A conserved subtilisin-like protein TgSUB1 in microneme organelles of Toxoplasma gondii

Proteolytic processing plays a significant role in the process of invasion by the obligate intracellular parasite Toxoplasma gondii. We have cloned a gene, TgSUB1, encoding for a subtilisin-type serine protease found in T. gondii tachyzoites. TgSUB1 protein is homologous to other Apicomplexan and bacterial subtilisins and is processed within the secretory pathway of the parasite. Initial cleavage occurs in the endoplasmic reticulum, after which the protein is transported to micronemes, vesicles that secrete early during host cell invasion. Upon stimulation of microneme secretion, TgSUB1 is cleaved into smaller products that are secreted from the parasite. This secondary processing is inhibited by brefeldin A and serine protease inhibitors. TgSUB1 is a candidate processing enzyme for several microneme proteins cleaved within the secretory pathway or during invasion.     

Heat shock protein-chaperoned peptides but not free peptides introduced into the cytosol are presented efficiently by major histocompatibility complex I molecules

The studies reported here bear on the events in the cytosol that lead to trafficking of peptides during antigen processing and presentation by major histocompatibility complex (MHC) I molecules. We have introduced free antigenic peptides or antigenic peptides bound to serum albumin or to cytosolic heat shock proteins hsp90 (and its endoplasmic reticular homologue gp96) or hsp70 into the cytosol of living cells and have monitored the presentation of the peptides by appropriate MHC I molecules. The experiments show that (i) free peptides or serum albumin-bound peptides, introduced into the cytosol, become ligands of MHC I molecules at a far lower efficiency than peptides chaperoned by any of the heat shock proteins tested and (ii) treatment of cells with deoxyspergualin, a drug that binds hsp70 and hsp90 with apparent specificity, abrogates the ability of cells to present antigenic peptides through MHC I molecules, and introduction of additional hsp70 into the cytosol overcomes this abrogation. These results suggest for the first time a functional role for cytosolic chaperones in antigen processing.     

NAB2, a corepressor of EGR-1, inhibits vascular endothelial growth factor-mediated gene induction and angiogenic responses of endothelial cells

In this study we have investigated the role of a specific corepressor of EGR-1, NAB2, to down-regulate vascular endothelial growth factor (VEGF)-induced gene expression in endothelial cells and to inhibit angiogenesis. Firstly, we show a reciprocal regulation of EGR-1 and NAB2 following VEGF treatment. During the initial phase EGR-1 is rapidly induced and NAB2 levels are down-regulated. This is followed by a reduction of EGR-1 and a concomitant increase of NAB2. Secondly, using the tissue factor gene as a readout for VEGF-induced and EGR-1-regulated gene expression we demonstrate that NAB2 can completely block VEGF-induced tissue factor reporter gene activity. Thirdly, by adenovirus-mediated expression we show that NAB2 inhibits up-regulation of tissue factor, VEGF receptor-1, and urokinase plasminogen activator mRNAs even when a combination of VEGF and bFGF is used for induction. In addition, NAB2 overexpression significantly reduced tubule and sprout formation in two different in vitro angiogenesis assays and largely prevented the invasion of cells and formation of vessel-like structures in the murine Matrigel model. These data suggest that NAB2 regulation represents a mechanism to guarantee transient EGR-1 activity following exposure of endothelial cells to VEGF and that NAB2 overexpression could be used to inhibit signals involved in the early phase of angiogenesis.     

Another fossil agaric from Dominican amber

We report the discovery of a fossil agaricoid homobasidiomycete from Dominican amber (ca 15-20 Ma). Aureofungus yaniguaensis appears to be a member of the euagarics clade, but its precise taxonomic placement is obscure. This is the fourth known fossil agaric and the third from Dominican amber.     

Charge-dependent translocation of the Trojan peptide penetratin across lipid membranes

We studied the interaction of the cell-penetrating peptide penetratin with mixed dioleoylphosphatidylcholine/dioleoylphoshatidylglycerol (DOPC/DOPG) unilamellar vesicles as a function of the molar fraction of anionic lipid, X(PG), by means of isothermal titration calorimetry. The work was aimed at getting a better understanding of factors that affect the peptide binding to lipid membranes and its permeation through the bilayer. The binding was well described by a surface partitioning equilibrium using an effective charge of the peptide of z(P) approximately 5.1 +/- 0.5. The peptide first binds to the outer surface of the vesicles, the effective binding capacity of which increases with X(PG). At X(PG) approximately 0.5 and a molar ratio of bound peptide-to-lipid of approximately 1/20 the membranes become permeable and penetratin binds also to the inner monolayer after internalization. The results were rationalized in terms of an "electroporation-like" mechanism, according to which the asymmetrical distribution of the peptide between the outer and inner surfaces of the charged bilayer causes a transmembrane electrical field, which alters the lateral and the curvature stress acting within the membrane. At a threshold value these effects induce internalization of penetratin presumably via inversely curved transient structures.     

