Kinetics of the reaction of yolk cell surface in the loach to puncture and mechanic deformation

Circumferential and radial components of the yolk cell surface movements were measured in the loach embryos at the late blastula stage within 40–50 min after puncture or indentation by an obliquely directed glass rod. The yolk cell surface was preliminarily marked by coal particles. It was shown that even closely located regions of the surface differed markedly in the rate and direction of their movements. In the vicinity of puncture, the yolk cell surface at first contracted in both circumferential and radial directions and then widened, but did not reach the initial values. In more remote areas, this surface continued to contract in the circumferential direction, but was extended in the radial direction. The degree of its contraction along different radii was unequal. The reaction to oblique indentation was anisotropic: the closest area of the yolk cell surface, located along the direction of indentation, contracted in both circumferential and radial directions and formed a fold “leaking” onto the rod, while the opposite area contracted in the circumferential direction, but extended in the radial direction. A conclusion was drawn that the yolk cell surface is a multivariant mechanosensitive system. Its active responses to mechanical influences obey the same patterns as multicellular embryonic tissues.     

The Mechanism of Supercoiled DNA Recognition by Eukaryotic Type I Topoisomerases: II. A Comparison of the Enzyme Interaction with Specific and Nonspecific Oligonucleotides

Data on the interaction of DNA type I topoisomerases from the murine and human placenta cells with specific and nonspecific oligonucleotides of various structures and lengths are summarized. The relative contributions of various contacts between the enzymes and DNA that have previously been detected by X-ray analysis to the total affinity of the topoisomerases for DNA substrates are estimated. Factors that determine the differences in the enzyme interactions with specific and nonspecific single- and double-stranded DNAs are revealed. The results of the X-ray analysis of human DNA topoisomerase I are interpreted taking into account data on the comprehensive thermodynamic and kinetic analysis of the enzyme interaction with the specific and nonspecific DNAs.     

Involvement of the Lipopolysaccharides of Azospirilla in the Interaction with Wheat Seedling Roots

The present study was undertaken to comparatively investigate the attachment capacities of Azospirillum brasilenseSp245 and its lipopolysaccharide-defective Omegon-Km mutants KM018 and KM252, as well as their activities with respect to the alteration of the morphology of wheat seedling root hairs. The adsorption dynamics of the parent Sp245 and mutant KM252 strains of azospirilla on the seedling roots of the soft spring wheat cv. Saratovskaya 29 were similar; however, the attachment capacity of the mutant KM252 was lower than that of the parent strain throughout the incubation period (15 min to 48 h). The mutation led to a considerable decrease in the hydrophobicity of the Azospirillumcell surface. The lipopolysaccharides extracted from the outer membrane of A. brasilenseSp245 and mutant cells with hot phenol and purified by chromatographic methods were found to induce the deformation of the wheat seedling root hairs, the lipopolysaccharide of the parent strain being the most active in this respect. The role of the carbohydrate moiety of lipopolysaccharides in the interaction of Azospirillumcells with plants is discussed.     

Optimized linker sequences for the expression of monomeric and dimeric bispecific single-chain diabodies.

Bispecific single-chain diabodies (scDb) consist of the variable heavy and light chain domains of two antibodies connected by three linkers. The structure of an scDb in the V(H)-V(L) orientation is V(H)A-linkerA-V(L)B-linkerM-V(H)B-linkerB-V(L)A, with linkers A and B routinely chosen to be 5-6 residues and linker M 15-20 residues. Here, we applied display of scDb on filamentous phage to analyse the composition of optimal linker sequences. The three linkers were randomized in length and sequence using degenerated triplets coding for only six hydrophilic or aliphatic amino acids (Thr, Ser, Asp, Asn, Gly, Ala). Antigen-binding clones were then isolated by one to two rounds of selection on the two different antigens recognized by the bispecific scDb. Using an scDb directed against carcinoembryonic antigen (CEA) and beta-galactosidase (Gal), we found that monomeric scDb had a preferred length of 15 or more amino acid residues for the middle linker M and of 3-6 residues for the linkers A and B. No obvious bias towards a preferred linker sequence was observed. Reduction of the middle linker below 13 residues led to the formation of dimeric scDb, which most likely results from interchain pairing between all the V(H) and V(L) domains. Dimeric scDb were also formed by fragments possessing a long linker M and linkers A and B of 0 or 1 residue. We assume that these dimeric scDb are formed by intrachain pairing of the central variable domains and interchain pairing of the flanking variable domains. Thus, the latter molecules represent a novel format of bispecific and tetravalent molecules. The described strategy allows for the isolation of both optimized and minimal linker sequences for the assembly of monomeric or dimeric single-chain diabodies.     

Ectopic expression of an osmotin gene leads to enhanced salt tolerance in transgenic chilli pepper (<Emphasis Type="Italic">Capsicum annum</Emphasis> L.)

Plants, when exposed to abiotic or biotic stress, produce several pathogenesis-related proteins to counteract the effects of stress. Osmotin is one of the important pathogenesis-related proteins induced during several stress conditions. We have developed improved salt stress tolerant transgenic chilli pepper plants (Capsicum annum L. var. Aiswarya 2103) by ectopic expression of the Nicotiana tabaccum osmotin gene using Agrobacterium tumefaciens EHA105 as a vector. Four-week-old chilli pepper leaves were used as an explant and A. tumefaciens EHA105 harboring pBINASCOSM plasmid that contains osmotin gene under the control of CaMV 35S promoter and npt II as a selectable marker was used in co-cultivation. Transgene integration and expression were analyzed using molecular, immunochemical, and biochemical assays. PCR and Southern blot analysis confirmed that osmotin gene has been successfully integrated into the genome of chilli pepper plants. The osmotin gene was stably segregated and expressed in T₂ generation transgenic chilli pepper plants, and it was confirmed by Western blot analysis. Biochemical assays of these putative transgenic plants revealed enhanced levels of chlorophyll, proline, glycinebetaine, APX, SOD, DHAR, MDHAR, GR, and relative water content. Yield potential of the putative transgenic chilli pepper plants was evaluated under salinity stress conditions in a green house. The putative transgenic chilli pepper plants overexpressing the osmotin gene were morphologically similar to wild-type plants and produced 3.32 kg chilli pepper fruits per plant at 300 mM NaCl concentration.     

