Aberrant E-cadherin, beta-catenin, and glial fibrillary acidic protein (GFAP) expression in canine choroid plexus tumors

Expression of E-cadherin and beta-catenin has been widely studied in various human and canine epithelial tumors and has been correlated with dedifferentiation, invasiveness, and metastasis. Choroid plexus tumors (CPTs) are of epithelial origin, and the most important prognostic factor in human medicine is the tumor grade. Limited information is available regarding E-cadherin and beta-catenin expression in human CPTs, and no information is found in the veterinary literature. In the current study, 42 canine CPTs (19 choroid plexus papillomas and 23 choroid plexus carcinomas) were retrospectively reviewed, and the intensity and cellular staining pattern of E-cadherin and beta-catenin were correlated with histological features, paying special attention to grade, invasion, and metastasis. In addition, cytokeratin and glial fibrillary acidic protein (GFAP) antibodies were evaluated as markers for canine CPTs. It was found that loss of E-cadherin and beta-catenin expression was uncommon in canine CPTs. Rather, membranous expression of both molecules was increased in CPTs compared to normal choroid plexus (NCP), regardless of tumor grade. Additionally, aberrant cytoplasmic or nuclear expression of both E-cadherin and beta-catenin was often observed in CPTs. GFAP was frequently expressed in CPTs in contrast to NCP. None of these parameters were correlated with malignancy, and therefore, do not appear to be useful for prognostic information. Nevertheless, a panel of antibodies including E-cadherin and GFAP might be useful to support the diagnosis of CPTs and help to differentiate them from other tumors, such as ependymomas and metastatic epithelial tumors.

Glutathione peroxidase overexpression causes aberrant ERK activation in neonatal mouse cortex after hypoxic preconditioning

Preconditioning of neonatal mice with nonlethal hypoxia (HPC) protects the brain from hypoxic-ischemic (HI) injury. Overexpression of human glutathione peroxidase 1 (GPx1), which normally protects the developing murine brain from HI injury, reverses HPC protection, suggesting that a certain threshold of hydrogen peroxide concentration is required for activation of HPC signaling.

Aberrant low expression level of bovine beta-lactoglobulin is associated with a C to A transversion in the BLG promoter region

Beta-lactoglobulin (beta-LG) is the major whey protein in cow's milk. It is well established that the predominant 2 genetic variants, beta-LG A and B, are differentially expressed. Extensive investigation of the genetic variation in the promoter region of the BLG gene revealed the existence of specific haplotypes associated with the A and B variants, respectively. However, the genetic basis for the differential expression of BLG A and B alleles is still elusive. We have previously reported a quantitative beta-LG B variant, characterized by a very low beta-LG protein expression level. Here, we report that the corresponding BLG allele (BLG B*) shows a correspondingly low mRNA expression level. Comparative DNA sequencing of 7,670 bp of the BLG B* allele and the established BLG B allele revealed a unique difference of a C to A transversion at position 215 bp upstream of the translation initiation site (g.-215C>A). This mutation segregated perfectly with the differential phenotypic expression in a paternal half-sib family and could be confirmed in 2 independent cases. The sequence of the BLG B allele in the region of the mutation is highly conserved among 4 related ruminant species. The site of the mutation corresponds to a putative consensus-binding sequence for the transcription factors c-Rel and Elk-1 as predicted by searching the TRANSFAC database. The beta-LG B* site might be relevant in the natural production of milk of low beta-LG content.

Effect of constant and variable domain glycosylation on pharmacokinetics of therapeutic antibodies in mice

Previous studies on the effect of glycosylation on the elimination rate of antibodies have produced conflicting results. Here, we performed pharmacokinetic studies in mice with two preparations of a monoclonal IgG1 antibody enriched for complex type or high mannose type oligosaccharides at the Fc glycosylation site. No significant difference in the serum half-life was found between the two antibody glycoforms, nor was any difference observed in the serum half-lives of different complex type glycoforms. To evaluate the influence of glycosylation within the variable domain, a second monoclonal antibody, glycosylated in both the Fc and Fv domains, was separated into fractions containing different amounts of Fv-associated sialic acid and administered to mice. Again, no significant difference was found in the clearance rates of variants carrying different amounts of Fv-associated sialic acid or lacking Fv-glycosylation. These results suggest that glycosylation has little or no impact on the pharmacokinetic behavior of these two monoclonal antibodies in mice.

