The Relationship between the Structure of the Tick-Borne Encephalitis Virus Strains and Their Pathogenic Properties

Tick-borne encephalitis virus (TBEV) is transmitted to vertebrates by taiga or forest ticks through bites, inducing disease of variable severity. The reasons underlying these differences in the severity of the disease are unknown. In order to identify genetic factors affecting the pathogenicity of virus strains, we have sequenced and compared the complete genomes of 34 Far-Eastern subtype (FE) TBEV strains isolated from patients with different disease severity (Primorye, the Russian Far East). We analyzed the complete genomes of 11 human pathogenic strains isolated from the brains of dead patients with the encephalitic form of the disease (Efd), 4 strains from the blood of patients with the febrile form of TBE (Ffd), and 19 strains from patients with the subclinical form of TBE (Sfd). On the phylogenetic tree, pathogenic Efd strains formed two clusters containing the prototype strains, Senzhang and Sofjin, respectively. Sfd strains formed a third separate cluster, including the Oshima strain. The strains that caused the febrile form of the disease did not form a separate cluster. In the viral proteins, we found 198 positions with at least one amino acid residue substitution, of which only 17 amino acid residue substitutions were correlated with the variable pathogenicity of these strains in humans and they authentically differed between the groups. We considered the role of each amino acid substitution and assumed that the deletion of 111 amino acids in the capsid protein in combination with the amino acid substitutions R16K and S45F in the NS3 protease may affect the budding process of viral particles. These changes may be the major reason for the diminished pathogenicity of TBEV strains. We recommend Sfd strains for testing as attenuation vaccine candidates.     

Germline integration of moloney murine leukemia virus at the Mov13 locus leads to recessive lethal mutation and early embryonic death

Thirteen mouse substrains genetically transmitting the exogenous Moloney murine leukemia virus (M-MuLV) at a single locus (Mov locus) have been derived previously. Experiments were performed to investigate whether homozygosity at the Mov loci would be compatible with normal development. Animals heterozygous at an Mov locus were mated, and the genotype of the offspring was analyzed. From parents heterozygous at the loci Mov1 to Mov12, respectively, homozygous offspring were obtained with the expected Mendelian frequency. In contrast, no homozygous offspring or embryos older than day 15 of gestation were obtained from parents heterozygous at the Mov13 locus. When pregnant Mov13 females at day 13 and day 14 of gestation were analyzed, approximately 25% of the embryos were degenerated. Genotyping revealed that these degenerated embryos were invariably homozygous and the normal appearing embryos were either heterozygous or negative for M-MuLV. These results suggest that integration of M-MuLV at the Mov13 locus leads to insertion mutagenesis, resulting in embryonic arrest between day 12 and day 13 of gestation. It is possible that the Mov13 locus represents a gene or gene complex involved in the early embryonic development of the mouse.     

Chronology of chromosome reduplication in Chinese hamster cells incubated at low temperature

Experiments have been carried out on Chinese hamster fibroblasts. Cultures at the log-stage of growth were incubated at 15 or 25° C for 24 hrs. In two groups of experiments the cells were labeled with H³-TdR for 6 hrs at the respective temperature, washed and further incubated at 37° C. In each group of experiments cultures labeled with H³-TdR at 37° C for 20 min were used as a control. It was found: 1. the delay in the onset of cell passage through the mitotic cycle at 37° in cultures exposed to 15 or 25° C was about equal to 1,5-1 hr resp. and cells proceeded through the life cycle without blockages at any phase of the cycle; 2. the patterns of chromosome reproduction during the second half of the S-phase were the same after labeling at 15, 25 and 37° C. — In the third group the cells were labelled with HP-TdR for 10–60 min and 6 hrs resp. at 25° C. The patterns of reproduction of chromosome pairs 1–4 and small metacentrics were found to be the same in cells labeled briefly and those labeled for 6 hrs. After brief labeling asynchronous reduplication of different segments in many chromosomes became evident. It was masked because of heavy labeling after 6 hrs treatment.     

