Intraaxonaler transport von Ethidium-Bromid-sensitiven RNS- und niedermolekularen 3H-Uridin-Verbindungen im tractus opticus von teleosteern

Zusammenfassung Am Sehtrakt von Carassius carassius und Scardinius erythrophthalmus wurde die axonale Ausbreitungsweise hoch- und niedermolekularer 3H-Uridin-Verbindungen untersucht. Dabei wurde nach intraocularer Injektion des Tracers für TCE-resistente Verbindungen eine intraaxonale Transportgeschwindigkeit von 2–4 mm/d bestimmt, für TCE-lösliche Verbindungen eine von ca. 30–50 mm/d. Durch Applikation des spezifisch mitochondrialen RNS-Synthese-Hemmers Ethidium-Bromid konnte die Einbaurate von 3H-Uridin in hochmolekulare RNS um 70–80% erniedrigt werden, was dafür spricht, daß die langsam im Axoplasma wandernden Mitochondrien einen Großteil der axonalen RNS synthetisieren.In der TCE-löslichen Fraktion konnten durch dünnschichtchromatographische Analyse noch nach 8d p. i. 3H-Uridin und 3H-UDPG nachgewiesen werden. Dieser Befund wird hinsichtlich eines transneuronalen Stoffübertritts von Uridin und der möglichen Bedeutung des UDPG-Transportes im Nervengewebe diskutiert.

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Intraaxonal transport of ethidium-bromide-sensitive RNA- and lowmolecular 3H-uridine-compounds in the optic tract of teleost

Summary In the optic system of teleosts (Carassius carassius and Scardinius erythrophthalmus) the axonal flow of high and low molecular 3H-uridine-compounds was investigated. After injection of the tracer into one eyeball and TCA-extraction of the samples a transport-rate of 2–4 mm/d was demonstrated. By the specific inhibitor of mitochondrial RNA-synthesis, Ethidium-Bromide, the amount of axonal radioactivity could be reduced to 20–30% of the control. This indicates the mitochondria as being the site of synthesis most of axonal RNA. Considering the TCA-soluble 3H-uridine-compounds, an intraaxonal flow also could be demonstrated, with a transport-rate of 30–50 mm/d, 16 times higher as the one of RNA. The analysis by thin layer chromatography indicated the existence of 3H-uridine and 3H-UDPG in the axonal fraction of TCA-soluble compounds still after an incorporation time of 8d. The possibility of a transneuronal convection of uridine and the function of UDPG-transport in the axon are discussed.

Frau Prof. Dr. H. Kersten (Erlangen) danke ich für die Überlassung einer Probe Ethidium-Bromid.     

Distribution of TT virus genomic groups 1-5 in Brazilian blood donors, HBV carriers, and HIV-1-infected patients

Human isolates of the highly prevalent TT virus (TTV) have been classified into five major genomic groups (1-5). The geographical distribution of the groups throughout the world is not well known. Five different PCR assays were developed in an attempt to amplify specifically TTV DNAs of each genomic group. Serum samples collected from 72 Brazilian adults (24 voluntary blood donors, 24 hepatitis B virus (HBV) carriers, and 24 human immunodeficiency virus type 1 (HIV-1)-infected patients) were tested. TTV DNA from at least one genomic group was detected in 11 (46%) blood donors, 13 (54%) HBV carriers, and 24 (100%) HIV-1 patients. All five genomic groups were detected in the three populations, with the exception of group 2 in blood donors. Some samples, negative with all five specific assays, were positive with the commonly used untranslated region (UTR) PCR system. On the other hand, TTV DNA was detected in some samples by using specific assays but not with the UTR PCR. Mixed infections with 2-5 TTV isolates from different groups were detected in 21% blood donors, 29% HBV carriers, and 71% HIV-1 patients. Fifteen PCR products (three obtained with each assay) were sequenced. Most sequences showed high (>86%) homology with those of TTV isolates belonging to their presumed groups. However, three sequences had low homology with all TTV sequences available from the DNA databanks. In conclusion, TTV isolates belonging to all five known genomic groups circulate in Brazil, and the results suggest the existence of new and as yet uncharacterised major genomic groups.     

Nucleotide sequence and in vitro expression of the capsid protein gene of tobacco ringspot virus

The nucleotide sequence of the 3' terminal 2022 nucleotides (nt) of tobacco ringspot virus (TobRV) RNA 2 has been determined. Protein microsequence analysis of the amino-terminal residues of purified capsid protein localized the capsid protein gene between nt 2014 and 583 (from the 3' terminus) of this sequence. The proteolytic cleavage site that is processed to liberate the capsid protein from the RNA 2-encoded polyprotein was identified as Cys-Ala. The predicted translation product from the gene is a 477 amino acid long polypeptide with a calculated MW of 53 kDa. The gene was modified at the 5' end to facilitate sub-cloning, and to provide it with a methionine initiation codon. The modified gene was sub-cloned, transcribed in vitro and expressed in a rabbit reticulocyte lysate translation system, where it directed the synthesis of a 53 kDa polypeptide. Garnier-Osguthorpe-Robson analyses of the secondary structure of the capsid protein predicted the presence of three beta sheet domains, which suggests that this nepovirus capsid may be structurally analogous to those of the como- and picornaviruses. These and other results from computer analyses of the nucleic acid and amino acid sequences, and comparisons with the capsid proteins of nepoviruses and other related viruses are discussed.     

