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.
Huntington’s disease (HD) is a hereditary neurodegenerative disease that is caused by polyglutamine expansion within the huntingtin (HTT) gene. One of the cellular activities that is dysregulated in HD is store-operated calcium entry (SOCE), a process by which Ca2+ release from the endoplasmic reticulum (ER) induces Ca2+ influx from the extracellular space. HTT-associated protein-1 (HAP1) is a binding partner of HTT. The aim of the present study was to examine the role of HAP1A protein in regulating SOCE in YAC128 mice, a transgenic model of HD. After Ca2+ depletion from the ER by the activation of inositol-(1,4,5)triphosphate receptor type 1 (IP3R1), we detected an increase in the activity of SOC channels when HAP1 protein isoform HAP1A was overexpressed in medium spiny neurons (MSNs) from YAC128 mice. A decrease in the activity of SOC channels in YAC128 MSNs was observed when HAP1 protein was silenced. In YAC128 MSNs that overexpressed HAP1A, an increase in activity of IP3R1 was detected while the ionomycin-sensitive ER Ca2+ pool decreased. 6-Bromo-N-(2-phenylethyl)-2,3,4,9-tetrahydro-1H-carbazol-1-amine hydrochloride (C20H22BrClN2), identified in our previous studies as a SOCE inhibitor, restored the elevation of SOCE in YAC128 MSN cultures that overexpressed HAP1A. The IP3 sponge also restored the elevation of SOCE and increased the release of Ca2+ from the ER in YAC128 MSN cultures that overexpressed HAP1A. The overexpression of HAP1A in the human neuroblastoma cell line SK-N-SH (i.e., a cellular model of HD (SK-N-SH HTT138Q)) led to the appearance of a pool of constitutively active SOC channels and an increase in the expression of STIM2 protein. Our results showed that HAP1A causes the activation of SOC channels in HD models by affecting IP3R1 activity.
Huntington's disease (HD) is a progressive neurodegenerative disorder that is characterized by motor, cognitive, and psychiatric problems. It is caused by a polyglutamine expansion in the huntingtin protein that leads to striatal degeneration via the transcriptional dysregulation of several genes, including genes that are involved in the calcium (Ca2+) signalosome. Recent research has shown that one of the major Ca2+ signaling pathways, store-operated Ca2+ entry (SOCE), is significantly elevated in HD. SOCE refers to Ca2+ flow into cells in response to the depletion of endoplasmic reticulum Ca2+ stores. The dysregulation of Ca2+ homeostasis is postulated to be a cause of HD progression because the SOCE pathway is indirectly and abnormally activated by mutant huntingtin (HTT) in γ-aminobutyric acid (GABA)ergic medium spiny neurons (MSNs) from the striatum in HD models before the first symptoms of the disease appear. The present review summarizes recent studies that revealed a relationship between HD pathology and elevations of SOCE in different models of HD, including YAC128 mice (a transgenic model of HD), cellular HD models, and induced pluripotent stem cell (iPSC)-based GABAergic medium spiny neurons (MSNs) that are obtained from adult HD patient fibroblasts. SOCE in MSNs was shown to be mediated by currents through at least two different channel groups, Ca2+ release-activated Ca2+ current (ICRAC) and store-operated Ca2+ current (ISOC), which are composed of stromal interaction molecule (STIM) proteins and Orai or transient receptor potential channel (TRPC) channels. Their role under physiological and pathological conditions in HD are discussed. The role of Huntingtin-associated protein 1 isoform A in elevations of SOCE in HD MSNs and potential compounds that may stabilize elevations of SOCE in HD are also summarized. Evidence is presented that shows that the dysregulation of molecular components of SOCE or pathways upstream of SOCE in HD MSN neurons is a hallmark of HD, and these changes could lead to HD pathology, making them potential therapeutic targets.
OCTN2 - the Organic Cation Transporter Novel family member 2 (SLC22A5) is known to be a xenobiotic/drug transporter. It transports as well carnitine - a compound necessary for oxidation of fatty acids and mutations of its gene cause primary carnitine deficiency. Octn2 regulation by protein kinase C (PKC) was studied in rat astrocytes - cells in which β-oxidation takes place in the brain. Activation of PKC with phorbol ester stimulated L-carnitine transport and increased cell surface presence of the transporter, although no PKC-specific phosphorylation of Octn2 could be detected. PKC activation resulted in an augmented Octn2 presence in cholesterol/sphingolipid-rich microdomains of plasma membrane (rafts) and increased co-precipitation of Octn2 with raft-proteins, caveolin-1 and flotillin-1. Deletion of potential caveolin-1 binding motifs pointed to amino acids 14–22 and 447–454 as the caveolin-1 binding sites within Octn2 sequence. A direct interaction of Octn2 with caveolin-1 in astrocytes upon PKC activation was detected by proximity ligation assay, while such an interaction was excluded in case of flotillin-1. Functioning of a multi-protein complex regulated by PKC has been postulated in rOctn2 trafficking to the cell surface, a process which could be important both under physiological conditions, when carnitine facilitates fatty acids catabolism and controls free Coenzyme A pool as well as in pathology, when transport of several drugs can induce secondary carnitine deficiency.
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