Biomedical subjects
Publications and source records attributed to P Zabel.
A 15-year-old girl suffering from acute dermatomyositis was treated with cyclosporin A after conventional therapy had failed. During this treatment there was a marked decrease of the myolysis and improvement of the paresis.
We describe a method for correlating polymerase activity with a particular polypeptide band in an SDS-polyacrylamide gel which does not require renaturation of the SDS-denatured enzyme. The method involves the following steps: (i) transfer of proteins from an SDS-polyacrylamide gel onto nitrocellulose; (ii) incubation with excess antiserum raised against a partially purified polymerase preparation to link one Fab site of an antibody molecule to the denatured enzyme on the nitrocellulose; (iii) binding of native polymerase to the other Fab site of the antibody molecule in the immune complex to generate a specific polymerase 'sandwich'; (iv) assaying of the nitrocellulose filter for antibody-linked native polymerase activity using an appropriate template and a radioactive substrate followed by treatment with trichloroacetic acid to precipitate in situ the radioactive product. The essential feature of this method is that the use of both non-specific anti-polymerase serum and a partially purified enzyme preparation is sufficient to allow identification of a specific protein following SDS-polyacrylamide gel electrophoresis. This antibody-linked polymerase assay has been developed to identify a 130,000-dalton RNA-dependent RNA polymerase from cowpea leaves. Possible applications of this type of assay as a tool for identifying a wide variety of proteins are discussed.
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We have prepared a rabbit antiserum specifically directed against the genome-linked protein (VPg) of cowpea mosaic virus by injecting an hydrolysate of purified virion RNA. Using this antiserum as a probe in combination with "Western" (protein) blots of subcellular fractions of cowpea mosaic virus-infected cowpea (Vigna unguiculata) cells, we have detected a bottom component RNA-encoded, 60,000-dalton polypeptide which is membrane bound and presumably represents the immediate precursor of VPg.
In cowpea protoplasts infected with cowpea mosaic virus, a bottom-component (B) RNA-encoded 60-kilodalton (60K) polypeptide is synthesized, which is membrane-bound and represents the direct precursor to the genome-bound protein VPg. The relationship between this VPg precursor and other B-RNA-encoded polypeptides was studied. Digestion of the B-RNA-encoded 170K and 84K polypeptides with Staphylococcus aureus protease V8 and subsequent analysis of the generated peptides with antiserum against VPg showed that a VPg sequence resides internally in these polypeptides. Furthermore, a new B-RNA-encoded polypeptide was detected, with a size of 58K, which differed from the 60K polypeptide only in the lack of VPg sequences. A model is presented in which the 60K VPg precursor is generated from the 200K primary translation product from B RNA and further processed to a 58K polypeptide and single VPg.
An enzyme which catalyzes the addition of a single UMP residue from UTP to the 3'-end of an RNA primer and which is referred to as terminal uridylyl transferase (TUT) has been extensively purified from the membrane fraction of vigna unguiculata leaves. The purification procedure involved (i) solubilization by cation depletion (ii) DEAE-Sepharose CL-6B column chromatography (iii) affinity chromatography of poly(U)-Sepharose 4B and (iv) glycerol gradient centrifugation. The molecular weight of the native enzyme was approximately 50,000 as determined by velocity sedimentation. Under conditions that were optimal for UMP-incorporation (5 mM Mg2+, low salt, 30 degrees C) TUT displayed a marked specificity for UTP as substrate, was unable to incorporate deoxyribonucleoside triphosphates and required a single-stranded oligo- or polyribonucleotide as primer. When oligoA20, tRNAasp of E. coli or alfalfa mosaic virus RNA 4 were used as primers at various substrate to primer ratio's, the vast majority of the product appeared to consist of primer molecules elongated with a single UMP residue as shown by polyacrylamide gelelectrophoresis and nearest neighbour analysis. We believe TUT to be a novel enzyme which has not been reported before and which may be a feasible tool in RNA sequencing as it enables the specific 3'-terminal labeling of RNA molecules.
Two poliovirus-specific RNA polymerase activities have been identified: a poly(U) polymerase that copies poly(A).oligo(U) and a replicase that copies natural heteropolymers with some preference for poliovirus RNA. Both activities purified together until a step of a salt gradient elution from poly(U)-agarose, when poly(U) polymerase but no replicase was recovered. Addition of a salt wash fraction from the ribisomes of uninfected cells to this poly(U) polymerase fraction reconstituted replicase activity, and a host factor was purified 50 fold from the ribosomal salt wash. None of the available initiation factors or elongation factors for protein synthesis were able to reconstitute replicase activity. Host factor activity could be supplied by adding oligo(U), suggesting that the factor acts at the initiation step of RNA replication. With the purified replicase-host factor combination, only poly(A)-containing RNAs were copied, and a preference for poliovirus RNA was shown.
