[Acute intestinal ischaemia]. / Akútna intestinálna ischémia.

In spite of introduction modern diagnostics and therapeutics modalities in clinical practice diagnostics of acute intestinal ischaemia is very difficult. Acute intestinal ischaemia is rare cause of acute abdominal dissease but results of surgical treatment and prognosis of the patients with acute intestinal ischaemia is very poor. The aim of study is occurence, diagnostics and therapeutics possibilities of acute intestinal ischaemia, because tretament of acute intestinal ischaemia have high rate of mortality. Authors claiming on own small set of patients that in diagnostics of acute abdominal pain everything in once mind on acute intestinal ischaemia.

[Postoperative adhesions, the everlasting topical subject]. / Pooperacné adhézie, stále aktuálny problém.

Creation of postoperative adhesions is a part of every abdominal operation. The authors analyse 320 patients operated for ileus in last 7 years. 118 patients were operated for adhesive ileus. Most common reoperations for ileus are after radical gynecological operations and inflammatory intraabdominal diseases. The creation of adhesiones depends on preoperative mechanical or chemical damage of tissues and peritoneum, bacterial infection and irradiation. The major complication of intraabdominal adhesions are disturbances of bowel function what leads to subileus or ileus. Authors present therapeutical possibilities and prefer laparoscopic operations.

Transgenic plants expressing cationic peptide chimeras exhibit broad-spectrum resistance to phytopathogens.

Here we describe a strategy for engineering transgenic plants with broad-spectrum resistance to bacterial and fungal phytopathogens. We expressed a synthetic gene encoding a N terminus-modified, cecropin-melittin cationic peptide chimera (MsrA1), with broad-spectrum antimicrobial activity. The synthetic gene was introduced into two potato (Solanum tuberosum L.) cultivars, Desiree and Russet Burbank, stable incorporation was confirmed by PCR and DNA sequencing, and expression confirmed by reverse transcription (RT)-PCR and recovery of the biologically active peptide. The morphology and yield of transgenic Desiree plants and tubers was unaffected. Highly stringent challenges with bacterial or fungal phytopathogens demonstrated powerful resistance. Tubers retained their resistance to infectious challenge for more than a year, and did not appear to be harmful when fed to mice. Expression of msrA1 in the cultivar Russet Burbank caused a striking lesion-mimic phenotype during leaf and tuber development, indicating its utility may be cultivar specific. Given the ubiquity of antimicrobial cationic peptides as well as their inherent capacity for recombinant and combinatorial variants, this approach may potentially be used to engineer a range of disease-resistant plants.

Interspecies transplacement of mitochondria in yeasts.

Protoplasts of a respiration-deficient rho(0)strain of Saccharomyces cerevisiae were incubated with mitochondria isolated from various respiration-competent yeast species under conditions enabling transplacement of mitochondria. Respiration-competent cybrids were selected by plating the protoplasts on agar media containing a non-fermentable energy source. The resulting cybrids contained nuclear DNA of the acceptor S. cerevisiae and mitochondrial DNA of the donor species, as detected by pulsed-field gel electrophoresis of chromosomes and restriction analysis of mitochondrial DNA, respectively. Successful restoration of respiration in the S. cerevisiae mutant was achieved by transplacement of mitochondria isolated from the following Saccharomyces species: S. bayanus, S. capensis, S. delbrueckii, S. exiguus, S. italicus and S. oviformis.

Autophosphorylation of purified c-Src at its primary negative regulation site.

The phosphorylating and transforming activities of c-Src are negatively regulated by phosphorylation at Tyr-527 near its carboxyl terminus. Previous studies have indicated that c-Src preferentially autophosphorylates Tyr-416, a residue in the middle of the catalytic domain, in vitro, and that Tyr-527 is phosphorylated by the carboxyl-terminal Src kinase, Csk. However, indirect evidence suggests that c-Src may also autophosphorylate Tyr-527 as part of a negative feedback loop. While some in vivo evidence suggests that Tyr-527 can be autophosphorylated in an intermolecular interaction, it has not previously been possible to directly demonstrate significant autophosphorylation in vitro. Here we show that c-Src purified from recombinant bacteria can autophosphorylate Tyr-527 to high levels in vitro when incubated with sufficiently high concentrations of ATP (KM(Mg2+/ATP) approximately equal to 20 microM) that are well above those that have been used previously. In vitro Tyr-527 autophosphorylation can occur both as an intra- and intermolecular interaction; higher enzyme concentrations are required for intermolecular Tyr-527 phosphorylation than for Tyr-416 autophosphorylation. These results support the possibility that, like G-proteins, c-Src can switch itself off in vivo by its own enzymatic activity.

