New phenotypes of the drought-tolerant cbp20 Arabidopsis thaliana mutant have changed epidermal morphology.

This paper provides a detailed phenotypic analysis of the abscisic acid (ABA) hypersensitive Cap Binding Protein 20 (cbp20) mutant. Some hitherto undescribed changes were found in the tissue structure and epidermal morphology of this mutant. These include more and smaller cells in the epidermis, a thicker cuticle and more frequent occurrence of trichomes on leaf surfaces. Some of these traits may contribute to the physiological processes responsible for the water-saving behaviour of the mutant. Abnormal spatial patterns between stomatal pore complexes were also found on various organs of the mutant. All these observations indicate profoundly disturbed development of epidermal tissue in the cbp20 mutant, which has not previously been reported for this class of mutants. A potential connection between the new phenotypes and disturbed miRNA metabolism and mRNA splicing of the mutant is discussed.

Herbal remedies of Solidago--correlation of phytochemical characteristics and antioxidative properties.

In this study the correlation of phytochemical characteristics and antioxidative properties of classical herbal tea extracts-Infusum solidaginis, Decoctum solidaginis, Maceratum solidaginis-and tinctures prepared by various concentration of ethanol (40, 70, 96% v/v) have been examined for the release of flavonoids and their antioxidant activity. Quantitative and composition determination of flavonoids were carried out by spectrophotometry, high-performance liquid chromatography and capillary electrophoresis, respectively. Hydrogen-donating ability and reducing power properties were used to define in vitro radical scavenging activity of Solidago extracts, but integral antioxidative capacity was determined by luminometry (Photochem), calculating the ascorbic acid equivalents. Chlorogenic acid, quercetin-3-O-beta-D-rutinoside, quercetin-3-O-beta-D-galactoside, quercetin-3-O-beta-D-glucoside, quercetin-3-O-beta-D-rhamnoside, kaempferol-3-O-alpha-L-rhamnoside and quercetin were confirmed by retention times and UV spectra. Based on the dissolution rate, variance of flavonoid release and ascorbic acid equivalents it was concluded, that Tinctura solidaginis (70% v/v ethanol) and Infusum solidaginis are the most appropriate preparations.

[Determination of the type and subtype of the hepatitis C virus in chronic viral hepatitis patients in Hungary]. / A magyarországi krónikus "C" vírushepatitises betegek vírustípus- és szubtípus-meghatározása.

The huge variability of hepatitis virus C is well-established. The geographical differences in its nucleotide sequence have important clinical significance by causing variant pathogenicities and affecting sensitivity to therapy. The authors pioneered the determination of the viral type and subtype in patients suffering from chronic viral hepatitis in Hungary. In this present work they report the applied methods and the results. In their virus serological laboratory was introduced the test "HCV Serotyping 1-6 Assay", which is based on the analysis of antibodies, in 1996. By this method they examined the samples of 127 patients and they found type 1 in 75.5%, type 4 in 25%, mixed types (type 1 + 2, 3, 4, 5, 6) in 9% and non-classifiable, non-reacting antibodies in 13% of the cases. Since 1999 they have used the combination of direct reverse-PCR of the viral antigen and reverse hybridization to type-specific specimens. These results, from 211 patients show that 6% belong to 1a, 85.5% to 1b, 3% to 1a + 1b, 1% to 1b + 2, 0.5% to 3 and 4% to mixed subtypes. Genotype 1b was associated with higher viremia. On the basis of the above they can conclude that 90% of the Hungarian population are infected by the most resistant 1b subtype of hepatitis virus C. It could explain the fact that only 20% of their patients with interferon monotherapy have become permanently virus-free. In view of these results they recommend combined, higher dose, long-lasting treatment in therapeutic protocols.

Identification of site-specific recombination genes int and xis of the Rhizobium temperate phage 16-3.

Phage 16-3 is a temperate phage of Rhizobium meliloti 41 which integrates its genome with high efficiency into the host chromosome by site-specific recombination through DNA sequences of attB and attP. Here we report the identification of two phage-encoded genes required for recombinations at these sites: int (phage integration) and xis (prophage excision). We concluded that Int protein of phage 16-3 belongs to the integrase family of tyrosine recombinases. Despite similarities to the cognate systems of the lambdoid phages, the 16-3 int xis att system is not active in Escherichia coli, probably due to requirements for host factors that differ in Rhizobium meliloti and E. coli. The application of the 16-3 site-specific recombination system in biotechnology is discussed.

Genetic interrelationships and genome organization of double-stranded RNA elements of Fusarium poae.

