The specific features of phononic and magnetic subsystems of type-VII clathrate EuNi2P4.

A type-VII clathrate with a Eu2+ guest embedded into a Ni-P covalent framework, EuNi2P4, was synthesized by a standard two-stage ampoule synthesis and confirmed to crystallize in the orthorhombic space group Fddd with unit cell parameters a = 5.1829(1) Å, b = 9.4765(1) Å, and c = 18.9900(1) Å. A general technique for studying the lattice and magnetic properties of REE containing compounds is proposed. The temperature and field dependences of electrical resistivity ρ(T,H), magnetization M(T,H), magnetic susceptibility χ(T,H), heat capacity Cp(T), and unit cell parameters a(T), b(T), c(T), and volume V(T) were experimentally studied and analyzed at different pressures in the temperature range of 2-300 K. A cascade of anomalies in the studied dependences was identified and attributed to the magnetic phase transformation and peculiar lattice contributions at temperatures below 20 K. As a result of comparison with an isostructural clathrate SrNi2P4, the parameters of the magnetic and lattice contributions were determined. It is characteristic that the phase transition from the paramagnetic to the magnetically ordered state is not reflected in the temperature changes of the lattice parameters due to weak bonds between guest europium atoms and the Ni-P host matrix. We have constructed a tentative H-T phase diagram based on the M(T) and M(H) data, which includes 6 different phases. It is established that the anomalous lattice contribution to the clathrate heat capacity CTLS(T) appears due to the effect of two-level systems (TLS) in the Eu2+ subsystem on the thermodynamic properties of EuNi2P4. The values of TLS parameters as well as the parameters of the magnetic subsystem of the clathrate were determined.

[Aortic thrombosis in a calf]. / Aortenthrombus bei einem Kalb.

A two month-old female Simmental calf was presented with sensomotoric dysfunction and recumbency. Neurologic examination revealed dysfunction of the cerebral cortex and paralysis of both hind limbs. Examination of the skeletal system revealed a marked reduction of the skin temperature of both hind limbs and the absence of femoral pulse. Examination of cerebrospinal fluid yielded physiological parameters. The radiographic examination of the vertebral column, hip and femur on both sides revealed no evidence of alteration of the bone structures. Thiamine pyrophosphate test indicated thiamine deficiency. Based on these findings a tentative diagnosis of cerebrocortical necrosis and aortic thrombosis were made and the animal was euthanised. Post mortem examination yielded thrombosis of the abdominal aorta cranial to the branching of the iliac arteries and consecutive necrosis of the skeletal muscle of the hind limbs. Possible causes of pathogenesis are discussed.

New method for density determination of nanoparticles using a CPS disc centrifuge™.

A model has been developed for the determination of the correct absolute size of nanoparticles using a disc centrifuge; the method does not require externally measured or literature derived particle densities. The principle of this method is the altered settling velocity of particles in fluids with different viscosities and/or densities, with the use of a linear regression analysis for the calculation of particle densities. This allows a fast particle density determination with at least two measurements using a disc centrifuge and a small subset of easily measurable parameters. Furthermore, correctness of the model is evaluated using viruses and nonbiological particles of known densities.

The double-resistance-breaking Tomato mosaic virus strain ToMV1-2 contains two independent single resistance-breaking domains.

Tomato mosaic virus (ToMV) causes a serious loss of yield and fruit quality in tomato crops. To control ToMV, three resistance genes, Tm-1, Tm-2, and Tm-2(2) from wild tomato species were introduced into commercial tomato cultivars. Soon after, however, single and, rarely, double-resistance-breaking virus strains for Tm-1 and Tm-2 emerged. Sequence analysis of a Tm-1/Tm-2 double-resistance-breaking virus, designated ToMV1-2, revealed 30 nucleotide substitutions compared with wild-type ToMV. Of these, six substitutions result in amino acid exchanges. Two exchanges are in the methyl transferase/helicase domain of the ToMV1-2 130/180-kDa proteins (D-1097 to V, R-1100 to Q), two exchanges are found in the 30-kDa movement protein (MP; E-52 to K, E-133 to K), and two exchanges are in the coat protein (I-22 to V, A-87 to S). Construction of chimeric full-length viral cDNA clones containing the base substitutions resulting in altered amino acids, either in the 130/180 kDa proteins or the MP, revealed that the amino acid exchanges in the 130/180-kDa proteins enable ToMV1-2 to break the Tm-1 resistance, while those in the MP enable ToMV1-2 to overcome the Tm-2 resistance. Furthermore, a novel Tm-1/Tm-2 double-resistance-breaking virus was generated by combining the Tm-1-breaking domain of ToMV1-2 and the MP of a new virus, ToMV2, containing the amino acid exchanges E-133 to K and N-135 to S. Together, the data suggest that double-resistance-breaking ToMV1-2 may have resulted from recombination between Tm-1 and Tm-2 single resistance-breaking virus strains.

