Translation enhancer in the 3′-untranslated region of rotavirus gene 6 mRNA promotes expression of the major capsid protein VP6

Summary. The eleven rotavirus mRNAs contain 5-cap structures and most end with the 3-consensus sequence 5-UGACC-3. The UGACC functions as a common translation enhancer (3-TE-con) that upregulates viral protein expression through a process mediated by the nonstructural protein NSP3. To address the possibility that gene-specific enhancers are also contained in the untranslated regions (UTRs) of the rotavirus mRNAs, we used rabbit reticulocyte lysates to investigate the translation efficiencies of analog RNAs containing viral-specific 5-and 3-UTRs and the open reading frame for chloramphenicol acetyltransferase. These experiments combined with the analysis of full-length viral RNAs and RNAs containing 3-truncations showed that a highly active enhancer was present near the 5-end of the 139-nucleotide 3-UTR of the gene 6 mRNA (3-TEg6). The 3-TEg6 represents a functionally independent enhancer, as no other portion of the gene 6 mRNA was required for its activity. The 3-TEg6 differs significantly from the 3-TE-con in that the gene 6-specific enhancer does not require viral protein for activity and is formed by a sequence unique to only one of the eleven viral mRNAs. Together, our findings suggest that the 3-UTR of the gene 6 mRNA contains two TEs, one is gene-specific (3-TEg6) and the other is common to nearly all rotavirus genes (3-TE-con). The activity of the 3-TEg6 is likely important for directing the efficient translation of the gene 6 mRNA at levels sufficient to provide the 780 copies of VP6 necessary for the assembly of each progeny virion.     

Completion of the Four Large Gene Sequences of Porcine Group C Cowden Rotavirus

The terminal nucleotide sequences of group C Cowden rotavirus gene segments 1-4 were determined. When compared with the published sequences, we found 14 to 29 additional nt at the 5 ends of the four reported gene sequences. For the 3 ends, we observed an additional 16 nt in gene 2 and 14 fewer nt in gene 4.     

Differences in antigenic properties of Fc-binding activity during infection with herpes simplex virus type 1

Summary The antigenic properties of the Fc receptor induced by herpes simplex virus type 1 (HSV-1) were studied with anti-HSV F(ab)₂ and pFc from infected rabbits.It appeared that the HSV-induced Fc-binding receptor had different antigenic characteristics at different times after infection. The Fc receptor present early in the infection (0.5 hours), during the adsorption period, most probably is the result of a fusion event between the virus envelope and the infected cell. We found that this Fc receptor reacted with anti-HSV F(ab)₂ and thus showed HSV-antigenic properties in such a way that binding of anti-HSV F(ab)₂ prevented the binding of pFc fragments.Later on in the infection (5 hours), the Fc-binding activity present on the surface of the infected cell is the result of newly synthesized and in the plasma membrane integrated polypeptides. The Fc-binding activity present on the cell surface of 5 hours infected cells could not be inhibited by anti-HSV F(ab)₂ and did not interfere with the binding of pFc to the Fc receptor.With 1 Figure     

