A MADS domain gene involved in the transition to flowering in Arabidopsis

Flowering time in many plants is triggered by environmental factors that lead to uniform flowering in plant populations, ensuring higher reproductive success. So far, several genes have been identified that are involved in flowering time control. AGL20 (AGAMOUS LIKE 20) is a MADS domain gene from Arabidopsis that is activated in shoot apical meristems during the transition to flowering. By transposon tagging we have identified late flowering agl20 mutants, showing that AGL20 is involved in flowering time control. In previously described late flowering mutants of the long-day and constitutive pathways of floral induction the expression of AGL20 is down-regulated, demonstrating that AGL20 acts downstream to the mutated genes. Moreover, we can show that AGL20 is also regulated by the gibberellin (GA) pathway, indicating that AGL20 integrates signals of different pathways of floral induction and might be a central component for the induction of flowering. In addition, the constitutive expression of AGL20 in Arabidopsis is sufficient for photoperiod independent flowering and the over-expression of the orthologous gene from mustard, MADSA, in the classical short-day tobacco Maryland Mammoth bypasses the strict photoperiodic control of flowering.     

Two-electron electrochemical oxidation of quercetin and kaempferol changes only the flavonoid C-ring

Bulk electrolysis of the antioxidant flavonoids quercetin and kaempferol in acetonitrile both yield a single oxidation product in two-electron processes. The oxidation products are more polar than their parent compounds, with an increased molecular weight of 16 g/mol, and were identified as 2-(3,4-dihydroxybenzoyl)-2,4,6-trihydroxy-3(2H)-benzofuranone and 2-(4-hydroxybenzoyl)-2,4,6-trihydroxy-3(2H)-benzofuranone for quercetin and kaempferol, respectively. Two-electron oxidation of the parent flavonoid is suggested to yield a 3,4-flavandione with unchanged substitution pattern in the A- and B-ring, which may rearrange to form the substituted 3(2H)-benzofuranone through the chalcan-trione ring-chain tautomer. The acidity of the 3-OH group is suggested to determine the fate of the flavonoid phenoxyl radical, originally formed by one-electron oxidation, as no well-defined oxidation product of luteolin (lacking the 3-OH group) could be isolated despite rather similar half-peak potentials: E_P/2 = 0.97 V, 0.98 V and 1.17 V vs. NHE for quercetin, kaempferol and luteolin, respectively, as measured by cyclic voltammetry in acetonitrile.     

Automated measurement of site‐specific N‐glycosylation occupancy with SWATH‐MS

Asparagine‐linked glycosylation is a common post‐translational modification of proteins catalyzed by oligosaccharyltransferase that is important in regulating many aspects of protein function. Analysis of protein glycosylation, including glycoproteomic measurement of the site‐specific extent of glycosylation, remains challenging. Here, we developed methods combining enzymatic deglycosylation and protease digestion with SWATH‐MS to enable automated measurement of site‐specific occupancy at many glycosylation sites. Deglycosylation with peptide‐endoglycosidase H, leaving a remnant N‐acetylglucosamine on asparagines previously carrying high‐mannose glycans, followed by trypsin digestion allowed robust automated measurement of occupancy at many sites. Combining deglycosylation with the more general peptide‐N‐glycosidase F enzyme with AspN protease digest allowed robust automated differentiation of nonglycosylated and deglycosylated forms of a given glycosylation site. Ratiometric analysis of deglycosylated peptides and the total intensities of all peptides from the corresponding proteins allowed relative quantification of site‐specific glycosylation occupancy between yeast strains with various isoforms of oligosaccharyltransferase. This approach also allowed robust measurement of glycosylation sites in human salivary glycoproteins. This method for automated relative quantification of site‐specific glycosylation occupancy will be a useful tool for research with model systems and clinical samples.     

The Breast Cancer-Associated Glycoforms of MUC1, MUC1-Tn and sialyl-Tn,Are Expressed in COSMC Wild-Type Cells and Bind the C-Type Lectin MGL

Aberrant glycosylation occurs in the majority of human cancers and changes in mucin-type O-glycosylation are key events that play a role in the induction of invasion and metastases. These changes generate novel cancer-specific glyco-antigens that can interact with cells of the immune system through carbohydrate binding lectins. Two glyco-epitopes that are found expressed by many carcinomas are Tn (GalNAc-Ser/Thr) and STn (NeuAcα2,6GalNAc-Ser/Thr). These glycans can be carried on many mucin-type glycoproteins including MUC1. We show that the majority of breast cancers carry Tn within the same cell and in close proximity to extended glycan T (Galβ1,3GalNAc) the addition of Gal to the GalNAc being catalysed by the T synthase. The presence of active T synthase suggests that loss of the private chaperone for T synthase, COSMC, does not explain the expression of Tn and STn in breast cancer cells. We show that MUC1 carrying both Tn or STn can bind to the C-type lectin MGL and using atomic force microscopy show that they bind to MGL with a similar deadadhesion force. Tumour associated STn is associated with poor prognosis and resistance to chemotherapy in breast carcinomas, inhibition of DC maturation, DC apoptosis and inhibition of NK activity. As engagement of MGL in the absence of TLR triggering may lead to anergy, the binding of MUC1-STn to MGL may be in part responsible for some of the characteristics of STn expressing tumours.     

