Rat dimethylglycine dehydrogenase (Me2GlyDH) was used as model protein to study the biogenesis of a covalently flavinylated mitochondrial enzyme. Here we show that: 1) enzymatically active holoenzyme correlated with trypsin resistance of the protein; 2) folding of the reticulocyte lysate-translated protein into the trypsin-resistant, holoenzyme form was a slow process that was stimulated by the presence of the flavin cofactor and was more efficient at 15 degrees C than at 30 degrees C; 3) the mitochondrial presequence reduced the extent but did not prevent holoenzyme formation; 4) covalent attachment of FAD to the Me2GlyDH apoenzyme proceeded spontaneously and did not require a mitochondrial protein factor; 5) in vitro only the precursor, but not the mature form, of the protein was imported into isolated rat liver mitochondria; in vivo, in stably transfected HepG2 cells, both the precursor and the mature form were imported into the organelle; 6) holoenzyme formation in the cytoplasm did not prevent the translocation of the proteins into the mitochondria in vivo; and 7) lack of vitamin B2 in the tissue culture medium resulted in a reduced recovery of the precursor and the mature form of Me2GlyDH from cell mitochondria, suggesting a decreased efficiency of mitochondrial protein import.
Zusammenfassung Die Versuche beweisen, daß man gegen Kanarienpocken nicht nur über das Hautorgan, sondern auch intramuskulär und über die Schleimhäute des Digestions‐ und Respirationstraktes immunisieren kann. Für einen Impferfolg sind dabei folgende Parameter maßgebend: 1. Attenuierungsgrad des Impfvirus, 2. Virusmenge pro Impfstoffdosis, 3. Anzahl der Revakzinationen. Für die intramuskuläre Immunisierung eignen sich nur Impfstoffstämme, die so stark attenuiert sind, daß sie schadlos vertragen werden. Diese Eigenschaft erfüllt der wenigstens 540mal in Hühnerembryo‐Fibroblasten‐Kulturen passierte Stamm KP 1 att. Zur aerogenen, nasalen, oralen, oesophagealen und kloakalen Immunisierung können sowohl hochattenuierte als auch weniger stark attenuierte Impfstoffstämme verwendet werden. Für eine wirksame aerogene, nasale oder kloakale Immunisierung sind dabei wesentlich geringere Virusmengen notwendig als für die orale Immunisierung bei Verwendung gleichartig attenuierter Virusstämme. Wird eine orale Immunisierung, bei der respiratorische Epithelien nicht beteiligt sind, angestrebt, müssen entweder die Virusmenge pro Impfstoffdosis erhöht und Revakzinationen durchgeführt werden oder man muß auf weniger stark attenuierte Impfstoffstämme zurückgreifen. Da der Unschädlichkeit bei jedem Impfverfahren der Vorzug zu geben ist, schlagen wir für die intramuskuläre, aerogene, nasale oder kloakale Vakzination den hochattenuierten Impfstoffstamm KP 1 att. vor, wobei obige quantitative Kriterien zu beachten sind. Summary Comparative studies on parenteral and non‐parenteral immunization against canary pox Experiments showed that immunization against canary pox can be effected no only via the skin but also intramuscularly and through the mucosa of the digestive and respiratory tracts. Successful protection by vaccine is influenced by the following parameters: 1. Degree of attenuation of the vaccine virus. 2. Amount of virus per inoculation dose. 3. Number of revaccinations. For intramuscular vaccination one must use only strains that have been so markedly attenuated as to be harmless. This necessitates at least the 540th passage of strain KP 1 att., in chick embryo fibroblast culture. For aerogenic, nasal, oral, oesophageal and cloacal vaccination one can use both highly attenuated and less attenuated strains. For an effective aerogenic, nasal or cloacal immunization appreciably smaller virus amounts are required than for oral immunization, when the same strain is employed. If oral immunization is desired without involvement of the respiratory epithelium it is necessary either to increase the amount of virus per dose and carry out revaccination or to use a less attenuated strain. Since the absence of ill effects is the prequisite of vaccination the authors advise intramuscular, aerogenic, nasal or cloacal vaccination with the highly attenuated strain KP 1 att., paying attention to the quantitative criteria mentioned above. Résumé Recherches comparées sur l'immunisation parentérale et non parentérale contre la variole du canari Les ...
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