Stochastic resonance as a possible mechanism of amplification of weak electric signals in living cells

The most important but still unresolved problem in bioelectromagnetics is the interaction of weak electromagnetic fields (EMFs) with living cells. Thermal and other types of noise pose restrictions in cell detection of weak signals. As a consequence, some extant experimental results that indicate low-intensity field effects cannot be accounted for, and this renders the results themselves questionable. One way out of this dead end is to search for possible mechanisms of signal amplification. In this paper, we discuss a general mechanism in which a weak signal is amplified by system noise itself. This mechanism was discovered several years ago in physics and is known, in its simplest form, as a stochastic resonance. It was shown that signal amplification may exceed a factor of 1000, which renders existing estimations of EMF thresholds highly speculative. The applicability of the stochastic resonance concept to cells is discussed particularly with respect to the possible role of the cell membrane in the amplification process. © 1994 Wiley-Liss, Inc.     

Attenuation effects on the kerma rates in air after cesium depositions on grasslands

Since the reactor accident of Chernobyl, cesium depth profiles and nuclide-specific kerma rates in air have been determined for various grassland sites in south Bavaria and in Ukraine. The sites are described by soil characteristics, annual precipitation, distance from release point, mode of deposition, and activity per unit area. The effects of surface roughness and migration of cesium into the soil on the kerma rate in air over grasslands was determined by two methods. The kerma rates in air obtained by the evaluations of in situ gamma-ray spectrometry results and of measured activity distributions in the soil showed only negligible differences for the observation period of 6 years after deposition. For the sites in Ukraine the kerma rate in air per activity per unit area was found to be systematically 40% higher than in Bavaria. The results from Bavaria on the attenuation of the kerma rate and a data set, including experiences from the weapons test fallout, are analytically approximated as a function of time up to 25 years after deposition.     

Analysis of peptides from known proteins: Clusterization in sequence space

A combinatorial sequence space (CSS) model was introduced to represent sequences as a set of overlapping k-tuples of some fixed length which correspond to points in the CSS. The aim was to analyze clusterization of protein sequences in the CSS and to test various hypotheses about the possible evolutionary basis of this clusterization. The authors developed an easy-to-use technique which can reveal and analyze such a clusterization in a multidimensional CSS. Application of the technique led to an unexpectedly high clusterization of points in the CSS corresponding to k-tuples from known proteins. The clusterization could not be inferred from nonuniform amino acid frequencies or be explained by the influence of homologous data. None of the tested possible evolutionary and structural factors could explain the clusterization observed either. It looked as if certain protein sequence variations occurred and were fixed in the early course of evolution. Subsequent evolution (predominantly neutral) allowed only a limited number of changes and permitted new variants which led to preservation of certain k-tuples during the course of evolution. This was consistent with the theory of exon shuffling and protein block structure evolution. Possible applications of sequence space features found were also discussed.Correspondence to: H.A. Lim     

Production of urinary tumour necrosis factors and soluble tumour necrosis factor receptors in bladder cancer patients afterbacillus Calmette-Guerin immunotherapy

Intravesical immunotherapy for bladder cancer is the most effective form of tumour immunotherapy. Following repeated instillations of bacillus Calmette-Guérin (BCG) organisms into the bladder large 0quantities of several cytokines are detected in the urine. These cytokines include interleukins IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, tumour necrosis factor α (TNFα), interferon γ (IFNγ) and also soluble intercellular adhesion molecule ICAM-1. In the work reported here we simultaneously quantified urinary levels of TNFα, TNFβ, TNF receptor I and TNF receptor II by enzyme-linked immunosorbent assay (ELISA) techniques and compared this with bioactive levels of TNF. This was undertaken with a limited number of patients throughout a course of six instillations of immuno therapy. Sequential instillations of BCG induced secretion of TNFα and TNFβ into urine. These cytokines were not always secreted simultaneously, perhaps suggesting differential regulation of their synthesis. Maximal concentrations of TNFα were 675 pg/ml and TNFβ 47 pg/ml. High levels of both species of soluble TNF receptor were readily identified in urine. Maximal levels of sTNF-RI were 6200 pg/ml (range from 0) and for sTNF-RII 7800 pg/ml (range from 0). Contrasting with earlier published observations concerning cytokine levels, the concentration of soluble receptor did not increase with repeated instillation. In apparent contrast with the ELISA data, very low levels of bioactive TNF were identified by the L929 bioassay (maximum concentration 1 U/ml) despite the elevated concen t ration of immunoreactive TNF. The large concentrations of soluble TNF receptor in patients’ urine samples could account for the apparently low bioactivity as determined by the L929 cytotoxicity assay. The precise nature of the role of TNF in BCG immunotherapy remains undetermined; however, it is thought that proinflammatory cytokines are in part responsible for the clinical efficacy of this therapeutic approach. Whether other cytokines are antogonised by soluble binding proteins remains to be determined. Furthermore, whether TNF is bioactive in the bladder wall and only neutralised in the urine also requires investigation. Received: 24 August 1994 / Accepted: 17 October 1994     