The similar sized double-stranded RNA (dsRNA) elements present in vegetatively compatible strains of Fusarium poae were always genetically related, while vegetatively incompatible strains of the fungus contained either homologous or non-homologous dsRNAs of the same size. Electron microscopic observations revealed the co-existence of encapsidated and naked dsRNA elements in the same host. A mycovirus, named FUPO-1 was purified from strain A-11 and was found to contain two kinds of dsRNA segments, dsRNA 1 and dsRNA 2. The dsRNA genome of these segments was converted to cDNA clones by reverse transcription and the clones were subjected to sequence analysis. The single long open reading frame deduced from the sequence of dsRNA 1 showed similarities to the putative coat protein genes known from other mycoviruses, while conserved motifs of an RNA-dependent RNA polymerase were identified in the predicted amino acid sequence of dsRNA 2. The genome organization and certain sequence motifs of FUPO-1 show similarities to that of the Atkinsonella hypoxylon 2H virus and the FusoV mycovirus, members of the Partitiviridae family.

Molecular and cytogenetic characterization of a transgene locus that induces silencing and methylation of homologous promoters in trans.

One type of homology-dependent gene silencing in transgenic plants involves a silencing locus that is able to transcriptionally inactivate and methylate an unlinked target locus with which it shares sequence identity in promoter regions. In a manner resembling paramutation of endogenous genes, the target locus reactivates and loses methylation progressively over several generations after segregating away from the silencing locus, which autonomously acquires stable methylation. To investigate the origins of trans-silencing ability and susceptibility, we have analyzed the structures, flanking DNA sequences and chromosomal locations of a nopaline synthase promoter silencing locus, H2, and a sensitive target locus, K81. A partially resistant target locus, K alpha has been characterized molecularly. The complex and scrambled H2 locus comprises six copies of the nopaline synthase promoter, two of which are collinear with prokaryotic non-T-DNA sequences, and is integrated close to a region of intercalary heterochromatin. These features probably contribute collectively to the silencing ability because H2 subclones reintroduced into random locations in the K81 genome did not frequently induce silencing. Both the K81 and K alpha loci have simple structures, although the former contains non-T-DNA prokaryotic sequences that are also present at H2, and they are flanked by low copy plant DNA. H2 and K81 might interact effectively because they are present on morphologically similar chromosomes from the T subgenome of allotetraploid tobacco.

Detection of Lawsonia intracellularis in Hungarian swine herds by polymerase chain reaction.

Pooled faecal samples and/or intestinal contents from pig carcasses were collected from 11 Hungarian swine farms and subjected to DNA extraction/ purification and subsequent polymerase chain reaction (PCR) in order to detect Lawsonia intracellularis, the aetiological agent of the porcine proliferative enteropathy complex. Specific PCR positivity was detected in 6 individual and 22 pooled samples out of 46, originating from eight herds. The PCR products of collected faecal samples hybridised in Southern blot hybridisation with the DNA of the type strain L. intracellularis NCTC 12657. This is the first confirmed detection of L. intracellularis by PCR in Hungary.

Molecular and cytogenetic analyses of stably and unstably expressed transgene loci in tobacco.

To study the influence of genomic context on transgene expression, we have determined the T-DNA structure, flanking DNA sequences, and chromosomal location of four independent transgene loci in tobacco. Two of these loci were stably expressed in the homozygous condition over many generations, whereas the other two loci became unstable after several generations of homozygosity. The stably expressed loci comprised relatively simple T-DNA arrangements that were flanked on at least one side by plant DNA containing AT-rich regions that bind to nuclear matrices in vitro. Of the unstably expressed loci, one consisted of multiple incomplete T-DNA copies, and the second contained a single intact T-DNA; in both cases, however, binary vector sequences were directly contiguous to a right T-DNA border. Fluorescence in situ hybridization demonstrated that the two stably expressed inserts were present in the vicinity of telomeres. The two unstably expressed inserts occupied intercalary and paracentromeric locations, respectively. Results on the stability of transgene expression in F1 progeny obtained by intercrossing the four lines and the sensitivity of the four transgene loci to inactivation in the presence of an unlinked "trans-silencing" locus are also presented. The findings are discussed in the context of repetitive DNA sequences and the allotetraploid nature of the tobacco genome.

[Practical experiences in the therapy of postweaning edema disease in piglets]. / Praktische Erfahrungen bei der Therapie der Odemkrankheit der abgesetzten Ferkel.

