Pathogenic role of a monoclonal IgA (κ) anti-IgG paraprotein associated with hemorrhagic diathesis,rheumatoid arthritis,vascular purpura,and acute membranoproliferative glomerulonephritis

Sixteen years earlier a 42-year-old woman with an IgA plasma cell neoplasm presented with bleeding disorder. Her prolonged course was complicated by subsequent development of rheumatoid arthritis, vascular purpura, and an acute membranoproliferative glomerulonephritis (MPGN). The paraprotein and its (Fab)₂ fragment showed affinity for a test myeloma IgG₂ () paraprotein. The patient's serum and the IgA-IgG complex separated by gel filtration did not exhibit cryoprecipitation. The complex also did not dissociate by ultracentrifugation. Electron microscopic and immunofluorescent studies of a renal biopsy sample taken during the episode of nephritis showed subendothelial deposits and a lacy fluorescent pattern strongly positive for IgA and IgG. The same immunoglobulins were eluted from the kidney at postmortem. A low concentration of monoclonal IgA (antibody) and excess unbound polyclonal IgG (antigen) were demonstrated in the patient's serum at the time of MPGN, apparently analogous to the conditions necessary for the induction of experimental immune complex nephritis.     

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.     

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 small subgenomic RNA of <Emphasis Type="Italic">Soybean dwarf virus</Emphasis>

Summary. We have characterized a small subgenomic RNA of Japanese strains of Soybean dwarf virus (SbDV). Northern blot analyses of SbDV-infected plants showed that the small RNA contained the 3 terminal sequence of the genome and was detected in four typical Japanese SbDV strains, YS, YP, DS and DP. In the case of SbDV-DS, the RNA was 220 nucleotides in length and was transcribed from the 3 terminal region of the genome. This RNA appeared at a similar time to genomic RNA and a large sgRNA, and thereafter persisted in the infected plant. Since no conserved open reading frame (ORF) among the strains was postulated in the 3 terminal region, the small subgenomic RNA may have some regulatory roles in SbDV infections.Received December 12, 2002; accepted April 16, 2003 Published online July 2, 2003     

The nucleotide sequence of the coat protein genes and 3′ non-coding regions of two resistance-breaking tobamoviruses in pepper shows that they are different viruses

Summary The nucleotide sequence of the coat protein genes and 3 non-coding regions of two different resistance-breaking tobamoviruses in pepper have been determined. The deduced coat protein of an Italian isolate of pepper mild mottle virus (PMMV-I) consists of 156 amino acids and its 3 non-coding region is 198 nucleotides long. They have been found to be very similar in sequence and structure to those previously reported for a Spanish isolate (PMMV-S). In contrast, a Dutch isolate termed P 11 codes for a coat protein of 160 amino acids and its 3 non-coding region is 291 nucleotides long, which may have arisen by duplication. The nucleotide and the predicted coat protein amino acid sequence analysis show that this isolate should be considered as a new virus within the tobamovirus group. The term paprika mild mottle virus (PaMMV) is proposed.     

DNA fingerprinting involving fluorescence-labeled termini of any enzymatically generated fragments of DNA

Summary We have developed a new fluorescence-based method for DNA fingerprinting that does not require a fluorescent linker or a synthetic oligonucleotide primer, both of which are normally used for labeling of DNA. Cosmid DNAs are digested with appropriate restriction enzymes and the 3 termini of DNA fragments are labeled with the corresponding, fluorescent dye-conjugated dideoxynucleotide triphosphate terminator (dye-ddNTP) by the Klenow fragment of DNA polymerase I fromEscherichia coli, which has 35 exonuclease and replacement activities as well as its main 53 polymerase activity. Samples are separated on a DNA-sequencing gel and data are analyzed by application of both the Version 0.3.8a mapper program (Applied Biosystem Inc., Foster City, CA) and our Overlap I program that facilitate rapid analysis of the frequency of overlapping of cosmid DNAs. Using this method we have determined the overlap frequency of DNA fragments of each cosmid clone from the mouse MHC class I gene cluster.     