Identification of bovine enteric Caliciviruses (BEC) from cattle in Baden-Württemberg.

Caliciviruses are known to cause different diseases in many animal species. The bovine enteric caliciviruses (BEC) are associated with diarrhoea in cattle. These viruses have been classified in the genus Norovirus and are closely related to human noroviruses, the leading cause of gastroenteritis in humans. This has raised questions about zoonotic transmission and an animal reservoir for the human viruses. Two samples from 41 stool samples collected for diagnostic purposes from diarrheic cattle in Aulendorf, Germany tested positive for BEC. The samples were amplified with new degenerate BEC specific primers, which amplify a 263 bp portion of the RNA polymerase region. Analysis of the nucleotide sequences showed that these viruses are most closely related to the Norovirus genogroup III/2 (Bo/NLV/Newbury-2/76/UK) viruses.

Periodontal dressing (Vocopac) influences outcomes in a two-step treatment procedure.

OBJECTIVES: It is not clear if periodontal dressing influences the long-term results in a non-surgical treatment procedure. MATERIAL AND METHODS: The periodontal parameters (pre-baseline) of 36 patients with aggressive periodontitis were obtained before the patients were treated initially (1st step) by a dental hygienist, who completely removed the supra- and subgingival concrements. Baseline parameters were raised 3 weeks after the 1st step, before the 2nd therapy step was conducted. It consisted of a non-surgical procedure, which comprised a closed full-mouth manual root curettage (root planing), immediate systemic application of metronidazole, and the placement of a periodontal dressing (Vocopac, Voco). The patients were randomized to two test groups having their periodontal packs removed after 3-4 days (group 1, n=12) and 7-8 days (group 2, n=12), respectively and a control group (n=12) without periodontal dressing. Clinical parameters were raised again after 6 and 24 months. RESULTS: Six and 24 months later, changes in probing pocket depth (PPD) and probing attachment level (PAL) were observed in all three groups compared with baseline, but the difference was significant in group 2 only. In addition, group 2 showed a greater reduction in mean PPD and also a significantly greater gain of attachment in comparison with the controls. CONCLUSION: Wound dressing has a positive effect on clinical long-term results using a two-step non-surgical procedure. Moreover, removing the dressing after 7-8 days leads to clearly better results than removing it earlier.

The Tomato mosaic virus 30 kDa movement protein interacts differentially with the resistance genes Tm-2 and Tm-2(2).

In tomato plants ( Lycopersicon esculentum Mill.), the genes Tm-2 and Tm-2(2) confer resistance to Tomato mosaic virus (ToMV). Sequence analysis of ToMV strains able to break the Tm-2 or Tm-2(2) resistance revealed distinct amino acid exchanges in the viral 30 kDa protein, suggesting that the movement protein is recognized by both resistance genes to induce the plant defense reaction. To analyze the interactions between the ToMV movement protein and the Tm-2 and Tm-2(2) genes in detail, we generated transgenic tomato lines expressing various movement protein gene constructs. Crosses of the transgenic tomato lines with cultivars containing either the Tm-2 or the Tm-2(2) gene demonstrated that both genes are able to elicit a hypersensitive reaction in response to movement proteins from resistance inducing ToMV strains. However, the domains and the structural requirements for induction of the necrotic response by the ToMV movement protein are completely different for either resistance gene. In the context of the Tm-2 gene, the resistance determinant lies within the N-terminal 188 amino acids of the ToMV movement protein. Interaction of the 30 kDa protein with the Tm-2(2) gene requires two distinct domains localized at the C-terminus and in a different region of the protein, respectively.

NIMIN-1, NIMIN-2 and NIMIN-3, members of a novel family of proteins from Arabidopsis that interact with NPR1/NIM1, a key regulator of systemic acquired resistance in plants.

