IncU plasmids are a distinctive group of mobile elements with highly conserved backbone functions and variable antibiotic resistance gene cassettes. The IncU archetype is conjugative plasmid RA3, whose sequence (45,909 bp) shows it to be a mosaic, modular replicon with a class I integron different from that of other IncU replicons. Functional analysis demonstrated that RA3 possesses a broad host range and can efficiently self-transfer, replicate, and be maintained stably in alpha-, beta-, and gammaproteobacteria. RA3 contains 50 open reading frames clustered in distinct functional modules. The replication module encompasses the repA and repB genes embedded in long repetitive sequences. RepA, which is homologous to antitoxin proteins from alpha-and gammaproteobacteria, contains a Cro/cI-type DNA-binding domain present in the XRE family of transcriptional regulators. The repA promoter is repressed by RepA and RepB. The minireplicon encompasses repB and the downstream repetitive sequence r1/r2. RepB shows up to 80% similarity to putative replication initiation proteins from environmental plasmids of beta-and gammaproteobacteria, as well as similarity to replication proteins from alphaproteobacteria and Firmicutes. Stable maintenance functions of RA3 are most like those of IncP-1 broad-host-range plasmids and comprise the active partitioning apparatus formed by IncC (ParA) and KorB (ParB), the antirestriction protein KlcA, and accessory stability components KfrA and KfrC. The RA3 origin of transfer was localized experimentally between the maintenance and conjugative-transfer operons. The putative conjugative-transfer module is highly similar in organization and in its products to transfer regions of certain broad-host-range environmental plasmids.Conjugative plasmids contribute greatly to the global spread of genetic information and gene exchange, as in some cases they can self-transfer even between distant bacterial species. Conjugative R factors assigned to the IncU incompatibility group have been isolated from a number of Aeromonas spp. and Escherichia coli strains from seawater fish hatcheries and diseased fish, as well as from clinical environments (2,4,6,15,26,30,31,42). Members of the IncU plasmid group are implicated particularly in the dissemination of antibiotic resistance in Aeromonas strains associated with aquatic environments (15).IncU representatives pAr-32 and pRAS1 contain resistance genes encoded within integrons, and on the basis of restriction enzyme analysis of both plasmid molecules, Sørum et al. (35) postulated a highly conserved backbone structure of IncU group members with variability in the region coding for antibiotic resistance. Similar observations were made with plasmids pASOT and pFBAOT (2,26,27). Plasmid pFBAOT6 (84,749 bp) has been sequenced recently and analyzed in silico (27). Plasmid Rms149 of the Pseudomonas IncP-6 group was assigned to the IncU group on the basis of incompatibility tests (12). Apart from homology between the replication genes of pFBAOT6 and Rms149, no conservati...

Huntington's disease (HD) is a hereditary neurodegenerative disease caused by the expansion of a polyglutamine stretch in the huntingtin (HTT) protein and characterized by dysregulated calcium homeostasis. We investigated whether these disturbances are correlated with changes in the mRNA level of the genes that encode proteins involved in calcium homeostasis and signaling (i.e., the calciosome). Using custom-made TaqMan low-density arrays containing probes for 96 genes, we quantified mRNA in the striatum in YAC128 mice, a model of HD, and wildtype mice. HTT mutation caused the increased expression of some components of the calcium signalosome, including calretinin, presenilin 2, and calmyrin 1, and the increased expression of genes indirectly involved in calcium homeostasis, such as huntingtin-associated protein 1 and calcyclin-binding protein. To verify these findings in a different model, we used PC12 cells with an inducible expression of mutated full-length HTT. Using single-cell imaging with Fura-2AM, we found that store-operated Ca2+ entry but not endoplasmic reticulum (ER) store content was changed as a result of the expression of mutant HTT. Statistically significant downregulation of the Orai calcium channel subunit 2, calmodulin, and septin 4 was detected in cells that expressed mutated HTT. Our data indicate that the dysregulation of calcium homeostasis correlates with changes in the gene expression of members of the calciosome. These changes, however, differed in the two models of HD used in this study. Our results indicate that each HD model exhibits distinct features that may only partially resemble the human disease.

Carnitine β‐hydroxy‐γ‐(trimethylammonio)butyrate – a compound necessary in the peripheral tissues for a transfer of fatty acids for their oxidation within the cell, accumulates in the brain despite low β‐oxidation in this organ. In order to enter the brain, carnitine has to cross the blood–brain barrier formed by capillary endothelial cells which are in close interaction with astrocytes. Previous studies, demonstrating expression of mRNA coding two carnitine transporters – organic cation/carnitine transporter 2 (OCTN2) and B0,+ in endothelial cells, did not give any information on carnitine transporters polarity in endothelium. Therefore more detailed experiments were performed on expression and localization of a high affinity carnitine transporter OCTN2 in an in vitro model of the blood–brain barrier by real‐time PCR, western blot analysis, and immunocytochemistry. The amount of mRNA was comparable in endothelial cells and kidney, when referred to house‐keeping genes, it was, however, significantly lower in astrocytes. Polarity of OCTN2 localization was further studied in an in vitro model of the blood–brain barrier with use of anti‐OCTN2 antibodies. Z‐axis analysis of the confocal microscope pictures of endothelial cells, with anti‐P‐glycoprotein antibodies as the marker of apical membrane, showed OCTN2 localization at the basolateral membrane and in the cytoplasmic region in the vicinity of nuclei. Localization of OCTN2 suggest that carnitine can be also transported from the brain, playing an important role in removal of certain acyl esters.

Huntington's disease (HD) is a hereditary neurodegenerative disease caused by a polyglutamine expansion within the huntingtin (HTT) gene. One of the cellular functions that is dysregulated in HD is store-operated calcium entry (SOCE), a process in which the depletion of Ca from the endoplasmic reticulum (ER) induces Ca influx from the extracellular space. We detected an enhanced activity of SOC channels in medium spiny neurons (MSNs) from YAC128 mice, a transgenic model of HD, and investigated whether this could be reverted by tetrahydrocarbazoles. The compound 6-bromo-N-(2-phenylethyl)-2,3,4,9-tetrahydro-1H-carbazol-1-amine hydrochloride was indeed able to restore the disturbed Ca homeostasis and stabilize SOCE in YAC128 MSN cultures. We also detected a beneficial effect of this compound on the mitochondrial membrane potential. Since dysregulated Ca homeostasis is believed to be one of the pathological hallmarks of HD, this compound might be a lead structure for HD treatment.

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