Control of the growth of Alicyclobacillus acidoterrestris in industrialized orange juice using rosemary essential oil and nisin

The use of rosemary essential oil (RO) and its combination with nisin (RO+N) in preventing the multiplication of Alicyclobacillus acidoterrestris in orange juice was evaluated. The minimum inhibitory and bactericidal concentrations (MIC and MBC) for RO were both 125 μg ml⁻¹ while RO+N displayed a synergistic effect. The use of RO and RO+N at concentrations of 1, 4 and 8× MIC in orange juice for 96 h was evaluated in terms of their sporicidal effectiveness. With regard to the action against A. acidoterrestris spores, RO at 8× MIC was sporostatic, whereas RO+N at 1× MIC was sporicidal. Morphological changes in the structure of the micro-organism after treatment were also observed by microscopy. Furthermore, flow cytometric analysis showed that most cells were damaged or killed after treatment. In general, the antioxidant activity after addition of RO+N decreased with time. The results demonstrate that using the combination of RO and nisin can prevent the A. acidoterrestris growth in orange juice.     

Free-living amoebae promote Candida auris survival and proliferation in water

Candida auris is an emerging species responsible for life-threatening infections. Its ability to be resistant to most systemic antifungal classes and its capacity to persist in a hospital environment have led to health concerns. Currently, data about environmental reservoirs are limited but remain essential in control of C. auris spread. The aim of our study was to explore the interactions between C. auris and two free-living amoeba (FLA) species, Vermamoeba vermiformis and Acanthamoeba castellanii, potentially found in the same water environment. Candida auris was incubated with FLA trophozoites or their culture supernatants. The number of FLA and yeasts was determined at different times and transmission electron microscopy (TEM) was performed. Supernatants of FLAs promoted yeast survival and proliferation. Internalization of viable C. auris within both FLA species was also evidenced by TEM. A water environmental reservoir of C. auris can therefore be considered through FLAs and contamination of the hospital water networks would consequently be possible.     

Fate of biomedical research protocols and publication bias in France: retrospective cohort study

Objectives To describe the fate of protocols approved by the French research ethics committees, a national system created by the French 1988 Huriet-Sérusclat Act; to assess publication bias at a national level.Design Retrospective cohort study.Setting Representative sample of 25/48 French research ethics committees in 1994.Protocols 649 research protocols approved by committees, with follow-up information.Main outcome measures Protocols'' initial characteristics (design, study size, investigator) abstracted from committees'' archives; follow-up information (rates of initiation, completion, and publication) obtained from mailed questionnaire to principal investigators.Results Completed questionnaires were available for 649/976 (69%) protocols. Of these, 581 (90%) studies were initiated, 501/581 (86%) were completed, and 190/501 (38%) were published. Studies with confirmatory results were more likely to be published as scientific papers than were studies with inconclusive results (adjusted odds ratio 4.59, 95% confidence interval 2.21 to 9.54). Moreover, studies with confirmatory results were published more quickly than studies with inconclusive results (hazard ratio 2.48, 1.36 to 4.55).Conclusion At a national level, too many research studies are not completed, and among those completed too many are not published. We suggest capitalising on research ethics committees to register and follow all authorised research on human participants on a systematic and prospective basis.     

Carcinogens can induce homologous recombination between duplicated chromosomal sequences in mouse L cells.

The ability of a series of DNA-damaging agents to induce homologous intrachromosomal recombination between duplicated genes in the chromosome of mouse cells was investigated. The target cells were the thymidine kinase-deficient mouse L-cell strain 333M, which contains a single integrated copy of a plasmid with two herpes simplex virus thymidine kinase (Htk) genes, each containing an 8-base-pair XhoI linker inserted at a unique site. Expression of a functional Htk enzyme requires a productive recombinational event between the two nonfunctional genes. The spontaneous rate of recombination in this strain is 3 per 10(6) cells per generation. The agents tested represent physical carcinogens (UV and ionizing radiation), a simple alkylating agent (N-methyl-N'-nitro-N-nitrosoguanidine), an alkylating cross-linking agent (mitomycin C), and a reactive metabolite of a polycyclic aromatic hydrocarbon ((+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene [BPDE] ). The background frequency of tk+ recombinants in the untreated population averaged 18 X 10(-6) +/- 5 X 10(-6). Ionizing radiation had little or no effect on recombination; exposure to mitomycin C, N-methyl-N'-nitro-N-nitrosoguanidine, BPDE, or UV, at doses that lowered the survival to between 90 and 10% of the control, caused a dose-dependent increase in frequency of recombinants, reaching 50 X 10(-6) to 100 X 10(-6). No tk+ cells could be generated with a control cell line that contained only one mutant copy of the Htk gene. Molecular hybridization analysis showed that 85 to 90% of the tk+ recombinants retained the Htk gene duplication, consistent with nonreciprocal transfer of wild-type genetic information, gene conversion. In the rest, only a single copy of the Htk gene remained, reflecting a single reciprocal exchange within a chromatid or a single unequal exchange between sister chromatids. Each recombinant tested contained an XhoI-resistant (wild-type) Htk gene.