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Abnormal amyloid-β (Aβ) production and deposition is believed to represent one of the main causes of Alzheimer's disease (AD). γ-Secretase is the enzymatic complex responsible for Aβ generation from its precursor protein. Inhibition or modulation of γ-secretase represents an attractive therapeutic approach. CHF5074 is a new γ-secretase modulator that has been shown to inhibit brain plaque deposition and to attenuate memory deficit in adult AD transgenic mice after chronic treatment. To date, it is not known whether the positive behavioral effects of this compound also occur in young transgenic mice without plaque deposition. Here, we evaluated the effects of acute and subchronic treatment with CHF5074 on contextual and recognition memory and on hippocampal synaptic plasticity in plaque-free Tg2576 mice. We found that at 5 months of age, contextual memory impairment was significantly attenuated after acute subcutaneous administration of 30 mg/kg CHF5074. At 6 months of age, recognition memory impairment was fully reversed after a 4-week oral treatment in the diet (≈60 mg/kg/day). These cognitive effects were associated with a reversal of long-term potentiation (LTP) impairment in the hippocampus. A significant reduction in brain intraneuronal AβPP/Aβ levels and hyperphosphorylated tau, but no change in soluble or oligomeric Aβ levels was detected in Tg2576 mice showing functional recovery following CHF5074 treatment. We conclude that the beneficial effects of CHF5074 treatment in young transgenic mice occurred at a stage that precedes plaque formation and were associated with a reduction in intraneuronal AβPP/Aβ and hyperphosphorylated tau.

We analyzed the results of periodic chromosome analyses performed on bone marrow of 22 patients with Shwachman-Diamond syndrome (SDS), 8 directly observed and 14 from the literature, selected because of changes in the cytogenetic picture during the course of the disease. This study points out some features of the cytogenetic evolution in SDS relevant for prognostic evaluation but never noted in the literature. In particular, the lack of any clonal progression and the frequent appearance of independent clones with chromosomal changes different from the one initially discovered, with possible severe prognostic implications, are reported.

Inositol-phosphorylceramide synthase 1 (Ipc1) is a fungal-specific enzyme that regulates the level of two bioactive molecules, phytoceramide and diacylglycerol (DAG). In previous studies, we demonstrated that Ipc1 regulates the expression of the antiphagocytic protein 1 (App1), a novel fungal factor involved in pathogenicity of Cryptococcus neoformans. Here, we investigated the molecular mechanism by which Ipc1 regulates App1. To this end, the APP1 promoter was fused to the firefly luciferase gene in the C. neofor-mans GAL7:IPC1 strain, in which the Ipc1 expression can be modulated, and found that the luciferase activity was indeed regulated when Ipc1 was modulated. Next, using the luciferase reporter assay in both C. neoformans wild-type and GAL7:IPC1 strains, we investigated the role of DAG and sphingolipids in the activation of the APP1 promoter and found that treatment with 1,2-dioctanoylglycerol does increase APP1 transcription, whereas treatment with phytosphingosine or ceramides does not. Two putative consensus sequences were found in the APP1 promoter for ATF and AP-2 transcription factors. Mutagenesis analysis of these sequences revealed that they play a key role in the regulation of APP1 transcription: ATF is an activator, whereas AP-2 in a negative regulator. Finally, we identified a putative Atf2 transcription factor, which is required for APP1 transcription and under the control of Ipc1-DAG pathway. These studies provide novel regulatory mechanisms of the sphingolipid pathway involved in the regulation of gene transcription of C. neoformans.

