Aspergillus fumigatus MedA governs adherence,host cell interactions and virulence

In medically important fungi, regulatory elements that control development and asexual reproduction often govern the expression of virulence traits. We therefore cloned the Aspergillus fumigatus developmental modifier MedA and characterized its role in conidiation, host cell interactions and virulence. As in the model organism Aspergillus nidulans, disruption of medA in A. fumigatus dramatically reduced conidiation. However, the conidiophore morphology was markedly different between the two species. Further, gene expression analysis suggested that MedA governs conidiation through different pathways in A. fumigatus compared with A. nidulans. The A. fumigatusΔmedA strain was impaired in biofilm production and adherence to plastic, as well as adherence to pulmonary epithelial cells, endothelial cells and fibronectin in vitro. The ΔmedA strain also had reduced capacity to damage pulmonary epithelial cells, and stimulate pro‐inflammatory cytokine mRNA and protein expression. Consistent with these results, the A. fumigatusΔmedA strain also exhibited reduced virulence in both an invertebrate and a mammalian model of invasive aspergillosis. Collectively, these results suggest that the downstream targets of A. fumigatus MedA mediate virulence, and may provide novel therapeutic targets for invasive aspergillosis.     

Dynamics and function of phospholipase D and phosphatidic acid during phagocytosis

Phospholipase D (PLD) produces phosphatidic acid (PA), an established intracellular signalling lipid that has been also implicated in vesicular trafficking, and as such, PLD could play multiple roles during phagocytosis. Using an RNA interference strategy, we show that endogenous PLD1 and PLD2 are necessary for efficient phagocytosis in murine macrophages, in line with results obtained with wild-type constructs and catalytically inactive PLD mutants which, respectively, enhance and inhibit phagocytosis. Furthermore, we found that PA is transiently produced at sites of phagosome formation. Macrophage PLD1 and PLD2 differ in their subcellular distributions. PLD1 is associated with cytoplasmic vesicles, identified as a late endosomal/lysosomal compartment, whereas PLD2 localizes at the plasma membrane. In living cells undergoing phagocytosis, PLD1 vesicles are recruited to nascent and internalized phagosomes, whereas PLD2 is only observed on nascent phagosomes. These results provide evidence that both PLD isoforms are required for phagosome formation, but only PLD1 seems to be implicated in later stages of phagocytosis occurring after phagosomal internalization.     

Inhibition of early steps of HIV-1 replication by SNF5/Ini1

To replicate, human immunodeficiency virus, type 1 (HIV-1) needs to integrate a cDNA copy of its RNA genome into a chromosome of the host cell, a step controlled by the viral integrase (IN) protein. Viral integration involves the participation of several cellular proteins. SNF5/Ini1, a subunit of the SWI/SNF chromatin remodeling complex, was the first cofactor identified to interact with IN. We report here that SNF5/Ini1 interferes with early steps of HIV-1 replication. Inhibition of SNF5/Ini1 expression by RNA interference increases HIV-1 replication. Using quantitative PCR, we show that both the 2-long terminal repeat circle and integrated DNA forms accumulate upon SNF5/Ini1 knock down. By yeast two-hybrid assay, we screened a library of HIV-1 IN random mutants obtained by PCR random mutagenesis using SNF5/Ini1 as prey. Two different mutants of interaction, IN E69G and IN K71R, were impaired for SNF5/Ini1 interaction. The E69G substitution completely abolished integrase catalytic activity, leading to a replication-defective virus. On the contrary, IN K71R retained in vitro integrase activity. K71R substitution stimulates viral replication and results in higher infectious titers. Taken together, these results suggest that, by interacting with IN, SNF5/Ini1 interferes with early steps of HIV-1 infection.     

Rate of oxygen consumption by isolated articular chondrocytes is sensitive to medium glucose concentration

Cartilage tissue engineering typically involves the culture of isolated chondrocytes within a scaffold material. The oxygen tension within the engineered tissue is known to be an essential parameter for implant success. This will be sensitive to the oxygen consumption behavior of the embedded chondrocytes, which remains to be characterized. We report that the oxygen consumption of bovine articular chondrocytes is sensitive to glucose deprivation below 2.7 mM, increasing from a basal level of 9.6x10(-16) to <18.4x10(-16) mol/cell.h in 1.3 mM glucose. Further studies examined the influence of selecting high (18.4 mM) or low (5.1 mM) glucose medium on the oxygen tension in 2 mm thick cellular agarose constructs. A relative upregulation of oxygen consumption was observed in constructs cultured in low glucose medium. This resulted in the near-anoxic oxygen concentration of 5 microM oxygen in constructs seeded with 40x10(6) cells/ml, compared to 57 microM in the corresponding high glucose culture. The upregulation of oxygen consumption generally corresponded to the inhibition of glycolysis, which is consistent with the Crabtree phenomenon. Medium osmolarity (316-600 mOsm) had minimal effects on chondrocyte oxygen consumption rate. In conclusion, glucose availability is a critical parameter that regulates the oxygen tension within tissue engineered constructs.     

Development and characterization of a small-scale bioreactor based on a bioartificial hepatic culture model for predictive pharmacological in vitro screenings

A vast majority of pharmacons are beset by possible interactions and side effects which have usually been tested in laboratory animals. However, better methods are needed to reduce the number of animal experiments and interspecies differences with respect to drug metabolism, as well as to provide a faster and more cost-effective way of analysis. These facts have led to the development of in vitro models based on isolated primary hepatocytes to better assess drug metabolism, interactions, and toxicity. A new small-scale bioreactor with the hepatic sandwich model and a gas-permeable membrane at the bottom allowing a definable oxygen exchange, has been constructed and compared with the conventional well plates. Compared to hepatocytes cultured in conventional systems, the cells exhibited a stronger liver-specific capacity and remained in a differentiated state in the small-scale bioreactor over a cultivation period of 17 days. This in vitro model could serve as a tool to predict the liver response to newly developed drugs.     