Dissociation of acetylcholine- and cyclic GMP-induced currents inXenopu oocytes

InXenopus follicular oocytes, activation of muscarinic receptors evokes a slow potassium current (H-response); a similar current is evoked by intracellular injection of cyclic guanosine 3,5-monophosphate, cGMP (Dascal et al. 1984). We have tested the hypothesis that cGMP may be the second messenger that mediates the opening of K channel by acetylcholine (ACh). ACh elevated the intracellular level of cGMP with a time course similar to that of the development of the muscarinic H-response; maximal increase in cGMP concentration above the control was about 0.2 pmole/oocyte. The amount of injected cGMP that produced a detectable K current (threshold dose) varied between 0.5 and 3 pmole/oocyte. At low doses of cGMP, the slope of log dose-log response curve was about 2.5, suggesting involvement of a biochemical process with a positive cooperativity of at least 3. Higher doses of cGMP evoked, in addition to the outward current, an irregular, rapidly developing, long-lasting inward current, that never reached amplitudes comparable to those of ACh-evoked Cl currents. The K current elicited by cGMP was insensitive to elevation or depletion of external Ca. It was potentiated by isobutylmethylxanthine (IBMX). ACh strongly inhibited the cGMP-evoked K current when applied at the plateau of the latter. 4-Phorbol 12,13-dibutyrate (PDBu) (1 M) rapidly and completely inhibited the cGMP response. It is concluded, that most of the results presented in this report contradict the hyothesis that cGMP is the intracellular mediator of ACh-induced changes in membrane conductance in the oocytes.Abbreviations ACh acetylcholine - cAMP cyclic adenosine 3,5-monophosphate - cGMP cyclic guanosine 3,5-monophosphate - EGTA ethylenediaminetetraacetic acid - Hepes N-2-hydroxyethyl-piperazinc-N-2-hydroxypropanesulphonic acid - IBMX 3-isobutyl-l-methylxanthine - IP₃ inositol 1,4,5-trisphosphate - PDBu 4-phorbol 12,13-dibutyrate     

The oligodendroglial reaction to brain stab wounds: An immunohistochemical study

Summary Myelin/oligodendrocyte specific protein was compared to glial fibrillary acidic protein and 23-cyclic nucleotide 3-phosphodiesterase expression in normal rat brains and following stab wounds to the cerebral cortex, corpus callosum and hippocampus. Animals with stab wounds were allowed to recover for 5,15,28, 45 and 70 days post-operation before fixation by perfusion. Sections were reacted with antibodies against myelin/oligodendrocyte specific protein, glial fibrillary acidic protein and 23-cyclic nucleotide 3-phosphodiesterase, and observed by light and electron microscopy. Normal cerebral cortex had very few myelin/oligodendrocyte specific protein-positive and 23-cyclic nucleotide 3-phosphodiesterasepositive cells, but some glial fibrillary acidic protein-positive cells. The myelinated fibres of the corpus callosum were heavily stained for myelin/oligodendrocyte specific protein but unstained by glial fibrillary acidic protein or 23-cyclic nucleotide 3-phosphodiesterase antibodies. Some immunopositive cells were present in the corpus callosum and hippocampus with all three antibodies. After stab wound myelin/oligodendrocyte specific protein-positive reactive cells had more and longer processes and stained more intensely than equivalent cells in normal brain. These cells were distributed along the wound track, including within the cerebral cortex. The numbers of these cells increased until 28 days post-operation and then decreased so that very few were found at 70 days post-operation except in the corpus callosum. Where demyelination occurred myelin/oligodendrocyte specific protein-staining was lost. Staining for 23-cyclic nucleotide 3-phosphodiesterase revealed a similar pattern. Glial fibrillary acidic protein-positive reactive cells, which were also more robust than the normal cells, were more widely distributed. They increased in number throughout the time periods studied and gliosis was evident on the contralateral side. The glial fibrillary acidic protein-positive astrocytes were also different from the myelin/oligodendrocyte specific protein-positive and 23-cyclic nucleotide 3-phosphodiesterase-positive oligodendrocytes in terms of cell shape. With electron microscopy myelin/oligodendrocyte specific protein-positive cells showed features typical of immature oligodendrocytes. We conclude that the injury caused a numerical increase in oligodendrocytes and that myelin/ oligodendrocyte specific protein is a good marker for the oligodendroglial response and demyelination in pathological conditions.     

Die seltenen Rh-Phänotypen rhy rh und rh′ rh′ in einer deutschen Familie

Zusammenfassung Nach einer Literaturübersicht wird das Vorkommen der seltenen Rh-Genotypen rr und rr in 2 Generationen einer deutschen Familie beschrieben.     