Aberrant expression of FGFRL1, a novel FGF receptor, in ovarian tumors

FGFRL1 is a novel member of the FGF receptor family. It is expressed at very low levels in a great variety of cell lines and at relatively high levels in SW1353 chondrosarcoma cells, MG63 osteosarcoma cells and A204 rhabdomyosarcoma cells. Screening of 241 different human tumors with the help of a cancer profiling array suggested major alterations in the relative expression of FGFRL1 in ovarian tumors. Five distinct ovary tumors were therefore analyzed by quantitative and competitive PCR. Several tumors were found to exhibit a significant decrease in the expression of FGFRL1 in the tumor tissue relative to the matched control tissue. One ovarian tumor showed a 25-fold increase in the relative expression. Since FGFRL1 appears to be involved in the control of cell proliferation and differentiation, its aberrant expression might contribute to the development and progression of ovarian tumors.

Glycosylation and pyranose-furanose isomerization of carbohydrates using HClO4-SiO2: Synthesis of oligosaccharides containing galactofuranose

A series of di- and trisaccharides containing galactofuranose were synthesized using HClO4-SiO2 catalyzed glycosylation and ring transformation for the conversion of galactopyranose to galactofuranose. Yields in glycosylations and ring transformation reactions were excellent.

β1- and β3- voltage-gated sodium channel subunits modulate cell surface expression and glycosylation of Nav1.7 in HEK293 cells

Voltage-gated sodium channels (Navs) are glycoproteins composed of a pore-forming α-subunit and associated β-subunits that regulate Nav α-subunit plasma membrane density and biophysical properties. Glycosylation of the Nav α-subunit also directly affects Navs gating. β-subunits and glycosylation thus comodulate Nav α-subunit gating. We hypothesized that β-subunits could directly influence α-subunit glycosylation. Whole-cell patch clamp of HEK293 cells revealed that both β1- and β3-subunits coexpression shifted V ½ of steady-state activation and inactivation and increased Nav1.7-mediated I Na density. Biotinylation of cell surface proteins, combined with the use of deglycosydases, confirmed that Nav1.7 α-subunits exist in multiple glycosylated states. The α-subunit intracellular fraction was found in a core-glycosylated state, migrating at ~250 kDa. At the plasma membrane, in addition to the core-glycosylated form, a fully glycosylated form of Nav1.7 (~280 kDa) was observed. This higher band shifted to an intermediate band (~260 kDa) when β1-subunits were coexpressed, suggesting that the β1-subunit promotes an alternative glycosylated form of Nav1.7. Furthermore, the β1-subunit increased the expression of this alternative glycosylated form and the β3-subunit increased the expression of the core-glycosylated form of Nav1.7. This study describes a novel role for β1- and β3-subunits in the modulation of Nav1.7 α-subunit glycosylation and cell surface expression.

Prion protein glycosylation is not required for strain-specific neurotropism.

In this study, we tested the hypothesis that the glycosylation of the pathogenic isoform of the prion protein (PrP(Sc)) might encode the selective neurotropism of prion strains. We prepared unglycosylated cellular prion protein (PrP(C)) substrate molecules from normal mouse brain by treatment with PNGase F and used reconstituted serial protein cyclic misfolding amplification reactions to produce RML and 301C mouse prions containing unglycosylated PrP(Sc) molecules. Both RML- and 301C-derived prions containing unglycosylated PrP(Sc) molecules were infectious to wild-type mice, and neuropathological analysis showed that mice inoculated with these samples maintained strain-specific patterns of PrP(Sc) deposition and neuronal vacuolation. These results show that PrP(Sc) glycosylation is not necessary for strain-dependent prion neurotropism.

