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

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

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

Epigenetic Bases of Aberrant Glycosylation in Cancer

In this review, the sugar portions of glycoproteins, glycolipids, and glycosaminoglycans constitute the glycome, and the genes involved in their biosynthesis, degradation, transport and recognition are referred to as "glycogenes". The extreme complexity of the glycome requires the regulatory layer to be provided by the epigenetic mechanisms. Almost all types of cancers present glycosylation aberrations, giving rise to phenotypic changes and to the expression of tumor markers. In this review, we discuss how cancer-associated alterations of promoter methylation, histone methylation/acetylation, and miRNAs determine glycomic changes associated with the malignant phenotype. Usually, increased promoter methylation and miRNA expression induce glycogene silencing. However, treatment with demethylating agents sometimes results in silencing, rather than in a reactivation of glycogenes, suggesting the involvement of distant methylation-dependent regulatory elements. From a therapeutic perspective aimed at the normalization of the malignant glycome, it appears that miRNA targeting of cancer-deranged glycogenes can be a more specific and promising approach than the use of drugs, which broad target methylation/acetylation. A very specific type of glycosylation, the addition of GlcNAc to serine or threonine (O-GlcNAc), is not only regulated by epigenetic mechanisms, but is an epigenetic modifier of histones and transcription factors. Thus, glycosylation is both under the control of epigenetic mechanisms and is an integral part of the epigenetic code

Selectin Ligands Sialyl-Lewis a and Sialyl-Lewis x in Gastrointestinal Cancers

The tetrasaccharide structures Sia\u3b12,3Gal\u3b21,3(Fuc\u3b11,4)GlcNAc and Sia\u3b12,3Gal\u3b21,4(Fuc\u3b11,3)GlcNAc constitute the epitopes of the carbohydrate antigens sialyl-Lewis a (sLe(a)) and sialyl-Lewis x (sLe(x)), respectively, and are the minimal requirement for selectin binding to their counter-receptors. Interaction of sLe(x) expressed on the cell surface of leucocytes with E-selectin on endothelial cells allows their arrest and promotes their extravasation. Similarly, the rolling of cancer cells ectopically expressing the selectin ligands on endothelial cells is potentially a crucial step favoring the metastatic process. In this review, we focus on the biosynthetic steps giving rise to selectin ligand expression in cell lines and native tissues of gastrointestinal origin, trying to understand whether and how they are deregulated in cancer. We also discuss the use of such molecules in the diagnosis of gastrointestinal cancers, particularly in light of recent data questioning the ability of colon cancers to express sLe(a) and the possible use of circulating sLe(x) in the early detection of pancreatic cancer. Finally, we reviewed the data dealing with the mechanisms that link selectin ligand expression in gastrointestinal cells to cancer malignancy. This promising research field seems to require additional data on native patient tissues to reach more definitive conclusions

Unexpected distribution of CA19.9 and other type 1 chain Lewis antigens in normal and cancer tissues of colon and pancreas: Importance of the detection method and role of glycosyltransferase regulation

