Novel spirochetes isolated from mosquitoes and black flies in the Czech Republic

During the years 1999–2002, a total of 4,898 individuals of 26 species of hematophagous insects (4,149 mosquitoes, 583 black flies, and 166 tabanid flies) was examined for the presence of spirochetes using dark‐field microscopy. There was an overall recovery of spirochetes from the midguts of Culicidae and Simuliidae of 23.5% and 11.4%, respectively. Spirochetes were not detected in Tabanidae. Seven spirochetal strains have been successfully recovered from mosquitoes and black flies: BR149 (Culex pipiens), BR151 (Cx. pipiens), BR173 (Cx. pipiens), BR177 (Cx. pipiens), BR193 (Aedes cinereus), BR208 (Cx. pipiens), and BR231 (Simulium noelleri). The strains have been adapted to laboratory conditions (BSK‐H Complete medium). Their preliminary determination based on 16S rRNA gene sequencing has shown that they differ from the Lyme disease spirochete Borrelia burgdorferi sensu lato as well as other members of the Order Spirochaetales indicating novel bacterial species in the Family Spirochaetaceae.     

The production of two Th2 cytokines, interleukin-4 and interleukin-10, is controlled independently by locus Cypr1 and by loci Cypr2 and Cypr3, respectively

 The strains BALB/cHeA (BALB/c) and STS/A (STS) differ in production of IL-4 and IL-10, two Th2 cytokines, after stimulation of spleen cells with Concanavalin A, STS being a low and BALB/c a high producer. We analyzed the genetic basis of this strain difference using the recombinant congenic (RC) strains of the BALB/c-c-STS/Dem (CcS/Dem) series. This series comprises 20 homozygous strains. Each CcS/Dem strain contains a different, random set of approximately 12.5% genes of the "donor" strain STS and approximately 87.5% of the "background" strain BALB/c. We selected for further analysis the RC strain production intermediate between BALB/c and STS. In (CcS-20×BALB/c)F₂ hybrids we found that different loci control expression of IL-4 and IL-10. Cypr1 (cytokine production 1) on chromosome 16 near D16Mit15 controls IL-4 production, whereas the production of IL-10 is influenced by loci Cypr2 near D1Mit14 and D1Mit227 on chromosome 1 and Cypr3 marked by D5Mit20 on chromosome 5. In addition, the relationship between the level of these two cytokines depends on the genotype of the F₂ hybrids at a locus cora1 (correlation 1) on chromosome 5. This differential genetic regulation may be relevant for the understanding of biological effects of T-helper cells in mice of different genotypes. Received: 2 March 1998 / Revised: 8 June 1998     

Sporulation as an effective strategy for the survival of bacteria in a biofilter with discontinuous operation

The microbial population of the biofilter developed for the treatment of air contaminated by solvent vapours was evaluated using plate count techniques. The number of total heterotrophic bacteria and bacteria utilizing contaminant as the only source of carbon and energy was estimated during the exchange of the filter-bed material and start-up of its operation. Spore-forming bacteria (Bacillus spp.) occupied a significant part of the filter-bed niche. It is proposed that sporulation helps bacteria to survive during the breaks in operation of the biofilter.     

Segregation of T-DNA Inserts in the Offspring of Arabidopsis Thaliana After Agrobacterium Transformation

Using various transformation methods, T-DNA constructions for insertional mutagenesis were introduced into Arabidopsis thaliana and the pattern of segregation of hygromycin resistance selectable marker was followed in succeeding generations in individual transgenic lines up to T₄ generation. Despite the low frequency of transformation, T-DNA was often inserted in two or more independent sites. Mendelian segregation ratios 3:1, 15:1, and irregular segregation ratios were observed. We have also shown continuous decrease of the expression of the resident hygromycin resistance transgenic trait in some lines. This revised version was published online in July 2006 with corrections to the Cover Date.     

Response of Calamagrostis Arundinacea and C. Epigeios to short- and long-term water stress

Water relations of two rapidly expanding species in deforested localities, Calamagrostis epigeios and Calamagrostis arundinacea, were compared. After short- or long-term water stress, water content and specific leaf area were more reduced in C. epigeios than in C. arundinacea. After short-term stress, osmotic potential was similar in both species, but after long-term stress, it was much lower in C. epigeios plants. Abscisic acid and proline contents were higher in stressed plants of C. epigeios than in those of C. arundinacea. This revised version was published online in July 2006 with corrections to the Cover Date.     

An antibody library for stabilizing and crystallizing membrane proteins - selecting binders to the citrate carrier CitS

Co-crystallization of membrane proteins with antibody fragments may emerge as a general tool to facilitate crystal growth and improve crystal quality. The bound antibody fragment enlarges the hydrophilic part of the mostly hydrophobic membrane protein, thereby increasing the interaction area for possible protein-protein contacts in the crystal. Additionally, it may restrain flexible parts or lock the membrane protein in a defined conformational state. For successful co-crystallization trials, the antibody fragments must be stable in detergents during the extended period of crystal growth and must be easily produced in amounts necessary for crystallography. Therefore, we constructed a library of antibody Fab fragments from a framework subset of the HuCAL GOLD library (Morphosys, Munich, Germany). By combining the most stable and well expressed frameworks, V(H)3 and V(kappa)3, with the further stabilizing constant domains, a Fab library with the desired properties was obtained in a standard phage display format. As a proof of principle, we selected binders with phage display against the detergent-solubilized citrate transporter CitS of Klebsiella pneumoniae. We describe efficient methods for the immobilization of the membrane protein during selection, for ELISA screening, and for BIAcore evaluation. We demonstrate that the selected Fab fragments form stable complexes with native CitS and recognize conformational epitopes with affinities in the low nanomolar range.     

