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.     

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.     

Micromolar Ca2+ from sparks activates Ca2+-sensitive K+ channels in rat cerebral artery smooth muscle

The goal of the present study was to testthe hypothesis that local Ca²⁺ release events(Ca²⁺ sparks) deliver high local Ca²⁺concentration to activate nearby Ca²⁺-sensitiveK⁺ (BK) channels in the cell membrane of arterial smoothmuscle cells. Ca²⁺ sparks and BK channels were examined inisolated myocytes from rat cerebral arteries with laser scanningconfocal microscopy and patch-clamp techniques. BK channels had anapparent dissociation constant for Ca²⁺ of 19 µM and aHill coefficient of 2.9 at 40 mV. At near-physiological intracellularCa²⁺ concentration ([Ca²⁺]_i; 100 nM) and membrane potential (40 mV), the open probability of a singleBK channel was low (1.2 × 10⁶). A Ca²⁺spark increased BK channel activity to 18. Assuming that 1-100% of the BK channels are activated by a single Ca²⁺ spark, BKchannel activity increases 6 × 10⁵-fold to 6 × 10³-fold, which corresponds to ~30 µM to 4 µM sparkCa²⁺ concentration.1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acidacetoxymethyl ester caused the disappearance of all Ca²⁺sparks while leaving the transient BK currents unchanged. Our resultssupport the idea that Ca²⁺ spark sites are in closeproximity to the BK channels and that local[Ca²⁺]_i reaches micromolar levels to activateBK channels.

     

Interaction of the lantibiotic nisin with mixed lipid bilayers: a 31P and 2H NMR study

Nisin is a positively charged antibacterial peptide which binds to the negatively charged membranes of Gram-positive bacteria. The initial interaction of the peptide with model membranes of neutral (phosphatidylcholine) and negatively charged (phosphatidylcholine/phosphatidylglycerol) model lipid membranes was studied using nonperturbing solid state magic angle spinning (MAS) (31)P NMR and (2)H wide-line NMR. In the presence of nisin, the coexistence of two bilayer lipid environments was observed both in charged and in neutral membranes. One lipid environment was found to be associated with lipid directly interacting with nisin and one with noninteracting lipid. Solid state (31)P MAS NMR results show that the acidic membrane lipid component partitions preferentially into the nisin-associated environment. Deuterium NMR ((2)H NMR) of the selectively headgroup-labeled acidic lipid provides further evidence of a strong interaction between the charged lipid component and the peptide. The segregation of acidic lipid into the nisin-bound environment was quantified from (2)H NMR measurements of selectively headgroup-deuterated neutral lipid. It is suggested that the observed lipid partitioning in the presence of nisin is driven, at least initially, by electrostatic interactions. (2)H NMR measurements from chain-perdeuterated neutral lipids indicate that nisin perturbs the hydrophobic region of both charged and neutral bilayers.     

Stereoselective disulfide formation stabilizes the local peptide conformation in nisin mimics

Nisin is a polymacrocyclic peptide antimicrobial with high activity against Gram-positive bacteria. Lanthionine and methyllanthionine bridges, closing the macrocycles, are stabilized by thioether bonds, formed between cysteines and dehydrated serine or threonine. The role of polypeptide backbone conformation in the formation of macrocycles A and B within cysteine mutants of nisin residues 1?12 is investigated here by molecular dynamics simulations. Enantiomeric combinational space of Cys3 and Cys7 and of Cys8 and Cys11 is examined for the preference of disulfide bond formation over helical turn formation within this region. A clear preference for spontaneous disulfide formation and closure of rings 3,7 and 8,11 is demonstrated for the D-Cys3, D-Cys7, L-Cys8, L-Cys11 nisin homologue, while interlinked rings A and B are obtained through disulfide bridges between L-Cys3 and D-Cys8 and between D-Cys7 and D-Cys11. This study offers a simple designer approach to solid phase synthesis of macrocyclic peptides and lantibiotic analogues.     

Genome-wide analysis of gene expression during Xenopus tropicalis tadpole tail regeneration

Background

During liver development, intrahepatic bile ducts are thought to arise by a unique asymmetric mode of cholangiocyte tubulogenesis characterized by a series of remodeling stages. Moreover, in liver diseases, cells lining the Canals of Hering can proliferate and generate new hepatic tissue. The aim of this study was to develop protocols for three-dimensional visualization of protein expression, hepatic portal structures and human hepatic cholangiocyte tubulogenesis.

