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.     

Synthesis and function of the mitochondrial intron —encoded bI4 RNA maturase from Saccharomyces cerevisiae

Summary We have analyzed the expression and function of the intron-encoded bI4 maturase when frame-shift mutations in the upstream exon alter the translational process. By constructing secondary cis-acting mutations within the b14 intron, we observed (1) that the bI4 maturase is still translated in the presence of the upstream mutation, albeit in very low amounts, and (2) that the limited amounts of bI4 maturase made under these conditions is no longer able to promote the splicing process of the aI4 intron. These observations, which further strengthen the maturase model, strongly suggest that bI4 maturase acts sequentially on the bI4 intron and then on the aI4 intron.     

Setting out the frame conditions for feasible use of FFPE derived RNA

Introduction

The usage of formalin-fixed paraffin embedded (FFPE) tissue is characterized by its long shelf-life and simple handling. Therefore it is the most commonly available tissue specimen in routine diagnostics and histological studies. Formaldehyde fixation may result in RNA degradation and cross linking with proteins, while storage conditions also affect RNA integrity. The present study was designed to investigate the influence of these factors on RNA analysis.

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.     

输血后丙型肝炎病毒非结构区抗体和丙氨酸转氨酶的动态分析

利用重组的丙型肝炎病毒非结构区（ＨＣＶＮＳ５）抗原建立了酶免疫试验（ＥＩＡ），对２５例输血后丙型肝炎进行了不同区抗体及丙氨酸转氨酶（ＡＬＴ）的动态研究，同时对１５６例慢性丙型肝炎患者血清进行ＨＣＶＲＮＡ和抗－ＮＳ５平行检测，两者符合率为６４．１％。抗－ＮＳ５抗体首次检出时间为３０～５７５天（１８２．９±１６８．５），晚于ＡＬＴ异常和其他区抗体的出现时间。在感染后１，３，６，１２和２４个月后抗－ＮＳ５的阳性率分别为２８％，４０％，５２％，６８％和７６％。抗－ＮＳ５的动态变化类型为四种：一过性阳性、间歇性阳性、持续性阳性和２年内持续阴性     

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     

血清HCV RNA检测在预防输血后丙型肝炎中的意义

目的 探讨血清HCV RNA检测在预防输血后丙型肝炎中的意义。方法 用ELISA法检测2000年1月至2003年12月末抗．HCV阴性的全血标本56400份次，再进行RT-PCR（荧光适时技术）检测HCV RNA，并对输过HCV RNA筛查阴性血液的患者进行追查。结果 HCVRNA阳性率为2．5‰（146／56400）。对输过HCVRNA筛查阴性血液的患者追查结果，无一例发生输血后丙型肝炎。结论 对抗-HCV阴性的血液标本用敏感的试剂进行HCV RNA检测在预防输血后丙型肝炎中不但有效，而且可行。     

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.