Isolation and RNA-binding analysis of NAD+-isocitrate dehydrogenases from Kluyveromyces lactis and Schizosaccharomyces pombe

Krebs cycle NAD⁺-isocitrate dehydrogenase (Idh) binds to the 5-UTRs of all mitochondrial mRNAs in Saccharomyces cerevisiae. We hypothesize that this leader-binding activity plays a role in translational regulation, thereby linking mitochondrial biogenesis to the need for respiratory function. Analysis of effects of leader binding on mitochondrial translation is complicated by the involvement of the enzyme in mitochondrial metabolism. We have therefore searched for an Idh altered in RNA binding, but retaining full enzyme activity. Idh from Kluyveromyces lactis and Schizosaccharomyces pombe was partially purified and examined for the ability to bind Cox2 mRNA. Sch. pombe Idh, like the S. cerevisiae enzyme, has high affinity for both its own, K. lactis and S. cerevisiaeCOX2 leaders. In contrast, Idh purified from K. lactis shows only low affinity for all mRNAs tested. To determine what distinguishes K. lactis Idh from S. cerevisiae Idh, genes encoding the two subunits of Idh in K. lactis were cloned and sequenced. Sequence comparison revealed high levels of similarity throughout the proteins, in particular in regions involved in enzyme activity, co-factor and regulator binding. Non-conserved residues between the subunits from the two yeasts are candidates for involvement in the interaction with RNA. Received: 19 January 2000 / 24 March 2000     

Clinical significance of antibodies to nonstructural and core proteins of hepatitis C virus in posttransfusion hepatitis patients during long-term follow-up

The prevalence of hepatitis C antibodies (anti- HCV) among multitransfused patients was studied and compared with predicted values obtained from a post-transfusion hepatitis study and from data on the prevalence of anti-HCV among blood donors. The prevalence of hepatitis B core antibodies (anti-HBc) was also studied to determine the routes of transmission of hepatitis C virus. The patients consisted of 65 dialysis patients (57 on haemodialysis and 8 on continuous ambulatory peritoneal dialysis) and 71 leukemia patients in long-term remission [49 with acute myeloid leukemia (AML) and 22 with acute lymphatic leukemia (ALL)]. The presence of anti-HCV was investigated using a second generation enzyme-linked immunosorbent assay. Reactive samples were confirmed by a second generation recombinant immunoblot assay. Anti-HBc was studied in the 65 dialysis patients and in 40 of the leukemia patients. Three (4.6%) of the 65 dialysis patients and 12 (24.5%) of the 49 AML patients were anti-HCV positive whereas all of the ALL patients were seronegative. The total number of blood units transfused to 134 patients (data on two dialysis patients were not available) was 18,148, out of which 17,575 units had been transfused prior to the initiation of anti- HCV screening of blood donors. On the basis of the anti-HCV prevalence among blood donors and the incidence of post-transfusion hepatitis, the predicted number of seropositive patients was 11 and 18, respectively. Five of the 65 dialysis patients were anti-HBc positive, compared with only one of the 40 leukemia patients. It is concluded that the anti-HCV prevalence among dialysis and leukemia patients is concordant with the risk of receiving contaminated blood products, whereas hepatitis B infection may have other routes of transmission in dialysis patients. © 1993 Wiley-Liss, Inc.     

The exclusion among the Ustilago viruses — A dsRNA restriction modification system?

Summary Specific exclusion relations are know among the three Ustilago maydis viruses that are associated with the cytoplasmically transmitted killer phemomenon. Of the three viruses P1, P4 and P6, only P1, and P4 cancoexist in one host cell. Mutual exclusion occurs between P1 and P6 and P4 unilaterally excludes P6. The exclusion relations were originally defined among the wild-type viruses. Those relations can be modified by two specific segments that are a part of the P4 dsRNA genome and were also found in some sensitive strains that contained part of the viral genome. Also, deletion of the dsRNA segment that is assumed to encode the toxin information permits the formation of hybrid genomes that otherwise cannot be formed. The data is interpreted in terms of a dsRNA restriction modification system in which the killer toxin or a toxin-linked function acts as the restriction factor and segments H3 and H4 or H4 alone contain the necessary information for the modification of certain sites on the M and L segments of the P1 and P4 viruses but not on the P6 segments.     

The asymmetries in morphology, desoxyribonucleic acid, protein and ribonucleic acid of the two hemispheres induced by monolateral removal of the olfactory bulb and retrobulbar structures in neonatal rats

Monolateral removal of the olfactory bulb and the olfactory penduncular structures in the neonatal rat resulted in a profound morphological and biochemical asymmetry between the two hemispheres. The experimental hemisphere, ipsilateral to the lesion, protruded into the space normally occupied by the olfactory bulb and showed enlarged ventricles. The brain loci were displaced rostrally in this hemisphere.The desoxyribonucleic acid, ribonucleic acid and protein contents of the experimental hemisphere at 25 and 60 days of age were all significantly lower than that found in the control hemisphere contralateral to the lesion.To resolve between the two possible causes of this asymmetry (i.e. atrophy of the experimental or hypertrophy of the control hemisphere) hemispheres of the asymmetric brain were compared with homonymous hemispheres of unoperated normal rats. This comparison revealed that the asymmetry is basically due to an excess of desoxyribonucleic acid synthesis in the control hemisphere, which continues even after postnatal day 25, on the one hand, and a dearth of protein in the experimental hemisphere on the other. This finding implies two separate mechanisms for the processes that underlie the asymmetries observed for these two substances.Our results demonstrate two important characteristics of the developing brain. Firstly they indicate that removal of the olfactory bulb and the olfactory peduncle can produce considerable changes in the hemispheres, and secondly they unravel the strong latent potential of the brain for cell proliferation beyond the usual period of cell division in the brain. As in these experiments regulation of cell division is affected, this system might serve as a model for the study of aberrant cell division found in tumor formation and the process of carcinogenesis.