VirE2-dependent pores for ssDNA transfer across artificial and cell membranes

The transfer of single-stranded (ss) T-DNA from soil bacteria of the genus Agrobacterium with the help of the VirE2 protein, which possibly mediates the delivery of ss-T-DNA across the cell membrane, was demonstrated earlier, but how VirE2 participates in ssDNA transfer across artificial and natural membranes is not known. Using computational methods, we reconstructed model structures composed of two and four VirE2 proteins and showed by the MOLE program the formation of pores with channel diameters of 1.2-1.6 and 1.4-4.6 nm in a model structure formed from two and four VirE2 molecules, respectively. Using light scattering, we recorded the size distribution for recombinant VirE2-dependent complexes in aqueous solutions and found that VirE2 in a buffer solution is present as a complex made up of two or more proteins. We revealed single, long-lived jumps in voltage-dependent membrane conductance during coincubation of planar black membranes with the VirE2 protein. On the addition of VirE2 and FAM-labeled oligonucleotides to HeLa cells, the fluorescence intensity for the cells increased by 56% as compared to that for cells incubated only with oligonucleotides.     

Production of miniantibodies to Agrobacterium tumefaciens VirE2 virulence protein by the method of phage display

The scFv miniantibodies to the recombinant protein VirE2 from Agrobacterium tumefaciens were obtained by the method of phage display. The miniantibodies were purified and tested using timmunodot method for binding to a recombinant protein from Escherichia coli and to the native protein VirE2 from A. tumefaciens. The functional activity of the miniantibodies was comparable to the activity of mouse polyclonal antibodies against the VirE2 protein.     

A computational tool for the genomic identification of regions of unusual compositional properties and its utilization in the detection of horizontally transferred sequences

Similarity Plot (S-plot) is a Windows-based application for large-scale comparisons and 2-dimensional visualization of compositional similarities between genomic sequences. This application combines 2 approaches widely used in genomics: window analysis of statistical characteristics along genomes and dot-plot visual representation. S-plot is effective in identifying highly similar regions between genomes as well as regions with unusual compositional properties (RUCPs) within a single genome, which may be indicative of horizontal gene transfer or of locus-specific selective forces. We use S-plot to identify regions that may have originated through horizontal gene transfer through a 2-step approach, by first comparing a genomic sequence to itself and, subsequently, comparing it to the genomic sequence of a closely related taxon. Moreover, by comparing these suspect sequences to one another, we can estimate a minimum number of sources for these putative xenologous sequences. We illustrate the uses of S-plot in a comparison involving Escherichia coli K12 and E. coli O157:H7. In O157:H7, we found 145 regions that have most probably originated through horizontal gene transfer. By using S-plot to compare each of these regions with 277 completely sequenced prokaryotic genomes, 1 sequence was found to have similar compositional properties to the Yersinia pseudotuberculosis genome, indicating a transfer from a Yersinia or Yersinia relative. Based upon our analysis of RUCPs in O157:H7, we infer that there were at least 53 sources of horizontally transferred sequences.     

Cytoplasm and organelle transfer between mesenchymal multipotent stromal cells and renal tubular cells in co-culture

Cell-to-cell interactions of human mesenchymal multipotent stromal cells (MMSC) and rat renal tubular cells (RTC) were explored under conditions of co-cultivation. We observed formation of different types of intercellular contacts, including so called tunneling nanotubes. These contacts were shown to be able to provide transfer of cell's contents, including organelles. We documented intercellular exchange with fluorescent probes specific to cytosol, plasmalemma and mitochondria. Initial transport of cellular components was revealed after 3 h of co-culturing, and occurred in two directions—both direct and retrograde as referred to RTC. However, transport of probes toward MMSC was more efficient. One significant result of such transport was appearance of renal-specific Tamm-Horsfall protein in MMSC, indicating induction of their differentiation into kidney tubular cells. We conclude that transfer of cellular compartments between renal and stem cells could provide differentiation of MMSC when transplanted into kidney and result in therapeutic benefits in renal failure.     