We investigated the role of β3Gal-T5, a member of the β1,3galactosyltransferase (β1,3Gal-T) family, in cancer-associated glycosylation, focusing on the expression of sialyl-Lewis a (sLea, the epitope of CA19.9 antigen), poly N-acetyllactosamines, and sialyl-Lewis x (sLex) antigen. A clone permanently expressing an antisense fragment of β3Gal-T5 was obtained from the human pancreas adenocarcinoma cell line BxPC3 and characterized. Both β1,3Gal-T activity and sLea expression are dramatically impaired in the clone. Analysis of the oligosaccharides synthesized in cells metabolically labelled with tritiated galactose shows that a relevant amount of radioactivity is associated to large O-glycans. Endo-β-galactosidase mostly releases NeuAcα2-3Galβ1-3[Fucα1-4]GlcNAcβ1-3Gal and NeuAcα2-3Galβ1-3GlcNAcβ1-3Gal from such O-glycans of BxPC3 membranes, but GlcNAcβ1-3Gal and type 2 chain oligosaccharides, including NeuAcα2-3Galβ1-4[Fucα1-3]GlcNAcβ1-3Gal, from those of the antisense clone. Furthermore, BxPC3 cells secrete sLea in the culture media but not sLex, while antisense clone secretes mostly sLex, and accumulation of both antigens is prevented by benzyl-α-GalNAc. These data indicate that β3Gal-T5 suppression turns synthesis of type 1 chain O-glycans to poly N-acetyllactosamine elongation and termination by sLex. In other cell lines and clones, β3Gal-T5 transcript, β1,3Gal-T activity, and sLea antigen are also correlated, but quantitatively the relative expression ratios are very different from cell type to cell type. We suggest that β3Gal-T5 plays a relevant role in gastrointestinal and pancreatic tissues counteracting the glycosylation pattern associated to malignancy, and is necessary for the synthesis and secretion of CA19.9 antigen, whose expression still depends on multiple interacting factors.

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 (NeuAcα2-3Galβ1-3[Fucα1-4]GlcNAc) sialyl-Lewis a studying the biosynthesis, expression, and secretion in colon cancers and related cancer cell lines. We found that the β1,3 galactosyltransferase β3Gal-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 β3Gal-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.

Background Chromosome changes in the bone marrow (BM) of patients with persistent cytopenia are often considered diagnostic for a myelodysplastic syndrome (MDS). Comprehensive cytogenetic evaluations may give evidence of the real pathogenetic role of these changes in cases with cytopenia without morphological signs of MDS. Results Chromosome anomalies were found in the BM of three patients, without any morphological evidence of MDS: 1) an acquired complex rearrangement of chromosome 21 in a boy with severe aplastic anaemia (SAA); the rearrangement caused the loss of exons 2–8 of the RUNX1 gene with subsequent hypoexpression. 2) a constitutional complex rearrangement of chromosome 21 in a girl with congenital thrombocytopenia; the rearrangement led to RUNX1 disruption and hypoexpression. 3) an acquired paracentric inversion of chromosome 1, in which two regions at the breakpoints were shown to be lost, in a boy with aplastic anaemia; the MPL gene, localized in chromosome 1 short arms was not mutated neither disrupted, but its expression was severely reduced: we postulate that the aplastic anaemia was due to position effects acting both in cis and in trans, and causing Congenital Amegakaryocytic Thrombocytopenia (CAMT). Conclusions A clonal anomaly in BM does not imply per se a diagnosis of MDS: a subgroup of BM hypoplastic disorders is directly due to chromosome structural anomalies with effects on specific genes, as was the case of RUNX1 and MPL in the patients here reported with diagnosis of SAA, thrombocytopenia, and CAMT. The anomaly may be either acquired or constitutional, and it may act by deletion/disruption of the gene, or by position effects. Full cytogenetic investigations, including a-CGH, should always be part of the diagnostic evaluation of patients with BM aplasia/hypoplasia and peripheral cytopenias.

Beta1,3-galactosyltransferase beta3Gal-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 beta3Gal-T5 mRNA, types A, B, and C, and found widespread 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 uniquebeta3Gal-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.