Molecular mechanisms of neuronal plasticity during learning: The role of secondary messengers

We present published data along with our own results concerning the role of second messengers and their intracellular receptors in molecular mechanisms associated with the plasticity of neurons during learning. The participation of cyclic 3,5-adenosine monophosphate, cyclic 35-guanosine monophosphate, calcium, calmodulin, and also the metabolic products of inositol phospholipids, inositol-1,4,5-triphosphate, diacylglycerol and the protein kinase C activated by it, arachidonic acid, and the products of its lipoxygenase oxidation during the regulation of neuronal plasticity over the course of prolonged potentiation, sensitization, habituation, and classical associative training are discussed.Translated from Zhurnal Vysshei Nervnoi Deyatel'nostiimeni I. P. Pavlova, Vol. 39, No. 2, pp. 195–214, March–April, 1989.     

Cyclic nucleotides in rheumatoid arthritis

Cyclic 3,5-adenosine monophosphate (cAMP) and cyclic 3,5-guanosine monophosphate (cGMP) may influence important regulatory mechanisms in the rheumatoid inflammatory process. It has been claimed that fasting imporves the condition of the patient with rheumatoid arthritis (RA). The present study was designed to measure cAMP in plasma and urine and cGMP in urine in medically untreated RA patients. 12 female patients were investigated in a cross-over study during a control and a fasting period. They received no other drugs than analgesics during these periods.Levels of plasma and urinary cAMP found during the control period were somewhat lower than previously reported. However, the ratio cAMP/cGMP in urine was 10 to 1 which is reported to be normal. Clinical and laboratory variables of inflammatory activity were significantly improved during the 7-day fasting period. The ratio of cAMP/cGMP in urine was significantly increased on days 2–4 and coincided in time with the maximum of clinical improvement. Cyclic AMP concentrations were lowered both in plasma and urine during fasting. This is in contrast to fasting in normal and obese subjects reported in previous studies.     

Predicted stem-loop structures and variation in nucleotide sequence of 3′ noncoding regions among animal calicivirus genomes

Caliciviruses are nonenveloped with a polyadenylated genome of approximately 7.6 kb and a single capsid protein. The RNA Fold computer program was used to analyze 3-terminal noncoding sequences of five feline calicivirus (FCV), rabbit hemorrhagic disease virus (RHDV), and two San Miguel sea lion virus (SMSV) isolates. The FCV 3-terminal sequences are 40–46 nucleotides in length and 72–91% similar. The FCV sequences were predicted to contain two possible duplex structures and one stem-loop structure with free energies of –2.1 to –18.2 kcal/mole. The RHDV genomic 3-terminal RNA sequences are 54 nucleotides in length and share 49% sequence similarity to homologous regions of the FCV genome. The RHDV sequence was predicted to form two duplex structures in the 3-terminal noncoding region with a single stem-loop structure, resembling that of FCV. In contrast, the SMSV 1 and 4 genomic 3-terminal noncoding sequences were 185 and 182 nucleotides in length, respectively. Ten possible duplex structures were predicted with an average structural free energy of –35 kcal/mole. Sequence similarity between the two SMSV isolates was 75%. Furthermore, extensive cloverleaflike structures are predicted in the 3 noncoding region of the SMSV genome, in contrast to the predicted single stem-loop structures of FCV or RHDV.Disclaimer: No endorsements are herein implied. Brand names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standards of the products, and the use of the names by the USDA implies no approval of the products to the exclusion of others that may also be suitable.     

