Purification and Properties of Deoxyribonucleic Acid Binding Factor Isolated from the Surface of Streptococcus sanguis Cells

Deoxyribonucleic acid (DNA) binding factor (BF) was found in surface fluids from competent and noncompetent cells of Streptococcus sanguis strains Challis, Wicky, and Blackburn. Fluids from noncompetent cells exhibited about 10% BF activity compared with extracts from competent cells. BF from competent Wicky cells was purified to homogeneity by electrophoresis and immunodiffusion. Purified BF preparations exhibited slight endonucleolytic activity, directed mainly against single-stranded DNA. Nucleolytic and DNA binding activities present in purified BF could be separated by polyacrylamide gel electrophoresis. Purified BF was sensitive to proteolytic enzymes and to phospholipase D, and its activity was stimulated in the presence of low Triton X-100 concentrations. The protein component of BF is a single, monomeric polypeptide with a molecular weight of 56,000 and an isoelectric point of pH 5.8. Binding of purified BF to DNA was a very rapid process at the optimum temperature, pH, and ionic strength and led to the formation of fast-sedimenting complexes. Purified BF was tested for several properties. It exhibited higher affinity to single- than to double-stranded DNA. It bound poorly to glucosylated phage T4 and single-stranded, synthetic polydeoxyribonucleotides and did not bind to RNA. It protected single-stranded DNA against nuclease S₁ action but did not protect native DNA against deoxyribonuclease I action. No evidence was found for unwinding activity, using double-stranded DNA as a substrate.     

Spreading of human fibroblasts in serum-free medium: Inhibition by dithiothreitol and the effect of cold insoluble globulin (plasma fibronectin)

We have tested the effect of dithiothreitol (DTT) treatment on the initial spreading of human fibroblasts in serum-free medium in tissue culture dishes. Cell spreading was inhibited following treatment of these cells with 10 mM DTT. Inhibition occurred when the cells were treated at 37°C but not at 4° and was reversible metabolically but not by the addition of sulfhydryl oxidizing reagents. The inhibition was overcome when DTT-treated human fibroblasts were plated on cold insoluble globulin (plasma fibronectin)—coated dishes. Under these conditions spreading appeared to be completely normal, including the formation of focal adhesions. Analysis of the fibronectin concentrations in the human fibroblasts following DTT treatment indicated that there was little decrease in the absolute level of activity as determined in a biological assay for BHK cell spreading on culture dishes. Analysis of the fibronectin distribution on the DTT-treated human fibroblasts by indirect immunofluorescence using a specific anti-CIG antiserum revealed that fibronectin was no longer deposited onto the culture dish surfaces. Even when the DTT-treated human fibroblasts spread in the presence of fetal calf serum, the cell fibronectin remained for the most part in a perinuclear location. These results indicate that DTT treatment of human fibroblasts prevents the normal translocation of fibronectin from a perinuclear location to the surface of the culture dish. This study further supports our hypothesis that the initial spreading in serum-free medium of fibroblasts from cell strains depends upon secretion of fibronectin onto the culture dish surface.     

Ancient Cytokines,the Role of Astakines as Hematopoietic Growth Factors

Hematopoiesis is the process by which hemocytes mature and subsequently enter the circulation. Vertebrate prokineticins (PKs) are known to take part in this process, as are the invertebrate prokineticin domain proteins, astakines. In Pacifastacus leniusculus, astakine 1 is essential for the release of new hemocytes into the open circulatory system of these animals. In addition to astakine 1, we have now cloned a homologue of astakine 1 with an insert of 13 amino acids, named as astakine 2. Both crustacean astakines lack the N-terminal AVIT motif, which is present in vertebrate PKs, and hence receptor binding differs from that of vertebrate PKs. We have found astakine-like sequences in 19 different invertebrate species, and the sequences show that some motifs are conserved among invertebrate groups. Previously we showed that astakine 1 is directly involved in hematopoiesis, and now we show that astakine 1 and astakine 2 have different roles in hemocyte lineage differentiation. Astakine 1 can stimulate proliferation of hematopoietic tissue (Hpt) cells (precursor of hemocytes) as well as specifically induce differentiation of Hpt cells along the semigranular cell lineage, whereas astakine 2 plays a role in granular cell differentiation. Moreover, we discuss the impact of the putative structures of different astakines in comparison with the vertebrate prokineticins.     

RNA polymerase heterogeneity in Streptomyces aureofaciens: characterization by antibody-linked polymerase assay

The ability of Escherichia coli with different receptor specificities to interact with meconium was studied. E. coli strains expressing P-fimbriae, specific for Gal alpha 1-4Gal beta-containing receptors, were agglutinated by meconium at high titres. This reaction was inhibited by globotetraosylceramide. The attachment of P-fimbriated E. coli to human colonic epithelial cells of the HT-29 cell line was inhibited by meconium. Some type 1 fimbriated strains were agglutinated by meconium, but the agglutination was rarely blocked by methyl alpha-D-mannoside. The attachment by type 1 fimbriated strains to HT-29 cells was reduced by meconium only in some cases. These results suggest that meconium interacts with the P-fimbriae of E. coli, in a way that may influence bacterial colonization of the neonatal intestine.     