NPR1/NIM1 is a key regulator of systemic acquired resistance (SAR) in Arabidopsis. Using the yeast two-hybrid system, we have identified three novel genes, NIMIN-1, NIMIN-2 and NIMIN-3 (NIMIN for NIM1-interacting) that encode structurally related proteins interacting physically with NPR1/NIM1. NIMIN-1 and NIMIN-2 both bind strongly to NPR1/NIM1 via a common binding motif interacting with the C-terminal moiety of NPR1/NIM1, whereas NIMIN-3 interacts with NPR1/NIM1 via the N-terminal part of NPR1/NIM1. In addition, NIMIN-1, NIMIN-2, and NIMIN-3 are able to interact via NPR1/NIM1 with basic leucine zipper transcription factors of the TGA family in a yeast tri-hybrid system. A mutant protein of NPR1/NIM1, npr1-2, which has been shown to be severely impaired in induction of SAR gene expression, failed to bind the NIMIN proteins. The NIMIN genes are expressed in Arabidopsis plants in response to SAR-inducing treatments, and the NIMIN proteins, like NPR1/NIM1, carry functional nuclear localization signals as revealed by expression of fusion proteins in yeast and in transgenic plants. Taken together, these data indicate that the NIMIN proteins, via physical interaction with NPR1/NIM1, are part of the signal transduction pathway leading to SAR gene expression in Arabidopsis.

The use of copper(I) halides as a preparative tool

The use of copper(I) halides as a preparative tool is discussed with respect to the synthesis of adduct compounds with new polymeric and oligomeric main group molecules. By using this approach new polymers of main group elements--some of which have been predicted by theoretical investigations--can be obtained in a crystalline state and are therefore accessible for a basic structural characterization. Thus, it becomes possible to compare the structural data, experimental data, and theoretical results. Mixed copper halide chalcogenides are accessible when complex copper thiometalates and copper halides are combined. These solid-state compounds are of special interest since they provide experimental access to new main group molecules. In addition, they exhibit a high copper ion conductivity when certain structural features are present in the compounds. A survey is given of basic synthetic and general structural aspects.

Tm-2(2) resistance in tomato requires recognition of the carboxy terminus of the movement protein of tomato mosaic virus.

The Tm-2(2) resistance gene is used in most commercial tomato cultivars for protection against infection with tomato mosaic virus (ToMV). It has been suggested that Tm-2(2) resistance interferes with viral cell-to-cell movement in plants; ToMV strain ToMV-2(2) requires two amino acid (aa) exchanges in the carboxy-terminal region of the viral 30-kDa movement protein (at positions 238 and 244) to overcome Tm-2(2) resistance. For further analysis of this region of the 30-kDa protein, two stop codons were introduced into ToMV movement proteins at aa positions 235 and 237, leading to deletion of the terminal 30 aa. The mutant virus strains were able to infect wild-type tomato plants systemically, suggesting the carboxy-terminal portion of the ToMV 30-kDa protein is dispensable for virus transport in tomato. Even more important, the deletion mutants overcame the Tm-2(2) resistance gene. These data indicate the carboxy-terminal domain of the ToMV movement protein serves as a recognition target in the context of the Tm-2(2) resistance gene. Furthermore, expression of the 30-kDa movement protein from wild-type ToMV, but not from ToMV-2(2), in transgenic tomato plants with the Tm-2(2) resistance gene led to elicitation of a necrotic reaction in tomato seedlings, showing that the 30-kDa protein on its own is able to induce the plant's defense reaction.

Two amino acid substitutions in the tomato mosaic virus 30-kilodalton movement protein confer the ability to overcome the Tm-2(2) resistance gene in the tomato.

The Tm-2(2) resistance gene is used in most commercial tomato cultivars for protection against infection with tobacco mosaic virus and its close relative tomato mosaic virus (ToMV). To study the mechanism of this resistance gene, cDNA clones encompassing the complete genome of a ToMV strain (ToMV-2(2)) that was able to break the Tm-2(2) resistance were generated. Chimeric full-length viral cDNA clones were constructed under the control of the cauliflower mosaic virus 35S RNA promoter, combining parts of the wild-type virus and ToMV-2(2). Using these clones in cDNA infection experiments, we showed that the 30-kDa movement protein of ToMV-2(2) is responsible for overcoming the Tm-2(2) resistance gene in the tomato. DNA sequence analysis revealed four amino acid exchanges between the 30-kDa proteins from wild-type ToMV and ToMV-2(2), Lys-130 to Glu, Gly-184 to Glu, Ser-238 to Arg, and Lys-244 to Glu. To clarify the involvement of the altered amino acid residues in the resistance-breaking properties of the ToMV-2(2) movement protein, different combinations of these amino acid exchanges were introduced in the genome of wild-type ToMV. Only one mutant strain which contained two amino acid substitutions, Arg-238 and Glu-244, was able to multiply in Tm-2(2) tomato plants. Both amino acid exchanges are found within the carboxy-terminal region of the movement protein, which displays a high variability among different tobamoviruses and has been shown to be dispensable for virus transport in tobacco plants. These observations suggest that the resistance conferred by the Tm-2(2) gene against ToMV depends on specific recognition events in this host-pathogen interaction rather than interfering with fundamental functions of the 30-kDa protein.

