Structures of the lectins from Pseudomonas aeruginosa: insight into the molecular basis for host glycan recognition

The opportunistic human pathogen Psuedomonas aeruginosa produces two lectins in close association with virulence factors: PA-IL adn PA-IIL, which bind to galactose- and fucose/mannose-containing glycoconjugates, respectively. We review here the structural aspects of these lectins relative to their putative roles in host recognition, cell surface adhesion and biofilm formation.     

Involvement of peptidorhamnomannan in the interaction of Pseudallescheria boydii and HEp2 cells

Pseudallescheria boydii is an emerging fungal pathogen that has a worldwide distribution. Virulence mechanisms of P. boydii are largely unknown. We studied the interaction between P. boydii and HEp2 cells and demonstrated that conidia of P. boydii attached to, and were ingested by, HEp2 cells in a time-dependent process. After 2 h of interaction, the conidia produced a germ-tube like projection, which was able to penetrate the epithelial cell membrane. Recently, our group characterized a peptidorhamnomannan (PRM) antigen on the cell surface of P. boydii. In order to better understand the role played by this surface glycoconjugate during cell adhesion and endocytosis, inhibition assays were performed using intact PRM and anti-PRM polyclonal antibody. When HEp2 cells were pre-treated with whole PRM molecule, the adhesion and endocytic indices were, respectively, 50% and 60% lower than in non-treated epithelial cells. Moreover, when the conidial cells were pre-incubated with anti-PRM antibodies, the adherence and endocytosis processes were inhibited in a dose-dependent manner. As PRM influenced the conidia P. boydii-HEp2 cell interaction, we also performed inhibition assays in order to observe which PRM moieties could be involved in this process. Treatment of PRM with proteinase K promoted a slight inhibition of adhesion. However, the de-O-glycosylated PRM molecule as well as the monosaccharide mannose was able to efficiently inhibit the adhesion and endocytic processes. In addition, our results indicate for the first time that P. boydii PRM binds to a polypeptide of 25 kDa on the HEp2 cell surface.     

An evolutionary change in telomere sequence motif within the plant section Asparagales had significance for telomere nucleoprotein complexes

In association with a phylogenetic tree of Asparagales, our previous results showed that a distinct clade included plant species where the ancestral, Arabidopsis-type of telomeric repeats (TTTAGGG)n had been partially, or fully, replaced by the human-type telomeric sequence (TTAGGG)n. Telomerases of these species synthesize human repeats with a high error rate in vitro. Here we further characterize the structure of telomeres in these plants by analyzing the overall arrangement of major and minor variants of telomeric repeats using fluorescence in situ hybridization on extended DNA strand(s). Whilst the telomeric array is predominantly composed of the human variant of the repeat, the ancestral, Arabidopsis-type of telomeric repeats was ubiquitously observed at one of the ends and/or at intercalary positions of extended telomeric DNAs. Another variant of the repeat typical of Tetrahymena was observed interspersed in about 20% of telomerics. Micrococcal nuclease digestions indicated that Asparagales plants with a human-type of telomere have telomeric DNA organised into nucleosomes. However, unexpectedly, the periodicity of the nucleosomes is not significantly shorter than bulk chromatin as is typical of telomeric chromatin. Using electrophoretic mobility shift assays we detected in Asparagales plants with a human type of telomere a 40-kDa protein that forms complexes with both Arabidopsis- and human-type G-rich telomeric strands. However, the protein shows a higher affinity to the ancestral Arabidopsis-type sequence. Two further proteins were found, a 25-kDa protein that binds specifically to the ancestral sequence and a 15-kDa protein that binds to the human-type telomeric repeat. We discuss how the organisation of the telomere repeats in Asparagales may have arisen and stabilised the new telomere at the point of mutation.     

Different action of killer toxins K1 and K2 on the plasma membrane and the cell wall of Saccharomyces cerevisiae

Study of Saccharomyces cerevisiae killer toxin-sensitive strains with the deltakre2 phenotype (resistant to toxin K1, sensitive to toxin K2) showed that the phenotype is complemented by the KRE2 gene not only in intact cells but also in spheroplasts, and resistance to K1 thus resides very probably in the plasma membrane. deltakre1 deletant displays a faulty interaction with both K1 and K2 toxin. Hence, Kre1p probably serves as plasma membrane receptor for both toxins. Deletants in seven other genes (GDA1, SAC1, LUV1, KRE23, SAC2, KRE21, ERG4) exhibit different degrees of the deltakre2-like resistance pattern, but the phenotype in deltagda1 and deltasac1 is not connected with a defect in K1 toxin interaction with the plasma membrane, similarly as in deltakre6 and deltakre11 strains with a higher resistance to K2 toxin. Differences between the K1 and K2 killer toxin thus occur on the level of both the plasma membrane and the cell wall.     

Isoamyl alcohol-induced morphological change in Saccharomyces cerevisiae involves increases in mitochondria and cell wall chitin content

Isoamyl alcohol reduced growth and induced filament formation in Saccharomyces cerevisiae. Isoamyl alcohol-induced filamentation was accompanied by an almost threefold greater increase in the specific activity of succinate dehydrogenase than in untreated cells, which suggested that isoamyl alcohol treatment caused the cells to produce more mitochondria than in normal yeast form proliferation. This was supported by measuring the dry weight of purified, isolated mitochondria. Filaments have an increased chitin content which is distributed over the majority of their surface, and is not confined to bud scars and the chitin ring between mother and daughter cells as in yeast-form cells.     

Activation and inhibition of cyclin-dependent kinase-2 by phosphorylation; a molecular dynamics study reveals the functional importance of the glycine-rich loop

Nanoseconds long molecular dynamics (MD) trajectories of differently active complexes of human cyclin-dependent kinase 2 (inactive CDK2/ATP, semiactive CDK2/Cyclin A/ATP, fully active pT160-CDK2/Cyclin A/ATP, inhibited pT14-; pY15-; and pT14,pY15,pT160-CDK2/Cyclin A/ATP) were compared. The MD simulations results of CDK2 inhibition by phosphorylation at T14 and/or Y15 sites provide insight into the structural aspects of CDK2 deactivation. The inhibitory sites are localized in the glycine-rich loop (G-loop) positioned opposite the activation T-loop. Phosphorylation of T14 and both inhibitory sites T14 and Y15 together causes ATP misalignment for phosphorylation and G-loop conformational change. This conformational change leads to the opening of the CDK2 substrate binding box. The phosphorylated Y15 residue negatively affects substrate binding or its correct alignment for ATP terminal phospho-group transfer to the CDK2 substrate. The MD simulations of the CDK2 activation process provide results in agreement with previous X-ray data.     

Synthesis and evaluation of phenylcarbamate derivatives as ligands for nicotinic acetylcholine receptors

Phenylcarbamate derivatives were synthesized and evaluated in radioligand binding assays for different nicotinic acetylcholine receptor (nAChR) subtypes. Carbamate derivatives bearing a pyrrolidine or piperidine moiety 8-20 exhibited much lower affinity for alpha7* nAChR than the analogues in the quinuclidine series 21-25, although the same structural elements are present. Furthermore, in contrast to the quinuclidine analogues 21-25, all (S)-pyrrolidine derivatives 8-12 and the piperidine analogues 15 and 16 exhibited higher affinities for alpha4beta2* nAChR.