Intergeneric Hybridization between Species of Leymus, Psathyrostachys and Hordeum (Poaceae, Triticeae)

A total of 132 intergeneric crossing attempts (49 combinations)involving species of Leymus Hochst., Psathyrostachys Nevskiand Hordeum L. were performed, of which 103 were between Hordeumand Leymus. Embryo rescue was used throughout the experiment.Hybrids between Leymus and Psathyrostachys were difficult toobtain. Hybrid progeny were relatively easily obtained whencrossing Hordeum and Leymus. Plants from 20 different combinationswere obtained. Nineteen of these have not previously been reported.Meiotic analysis of three hybrid combinations of Hordeum x Leymusis reported. The high frequency of univalents in meiotic interphase(MI) indicates that allosyndetic chromosome pairing did notoccur, supporting the assumption that the genomes of Leymusare non-homologous to the H genomes of Hordeum.Copyright 1994,1999 Academic Press Taxonomy, Triticeae, Leymus, Psathyrostachys, Hordeum, intergeneric hybridization     

Positive selection and convergent evolution shape molecular phenotypic traits of innate immunity receptors in tits (Paridae)

Despite widespread variability and redundancy abounding animal immunity, little is currently known about the rate of evolutionary convergence (functionally analogous traits not inherited from a common ancestor) in host molecular adaptations to parasite selective pressures. Toll‐like receptors (TLRs) provide the molecular interface allowing hosts to recognize pathogenic structures and trigger early danger signals initiating an immune response. Using a novel combination of bioinformatic approaches, here we explore genetic variation in ligand‐binding regions of bacteria‐sensing TLR4 and TLR5 in 29 species belonging to the tit family of passerine birds (Aves: Paridae). Three out of the four consensual positively selected sites in TLR4 and six out of 14 positively selected positions in TLR5 were located on the receptor surface near the functionally important sites, and based on the phylogenetic pattern evolved in a convergent (parallel) manner. This type of evolution was also seen at one N‐glycosylation site and two positively selected phosphorylation sites, providing the first evidence of convergence in post‐translational modifications in evolutionary immunology. Finally, the overall mismatch between phylogeny and the clustering of surface charge distribution demonstrates that convergence is common in overall TLR4 and TLR5 molecular phenotypes involved in ligand binding. Our analysis did not reveal any broad ecological traits explaining the convergence observed in electrostatic potentials, suggesting that information on microbial symbionts may be needed to explain TLR evolution. Adopting state‐of‐the‐art predictive structural bionformatics, we have outlined a new broadly applicable methodological approach to estimate the functional significance of positively selected variation and test for the adaptive molecular convergence in protein‐coding polymorphisms.     

Phenotypic and Molecular Assessment of Wheat Genotypes Tolerant to Leaf Blight,Rust and Blast Diseases

Globally among biotic stresses, diseases like blight, rust and blast constitute prime constraints for reducing wheat productivity especially in Bangladesh. For sustainable productivity, the development of disease-resistant lines and high yielding varieties is vital and necessary. This study was conducted using 122 advanced breeding lines of wheat including 21 varieties developed by Bangladesh Wheat and Maize Research Institute (BAMRI) with aims to identify genotypes having high yield potential and resistance to leaf blight, leaf rust and blast diseases. These genotypes were evaluated for resistance against leaf blight and leaf rust at Dinajpur and wheat blast at Jashore under field condition. Out of 122 genotypes tested, 20 lines were selected as resistant to leaf blight based on the area under the diseases progress curve (AUDPC) under both irrigated timely sown (ITS) and irrigated late sown (ILS) conditions. Forty-two genotypes were found completely free from leaf rust infection, 59 genotypes were identified as resistant, and 13 genotypes were identified as moderately resistant to leaf rust. Eighteen genotypes were immune against wheat blast, 42 genotypes were categorized as resistant, and 26 genotypes were identified as moderately resistant to wheat blast. Molecular data revealed that the 16 genotypes showed a positive 2NS segment among the 18 immune genotypes selected against wheat blast under field conditions. The genotypes BAW 1322, BAW 1295, and BAW 1203 can be used as earlier maturing genotypes and the genotypes BAW 1372, BAW 1373, BAW 1297 and BAW 1364 can be used for lodging tolerant due to short plant height. The genotypes WMRI Gom 1, BAW 1349 and BAW 1350 can be selected for bold grain and the genotypes WMRI Gom 1, BAW 1297, BAW 1377 can be used as high yielder for optimum seeding condition but genotypes BAW 1377 and BAW 1366 can be used for late sown condition. The selected resistant genotypes against specific diseases can be used in the further breeding program to develop wheat varieties having higher disease resistance and yield potential.     

Crop photosynthesis for the twenty-first century

Photosynthesis Research -