Functional domains of human tryptophan hydroxylase 2 (hTPH2)

Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in serotonin biosynthesis. A novel gene, termed TPH2, has recently been described. This gene is preferentially expressed in the central nervous system, while the original TPH1 is the peripheral gene. We have expressed human tryptophan hydroxylase 2 (hTPH2) and two deletion mutants (NDelta150 and NDelta150/CDelta24) using isopropyl beta-D-thiogalactopyranoside-free autoinduction in Escherichia coli. This expression system produced active wild type TPH2 with relatively low solubility. The solubility was increased for mutants lacking the NH(2)-terminal regulatory domain. The solubility of hTPH2, NDelta150, and NDelta150/CDelta24 are 6.9, 62, and 97.5%, respectively. Removal of the regulatory domain also produced a more than 6-fold increase in enzyme stability (t((1/2)) at 37 degrees C). The wild type hTPH2, like other members of the aromatic amino acid hydroxylase superfamily, exists as a homotetramer (236 kDa on size exclusion chromatography). Similarly, NDelta150 also migrates as a tetramer (168 kDa). In contrast, removal of the NH(2)-terminal domain and the COOH-terminal, putative leucine zipper tetramerization domain produces monomeric enzyme (39 kDa). Interestingly, removal of the NH(2)-terminal regulatory domain did not affect the Michaelis constants for either substrate but did increase V(max) values. These data identify the NH(2)-terminal regulatory domain as the source of hTPH2 instability and reduced solubility.     

Purification and characterization of the thyrotropin-releasing hormone (TRH)-degrading serum enzyme and its identification as a product of liver origin.

Previous biochemical studies have indicated that the membrane-bound thyrotropin-releasing hormone (TRH)-degrading enzyme (TRH-DE) from brain and liver and the serum TRH-DE are derived from the same gene. These studies also suggested that the serum enzyme is of liver origin. The present study was undertaken to verify these hypotheses. In different species, a close relationship between the activities of the serum enzyme and the particulate liver enzyme was noticed. The activity of the serum enzyme decreased when rats were treated with thioacetamide, a known hepatotoxin. With hepatocytes cultured in a sandwich configuration, release of the TRH-DE into the culture medium could also be demonstrated. The trypsin-solubilized particulate liver TRH-DE and the serum TRH-DE were purified to electrophoretic homogeneity. Both enzymes and the brain TRH-DE were recognized by a monoclonal antibody generated with the purified brain enzyme as antigen. Lectin blot analysis indicated that the serum enzyme and the liver enzyme are glycoproteins containing a sugar structure of the complex type, whereas the brain enzyme exhibits an oligomannose/hybrid glycostructure. A molecular mass of 97 000 Da could be estimated for all three enzymes after deglycosylation and SDS/PAGE followed by Western blotting. Fragment analysis of the serum TRH-DE revealed that the peptide sequences correspond to the cDNA deduced amino-acid sequences of the membrane-bound brain TRH-DE, whereby two peptides were identified that are encoded by exon 1. These data strongly support the hypothesis that the TRH-DEs are all derived from the same gene, whereby the serum enzyme is generated by proteolytic cleavage of the particulate liver enzyme.     

SeqHound: biological sequence and structure database as a platform for bioinformatics research

Background  

SeqHound has been developed as an integrated biological sequence, taxonomy, annotation and 3-D structure database system. It provides a high-performance server platform for bioinformatics research in a locally-hosted environment.     

Comparisons of photosynthesis‐related traits of 27 abundant or subordinate bryophyte species in a subalpine old‐growth fir forest

Bryophyte communities can exhibit similar structural and taxonomic diversity as vascular plant communities, just at a smaller scale. Whether the physiological diversity can be similarly diverse, and whether it can explain local abundance patterns is unknown, due to a lack of community‐wide studies of physiological traits. This study re‐analyzed data on photosynthesis‐related traits (including the nitrogen, phosphorus and chlorophyll concentrations, photosynthetic capacities, and photosynthetic nutrient use efficiencies) of 27 bryophyte species in a subalpine old‐growth fir forest on the eastern Tibetan Plateau. We explored differences between taxonomic groups and hypothesized that the most abundant bryophyte species had physiological advantages relative to other subdominant species. Principal component analysis (PCA) was used to summarize the differences among species and trait values of the most abundant and other co‐occurring subdominant species. Species from the Polytrichaceae were separated out on both PCA axes, indicating their high chlorophyll concentrations and photosynthetic capacities (axis 1) and relatively high‐light requirements (axis 2). Mniaceae species also had relatively high photosynthetic capacities, but their light saturation points were low. In contrast, Racomitrium joseph‐hookeri and Lepidozia reptans, two species with a high shoot mass per area, had high‐light requirements and low nutrient and chlorophyll concentrations and photosynthetic capacities. The nutrient concentrations, photosynthetic capacities, and photosynthetic nutrient use efficiencies of the most abundant bryophyte species did not differ from co‐occurring subdominant species. Our research confirms the links between the photosynthesis‐related traits and adaptation strategies of bryophytes. However, species relative abundance was not related to these traits.