The role of sensitive bacteria in determining the count of phage negative colonies on solid growth media

The purpose of the work was to study how the number of phage negative colonies on a solid growth medium depended on the concentration of a sensitive bacterial strain. For the system of phage lambda and a sensitive E. coli cell, the number of negative colonies was maximal at a concentration of ca. 4 X 10(8) cells/ml.     

Localization of vitamin D3-responsive alkaline phosphatase in cultured chondrocytes

Alkaline phosphatase activity appears to be altered when chondrocyte cultures are incubated with 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). This study examined whether the hormone-responsive enzyme activity is associated with alkaline phosphatase-enriched extracellular membrane organelles called matrix vesicles. Confluent, third passage cultures of rat costochondral growth cartilage (GC) or resting zone chondrocytes (RC) were incubated with 1,25-(OH)2D3 or 24,25-dihydroxyvitamin D3 (24,25-(OH)2D3) and enzyme specific activity was assayed in the cell layer or in isolated matrix vesicle and plasma membrane fractions. Alkaline phosphatase-specific activity in the matrix vesicles was enriched at least 2-fold over that of the plasma membrane and 10-fold over that of the cell layer. Matrix vesicle alkaline phosphatase was stimulated by 1,25-(OH)2D3 in GC cultures and by 24,25-(OH)2D3 in RC cultures. The cell layer failed to reveal these subtle differences. 1,25-(OH)2D3 increased GC enzyme activity but the effect was one-half that observed in the matrix vesicles alone. No effect of 1,25-(OH)2D3 on enzyme activity of the RC cell layer or of 24,25-(OH)2D3 on either GC or RC cell layers was detected. Thus, response to the metabolites is dependent on chondrocytic differentiation and is site specific: the matrix vesicle fraction is targeted and not the cells per se.     

Neural mechanisms of auditory information processing by identified interneurones in Orthoptera

Three qualities of sound—the directional, the temporal and the spectral—are important for intraspecific communication in Orthoptera. The neural mechanisms employed by identified interneurones for encoding these sound qualities are illustrated by examples of physiological processes found at different levels of the CNS. Discussed are: (1) the creation of directional information by local interneurones in the thorax, and the use of time-intensity trading in sound location; (2) mechanisms for encoding the temporal parameters of sound by interneurones ascending to the brain; (3) frequency-dependent neural filtering of auditory information by local interneurones.     

Binding of vanadate to human erythrocyte ghosts and subsequent events

Spectroscopic techniques were used to investigate the interaction between vanadate and human erythrocyte ghosts. Direct evidence from ⁵¹V nuclear magnetic resonance (NMR) studies suggested that the monomeric and polymeric vanadate species may bind to the anion binding sites of band 3 protein of the erythrocyte membrane. The results of ⁵¹V NMR studies and the quenching effect of vanadate on the intrinsic fluorescence of the membrane proteins indicated that in the low concentration range of vanadate (<0.6 mm), monomeric vanadate binds mostly to the anion sites of band 3 protein with the dissociation constant close to 0.23 mm. The experiments of sulfhydryl content titration by the method of Ellman and residue sulfhydryl-labeled fluorescence spectroscopies clearly displayed that vanadate reacts directly with sulfhydryl groups. The appearance of the anisotropic election spin resonance (ESR) signal of vanadyl suggests that a small (c. 3%) amount of vanadate was reduced by sulfhydryl groups of membrane proteins. The fluidity and order of intact ghost membrane were reduced by the reaction with vanadate, as shown by the ESR studies employing the protein- and lipid-specific spin labels. It was concluded that although vanadates mainly bind to band 3 protein, a minor part of vanadate may oxidize the residue sulfhydryl groups of membrane proteins, and thus decrease the fluidity of erythrocyte membrane.     

