The structure of the carbohydrate backbone of the LPS from Myxococcus xanthus strain DK1622

Gram-negative rod shaped bacterium Myxococcus xanthus DK1622 produces a smooth-type LPS. The structure of the polysaccharide O-chain and the core-lipid A region of the LPS has been determined by chemical and spectroscopic methods. The O-chain was built up of disaccharide repeating units having the following structure: -->6)-alpha-D-Glcp-(1-->4)-alpha-D-GalpNAc6oMe*-(1--> with partially methylated GalNAc residue. The core region consisted of a phosphorylated hexasaccharide, containing one Kdo residue, unsubstituted at O-4, and no heptose residues. The lipid A component consisted of beta-GlcN-(1-->6)-alpha-GlcN1P disaccharide, N-acylated with 13-methyl-C14-3OH (iso-C15-3OH), C16-3OH, and 15-methyl-C16-3OH (iso-C17-3OH) acids. The lipid portion contained O-linked iso-C16 acid.     

Cyclophilin A produced by thymocytes regulates the migration of murine bone marrow cells

Supernatant obtained from high dose hydrocortisone resistant thymocytes can induce migration of the bone marrow cell precursors to the periphery. This biological activity depends on the presence of the 18 kDa protein, whose amino acid sequence fits with the sequence of the secretory form of murine cyclophilin A (SP-18). Cyclophilin A isolated from the supernatant of the cortisone-resistant thymoma EL-4 shows its characteristic functional features as it demonstrates isomerase activity and binds with cyclosporine A. The cyclophilin A obtained manifests chemotactic activity that regulates migration of bone marrow cell precursors of neutrophils, T-, B- and dendritic cells.     

Asymmetry of syringomycin E channel studied by polymer partitioning

To probe the size of the ion channel formed by Pseudomonas syringae lipodepsipeptide syringomycin E, we use the partial blockage of ion current by penetrating poly(ethylene glycol)s. Earlier experiments with symmetric application of these polymers yielded a radius estimate of approximately 1 nm. Now, motivated by the asymmetric non-ohmic current-voltage curves reported for this channel, we explore its structural asymmetry. We gauge this asymmetry by studying the channel conductance after one-sided addition of differently sized poly(ethylene glycol)s. We find that small polymers added to the cis-side of the membrane (the side of lipodepsipeptide addition) reduce channel conductance much less than do the same polymers added to the trans-side. We interpret our results to suggest that the water-filled pore of the channel is conical with cis- and trans-radii differing by a factor of 2-3 and that the smaller cis-radius is in the 0.25-0.35 nm range. In symmetric, two-sided addition, polymers entering the pore from the larger opening dominate blockage.     

N-terminal extension of Saccharomyces cerevisiae translation termination factor eRF3 influences the suppression efficiency of sup35 mutations

The eukaryotic translation termination factor eRF3 stimulates release of nascent polypeptides from the ribosome in a GTP-dependent manner. In most eukaryotes studied, eRF3 consists of an essential, conserved C-terminal domain and a nonessential, nonconserved N-terminal extension. However, in some species, this extension is required for efficient termination. Our data show that the N-terminal extension of Saccharomyces cerevisiae eRF3 also participates in regulation of termination efficiency, but acts as a negative factor, increasing nonsense suppression efficiency in sup35 mutants containing amino acid substitutions in the C-terminal domain of the protein.     

Study of localization of the protein-synthesizing machinery along actin filament bundles.

Indirect immunofluorescent microscopy was used to study the distribution of elongation factor 2 (eEF-2) in fixed human skin diploid and mouse embryo fibroblasts. It was found earlier that some of the eEF-2 ribosomes and initiation factor 2 (eIF-2) are co-localized with a part of the actin microfilament bundles in these cells (Gavrilova et al., 1987; Shestakova et al., 1991). Here it has been shown that inhibition of protein synthesis either by inactivation of eEF-2 itself with diphtheria toxin or by inactivation of ribosomes with ricin does not abolish the distribution of eEF-2 along the actin microfilament bundles. At the same time, the disassembly of actin microfilaments by cytochalasin D results also in the disappearance of eEF-2-carrying threads. This means that the eEF-2-carrying threads do not exist per se, and that the organization of eEF-2 in visible "filaments" depends upon the integrity of the actin cytoskeleton.     