Cowpea mosaic virus (CPMV) RNA replicase has been purified about 200-fold from CPMV-infected Vigna unguiculata leaves. Optimal reaction conditions for replicase activity have been established that allow RNA synthesis to proceed for at least 15 h. Using a polymerase assay under conditions optimal for CPMV RNA-directed RNA synthesis, all natural RNA species tested appeared to be able to direct the incorporation of labeled ribonucleotides, whereas synthetic homoribopolymers were either inactive or only slightly active. Using a nitrocellulose membrane filter assay to measure complex formation between the replicase preparation and various RNA species, all natural RNA species tested, except that of the comovirus radish mosaic virus, appeared to be unable to compete with (32)P-labeled CPMV RNA in binding to replicase. We propose that CPMV replicase is actually template specific but does not display this property in a polymerase assay, since labile complexes between heterologous templates and replicase become stabilized by the formation of phosphodiester bonds. From homoribopolymer competition binding experiments we conclude that the polyadenylic acid on the CPMV genome might be a part of the replicase binding site.
Evidence is presented for the presence of a protein covalently bound to the 5' termini of both M and B RNA components of CPMV. The protein is found to be linked in both cases to the 5' phosphate of the dinucleotide pUpAp, derived by ribonuclease digestion of the RNA. The intact protein is not required for infectivity or for in vitro translation of the RNA in cell-free extracts.
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32P-labeled cowpea mosaic virus (CPMV) RNAs were analyzed for their 5' termini by three different fingerprinting standard procedures. These analyses showed unequivocally that CPMV RNAs lack a structure like m7GpppN ... at their 5' termini. In addition, neither mono-, di- or triphosphorylated [(p)(p)pN ....] termini were found in the respective fingerprints. The inability to label the 5' termini treatment with gamma-32P-ATP and polynucleotide kinase, either per se or after pretreatment with alkaline phosphatase, strongly suggests the absence of a free 5'-OH and a free 5'-phosphate at the 5' terminal nucleotide in CPMV RNAs. The various analyses also showed that no 2' -O-methylated nucleotides, N6-methyladenosines or pseudo-uridines are present in CPMV RNAs.
A method for the solubilization of membrane-bound Cowpea mosaic virus RNA replicase has been developed by bypassing the use of detergents. Solubilization has been achieved by washing the 31,000 x g-pellet containing the bound replicase with a Mg2+-deficient buffer. This procedure had several advantages as compared to treatments with nonionic or ionic detergents: (i) the solubilized enzyme was stable at 4 C, (ii) more than 80% of the replicase could be solubilized without loss of total enzyme activity, (iii) the replicase was rather selectively released resulting in a two- to threefold increase in specific activity per se, and (iv) most of the green color from chloroplast fragments present in the crude replicase fraction remained membrane bound resulting in only slightly colored preparations of solubilized enzyme. The solubilized replicase has been further purified by DEAE-Bio Gel column chromatography. RNA synthesis directed by the DEAE-purified enzyme was template dependent and proceeded at a linear rate for at least 9 h.
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Cardiovascular operations are connected with an increased risk of infection. Postoperatively there happened a temporary fall of all immunglobulin levels. Concerning the cellular immunity, we found an impressive decrease of the transformation of the lymphocytes in the presence of polyclonal mitogens which normalises only partially after the eighth day. These results refer to a complex alteration of the immunological apparatus, imposing as a temporary immundeficiency, and could contribute to an elucidation of the increased risk of infections.
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A fraction which contained the membrane-bound cowpea mosaic virus RNA replicase was isolated from cowpea mosaic virus-infected cowpea leaves. The replicase activity appeared on day 1 after inoculation, then increased to reach a maximal on day 4. The increase in enzyme activity preceded the most-rapid virus multiplication. The membrane-bound replicase activity was almost completely insensitive to actinomycin D and DNase. The corresponding fraction from healthy leaves had no RNA-dependent RNA polymerase activity. The viral RNA synthesis in vitro proceeded linearly for 20 min and required all four ribonucleoside triphosphates and Mg(2+) ions. Mn(2+) was a poor substitute for Mg(2+). The reaction was optimal at pH 8.2. During the whole period of RNA synthesis the in vitro synthesized RNA was at least 70% resistant against RNase in 2 x SSC (0.15 M NaCl plus 0.015 M sodium citrate), but completely digestable by RNase in 0.1 x SSC. Analysis of the products by sucrose gradient centrifugation followed by treatment of separate fractions with RNase demonstrated that both single-and double-stranded RNA were present. Double-stranded RNA sedimented at about 20S, with a shoulder at 16S to 17S. A minor part of the double-stranded RNA sedimented below 10S. Single-stranded RNA sedimented with the same rate as the two viral RNAs, 26S and 34S.
Oxpentifylline (pentoxifylline), which is known to have pharmacological effects in animal models of respiratory distress syndrome, multiorgan failure, and shock, was tested in human beings after injection of endotoxin. Of ten healthy volunteers, nine met the inclusion criterion of a rise in body temperature of at least 1.0 degrees C after 100 ng endotoxin (Salmonella abortus equi) as a bolus injection. Serum levels of tumour necrosis factor alpha (TNF) and interleukin-6 (IL-6) were both significantly higher than baseline levels 2 h and 3 h after endotoxin injection. 3 weeks later the nine volunteers were again injected with 100 ng endotoxin and oxpentifylline (500 mg over 4 h) was also infused. There was no rise in TNF levels, though IL-6 levels rose in parallel with body temperature. These data suggest that oxpentifylline blocks the endotoxin-induced synthesis of TNF in man and, therefore, could possibly have beneficial effects in clinical endotoxaemia.