The binding of precipitant ions in the tetragonal crystals of hen egg white lysozyme

Abstract

The bonds between lysozyme molecules and precipitant ions in single crystals grown with chlorides of several metals are analysed on the basis of crystal structure data. Crystals of tetragonal hen egg lysozyme (HEWL) were grown with chlorides of several alkali and transition metals (LiCl, NaCl, KCl, NiCl₂ and CuCl₂) as precipitants and the three-dimensional structures were determined at 1.35?Å resolution by X-ray diffraction method. The positions of metal and chloride ions attached to the protein were located, divided into three groups and analysed. Some of them, in accordance with the recently proposed and experimentally confirmed crystal growth model, provide connections in protein dimers and octamers that are precursor clusters in the crystallization lysozyme solution. The first group, including Cu⁺², Ni⁺² and Na⁺¹ cations, binds specifically to the protein molecule. The second group consists of metal and chloride ions bound inside the dimers and octamers. The third group of ions can participate in connections between the octamers that are suggested as building units during the crystal growth. The arrangement of chloride and metal ions associated with lysozyme molecule at all stages of the crystallization solution formation and crystal growth is discussed.

Communicated by Ramaswamy H. Sarma     

Isolation of Allium pollen protoplasts

Studies on protoplasts isolation were carried out with mature pollen grains of 29 samples of species of Allium aflatunense, A. cepa, A. fistulosum, A. karataviense, A. longicuspis, A. nutans, A. odorum, A. sativum and A. schoenoprasum. Surface sterilized pollen grains drifted from crushed anthers were incubated in an enzyme solution containing 1% (w/v) cellulase Onozuka R-10, 1% (w/v) Macerozyme R-10, 0,5 mol l^-1 sucrose and the basal salts of Nitsch medium. Protoplasts were released within 3 to 120 min, either from the pollen grain, through a slightly disturbed germination pore (narrow aperture), or through a wider aperture, when the exine surrounding the germination pore was disturbed. For the first time, protoplasts were obtained from 13 genotypes of 6 Allium species, at a rate of 1 to 30% of the digested intact pollen grains, depending on the genotype.     

Phosphatidylinositol ether lipid analogues that inhibit AKT also independently activate the stress kinase, p38alpha, through MKK3/6-independent and -dependent mechanisms

Previously, we identified five active phosphatidylinositol ether lipid analogues (PIAs) that target the pleckstrin homology domain of Akt and selectively induce apoptosis in cancer cells with high levels of Akt activity. To examine specificity, PIAs were screened against a panel of 29 purified kinases. No kinase was inhibited, but one isoform of p38, p38alpha, was uniformly activated 2-fold. Molecular modeling of p38alpha revealed the presence of two regions that could interact with PIAs, one in the activation loop and a heretofore unappreciated region in the upper lobe that resembles a pleckstrin homology domain. In cells, two phases of activation were observed, an early phase that was independent of the upstream kinase MKK3/6 and inhibited by the p38 inhibitor SB203580 and a latter phase that was coincident with MKK3/6 activation. In short term xenograft experiments that employed immunohistochemistry and immunoblotting, PIA administration increased phosphorylation of p38 but not MKK3/6 in tumors in a statistically significant manner. Although PIAs rapidly activated p38 with similar time and dose dependence as Akt inhibition, p38 activation and Akt inhibition were independent events induced by PIAs. Using SB203580 or p38alpha(-/-) cells, we showed that p38alpha is not required for PIA-induced apoptosis but is required for H(2)O(2)- and anisomycin-induced apoptosis. Nonetheless, activation of p38a contributes to PIA-induced apoptosis, because reconstitution of p38a into p38alpha(-/-) cells increased apoptosis. These studies indicate that p38alpha is activated by PIAs through a novel mechanism and show that p38alpha activation contributes to PIA-induced cell death. Independent modulation of Akt and p38alpha could account for the profound cytotoxicity of PIAs.     

Increased Expression of a Phloem Membrane Protein Encoded by NHL26 Alters Phloem Export and Sugar Partitioning in Arabidopsis

The complex process of phloem sugar transport involves symplasmic and apoplasmic events. We characterized Arabidopsis thaliana lines ectopically expressing a phloem-specific gene encoding NDR1/HIN1-like26 (NHL26), a putative membrane protein. NHL26 overexpressor plants grew more slowly than wild-type plants, accumulated high levels of carbohydrates in mature leaves, and had a higher shoot biomass, contrasting with slower root growth and a lower seed yield. Similar effects were observed when NHL26 was overexpressed in companion cells, under the control of a companion cell–specific promoter. The soluble sugar content of the phloem sap and sink organs was lower than that in the wild type, providing evidence of a sugar export defect. This was confirmed in a phloem-export assay with the symplastic tracer carboxyfluorescein diacetate. Leaf sugar accumulation was accompanied by higher organic acid, amino acid, and protein contents, whereas analysis of the metabolite profile of phloem sap exudate revealed no change in amino acid or organic acid content, indicating a specific effect on sugar export. NHL26 was found to be located in the phloem plasmodesmata and the endoplasmic reticulum. These findings reveal that NHL26 accumulation affects either the permeability of plasmodesmata or sugar signaling in companion cells, with a specific effect on sugar export.     

