Biomedical subjects
Publications and source records attributed to E Wimmer.
Poliovirus RNA that had been labelled with 125I in the 5'-terminal protein (VPg) was found competent to form an initiation complex of translation in a cell-free reticulocyte lysate. In conditions of ribosome binding, no cleavage occurred between VPg and RNA. We conclude that removal of VPg from poliovirus RNA is not a prerequisite for this RNA to initiate translation in vitro.
The purpose of the present study was the collection of data concerning digitalis treatment of patients outside hospital. The investigation was carried out on 200 patients over 60 years of age, consecutively admitted to a 750-bed general hospital (35% ot a medical ward, 65% to other specialities). An ECG and serum creatinine and digoxin determinations were performed on the day of admission and a careful history taken of the drugs administered before admission. 30 patients reported the use of digoxin or other digitalis drugs, whilst another 48 patients reported taking cardiac drugs in general. The prevalence of digoxin levels exceeding 2 ng/ml was 11.5% in the group of patients known to have taken digitalis preparations, 18% among the 92 patients with positive serum digoxin levels and highest (30%) in the subgroup admitted due to cardiac failure. In patients with digoxin levels above 2 ng/ml the prevalence of elevated serum creatinine values (greater than 1.2 mg%) was markedly increased. Among patients with normal serum creatinine levels the mean age of patients with digoxin levels above 2 ng/ml was significantly higher than of those with digoxin levels below 2 ng/ml. A clear correlation between digoxin levels and ECG changes was not demonstrable; simultaneous administration of diuretics promotes the appearance of electrocardiographic signs of digitalis toxicity.
The structure of polio replicative form (RF) has been investigated by 3' end labeling and the use of polynucleotide phosphorylase to now allow a complete composite of the RF structure. The evidence presented indicates that the 3' terminal sequence of the minus strand is an exact complement to the 5' end of polio RNA. This suggests that the 5' terminal U of polio RNA is genetically coded. Other data is presented to show that in addition to the genetically coded poly(A) tract of the plus strand in RF, a single-stranded poly(A) tail protrudes beyond the double-stranded RNA.
The poliovirus genome-linked protein (VPg) has been subjected to radiochemical microsequence analysis. Sequence studies of virion RNA by a modification of Sanger's dideoxy method have revealed a base sequence corresponding to the amino acid analysis. This result proves that VPg is virus-encoded. The RNA sequence has allowed us to predict the total amino acid sequence of VPg and part of its precursor. VPg is, at most, 27 amino acids long. It maps within the 3' terminal segment of the viral genome that encodes the precursor polypeptide NCVP1b for the virus-specific RNA polymerase NCVP4.
The dideoxynucleotide method for sequencing DNA developed by Sanger et al. [Sanger, F., Nicklen, S. & Coulson, A. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467] was modified to allow sequence analysis of poliovirus RNA without recourse to cloning. Our method involves reverse transcription of poliovirus RNA followed by cDNA-dependent DNA synthesis in the presence of unlabeled dNTPs and 2',3'-dideoxynucleoside triphosphates, with Escherichia coli DNA polymerase I (Klenow) used to catalyze the reaction. DNA synthesis is primed by 5'-32P-labeled RNase T1- or RNase A-resistant oligonucleotides generated from poliovirus RNA. The sequence of 1060 nucleotides preceding the 3'-terminal poly(A) is presented. Based on the position of termination codons we propose that viral translation terminates at nucleotide -562.
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It has previously been shown that the RNA replicase of encephalomyocarditis virus contains two virus-coded proteins, D and E, which are produced in two successive proteolytic steps: (i) C leads to D + ?; and (ii) D leads to p22 + E. It is here shown (i) that virus protein H (molecular weight, 12,000) is the previously unidentified product of the first step and (ii) that VPg, a protein linked covalently to the virion RNA, yields two tryptic peptides found in protein C but not in protein D. The results suggest that VPg is derived by cleavage of protein C and that protein H may be intermediate. Preliminary experiments with VPg sequences in polioviral noncapsid protein 1b, the counterpart of encephalomyocarditis viral protein C, were inconclusive.
Digestion of poliovirus type 2[32P]-RNA with enzymes and analysis of the products by column chromatography and paper electrophoresis provides evidence that the RNA is covalently linked to a small, basic protein, VPg, via a 5'-terminal pU residue. Digestion of the RNA with proteinase K followed by labeling of the peptidyl-RNA with the Bolton and Hunter reagent [iodinated 3-(4-hydroxyphenyl)propionic acid N-hydroxysuccinimide ester] yields 5'-labeled material suitable for rapid sequencing. The 5'-terminal sequence of poliovirion type 2 RNA was determined to be VPg-pUUAAAACAG... which is identical to the sequence at the 5'-terminus of the poliovirus type 1 genome.