Interactions between meat intake and genetic variation in relation to colorectal cancer

Meat intake is associated with the risk of colorectal cancer. The objective of this systematic review was to evaluate interactions between meat intake and genetic variation in order to identify biological pathways involved in meat carcinogenesis. We performed a literature search of PubMed and Embase using “interaction”, “meat”, “polymorphisms”, and “colorectal cancer”, and data on meat–gene interactions were extracted. The studies were divided according to whether information on meat intake was collected prospectively or retrospectively. In prospective studies, interactions between meat intake and polymorphisms in PTGS2 (encoding COX-2), ABCB1, IL10, NFKB1, MSH3, XPC (P_int = 0.006, 0.01, 0.04, 0.03, 0.002, 0.01, respectively), but not IL1B, HMOX1, ABCC2, ABCG2, NR1I2 (encoding PXR), NR1H2 (encoding LXR), NAT1, NAT2, MSH6, or MLH1 in relation to CRC were found. Interaction between a polymorphism in XPC and meat was found in one prospective and one case–control study; however, the directions of the risk estimates were opposite. Thus, none of the findings were replicated. The results from this systematic review suggest that genetic variation in the inflammatory response and DNA repair pathway is involved in meat-related colorectal carcinogenesis, whereas no support for the involvement of heme and iron from meat or cooking mutagens was found. Further studies assessing interactions between meat intake and genetic variation in relation to CRC in large well-characterised prospective cohorts with relevant meat exposure are warranted.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-014-0448-9) contains supplementary material, which is available to authorized users.     

Overexpression of heat shock protein 27 (HSP27) increases gemcitabine sensitivity in pancreatic cancer cells through S‐phase arrest and apoptosis

We previously established a role for HSP27 as a predictive marker for therapeutic response towards gemcitabine in pancreatic cancer. Here, we investigate the underlying mechanisms of HSP27‐mediated gemcitabine sensitivity. Utilizing a pancreatic cancer cell model with stable HSP27 overexpression, cell cycle arrest and apoptosis induction were analysed by flow cytometry, nuclear staining, immunoblotting and mitochondrial staining. Drug sensitivity studies were performed by proliferation assays. Hyperthermia was simulated using mild heat shock at 41.8°C. Upon gemcitabine treatment, HSP27‐overexpressing cells displayed an early S‐phase arrest subsequently followed by a strongly increased sub‐G1 fraction. Apoptosis was characterized by PARP‐, CASPASE 3‐, CASPASE 8‐, CASPASE 9‐ and BIM‐ activation along with a mitochondrial membrane potential loss. It was reversible through chemical caspase inhibition. Importantly, gemcitabine sensitivity and PARP cleavage were also elicited by heat shock‐induced HSP27 overexpression, although to a smaller extent, in a panel of pancreatic cancer cell lines. Finally, HSP27‐overexpressing pancreatic cancer cells displayed an increased sensitivity also towards death receptor‐targeting agents, suggesting another pro‐apoptotic role of HSP27 along the extrinsic apoptosis pathway. Taken together, in contrast to the well‐established anti‐apoptotic properties of HSP27 in cancer, our study reveals novel pro‐apoptotic functions of HSP27—mediated through both the intrinsic and the extrinsic apoptotic pathways—at least in pancreatic cancer cells. HSP27 could represent a predictive marker of therapeutic response towards specific drug classes in pancreatic cancer and provides a novel molecular rationale for current clinical trials applying the combination of gemcitabine with regional hyperthermia in pancreatic cancer patients.     

High ABCC2 and Low ABCG2 Gene Expression Are Early Events in the Colorectal Adenoma-Carcinoma Sequence

Development of colorectal cancer (CRC) may result from a dysfunctional interplay between diet, gut microbes and the immune system. The ABC transport proteins ABCB1 (P-glycoprotein, Multidrug resistance protein 1, MDR1), ABCC2 (MRP2) and ABCG2 (BCRP) are involved in transport of various compounds across the epithelial barrier. Low mRNA level of ABCB1 has previously been identified as an early event in colorectal carcinogenesis (Andersen et al., PLoS One. 2013 Aug 19;8(8):e72119). ABCC2 and ABCG2 mRNA levels were assessed in intestinal tissue from 122 CRC cases, 106 adenoma cases (12 with severe dysplasia, 94 with mild-moderate dysplasia) and from 18 controls with normal endoscopy.We found significantly higher level of ABCC2 in adenomas with mild to moderate dysplasia and carcinoma tissue compared to the levels in unaffected tissue from the same individual (P = 0.037, P = 0.037, and P<0.0001) and in carcinoma and distant unaffected tissue from CRC cases compared to the level in the healthy individuals (P = 0.0046 and P = 0.036). Furthermore, ABCG2 mRNA levels were significantly lower in adenomas and carcinomas compared to the level in unaffected tissue from the same individuals and compared to tissue from healthy individuals (P<0.0001 for all). The level of ABCB2 in adjacent normal tissue was significantly higher than in tissue from healthy individuals (P = 0.011).In conclusion, this study found that ABCC2 and ABCG2 expression levels were altered already in mild/moderate dysplasia in carcinogenesis suggesting that these ABC transporters are involved in the early steps of carcinogenesis as previously reported for ABCB1. These results suggest that dysfunctional transport across the epithelial barrier may contribute to colorectal carcinogenesis.