Genetic analysis of autosomal and X-linked markers across a mouse hybrid zone

In this paper, we present results of the first comprehensive study of the introgression of both autosomal and sex-chromosome markers across the central European portion of the hybrid zone between two house mouse subspecies, Mus musculus musculus and M. m. domesticus. More than 1800 individuals sampled from 105 sites were analyzed with a set of allozyme loci (hopefully representing neutral or nearly neutral markers) and X-linked loci (which are assumed to be under selection). The zone center is best modeled as a single straight line independent of fine-scale local geographic or climatic conditions, being maintained by a balance between dispersal and selection against hybrids. The width (w) of the multilocus autosomal cline was estimated as 9.6 km whereas the estimate for the compound X-chromosome cline was about 4.6 km only. As the former estimate is comparable to that of the Danish portion of the zone (assumed to be much younger than the central European one), zone width does not appear to be related to its age. The strength (B) of the central barrier was estimated as about 20 km; with dispersal (sigma) of about 1 km/gen(1/2), this means effective selection (s*) is approximately 0.06-0.09 for autosomal loci and about 0.25 for X-linked loci. The number of loci under selection was estimated as N= 56-99 for autosomes and about 380 for X-linked loci. Finally, we highlight some potential pitfalls in hybrid zone analyses and in comparisons of different transects. We suggest that conclusions about parts of the mouse genome involved in reproductive isolation and speciation should be drawn with caution and that analytical approaches always providing some estimates should not be used without due care regarding the support or confidence of such estimates, especially if conclusions are based on the difference between these estimates. Finally, we recommend that analysis in two-dimensional space, dense sampling, and rigorous treatment of data, including inspection of likelihood profiles, are essential for hybrid zone studies.     

The effects of membrane compartmentalization of csk on TCR signaling

The TCR signal transduction is initiated by the activation of Src-family kinases (SFK) which phosphorylate Immunoreceptor tyrosine-based activation motifs (ITAM) present in the intracellular parts of the T-cell receptor (TCR) signaling subunits. Numerous data suggest that after stimulation TCR interacts with membrane rafts and thus it gains access to SFK and other important molecules involved in signal transduction. However, the precise mechanism of this process is unclear. One of the key questions is how SFK access TCR and what is the importance of non-raft and membrane raft-associated SFK for the initiation and maintenance of the TCR signaling. To answer this question we targeted a negative regulator of SFK, C-terminal Src kinase (Csk) to membrane rafts, recently described “heavy rafts” or non-raft membrane. Our data show that only Csk targeted into “classical” raft but not to “heavy raft” or non-raft membrane effectively inhibits TCR signaling, demonstrating the critical role of membrane raft-associated SFK in this process.     

Linoleic acid-induced ultra-weak photon emission from Chlamydomonas reinhardtii as a tool for monitoring of lipid peroxidation in the cell membranes

Reactive oxygen species formed as a response to various abiotic and biotic stresses cause an oxidative damage of cellular component such are lipids, proteins and nucleic acids. Lipid peroxidation is considered as one of the major processes responsible for the oxidative damage of the polyunsaturated fatty acid in the cell membranes. Various methods such as a loss of polyunsaturated fatty acids, amount of the primary and the secondary products are used to monitor the level of lipid peroxidation. To investigate the use of ultra-weak photon emission as a non-invasive tool for monitoring of lipid peroxidation, the involvement of lipid peroxidation in ultra-weak photon emission was studied in the unicellular green alga Chlamydomonas reinhardtii. Lipid peroxidation initiated by addition of exogenous linoleic acid to the cells was monitored by ultra-weak photon emission measured with the employment of highly sensitive charged couple device camera and photomultiplier tube. It was found that the addition of linoleic acid to the cells significantly increased the ultra-weak photon emission that correlates with the accumulation of lipid peroxidation product as measured using thiobarbituric acid assay. Scavenging of hydroxyl radical by mannitol, inhibition of intrinsic lipoxygenase by catechol and removal of molecular oxygen considerably suppressed ultra-weak photon emission measured after the addition of linoleic acid. The photon emission dominated at the red region of the spectrum with emission maximum at 680 nm. These observations reveal that the oxidation of linoleic acid by hydroxyl radical and intrinsic lipoxygenase results in the ultra-weak photon emission. Electronically excited species such as excited triplet carbonyls are the likely candidates for the primary excited species formed during the lipid peroxidation, whereas chlorophylls are the final emitters of photons. We propose here that the ultra-weak photon emission can be used as a non-invasive tool for the detection of lipid peroxidation in the cell membranes.     

Secondary alcohol dehydrogenase catalyzes the reduction of exogenous acetone to 2-propanol in Trichomonas vaginalis

Secondary alcohols such as 2-propanol are readily produced by various anaerobic bacteria that possess secondary alcohol dehydrogenase (S-ADH), although production of 2-propanol is rare in eukaryotes. Specific bacterial-type S-ADH has been identified in a few unicellular eukaryotes, but its function is not known and the production of secondary alcohols has not been studied. We purified and characterized S-ADH from the human pathogen Trichomonas?vaginalis. The kinetic properties and thermostability of T.?vaginalis S-ADH were comparable with bacterial orthologues. The substantial activity of S-ADH in the parasite's cytosol was surprising, because only low amounts of ethanol and trace amounts of secondary alcohols were detected as metabolic end products. However, S-ADH provided the parasite with a high capacity to scavenge and reduce external acetone to 2-propanol. To maintain redox balance, the demand for reducing power to metabolize external acetone was compensated for by decreased cytosolic reduction of pyruvate to lactate and by hydrogenosomal metabolism of pyruvate. We speculate that hydrogen might be utilized to maintain cytosolic reducing power. The high activity of Tv-S-ADH together with the ability of T.?vaginalis to modulate the metabolic fluxes indicate efficacious metabolic responsiveness that could be advantageous for rapid adaptation of the parasite to changes in the host environment.