A broad bean mitochondrial atp6 gene with an unusually simple,non-conserved 5′ region

Summary A nucleotide sequence of broad bean mitochondrial DNA (mtDNA) that contains an atp6 gene of 876 ntp is presented. Relative to other plant atp6 genes, this broad bean gene comprises a 90 ntp non-conserved 5 region, a 759 ntp highly conserved central region and a 27 ntp non-conserved 3 region. The non-conserved, 5 region of the broad bean atp6 gene differs from the corresponding regions of most other plant atp6 genes in that it contains only one potential translation initiation codon and, following this codon, a 63 ntp segment that predicts an amino acid sequence with a predominance of alternating leucines.     

Genetic Analysis of Pestiviruses at the 3′ End of the Genome

Specific PCR primers were selected for each pestivirus genotype which flanked the 3-part of the NS5B gene and more than three quarters of the 3-UTR. PCR products were sequenced in both directions using an automatic sequencing device and analyzed by computer package program DNASTAR. A comparative analysis of the 3 untranslated region (3-UTR) of 82 viruses, representing the four genotypes of the Pestivirus genus, provided a similar phylogenetic grouping as other genomic regions. Intertypic recombination was not observed, but Border disease virus (BDV) and Bovine viral diarrhoea virus type 1 (BVDV I) showed great intragenotypic variability. In most pestiviruses the stop codon is TGA, but BDV isolates were found to have either a TAG or a TAA stop codon. Various deletions and insertions were observed in the 3-UTR region. Viruses of the BVDV Ib group contained a characteristic deletion of 41 nucleotides. Compared to the 5-UTR, the 3-UTR was less conserved. The first 50–60 nucleotides were particularly variable, whilst the most conserved part was found at the 3 end of the studied region. All 82 viruses contained AT-rich stretches, the positions and sizes of which differed between the genotypes.     

Proteins specifically binding to the 3′ untranslated region of hepatitis A virus RNA in persistently infected cells

Summary Establishment of persistency is the common result of hepatitis A virus (HAV) infection in most HAV/cell culture systems. Previous studies provided evidence that shortly before or concomitantly with establishment of persistent infections synthesis of viral RNA is down-regulated. This may be an effect of regulating factors. Using RNA/protein binding assays it was shown that, at the critical time during virus replication, proteins accumulate which interact specifically with a distinct nucleotide sequence (HPE) within the 3 non-coding region of the HAV genome and/or (HME) within the 5 terminal region of the HAV antigenome. The sequences consist of 23 nucleotides (HPE: 5-AAAUUUUCUUAAAAUUUCUGAGG-3; HME: 5-CCUCAGAAAUUUUAAGAAAAUUU-3). A sequence with 79% similarity was found in the corresponding 3 non-coding region of poliovirus type I (Sabin) RNA. The latter sequence was shown to bind proteins from HAV infected cells but comparable proteins were absent in cells infected poliovirus.     

Adenosine 3′,5′-cyclic monophosphate in rabbit cerebrospinal fluid during fever induced byE. coli-endotoxin

In rabbit cerebrospinal fluid during fever induced byE. coli-endotoxin adenosine 3,5-cyclic monophosphate concentrations are about 2-fold higher in comparison to normal values. In addition to prostaglandins of the E series adenosine 3,5-cyclic monophosphate might act as another mediator in the genesis of fever during infectious diseases.This investigation was supported by the Deutsche Forschungsgemeinschaft.     

Komplement- und Antikörper-Titer bei Patienten mit chronischer Bronchitis

Zusammenfassung Bei insgesamt 76 Patienten mit chronischer Bronchitis wurden Komplement(C)- und Antikörper(Ak)Titer (gegen die jeweiligen aus Sputumproben gezüchteten Keime) bestimmt. Die C-Titer (CH50/ml) lagen bei 57 Patienten (= 75%) im Normbereich, bei 17 Patienten (= 22,4%) oberhalb und bei 2 Patienten (= 2,6%) unterhalb der Grenzwerte bei Kontrollpersonen. Dabei tendierten bei höheren Ak-Titern die C-Titer zu niedrigeren Werten. Es wird vermutet, daß kontinuierliche Immunreaktionen den relativen Abfall der C-Titer bei gleichzeitig hohen Ak-Titern bedingen.

