A population-based epidemiological study of neuromyelitis optica spectrum disorder in Hungary.

BACKGROUND AND PURPOSE: The goal of this study was to determine the prevalence and incidence of neuromyelitis optica spectrum disorder (NMOSD) in Hungary based on the 2015 International Panel of NMO Diagnosis (IPND) criteria. METHODS: A retrospective population-based cohort study was conducted of 6.4 million Hungarians (age ≥ 16 years) between 1 January 2006 and 31 December 2016. Possible NMOSD patients were selected via multistage re-evaluation from multiple sources. Crude and sex- and serostatus-specific prevalence (per 100 000 persons) and incidence rates (per 1 000 000 person-years) from 2006 to 2015 were estimated and age-adjusted rates were determined. RESULTS: Of 2262 study candidates, 154 NMOSD patients (age ≥ 16 years) with onset until 31 December 2016 were identified based on 2015 IPND criteria. The prevalence analysis on 1 January 2016 included 123 NMOSD living cases, resulting in a prevalence of 1.91 [95% confidence interval (CI) 1.52-2.28] per 100 000 persons. The 101 incident cases emerging from the observed 76 394 288 person-years provided an incidence rate of 1.32 (95% CI 1.08-1.61) per 1 000 000 person-years. Age-adjusted prevalence was 1.87 (95% CI 1.56-2.23) per 100 000 persons and incidence was 1.20 (95% CI 0.98-1.46) per 1 000 000 person-years. CONCLUSIONS: In this first report of a large population-based epidemiological study from an Eastern European Caucasian population using robust case validation, a greater prevalence and incidence of NMOSD was found compared to previous large studies in Caucasian populations.

Expression of CDX2 in metastatic prostate cancer.

The intestinal marker CDX2 has recently been found to stain a small percentage of primary prostate adenocarcinomas, but little is known of its expression in metastatic prostate cancers. We present a case of metastatic prostate adenocarcinoma that stained for CDX2 and highlight the confusion this may create when evaluating a carcinoma of unknown primary.

Subcellular redistribution of Pit-2 P(i) transporter/amphotropic leukemia virus (A-MuLV) receptor in A-MuLV-infected NIH 3T3 fibroblasts: involvement in superinfection interference.

Amphotropic murine leukemia virus (A-MuLV) utilizes the Pit-2 sodium-dependent phosphate transporter as a cell surface receptor to infect mammalian cells. Previous studies established that infection of cells with A-MuLV resulted in the specific down-modulation of phosphate uptake mediated by Pit-2 and in resistance to superinfection with A-MuLV. To study the mechanisms underlying these phenomena, we constructed plasmids capable of efficiently expressing epsilon epitope- and green fluorescent protein (GFP)-tagged human Pit-2 proteins in mammalian cells. Overexpression of epsilon-epitope-tagged Pit-2 transporters in NIH 3T3 cells resulted in a marked increase in sodium-dependent P(i) uptake. This increase in P(i) uptake was specifically blocked by A-MuLV infection but not by infection with ecotropic MuLV (E-MuLV) (which utilizes a cationic amino acid transporter, not Pit-2, as a cell surface receptor). These data, together with the finding that the tagged Pit-2 transporters retained their A-MuLV receptor function, indicate that the insertion of epitope tags does not affect either retrovirus receptor or P(i) transporter function. The overexpressed epitope-tagged transporters were detected in cell lysates, by Western blot analysis using both epsilon-epitope- and GFP-specific antibodies as well as with Pit-2 antiserum. Both the epitope- and GFP-tagged transporters showed almost exclusive plasma membrane localization when expressed in NIH 3T3 cells, as determined by laser scanning confocal microscopy. Importantly, when NIH 3T3 cells expressing these proteins were productively infected with A-MuLV, the tagged transporters and receptors were no longer detected in the plasma membrane but rather were localized to a punctate structure within the cytosolic compartment distinct from Golgi, endoplasmic reticulum, endosomes, lysosomes, and mitochondria. The intracellular Pit-2 pool colocalized with the virus in A-MuLV-infected cells. A similar redistribution of the tagged Pit-2 proteins was not observed following infection with E-MuLV, indicating that the redistribution of Pit-2 is not directly attributable to general effects associated with retroviral infection but rather is a specific consequence of A-MuLV-Pit-2 interactions.

Up-regulation of the Pit-2 phosphate transporter/retrovirus receptor by protein kinase C epsilon.

