Membrane particle changes attending the acrosome reaction in guinea pig spermatozoa

To examine the freeze-fracture appearance of membrane alterations accompanying the preparation of sperm membranes for fusions-the first preparatory stage occurring before physiological release of the acrosomal content, the second afterward-we induced the acrosome reaction in capacitated guinea pig spermatozoa by adding calcium to the mixture. The most common features observed before fusion of the acrosomal and plasma membranes were the deletion of fibrillar intramembranous particles from the E-fracture faces of both membranes, and the clearance of globular particles from the P face of the plasma membrane-events taking place near the terminus of the equatorial segment. Large particles, >12nm, remained not far from the cleared E-face patches. The P face of the outer acrosomal membrane is virtually clear from the outset. In addition, when fusion was completed, occasional double lines of large particles transiently embossed the P face of the plasma membrane (postacrosomal) side of the fusion zone. Behind the line of fusion, another series of particle-cleared foci emerged. We interpreted these postfusion membrane clearances as a second adaptation for sperm-egg interaction. Induction of the acrosome reaction in media containing phosphatidylcholine liposomes resulted in their apparent attachment, incorporation, or exchange in both the originally and secondarily cleared regions. Our observations support the concepts that membranes become receptive to union at particle- deficient interfaces, and that the physiologically created barren areas in freeze-fracture replicas may herald incipient membrane fusion.     

Modifications of anionic-lipid domains preceding membrane fusion in guinea pig sperm

The relationship between anionic-lipid concentration and the functional properties of plasma-membrane domains was explored using the guinea-pig sperm membrane as a model, with polymyxin B (PXB) as a probe. Areas of plasmalemma specialized for fusion during the acrosome reaction had a higher affinity for the probe than adjacent nonfusigenic regions. In addition, capacitation--a process preceding acrosome:plasma-membrane fusion--markedly enlarged the area susceptible to PXB binding over the acrosomal cap. Protease treatment mimicked capacitation by increasing the acrosome-reaction incidence as well as PXB binding, at enzyme concentrations not affecting the surface coat nor altering filipin/sterol localization. Both proteolytic digestion and capacitation failed to augment PXB- or filipin-affinity in nonfusigenic zones, such as the post-acrosomal segment, including its particle-free maculae. Incubation of sperm in capacitating medium supplemented with 32P-labeled phosphate, followed by lipid extraction, thin-layer chromatography, and autoradiography, revealed a radioactive band comigrating with cardiolipin and phosphatidic acid. Vermiform protrusions elicited by PXB in the outer lamellae of cardiolipin- phosphatidylcholine liposomes resembled those seen in fusional regions of sperm membrane. We conclude that (a) differing concentrations of anionic lipids are found in adjacent domains of the sperm plasma membrane; (b) these domains mirror the functional regions of the membrane, with higher anionic-lipid concentrations localized over fusional zones; (c) the surface coat does not participate in the maintenance of such domains; (d) anionic-lipid synthesis may contribute to their formation; and (e) anionic-lipid concentrations increase as the membrane becomes fusionally competent, indicating that cellular modulation of lipid domains accompanies regulation of membrane function.     

Monoclonal antibodies to rabbit liver cytochrome P-448

Cytochrome P-448 (mol wt 55,000 Daltons) from rabbit liver was purified to a specific content of 16.6 nmol/mg. Mice were immunised with this preparation, their spleens removed and dissociated lymphocytes hybridised with myeloma cells. Four monoclonal antibodies against cytochrome P-448 were raised and partially characterised. All four antibodies interacted with cytochrome P-448 in intact microsomal fractions and selectively immunoadsorbed cytochrome P-448 from solubilised microsomal preparations. One of the antibodies inhibited benzo[a] pyrene hydroxylase activity in a reconstituted system, one had no effect on activity and two increased activity. The possible applications of such antibodies are discussed.     

Nanocrystalline SrS:Ce3+ system for the generation of white light‐emitting diodes

Nanocrystalline SrS phosphors doped with Ce³⁺ ions at different concentrations (0.5, 1, 1.5 and 2 mol%) are synthesized via the solid‐state diffusion method (SSDM), which is suitable for the large‐scale production of phosphors in industrial applications. The as‐prepared samples are characterized using an X‐ray diffraction (XRD) technique, field emission scanning electron microscopy (FESEM), high‐resolution transmission electron microscopy (HRTEM) and energy‐dispersive X‐ray (EDX) analysis. The optical properties of these phosphors are analyzed using reflectance spectra, photoluminescence spectra and afterglow decay curves. The cubic structure of the SrS phosphor is confirmed by XRD analysis and the crystallite size calculated by Scherer's formula using XRD data shows the nanocrystalline nature of the phosphors. No phase change is observed with increasing concentrations of Ce³⁺ ions. The surface morphology of the prepared phosphors is determined by FESEM, which shows a sphere‐like structure and good connectivity of the grains. The authenticity of the formation of nanocrystalline phosphors is examined by HRTEM analysis. Elemental compositional information for the prepared phosphors is gathered by EDX analysis. Photoluminescence studies reveal that the emission spectra of the prepared phosphor shows broad band emission centered at 458 and 550 nm due to the transition of electrons from the 5d → 4f energy levels. The afterglow decay characteristics of different as‐synthesized SrS:Ce³⁺ nanophosphors are conceptually described. Copyright © 2016 John Wiley & Sons, Ltd.     

