Cryopreservation of immature spring wheat zygotic embryos using an abscisic acid pretreatment

Spring wheat (Triticum aestivum L.) zygotic embryos were successfully cryopreserved, without the addition of exogenous cryoprotectants, using only an abscisic acid (ABA) pretreatment. Optimum survival was obtained when embryos were cultured in vitro for 10 days on semisolid Murashige and Skoog (MS) nutrient medium supplemented with 0.5 mg/L (±) ABA prior to cryopreservation. The embryos resumed growth within three days when returned to MS medium devoid of ABA but containing 2mg/L 2,4-dichlorophenoxyacetic acid. The embryogenic calli produced from these embryos exhibited normal plant regeneration on auxin-free media. Changes in dw/fw ratio, as well as the esterified fatty acid and sucrose concentrations correlated positively with the development of tolerance to cryopreservation.NRCC Publication No. 33519     

Degradation of (+/-)-synephrine by Arthrobacter synephrinum. Oxidation of 3,4-dihydroxyphenylacetate to 2-hydroxy-5-carboxymethyl-muconate semialdehyde.

1. Cell-free extracts of Arthrobacter synephrinum catalyse the oxidation of 3,4-dihydroxy-phenylacetate. 2. The product of oxidation was characterized as 2-hydroxy-5-carboxymethylmuconate semialdehyde from its chemical behaviour as well as from nuclear-magnetic-resonance spectra. 3. A 3,4-dihydroxyphenylacetate 2,3-dioxygenase (EC 1.13.11.15) was partially purified from A. synephrinum. 4. The enzyme had a Km of 25 micrometer towards its substrate and exhibited typical Michaelis-Menten kinetics. 5. The enzyme also catalysed the oxidation of 3,4-dihydroxymandelate and 3,4-dihydroxyphenylpropionate, at reaction rates of 0.5 and 0.04 respectively of that for 3,4-dihydroxyphenylacetate. 6. The enzyme was sensitive to treatment with thiol-specific reagents. 7. The molecular weight of the enzyme as determined by Sephadex G-200 chromatography was approx. 282000.     

Microbial metabolism of phenolic amines: degradation of dl-synephrine by an unidentified arthrobacter.

Microorganisms capable of degrading dl-synephrine were isolated from soil of Citrus gardens by enrichment culture, with dl-synephrine as the sole source of carbon and nitrogen. An organism which appears to be an arthrobacter, but which cannot be identified with any of the presently recognized species was predominant in these isolates. It was found to metabolize synephrine by a pathway involving p-hydroxyphenylacetaldehyde, p-hydroxyphenylacetic acid, and 3,4-dihydroxyphenylacetic acid as intermediates. Some of the enzymes of this pathway were demonstrated in cell-free extracts. An aromatic oxygenase, which could also be readily obtained in a cell-free system, was found to degrade 3,4-dihydroxyphenylacetic acid by meta cleavage.     

Wound healing activity of NOE-aspirin: a pre-clinical study.

Aspirin, one of the oldest non-steroidal anti-inflammatory drugs, impedes tissue repair by virtue of retarding inflammation. The present study was undertaken to find out if linking of nitrooxyethyl ester to aspirin reverses its healing-depressant propensity. Nitrooxyethyl ester of aspirin (NOE-Asp) was synthesized in our laboratory through well-established synthetic pathway, starting from aspirin through esterification with ethylene glycol and nitration with a mixture of nitric and sulfuric acids at 0 degrees C. The effect of NOE-Asp on phases of healing such as collagenation, wound contraction and epithelialization and on scar size of healed wound was evaluated in three wound models-incision, dead space, and excision wounds. To assess its influence on the oxidative stress, the levels of glutathione (GSH) and thiobarbiturate reactive species (TBARS) were also determined in 10-day-old granulation tissue. NOE-Asp was further screened for its anti-inflammatory activity in rat paw edema model. NOE-Asp promoted collagenation (increase in breaking strength, granulation weight, and collagen content), wound contraction, and epithelialization phases of healing. NOE-Asp also showed a significant antioxidant effect in 10-day-old granulation tissue as compared to aspirin. The results vindicate our assumption that the esterification of aspirin with nirooxyethyl group reverses the healing-suppressant effect of aspirin. The compound also showed equipotent anti-inflammatory activity as aspirin.     