Human-blind probes and primers for dengue virus identification

Reliable detection and identification of pathogens in complex biological samples, in the presence of contaminating DNA from a variety of sources, is an important and challenging diagnostic problem for the development of field tests. The problem is compounded by the difficulty of finding a single, unique genomic sequence that is present simultaneously in all genomes of a species of closely related pathogens and absent in the genomes of the host or the organisms that contribute to the sample background. Here we describe 'host-blind probe design'- a novel strategy of designing probes based on highly frequent genomic signatures found in the pathogen genomes of interest but absent from the host genome. Upon hybridization, an array of such informative probes will produce a unique pattern that is a genetic fingerprint for each pathogen strain. This multiprobe approach was applied to 83 dengue virus genome sequences, available in public databases, to design and perform in silico microarray experiments. The resulting patterns allow one to unequivocally distinguish the four major serotypes, and within each serotype to identify the most similar strain among those that have been completely sequenced. In an environment where dengue is indigenous, this would allow investigators to determine if a particular isolate belongs to an ongoing outbreak or is a previously circulating version. Using our probe set, the probability that misdiagnosis at the serotype level would occur is approximately 1 : 10(150).     

The sodium cycle. III. Vibrio alginolyticus resembles Vibrio cholerae and some other vibriones by flagellar motor and ribosomal 5S-RNA structures

An electron microscopic study of the basal bodies of the Vibrio albinolyticus flagellum revealed a four-disc structure. The diameters of the two discs localized closer to the cytoplasmic membrane proved to be about 2-fold shorter than those of the two others. In this respect the basal body of V. alginolyticus resembles very much that of V. cholerae described by Ferris and co-workers. The sequence of the V. alginolyticus ribosomal 5S-RNA showed that it is similar to those of V. cholerae, V. harveyi and some other vibriones. On the basis of the 5S-RNA sequences, a dendrogram of prokaryotes is presented. It confirmed the suggestion that V. alginolyticus is a typical representative of Vibrionaceae rather than a 'monster' greatly differing from other vibriones. Possible evolutionary relation of various bacterial species possessing the primary Na+ pumps is discussed.     

p53 does not control the spindle assembly cell cycle checkpoint but mediates G1 arrest in response to disruption of microtubule system

p53 plays a critical role as a tumour-suppressor in restricting the proliferation of damaged cells, thus preventing formation of genetically altered cell clones. Its inactivation leads, in particular, to accumulation of polyploid and aneuploid cells. To elucidate the role of p53 in control of chromosome number, we analysed its participation in the cell cycle checkpoints controlling: (1) spindle assembly; and (2) G1-to-S transitions in cells with disintegrated microtubule cytoskeleton. Treatment with 8-10 ng/ml of colcemid causing no visible destruction of the spindle leads to arrest of metaphase-to-anaphase transition in both p53-positive and p53-negative murine fibroblasts, as well as in p53-positive REF52 cells and their counterparts (where the p53 function was inactivated by transduction of dominant-negative p53 fragment). Furthermore, p53-positive and p53-defective rodent and human cells showed no significant difference in kinetics of metaphase-to-interphase transitions in cultures treated with high colcemid doses preventing spindle formation. These data argue against the hypothesis that p53 is a key component of the spindle-assembly checkpoint. However, p53 mediates activation of the G1 checkpoint in response to depolymerization of microtubules in interphase cells. Treatment of synchronized G0/G1 cells with colcemid causes arrest of G1-to-S transition. Inactivation of the p53 function by transduction of dominant-negative p53 fragment abolishes the G1 checkpoint that prevents entry into S phase of cells with disrupted microtubules. Transduction of kinase-defective dominant-negative c- raf mutant or application of PD 098059, a specific inhibitor of MEK1, also abrogates the G1 cell cycle arrest in cells with disintegrated microtubule system. It seems that Raf-MAP-kinase signalling pathways are responsible for p53 activation induced by depolymerization of microtubules.     

Agrobacterial Transformation of Uninjured Plants

The capacity for agrobacterial transformation (using Agrobacterium tumefaciens strain GV3101 with the activated vir genes) was investigated in uninjured seedlings of tobacco (Nicotiana tabacum L.), rice (Oryza sativa L.), and wheat (Triticum aestivum L.) along with the effect of various factors on the frequency of tobacco seedling transformation. Transformed cells were found at the base of the rice leaf sheath and in the upper part of tobacco leaves, whereas they were absent from wheat leaves. Illumination and temperature, which were below and over the optimum, were the factors limiting agrobacterial transformation of uninjured plants. Preincubation of tobacco seedlings in 100 M ABA, 10 mM EGTA, and 0.1 mM colchicine decreased the frequency of agrobacterial T-DNA penetration into plant cells. The authors conclude that transformation occurs predominantly in young growing cells, and its frequency is related to the state of stomata and plasmodesmata of the target plant cells. The elements of the cytoskeleton probably participate in T-complex transport.