Regions of homology in small colicinogenic plasmids

Restriction maps have been constructed for the colicinogenic plasmids (ColA, ColD, and ColK. Their regions of homology with the ColE1 plasmid and its deletion derivative pAO3 carrying the region responsible for autonomous replication of ColE1 plasmid were determined by means of blotting hybridization and heteroduplex analysis. The plasmids ColA, ColD, and ColK were shown to contain DNA fragments homologous to the region of ColE1 involved in the regulation of replication.     

Incidence of Q fever in two inadequately investigated occupational groups.

In 1973 the authors investigated the incidence of Q fever serologically by means of the reaction of complement fixation (RCF) and the method of immunofluorescent titration (MIFT) in two inadequately investigated occupational groups--communal workers from the town of Russe and medical workers in obstetric departments of several towns in North Bulgaria. In addition, they carried out comparative studies in order to characterize the incidence and the degree of affection from the same disease in other persons exposed and not exposed at work in the same area--transport workers and blood donors. Out of 198 communal workers, 91 (45.95 +/- 3.54%) had positive titres for Q fever (1:8--1:512). A high incidence of Q fever was established in dustmen (61.40%), sweepers (46.55%) and drivers of dust cars (38.00%), i.e. persons collecting and rendering harmless the garbage of big town. Out of 174 medical workers in obstetric departments 65 (37.36% +/- 3.78%) were positive in titres 1:8--1:512. A high incidence of Q fever was established in obstetricians (57.14%), midwives (38.11%) and hospital attendants (34.38%), i.e. persons providing medical care for pregnant women or women in childbirth. In both groups the occupational hazard increases with the length of service. Out of 244 transport workers 82 (33.60% +/- 3.02%) were positive for Q fever, and out of 237 blood donors 19 (8.01 +/- 2.54%) were serologically positive for Q fever. The authors suggest continued investigation of these two occupational groups.     

Genetic control and expression of the major ejaculatory bulb protein (PEB-me) inDrosophila melanogaster

PEB-me is a predominant protein of matureDrosophila melanogaster ejaculatory bulbs. It is resolved into four or five closely spaced subfractions (apparent molecular weight 35–39 kD) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Four electrophoretic variants of PEB-me differing in apparent molecular weight by 200–800 daltons were found. These appear to be controlled by four alleles of a gene (peb) located by recombination and deletion mapping to the 60F1-2 region of chromosome 2. A minor ejaculatory bulb protein of ca. 80 kD (hPEB) was found to be immunochemically related to PEB and possibly encoded bypeb. PEB is not detected by immunoblotting techniques in virgin females, in male tissues other than the ejaculatory bulb, or during developmental stages preceding the formation of this organ. The results of transplantations of genital imaginal discs and of immature ejaculatory bulbs between two strains having different PEB alleles suggest that the ejaculatory bulb is the site of PEB synthesis. In flies mutant fortra, tra-2, dsx, orix, tissue specificity of PEB localization is retained and the protein is found whenever the ejaculatory bulb is formed, regardless of the chromosomal sex of the fly. The protein is transferred into the female genital duct during mating, where it can be detected for up to 12 hr. Possible functions of PEB inDrosophila reproduction are discussed.