Zur Bedeutung des cyclischen Adenosin-3′:5′-monophosphat als „second messenger“ bei der Säuresekretion des Magens

Zusammenfassung Ob bei einer bestimmten Hormonwirkung cyclisches Adenosin-3:5-monophosphat (cAMP) als intracellulärer Vermittler fungiert, läßt sich nach den von Sutherland entwickelten Kriterien beurteilen; dabei sind die qualitativen, quantitativen und zeitlichen Beziehungen zwischen dem Hormoneffekt auf den intracellulären cAMP-Spiegel und die jeweilige physiologische Antwort zu prüfen. Im Falle der Histamin- bzw. Pentagastrin-stimulierten gastralen Säuresekretion des Frosches (Necturus maculosus) und der Ratte erfüllt das cAMP die Bedingungen eines second messenger. Dagegen ließ sich beim Hund und Menschen eine derartige Funktion des cAMP bisher nicht verifizieren; ob bei diesen Species cyclisches Guanosin-3:5-monophosphat anstelle des cAMP die intracelluläre Vermittlerrolle übernimmt, werden erst noch systematische Untersuchungen erweisen müssen.Die in diesem Zusammenhang zitierten eigenen Arbeiten wurden von der Deutschen Forschungsgemeinschaft, Bad Godesberg, unterstützt.     

Studien über den Mechanismus der Immunhämolyse: Der Bindungsmodus der vierten Komplementkomponente

Zusammenfassung Die Bindung der vierten Komplementkomponente (C₄) an sensibilisierte Hammelblutzellen (EA) ist von der vorhergehenden Bindung der ersten Komplementkomponente abhängig. Das Präparat EAC₁ kann, wenn ihm C₄ in geeigneter Form angeboten wird, in den Komplex EAC_1,4 überführt werden. Hierbei verschwindet der titrierbare Gehalt der flüssigen Phase an C₄; gleichzeitig acquirieren die Zellen die Fähigkeit, mit R₄ zu lysieren. Der zeitliche Ablauf dieser Veränderung wird untersucht. Die Eeaktion zwischen EAC₁ und C₄ verläuft äußerst schnell, und ihre Geschwindigkeit wird durch die Reaktionstemperatur nicht beeinflußt. Während die Bindung von C₁ an EA nur in Gegenwart von Ca⁺⁺ erfolgt, läuft die Bindung von C₄ an EAC₄ auch in Abwesenheit von zweiwertigen Metallionen ab. Die vonLevine u.Mayer als ein Ganzes betrachtete Ca⁺⁺-abhängige Überführung von EA in EAC_1,4 besteht mithin aus zwei aufeinanderfolgenden Teilreaktionen, von denen die erste (C₁-Bindung) calciumabhängig und die zweite (C₄-Bindung) von zweiwertigen Ionen unabhängig ist.Mit Unterstützung der Deutschen Forschungsgemeinschaft sowie der Gesellschaft der Freunde und Förderer der Medizinischen Akademie Düsseldorf.     

Effect of isometric exercise on catecholamines in the coronary circulation

Summary Arterial and coronary sinus blood levels of catecholamines, adenosine 3, 5-cyclic monophosphate (c-AMP) and lactate were measured during isometric exercise in fourteen patients. In no patient did lactate production occur. Mean resting total catecholamine levels both arterial (0.53±0.07 ng/ml; 2.94±0.38 nmol/l) and coronary sinus (0.4±0.08 ng/ml; 2.22±0.44 nmol/l), did not change significantly on exercise. Coronary sinus c-AMP levels fell on exercise from 11.5±0.8 nmol/l (resting) to 9.9±0.8 nmol/l (exercise) (P<0.01) with an arterial-coronary sinus difference of 1.2 nmol/l (P<0.01) on exercise.Our findings suggest that isometric exercise does not normally result in excessive cardiac symphathetic activity.     

Synergistic effects of tumour necrosis factor-α and interferon-γ on feline haemopoietic progenitors

