Functional and genomic analyses of blocked protein O-mannosylation in baker's yeast.

O-mannosylation is a crucial protein modification in eukaryotes that is initiated by the essential family of protein O-mannosyltransferases (PMTs). Here we demonstrate that in the model yeast Saccharomyces cerevisiae rhodanine-3-acetic acid derivatives affect members of all PMT subfamilies. Specifically, we used OGT2468 to analyse genome-wide transcriptional changes in response to general inhibition of O-mannosylation in baker's yeast. PMT inhibition results in the activation of the cell wall integrity (CWI) pathway. Coinciding, the mitogen-activated kinase Slt2p is activated in vivo and CWI pathway mutants are hypersensitive towards OGT2468. Further, induction of many target genes of the unfolded protein response (UPR) and ER-associated protein degradation (ERAD) is observed. The interdependence of O-mannosylation and UPR/ERAD is confirmed by genetic interactions between HAC1 and PMTs, and increased degradation of the ERAD substrate Pdr5p* in pmtΔ mutants. Transcriptome analyses further suggested that mating and filamentous growth are repressed upon PMT inhibition. Accordingly, in vivo mating efficiency and invasive growth are considerably decreased upon OGT2468 treatment. Quantitative PCR and ChIP analyses suggest that downregulation of mating genes is dependent on the transcription factor Ste12p. Finally, inhibitor studies identified a role of the Ste12p-dependent vegetative signalling cascade in the adaptive response to inhibition of O-mannosylation.

The genetic interaction network of CCW12, a Saccharomyces cerevisiae gene required for cell wall integrity during budding and formation of mating projections.

BACKGROUND: Mannoproteins construct the outer cover of the fungal cell wall. The covalently linked cell wall protein Ccw12p is an abundant mannoprotein. It is considered as crucial structural cell wall component since in baker's yeast the lack of CCW12 results in severe cell wall damage and reduced mating efficiency. RESULTS: In order to explore the function of CCW12, we performed a Synthetic Genetic Analysis (SGA) and identified genes that are essential in the absence of CCW12. The resulting interaction network identified 21 genes involved in cell wall integrity, chitin synthesis, cell polarity, vesicular transport and endocytosis. Among those are PFD1, WHI3, SRN2, PAC10, FEN1 and YDR417C, which have not been related to cell wall integrity before. We correlated our results with genetic interaction networks of genes involved in glucan and chitin synthesis. A core of genes essential to maintain cell integrity in response to cell wall stress was identified. In addition, we performed a large-scale transcriptional analysis and compared the transcriptional changes observed in mutant ccw12Δ with transcriptomes from studies investigating responses to constitutive or acute cell wall damage. We identified a set of genes that are highly induced in the majority of the mutants/conditions and are directly related to the cell wall integrity pathway and cell wall compensatory responses. Among those are BCK1, CHS3, EDE1, PFD1, SLT2 and SLA1 that were also identified in the SGA. In contrast, a specific feature of mutant ccw12Δ is the transcriptional repression of genes involved in mating. Physiological experiments substantiate this finding. Further, we demonstrate that Ccw12p is present at the cell periphery and highly concentrated at the presumptive budding site, around the bud, at the septum and at the tip of the mating projection. CONCLUSIONS: The combination of high throughput screenings, phenotypic analyses and localization studies provides new insight into the function of Ccw12p. A compensatory response, culminating in cell wall remodelling and transport/recycling pathways is required to buffer the loss of CCW12. Moreover, the enrichment of Ccw12p in bud, septum and mating projection is consistent with a role of Ccw12p in preserving cell wall integrity at sites of active growth.The microarray data produced in this analysis have been submitted to NCBI GEO database and GSE22649 record was assigned.

Anthocyanins and anthocyanidins are poor inhibitors of CYP2D6.

The cytochrome P450 CYP2D6 isoform is involved in the metabolism of about 50% of all psychoactive drugs, including neuroleptic agents, selective serotonin reuptake inhibitors, selective norepinephrine reuptake inhibitors and tricyclic antidepressants. Therefore, inhibition of cytochrome P450 activity by foodstuffs has implications for drug safety. The present study addresses inhibitory effects of polyphenolic anthocyanins and their aglycons that are found in many dietary fruits and vegetables. Using a chemiluminescent assay, we obtained IC(50) values ranging from 55 microM to > 800 microM for 17 individual compounds. According to earlier data on furanocoumarins from grapefruit extract, CYP2D6 inhibition is achieved in the range of 190-900 nM. As the tested anthocyanins and anthocyanidins were shown to be about 1,000-fold less potent, they are unlikely to interfere with drug metabolism by CYP2D6. Further studies are warranted to examine the effects of the above flavonoids on other CYP isoforms for more detailed toxicity profiles.

