Comparative analysis of retroviral and native promoters driving expression of β1,3-galactosyltransferase β3Gal-T5 in human and mouse tissues

β1,3-Galactosyltransferase β3Gal-T5 is highly expressed in the colons of humans and certain primates due to a retroviral long terminal repeat (LTR) acting as a strong promoter. Because this promoter is inactive in other human tissues or mice, we attempted to understand how adoption of a retrotransposon allowed the gene to acquire tissue-specific expression. We identified three novel 5′-UTRs of 3Gal-T5 mRNA, types A, B, and C, and found wide-spread expression of the type A transcript at much lower levels than the LTR transcript, the expression of which is restricted to organs of the gastrointestinal tract. Expression of the type C 5′-UTR transcript was mostly restricted to the ileum, where it was expressed at high levels. We cloned the 5′-flanking regions of both types A and B 5′-UTRs, found deletion constructs functionally active as promoters, and identified CCAAT-binding factor (CBF) and hepatocyte nuclear factor 1 (HNF-1) as the principal nuclear factors controlling the promoters of types A and B 5′-UTR transcripts, respectively. The CCAAT-binding factor binding site and the entire downstream sequence driving the expression of type A transcripts in humans are structurally and functionally conserved in mice, where they constitute a unique β3Gal-T5 promoter that appears to be the ancestral promoter of the gene. The HNF-1 binding motif of the second human promoter is identical to the HNF-1/Cdx binding motif of the LTR promoter but is in the antisense orientation, resulting in much lower binding affinity and promoter strength. These data may explain the successful insertion of the transposon during evolution

Filtrate seaweed extract as biostimulant in nursery organic horticulture

Many naturally-derived products are used as growth promoters or biostimulants on vegetables, even if their mechanisms of action are not still now completely clarified. Among them, seaweeds and seaweed products are inserted as admissible organic fertilizers in the Annex I of the Reg. (EC) No. 889/2008, even if their biostimulant properties are not recognized in organic farming. After a previous bioassay on maize germination for defining the optimal water dilution ratios of the filtrate seaweed extract (FSE), in the present research we applied the FSE at two diluted doses to a greenhouse organic lettuce, for assessing its biostimulant effect on vegetable growth and nutrient uptake. The greenhouse pot trials were conducted on Lactuca sativa L., by applying FSE at 1/250 and 1/500 v/v water dilutions. After 90 days, root fresh/dry weight, shoot fresh/dry weight and dry matter, number of leaves, LAI, specific leaf fresh/dry weight, total dry biomass, leaf macro and micro elements (P, K, Ca, Mg, Fe, Na, Mn, Cu, Zn, B) content and total chlorophyll were determined. Obtained results indicated that FSE plays a role in promoting the increase of lettuce shoot dry weight and related nutrient uptake, with particular efficacy in relation to P and K macroelements. This property is better expressed at the highest dilution, confirming that the biostimulating activity of the formulate acts without any nutritional effect: thus, the FSE appears to be a good opportunity for promoting the early-stage development for organic nursery productio

CA19.9 antigen circulating in the serum of colon cancer patients: Where is it from?

CA19.9 antigen is a glycoprotein present in human serum and found elevated in various diseases. It is intensively studied since long time as a potential marker for managing cancers of the gastrointestinal tract, but its reliability is widely accepted only for pancreatic cancers. Here, we focused on the tetrasaccharide epitope (NeuAc\u3b12-3Gal\u3b21-3[Fuc\u3b11-4]GlcNAc) sialyl-Lewis a studying the biosynthesis, expression, and secretion in colon cancers and related cancer cell lines. We found that the \u3b21,3 galactosyltransferase \u3b23Gal-T5, responsible for sialyl-Lewis a synthesis, is dramatically reduced in colon adenocarcinomas, in terms of both transcript and enzyme activity levels. Moreover, no or very faint antigen is detectable in colon cancer homogenates, by dot-blot or enzyme immunoassay, while it is commonly evident in sera from different patients. In cancer cell lines synthesizing CA19.9, the amount of antigen secreted is proportional to that expressed on the cell surface, and depends on appreciable levels of \u3b23Gal-T5, which appear much higher than those measured in colon cancer specimens. Since colon cancers appear unable to synthesize relevant amount of CA19.9, we suggest that the antigen circulating in the serum of colon cancer patients may have a different and more complex origin than expected so far

Convenient and Sensitive Measurement of Lactosylceramide Synthase Activity Using Deuterated Glucosylceramide and Mass Spectrometry