Pathogenic mechanisms that underlie feline leukaemia virus subgroup-C (FeLV-C) induced erythroid aplasia are unknown. FeLV-C infection is associated with higher serum levels of interferon- (IFN-) and tumour necrosis factor- (TNF-), which may act synergistically to cause haemopoietic suppression. In the present studies, the synergistic effects of TNF- and IFN- on feline bone marrow progenitors in vitro were evaluated. Bone marrow mononuclear cells from specific-pathogen-free cats were exposed to TNF- (100 and 200 pg/ml) and IFN- (100 or 200 units/ml), alone or in combination, for 2 h before plating for clonal assays of colony forming units. Our results show that TNF- and IFN- in combination caused marked suppression of feline colony forming units-erythroid (CFU-E), burst forming units-erythroid (BFU-E), and colony forming units-fibroblasts (CFU-F), whereas colony forming units-granulocyte/macrophage (CFU-GM) were minimally affected. The same concentrations of TNF- and IFN- alone had minimal effects on CFU-E, BFU-E and CFU-F. These results suggest that TNF- and IFN- may play a significant role in regulating haemopoiesis in cats and may be involved in the pathogenesis of erythroid aplasia in cats infected with feline leukaemia virus.     

Effect of molsidomine on ex vivo platelet aggregation and plasma guanosine 3′∶5′-cyclic monophosphate levels in healthy volunteers

Summary To find out whether 3-morpholino-sydnonimine (SIN 1), the active metabolite of molsidomine, exerts its antiaggregatory effects not only in vitro but also in vivo, we tested ex vivo aggregation before and after intravenous application of molsidomine in healthy volunteers. We also measured plasma levels of guanosine 35-cyclic monophosphate (cyclic GMP) as SIN 1, the bioactive metabolite of molsidomine, becomes effective via activation of soluble guanylate cyclase. In eight out of ten subjects molsidomine had an inhibitory effect on platelet aggregation and a higher threshold concentration of platelet-activating factor was required after molsidomine application to induce irreversible aggregation. Despite the effect on platelets, plasma cyclic GMP levels did not increase. These results suggest that the nitric oxide-containing SIN 1 inhibits platelet aggregation not only in vitro but also in vivo and that this property can be a beneficial effect in antianginal therapy.Abbreviations Cyclic GMP guanosine 35-cyclic monophosphate - NO nitric oxide - PAF platelet-activating factor - PRP platelet-rich plasma - SIN 1 3-morpholino-sydnonimine     

Characterization of the hog cholera virus 5′ terminus

Hog cholera virus (HoCV) 5 terminus of the ALD and GPE(–) strains were analyzed by using rapid amplification of cDNA end method (5RACE). An additional nine nucleotides were found at the 5 termini of genomic RNA in the ALD and GPE(–) strains of HoCV. These nine nucleotides were also conserved in BVDV and were suggested to form a hairpin structure at the 5 terminus by computer-assisted analysis. It seems possible that the secondary structure and/or the 5 terminus sequence has a significant role in the HoCV virus genome.     

Sequence determination of the extreme 5′ end of equine arteritis virus leader region

The extreme 5 end of the leader sequence of four equine arteritis virus (EAV) strains was obtained by using rapid amplification of cDNA end method (5 RACE), and sequenced. Seventeen more nucleotides were added upstream of the 5 end of the EAV published genomic sequence. A common feature among the analyzed EAV isolates was the presence of an AUG start codon within the added sequence and the appearance of an intraleader open reading frame (ORF) of 111 nucleotides which was predicted to encode a peptide of 37 amino acids. The role of this putative intraleader ORF has yet to be determined.     

Bean yellow mosaic,clover yellow vein,and pea mosaic are distinct potyviruses: evidence from coat protein gene sequences and molecular hybridization involving the 3′ non-coding regions