The phylogeny and evolution of host choice in the Hippoboscoidea (Diptera) as reconstructed using four molecular markers

Hippoboscoidea is a superfamily of Diptera that contains the Glossinidae or tsetse flies, the Hippoboscidae or louse flies, and two families of bat flies, the Streblidae and the Nycteribiidae. We reconstruct the phylogenetic relationships within Hippoboscoidea using maximum parsimony and Bayesian methods based on nucleotide sequences from fragments of four genes: nuclear 28S ribosomal DNA and the CPSase domain of CAD, and mitochondrial 16S rDNA and cytochrome oxidase I. We recover monophyly for most of the presently recognized groups within Hippoboscoidea including the superfamily as a whole, the Hippoboscidae, the Nycteribiidae, the bat flies, and the Pupipara (=Hippoboscidae+Nycteribiidae+Streblidae), as well as several subfamilies within the constituent families. Streblidae appear to be paraphyletic. Our phylogenetic hypothesis is well supported and decisive in that most competing topological hypotheses for the Hippoboscoidea require significantly longer trees. We confirm a single shift from a free-living fly to a blood-feeding ectoparasite of vertebrates and demonstrate that at least two host shifts from mammals to birds have occurred. Wings have been repeatedly lost, but never regained. The hippoboscoid ancestor also evolved adenotrophic viviparity and our cladogram is consistent with a gradual reduction in the motility of the deposited final instar larvae from active burrowing in the soil to true pupiparity where adult females glue the puparium within the confines of bat roosts.     

Identification and characterization of rad51 of Pneumocystis

Rad51, a eukaryotic homolog of RecA, is an important protein involved in DNA recombination and repair. We have characterized rad51 of Pneumocystis carinii and Pneumocystis murina. rad51 is a single copy gene that encodes a 1.2 kb mRNA, which contains an open reading frame encoding 343 amino acids. Rad51 from Pneumocystis showed high homology to those from yeast. ATP binding motifs GEFRTGKS and LLIVD, similar to those of Saccharomyces cerevisiae and Schizosaccharomyces pombe, are conserved in Pneumocystis Rad51. The recombinant protein when expressed in E. coli showed DNA-dependent ATPase activity. Since Rad51 is a key enzyme in DNA repair and recombination, it potentially plays an important role in the recombination process leading to antigenic variation and thereby resistance to host immune responses in Pneumocystis.     

Affinity binding of cells to cryogel adsorbents with immobilized specific ligands: effect of ligand coupling and matrix architecture

The capture of human acute myeloid leukemia KG-1 cells expressing the CD34 surface antigen and the fractionation of human blood lymphocytes were evaluated on polyvinyl alcohol (PVA)-cryogel beads and dimethyl acrylamide (DMAAm) monolithic cryogel with immobilized protein A. The affinity ligand (protein A) was chemically coupled to the reactive PVA-cryogel beads and epoxy-derivatized monolithic cryogels through different immobilization techniques and the binding efficiency of the cell surface receptors specific antibody-labeled cells to the gels/beads was determined. The binding of cells to monolithic cryogel was higher (90-95%) compared with cryogel beads (76%). B-lymphocytes, which bound to the protein A-cryogel beads, were separated from T-lymphocytes with yields for the two cell types 74 and 85%, respectively. About 91% of the bound B-cells could be recovered without significantly impairing their viability. Our results show differences in the percentage of cell-binding to the immunosorbents caused by ligand density, flow shear forces and bond strength between the cells and the affinity surface once distinct chemical coupling of protein A, size of beads, sequence of antibody binding to protein A adsorbents, morphology and geometry of surface matrices were compared.     

Antibody-induced arthritis: disease mechanisms and genes involved at the effector phase of arthritis

During the development of rheumatoid arthritis (RA) autoantibodies to IgG-Fc, citrullinated proteins, collagen type II (CII), glucose 6 phosphoisomerase (G6PI) and some other self-antigens appear. Of these, a pathogenic effect of the anti-CII and anti-G6PI antibodies is well demonstrated using animal models. These new antibody mediated arthritis models have proven to be very useful for studies involved in understanding the molecular pathways of the induction of arthritis in joints. Both the complement and FcgammaR systems have been found to play essential roles. Neutrophils and macrophages are important inflammatory cells and the secretion of tumour necrosis factor-alpha and IL-1beta is pathogenic. The identification of the genetic polymorphisms predisposing to arthritis is important for understanding the complexity of arthritis. Disease mechanisms and gene regions studied using the two antibody-induced arthritis mouse models (collagen antibody-induced arthritis and serum transfer-induced arthritis) are compared and discussed for their relevance in RA pathogenesis.