Hemophilia A is one of the major inherited bleeding disorders caused by a deficiency or abnormality in coagulation factor VIII (FVIII). Hemophiliacs have been treated with whole plasma or purified FVIII concentrates. The risk of transmitting blood-borne viruses and the cost of highly purified FVIII are the major factors that restrict prophylaxis in hemophilia therapy. One of the challenges created by the biotechnology revolution is the development of methods for the economical production of highly purified proteins in large scales. Recent developments indicate that manipulating milk composition using transgenesis has focused mainly on the mammary gland as a bioreactor to produce pharmaceuticals. In the present study, a hybrid gene containing bovine -lactalbumin and human FVIII cDNA was constructed for microinjection into the pronuclei of newly fertilized mouse eggs. The LA-hFVIII hybrid gene was confirmed to be successfully integrated and stably germ-line transmitted in 12 (seven females/five males) lines. Western-blot analysis of milk samples obtained from eight of the transgenic founders and F1 offspring indicated that the recombinant hFVIII was secreted into the milk of the transgenic mice. The concentrations of rFVIII ranged from 7.0 to 50.2g/ml, over 35–200-fold higher than that in normal human plasma. Up to 13.4U/ml of rFVIII was detected in an assay in which rFVIII restored normal clotting activity to FVIII-deficient human plasma. 相似文献
We all take our visual systems for granted, and often assume we are always ‘near perfect’ observers. This is not the case; expert visual recognition is complex and can be error prone. Starting with examples that define the problem I will explore some of the issues of recognition where expert judgements are required.
In addition to ‘expert’ effects, there are a number of cognitive factors that can severely affect performance, including fatigue, boredom, recency effects, positivity bias and short-term memory effects. Experimental evidence of the impact of these on performance are presented and discussed.
The specimen identifications generated by experts are useful not only to ecology, but to researchers developing systems for automatic labelling of marine plankton. Comparisons of performance are presented, where human experts have been pitted against machines to label plankton. Consensus of opinion is important in reducing errors, yet it is the norm for experts to operate alone. The shortcomings of man and machines engaged in plankton recognition are reviewed and the future of automation is assessed. 相似文献
Hemophilia A, a life-threatening bleeding disorder, is caused by deficiency of factor VIII (FVIII). Replacement therapy using rFVIII is the first line therapy for hemophilia A. However, 15-30% of patients develop neutralizing antibody, mainly against the C2, A3 and A2 domains. It has been reported that PS-FVIII complex reduced total and neutralizing anti-rFVIII antibody titers in hemophilia A murine models. Here, we developed FVIII-containing cochleate cylinders, utilizing PS-Ca2+ interactions and characterized these particles for optimal in vivo properties using biophysical and biochemical techniques. Approximately 75% of the protein was associated with cochleate cylinders. Sandwich ELISA, acrylamide quenching and enzymatic digestion studies established that rFVIII was shielded from the bulk aqueous phase by the lipidic structures, possibly leading to improved in vivo stability. Freeze-thawing and rate-limiting diffusion studies revealed that small cochleate cylinders with a particle size of 500 nm or less could be generated. The release kinetics and in vivo experiments suggested that there is slow and sustained release of FVIII from the complex upon systemic exposure. In vivo studies using tail clip method indicated that FVIII-cochleate complex is effective and protects hemophilic mice from bleeding. Based on these studies, we speculate that the molecular interaction between FVIII and PS may provide a basis for the design of novel FVIII lipidic structures for delivery applications. 相似文献
The immobilization of proteins to surfaces is an active area of research due to strong interest in protein-based sensors. Here, we describe a novel method for immobilizing ligand proteins onto Biacore sensor chips using the transpeptidase activity of Staphylococcus aureus sortase A (SrtA). This method provides a robust and gentle approach for the site-directed, covalent coupling of proteins to biosensor chips. Notably, the high specificity of the sortase allows immobilization of proteins from less than pure protein samples allowing short cuts in protein purification protocols. 相似文献
Human factor IX is synthesized in the liver and secreted in the blood, where it participates in a group of reactions involving
coagulation factors and proteins that permit sanguinary coagulation. In this work two lines of transgenic mice were developed
to express the FIX gene in the mammalian glands under control of milk β-casein promoter. The founding females secreted the FIX in their milk
(3% total soluble protein). The stable integration of transgene was confirmed by southern blot analysis. The presence of the
FIX recombinant protein in the milk of transgenic females was confirmed by western blot and the clotting activity was revealed
in blood-clotting assays. The coagulation activity in human blood treated with recombinant FIX increased while the time of
coagulation decreased. Our results confirm the production of a large amount of recombinant biologically active FIX in the
mammary gland of transgenic mice. 相似文献
The Protein Data Bank (PDB) is a widely used biological databaseof macromolecular structures with a long history. This historyis treated as lessons learned and is used to highlight whatare believed to be the best practices important to developersof biological databases today. While the focus is on data quality,data representation and the information technology to supportthese data, the non-data and technology issues cannot be ignored.The role of the human factor in the form of users, collaborators,scientific society and ad hoc committees is also included. 相似文献
There is interest in the global community on how fire regimes are changing as a function of changing demographics and climate. The ground-based data to monitor such trends in fire activity are inadequate at the global scale. Satellite observations provide a basis for such a monitoring system. In this study, a set of metrics were developed from 6 years of MODIS active fire data. The metrics were grouped into eight classes representing three axes of fire activity: density, season duration and interannual variability. These groups were compared with biophysical and human explanatory variables on a global scale. We found that more than 30% of the land surface has a significant fire frequency. The most extensive fire class exhibited high fire density, low duration and high variability and was found in boreal and tropical wet and dry environments. A high association was found between population distribution and fire persistence. Low GDP km−2 was associated with fire classes with high interannual variability and low seasonal duration. In areas with more economic resources, fires tend to be more regular and last longer. High fire duration and low interannual variability were associated with croplands, but often with low fire density. The study was constrained by the limited length of satellite data record but is a first step toward developing a comprehensive global assessment of fire regimes. However, more attention is needed by the global observing systems to provide the underpinning socio-economic observations to better quantify and analyze the human characteristics of fire regimes. 相似文献