ABSTRACT
Pokeweed antiviral protein (PAP), a ribosome-inactivating protein isolated from the leaves of Phytolacca americana, reveals potent antiviral activity against viruses or cytotoxic action against cells once inside the cytoplasm. Therefore PAP is a good candidate to be used as an immunotoxin. We constructed a bacterial expression plasmid encoding PAP as a fusion protein with gonadotropin-releasing hormone (GnRH), a neuropeptide with receptor sites on several gynaecologic tumors. The resulting recombinant toxin was produced in Escherichia coli and accumulated in inclusion bodies. After purification under denaturing conditions, renaturated GnRH-PAP shows an IC(50) of 3 nM on in vitro translation assays and selectively inhibits the growth of the GnRH receptor positive Ishikawa cell line (ID(50) of 15 nM); on the other hand, neither GnRH nor PAP alone had any effect.
Subject(s)
Antineoplastic Agents/pharmacology , Gonadotropin-Releasing Hormone/pharmacology , Immunotoxins/pharmacology , N-Glycosyl Hydrolases , Plant Proteins/pharmacology , Drug Screening Assays, Antitumor , Gene Expression , Gonadotropin-Releasing Hormone/genetics , Humans , Immunotoxins/genetics , Immunotoxins/isolation & purification , Plant Proteins/genetics , Receptors, LHRH/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/pharmacology , Ribosome Inactivating Proteins, Type 1 , Tumor Cells, CulturedABSTRACT
Pokeweed antiviral protein (PAP) inactivates both eukaryotic and prokaryotic ribosomes via a specific depurination of rRNA. The sensitivity of pokeweed ribosomes to PAP implies the existence of a mechanism to protect the plant. Using monoclonal antibodies specific to PAP, a protein complex (PAPi) which contained PAP was identified in leaf extract. In this complex, the enzymatic activity of the toxin was strongly inhibited. This protein complex had a pI lower than that of PAP and was separated from free PAP by a preparative native gel electrophoresis. PAPi had an apparent molecular mass of 57 kDa and was dissociated by heating for 5 min at 80 degrees C or by treatment by alkaline or acidic pH or by 7 M urea. The other components involved in the complex remain unknown.
Subject(s)
Antiviral Agents/analysis , N-Glycosyl Hydrolases , Plant Proteins/analysis , Animals , Antiviral Agents/pharmacology , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Mice , Molecular Weight , Plant Proteins/pharmacology , Plants/chemistry , Protein Denaturation , Ribosome Inactivating Proteins, Type 1ABSTRACT
To verify the suspected diagnosis of deep thrombosis in the pelvic and/or the leg veins or to clarify the causes of pulmonary embolism, 84 patients were examined prospectively by color-coded duplex-sonography. The findings in the 103 legs examined were subsequently compared to those of an ascending phlebography. The deep veins from the region of the vena iliaca communis down to the lower leg were visualized, and blood flow, lumen and compressibility were checked. This procedure enabled the identification of totally occluding thrombi as well as partial thrombi with the circumfluent blood. The sensitivity of thrombus identification was 92.9%, the specificity was about 97% in the different segments. The results show that color-coded duplex sonography is a sensitive diagnostic instrument for follow-up in conservative therapy. Also, in postthrombotic changes, fresh clots can be identified for the most part. Color-coded duplex sonography can replace phlebography in most cases, if special attention is paid to regions which are difficult to visualize, such as the pelvic veins, the Hunter's channel and the veins of the lower leg.