Transfer of a male-sterility-inducing cytoplasm from onion to leek ( Allium ampeloprasum)

Two interspecific triploid (AAC) hybrids (84/1-94 and 99/1-94) from crosses between onion [ Allium cepa (2 n=2 x=16, CC)] and leek [ A. ampeloprasum (2 n=4 x=32, AAAA)] were backcrossed to leek in order to transfer a male-sterility-inducing cytoplasm from onion that would enable the production of hybrid leek. GISH evaluations of meiosis in the interspecific hybrids revealed irregularities due to univalent onion chromosomes producing micronuclei from onion chromatin, whereas the pairing of the two sets of leek chromosomes was nearly normal. Attempts to use colchicine to double the chromosome number of the hybrids failed. Backcrosses of 84/1-94 to leek as the pollen parent were not successful. The first backcross of 99/1-94 to tetraploid leek produced 11 BC(1) plants with chromosome numbers between 38 and 41. Identification of parental chromosomes by GISH showed that all eight onion chromosomes and 30-33 leek chromosomes were transmitted to the backcross progenies due to unreduced egg cells. Onion chromosomes were eliminated during the second backcross. Southern hybridization confirmed the transfer of the T-cytoplasm like source of CMS from onion to the BC(2) progenies. After the third backcross to leek, 158 plants were obtained with varying numbers of onion chromosomes and some intergenomic recombinant chromosomes. Alloplasmic leek plants without onion chromatin were selected for further characterization of male sterility and quality traits.     

Diabetic neuropathy and surface sway-referencing disrupt somatosensory information for postural stability in stance

In order to determine the type of somatosensory information for postural control that is most affected by neuropathy, we compared the relative effects of three methods of sway-referencing the surface in a group of subjects with profound loss of somatosensory function associated with sensory polyneuropathy from diabetes with age-matched control subjects. Sway-referencing disrupted somatosensory feedback for postural control by servo-controlling the dorsi- and plantar-flexion rotation of the support surface in proportion to anterior-posterior excursion of (1) ankle angle, (2) center of body mass (CoM) angle or (3) filtered center of pressure (CoP). Postural sway in subjects with somatosensory loss was significantly larger than normal on a firm surface but not on the sway-referenced surfaces, suggesting that sway-referencing disrupts somatosensory information for postural control already disrupted by neuropathy. Control subjects standing on any sway-referenced surface swayed significantly more than neuropathy subjects who stood on a firm surface, suggesting that sway-referencing disrupts more somatosensory information than disrupted by severe neuropathy. CoP sway-referencing was less sensitive than ankle or CoM sway-referencing for distinguishing postural sway in subjects with somatosensory loss from age-matched control subjects. Given that filtered CoP sway-referencing disrupts the ability to utilize somatosensory information related to surface reactive force to a greater extent than the other two methods of sway-referencing, then these results support the hypothesis that subjects with diabetic peripheral neuropathy have lost more CoP information, than ankle or CoM angle information, for controlling postural sway in stance.     

Unravelling the genetic diversity of ruminal bacteria belonging to the CFB phylum

Molecular biology approaches were employed to examine the genetic diversity of bacteria from the Cytophaga/Flexibacter/Bacteroides (CFB) phylum in the rumen of cattle. By this means we were able to identify cultured strains that represent some of the larger CFB clusters previously identified only by PCR amplification and sequencing. Complete 16S rDNA sequences were obtained for 16 previously isolated rumen strains, including the type strains of Prevotella ruminicola, P. bryantii, P. brevis and P. albensis to represent a wide range of diversity. Phylogenetic analysis of cultured strains revealed the existence of three clusters of ruminal CFB: (i) a cluster of Prevotella strains, which have been found only in the rumen, including the two type strains, P. brevis GA33(T) and P. ruminicola 23(T); (ii) Prevotella spp. that cluster with prevotellas from other ecological niches such as the oral cavity and which include the type strains, P. bryantii B(1)4(T) and P. albensis M384(T); (iii) two Bacteroides spp. strains clustering with B. forsythus of oral origin. In order to establish whether the cultivated isolates cover the whole range of ruminal CFB genetic diversity, 16S rRNA gene sequences were amplified and cloned from DNA extracted from the same rumen samples (one cow in Slovenia, one in Scotland and three in Japan). Sequencing and phylogenetic analysis of 16S rRNA genes confirmed the existence of two superclusters of ruminal Prevotella, one exclusively ruminal and the other including non-ruminal species. In the case of ruminal Bacteroides spp., however, phylogenetic analysis revealed the existence of three new superclusters, one of which has as yet no cultivable counterpart. Interestingly, these Bacteroides clusters were represented almost exclusively by clone libraries from the Japanese cattle and only three sequences were from the European cattle. This study agrees with previous analyses in showing that rumen Prevotella/Bacteroides strains exhibit a remarkable degree of genetic diversity and suggests that different strain groupings may differ greatly in their recovery by cultural methods. The most important conclusion, however, is that cultured strains can be identified that represent some of the larger clusters previously identified only by PCR amplification and sequencing.     

Bacteria in cancer therapy: a novel experimental strategy

Resistance to conventional anticancer therapies in patients with advanced solid tumors has prompted the need of alternative cancer therapies. Moreover, the success of novel cancer therapies depends on their selectivity for cancer cells with limited toxicity to normal tissues. Several decades after Coley's work a variety of natural and genetically modified non-pathogenic bacterial species are being explored as potential antitumor agents, either to provide direct tumoricidal effects or to deliver tumoricidal molecules. Live, attenuated or genetically modified non-pathogenic bacterial species are capable of multiplying selectively in tumors and inhibiting their growth. Due to their selectivity for tumor tissues, these bacteria and their spores also serve as ideal vectors for delivering therapeutic proteins to tumors. Bacterial toxins too have emerged as promising cancer treatment strategy. The most potential and promising strategy is bacteria based gene-directed enzyme prodrug therapy. Although it has shown successful results in vivo yet further investigation about the targeting mechanisms of the bacteria are required to make it a complete therapeutic approach in cancer treatment.