Expression of a viral avirulence gene in transgenic plants is sufficient to induce the hypersensitive defense reaction.

Tobacco plants containing the N' resistance gene exhibit a hypersensitive defense reaction when infected with tomato mosaic virus (ToMV); infection results in necrotic lesions at the primary infection sites. In an attempt to investigate the molecular mechanism(s) underlying this plant-pathogen interaction, the ToMV coat protein gene was joined by a transcriptional fusion to the strong constitutive 35S RNA promoter from cauliflower mosaic virus. This chimeric gene was introduced via Agrobacterium-mediated transformation into isogenic tobacco cultivars differing only with respect to the N' gene. Strong necrotic reactions were observed on most emerging calli of the N' genotype, but never on calli lacking the N' resistance gene. These data indicate that the coat protein of ToMV is, on its own, sufficient to induce a hypersensitive reaction in tobacco. Thus, recognition of a single viral gene product may be the only prerequisite for the induction of a specific defense reaction in higher plants.

A cDNA clone of tomato mosaic virus is infectious in plants.

A cDNA clone of tomato mosaic virus (ToMV) genomic RNA was fused to the cauliflower mosaic virus 35S RNA promoter and the nopaline synthase gene polyadenylation signal. The transcriptional initiation site of the 35S RNA promoter was altered by in vitro mutagenesis so that the resulting transcripts start at the first nucleotide of the ToMV sequence. In addition, 12 nucleotides were inserted in the 5' untranslated region of the ToMV genome. This plasmid, pSLN, was used to inoculate several host plants of ToMV. Among five plant species tested, only Chenopodium quinoa accumulated large amounts of viral particles. The infectivities and systemic movements of the resulting viruses were the same as those of virus preparations obtained from a ToMV infection of C. quinoa. Primer extension analyses revealed that the 5' end of the viral genomic RNA was identical to those of RNAs isolated from virus progeny of an infection with T7 transcripts analogous to pSLN. Moreover, the insertion in the 5' untranslated region of the viral genome was stably maintained through several systemic passages of the virus. Thus, inoculation of plants with a plasmid containing a cDNA clone of an RNA virus under the control of a eukaryotic promoter seems to be a convenient alternative to the generation of in vitro transcripts and should facilitate the analysis of viral mutants generated at the DNA level.

Functional analysis of the pathogenesis-related 1a protein gene minimal promoter region. Comparison of reporter gene expression in transient and in stable transfections.

Pathogenesis-related (PR) proteins are a heterogeneous group of host encoded, low-molecular-mass proteins that are induced in plants by various external stimuli, such as pathogen attack or exposure of the plants to certain chemicals. To examine the regulation of these genes, the 5'-flanking region of the tobacco PR-1a gene [Pfitzner U.M., Pfitzner, A.J.P. & Goodman, H.M. (1988) Mol. Gen. Genet. 211, 290-295] was joined by a transcriptional fusion to the Escherichia coli beta-glucuronidase (GUS) gene. Expression of the reporter gene was monitored in transient expression assays as well as in stable transformants. The PR-1a 5'-flanking sequences from -335, -149 or -71 to +28 are functional promoter elements in tobacco and carrot protoplasts, as determined by transient expression. These constructs direct correct initiation at the normal transcription-start site of the PR-1a gene. The level of gene expression was about twofold less than that obtained with the cauliflower mosaic virus 35S RNA promoter. Regulation of gene expression by acetylsalicylic acid, however, could not be detected in the transient assays. When the same constructs were stably integrated into the tobacco genome, neither constitutive nor induced beta-glucuronidase activity was observed. A comparison of the results from the transient and the stable transfection experiments suggests that expression of the reporter gene may be due to a constitutive transcriptional activity of the PR-1a 5'-flanking regions under the conditions of the transient assays and that the PR-1a promoter may contain at least two functional domains.