In a field trial 23 weeks old weaned piglets were orally inoculated with E. coli O139 K12 H1. The piglets received the same food and were kept under the same management regime. They were selected randomly into four groups and treated after the first clinical signs of CNS involvement of edema disease as follows during three days: Group 1: received a single daily i.m. application of 4 mg/kg body weight Melperone. Group 2: received a single daily i.m. application of 2 mg/kg body weight Amperozide. Group 3: was treated orally (intranasally) with 2 mg/kg body weight Amphetamin in a single daily application. Group 4: untreated control. The following parameters were evaluated before the begin of the therapy (day 0-4). A: Occurrence of diarrhea in a group B: Food consumption per piglet per day C: Death of piglets per group After the begin of the therapy (day 5-32) D: Death of piglets per group E: Average daily feed intake per piglet The results showed that the Melperone and Amphetamin treatment was superior regarding all examined parameters when compared to Amperozide treatment and to the control group.

Structural instability of a transgene locus in tobacco is associated with aneuploidy.

This paper describes molecular and cytogenetic evidence for the stability of a transgene locus that is present on the triplicated chromosome in an aneuploid tobacco line. This instability was manifested in several ways in trisomics including a major chromosome rearrangement that was detectable cytogenetically, smaller scale DNA rearrangements that occurred both germinally and somatically, and methylation/epigenetic silencing. In a deletion derivative of the locus, DNA breakpoints were found in AT-rich regions. One of these regions binds to nuclear scaffolds in vitro, suggesting a possible role for aberrant topoisomerase II cleavage in destabilization of the locus. The implications of increased chromosome instability in aneuploids for plant karyotype evolution and human carcinogenesis are discussed.

Gene silencing mediated by promoter homology occurs at the level of transcription and results in meiotically heritable alterations in methylation and gene activity.

The promoter homology-dependent inactivation of a 35Spro-hygromycin phosphotransferase (hpt) gene, which is present at the H2 locus, by the multipurpose 271 silencing locus has been studied. The 271 locus can silence any gene under the control of the 35Spro as well as endogenous nitrite reductase (NiR) genes of tobacco because of the presence of a chimeric antisense gene (35Spro-RiN). All F1 progeny of a cross between homozygous H2 and 271 lines were sensitive to hygromycin and were chlorotic (a symptom of nitrogen deficiency). These phenotypes were accompanied by a reduction in the steady-state levels of Hyg and NiR transcripts. Transcriptional run-on experiments indicated, however, that while NiR silencing occurred post-transcriptionally, the hpt gene was inactivated at the transcriptional level; this was associated with increased methylation of the 35Spro of the hpt gene. NiR gene expression recovered uniformly to wild-type levels in first generation backcross (BC1) progeny that did not inherit the 271 locus. In contrast, hygromycin resistance was only partially and non-uniformly regained among adult BC1 plants. Moreover, substantial silencing of the hpt gene could persist into the BC2 generation. Genomic sequencing demonstrated that the meiotic heritability of hpt silencing in the absence of the 271 locus was correlated with cytosine methylation primarily at CpG and CpNpG residues. Despite this residual methylation, H2 loci weakened by an association with 271 did not acquire the ability to silence a 'naive' H2 locus. Fluorescence in situ hybridization revealed that the 271 locus was located at a telomere. The results strengthen the distinction between silencing effects involving homology restricted to coding or promoter regions, respectively. The former is a post-transcriptional process that is meiotically reversible; the latter is due to transcriptional inactivation and is associated with increased promoter methylation, which can lead to meiotically heritable reductions in target gene activity. The relevance of these data for the meiotic heritability of silencing, the non-transferability of silencing activity, and the basis of 271 silencing effects is discussed.

High-frequency occurrence of virus-like particles with double-stranded RNA genome in Fusarium poae.

Fifty-five geographically different strains of Fusarium poae were assayed for the presence of extrachromosomal nucleic acid elements. All strains were found to harbour double-stranded RNA (dsRNA) elements and encapsidated virus-like particles (VLP). There were great individual differences in dsRNA patterns of the various strains, but numbers and sizes characteristic for a given isolate remained unchanged after repeated subculturing of the fungi. Morphological alterations or signs of degeneration were not observed in dsRNA-containing isolates. This is the first report on the ubiquitous occurrence of dsRNAs in a hyphomycete fungus species.

Inheritance and expression of a transgene insert in an aneuploid tobacco line.

