Altered glycosylation profile of purified plasma ACT from Alzheimer’s disease

Background

Alzheimer’s disease (AD) is one of the most frequent cause of neurodegenerative disorder in the elderly. Inflammation has been implicated in brain degenerative processes and peripheral markers of brain AD related impairment would be useful. Plasma levels of alpha-1-antichymotrypsin (ACT), an acute phase protein and a secondary component of amyloid plaques, are often increased in AD patients and high blood ACT levels correlate with progressive cognitive deterioration. During inflammatory responses changes in the micro-heterogeneity of ACT sugar chains have been described.

Methods

N-Glycanase digestion from Flavobacterium meningosepticum (PNGase F) was performed on both native and denatured purified ACT condition and resolved to Western blot with the purpose to revealed the ACT de-glycosylation pattern.Further characterization of the ACT glycan profile was obtained by a glycoarray; each lectin group in the assay specifically recognizes one or two glycans/epitopes. Lectin-bound ACT produced a glyco-fingerprint and mayor differences between AD and controls samples were assessed by a specific algorithms.

Results

Western blot analysis of purified ACT after PNGase F treatment and analysis of sugar composition of ACT showed significantly difference in “glyco-fingerprints” patterns from controls (CTR) and AD; ACT from AD showing significantly reduced levels of sialic acid. A difference in terminal GlcNac residues appeared to be related with progressive cognitive deterioration.

Conclusions

Low content of terminal GlcNac and sialic acid in peripheral ACT in AD patients suggests that a different pattern of glycosylation might be a marker of brain inflammation. Moreover ACT glycosylation analysis could be used to predict AD clinical progression and used in clinical trials as surrogate marker of clinical efficacy.

     

Cell entry and cAMP imaging of anthrax edema toxin

The entry and enzymatic activity of the anthrax edema factor (EF) in different cell types was studied by monitoring EF-induced changes in intracellular cAMP with biochemical and microscopic methods. cAMP was imaged in live cells, transfected with a fluorescence resonance energy transfer biosensor based on the protein kinase A regulatory and catalytic subunits fused to CFP and YFP, respectively. The cAMP biosensor was located either in the cytosol or was membrane-bound owing to the addition of a tag determining its myristoylation/palmitoylation. Real-time imaging of cells expressing the cAMP biosensors provided the time course of EF catalytic activity and an indication of its subcellular localization. Bafilomycin A1, an inhibitor of the vacuolar ATPase proton pump, completely prevented EF activity, even when added long after the toxin. The time course of appearance of the adenylate cyclase activity and of bafilomycin A1 action suggests that EF enters the cytosol from late endosomes. EF remains associated to these compartments and its activity shows a perinuclear localization generating intracellular cAMP concentration gradients from the cell centre to the periphery.     

Infant hybrids in a newly formed mixed-species group of howler monkeys (Alouatta guariba clamitans and Alouatta caraya) in northeastern Argentina

Natural hybridisation between species has been reported in several primate taxa. In the Neotropics, there is increasing evidence of this phenomenon in howler monkeys (genus Alouatta) in contact zones between species. We describe the first known case of formation of a mixed-species group, and two cases of putative infant hybrids between the brown howler (Alouatta guariba clamitans) and the black howler (A. caraya) in Misiones, Argentina. For 2 years, we followed a group consisting of one adult male and two adult female brown howlers and one adult female black howler. The adult female black howler was observed to copulate twice with brown howler males, and never with black howler males. In December 2006, this female was carrying an infant with a hybrid morphotype. This infant died at approximately 1.5 months of age. In November 2007, the same female had another putative hybrid newborn. This infant male died together with all members of his group during a yellow fever outbreak in early 2008. The lower frequency of mixed-species groups and hybrids at our site compared with other contact zones reported in the literature, suggests that the incidence of natural hybridisation between howler species differs depending on local factors such as population demography and landscape fragmentation.     

The Tm7sf2 Gene Deficiency Protects Mice against Endotoxin-Induced Acute Kidney Injury

Cholesterol is essential for diverse cellular functions and cellular and whole-body cholesterol homeostasis is highly controlled. Cholesterol can also influence cellular susceptibility to injury. The connection between cholesterol metabolism and inflammation is exemplified by the Tm7sf2 gene, the absence of which reveals an essential role in cholesterol biosynthesis under stress conditions but also results in an inflammatory phenotype, i.e. NF-κB activation and TNFα up-regulation. Here, by using Tm7sf2^+/+and Tm7sf2^−/− mice, we investigated whether the Tm7sf2 gene, through its role in cholesterol biosynthesis under stress conditions, is involved in the renal failure induced by the administration of LPS. We found that the loss of Tm7sf2 gene results in significantly reduced blood urea nitrogen levels accompanied by decreased renal inflammatory response and neutral lipid accumulation. The increased expression of fatty acids catabolic enzymes reduces the need of the renal autophagy, a known crucial nutrient-sensing pathway in lipid metabolism. Moreover, we observed that the Tm7sf2 insufficiency is responsible for the inhibition of the NF-κB signalling thus dampening the inflammatory response and leading to a reduced renal damage. These results suggest a pivotal role for Tm7sf2 in renal inflammatory and lipotoxic response under endotoxemic conditions.     

