ΔcptA deletion mutants were constructed. Compared to the wild type, most mutant strains were found to be more susceptible to antibiotics that must diffuse across the LPS layer of the OM. All mutant strains also showed increased influx rates of ethidium dye across their OM, suggesting that PmrAB-regulated pEtN modifications affect OM permeability. This was confirmed by increased partitioning of the fluorescent dye 1-N-phenylnaphthylamine (NPN) into the OM phospholipid layer of the mutant strains. In addition, substantial release of periplasmic β-lactamase was observed for the ΔpmrAB and ΔpmrC ΔcptA strains, indicating a loss of OM integrity. This study attributes a new role for PmrAB-mediated pEtN LPS modifications in the maintenance of C. rodentium OM integrity.