Derivation of a polymorphic lineage of Gasteromycetes from boletoid ancestors

The phylogeny of selected gasteromycetes and hymenomycetes was inferred from partial nuclear large subunit rDNA (nuc-lsu, 28S) sequences, delimited by primers LR0R and LR5. Taxon sampling with emphasis on relationships within the Boletales further included some gasteroid groups, which obviously have evolved convergent fruiting body morphology, and therefore remained controversial in taxonomy. This study confirms the close relationship of Geastrales, Gauteriales and Phallales and the presumable derivation of Nidulariales and Tulostomatales within the euagarics clade, as widely accepted. In addition, four Hymenogaster species investigated were found to be in the euagarics clade and a relationship to the Cortinariaceae was indicated. The gasteroid fungus Zelleromyces stephensii is an example for maintaining morphological linkage by a lactiferous hyphal system to the genus Lactarius in the Russulales, and this relationship was affirmed in the sequence analysis. Several previously suggested relationships of gasteromycetes and Boletales were reproducible by analyzing nuc-lsu sequences. As a new result, Astraeus hygrometricus, the barometer earth star, is an additional representative of the Boletales. Together with Boletinellus, Phlebopus, Pisolithus, Calostoma, Gyroporus, Scleroderma, and Veligaster, Astraeus forms an unusual group comprising pileate-stipitate hymenomycetes and polymorphic gasteromycetes. This group is a major lineage within the Boletales and we propose the new suborder Sclerodermatineae, including the six families Boletinellaceae fam. nov. (Boletinellus and Phlebopus), Gyroporaceae (Singer) fam. nov. (Gyroporus), Pisolithaceae (Pisolithus), Astraeaceae (Astraeus), Calostomataceae (Calostoma), and the typus subordinis Sclerodermataceae (Scleroderma and Veligaster). Morphological and ecological characters, and pigment synthesis support the delimitation of the Sclerodermatineae, and indicate the radiation of different lineages in the Boletales originating from fungi with primitive tubular hymenophores. We regard such boletes with gyroid-boletinoid hymenophores, like Boletinellus, Gyrodon, Gyroporus, Paragyrodon and Phlebopus as key taxa in the evolution of Paxillineae, Sclerodermatineae and Boletineae.     

Influence of irradiation and pentoxifylline on histone H3 phosphorylation in human tumour cell lines

Phosphorylation of histone H3 at Ser-10 correlates with chromatin condensation and this amino terminal modification is now recognized as a specific marker of mitosis. We have monitored the appearance of cells showing histone H3 phosphorylation in four human tumour cell lines to identify cell cycle progression after irradiation. In the human melanoma cell lines Be11 and MeWo and in the squamous cell carcinoma lines 4197 and 4451 a dose of 7 Gy of Co-gamma irradiation increases the number of cells binding anti-histone H3-P antibody 1-8-fold in a p53-independent manner. In the p53 mutant cell lines MeWo and 4451 H3-P phosphorylated cells can be detected as early as 30 min and show a maximum 1 h post-irradiation. In the cell lines Be11, 4197 and 4451 the early wave of H3 phosphorylated cells is followed by a second wave, which reaches a maximum 4.5-7 h post-irradiation and then declines. These events are attributed to damage-induced cell cycle blocks in the G1 and G2 phase of the cell cycle. Addition of the dose modifying drug pentoxifylline before irradiation increases the appearance of cells showing early and the late H3 phosphorylation. When pentoxifylline is added 12-24 h post-irradiation when the cell cycle blocks have reached their maximum the appearance of cells with phosphorylated H3 increases 3-5-fold in the p53 mutant cell lines MeWo and 4451. These observations are consistent with the function of the drug as a G2 block abrogator. The large H3 phosphorylation signal in p53 mutant cells is consistent with early entry of a cohort of G2 cells into mitosis. The smaller H3-P signal in p53 wild type cells correlates with the lower proportion of stable G2 populations in G1 blocked cells. These results indicate that pentoxifylline influences the appearance of histone H3 phosphorylated cells in a manner strongly dependent on the number of cells in G2 phase. This suggests that addition of pentoxifylline indeed abrogates the G2 block and thereby facilitates early entry into mitosis.