LmxPK4, a mitogen-activated protein kinase kinase homologue of Leishmania mexicana with a potential role in parasite differentiation

Members of the mitogen-activated protein (MAP) kinase cascade are important for the establishment of a Leishmania mexicana infection and are involved in flagellar length control, although the underlying molecular mechanisms remain to be elucidated. This study reports the cloning and characterization of LmxPK4, a MAP kinase kinase homologue of L. mexicana displaying putative plant-like regulatory phosphorylation sites. The recombinant protein has autophosphorylating activity and phosphorylates myelin basic protein. An LmxPK4 gene deletion mutant showed a proliferation defect after infection of macrophages and no or delayed lesion development in mice. Irrespective of the onset of lesion development parasites showed an early and homogeneous lesion development in re-infection experiments. This is indicative for a compensation of the null mutant phenotype. Additionally, this phenotype could be reverted by reintroduction of the wild-type gene into the deletion background. Mutants expressing loss-of-function or N-terminally truncated versions of LmxPK4 retained the null mutant phenotype. LmxPK4 is stage-specifically expressed in promastigotes and during differentiation to amastigotes, but is not detectable in amastigotes isolated from the mammalian host. Moreover, its in vitro kinase activity increases with temperature rise up to 40 degrees C. Our results suggest that LmxPK4 is involved in the differentiation process and affects virulence of Leishmania mexicana.     

Cytoplasmic membrane of a sensitive yeast is a primary target for Cryptococcus humicola mycocidal compound (microcin)

A basidiomycetous yeast strain, Cryptococcus humicola 9-6, secretes a mycocidal compound (microcin) which is lethal for many yeasts. In this study a new protocol for microcin purification has been developed, and TLC-purity product was obtained. Using fluorescein as a pH-sensitive probe it was found that microcin treatment of Cryptococcus terreus, a model microcin-sensitive yeast, immediately caused transient alkalization followed by acidification of the cells' cytoplasm. Upon completion of this process, endogenous respiration as well as activity of unspecific esterases were inhibited, and alterations in cell wall and/or capsule started. Microcin was shown to make the cells leaky for intracellular ATP. The mycocidal effect of microcin did not depend on the cell cycle phase of Cr. terreus. Based on these observations and on electrical measurements on planar phospholipid bilayers, which indicated a microcin-induced membrane permeabilization, it is suggested that the cytoplasmic membrane of the sensitive yeast is a primary target of microcin action. The conjectured mode of microcin action involves gradual increase of the cytoplasmic membrane's unspecific permeability. Intracellular ion homeostasis changes induced by microcin are considered to be the main cause of enzyme inhibition, alterations in the outer layers of the cell envelope and, finally, division arrest.     

Comparison of the usefulness of the MTT, ATP, and calcein assays to predict the potency of cytotoxic agents in various human cancer cell lines

Cell viability assays are important tools in oncological research and clinical practice to assess the tumor cell sensitivity of individual patients. The purpose of this study was to demonstrate the comparability of 3 widely used assays (MTT, ATP, calcein assays) by principal component analysis. The study included 4 different cytostatics (cisplatin, docetaxel, doxorubicin, vinblastine) and 3 different human cancer cell lines (MCF-7, A2780, doxorubicin resistant A2780adr). Ninety-three percent of the total variance of all variables included in the principal component analysis (resulting from 3 cell lines and 3 assays) could be explained by 1 principal component. Factor loadings were > 0.937 except for the variable MTT-A2780adr, which was 0.872. These results indicate the similarity of the 3 assays. A 2nd principal component analysis included literature data and showed accordance of data from this study and the literature. The MTT assay was further improved as a high-throughput screening-capable assay. The ATP assay is able to detect effects of cytostatics already after 1 h incubation. The determination of resistance factors allowed to differentiate cytostatics into P-gp or non-P-gp substrates. In conclusion, this study provides improved microplate reader-based cell viability assays and sets a statistically solid basis for a future comparison of data obtained in different laboratories by any of the 3 assays.     

Nearly instantaneous, cation-independent, high selectivity nucleic acid hybridization to DNA microarrays

Hybridization rate enhancement has been demonstrated for high molecular weight DNA target binding to a microarray. Microarrays were fabricated using biotin-modified oligonucleotides complexed with streptavidin (SA), which serves as an attachment to the underlying surface. It is shown that at low salt and pH 5, where SA develops a positive charge, duplex formation becomes at least 80-fold faster than seen under standard conditions, where SA is neutral or anionic. Duplex formation becomes independent of solution state cation concentration in the low pH state, under conditions where specificity remains high. The utility of such applied surface science is discussed.     

Interaction between lipopolysaccharide (LPS), LPS-binding protein (LBP), and planar membranes

The mechanism of interaction of the lipopolysaccharide (LPS)-binding protein, LBP, with differently composed symmetric and asymmetric planar lipid bilayers was investigated in electrical measurements (membrane current, potential, capacitance). From a change of the inner membrane potential difference, binding of LBP to membranes was deduced. After addition of LBP to one side of the membrane, binding of anti-LBP antibodies and LPS to LBP on both sides of the bilayer was observed. Effects resulting from an interaction of anti-LBP antiserum with membrane-bound LBP depend on the side of addition of the antiserum, indicating a directed intercalation of LBP into the membrane. Addition of LPS to the same side as LBP may induce a change of the conformation of LBP or its orientation in the membrane. Based on these observations, we propose that LBP intercalates in a directed orientation into negatively-charged membranes and assumes a transmembrane configuration. Moreover, pre-incubated complexes of LPS and LBP do not interact with membranes. These experiments show that reconstituted planar membranes are a suitable tool for investigations of the interaction of non pore-forming proteins that are involved in signal transduction.     