Summary The sequences of the 3 1019 nucleotides of the genome of an atypical strain of bean yellow mosaic virus (BYMV-S) and of the 3 1018 nucleotides of the clover yellow vein virus (CYVV-B) genome have been determined. These sequences contain the complete coding region of the viral coat protein followed by a 3 non-coding region of 173 and 178 nucleotides for BYMV-S and CYVV-B, respectively. When the deduced amino acid sequences of the coat protein coding regions were compared, a sequence identity of 77% was found between the two viruses, and optimal alignment of the 3 untranslated regions of BYMV-S and CYVV-B gave a 65% identity. However, the degree of homology of the amino acid sequences of coat proteins of BYMV-S with the published sequences for three other strains of BYMV ranged from 88% to 94%, while the sequence homology of the 3 untranslated regions between the four strains of BYMV ranged between 86% and 95%. Amplified DNA probes corresponding to the 3 non-coding regions of BYMV-S and CYVV-B showed strong hybridization only with the strains of their respective viruses and not with strains of other potyviruses, including pea mosaic virus (PMV). The relatively low sequence identities between the BYMV-S and CYVV-B coat proteins and their 3 non-coding regions, together with the hybridization results, indicate that BYMV, CYVV, and PMV are distinct potyviruses.     

Experimentelle Pyelonephritis

Zusammenfassung Gesamtlytische Serumkomplement (C)-Aktivität sowie Antikörper(Ak)-Titer wurden zu verschiedenen Zeiten bei experimenteller nichtobstruktiver Enterokokken-Pyelonephritis untersucht. In der akuten Infektionsperiode ergibt sich nach anfänglichem C-Titerabfall ein ausgeprägter Anstieg der antibakteriellen Serumantikörper mit dann entgegengesetztem Verhalten der C-Titerkurve. Bei fortgeschrittener chronischer Pyelonephritis sind bei weiterhin erhöhten Ak-Titern unauffällige C-Titer nachzuweisen.Die C-Titerhöhe ergibt damit keine diagnostischen oder prognostischen Hinweise für das entzündliche Geschehen einer chronischen Pyelonephritis.

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Summary Total lytic C activity and antibody titer in experimental non-obstructive enterococcal pyelonephritis was measured at different times. In the period of acute infection an initial C-titer decrease with a definite rise of antibacterial serum antibodies is found with a subsequent increase to near normal levels of the C-titer curve. In progressive chronic pyelonephritis increased bacterial antibody titers with normal C-titers are found. — The height of the C-titer level therefore does not give any diagnostic or prognostic leads about the inflammatory state of a chronic pyelonephritis.

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Die vorliegenden Untersuchungen wurden mit Hilfe der Deutschen Forschungsgemeinschaft durchgeführt.

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Herrn Prof. Dr.R. Brühl, Trier, zum 70. Geburtstag gewidmet.     

Specific Interaction of a 25-kilodalton Cellular Protein,a 40S Ribosomal Subunit Protein,with the Internal Ribosome Entry Site of Hepatitis C Virus Genome

Translation initiation of hepatitis C virus (HCV) RNA is controlled by an internal ribosome entry site (IRES) contained in 5 noncoding region (NCR) and in several nucleotides of the coding region. The ability of a 25-kilodalton cellular protein (p25) to bind the HCV 5 NCR is correlated with the efficiency of translation initiation of HCV RNA, indicating that this protein plays a critical role in HCV translation (S. Fukushi, C. Kurihara, N. Ishiyama, F. B. Hoshino, A. Oya, and K. Katayama, J Virol 71, 1662–1666, 1997). We have extended the study for identification of the IRES region required for p25 binding. For this purpose, we have performed UV cross-linking competition analyses using 5- or 3- deleted mutants of the HCV 5 NCR as competitor RNAs for binding of p25 to wild-type HCV 5 NCR. Competitor RNAs lacking nucleotides (nt) 47–74 or nt 279–331 did not inhibit p25 binding to the HCV IRES, indicating that these regions are necessary for interaction of the p25 and HCV IRES. Since p25 binding was not observed in the IRES elements of encephalomyocarditis virus and poliovirus in UV cross-linking competition analyses, the p25 binding may be specific for the HCV IRES. p25 bound to the HCV IRES was detected when a purified 40S ribosomal subunit was used for UV cross-linking experiment, indicating that p25 is one of 40S ribosomal subunit proteins. These results reveal an unique interaction between the 40S ribosomal subunit and HCV IRES to contribute to translation initiation of the HCV genome.