Results

Protocols were developed to digitally visualize portal vessel branching and protein expression of hepatic cell lineage and extracellular matrix deposition markers in three dimensions. Samples from human prenatal livers ranging from 7 weeks + 2 days to 15½ weeks post conception as well as adult normal and acetaminophen intoxicated liver were used. The markers included cytokeratins (CK) 7 and 19, the epithelial cell adhesion molecule (EpCAM), hepatocyte paraffin 1 (HepPar1), sex determining region Y (SRY)-box 9 (SOX9), laminin, nestin, and aquaporin 1 (AQP1). Digital three-dimensional reconstructions using CK19 as a single marker protein disclosed a fine network of CK19 positive cells in the biliary tree in normal liver and in the extensive ductular reactions originating from intrahepatic bile ducts and branching into the parenchyma of the acetaminophen intoxicated liver. In the developing human liver, three-dimensional reconstructions using multiple marker proteins confirmed that the human intrahepatic biliary tree forms through several developmental stages involving an initial transition of primitive hepatocytes into cholangiocytes shaping the ductal plate followed by a process of maturation and remodeling where the intrahepatic biliary tree develops through an asymmetrical form of cholangiocyte tubulogenesis.

Conclusions

The developed protocols provide a novel and sophisticated three-dimensional visualization of vessels and protein expression in human liver during development and disease.     

Ca2+-activated K+ channels in murine endothelial cells: block by intracellular calcium and magnesium

The intermediate (IK(Ca)) and small (SK(Ca)) conductance Ca(2+)-sensitive K(+) channels in endothelial cells (ECs) modulate vascular diameter through regulation of EC membrane potential. However, contribution of IK(Ca) and SK(Ca) channels to membrane current and potential in native endothelial cells remains unclear. In freshly isolated endothelial cells from mouse aorta dialyzed with 3 microM free [Ca(2+)](i) and 1 mM free [Mg(2+)](i), membrane currents reversed at the potassium equilibrium potential and exhibited an inward rectification at positive membrane potentials. Blockers of large-conductance, Ca(2+)-sensitive potassium (BK(Ca)) and strong inward rectifier potassium (K(ir)) channels did not affect the membrane current. However, blockers of IK(Ca) channels, charybdotoxin (ChTX), and of SK(Ca) channels, apamin (Ap), significantly reduced the whole-cell current. Although IK(Ca) and SK(Ca) channels are intrinsically voltage independent, ChTX- and Ap-sensitive currents decreased steeply with membrane potential depolarization. Removal of intracellular Mg(2+) significantly increased these currents. Moreover, concomitant reduction of the [Ca(2+)](i) to 1 microM caused an additional increase in ChTX- and Ap-sensitive currents so that the currents exhibited theoretical outward rectification. Block of IK(Ca) and SK(Ca) channels caused a significant endothelial membrane potential depolarization (approximately 11 mV) and decrease in [Ca(2+)](i) in mesenteric arteries in the absence of an agonist. These results indicate that [Ca(2+)](i) can both activate and block IK(Ca) and SK(Ca) channels in endothelial cells, and that these channels regulate the resting membrane potential and intracellular calcium in native endothelium.     

Frequency modulation of Ca2+ sparks is involved in regulation of arterial diameter by cyclic nucleotides

Forskolin, which elevates cAMP levels, and sodium nitroprusside(SNP) and nicorandil, which elevate cGMP levels, increased, by two- tothreefold, the frequency of subcellularCa²⁺ release("Ca²⁺ sparks") throughryanodine-sensitive Ca²⁺ release(RyR) channels in the sarcoplasmic reticulum (SR) of myocytes isolatedfrom cerebral and coronary arteries of rats. Forskolin, SNP,nicorandil, dibutyryl-cAMP, and adenosine increased the frequency ofCa²⁺-sensitiveK⁺(K_Ca) currents["spontaneous transient outward currents" (STOCs)] bytwo- to threefold, consistent withCa²⁺ sparks activating STOCs.These agents also increased the mean amplitude of STOCs by 1.3-fold, aneffect that could be explained by activation ofK_Ca channels, independent ofeffects on Ca²⁺ sparks. To testthe hypothesis that cAMP could act to dilate arteries throughactivation of the Ca²⁺sparkK_Ca channel pathway,the effects of blockers of K_Cachannels (iberiotoxin) and of Ca²⁺sparks (ryanodine) on forskolin-induced dilations of pressurized cerebral arteries were examined. Forskolin-induced dilations were partially inhibited by iberiotoxin and ryanodine (with no additive effects) and were entirely prevented by elevating externalK⁺. Forskolin lowered averageCa²⁺ in pressurized arteries whileincreasing ryanodine-sensitive, caffeine-inducedCa²⁺ transients. These experimentssuggest a new mechanism for cyclic nucleotide-mediated dilationsthrough an increase in Ca²⁺ sparkfrequency, caused by effects on SRCa²⁺ load and possibly on the RyRchannel, which leads to increased STOC frequency, membrane potentialhyperpolarization, closure of voltage-dependentCa²⁺ channels, decrease inarterial wall Ca²⁺, and,ultimately, vasodilation.