Lactosylceramide is necessary for the biosynthesis of almost all classes of glycosphingolipids and plays a relevant role in pathways involved in neuroinflammation. It is synthesized by the action of galactosyltransferases B4GALT5 and B4GALT6, which transfer galactose from UDP-galactose to glucosylceramide. Lactosylceramide synthase activity was classically determined in vitro by a method based on the incorporation of radiolabeled galactose followed by the chromatographic separation and quantitation of the product by liquid scintillation counting. Here, we used deuterated glucosylceramide as the acceptor substrate and quantitated the deuterated lactosylceramide product by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). We compared this method with the classical radiochemical method and found that the reactions have similar requirements and provide comparable results in the presence of high synthase activity. Conversely, when the biological source lacked lactosylceramide synthase activity, as in the case of a crude homogenate of human dermal fibroblasts, the radiochemical method failed, while the other provided a reliable measurement. In addition to being very accurate and sensitive, the proposed use of deuterated glucosylceramide and LC-MS/MS for the detection of lactosylceramide synthase in vitro has the relevant advantage of avoiding the costs and discomforts of managing radiochemicals

Dynamics of the Fermentation Process and Chemical Profiling of Pomegranate (Punica granatum L.) Wines Obtained by Different Cultivar×Yeast Combinations

Pomegranate (Punica granatum L.) is one of the historical tree crops in the Mediterranean region and is nowadays commercialized for its beneficial properties in the form of fruits, juice, jams and, in some East countries, as fermented juice (pomegranate wine). However, pomegranate wines are not established as a common beverage in Western countries. In this work, we produced pomegranate wines using two cultivars and two yeasts (Saccharomyces cerevisiae strain Clos and S. cerevisiae ex-bayanus strain EC1118) with contrasting characteristics. A comprehensive chemical profile of the wines was obtained. Notable differences were observed in the function of the cultivars and the yeasts. Different cultivar×yeast combinations provided wines with clearly different chemical profiles and specific features in the patterns of organic acids, phenolics, and volatile compounds. This highlights the opportunity to obtain tailored pomegranate wines with desired chemical profiles and, consequently, sensory properties, through management optimization of pomegranate winemaking. In this view, pomegranate wines have the potential to become an established beverage in Western countries

Assessing the Origin of Phosphonic Acid Residues in Organic Vegetable and Fruit Crops: The Biofosf Project Multi-Actor Approach

Recently, on the EU market, phosphonic acid residues were detected in many organic goods, although fosetyl-derivates and phosphite salts are not allowed by Reg. EC n. 889/2009. The BIOFOSF project "Solving phosphite issue in organic fruit and horticultural crops" aimed at verifying whether the phosphonic acid contamination could be due to unproper use of fertilizers/plant protection products by organic farmers, or to the plant's ability to self-produce it spontaneously. Applying a participative approach, field case-studies on potato, rocket lettuce, and pears were carried out (organic vs. integrated systems). The ethyl-phosphonic acid and phosphonic acid were determined in soil, tubers, leaves, fruits, tree woody organs, used fertilizers, and plant protection products to correlate them to the applied farming management. Tested crops were not able to self-synthetize phosphonic acid, being its detection due to: (i) external inputs not allowed in organic farming; (ii) fertilizers/plant protection products allowed in organic farming, contaminated by fosetyl or phosphite. In addition, it was found that tree crops can stock the phosphite in their woody organs, then translocate it from branches to leaves and fruits over time. Regression models applied to field data showed that fruit trees decontamination could take more than 5 years, depending on the starting value of phosphonic acid contamination, useful to define the phosphite maximum residue limit in organic fruit crops

Analysis of the proximal promoter of the human colon-specific B4GALNT2 (Sda synthase) gene: B4GALNT2 is transcriptionally regulated by ETS1

13siopenBackground: The Sda antigen and corresponding biosynthetic enzyme B4GALNT2 are primarily expressed in normal colonic mucosa and are down-regulated to a variable degree in colon cancer tissues. Although their expression profile is well studied, little is known about the underlying regulatory mechanisms. Methods: To clarify the molecular basis of Sda expression in the human gastrointestinal tract, we investigated the transcriptional regulation of the human B4GALNT2 gene. The proximal promoter region was delineated using luciferase assays and essential trans-acting factors were identified through transient overexpression and silencing of several transcription factors. Results: A short cis-regulatory region restricted to the −72 to +12 area upstream of the B4GALNT2 short-type transcript variant contained the essential promoter activity that drives the expression of the human B4GALNT2 regardless of the cell type. We further showed that B4GALNT2 transcriptional activation mostly requires ETS1 and to a lesser extent SP1. Conclusions: Results presented herein are expected to provide clues to better understand B4GALNT2 regulatory mechanisms.openWavelet-Vermuse C.; Groux-Degroote S.; Vicogne D.; Cogez V.; Venturi G.; Trinchera M.; Brysbaert G.; Krzewinski-Recchi M.-A.; Bachir E.H.; Schulz C.; Vincent A.; Van Seuningen I.; Harduin-Lepers A.Wavelet-Vermuse, C.; Groux-Degroote, S.; Vicogne, D.; Cogez, V.; Venturi, G.; Trinchera, M.; Brysbaert, G.; Krzewinski-Recchi, M. -A.; Bachir, E. H.; Schulz, C.; Vincent, A.; Van Seuningen, I.; Harduin-Lepers, A

