Cu XAS shows a change in the ligation of CuB upon reduction of cytochrome bo3 from Escherichia coli

Copper X-ray absorption spectroscopy (XAS) has been used to examine the structures of the Cu(II) and Cu(I) forms of the cytochrome bo3 quinol oxidase from Escherichia coli. Cytochrome bo3 is a member of the superfamily of heme-copper respiratory oxidases. Of particular interest is the fact that these enzymes function as redox-linked proton pumps, resulting in the net translocation of one H+ per electron across the membrane. The molecular mechanism of how this pump operates and the manner by which it is linked to the oxygen chemistry at the active site of the enzyme are unknown. Several proposals have featured changes in the coordination of CuB during enzyme turnover that would result in sequential protonation or deprotonation events that are key to the functioning proton pump. This would imply lability of the ligands to CuB. In this work, the structure of the protein in the immediate vicinity of CuB, in both the fully oxidized and fully reduced forms of the enzyme, has been examined by Cu XAS, a technique that is particularly sensitive to changes in metal coordination. The results show that in the oxidized enzyme, CuB(II) is four-coordinate, consistent with three imidazoles and one hydroxyl (or water). Upon reduction of the enzyme, the coordination of CuB(I) is significantly altered, consistent with the loss of one of the histidine imidazole ligands in at least a substantial fraction of the population. These data add to the credibility that changes in the ligation of CuB might occur during catalytic turnover of the enzyme and, therefore, could, in principle, be part of the mechanism of proton pumping.     

Liposomes composed of a double-chain cationic amphiphile (vectamidine) induce their own encapsulation into human erythrocytes

Vectamidine is a liposome-forming double-chain cationic amphiphile. The present work was aimed to microscopically study the interactions of Vectamidine liposomes with the human erythrocyte plasma membrane. Vectamidine rapidly induced stomatocytic shapes. Attachment of Vectamidine liposomes to the erythrocyte induced a strong local invagination of the membrane. This frequently resulted in a complete encapsulation of the liposome. Liposomes composed of phosphatidylcholine (neutral) or phosphatidylserine/phosphatidylcholine (anionic) did not perturb the erythrocyte shape. Our results indicate that besides an attraction of Vectamidine liposomes to the plasma membrane, there is a preference of Vectamidine for the inner bilayer leaflet. We suggest that cationic amphiphiles may transfer from membrane-attached liposomes to the plasma membrane and then translocate to the inner bilayer leaflet where they induce a strong local inward bending of the plasma membrane resulting in an encapsulation of the liposome.     

Green fluorescent protein (GFP) as a marker during pollen development

The transient expression of three mutant forms of green fluorescent protein (GFP) genes, GFP4, GFP5ER, and GFP4S65C, under several constitutive and pollenspecific promoters throughout pollen development in Nicotianatabacum, thaliana and Antirrhinummajus is described. Immature pollen of tobacco, Arabidopsis and snapdragon, isolated at different developmental stages, were bombarded with plasmids containing the GFP and cultured in vitro for several days until maturity. The expression of GFP was monitored every day during in vitro maturation, germination and pollination, as well as after in situ pollination. The expression pattern of each construct was compared in parallel experiments to that of ßglucuronidase (GUS) constructs expressed by the same promoters. The results show that the expression level of all three GFP mutant forms was dependent on the strength of the promoter used. The strongest promoter was the DC3 promoter, and no notable differences in the intensity and brightness of all three versions of GFP were observed. GFPexpressing pollen from tobacco and snapdragon developed in vitro for several days until maturity and germinated in vitro as well as on the surface of stigmata, strongly suggesting that all three GFPs are not toxic for the development of functional pollen. Furthermore, stably transformed tobacco plants expressing GFP under the control of the strong pollenexpressed DC3 and LAT52 promoters were not impaired in reproductive function, confirming that GFP can be used as a nondestructive marker for plant reproductive biology and development.     

Simultaneous detection of IFN-gamma and IL-4 mRNAs using RT-PCR and time-resolved fluorometry

Time-resolved fluorometry was applied in the detection of RT-PCR amplified mRNAs for the Th1 and Th2 cell-derived cytokines interferon gamma (IFN-gamma) and interleukin (IL-)4, respectively. RNA stimulated cells was reverse transcribed and the cDNAs for the cytokine mRNAs and the constantly expressed beta-actin (beta-ACT) mRNA were simultaneously amplified in one multiplex PCR reaction. The PCR conditions were optimized to minimize mutual inhibition of individual amplifications. One of the PCR primers in each primer pair was biotinylated, and the PCR products were captured onto streptavidin-coated microtitre plates. The three PCR products were detected with three different lanthanide labelled target-specific probes in solution hybridization. IFN-gamma, IL-4 and beta-ACT were detected with europium (Eu), terbium (Tb) and samarium (Sm) labelled probes, respectively, using time-resolved fluorometry. Small cell numbers used in microtitre plate cultures were sufficient to detect cytokine messages after mitogen stimulation. This sequence-based method provides a sensitive, specific, fast and nonisotopic alternative to conventional blotting and hybridisation with radioactive probes. In addition, the multiplex fluorogenic dye detection facilitates relative quantification of target mRNAs.     