Supplementary motor area (SMA) volume is associated with psychotic aberrant motor behaviour of patients with schizophrenia

We aimed to investigate whether aberrant motor behavior in schizophrenia was associated with structural alterations in the motor system. Whole brain voxel based morphometry of patients with different severity of motor symptoms identified altered gray matter volume in the supplementary motor area (SMA), a key region of the motor system.

Glycosylation of TRPM4 and TRPM5 channels: molecular determinants and functional aspects

The transient receptor potential channel, TRPM4, and its closest homolog, TRPM5, are non-selective cation channels that are activated by an increase in intracellular calcium. They are expressed in many cell types, including neurons and myocytes. Although the electrophysiological and pharmacological properties of these two channels have been previously studied, less is known about their regulation, in particular their post-translational modifications. We, and others, have reported that wild-type (WT) TRPM4 channels expressed in HEK293 cells, migrated on SDS-PAGE gel as doublets, similar to other ion channels and membrane proteins. In the present study, we provide evidence that TRPM4 and TRPM5 are each N-linked glycosylated at a unique residue, Asn(992) and Asn(932), respectively. N-linked glycosylated TRPM4 is also found in native cardiac cells. Biochemical experiments using HEK293 cells over-expressing WT TRPM4/5 or N992Q/N932Q mutants demonstrated that the abolishment of N-linked glycosylation did not alter the number of channels at the plasma membrane. In parallel, electrophysiological experiments demonstrated a decrease in the current density of both mutant channels, as compared to their respective controls, either due to the Asn to Gln mutations themselves or abolition of glycosylation. To discriminate between these possibilities, HEK293 cells expressing TRPM4 WT were treated with tunicamycin, an inhibitor of glycosylation. In contrast to N-glycosylation signal abolishment by mutagenesis, tunicamycin treatment led to an increase in the TRPM4-mediated current. Altogether, these results demonstrate that TRPM4 and TRPM5 are both N-linked glycosylated at a unique site and also suggest that TRPM4/5 glycosylation seems not to be involved in channel trafficking, but mainly in their functional regulation.

A Missense Change in the ATG4D Gene Links Aberrant Autophagy to a Neurodegenerative Vacuolar Storage Disease.

Inherited neurodegenerative disorders are debilitating diseases that occur across different species. We have performed clinical, pathological and genetic studies to characterize a novel canine neurodegenerative disease present in the Lagotto Romagnolo dog breed. Affected dogs suffer from progressive cerebellar ataxia, sometimes accompanied by episodic nystagmus and behavioral changes. Histological examination revealed unique pathological changes, including profound neuronal cytoplasmic vacuolization in the nervous system, as well as spheroid formation and cytoplasmic aggregation of vacuoles in secretory epithelial tissues and mesenchymal cells. Genetic analyses uncovered a missense change, c.1288G>A; p.A430T, in the autophagy-related ATG4D gene on canine chromosome 20 with a highly significant disease association (p = 3.8 x 10-136) in a cohort of more than 2300 Lagotto Romagnolo dogs. ATG4D encodes a poorly characterized cysteine protease belonging to the macroautophagy pathway. Accordingly, our histological analyses indicated altered autophagic flux in affected tissues. The knockdown of the zebrafish homologue atg4da resulted in a widespread developmental disturbance and neurodegeneration in the central nervous system. Our study describes a previously unknown canine neurological disease with particular pathological features and implicates the ATG4D protein as an important autophagy mediator in neuronal homeostasis. The canine phenotype serves as a model to delineate the disease-causing pathological mechanism(s) and ATG4D function, and can also be used to explore treatment options. Furthermore, our results reveal a novel candidate gene for human neurodegeneration and enable the development of a genetic test for veterinary diagnostic and breeding purposes.