Demethoxy-Q, an intermediate of coenzyme Q biosynthesis, fails to support respiration in Saccharomyces cerevisiae and lacks antioxidant activity

Caenorhabditis elegans clk-1 mutants cannot produce coenzyme Q(9) and instead accumulate demethoxy-Q(9) (DMQ(9)). DMQ(9) has been proposed to be responsible for the extended lifespan of clk-1 mutants, theoretically through its enhanced antioxidant properties and its decreased function in respiratory chain electron transport. In the present study, we assess the functional roles of DMQ(6) in the yeast Saccharomyces cerevisiae. Three mutations designed to mirror the clk-1 mutations of C. elegans were introduced into COQ7, the yeast homologue of clk-1: E233K, predicted to disrupt the di-iron carboxylate site considered essential for hydroxylase activity; L237Stop, a deletion of 36 amino acid residues from the carboxyl terminus; and P175Stop, a deletion of the carboxyl-terminal half of Coq7p. Growth on glycerol, quinone content, respiratory function, and response to oxidative stress were analyzed in each of the coq7 mutant strains. Yeast strains lacking Q(6) and producing solely DMQ were respiratory deficient and unable to support (6)either NADH-cytochrome c reductase or succinate-cytochrome c reductase activities. DMQ(6) failed to protect cells against oxidative stress generated by H(2)O(2) or linolenic acid. Thus, in the yeast model system, DMQ does not support respiratory activity and fails to act as an effective antioxidant. These results suggest that the life span extension observed in the C. elegans clk-1 mutants cannot be attributed to the presence of DMQ per se.     

Levels of apolipoprotein A-II in cerebrospinal fluid in patients with neuroborreliosis are associated with lipophagocytosis

Levels of most of the examined proteins in cerebrospinal fluid (CSF) of 107 patients with neuroborreliosis were associated with cytological findings, the status of hematoencephalic barrier as evaluated by Qalb (cerebrospinal fluid to serum quotient) and the intrathecal synthesis of immunoglobulins. Cytological findings consisted of normal cytology, or both oligocytosis and pleocytosis of monocytes or lymphocytes. The lipophagic elements were present in 20% of samples. Concentrations of apolipoproteins A-I and A-II in the CSF were correlated with the concentration of albumin without regard to the CSF cytology. The levels of apolipoprotein B were increased only in samples with lymphocytic pleocytosis and Qalb > 7.4. The presence of lipophages in the CSF was significantly associated with the CSF concentration of apolipoprotein A-II.     

Potential risk of infection transmission during storage and transplantation of hematological progenitor cells. Safety assurance

In a group of 71 patients and 22 donors the danger of infection transmission by infusion of cryopreserved peripheral blood progenitor cells to the patient and/or cross contamination of stored grafts was evaluated. No laboratory signs of active infection were found in 15 donors (13 related, 2 unrelated; 68%) and in 55 patients (77%). Active infection by herpesviruses was the most common (in 13 patients and 7 donors), hepatitis B being found in only one case. The cytomegalovirus IgG test was the most common marker of previous infection; it was found in 14 donors and 55 patients. The rate of clinically unsuspected infections in donors and patients including cases requiring immediate treatment among the patients group is relatively high and fully justifies the practice of prophylactic serological testing in the whole range of tests according to the European Blood and Marrow Transplantation Group and International Society for Hematotherapy and Graft Engineering in both autologous and allogeneic transplantations of hematopoietic stem cells.     

Moderately Elevated Temperature Eliminates Resistance of Rice Plants with Enhanced Expression of Glutathione Reductase to Intensive Photooxidative Stress

We developed transgenic rice plants (Oryza sativa L. cv. Daeribbyeo) overproducing cytosolic glutathione reductase (GR) using a GR gene from Brassica campestris and studied their response to photo-oxidative stress in the presence of methyl viologen (MV, 10 and 50 M concentrations) under room (25 °C) and moderately elevated (35 °C) temperature by analysis of chlorophyll (Chl) a fluorescence parameters (F_V/F_M, q_N, and q_P) and of Chl content. Elevated temperature enhanced and accelerated the photo-oxidative damage to photosynthetic apparatus expressed mainly by a fast decrease of q_N. Higher temperature supported the protective reaction in transformed rice plants for lower MV concentration (10 M) and eliminated the enhanced tolerance of photosystem 2 photochemistry to photooxidative stress for higher (50 M) MV concentration. Different mechanisms and temperature dependence of oxidative and protective reactions explain the results.