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Summary In 76 patients with chronic bronchitis total complement (C) activity and antibody titer (versus correspondent microorganisms cultured from sputum samples) were determined. 57 patients (= 75%) were found to have normal C titers (CH50/ml), in 17 cases (= 22,4%) the C titers were elevated above and in 2 cases (= 2,6%) reduced under limiting values of control persons. The C titers tended to lower levels when antibody titers increased. It is supposed that continuous immune reactions cause the relative decrease of complement activity when antibody titers are increased.

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Mit finanzieller Beihilfe der Europäischen Gemeinschaft für Kohle und Stahl durchgeführte Forschungsarbeit.     

Two polymorphicAvaI andHhaI sites in a differentially methylated region of the human H19 gene

Summary The H19 gene is paternally imprinted both in the human and mouse (Bartolomeiet al., 1991; Zhang and Tycko, 1992), although its expression pattern seems somewhat different between the two species (Jinno,et al., 1995). DNA-methylation is a promising candidate for a parent-of-origin mark of the gene, and a paternal allele-specific methylation imprint was recently identified at the mouse H19 locus (Tremblayet al., 1995). We found a 50% methylated region in the human H19 gene (Jinno, unpublished data). A search for polymorphisms in this region revealed two novelAvaI andHhaI RFLPs, which contribute to the detection of allele-specific methylation at the human H19 locus.PCR primers for the AvaI-site PANL2 5-GAGCCTGCCAAGCAGAGCG-3 - PANR2 5-CACATAAGTAGGCGTGACTTGA-3 PCR primers for the HhaI-site ASMA 5-CAATGAGGTGTCCCAGTTCCA-3 - PANR2 5-CACATAAGTAGGCGTGACTTGA-3     

Characterization of human larynx carcinoma cell lines HLaC′79 and HLaC′82: a common origin but diverged malignancies

As part of a study on the relationship of tumour phenotype and behaviour, we have characterized two head and neck squamous cell carcinoma cell lines, derived from human laryngeal carcinomas and designated HLaC79 and HLaC82. Cytogenetic analysis revealed that HLaC79 and HLaC82 shared 10 major chromosome rearrangements indicating that the cell lines had a common origin. In the extremely complex chromosomal patterns, abnormalities were found in chromosomes 1, 3 (surplus 3q) and 5 (i(5p) × 2). Both cell lines displayed constitutive expression of vimentin and were capable of anchorage-independent growth in agarose gels. However, in spite of their common origin specific differences were found. Cells of HLaC79 were spindle shaped and formed tumours in athymic mice. In contrast, cells of HLaC82 had a compact morphology, contained less vimentin, were more contact inhibited and were not tumorigenic. These results indicate that malignant transformation in HLaC82 was partially reversed.     

Studien über den Mechanismus der Immunhämolyse: Die Bindung der zweiten Komplementkomponente

Zusammenfassung Es wird die Reaktion EAC_1,4 + C₂ isoliert untersucht. Hierzu bietet man einer Charge von stufenweise aufgebautem EAC_1,4 ein von allen übrigen Komplementkomponenten befreites C₂-Präparat an. Bei der Reaktion kommt es zu einem meßbaren Schwund von C₂ im Überstand; gleichzeitig erwerben die Zellen die Fähigkeit, mit cheliertem Komplement zu lysieren. Die Reaktionsgeschwindigkeit ist bei 37° C hoch und bei 0° C gering. Für die Fähigkeit der Zellen, C₂ zu binden, ist nicht allein das gebundene C₄ maßgebend, sondern ein von C₁ nicht abgrenzbarer Zusatzfaktor. Demnach kommen dem C₁ nach seiner Bindung zwei Funktionen zu: Einmal vermittelt es die Bindung von C₄, zum anderen vermittelt es zusammen mit dem gebundenen C₄ die Bindung von C₂.Mit Unterstützung der Deutschen Forschungsgemeinschaft sowie der Gesellschaft der Freunde und Förderer der Medizinischen Akademie Düsseldorf.     

Determination of the 5′ and 3′ terminal noncoding sequences of the bi-segmented genome of the avibirnavirus infectious bursal disease virus

Summary Terminal sequences of the bi-segmented dsRNA genome of 3 different strains of infectious bursal disease virus (IBDV) were analyzed by the rapid amplification of cDNA 5 ends (5RACE) procedure. Both segments are 85% homologous in a 32-nucleotide sequence comprising the 5 end, whereas the 3 end has a conserved pentamer. Comparison to published terminal sequences of other IBDV strains revealed high conservation between the two segments but more serotype-specific nucleotide changes (5 on segment A and 3 on segment B) in the 5 noncoding region compared to the 3 noncoding region (none on segment A and 1 on segment B).     