Vectors with the fd replicon for in vivo cloning and analysis of genes.

We have constructed two new mini-Mu derivatives, pMRfP and pBEf, that combine the properties of known mini-Mu vectors and the advantages of the replication origin (orifd) of filamentous phage fd. Mini-Mu pMRfP consists of the left (850 bp) and the right (216 bp) ends of the Mu genome, orifd, packaging signal of fd, and the gene conferring resistance to chloramphenicol. The second mini-Mu, termed pBEf, carries the left end of Mu (1001 bp), which contains the so-called internal activation sequence (enhancer of transposition), required for a higher frequency of transposition, the right end (116 bp) and the gene conferring resistance to kanamycin. These new mini-Mu vectors are suitable for in vivo cloning with the ability of single-stranded DNA preparation using one of the helper phages (M13K07, rv1, IR1, R408) and with a large cloning capacity (the size of the cloned fragment can be up to 35 kb). They can also be used as the hoppers (a transposable ori that can be turned on or off depending on the presence of the fd gene 2 product). Thus, these mini-Mu derivatives can be employed as vectors for in vivo cloning, and as regulated transposons or mobile replicons.

Characterization of mitochondrial DNA topoisomerase I from Neurospora crassa.

DNA topoisomerase I isolated from the lower eukaryote Neurospora crassa mitochondria was characterized. Molar mass of the enzyme in the native state is 120 kDa and 60-65 kDa when denatured. The pH optimum of the enzyme is 7.8 and the KCl optimum concentration is 40 mmol/L. This topoisomerase is independent of ATP and Mg2+. N-Ethylmaleimide, 4-chloromercuribenzoate, SDS, guanidinium chloride, polyethylene glycol, heparin and ethidium bromide inhibit its activity, while novobiocin, nalidixic acid, Triton X-100 and chloroquine do not. Polyamines and histone H1 stimulate the topoisomerase activity. We classify this DNA topoisomerase as type I and eukaryotic. Conversion of the topoisomerase to a nonspecific endonuclease at increased temperature is proposed.

Substrate-specific modulation of Src-mediated phosphorylation of Ras and caseins by sphingosines and other substrate modulators.

It is important for the understanding of protein kinase action to differentiate between regulation at the enzyme and at the substrate levels. For example, the inhibitors dinitrophenol-tyrosine and tyrphostins act at the enzyme level to inhibit phosphorylation of all substrates by c-Src and v-Src kinases. In contrast, polylysine acts at the substrate level to stimulate Src-mediated phosphorylation of beta-casein but to inhibit phosphorylation of alpha-casein. Here we demonstrate novel enzyme-specific and substrate-specific modulations of Src kinase activity of potential physiological significance. At the enzyme level, we observed that c-Src kinase preferentially phosphorylates alpha-casein, while the v-Src kinase prefers beta-casein. At the substrate level we observed substrate-specific modulation by physiological factors including sphingosine, sphingosine derivatives and the ganglioside GM3. Galactosyl-sphingosine (psychosine) was more effective in stimulating phosphorylation of beta-casein and poly(E1A1Y1) than sphingosine. Glucosyl- and lactosyl-sphingosine were ineffective. Rat was extensively phosphorylated by c-Src in the presence of polylysine, and to a lesser extent in the sphingosine and galactosyl-sphingosine. These unexpected differences point out another potential mechanism for regulation of c-Src and v-Src kinase activities and may help to explain some of the pleotyptic manifestations of protein tyrosine kinase actions.

Transgenic plants of tall fescue (Festuca arundinacea Schreb.) obtained by direct gene transfer to protoplasts.

Chimeric hygromycin phosphotransferase (hph) and phosphinothricin acetyltransferase (bar) genes were introduced, using polyethylene glycol treatment, into protoplasts isolated from embryogenic cell suspension cultures of tall fescue (Festuca arundinacea Schreb.), a graminaceous plant that is an important forage crop in temperate pastures. Colonies resistant to either 200 mg/l hygromycin or 100 mg/l phosphinothricin, respectively, were recovered upon selection using bead-type culture systems. Stable integration of the transgenes in the genomes of plants regenerated from resistant callus clones was shown by Southern hybridization analysis. In situ hybridization of a labeled transgene-probe to metaphase chromosomes is shown for one transgenic primary regenerant. Expression of the transgenes in mature plants was demonstrated by HPH enzyme assay or by phosphinothricin-herbicide spraying.