DNA microarrays based on noncovalent oligonucleotide attachment and hybridization in two dimensions

Short oligonucleotide probes have been linked to a solid support by simple electrostatic adsorption onto a positively charged surface film. Attachment was obtained by microfluidic application of unmodified oligonucleotides in distilled water onto amino-silanized glass. It has been demonstrated that an extremely stable monolayer of oligonucleotide is obtained by this method, at a density of about 10(11) molecules/mm(2), which approaches the limit expected for a two-dimensional closest-packed array. Application of oligonucleotide by adsorption is followed by capping with acetic anhydride in the vapor phase, and then capping with succinic anhydride in solution to form a surface with weak negative charge. The capping method has been successfully employed for microarray fabrication and for the analysis of single nucleotide polymorphisms in the k-ras gene. The data reveal that, subsequent to capping, the adsorptive association of oligonucleotide to the surface yields a probe layer which is capable of single nucleotide base mismatch discrimination and high apparent binding affinity.     

Relaxation of variable chlorophyll fluorescence after illumination of dark-adapted barley leaves as influenced by the redox states of electron carriers

Kinetics of the dark relaxation of variable chlorophyll fluorescence, Fv, were studied after brief illumination of dark-adapted barley leaves in order to understand the rapid reversibility of pulse-induced fluorescence increases, which is observed even when fast linear electron transport to an external electron acceptor is not possible. Four kinetically distinct components were observed which reveal complexity in the oxidation of the reduced primary quinone acceptor of Photosystem II, Q_A ⁻: the slowest component accounted for 4–5% of maximal Fv and had a life-time of several seconds. It is suggested to represent a minor population of inactive Photosystem II centers. The other three components displayed first-order kinetics with half-time of 6–8 ms (`fast' component), 60–80 ms (`middle' component) and 650–680 ms (`slow' component). The fast component dominated Fv when methyl viologen or far-red light accelerated oxidation of plastohydroquinone. It shows rapid oxidation of Q_A ⁻ during electron flow to plastoquinone commensurate with maximum linear electron flow through the electron transport chain. The other two components were observed under conditions of restricted electron flow and excessive reduction of electron carriers. Unexpectedly, the slow component, which is interpreted to reflect the recombination between Q_A ⁻ and an intermediate on the oxidizing side of Photosystem II, saturated already at low irradiances of actinic light when plastoquinone was not yet strongly reduced suggesting that dark-adaptation of leaves results not only in the loss of activity of light-regulated enzymes of the carbon cycle but affects also electron flow from Q_A ⁻ to plastoquinone. KCN poisoning or high temperature treatment of leaves produced a nonexponential pattern of slow Fv relaxation. This effect was largely (heat treatment) or even completely (KCN) abolished by far-red light. This revised version was published online in August 2006 with corrections to the Cover Date.     

On sexual agglutination and mating type substances in isogamous dioecious Chlamydomonads: IV. Unilateral inactivation of the sex contact capacity in compatible and incompatible taxa by α-mannosidase and snake venom protease

Sex cell contact at fertilization is analysed in the mating type reaction of isogamous Chlamydomonas species. Contact is based upon a complementarity between special mating type substances, sex and species specific glycoprotein complexes. In three related taxa, the contact capactiy of their (+) gamete type is sensitive to snake venom protease (α-protease) and depends decisively on terminal α-glycosidically bound mannose residues. Enzymatic removal of these residues by α-mannosidase incapacitates live (+) gametes and inactivates isolated (+) mating type substance. (+) gametes inactivated by α-mannosidase or α-protease do not agglutinate with (?) gametes nor respond to isolated (?) mating type substance. Isolated (?) substance is adsorbed to and inactivated by the homologous (+) gametes. (+) gametes incapacitated by α-mannosidase or α-protease do not adsorb nor inactivate the isolated (?) substance. The agglutinability of live (?) gametes and the contact capacity of isolated (?) mating type substance is not affected by α-mannosidase or α-protease. The mannose residues react only within the species-typical complementarity. Some additional feature(s) of the (+) mating type substances must effect their species specificity and account for gametic isolation and sexual incompatibility between species.     

Dual role of mitofilin in mitochondrial membrane organization and protein biogenesis

The mitochondrial inner membrane consists of two domains, inner boundary membrane and cristae membrane that are connected by crista junctions. Mitofilin/Fcj1 was reported to be involved in formation of crista junctions, however, different views exist on its function and possible partner proteins. We report that mitofilin plays a dual role. Mitofilin is part of a large inner membrane complex, and we identify five partner proteins as constituents of the mitochondrial inner membrane organizing system (MINOS) that is required for keeping cristae membranes connected to the inner boundary membrane. Additionally, mitofilin is coupled to the outer membrane and promotes protein import via the mitochondrial intermembrane space assembly pathway. Our findings indicate that mitofilin is a central component?of MINOS and functions as a multifunctional regulator of mitochondrial architecture and protein biogenesis.