Two families of extracellular phospholipase C genes are present in aspergilli.

Fungi secrete extracellular enzymes to enable them to harvest nutrients from the environment. In the case of pathogenic fungi these enzymes can also be pathogenesis factors. Here we report the identification in fungi of a complex family of extracellular phospholipase C (PLC) enzymes, homologous to the Pseudomonas aeruginosa PLCH_PSEAE. Database searches and phylogenetic analysis showed that the PLCs clustered into two groups with different evolutionary histories. One group, subdivided into PLC-A, -B, -C and -D, was found only in aspergilli and Neosartorya fischeri. Each species only ever showed three of the four PLCs except N. fischeri which had all four PLCs plus duplicate PLC-A, -B and -C genes. Modelling studies indicated that these PLCs had mechanistic similarities to phosphoesterases and aryl sulphatases, but that they probably did not differ in substrate specificity. The second group, PLC-E, was seen in a wider range of fungi including some species of aspergilli and was always found in a head-to-head arrangement with a copper oxidase, similar to the laccases. The PLC genes appear to have arisen from separate gene transfer events from bacteria or lower eukaryotes. Thus, aspergilli have acquired PLCs twice in the course of evolution.

ADAMs are present in fungi: identification of two novel ADAM genes in Aspergillus fumigatus.

The ADAMs are a family of integral membrane proteases involved in shedding and fusion events in animal tissues. Here, we report the identification of two ADAMs, ADM-A and ADM-B, in the pathogenic fungus Aspergillus fumigatus. The domain structure of metazoan ADAMs was seen in ADM-A and -B, although with some differences. ADAMs were identified in other filamentous fungi and phylogenetic analysis indicated that the fungal ADAMs were monophyletic and most closely related to metazoan ADAM 10 and 17. Recombinant ADM-B protease specifically cleaved casein and albumin while recombinant propeptide+protease was inactive. A sheddase function is therefore proposed for fungal ADAMs.

Identification of protein tyrosine kinases required for B-cell- receptor-mediated activation of an Epstein-Barr Virus immediate-early gene promoter.

Epstein-Barr Virus (EBV) is a potentially oncogenic herpesvirus that infects >90% of the world's population. EBV exists predominantly as a latent infection in B lymphocytes, with periodic lytic-cycle reactivation essential for cellular and host transmission. Viral reactivation can be stimulated by ligand-induced activation of B-cell-receptor (BCR)-coupled signaling pathways. The critical first step in the transition from latency to the lytic cycle is the expression of the viral immediate-early gene BZLF1 through the transcription activation of its promoter, Zp. However, the BCR-coupled signal transduction cascade(s) leading to the induction of Zp and the expression of the BZLF1 gene product, Zta, is currently unclear. A major obstacle to delineating the relevant signal transduction events has been the lack of a model of EBV infection that is amenable to genetic manipulation. The use of the avian B-cell line DT40 has proven to be a powerful tool for delineating BCR-mediated signal transduction pathways that appear to be highly conserved between avian and mammalian systems. We demonstrate that the DT40 cell line is a robust and genetically tractable system for the study of BCR-mediated signaling pathways leading to transcriptional activation of BZLF1. Using this system, we demonstrate that activation of Zp requires the BCR-coupled protein tyrosine kinases Syk and Btk and that it is positively regulated by Lyn. Thus, the use of DT40 cells has allowed us to delineate the early signaling components required for BCR-dependent reactivation of latent EBV, and this system is likely to prove useful for further dissection of the downstream signaling cascades involved.

Role of Tyrosine Kinases in IgE-Mediated Signal Transduction in Human Lung Mast Cells and Basophils.

Tyrosine kinases are now thought to play an important role in IgE signal transduction in rodent cell lines. We have assessed the effects of four different inhibitors of tyrosine kinases on human lung mast cells and basophils to see if there is a similar requirement in human cells. Genistein proved to be a potent inhibitor of anti-IgE-induced histamine release in human basophils, though less potent in human lung mast cells. While tyrphostin produced a significant inhibition of histamine release neither lavendustin nor methyl-2,5-dihydroxy cinnamate (MDC) affected degranulation. Human lung mast cells showed a different inhibitory profile with MDC proving to be a potent inhibitor, genistein was much less effective and neither tyrphostin nor lavendustin A significantly affected histamine release. These results suggest that tyrosine kinases may have a role in IgE-dependent signal transduction in human lung mast cells and basophils but indicates that the two cell types have distinct inhibitory profiles.