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Urinary amylase was estimated by chromogenic (amylochrome Roche) as well as enzymatic methods (SKI and Beckman: substrate starch and substrate maltotetraose respectively). Random and timed urines (24 hour collections) were analysed. Clearances of amylase gave different results dependent upon the amylase-test used and the glomerular filtration rate. Correlation between chromogenic and enzymatic methods (starch as substrate) was poor. The ratio of amylase and creatinine clearance was used to test different methods. Reference values for this ratio for the amylochrome method (N = 106) were 2.85 +/- 0.99% and for the Beckman-DS method (N = 60) 2.82 +/- 0.87%.
The 32P-labelled genomes of poliovirus type 1, 2 and 3 have been digested with RNase T1 and the products separated by two-dimensional gel electrophoresis. All three fingerprints differ in the separation pattern of the large oligonucleotides. The molar yields of the large RNase T1-resistant oligonucleotides of type 1 and type 2 RNA of poliovirus RNA are close to one. By comparing the yields of these oligonucleotides to the amount of RNA from which they originated, the chain length of type 1 poliovirus RNA was found to be 7851 +/- 567 nucleotides (mol. wt. 2.66 +/- 0.19 x 10(6) and that of poliovirus type 2, 8181 +/- 578 nucleotides (mol. wt. 2.77 +/- 0.19 x 10(6). The chain length of two defective interfering particle (DI) RNAs of poliovirus type 1 were determined to be 7042 +/- 999 nucleotides for DI(1) and 6639 +/- 674 nucleotides for DI(2).
The enzymatic method of H. W. Schiwara (1972) Z. Klim. Chem. Klin. Biochem. 10,12--16 (reagents by Smith Kline Instruments), using the enzymatic reaction sequence alpha-amylase -- alpha-glucosidase -- hexokinase/glucose 6-phosphate dehydrogenase for the determination of alpha-amylase was evaluated on the ABA-100. The coefficient of variation for control sera and human pooled serum was 0.9--4.2% within series, and 1.4--6.6% day to day. Reference values for a healthy population (212 blood donors) in sera were 13--79 U/1 (+/- 2 SD), mean 46 U/1. In catch urines the values did not show a normal distribution; the minimal and maximal range for men was 58--385 U/1, for women 7--318 U/1. The kinetic curve of the enzymatic test was measured and the influence of glucose and linearity studied. In comparison with the enzymatic test, the chromogenic method Amlyochrom Roche was tested on the sera and urine of patients. The coefficient of correlation in sera was r = 0.975, in urine r = 0.965.
44 male and 103 female subjects were given an oral load of 1 g or 2 sucrose/kg body weight. Glucose, fructose and sucrose were determined in capillary blood and urine samples before and after sucrose ingestion. Sucrose was detected in the blood only in 10 cases, but was present in the urine in each subject (max. 1132 mg sucrose/100 ml). Urinary excretion of sucrose is considered to be a physiological phenomenon since none of the subjects had any metabolic disorder.
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Virion RNA of poliovirus type 1 has been analyzed for the linkage between genome-protein VPg and the polyribonucleotide chain. Hydrolysis of the linkage with acid or alkali and enzymatic degradation lead to the conclusion that the bond is neither a phosphodiester such as nucleotidyl-(P-O)-serine (or threonine) nor a phosphoramidate such as nucleotidyl-(P-N)-amino acid. VPg-RNA can be iodinated by the Bolton and Hunter reagent [iodinated 3-(4-hydroxyphenyl)propionic acid N-hydroxysuccinimide ester] but not by the chloramine-T or lactoperoxidase procedures, an observation suggesting that VPg does not contain accessible tyrosine. However, VPg can be labeled with [3H]tyrosine in vivo. Hydrolysis of VPg-[32P]pUp with 5.6 M HCl at 110 degrees yielded 32P-labeled O4-(3'-phospho-5'-uridylyl)tyrosine that could be cleaved with micrococcal nuclease to O4-[32P]phosphotyrosine and uridine 3'-[32P]phosphate. These data establish that VPg is linked to the poliovirus genome by a bond between the O4 of tyrosine and the 5'-P atom of the terminal uridylic acid residue. The 5' end of polio genome RNA can now be described as VPg(Tyr-O)-pU-U-A-A-A-A-C-A-G.