Different integrins mediate cell spreading, haptotaxis and lateral migration of HaCaT keratinocytes on fibronectin

Collaborative role of various fibronectin-binding integrins (alpha5beta1, alphavbeta1 and alphavbeta6) as mediators of cell adhesion and migration on fibronectin was studied using cultured HaCaT keratinocytes. This cell line spontaneously expressed all three fibronectin-binding integrins. In addition, the expression of alphavbeta6 integrin was strongly and specifically upregulated by transforming growth factor-beta1 (TGFbeta1) whereas the amount of other integrins remained practically unchanged on the cell surface. Adhesion, spreading and motility of HaCaT keratinocytes on fibronectin were promoted by TGFbeta1. Based on antibody blocking experiments, both untreated and TGFbeta1-treated HaCaT cells used alphavbeta6 integrin as their main fibronectin receptor for cell spreading. In contrast to TGFbeta1-treated cells, the untreated cells also needed alpha5beta1 integrin for maximal cell spreading on fibronectin. Combinations of antibodies blocking both of these receptors totally prevented spreading of both untreated and TGFbeta1-treated cells. Haptotactic motility of individual HaCaT cells through fibronectin-coated membranes was again mainly dependent on alphavbeta6 integrin, while alphavbeta1 and alpha5beta1 integrins played a lesser role both in untreated and TGFbeta1-treated HaCaT cells. However, unlike haptotaxis, lateral migration of HaCaT cell sheet was mainly mediated by beta1 integrins, and alphavbeta6 integrin showed a minor role. The migration process appeared to involve a number of beta1 integrins that could adaptively replace each other when blocking antibodies were present. Thus, keratinocytes appear to use different fibronectin receptors for different functions, such as cell spreading, haptotaxis and lateral migration. The cells can also adapt to a situation where one receptor is unfunctional by switching to another receptor of the same ligand.     

Transformation of nuclear and plastomic plant genomes by biolistic particle bombardment

Microprojectile bombardment is a powerful method for the transformation of various organisms and tissues. For plants, the biolistic approach is primarily used for transformation of cereals and other monocotyledons, as well as for dicotyledonous plants shown to be recalcitrant to Agrobacterium-based transformation of organellar genomes, and transformation of plant and algal chloroplasts has recently been reported. In this protocol paper we provide methods for nuclear and plastomic transformation of plants using the biolistic technique.     

Comparison of recovery of anaerobic bacteria using the Anoxomat, anaerobic chamber, and GasPak jar systems

The Anoxomat system provides an automated evacuation-replacement technique to create an anaerobic or microaerophilic environment in a jar. We evaluated the Anoxomat system for the growth of obligate anaerobes and for the recovery of anaerobic organisms from clinical specimens, and compared its performance to that of an anaerobic chamber and the GasPak System. Of the 54 stock strains tested, the Anoxomat, the chamber, and the GasPak recovered 95%, 95% and 93% at 24 h, respectively. On 29 occasions (51%), the colonies on the Anoxomat plates were slightly larger than those in the chamber and on 17 (30%) occasions larger than the colonies on the GasPak jar plates. At 48 h, the Anoxomat, the chamber, and the GasPak recovered 93.5%, 94.4% and 88.9%, respectively; of 108 anaerobes isolated from 31 clinical specimens. Methylene blue indicators became decolorized (average of 10 tests) within 2 h inside the Anoxomat jars, 2 h 10 min inside the anaerobic chamber, and 2 h 30 min inside the GasPak jars.     

Emerging roles of caspase-3 in apoptosis

Caspases are crucial mediators of programmed cell death (apoptosis). Among them, caspase-3 is a frequently activated death protease, catalyzing the specific cleavage of many key cellular proteins. However, the specific requirements of this (or any other) caspase in apoptosis have remained largely unknown until now. Pathways to caspase-3 activation have been identified that are either dependent on or independent of mitochondrial cytochrome c release and caspase-9 function. Caspase-3 is essential for normal brain development and is important or essential in other apoptotic scenarios in a remarkable tissue-, cell type- or death stimulus-specific manner. Caspase-3 is also required for some typical hallmarks of apoptosis, and is indispensable for apoptotic chromatin condensation and DNA fragmentation in all cell types examined. Thus, caspase-3 is essential for certain processes associated with the dismantling of the cell and the formation of apoptotic bodies, but it may also function before or at the stage when commitment to loss of cell viability is made.