Developing efficient strategies for the generation of transgenic cattle which produce biopharmaceuticals in milk

At the close of the millennium, a revolution in the treatment of disease is taking shape due to the emergence of new therapies based on human recombinant proteins. The ever-growing demand for such pharmaceutical proteins is an important driving force for the development of safe and large-scale production platforms. Since the efficacy of a human protein is generally dependent on both its amino acid composition as well as various post-translational modifications, many recombinant human proteins can only be obtained in a biologically active conformation when produced in mammalian cells. Hence, mammalian cell culture systems are often used for expression. However, this approach is generally known for limited production capacity and high costs. In contrast, the production of (human) recombinant proteins in milk of transgenic farm animals, particularly cattle, presents a safe alternative without the constraint of limited protein output. Moreover, compared to cell culture, production in milk is very cost-effective. Although transgenic farm animal technology was still in its infancy a decade ago, today it is on the verge of fulfilling its potential of providing therapeutic proteins that can not be produced otherwise in sufficient quantities or at affordable cost. Since 1989, we have been at the forefront of this development, as illustrated by the birth of Herman, the first transgenic bull. In this communication, we will present an overview of approaches we have taken over the years to generate transgenic founder animals and production herds. Our initial strategies were based on microinjection; at the time the only viable option to generate transgenic cattle. Recently, we have adopted a more powerful approach founded on the application of nuclear transfer. As we will illustrate, this strategy presents a breakthrough in the overall efficiency of generating transgenic animals, product consistency, and time of product development.     

Veno-arterial extracorporeal membrane oxygenation in addition to primary PCI in patients presenting with ST-elevation myocardial infarction

Introduction

Primary percutaneous coronary intervention (pPCI) in ST-elevation myocardial infarction (STEMI) can cause great haemodynamic instability. Veno-arterial extracorporeal membrane oxygenation (VA-ECMO) can provide haemodynamic support in patients with STEMI but data on outcome and complications are scarce.

Methods

An in-hospital registry was conducted enrolling all patients receiving VA-ECMO. Patients were analysed for medical history, mortality, neurological outcome, complications and coronary artery disease.

Results

Between 2011 and 2016, 12 patients underwent pPCI for STEMI and received VA-ECMO for haemodynamic support. The majority of the patients were male (10/12) with a median age of 63 (47–75) years and 4 of the 12 patients had a history of coronary artery disease. A cardiac arrest was witnessed in 11 patients. The left coronary artery was compromised in 8 patients and 4 had right coronary artery disease. All patients were in Killip class IV. Survival to discharge was 67% (8/12), 1?year survival was 42% (5/12), 2 patients have not yet reached the 1?year survival point but are still alive and 1 patient died within a year after discharge. All-cause mortality was 42% (5/12) of which mortality on ECMO was 33% (4/12). Patient-related complications occurred in 6 of the 12 patients: 1 patient suffered major neurological impairment, 2 patients suffered haemorrhage at the cannula site, 2 patients had limb ischaemia and 1 patient had a haemorrhage elsewhere. There were no VA-ECMO hardware malfunctions.

Conclusion

VA-ECMO in pPCI for STEMI has a high survival rate and neurological outcome is good, even when the patient is admitted with a cardiac arrest.

     

Clonal variants of differentiated P19 embryonal carcinoma cells exhibit epidermal growth factor receptor kinase activity

Differentiated clonal cell lines were isolated from pluripotent P19 embryonal carcinoma (EC) cells treated as aggregates with retinoic acid. Two were characterized in detail. The lines differ in morphology, proliferation rate, the production of plasminogen activator, and in their mitogenic response to insulin but both produce extracellular matrix proteins and can be serially passaged over extended periods, in contrast to differentiated derivatives of many other EC lines. Further, both lines have receptors for and respond mitogenically to epidermal growth factor (EGF). Endogenous phosphorylation of several proteins, including the EGF receptor (150 kDa) and a 38-kDa protein, is induced by EGF in membranes isolated from these cells. Preincubation of membranes with EGF renders them able to catalyze phosphorylation of tyrosine residues in exogenously added peptide substrates. High voltage electrophoresis confirmed the tyrosine specificity of the phosphorylation on the 150- and 38-kDa bands. By contrast, similar experiments in undifferentiated cells showed that intact P19 EC neither bind nor respond to EGF mitogenically and EGF induces no changes in phosphorylation in isolated membranes.     