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.     

Attenuated Lapinized Chinese Strain of Classical Swine Fever Virus: Complete Nucleotide Sequence and Character of 3′-Noncoding Region

The complete nucleotide sequence including precise 5- and 3-terminal non-coding regions (NCRs) of the attenuated lapinized Chinese strain (HCLV) of Classical Swine Fever Virus (CSFV) was determined from overlapping cDNA clones constructed by separated RT-PCR and rapid amplification of cDNA ends (RACE) methods. The genomic RNA of the HCLV strain consists of 12,310 nucleotides (nts) including 374nts and 242nts in the 5- and 3-NCRs, respectively. It contains one large open reading frame (ORF) encoding a polyprotein of 3,898 amino acids with a calculated molecular weight of 437.6kDa. There is one notable insertion of 12 continuous nts, CTTTTTTCTTTT in the 3-NCR of HCLV genomic cDNA when compared with its parental virulent Shimen strain. Sequence alignment of partial 3-NCR reveals two groups of CSFV vaccine strains carrying similar T-rich insertions at different positions in this region. Computer-predicted secondary structures suggest that T-rich insertion greatly change the structures and thus decrease the promoter functions of 3-NCRs during the replications of these two groups of CSFV vaccine strains.     

Effects of 5-hydroxytryptamine,dopamine, and acetylcholine on accumulation of cyclic AMP and cyclic GMP in the anterior byssus retractor muscle ofMytilus edulis L. (Mollusca)

We investigated in vitro accumulation of adenosine 3,5-monophosphate (induced by 5-hydroxytryptamine and dopamine) and of guanosine 3,5-monophosphate (induced by acetylcholine) in the anterior byssus retractor muscle ofMytilus.The response to 5-hydroxytryptamine exceeded that induced by equimolar concentrations of dopamine.1-methyl lysergic acid, a 5-hydroxytryptamine-blocking agent, diminished the 5-hydroxytryptamine-induced increase of cyclic AMP level. This parallels the effect of this amine on the contracted muscle.Acetylcholine, which causes a tonic contraction of the muscle, increased intracellular levels of cyclic GMP in a dose-dependent (max. 45-fold at 10^–4 M ACh) manner. The time course of the rise in cyclic GMP level was rapid and transient (peak concentration of cyclic GMP at 2 min).Mytolon was the most effective of all cholinergic blockers tested. It was concluded that cyclic nucleotides may play a role in the modulatory process of the transmitters. A direct relation to the relaxation-contraction process could not be established.Abbreviations ABRM anterior byssus retractor muscle - cyclic AMP adenosine 3,5-monophosphate - cyclic GMP guanosine 3,5-monophosphate - ACh acetylcholine - 5-HT 5-hydroxytryptamine, serotonin - DA dopamine - TCA trichloroacetic acid - UML 1-methyl lysergic acid hydrogen maleinate     

Association of neutropenia in systemic lupus erythematosus (SLE) with anti-Ro and binding of an immunologically cross-reactive neutrophil membrane antigen

SLE is associated with the production of autoantibodies to self-constituents. In particular, certain ribonucleoprotein particles are targeted. Despite the multitude of autoantibodies produced and the remarkable concentrations of these antibodies in the sera of SLE patients, there have been little data that the autoantibodies found in SLE are involved in the pathogenesis of disease or its manifestations. The present work demonstrates that anti-Ro (or SSA) is associated with granulocytopenia, binds the surface of granulocytes and fixes complement to this membrane surface. Binding is a property of anti-Ro Fab fragments and can be inhibited by 60-kD Ro. However, the antigen bound on the surface of granulocytes is a 64 000 mol. wt protein that is a novel autoantigen in SLE. As suggested by inhibition studies, sequence identity between 60-kD Ro and eight tandem repeats in the 64-kD antigen may be responsible for the observed serologic cross-reactivity. These data imply that anti-Ro antibodies that also bind the 64-kD protein mediate neutropenia in patients with SLE.     

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.