Molecular and cytogenetic characterization of repetitive DNA sequences from Lolium and Festuca: applications in the analysis of Festulolium hybrids.

A set of species-specific repetitive DNA sequences was isolated from Lolium multiflorum and Festuca arundinacea. The degree of their species specificity as well as possible homologies among them were determined by dot-blot hybridization analysis. In order to understand the genomic organization of representative Lolium and Festuca-specific repetitive DNA sequences, we performed Southern blot hybridization and in situ hybridization to metaphase chromosomes.Southern blot hybridization analysis of eight different repetitive DNA sequences of L. multiflorum and one of F. arundinacea indicated either tandem and clustered arrangements of partially dispersed localization in their respective genomes. Some of these sequences, e.g. LMB3, showed a similar genomic organization in F. arundinacea and F. pratensis, but a slightly different organization and degree of redundancy in L. multiflorum. Clones sequences varied in size between 100 bp and 1.2 kb. Estimated copy number in the corresponding haploid genomes varied between 300 and 2×10(4). Sequence analysis of the highly species-specific sequences from plasmids pLMH2 and pLMB4 (L. multiflorum specific) and from pFAH1 (F. arundinacea specific) revealed some internal repeats without higher order. No homologies between the sequences or to other repetitive sequences were observed. In situ hybridization with these latter sequences to metaphase chromosomes from L. multiflorum, F. arundinacea and from symmetric sexual Festulolium hybrid revealed their relatively even distribution in the corresponding genomes. The in situ hybridization thus also allowed a clearcut simple identification of parental chromosomes in the Festulolium hybrid.The potential use of these species-specific clones as hybridization probes in quantitative dot-blot analysis of the genomic make-up of Festulolium (sexual and somatic) hybrids is also demonstrated.

Organelle transfer by microfusion of defined protoplast-cytoplast pairs.

Defined cybridization was performed by one-to-one electrofusion (microfusion) of preselected protoplast-cytoplast pairs of male-fertile, streptomycin-resistant Nicotiana tabacum and cytoplasmic male-sterile, streptomycin-sensitive N. tabacum cms (N. bigelovii), followed by microculture of the fusion products until plant regeneration. Dominant selectable markers, namely, kanamycin resistance (nptII) and hygromycin B resistance (hpt) genes had been previously integrated in the nuclear genomes of the otherwise almost fully isogenic parental strains using direct gene transfer to protoplasts. In addition to chromosome counts indicating the expected allotetraploid tobacco count of 48, the absence of the nucleus from the cytoplast donor line was confirmed by Southern blot hybridization using nptII and hpt probes, as well as by an in vitro selection test with leaf expiants and the corresponding enzyme assays for 30 cybrids. The cytoplasmic composition of the cybrids obtained was analyzed for chloroplast type using the streptomycin resistance/sensitivity locus. The fate of mitochondria in cybrids was checked by species-specific patterns in Southern analysis of restriction endonuclease digests of total DNA with N. sylvestris mitochondrial DNA probes.

Somatic hybridization by microfusion of defined protoplast pairs in Nicotiana: morphological, genetic, and molecular characterization.

Somatic hybrid/cybrid plants were obtained by microfusion of defined protoplast pairs from malefertile, streptomycin-resistant Nicotiana tabacum and cytoplasmic male-sterile (cms), streptomycin-sensitive N. tabacum cms (N. bigelovii) after microculture of recovered fusants. Genetic and molecular characterization of the organelle composition of 30 somatic hybrid/cybrid plants was performed. The fate of chloroplasts was assessed by an in vivo assay for streptomycin resistance/ sensitivity using leaf explants (R0 generation and R1 seedlings). For the analysis of the mitochondrial (mt) DNA, species-specific patterns were generated by Southern hybridization of restriction endonuclease digests of total DNA and mtDNA, with three DNA probes of N. sylvestris mitochondrial origin. In addition, detailed histological and scanning electron microscopy studies on flower ontogeny were performed for representative somatic hybrids/cybrids showing interesting flower morphology. The present study demonstrates that electrofusion of individually selected pairs of protoplasts (microfusion) can be used for the controlled somatic hybridization of higher plants.