We investigated the role of beta 3 Gal-T5, a member of the beta 1,3galactosyltransferase (beta 1,3Gal-T) family, in cancer-associated glycosylation, focusing on the expression of sialyl-Lewis a (sLea, the epitope of CA19.9 antigen), poly N-acetyllactosamines, and sialyl-Lewis x (sLex) antigen. A clone permanently expressing an antisense fragment of beta 3Gal-T5 was obtained from the human pancreas adenocarcinoma cell line BxPC3 and characterized. Both beta 1,3Gal-T activity and sLea expression are dramatically impaired in the clone. Analysis of the oligosaccharides synthesized in cells metabolically labelled with tritiated galactose shows that a relevant amount of radioactivity is associated to large O-glycans. Endo-beta-galactosidase mostly releases NeuAc alpha 2-3Gal beta 1-3[Fuc alpha 1-4]GlcNAc beta 1-3Gal and NeuAc alpha 2-3Gal beta 1-3GlcNAc beta 1-3Gal from such O-glycans of BxPC3 membranes, but GlcNAc beta 1-3Gal and type 2 chain oligosaccharides, including NeuAc alpha 2-3Gal beta 1-4[Fuc alpha 1-3]GlcNAc beta 1-3Gal, from those of the antisense clone. Furthermore, BxPC3 cells secrete sLea in the culture media but not sLex, while antisense clone secretes mostly sLex, and accumulation of both antigens is prevented by benzyl-alpha-GalNAc. These data indicate that beta 3Gal-T5 suppression turns synthesis of type 1 chain O-glycans to poly N-acetyllactosamine elongation and termination by sLex. In other cell lines and clones, beta 3Gal-T5 transcript, beta 1,3Gal-T activity, and sLea antigen are also correlated, but quantitatively the relative expression ratios are very different from cell type to cell type. We suggest that beta 3Gal-T5 plays a relevant role in gastrointestinal and pancreatic tissues counteracting the glycosylation pattern associated to malignancy, and is necessary for the synthesis and secretion of CA19.9 antigen, whose expression still depends on multiple interacting factors.

Introduction The thrombocytopenia of the Paris-Trousseau (TCPT) type is a contiguous gene syndrome characterized by mild bleeding tendency, variable thrombocytopenia (THC), abnormal giant alpha-granules in platelets and dysmegakaryopoiesis: it derives from a constitutional deletion of chromosome 11 leading to the loss of FLI1, a transcription factor involved in megakaryocyte differentiation and maturation. Case report A women with an acquired, isolated THC developing over 10 yr showed morphological features typical of TCPT in platelets and bone marrow (BM). Twenty years after the onset of THC, the other hematological parameters are still normal and the patient is well. Results Clonal hemopoiesis was shown and chromosome analyses performed on BM revealed a clone with 45 chromosomes and a complex unbalanced translocation involving chromosomes 2, 3, and 11. The anomaly was present in the majority of bone marrow cells but only in a few peripheral blood elements. A microarray-based comparative genomic hybridization defined the deleted region of chromosome 11 including the FLI1 locus that was missing. Conclusion Although our patient presented with nearly all the characteristics of TCPT, her illness was acquired instead of being inherited and the most appropriate diagnosis is that of the unilineage dysplasia ‘refractory THC.' This observation suggests that appropriate cytogenetic investigations should be always considered in patients with acquired THC of unknown origin.

Background An interstitial deletion of the long arms of chromosome 20, del(20)(q), is frequent in the bone marrow (BM) of patients with myelodysplastic syndromes (MDS), acute myeloid leukemia (AML), and myeloproliferative neoplasms (MPN), and it is recurrent in the BM of patients with Shwachman-Diamond syndrome (SDS), who have a 30-40% risk of developing MDS and AML. Results We report the results obtained by microarray-based comparative genomic hybridization (a-CGH) in six patients with SDS, and we compare the loss of chromosome 20 material with one patient with MDS, and with data on 92 informative patients with MDS/AML/MPN and del(20)(q) collected from the literature. Conclusions The chromosome material lost in MDS/AML/MPN is highly variable with no identifiable common deleted regions, whereas in SDS the loss is more uniform: in 3/6 patients it was almost identical, and the breakpoints that we defined are probably common to most patients from the literature. In some SDS patients less material may be lost, due to different distal breakpoints, but the proximal breakpoint is in the same region, always leading to the loss of the EIF6 gene, an event which was related to a lower risk of MDS/AML in comparison with other patients.