Molecular analysis of two PR-1 pseudogenes from tobacco.

Two independent PR-1 lambda genomic clones (W38/1 and W38/3) were isolated and characterized from a tobacco (Nicotiana tabacum cv. Wisconsin 38) library. Neither clone is identical to the previously described PR-1 cDNA clones, and both clones carry mutations within the highly conserved PR-1 protein coding region. For example, clone W38/1 has a GAA Glu codon instead of the translation stop codon thus harbouring an open reading frame extended by 16 additional amino acids. Furthermore, both clones display considerable variations in the genomic flanking sequences when compared to the PR-1a gene. In order to test whether the encoded genes are active, their upstream sequences were fused to the E. coli beta-glucuronidase (GUS) reporter gene. While significant GUS activities as compared to the 35S RNA promoter from cauliflower mosaic virus (CaMV) were obtained with the W38/1 and W38/3 sequences in transient gene expression assays, no transcriptional activities could be observed upon stable transformation of the same constructs. In addition, the protein coding region of W38/1 was joined to the CaMV 35S RNA promoter and transgenic tobacco plants were generated. However, neither transcripts nor a protein could be detected deriving from the W38/1 structural gene with this chimaeric construct in the transformants. Taken together, these data indicate that the genes contained in lambda clones W38/1 and W38/3 are not active in planta.

Characterization of a cDNA clone corresponding to a transcript from the Epstein-Barr virus BamHI M fragment: evidence for overlapping mRNAs.

A 1.95-kilobase cDNA clone was isolated by screening a size-selected lambda gt10 cDNA library prepared from an Epstein-Barr virus-transformed B-cell line, IB4, with the Epstein-Barr virus BamHI M fragment. Sequence analysis revealed that this clone contains about 75% of the BMRF1 and the complete BMRF2 open reading frames. The transcript is not spliced, and the polyadenylation signal at base pair 2641 of the BamHI M fragment is used. Northern blots (RNA blots) indicate that this polyadenylation signal is used for three overlapping mRNAs. The sizes of these transcripts are 3.5, 2.6, and 1.5 kilobases.

Isolation and characterization of cDNA clones corresponding to transcripts from the BamHI H and F regions of the Epstein-Barr virus genome.

The Epstein-Barr virus (EBV) mutant P3HR1 is incapable of immortalizing B lymphocytes because of a 6.8-kilobase deletion in the BamHI W, Y, and H regions of the viral genome (M. Rabson, L. Gradoville, L. Heston, and G. Miller, J. Virol. 44:834-844, 1982). To characterize transcripts that are encoded in this region, poly(A)+ RNA from the EBV-transformed lymphoblastoid cell line JY was isolated, and this RNA was used to generate a cDNA library in lambda gt10. By screening 500,000 recombinant bacteriophages with the BamHI H fragment, we isolated 10 cDNA clones and characterized them in detail. One group of six cDNA clones was derived from a 2.9-kilobase early transcript encoded by the IR2 repeat element and showed restriction site polymorphism for the enzyme SmaI. The second group consisted of four cDNA clones, all of which contained the BamHI-H right reading frame (BHRF1), and used the polyadenylation signal at base pair 662 in the BamHI F fragment. Computer analysis of the hydrophobicity of the BHRF1 protein revealed that it is likely to be a membrane protein. Northern blotting experiments with RNA from an EBV producer line, B95-8, and a tightly latent lymphoblastoid B-cell line, IB4, revealed that BHRF1 is contained in at least two different mRNA species which can be detected during the latent cycle of EBV. These data and the recent characterization of a spliced transcript (containing five exons in common with other known latent messages [M. Bodescot and M. Perricaudet, Nucleic Acids Res. 14:7103-7113, 1986]) suggest that alternative splicing is used to generate transcripts containing BHRF1, as for the EBV nuclear antigen 1 transcripts. Furthermore, the observation that a potential oncogene activated in human follicular lymphomas is homologous to the BHRF1-encoded polypeptide (M. L. Cleary, S.D. Smith, and J. Sklar, Cell 47:19-28, 1986) suggests a possible role for this putative viral protein in EBV-induced growth transformation of B lymphocytes.