Chemogenetic ON and OFF switches for RNA virus replication.

Therapeutic application of RNA viruses as oncolytic agents or gene vectors requires a tight control of virus activity if toxicity is a concern. Here we present a regulator switch for RNA viruses using a conditional protease approach, in which the function of at least one viral protein essential for transcription and replication is linked to autocatalytical, exogenous human immunodeficiency virus (HIV) protease activity. Virus activity can be en- or disabled by various HIV protease inhibitors. Incorporating the HIV protease dimer in the genome of vesicular stomatitis virus (VSV) into the open reading frame of either the P- or L-protein resulted in an ON switch. Here, virus activity depends on co-application of protease inhibitor in a dose-dependent manner. Conversely, an N-terminal VSV polymerase tag with the HIV protease dimer constitutes an OFF switch, as application of protease inhibitor stops virus activity. This technology may also be applicable to other potentially therapeutic RNA viruses.

An inherited bleeding disorder linked to a defective interaction between ADP and its receptor on platelets. Its influence on glycoprotein IIb-IIIa complex function.

Much discussion has concerned the central role of ADP in platelet aggregation. We now describe a patient (M.L.) with an inherited bleeding disorder whose specific feature is that ADP induces a limited and rapidly reversible platelet aggregation even at high doses. Platelet shape change and other hemostatic parameters were unmodified. A receptor defect was indicated, for, while epinephrine normally lowered cAMP levels of PGE1-treated (M.L.) platelets, ADP was without effect. The binding of [3H]2-methylthio-ADP decreased from 836 +/- 126 molecules/platelet for normals to 30 +/- 17 molecules/platelet for the patient. Flow cytometry confirmed that ADP induced a much lower fibrinogen binding to (M.L.) platelets. Nonetheless, the binding in whole blood of activation-dependent monoclonal antibodies showed that some activation of GP IIb-IIIa complexes by ADP was occurring. Platelets of a patient with type I Glanzmann's thrombasthenia bound [3H]2-methylthio-ADP and responded normally to ADP in the presence of PGE1. Electron microscopy showed that ADP-induced aggregates of (M. L.) platelets were composed of loosely bound shape-changed platelets with few contact points. Thus this receptor defect has a direct influence on the capacity of platelets to bind to each other in response to ADP.

Protein synthesis and storage in human platelets: a defective storage of fibrinogen in platelets in Glanzmann's thrombasthenia.

In vivo metabolic labelling experiments were performed to investigate the ability of human platelets to synthesize and store fibrinogen and thrombospondin. Newly synthesized proteins were analyzed by SDS-polyacrylamide gel electrophoresis. Results were compared with those obtained for the platelets of a patient with Glanzmann's thrombasthenia where endogenous fibrinogen levels were severely reduced. Normal human platelets were able to synthesize the different subunits of fibrinogen and thrombospondin and to assemble them into native fibrinogen and thrombospondin molecules. This synthesis was inhibited by cycloheximide. Synthesis of both fibrinogen and thrombospondin was observed in the platelets of the Glanzmann's thrombasthenia patient. However, radiolabelled fibrinogen was no longer detected after an 18-h non-radioactive chase, although it was retained in the control platelets. Neosynthesized thrombospondin of the patient was normally preserved during the same chase period. When the fate of the radioactive fibrinogen was studied, it was found to be degraded in Glanzmann's thrombasthenia platelets to the same extent as neosynthesized cytoplasmic proteins, whereas in control platelets less degradation had occurred. We conclude that human platelets maintain a residual capacity to synthesize fibrinogen and that its deficiency in Glanzmann's thrombasthenia results from a storage abnormality and not from a synthesis defect.

Studies in polycythemia after kidney transplantation.

Mononuclear cells from peripheral blood of six patients with polycythemia after kidney transplantation were studied in methyl cellulose cultures using fetal calf serum with and without adding erythropoietin. Parallel tests were run with "synthetic medium". The data obtained were compared to those of patients with polycythemia vera and normal subjects. The erythropoietin independent proliferation in synthetic medium typical for polycythemia vera could not be found in patients with renal grafts. However, in the preparations with fetal calf serum without erythropoietin, a formation of erythroid colonies could be detected suggesting an increased sensitivity or proliferation of erythropoietically determined stem cells in patients with transplanted kidneys.

[Do oral contraceptives affect the folic acid level in blood?]. / Beeinflussen orale Kontrazeptiva den Folsäure-Spiegel im Plasma?

PIP: 20 women, aged 20-39, who had taken oral contraceptives (with estrogen content of .03-.1 mg.) for 6 months to 5 years were compared with a control group of 24 women,a aged 19-42, who had taken no medication. The results were analyzed in order to determine the effect of oral contraceptives on the folic acid level. 6 patients with idiopathic sprue were also studied. Use of contraceptives did not cause a significant difference in folic acid levels. The women suffering from sprue had significantly lower levels of folic acid after contraceptive use. No relationship between the length of contraceptive use and folic acid levels was found. Women using contraceptives with higher estrogen content had lower folic acid levels.^ieng