Background: CA19.9 antigen has been assumed as an abundant product of cancer cells, due to the reactivity found by immunohistochemical staining of cancer tissues with anti-CA19.9 antibody. Methods: Expression and biosynthesis of type 1 chain Lewis antigens in the colon and the pancreas were studied by immunodetection in tissue sections and lysates, quantification of glycosyltransferase transcripts, bisulfite sequencing, and chromatin immunoprecipitation assays. Results: CA19.9 was poorly detectable in normal colon mucosa and almost undetectable in colon cancer, while it was easily detected in the pancreatic ducts, togetherwith Lewis b antigen, under both normal and cancer conditions. B3GALT5 transcripts were down-regulated in colon cancer, while they remained expressed in pancreatic cancer. Even ST3GAL3 transcript appeared well expressed in the pancreas but poorly in the colon, irrespective of normal or cancer conditions. CpG islands flanking B3GALT5 native promoter presented an extremely low degree of methylation in pancreatic cancer with respect to colon cancer. In a DNA region about 1 kb away fromthe B3GALT5 retroviral promoter, a stretch of CG dinucleotides presented a methylation pattern potentially associated with transcription. Such a DNA region and the transcription factor binding site provided overlapping results by chromatin immunoprecipitation assays, corroborating the hypothesis. Conclusions: CA19.9 appears as a physiological product whose synthesis strongly depends on the tissue specific and epigenetically-regulated expression of B3GALT5 and ST3GAL3. General significance: CA19.9 and other Lewis antigens acquire tumor marker properties in the pancreas due to mechanisms giving rise to reabsorption into vessels and elevation in circulating levels.Background CA19.9 antigen has been assumed as an abundant product of cancer cells, due to the reactivity found by immunohistochemical staining of cancer tissues with anti-CA19.9 antibody. Methods Expression and biosynthesis of type 1 chain Lewis antigens in the colon and the pancreas were studied by immunodetection in tissue sections and lysates, quantification of glycosyltransferase transcripts, bisulfite sequencing, and chromatin immunoprecipitation assays. Results CA19.9 was poorly detectable in normal colon mucosa and almost undetectable in colon cancer, while it was easily detected in the pancreatic ducts, together with Lewis b antigen, under both normal and cancer conditions. B3GALT5 transcripts were down-regulated in colon cancer, while they remained expressed in pancreatic cancer. Even ST3GAL3 transcript appeared well expressed in the pancreas but poorly in the colon, irrespective of normal or cancer conditions. CpG islands flanking B3GALT5 native promoter presented an extremely low degree of methylation in pancreatic cancer with respect to colon cancer. In a DNA region about 1\ua0kb away from the B3GALT5 retroviral promoter, a stretch of CG dinucleotides presented a methylation pattern potentially associated with transcription. Such a DNA region and the transcription factor binding site provided overlapping results by chromatin immunoprecipitation assays, corroborating the hypothesis. Conclusions CA19.9 appears as a physiological product whose synthesis strongly depends on the tissue specific and epigenetically-regulated expression of B3GALT5 and ST3GAL3. General significance CA19.9 and other Lewis antigens acquire tumor marker properties in the pancreas due to mechanisms giving rise to reabsorption into vessels and elevation in circulating levels

Effects of chronic desipramine treatment on alpha(2)-adrenoceptors and mu-opioid receptors in the guinea pig cortex and hippocampus

The existence of a close relation between presynaptic inhibitory alpha(2)-adrenoceptor and mu-opioid receptor pathways is well established. Such interplay may occur during chronic conditions that give rise to neuroadaptive changes involving both receptor systems. The aim of this study was to examine the effect of chronic treatment with the tricyclic antidepressant drug, desipramine, on alpha(2)-adrenoceptors and mu-opioid receptors in the guinea pig brain. Guinea pigs were treated with 10 mg/kg desipramine, injected i.p. for 21 days, every 24 h. The levels of expression of alpha(2)-adrenoceptors and mu-opioid receptors, the G protein receptor regulatory kinase, GRK2/3 and signal transduction inhibitory G proteins in synaptosomes of the guinea pig hippocampus and cortex were evaluated by immunoblotting. Quantitative analysis of alpha(2)-adrenoceptor and mu-opioid receptor mRNA levels has been carried out by competitive reverse transcriptase polymerase chain reaction. The expression levels of alpha(2)-adrenoceptors and mu-opioid receptors and the respective mRNAs were found unchanged in the cortex, after chronic desipramine treatment. In these experimental conditions alpha(2)-adrenoceptor and mu-opioid receptor levels decreased, while the relevant transcripts increased, in the hippocampus. GRK2/3 levels remained unchanged and increased, respectively, in the cortex and the hippocampus, after chronic exposure to desipramine. In the same experimental conditions, Galpha(i1), Galpha(i2), Galpha(o) and Galpha(z) levels remained unchanged, while Galpha(i3) levels decreased, in the cortex; whereas, Galpha(i1), Galpha(i2) and Galpha(i3) levels significantly increased, and Galpha(o) and Galpha(z) levels remained unchanged, in the hippocampus. On the whole, the present data suggest that alpha(2)-adrenoceptor and mu-opioid receptor expression and transcription are similarly influenced by chronic treatment with desipramine, in the guinea pig cortex and hippocampus. Furthermore, alterations in the levels of regulatory GRK2/3 and of inhibitory signal transduction G proteins, relevant to activation of both receptor pathways, have been documented. The distinct pattern of adaptations of the different protein studied in response to chronic desipramine treatment in both regions is discussed