Triamcinolone regulated apopto-phagocytic gene expression patterns in the clearance of dying retinal pigment epithelial cells. A key role of Mertk in the enhanced phagocytosis.

BACKGROUND: The apopto-phagocytic gene expression patterns during clearance of dying cells in the retina and the effect of triamcinolone (TC) upon these processes have relevance to development of age-related macular degeneration (AMD). METHODS: ARPE-19 cells and primary human retinal pigment epithelium (hRPE) were induced to undergo cell death by anoikis and the clearance of these cells by living hRPE/ARPE-19 or human monocyte-derived macrophages (HMDMs) in the presence or absence of TC was quantified by flow cytometry. TaqMan low-density gene expression array determining known markers of phagocytosis and loss-of-function studies on selected apopto-phagocytic genes was carried out in HMDM engulfing anoikic cells. RESULTS: The glucocorticoid TC had a profound phagocytosis-enhancing effect on HMDM engulfing anoikic ARPE-19 or hRPE cells, causing a selective upregulation of the Mer tyrosine kinase (MERTK) receptor, while decreasing the expression of the AXL receptor tyrosine kinase and thrombospondin-1 (THSB-1). The key role of the MERTK could be demonstrated in HMDM engulfing dying cells using gene silencing as well as blocking antibodies. Similar pathways were found upregulated in living ARPE-19 engulfing anoikic ARPE-19 cells. Gas6 treatment enhanced phagocytosis in TC-treated HMDMs. CONCLUSIONS: Specific agonists of the Mertk receptor may have a potential role as phagocytosis enhancers in the retina and serve as future targets for AMD therapy. GENERAL SIGNIFICANCE: The use of Gas6 as enhancer of retinal phagocytosis via the MerTK receptor, alone or in combination with other specific ligands of the tyrosine kinase receptors' family may have a potential role in AMD therapy.

Adenylyl cyclase regulates heavy metal sensitivity, bikaverin production and plant tissue colonization in Fusarium proliferatum.

A homologue of the adenylyl cyclase (AC) gene of Neurospora crassa, named Fpacy1 was cloned from the genomic library of Fusarium proliferatum ITEM 2287 by screening the library with a DNA fragment amplified by using PCR primers designed from conserved sequences of the catalytic domain of AC genes from other fungi. The deduced FPACY1 protein had 53-77% identity with the AC proteins of other fungi. DeltaFpacy1 mutants obtained by targeted gene disruption showed retarded vegetative growth, increased conidiation and delayed conidial germination. Colonization capability of the mutants, assessed on maize seedlings and tomato fruits also was adversely affected. In sexual crosses the AC mutants retained full male fertility, but their female fertility decreased significantly. Disruption of Fpacy1 abolished vegetative self-incompatibility, suggesting that the AC gene is involved in multiple developmental processes related to vegetative growth, as well as sexual and parasexual events. The elevated thermo- and H(2)O(2)-tolerance of the DeltaFpacy1 mutants was coupled to an increased sensitivity towards Cd and Cu, indicating that the cAMP signaling pathway may have both negative and positive regulatory roles on the stress response mechanisms of fungal cells. When grown under nitrogen limitation conditions, the DeltaFpacy1 mutants produced an average of approximately 274 microg g(-1) bikaverin, whereas only traces of this metabolite was detected in the wild type. This finding provides further evidence of the role of the cAMP-PKA pathway in regulating bikaverin production.

Does the detoxification of penicillin side-chain precursors depend on microsomal monooxygenase and glutathione S-transferase in Penicillium chrysogenum?

The glutathione (GSH) S-conjugation of 1,2-epoxy-3-(4'-nitrophenoxy)propane was catalysed predominantly by microsomal glutathione S-transferase (mGST) in Penicillium chrysogenum. The specific mGST activity unlike the cytosolic GST (cGST) activity increased substantially when the penicillin side-chain precursor phenoxyacetic acid (POA) was included in the culture medium. Therefore, a microsomal monooxygenase (causing possible release of epoxide intermediates) and mGST-dependent detoxification pathway may exist for the side-chain precursors as an alternative to microsomal activation to acyl-CoA and subsequent transfer to beta-lactam molecules. The P. chrysogenum pahA and Aspergillus nidulans phacA gene products, which are cytochrome p450 monooxygenases and are able to hydroxylate phenylacetic acid (PA) at position 2 on the aromatic ring, are unlikely to release toxic epoxide intermediates but epoxidation of PA and POA due to the action of other microsomal monooxygenases cannot be excluded. The GSH-dependent detoxification of POA was provoked by a well-controlled transient lowering of pH (down to 5.0) at the beginning of the production phase in a fed-batch fermentation system. Both the specific GST and gammaGT activities were increased but the intracellular GSH concentrations remained unaltered unless the pH of the feed was transiently lowered below 5.0. At pH 4.6, the GSH pool was depleted rapidly but no antibiotic production was observed. Although sucrose was taken up effectively by the cells, cell death and autolysis were progressing. Therefore, the industrial exploitation of the GSH-dependent detoxification of penicillin side-chain precursors to reduce intracellular GSH-levels in order to avoid the GSH inhibition of the beta-lactam biosynthetic enzymes seems to be rather unlikely. P. chrysogenum mGST and cGST were separated using GSH-Sepharose 6B affinity chromatography. The purified cGST possessed a homodimer (alpha(2)) tertiary structure with M(r) (, alpha) = 29500.