Manipulation of division symmetry and developmental fate in cultures of potato microspores

The effect of media composition on microspore culture was investigated in one tetraploid and two diploid potatoes. The viability of microspores isolated from 4.5 to 5 mm buds was in the range of 33 to 52%. In media for anther culture, microspores showed no further development and lost viability within 2 days. In M1 medium containing mineral components, sucrose, uridine, cytidine, myo-inositol, glutamine and lactalbumin hydrolysate, 18 to 37% of microspores underwent mitosis within 14 days. Up to 95% of the divisions were symmetric and produced equal nuclei. Some symmetrically divided microspores eventually produced structures with 3 to 10 nuclei. The proportion of the total microspore population producing multinuclear structures reached 9% in diploid clones responsive to anther culture and 1 to 2% in recalcitrant cv. Borka. Symmetric mitoses in M1 medium were induced in the presence of glutamine and lactalbumin hydrolysate. Nucleosides and myo-inositol had no effect on microspore division. In the absence of all organic components except sucrose, most mitoses were asymmetric, formation of multinuclear structures was reduced and most pollen accumulated starch indicative of gametophytic fate. In complete M1 medium, starch accumulation was suppressed. Suppression also occurred in asymmetrically divided microspores, indicating a direct inhibition of pollen development independent of the mode of microspore division. This inhibitory effect of M1 medium might present a stress which triggers the induction of symmetric microspore division and subsequent formation of multinuclear structures. This revised version was published online in June 2006 with corrections to the Cover Date.     

Rhizobial nod gene-inducing activity in pea nodulation mutants: dissociation of nodulation and flavonoid response

With the characterization of the total genomes of Arabidopsis thaliana and Oryza sativa , several putative plasma membrane components have been identified. However, a lack of knowledge at the protein level, especially for hydrophobic proteins, have hampered analyses of physiological changes. To address whether protein complexes may be present in the native membrane, we subjected plasma membranes isolated from Spinacia oleracea leaves to blue-native polyacrylamide gel electrophoresis (BN-PAGE). BN-PAGE is well established in the separation of functional membrane protein complexes from mitochondria and chloroplasts, but a resolved protein complex pattern from PM of eukaryotic cells has previously not been reported. Using this method, protein complexes from Spinacia oleracea PM could be efficiently solubilized and separated, including the highly hydrophobic aquaporin (apparent molecular mass 230 kDa), a putative tetramer of H⁺-ATPase, and several less abundant complexes with apparent masses around or above 750 kDa. After denaturation and separation of the complexes into their subunits in a second dimension (SDS-PAGE), several of the complexes were identified as hydrophobic membrane proteins. Large amounts of protein (up to 1 mg) can be resolved in each lane, which suggests that the method could be used to study also low-abundance protein complexes, e.g. under different physiological conditions.     

A key role for the mRNA leader structure in translational control of ribosomal protein S1 synthesis in gamma-proteobacteria

The translation initiation region (TIR) of the Escherichia coli rpsA mRNA coding for ribosomal protein S1 is characterized by a remarkable efficiency in driving protein synthesis despite the absence of the canonical Shine–Dalgarno element, and by a strong and specific autogenous repression in the presence of free S1 in trans. The efficient and autoregulated E.coli rpsA TIR comprises not less than 90 nt upstream of the translation start and can be unambiguously folded into three irregular hairpins (HI, HII and HIII) separated by A/U-rich single-stranded regions (ss1 and ss2). Phylogenetic comparison revealed that this specific fold is highly conserved in the γ-subdivision of proteobacteria (but not in other subdivisions), except for the Pseudomonas group. To test phylogenetic predictions experimentally, we have generated rpsA′–′lacZ translational fusions by inserting the rpsA TIRs from various γ-proteobacteria in-frame with the E.coli chromosomal lacZ gene. Measurements of their translation efficiency and negative regulation by excess protein S1 in trans have shown that only those rpsA TIRs which share the structural features with that of E.coli can govern efficient and regulated translation. We conclude that the E.coli-like mechanism for controlling the efficiency of protein S1 synthesis evolved after divergence of Pseudomona