Molecular recognition of lipopolysaccharide by the lantibiotic nisin

Nisin is a lanthionine antimicrobial effective against diverse Gram-positive bacteria and is used as a food preservative worldwide. Its action is mediated by pyrophosphate recognition of the bacterial cell wall receptors lipid II and undecaprenyl pyrophosphate. Nisin/receptor complexes disrupt cytoplasmic membranes, inhibit cell wall synthesis and dysregulate bacterial cell division. Gram-negative bacteria are much more tolerant to antimicrobials including nisin. In contrast to Gram-positives, Gram-negative bacteria possess an outer membrane, the major constituent of which is lipopolysaccharide (LPS). This contains surface exposed phosphate and pyrophosphate groups and hence can be targeted by nisin. Here we describe the impact of LPS on membrane stability in response to nisin and the molecular interactions occurring between nisin and membrane-embedded LPS from different Gram-negative bacteria. Dye release from liposomes shows enhanced susceptibility to nisin in the presence of LPS, particularly rough LPS chemotypes that lack an O-antigen whereas LPS from microorganisms sharing similar ecological niches with antimicrobial producers provides only modest enhancement. Increased susceptibility was observed with LPS from pathogenic Klebsiella pneumoniae compared to LPS from enteropathogenic Salmonella enterica and gut commensal Escherichia coli. LPS from Brucella melitensis, an intra-cellular pathogen which is adapted to invade professional and non-professional phagocytes, appears to be refractory to nisin. Molecular complex formation between nisin and LPS was studied by solid state MAS NMR and revealed complex formation between nisin and LPS from most organisms investigated except B. melitensis. LPS/nisin complex formation was confirmed in outer membrane extracts from E. coli.     

Interfacial anchor properties of tryptophan residues in transmembrane peptides can dominate over hydrophobic matching effects in peptide-lipid interactions

Membrane model systems consisting of phosphatidylcholines and hydrophobic alpha-helical peptides with tryptophan flanking residues, a characteristic motif for transmembrane protein segments, were used to investigate the contribution of tryptophans to peptide-lipid interactions. Peptides of different lengths and with the flanking tryptophans at different positions in the sequence were incorporated in relatively thick or thin lipid bilayers. The organization of the systems was assessed by NMR methods and by hydrogen/deuterium exchange in combination with mass spectrometry. Previously, it was found that relatively short peptides induce nonlamellar phases and that relatively long analogues order the lipid acyl chains in response to peptide-bilayer mismatch. Here it is shown that these effects do not correlate with the total hydrophobic peptide length, but instead with the length of the stretch between the flanking tryptophan residues. The tryptophan indole ring was consistently found to be positioned near the lipid carbonyl moieties, regardless of the peptide-lipid combination, as indicated by magic angle spinning NMR measurements. These observations suggest that the lipid adaptations are not primarily directed to avoid a peptide-lipid hydrophobic mismatch, but instead to prevent displacement of the tryptophan side chains from the polar-apolar interface. In contrast, long lysine-flanked analogues fully associate with a bilayer without significant lipid adaptations, and hydrogen/deuterium exchange experiments indicate that this is achieved by simply exposing more (hydrophobic) residues to the lipid headgroup region. The results highlight the specific properties that are imposed on transmembrane protein segments by flanking tryptophan residues.     

Effects of the eukaryotic pore-forming cytolysin Equinatoxin II on lipid membranes and the role of sphingomyelin

Equinatoxin II (EqtII), a protein toxin from the sea anemone Actinia equina, readily creates pores in sphingomyelin-containing lipid membranes. The perturbation by EqtII of model lipid membranes composed of dimyristoylphosphatidycholine and sphingomyelin (10 mol %) was investigated using wideline phosphorus-31 and deuterium NMR. The preferential interaction between EqtII (0.1 and 0.4 mol %) and the individual bilayer lipids was studied by (31)P magic angle spinning NMR, and toxin-induced changes in bilayer morphology were examined by freeze-fracture electron microscopy. Both NMR and EM showed the formation of an additional lipid phase in sphingomyelin-containing mixed lipid multilamellar suspensions with 0.4 mol % EqtII. The new toxin-induced phase consisted of small unilamellar vesicles 20-40 nm in diameter. Deuterium NMR showed that the new lipid phase contains both dimyristoylphosphatidycholine and sphingomyelin. Solid-state (31)P NMR showed an increase in spin-lattice and a decrease in spin-spin relaxation times in mixed-lipid model membranes in the presence of EqtII, consistent with an increase in the intensity of low frequency motions. The (2)H and (31)P spectral intensity distributions confirmed a change in lipid mobility and showed the creation of an isotropic lipid phase, which was identified as the small vesicle structures visible by electron microscopy in the EqtII-lipid suspensions. The toxin appears to enhance slow motions in the membrane lipids and destabilize the membrane. This effect was greatly enhanced in sphingomyelin-containing mixed lipid membranes compared with pure phosphatidylcholine bilayers, suggesting a preferential interaction between the toxin and bilayer sphingomyelin.     

