Effects of calcium,magnesium, potassium and boron on sperm cells isolated from pollen of Zea mays L.

Our previous studies showed that Brewbaker and Kwack salts, which have been widely used in pollen germination and sperm isolation, are not appropriate for the maintenance of isolated maize (Zea mays L.) sperm cells. In the present study, we have characterized the effects of each BKS component salt on the integrity of isolated sperm cells using hemacytometry. At 0.01 and 0.1 mM, there were no differences in cell number between control and any salt-treated cells except a 22% decrease with 0.1 mM MgSO₄ at 48 h. At the 1 mM level, cell number decreased with time in the presence of Ca(NO₃)₂ and MgSO₄, with loss of integrity of most cells at 48 h, while KNO₃ and H₃BO₃ had little or no effect. Further characterization of calcium-induced reduction in cell integrity using flow cytometry showed that depletion of possible residual free calcium by addition of EGTA to the suspension medium improved cell longevity and viability. Exposure of isolated sperm cells to 1 mM calcium had no effect on cell integrity and viability in 5 h; however, only 12% of cells remained intact at 24 h. The reduction in cell integrity was hastened when cells were pretreated with the calcium ionophore A23187 prior to exposure to 1 mM calcium, with a 54% reduction in cell number at 1 h and complete cell lysis at 24 h. However, depletion of cytosolic free calcium by pretreatment of cells with the calcium ionophore followed by resuspension in the presence of EGTA resulted in rapid reduction of cell integrity as well. These results collectively suggest that maize sperm cells are sensitive to exogenous free calcium; however, a certain level of cytosolic free calcium is necessary for maintenance of integrity. Mechanisms of calcium-induced reduction in cell integrity are discussed along with possible roles of the sensitivity of sperm cells to calcium in fertilization.     

Chromosomal localization and molecular characterization of 53 cosmid-derived bovine microsatellites

Gene mapping in cattle has progressed rapidly in recent years largely owing to the introduction of powerful genetic markers, such as the microsatellites, and through advances in physical mapping techniques such as synteny mapping and fluorescence in situ hybridization (FISH). Microsatellite markers are often not physically mapped because they are generally isolated from small insert plasmid libraries, which makes their chromosomal localization inefficient. In this report we describe the FISH mapping of a large group of cosmid-derived bovine microsatellite markers, as our contribution to the European mapping initiative, BovMap. One objective of BovMap is to develop a set of anchored loci for the cattle genome map.Two cosmid libraries were screened with probes corresponding to the (AC) _n microsatellite motif. Positive clones were mapped by FISH, and then a subset was further analyzed by sequencing the region flanking the microsatellite repeat. In total, 58 clones were hybridized with chromosomes and identified loci on 22 of the 31 different bovine chromosomes. Three clones contained satellite DNA. Two or more markers were placed on 12 chromosomes. Sequencing of the microsatellites and flanking regions was performed directly from 43 cosmids, as previously reported (Ferretti et al. Anim. Genet. 25, 209–214, 1994). Primers were developed for 39 markers and used to describe the polymorphism associated with the corresponding loci.     

Kinetic study on starch hydrolysis using a glucose/maltose sensing system

Summary A dual-enzyme electrode flow injection system that can simultaneously determine glucose and maltose is used for an on-line study of starch hydrolyses catalysed by amylases. With the working system, determinations can be made every 2 minutes. A 10 L sample size with recycled back-flow minimises any loss of the reaction medium. The production, growth and decay of glucose and maltose concentrations during starch hydrolysis under various enzymatic conditions can thus be closely monitored, making it useful for the study of the catalytic kinetics of amylases and in screening and analysing enzyme systems.     

Rapid production and field testing of homozygous transgenic tobacco lines with virus resistance conferred by expression of satellite RNA and coat protein of cucumber mosaic virus

A procedure for the fast production of homozygotic transgenic plants was developed. Leaf discs of haploid tobacco plants from anther cultures were transformed with a chimaeric vector containing coat protein (CP) and satellite RNA (Sat-RNA) genes from cucumber mosaic virus (CMV). One-hundred-and-twelve Kanamycin-resistant transformed haploid plants were subjected to selection based on the expression of both CP and Sat-RNA. Eighty-nine transgenic plants expressing both genes were selected and tested for their resistance to CMV by inoculation with high concentration of CMV (200 g ml^–1). Only five plants showed no symptoms of viral infection 30 days after inoculation. These plants were then diploidized by colchicine treatment. Three homozygous diploid lines with high levels of resistance to CMV were obtained after only one generation. The three transgenic lines were further tested under field conditions. The results showed that the progenies of these transgenic lines were homozygous and were highly resistant to CMV under natural field infection and manual inoculation conditions.     

Inducible nitric oxide synthase and guinea-pig ileitis induced by adjuvant

We sought to establish a model of inflammatory bowel disease by augmenting the activity of the local immune system with Freund's complete adjuvant, and to determine if inducible nitric oxide synthase (iNOS) expression and peroxynitrite formation accompanied the inflammatory condition. In anaesthetized guinea-pigs, a loop of distal ileum received intraluminal 50% ethanol followed by Freund's complete adjuvant. Control animals were sham operated. When the animals were killed 7 or 14 days later, loop lavage fluid was examined for nitrite and PGE(2) levels; mucosal levels of granulocyte and macrophages were estimated by myeloperoxidase (MPO) and N-acetyl-D-glucosaminidase (NAG) activity, respectively. Cellular localization if iNOS and peroxynitrite formation were determined by immunohistochemistry with polyclonal antibodies directed against peptide epitopes of mouse iNOS and nitrotyrosine, respectfully. Adjuvant administration resulted in a persistent ileitis, featuring gut thickening, crypt hyperplasia, villus tip swelling and disruption, and cellular infiltration. Lavage levels of PGE(2) and nitrite were markedly elevated by adjuvant treatment. Immunoreactive iNOS and nitrotyrosine bordered on detectability in normal animals but were markedly evident with adjuvant treatment at day 7 and particularly day 14. Immunohistochemistry suggested that enteric neurons and epithelia were major sites of iNOS activity and peroxynitrite formation. We conclude that local administration of adjuvant establishes a chronic ileitis. Inducible nitric oxide synthase may contribute to the inflammatory process.