Association of kidney disease measures with risk of renal function worsening in patients with type 1 diabetes

Background: Albuminuria has been classically considered a marker of kidney damage progression in diabetic patients and it is routinely assessed to monitor kidney function. However, the role of a mild GFR reduction on the development of stage 653 CKD has been less explored in type 1 diabetes mellitus (T1DM) patients. Aim of the present study was to evaluate the prognostic role of kidney disease measures, namely albuminuria and reduced GFR, on the development of stage 653 CKD in a large cohort of patients affected by T1DM. Methods: A total of 4284 patients affected by T1DM followed-up at 76 diabetes centers participating to the Italian Association of Clinical Diabetologists (Associazione Medici Diabetologi, AMD) initiative constitutes the study population. Urinary albumin excretion (ACR) and estimated GFR (eGFR) were retrieved and analyzed. The incidence of stage 653 CKD (eGFR < 60 mL/min/1.73 m2) or eGFR reduction > 30% from baseline was evaluated. Results: The mean estimated GFR was 98 \ub1 17 mL/min/1.73m2 and the proportion of patients with albuminuria was 15.3% (n = 654) at baseline. About 8% (n = 337) of patients developed one of the two renal endpoints during the 4-year follow-up period. Age, albuminuria (micro or macro) and baseline eGFR < 90 ml/min/m2 were independent risk factors for stage 653 CKD and renal function worsening. When compared to patients with eGFR > 90 ml/min/1.73m2 and normoalbuminuria, those with albuminuria at baseline had a 1.69 greater risk of reaching stage 3 CKD, while patients with mild eGFR reduction (i.e. eGFR between 90 and 60 mL/min/1.73 m2) show a 3.81 greater risk that rose to 8.24 for those patients with albuminuria and mild eGFR reduction at baseline. Conclusions: Albuminuria and eGFR reduction represent independent risk factors for incident stage 653 CKD in T1DM patients. The simultaneous occurrence of reduced eGFR and albuminuria have a synergistic effect on renal function worsening

The Link between Gaucher Disease and Parkinson’s Disease Sheds Light on Old and Novel Disorders of Sphingolipid Metabolism

Sphingolipid metabolism starts with the biosynthesis of ceramide, a bioactive lipid and the backbone for the biosynthesis of complex sphingolipids such as sphingomyelin and glycosphingolipids. These are degraded back to ceramide and then to sphingosine, which enters the ceramide–sphingosine-1-phosphate signaling pathway or is further degraded. Several enzymes with multiple catalytic properties and subcellular localizations are thus involved in such metabolism. Hereditary defects of lysosomal hydrolases have been known for several years to be the cause of lysosomal storage diseases such as gangliosidoses, Gaucher disease, Niemann–Pick disease, Krabbe disease, Fabry disease, and Farber disease. More recently, many other inborn errors of sphingolipid metabolism have been recognized, involving enzymes responsible for the biosynthesis of ceramide, sphingomyelin, and glycosphingolipids. Concurrently, epidemiologic and biochemical evidence has established a link between Gaucher disease and Parkinson’s disease, showing that glucocerebrosidase variants predispose individuals to α-synuclein accumulation and neurodegeneration even in the heterozygous status. This appears to be due not only to lysosomal overload of non-degraded glucosylceramide, but to the derangement of vesicle traffic and autophagy, including mitochondrial autophagy, triggered by both sphingolipid intermediates and misfolded proteins. In this review, old and novel disorders of sphingolipid metabolism, in particular those of ganglioside biosynthesis, are evaluated in light of recent investigations of the link between Gaucher disease and Parkinson’s disease, with the aim of better understanding their pathogenic mechanisms and addressing new potential therapeutic strategies

Epigenetic Regulation of Glycosylation in Cancer and Other Diseases

In the last few decades, the newly emerging field of epigenetic regulation of glycosylation acquired more importance because it is unraveling physiological and pathological mechanisms related to glycan functions. Glycosylation is a complex process in which proteins and lipids are modified by the attachment of monosaccharides. The main actors in this kind of modification are the glycoenzymes, which are translated from glycosylation-related genes (or glycogenes). The expression of glycogenes is regulated by transcription factors and epigenetic mechanisms (mainly DNA methylation, histone acetylation and noncoding RNAs). This review focuses only on these last ones, in relation to cancer and other diseases, such as inflammatory bowel disease and IgA1 nephropathy. In fact, it is clear that a deeper knowledge in the fine-tuning of glycogenes is essential for acquiring new insights in the glycan field, especially if this could be useful for finding novel and personalized therapeutics