Inhibition of proteasome activity by anthocyanins and anthocyanidins.

Recent reports have demonstrated multiple benefits associated with the consumption of berry fruits, including a decreased vulnerability to oxidative stress, reduced ischemic brain damage, protection of neurons from stroke-induced damage and the reversal of age-related changes in brain and behaviour. Berry fruits contain high amounts of anthocyanins, which play a major role as free radical scavengers. The present study addresses proteasome inhibition as a further mechanism by which anthocyanins and their aglycons, the anthocyanidins, may exert health-promoting effects. HL-60 cells were incubated with 19 test substances and inhibition of the chymotrypsin-like enzyme activity was determined in a chemiluminescent assay. Anthocyanins and their aglycons achieved IC(50) values ranging from 7.8 microM for kaempferidinidin and pelargonidin, to 32.4 microM for delphinidin. Thus proteasome inhibitory properties of anthocyanins may contribute to their known anticarcinogenic, antioxidative, anti-inflammatory and neuroprotective activities, rationalizing dietary supplementations with anthocyanins in the prevention and treatment of chronic diseases, including neurodegenerative disorders.

Role of soluble factors and three-dimensional culture in in vitro differentiation of intestinal macrophages.

AIM: To examine the factor(s) involved in differentiation of intestinal macrophages (IMACs) using a recently established in vitro model. METHODS: To test whether soluble or membrane bound factors induce IMAC-differentiation, freshly elutriated monocytes (MO) were incubated with conditioned media or cell membranes of intestinal epithelial cells (IEC) or cultured with IEC in transwell systems. To determine the importance of an active migration of MO, three-dimensional aggregates from a 1:1-mixture of MO and IEC were examined by immunohistochemistry and flow cytometry. Apoptosis was examined by caspase-3 Western blots. Extracellular matrix production in differentiation models was compared by immunohistochemistry. RESULTS: IMAC differentiation was observed in a complex three-dimensional co-culture model (multicellular spheroid, MCS) with IEC after migration of MO into the spheroids. By co-culture of MO with conditioned media or membrane preparations of IEC no IMAC differentiation was induced. Co-culture of MO with IEC in transwell-cultures, with the two cell populations separated by a membrane also did not result in intestinal-like differentiation of MO. In contrast to IEC-spheroids with immigrating MO in mixed MCS of IEC and MO only a small subpopulation of MO was able to survive the seven day culture period. CONCLUSION: Intestinal-like differentiation of MO in vitro is only induced in the complex three-dimensional MCS model after immigration of MO indicating a role of cell-matrix and/or cell-cell interactions during the differentiation of IMACs.

Bilberries and their anthocyanins ameliorate experimental colitis.

Bilberries have positive effects in acute and chronic diarrhea. Patients with inflammatory bowel disease (IBD) report on improved symptoms upon ingestion. Bilberries contain approximately 10% of anthocyanins (ACs), which have anti-oxidative, anti-carcinogenic, and anti-inflammatory properties. We investigated whether experimental colitis can be ameliorated by dried bilberries or ACs. Acute and chronic dextrane sodium sulphate (DSS) colitis were induced in Balb/c mice by 2.5% DSS in the drinking water. Mice were fed with dried bilberries or ACs, respectively. Cytokines were determined in supernatants from mesenteric lymph nodes (MLNs) by ELISA and apoptosis was investigated by terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling assays. Oral administration of bilberries during acute DSS-induced colitis ameliorated disease severity and reduced secretion of IFN-γ and tumor necrosis factor from mesenteric lymph node cells. Dried bilberries also improved chronic DSS-colitis. Ingestion of ACs reduced intestinal inflammation in acute and chronic DSS-colitis with decreased histological scores and cytokine secretion. Both bilberries and ACs prevented inflammation-induced apoptosis in colonic epithelial cells. Taken together, ingestion of dried bilberries had positive effects on various parameters especially in acute DSS-colitis. Oral administration of ACs resulted in an amelioration of acute colitis as well as chronic colitis. These promising results justify a clinical study on their therapeutic effect in inflammatory bowel disease patients.