17 beta-estradiol-BSA conjugates and 17 beta-estradiol regulate growth plate chondrocytes by common membrane associated mechanisms involving PKC dependent and independent signal transduction

Nuclear receptors for 17 beta-estradiol (E(2)) are present in growth plate chondrocytes from both male and female rats and regulation of chondrocytes through these receptors has been studied for many years; however, recent studies indicate that an alternative pathway involving a membrane receptor may also be involved in the cell response. E(2) was found to directly affect the fluidity of chondrocyte membranes derived from female, but not male, rats. In addition, E(2) activates protein kinase C (PKC) in a nongenomic manner in female cells, and chelerythrine, a specific inhibitor of PKC, inhibits E(2)-dependent alkaline phosphatase activity and proteoglycan sulfation in these cells, indicating PKC is involved in the signal transduction mechanism. The aims of the present study were: (1) to examine the effect of a cell membrane-impermeable 17 beta-estradiol-bovine serum albumin conjugate (E(2)-BSA) on chondrocyte proliferation, differentiation, and matrix synthesis; (2) to determine the pathway that mediates the membrane effect of E(2)-BSA on PKC; and (3) to compare the action of E(2)-BSA to that of E(2). Confluent, fourth passage resting zone (RC) and growth zone (GC) chondrocytes from female rat costochondral cartilage were treated with 10(-9) to 10(-7) M E(2) or E(2)-BSA and changes in alkaline phosphatase specific activity, proteoglycan sulfation, and [(3)H]-thymidine incorporation measured. To examine the pathway of PKC activation, chondrocyte cultures were treated with E(2)-BSA in the presence or absence of GDP beta S (inhibitor of G-proteins), GTP gamma S (activator of G-proteins), U73122 or D609 (inhibitors of phospholipase C [PLC]), wortmannin (inhibitor of phospholipase D [PLD]) or LY294002 (inhibitor of phosphatidylinositol 3-kinase). E(2)-BSA mimicked the effects of E(2) on alkaline phosphatase specific activity and proteoglycan sulfation, causing dose-dependent increases in both RC and GC cell cultures. Both forms of estradiol inhibited [(3)H]-thymidine incorporation, and the effect was dose-dependent. E(2)-BSA caused time-dependent increases in PKC in RC and GC cells; effects were observed within three minutes in RC cells and within one minute in GC cells. Response to E(2) was more robust in RC cells, whereas in GC cells, E(2) and E(2)-BSA caused a comparable increase in PKC. GDP beta S inhibited the activation of PKC in E(2)-BSA-stimulated RC and GC cells. GTP gamma S increased PKC in E(2)-BSA-stimulated GC cells, but had no effect in E(2)-BSA-stimulated RC cells. The phosphatidylinositol-specific PLC inhibitor U73122 blocked E(2)-BSA-stimulated PKC activity in both RC and GC cells, whereas the phosphatidylcholine-specific PLC inhibitor D609 had no effect. Neither the PLD inhibitor wortmannin nor the phosphatidylinositol 3-kinase inhibitor LY294022 had any effect on E(2)-BSA-stimulated PKC activity in either RC or GC cells. The classical estrogen receptor antagonist ICI 182780 was unable to block the stimulatory effect of E(2)-BSA on PKC. Moreover, the classical receptor agonist diethylstilbestrol (DES) had no effect on PKC, nor did it alter the stimulatory effect of E(2)-BSA. The specificity of the membrane response to E(2) was also demonstrated by showing that the membrane receptor for 1 alpha,25-(OH)(2)D(3) was not involved. These data indicate that the rapid nongenomic effect of E(2)-BSA on PKC activity in RC and GC cells is dependent on G-protein-coupled PLC and support the hypothesis that many of the effects of E(2) involve membrane-associated mechanisms independent of classical estrogen receptors. (c) 2001 Wiley-Liss, Inc.