Phylogenetic distribution in the genus Mus of t-complex-specific DNA and protein markers: inferences on the origin of t-haplotypes

We have examined the phylogenetic distribution of two t-specific markers among representatives of various taxa belonging to the genus Mus. The centromeric TCP-1a marker (a testicular protein variant specific for all t-haplotypes so far studied) has also been apparently detected in several non-t representatives of the Mus IVA, Mus IVB, and probably M. cervicolor species. By contrast, a t-specific restriction- fragment-length polymorphism allele (RFLP) of the telomeric alpha- globin pseudogene DNA marker alpha-psi-4 was found only in animals belonging to the M. musculus-complex species either bearing genuine t- haplotypes or, like the M. m. bactrianus specimen studied here, likely to do so. This t-specific alpha-psi-4 RFLP allele was found to be as divergent from the RFLP alleles of the latter, non-t, taxonomical groups as it is from Mus 4A, Mus 4B, or M. spretus ones. These results suggest the presence of t-haplotypes and of t-specific markers in populations other than those belonging to the M. m. domesticus and M. m. musculus subspecies, implying a possible origin for t-haplotypes prior to the radiation of the most recent offshoot of the Mus genus (i.e., the spretus/domesticus divergence), some 1-3 Myr ago.      

The contractile basis of amoeboid movement: V. The control of gelation, solation, and contraction in extracts from dictyostelium discoideum

Motile extracts have been prepared from Dictyostelium discoideum by homogenization and differential centrifugation at 4 degrees C in a stabilization solution (60). These extracts gelled on warming to 25 degrees Celsius and contracted in response to micromolar Ca++ or a pH in excess of 7.0. Optimal gelation occurred in a solution containing 2.5 mM ethylene glycol-bis (β-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA), 2.5 mM piperazine-N-N'-bis [2-ethane sulfonic acid] (PIPES), 1 mM MgC1(2), 1 mM ATP, and 20 mM KCI at ph 7.0 (relaxation solution), while micromolar levels of Ca++ inhibited gelation. Conditions that solated the gel elicited contraction of extracts containing myosin. This was true regardless of whether chemical (micromolar Ca++, pH >7.0, cytochalasin B, elevated concentrations of KCI, MgC1(2), and sucrose) or physical (pressure, mechanical stress, and cold) means were used to induce solation. Myosin was definitely required for contraction. During Ca++-or pH-elicited contraction: (a) actin, myosin, and a 95,000-dalton polypeptide were concentrated in the contracted extract; (b) the gelation activity was recovered in the material sqeezed out the contracting extract;(c) electron microscopy demonstrated that the number of free, recognizable F-actin filaments increased; (d) the actomyosin MgATPase activity was stimulated by 4- to 10-fold. In the absense of myosin the Dictyostelium extract did not contract, while gelation proceeded normally. During solation of the gel in the absense of myosin: (a) electron microscopy demonstrated that the number of free, recognizable F- actin filaments increased; (b) solation-dependent contraction of the extract and the Ca++-stimulated MgATPase activity were reconstituted by adding puried Dictyostelium myosin. Actin purified from the Dictyostelium extract did not gel (at 2 mg/ml), while low concentrations of actin (0.7-2 mg/ml) that contained several contaminating components underwent rapid Ca++ regulated gelation. These results indicated : (a) gelation in Dictyostelium extracts involves a specific Ca++-sensitive interaction between actin and several other components; (b) myosin is an absolute requirement for contraction of the extract; (c) actin-myosin interactions capable of producing force for movement are prevented in the gel, while solation of the gel by either physical or chemical means results in the release of F-actin capable of interaction with myosin and subsequent contraction. The effectiveness of physical agents in producting contraction suggests that the regulation of contraction by the gel is structural in nature.     