The membrane receptors for the gibbon ape leukemia retrovirus and the amphotropic murine retrovirus serve normal cellular functions as sodium-dependent phosphate transporters (Pit-1 and Pit-2, respectively). Our earlier studies established that activation of protein kinase C (PKC) by treatment of cells with phorbol 12-myristate 13-acetate (PMA) enhanced sodium-dependent phosphate (Na/Pi) uptake. Studies now have been carried out to determine which type of Na/Pi transporter (Pit-1 or Pit-2) is regulated by PKC and which PKC isotypes are involved in the up-regulation of Na/Pi uptake by the Na/Pi transporter/viral receptor. It was found that the activation of short term (2-min) Na/Pi uptake by PMA is abolished when cells are infected with amphotropic murine retrovirus (binds Pit-2 receptor) but not with gibbon ape leukemia retrovirus (binds Pit-1 receptor), indicating that Pit-2 is the form of Na/Pi transporter/viral receptor regulated by PKC. The PKC-mediated activation of Pit-2 was blocked by pretreating cells with the pan-PKC inhibitor bisindolylmaleimide but not with the conventional PKC isotype inhibitor Gö 6976, suggesting that a novel PKC isotype is required to regulate Pit-2. Overexpression of PKCepsilon, but not of PKCalpha, -delta, or -zeta, was found to mimic the activation of Na/Pi uptake. To further establish that PKCepsilon is involved in the regulation of Pit-2, cells were treated with PKCepsilon-selective antisense oligonucleotides. Treatment with PKCepsilon antisense oligonucleotides decreased the PMA-induced activation of Na/Pi uptake. These results indicate that PMA-induced stimulation of Na/Pi uptake by Pit-2 is specifically mediated through activation of PKCepsilon.

Cloning and characterization of the genes of the CeqI restriction-modification system.

Two genes from Corynebacterium equii, a Gram-positive bacterium producing the CeqI restriction-modification enzymes were cloned and sequenced. In vivo restriction experiments, DNA and amino acid sequence data suggest that the two genes code for the endonuclease and the methyltransferase enzymes. However, when the two genes are expressed in E. coli, practically no enzyme activity can be detected in the supernatants of sonicated cells. Based on the DNA sequence data CeqI restriction endonuclease (an EcoRV izoschizomer) consists of 270 amino acid residues with a predicted molecular mass of 31.6 kDa, in good agreement with the previously measured 32 +/- 2 kDa. The methyltransferase is 517 residues long (approx. 60 kDa). The two genes are in opposite orientation and overlap by 37 base pairs on the chromosome. The deduced amino acid sequence of the putative endonuclease gene revealed long stretches of hydrophobic amino acids, that may form the structural basis of the unusual aggregation properties of the restriction endonuclease. The amino acid sequence of the methylase shows homologies with other type II methyltransferases.

Purification and characterization of CeqI restriction endonuclease.

CeqI, a type II restriction endonuclease, an isoschizomer of EcoRV was purified to apparent homogeneity by a combination of salt precipitation, ion exchange, dye affinity and hydrophobic interaction chromatographies. The crude enzyme was present in the form of large aggregates that could be pelleted by high speed centrifugation. The enzyme was not associated with cellular membranes, though non-ionic detergents lowered the apparent size of the aggregates. The purified enzyme also showed a tendency to form large molecular mass (66-600 kDa) complexes under physiological conditions, in the absence of cleavable DNA. The enzyme formed smaller complexes in the presence of DNA and non-ionic detergents and dissociated into subunits (and undergoes reversible loss of activity) in the presence of high concentrations of salts. According to SDS gel electrophoresis and sedimentation analysis the molecular mass of the monomer 32 +/- 2 kDa. The enzyme had a rather broad PH optimum, extending into the alkaline range and lost specificity and activity in buffers below pH 6.

Positive co-operative interaction between the subunits of CeqI restriction endonuclease.

CeqI restriction endonuclease, an isoschizomer of EcoRV, forms complexes of 12-20 subunits under physiological conditions, in the absence of DNA. These molecules partially dissociate in the presence of DNA sequences recognized by CeqI or in the presence of non-ionic detergents. In solutions containing high concentrations of salts (e.g. 1 M-NaCl), the enzyme dissociates into subunits, concomitantly losing its activity. According to our experiments, it is the tetrameric form of the enzyme that binds the DNA and represents the catalytically active molecule. Analysis of the enzyme kinetics revealed a positive co-operative interaction between the subunits of the enzyme. Computer-assisted analysis of these data yielded a Hill coefficient of approx. 1.35, suggesting two binding sites per tetrameric enzyme molecule, two subunits per palindromic recognition site.