A novel ubiquitin-specific protease, UBP43, cloned from leukemia fusion protein AML1-ETO-expressing mice, functions in hematopoietic cell differentiation

Using PCR-coupled subtractive screening-representational difference analysis, we have cloned a novel gene from AML1-ETO knockin mice. This gene is highly expressed in the yolk sac and fetal liver of the knockin mice. Nucleotide sequence analysis indicates that its cDNA contains an 1,107-bp open reading frame encoding a 368-amino-acid polypeptide. Further protein sequence and protein translation analysis shows that it belongs to a family of ubiquitin-specific proteases (UBP), and its molecular mass is 43 kDa. Therefore, we have named this gene UBP43. Like other ubiquitin proteases, the UBP43 protein has deubiquitinating enzyme activity. Protein ubiquitination has been implicated in many important cellular events. In wild-type adult mice, UBP43 is highly expressed in the thymus and in peritoneal macrophages. Among nine different murine hematopoietic cell lines analyzed, UBP43 expression is detectable only in cell lines related to the monocytic lineage. Furthermore, its expression is regulated during cytokine-induced monocytic cell differentiation. We have investigated its function in the hematopoietic myeloid cell line M1. UBP43 was introduced into M1 cells by retroviral gene transfer, and several high-expressing UBP43 clones were obtained for further study. Morphologic and cell surface marker examination of UBP43/M1 cells reveals that overexpression of UBP43 blocks cytokine-induced terminal differentiation of monocytic cells. These data suggest that UBP43 plays an important role in hematopoiesis by modulating either the ubiquitin-dependent proteolytic pathway or the ubiquitination state of another regulatory factor(s) during myeloid cell differentiation.     

HDAC modulation and cell death in the clinic

Histone acetyltransferases (HATs) and histone deacetylases (HDACs) are two opposing classes of enzymes, which finely regulate the balance of histone acetylation affecting chromatin packaging and gene expression. Imbalanced acetylation has been associated with carcinogenesis and cancer progression. In contrast to genetic mutations, epigenetic changes are potentially reversible. This implies that epigenetic alterations are amenable to pharmacological interventions. Accordingly, some epigenetic-based drugs (epidrugs) have been approved by the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for cancer treatment. Here, we focus on the biological features of HDAC inhibitors (HDACis), analyzing the mechanism(s) of action and their current use in clinical practice.     

HDAC Inhibitors Repress BARD1 Isoform Expression in Acute Myeloid Leukemia Cells via Activation of miR-19a and/or b

Over the past years BARD1 (BRCA1-associated RING domain 1) has been considered as both a BRCA1 (BReast Cancer susceptibility gene 1, early onset) interactor and tumor suppressor gene mutated in breast and ovarian cancers. Despite its role as a stable heterodimer with BRCA1, increasing evidence indicates that BARD1 also has BRCA1-independent oncogenic functions. Here, we investigate BARD1 expression and function in human acute myeloid leukemias and its modulation by epigenetic mechanism(s) and microRNAs. We show that the HDACi (histone deacetylase inhibitor) Vorinostat reduces BARD1 mRNA levels by increasing miR-19a and miR-19b expression levels. Moreover, we identify a specific BARD1 isoform, which might act as tumor diagnostic and prognostic markers.     

A single amino acid residue in bank vole prion protein drives permissiveness to Nor98/atypical scrapie and the emergence of multiple strain variants

Prions are infectious agents that replicate through the autocatalytic misfolding of the cellular prion protein (PrP^C) into infectious aggregates (PrP^Sc) causing fatal neurodegenerative diseases in humans and animals. Prions exist as strains, which are encoded by conformational variants of PrP^Sc. The transmissibility of prions depends on the PrP^C sequence of the recipient host and on the incoming prion strain, so that some animal prion strains are more contagious than others or are transmissible to new species, including humans. Nor98/atypical scrapie (AS) is a prion disease of sheep and goats reported in several countries worldwide. At variance with classical scrapie (CS), AS is considered poorly contagious and is supposed to be spontaneous in origin. The zoonotic potential of AS, its strain variability and the relationships with the more contagious CS strains remain largely unknown. We characterized AS isolates from sheep and goats by transmission in ovinised transgenic mice (tg338) and in two genetic lines of bank voles, carrying either methionine (BvM) or isoleucine (BvI) at PrP residue 109. All AS isolates induced the same pathological phenotype in tg338 mice, thus proving that they encoded the same strain, irrespective of their geographical origin or source species. In bank voles, we found that the M109I polymorphism dictates the susceptibility to AS. BvI were susceptible and faithfully reproduced the AS strain, while the transmission in BvM was highly inefficient and was characterized by a conformational change towards a CS-like prion strain. Sub-passaging experiments revealed that the main strain component of AS is accompanied by minor CS-like strain components, which can be positively selected during replication in both AS-resistant or AS-susceptible animals. These findings add new clues for a better comprehension of strain selection dynamics in prion infections and have wider implications for understanding the origin of contagious prion strains, such as CS.