A T-DNA locus comprising nptII, uidA and nos genes--all under the control of the nos promoter (this locus was designated K because it encodes resistance to Kanamycin)--was found to be inherited erratically in a transgenic tobacco line. This anomalous behavior was partially explained following a karyotype analysis of plants representing several generations: these plants were aneuploids, presumably for the K-containing chromosome. During four generations of sexual propagation, transgenic plants that were either trisomic or tetrasomic for the K-containing chromosome (i.e. 2n = 49 or 2n = 50, respectively) were obtained. The trisomic plants (2n = 48 + 1) were virtually indistinguishable phenotypically from normal euploids (2n = 4x = 48), whereas the tetrasomic plants (2n = 48 + 2) were smaller, had somewhat misshapen leaves and exhibited reduced fertility. Although the amount of NPTII protein in different trisomic (K--, KK-, KKK) and tetrasomic (KK--, KKK-) plants was generally consistent with a K dosage effect, the genetic behavior of each trisomic--with respect to segregation of KanR and marker gene activity in progeny--was unique and not completely explicable by invoking aneuploidy. Specifically, unexpected gains or losses of K could occur, suggesting the formation of double reductional gametes and/or frequent gene conversion at this locus. The susceptibility of K locus marker genes to trans-inactivation in the trisomic and tetrasomic lines was tested by crossing in partially homologous silencing loci. In all transgenotypes tested, the three K marker genes were sensitive to trans-silencing, which was accompanied by methylation in all copies of the nos promotor. In addition to this directed inactivation/methylation, the K locus could also undergo infrequent, spontaneous partial methylation, which produced stable epialleles. In most plants, however, the multiple copies of the nos promoter at this locus remained unmethylated and active through four generations in all transgenotypes examined. The significance of these results for irregular inheritance patterns, aneuploid syndromes and homology-dependent gene silencing is discussed.

CD4 mAb therapy modulates alloantibody production and intracardiac graft deposition in association with selective inhibition of Th1 lymphokines.

The accelerated (24 h) rejection of (LEWxBN)F1 cardiac allografts (Tx) in LEW rats sensitized with BN skin grafts, is abrogated with CD4 mAb (BWH-4) administration between skin (day -7) and heart (day 0) transplantation (Tx survival ca. 11 days, p < 0.0001). This study analyzed the effects of CD4-targeted therapy upon host IgG and IgM alloantibody (allo-Ab) within the serum by two-color flow cytometry, and within the Tx, by immunohistology. These data were correlated with mRNA and protein production profiles of Th1 (IL-2, IFN-gamma) vs Th2 (IL-4) specific cytokines (polymerase chain reaction and/or immunohistology). Skin grafts elicited a strong systemic IgM allo-Ab response, which peaked at the time of cardiac Tx rejection at 24 h. It was associated with extensive deposits of IgM on Tx endothelium. Treatment with BWH-4 mAb diminished circulating IgM allo-Ab levels, and only low levels of IgM could be detected at the Tx site. Conversely, the low circulating IgG allo-Ab levels during rejection at 24 h in untreated recipients were accompanied by a strong labeling for intra-Tx IgG. BWH-4 mAb therapy did not prevent totally the switch of the IgM to IgG, but the IgG allo-Ab response was earlier, less intense and more transient than in untreated recipients. Accelerated rejection triggered sequential lymphokine mRNA expression in cardiac Tx, with the peak of transcription for IL-2 (6-12 h) preceding that for IL-4 (24 h). Interestingly, although CD4 targeted therapy virtually ablated the induction of IL-2 mRNA, it preserved transcription of the IL-4 gene. BWH-4 mAb therapy decreased otherwise abundant intra-Tx IL-2 and IFN-gamma, but allowed a vigorous elaboration of IL-4, confirming the translation of mRNA to the protein in vivo. Thus, CD4 mAb-mediated abrogation of accelerated cardiac Tx injury correlates with suppression of Th1 responses (depressed IL-2 and IFN-gamma production), but sparing of the Th2 function (enhanced IL-4 elaboration). Indeed, CD4 mAb-induced allo-Ab depression and immunosuppressive effects may reflect selective targeting of proinflammatory Th1-like cells and the multifaceted effects of IL-4 produced by unopposed Th2-like cells.

The bacterial attachment site of the temperate Rhizobium phage 16-3 overlaps the 3' end of a putative proline tRNA gene.

Bacteriophage 16-3 inserts its genome into the chromosome of Rhizobium meliloti strain 41 (Rm41) by site-specific recombination. The DNA regions around the bacterial attachment site (attB) and one of the hybrid attachment sites bordering the integrated prophage (attL) were cloned and their nucleotide sequences determined. We demonstrated that the 51 bp region, where the phage and bacterial DNA sequences are identical, is active as a target site for phage integration. Furthermore it overlaps the 3' end of a putative proline tRNA gene. This gene shows 79% similarity to the corresponding proline tRNA-like genomic target sequence of certain integrative plasmids in Actinomycetes.