Isolation and stability of histidine-tagged proteins produced in plants via potyvirus gene vectors

A system for the expression and purification of histidine-tagged proteins from plants has been developed using a tobacco etch potyvirus (TEV)-derived gene vectors. The vectors offered a convenient polylinker and a choice of histidine tagging at the recombinant proteins' N or C termini. These vectors were utilized for expression of proteins encoded by beet yellows closterovirus (BYV). Approximately 4 micrograms/g of 20-kDa BYV protein was readily isolated from plants systemically infected by hybrid TEV. In contrast, only minute quantities of 22-kDa BYV capsid protein (CP) histidine-tagged at its N or C terminus could be purified. Rapid degradation of the recombinant CP has been implicated in its failure to accumulate in infected plants. Fusion with TEV HC-Pro stabilized the histidine-tagged BYV CP and facilitated purification of the fusion product from infected plants. This same fusion approach was successfully used with the 24-kDa minor BYV CP. The recombinant proteins were recognized by histidine-tag-specific monoclonal antibody in immunoblot analysis. These results demonstrate the utility of a designed series of TEV vectors for expression, detection, and purification of the recombinant proteins and suggest that intrinsic protein stability is a major factor in a recovery of recombinant proteins from plants.     

Identification of an insulin-responsive, slow endocytic recycling mechanism in Chinese hamster ovary cells

In adipocytes, the insulin-regulated aminopeptidase (IRAP) is trafficked through the same insulin-regulated recycling pathway as the GLUT4 glucose transporter. We find that a chimera, containing the cytoplasmic domain of IRAP fused to transmembrane and extracellular domains of the transferrin receptor, is slowly recycled and rapidly internalized in Chinese hamster ovary cells. Morphological studies indicate that the chimera is slowly trafficked through the general endosomal recycling compartment rather than being sorted to a specialized recycling pathway. A chimera in which a di-leucine sequence within the cytoplasmic domain of IRAP has been mutated to alanines is rapidly internalized and rapidly recycled, indicating that this di-leucine is required for the slow recycling but not for the rapid internalization. Insulin stimulates a 2-3-fold increase in the recycling of the chimera and only a 1.2-fold increase in the recycling of the transferrin receptor. The effect of insulin on the recycling of the chimera is blocked by wortmannin, a phosphatidylinositol 3'-kinase inhibitor. GTPgammaS (guanosine 5'-3-O-(thio)triphosphate) increases the recycling of the chimera by 50% but has no effect on the recycling of the transferrin receptor. In these studies, we have identified in Chinese hamster ovary cells a novel, slow endocytic recycling mechanism that is regulated by insulin.     

A study on the orientation effects in polyethylene in the light of crystalline texture

Special sample types established and characterised in Parts 1 and 2 [1,2] were examined systematically under different modes of external deformation at constant temperatures. The structural changes recorded by means of wide- and low-angle X-ray patterns fully substantiated the bodily rotation of the otherwise unaltered crystallites in accordance with the interlamellar slip mechanism postulated in Parts 1 and 2, both in tension and under two modes of compression. Dimensional changes, however, were significantly in excess of those expected from the rotation of crystallites, but were uniquely correlated with this relation in the different samples under different conditions of deformation. This points to a unique structural and mechanistic interrelation between the crystallites and more compliant amorphous regions. As the X-ray long periods were unaffected by the deformation, the more extensible material cannot be identical to that associated with the lamellar periodicity, a fact which points to a previously unsuspected superlamellar regularity in the texture. A simple structural suggestion is made.     

Intracondylar knee joint prosthesis with rotation capacity- Endo model

The St. Georg total knee prosthesis has been modified to incorporate axial rotation of the tibial component. The kinematics approximate more those of the normal knee and the stress imposed at the bone-cement interface are attenuated, thus reducing the risk of mechanical failure. The construction and laboratory testing are described.     

Lymphography in testicular tumors (author's transl)

Results of lymphography in 101 cases of testicular tumor are described. Secondaries could be shown in 43% of seminomas and 46% of teratomas. Examples show the criteria of lymphographic diagnosis of secondaries. Lymphography permits a) more exact classification of stages b) better planning and control of treatment and c) prognosis.     

Formation and properties of single texture polyethylene produced by rolling

The formation of single textured low density polyethylene by unidirectional rolling has been investigated. It was found to be associated with the shear of the sample during annealing, which is a reversal of the shear imparted by the rolling process. Single texture could be produced from double texture by compressing it obliquely in such a way as to simulate this shear. It is concluded that the material probably contains another component besides the single orientation of lamellae which would need to be taken into account when using it as a model for the study of lamellar properties.     

Superconductors with crystalline-field split impurities: Upper critical field and specific heat

We continue the investigation of crystalline-field effects on the properties of superconductors containing rare-earth impurities. Attention is focused on impurities with a nonmagnetic ground state. We calculate the upper critical fieldH _{c 2} and the jump of the specific heat at the phase transition.