Culture and fusion of pollen protoplasts of Brassica oleracea L. var. italica with haploid mesophyll protoplasts of B. rapa L. ssp. pekinensis

Hybrid callus was formed from the successful protoplast fusion between pollen protoplasts of Brassica oleracea var. italica and haploid mesophyll protoplasts of Brassica rapa. The pollen protoplast isolation frequency in broccoli was highly related to the ratio of trinucleate pollens in the male gametophyte population. Large quantities of pollen protoplasts with high vigor could be isolated, and the isolation frequency reached up to 90% in 6.0-7.0 mm long flower buds with about 94.7% trinucleate-stage pollens. Pollen protoplasts could be collected and purified by discontinuous gradient centrifugation. In 1% Na-alginate embedding culture, cell divisions were observed but no further development was found. The haploid mesophyll protoplasts were isolated from in vitro haploid plants of B. rapa. Results strongly showed the variability in culturability of mesophyll protoplasts from different haploid lines. Both pollen protoplasts and haploid mesophyll protoplasts retained a stable round shape in the designed prefusion solution with an osmotic pressure of 0.74 osmol/kg. Polyethylene glycol was used for the protoplast fusion, and 40% polyethylene glycol 4000 enabled the highest fusion frequency of about 20%. Some postfusion protoplasts showed cell divisions up to callus proliferation. Calli were screened by random amplified polymorphic DNA analysis for their hybrid character. Results revealed the existence of the hybrid calli. Some of the hybrid calli grew well with green color and shoot primordia. According to our knowledge, this is the first report about a hybrid formation between two haploid protoplasts. Potential comprehensive applications, as well as problems of this technique, are discussed.     

Structural biology of human cannabinoid receptor-2 helix 6 in membrane-mimetic environments

We detail the structure and dynamics of a synthetic peptide corresponding to transmembrane helix 6 (TMH6) of human cannabinoid receptor-2 (hCB2) in biomembrane-mimetic environments. The peptide’s NMR structural biology is characterized by two α-helical domains bridged by a flexible, nonhelical hinge region containing a highly-conserved CWFP motif with an environmentally sensitive, Pro-based conformational switch. Buried within the peptide’s flexible region, W²⁵⁸ may hydrogen-bond with L²⁵⁵ to help stabilize the Pro-kinked hCB2 TMH6 structure and position C²⁵⁷ advantageously for interaction with agonist ligands. These characteristics of hCB2 TMH6 are potential structural features of ligand-induced hCB2 activation in vivo.     

The effect of spray volume on spray partitioning between plant and soil

Summary Applications of chlorsulfuron (11.25 g ai ha⁻¹) were made to wheat, flax, canola and lentils at spray volumes of 48, 108 and 2171 ha⁻¹, and with metsulfuron methyl (6.00 g ai ha⁻¹) at spray volumes of 48 and 2171 ha⁻¹. Applications were made to the shoot only, the soil only and to the plant plus soil. Spectrofluorometric analysis was used to determine spray partitioning within the plant-soil system and foliar retention was related to efficacy. Fresh and dry weights of shoot material were determined 3 weeks after treatment. Flax and wheat were more tolerant of restricted-foliar applications than those made to the soil, the converse being true to canola and lentils. Applications made to the plant and soil were always the most deleterious. Foliar retention and efficacy did not correlate directly. Applications in 2171 ha⁻¹ were generally more efficacious than those at 481 ha⁻¹.     

Application of spectrofluorometry to the prediction of PHB concentrations in a fed-batch process

On-line estimation of biopolymer production during fermentation would be a useful adjunct to the development of strategies for process control and optimization. This study examined the applicability of spectrofluorometry, along with other on-line measurements, for the prediction of poly-ß-hydroxybutyric acid (PHB) concentrations in a high-cell density fed-batch fermentation of Ralstonia eutropha. Models previously used for modelling PHB evolution with time are not sufficiently accurate for situations where transient intermediate accumulations or PHB degradation occur. Thus, the mass balance in the model was modified to account for these situations. An estimation algorithm was developed that is based on a hybrid model consisting of a dynamic mass balance of PHB where the main reaction coefficient was regressed with respect to spectrofluorometric data. The regression between the kinetic parameter and the spectrofluorometric data was accomplished using partial least squares (PLS) regression to avoid high sensitivity to noise expected from highly correlated data, such as the spectrofluorometric readings. The model accounts for dynamics of intermediates and in this way allows the prediction of dynamic behaviour in PHB concentrations that cannot be predicted with other reported mathematical models.     

The Staphylococcal Pore-forming Leukotoxins Open Ca2+ Channels in the Membrane of Human Polymorphonuclear Neutrophils

The ability of leukotoxins secreted by Staphylococcus aureus to modify the permeability of the membrane of human polymorphonuclear neutrophils has been studied by spectrofluorometry and appropriate fluorescent probes. This family of bicomponent leukotoxins is constituted by, at least, three pairs of proteins: LukS-PV/LukF-PV, HlgA/HlgB, HlgC/HlgB. After binding of both components to the membrane, each pair induces influxes of divalent cations and ethidium in polymorphonuclear neutrophils, although with different intensities. The influx of divalent cations appears sooner than the influx of ethidium. The pathway for divalent cations is not permeable to monovalent cations (Na⁺, K⁺, ethidium⁺) and is blocked by Ca²⁺ channel inhibitors that do not block the fluxes of ethidium and monovalent cations. It is concluded that the leukotoxins bind to a receptor linked to a divalent cation-selective channel or to the channel itself which is activated. Then, the leukotoxins open a second pathway by insertion into the membrane and subsequent formation of aspecific pores allowing an influx of ethidium. Received: 8 May 1997/Revised: 22 December 1997