Continental estimates of forest cover and forest cover changes in the dry ecosystems of Africa between 1990 and 2000

Aim

This study provides regional estimates of forest cover in dry African ecoregions and the changes in forest cover that occurred there between 1990 and 2000, using a systematic sample of medium‐resolution satellite imagery which was processed consistently across the continent.

Location

The study area corresponds to the dry forests and woodlands of Africa between the humid forests and the semi‐arid regions. This area covers the Sudanian and Zambezian ecoregions.

Methods

A systematic sample of 1600 Landsat satellite imagery subsets, each 20 km × 20 km in size, were analysed for two reference years: 1990 and 2000. At each sample site and for both years, dense tree cover, open tree cover, other wooded land and other vegetation cover were identified from the analysis of satellite imagery, which comprised multidate segmentation and automatic classification steps followed by visual control by national forestry experts.

Results

Land cover and land‐cover changes were estimated at continental and ecoregion scales and compared with existing pan‐continental, regional and local studies. The overall accuracy of our land‐cover maps was estimated at 87%. Between 1990 and 2000, 3.3 million hectares (Mha) of dense tree cover, 5.8 Mha of open tree cover and 8.9 Mha of other wooded land were lost, with a further 3.9 Mha degraded from dense to open tree cover. These results are substantially lower than the 34 Mha of forest loss reported in the FAO's 2010 Global Forest Resources Assessment for the same period and area.

Main conclusions

Our method generates the first consistent and robust estimates of forest cover and change in dry Africa with known statistical precision at continental and ecoregion scales. These results reduce the uncertainty regarding vegetation cover and its dynamics in these previously poorly studied ecosystems and provide crucial information for both science and environmental policies.     

Cysteinyl-leukotriene receptors in pulmonary vessels.

Two categories of cysteinyl-leukotrienes have been proposed, namely, CysLT1 and CysLT2. These receptors are found not only on the vascular smooth muscle but also on the endothelium. Activation of the receptor(s) on vascular smooth muscle provokes contraction whereas activation of the receptors on the endothelium produces contraction and/or relaxation. These endothelium dependent effects are due to the release of both contractile and relaxant factors derived from the endothelium. While factors derived from either the cyclooxygenase or nitric oxide pathways are involved, in some vascular preparations other mediators such as endothelin may be involved. However, in isolated human pulmonary vascular preparations, this appears not to be the case and presently the nature and origin of the contractile factor remains to be established.     

The Origins of Hypertrophic Cardiomyopathy–Causing Mutations in Two South African Subpopulations: A Unique Profile of Both Independent and Founder Events

Hypertrophic cardiomyopathy (HCM) is an autosomal dominantly inherited disease of the cardiac sarcomere, caused by numerous mutations in genes encoding protein components of this structure. Mutation carriers are at risk of sudden cardiac death, mostly as adolescents or young adults. The reproductive disadvantage incurred may explain both the global occurrence of diverse independent HCM-associated mutations and the rare reports of founder effects within populations. We have investigated whether this holds true for two South African subpopulations, one of mixed ancestry and one of northern-European descent. Previously, we had detected three novel mutations-Ala797Thr in the beta-myosin heavy-chain gene (betaMHC), Arg92Trp in the cardiac troponin T gene (cTnT), and Arg645His in the myosin-binding protein C gene (MyBPC)-and two documented betaMHC mutations (Arg403Trp and Arg249Gln). Here we report three additional novel mutations-Gln499Lys in betaMHC and Val896Met and Deltac756 in MyBPC-and the documented betaMHC Arg719Gln mutation. Seven of the nine HCM-causing mutations arose independently; no conclusions can be drawn for the remaining two. However, the betaMHC Arg403Trp and Ala797Thr and cTnT Arg92Trp mutations were detected in another one, eight, and four probands, respectively, and haplotype analysis in families carrying these recurring mutations inferred their origin from three common ancestors. The milder phenotype of the betaMHC mutations may account for the presence of these founder effects, whereas population dynamics alone may have overridden the reproductive disadvantage incurred by the more lethal, cTnT Arg92Trp mutation.