Chronic antidepressant treatment rescues abnormally reduced REM sleep theta power in socially defeated rats.

The effects of chronic antidepressant (AD) treatment on sleep disturbances in rodent chronic stress models have not been thoroughly investigated. Here, we show that chronic social defeat stress (SDS) in rats induces prolonged social avoidance, alterations in sleep architecture (increased total rapid eye movement [REM] sleep duration, bout, and shortened REM latency), and contextual but not cued fear memory deficits, even 1 month after the last SDS. These abnormalities were associated with changes in electroencephalography (EEG) spectral powers, including reduced REM sleep theta power during the light phase. Chronic treatment with two different classes of antidepressants (ADs), imipramine and fluoxetine, significantly ameliorated these behavioral, sleep, and EEG abnormalities. Interestingly, REM theta power was normalized by chronic (1 month) but not 1 week AD administration and solely correlated with the ratio (an objective indicator) of social interaction 1 month after the last SDS. These data suggest that reductions in REM sleep theta power, an EEG parameter that has never been directly investigated in humans, is a core sleep symptom in socially defeated rats, and, potentially, also in patients with stress-related psychiatric disorders, including major depressive and posttraumatic stress disorders.

BTB-ZF Protein Znf131 Regulates Cell Growth of Developing and Mature T Cells.

Many members of the BTB-ZF family have been shown to play important roles in lymphocyte development and function. The role of zinc finger Znf131 (also known as Zbtb35) in T cell lineage was elucidated through the production of mice with floxed allele to disrupt at different stages of development. In this article, we present that Znf131 is critical for T cell development during double-negative to double-positive stage, with which significant cell expansion triggered by the pre-TCR signal is coupled. In mature T cells, Znf131 is required for the activation of effector genes, as well as robust proliferation induced upon TCR signal. One of the cyclin-dependent kinase inhibitors, p21(Cip1) encoded by cdkn1a gene, is one of the targets of Znf131. The regulation of T cell proliferation by Znf131 is in part attributed to its suppression on the expression of p21(Cip1).

Amino acid residues important for CMP-sialic acid recognition by the CMP-sialic acid transporter: analysis of the substrate specificity of UDP-galactose/CMP-sialic acid transporter chimeras.

In our previous studies, we demonstrated that chimeric molecules of the CMP-sialic acid (CMP-Sia) transporter (CST) and the UDP-galactose (Gal) transporter (UGT) in which the seventh transmembrane helix-containing segment was derived from the CST could transport both CMP-Sia and UDP-Gal and that the CST-derived seventh transmembrane helix segment was sufficient for the chimera to recognize CMP-Sia in the otherwise UGT context. In this study, we continued to more precisely define the submolecular region that is necessary for CMP-Sia recognition, and we demonstrated that the N-terminal half of the seventh transmembrane helix of CST is essential for the CMP-Sia transport mediated by the chimeric transporters. We further showed that Tyr214Gly and Ser216Phe mutations of a chimeric transporter that was capable of transporting both CMP-Sia and UDP-Gal led to the selective loss of CMP-Sia transport activity without affecting UDP-Gal transport activity. Conversely, when a residue in a chimeric transporter that was active for UDP-Gal transport but not CMP-Sia transport was replaced by Tyr, so that Tyr occupied the same position as in the CMP-Sia transporter, the resulting mutant chimera acquired the ability to transport CMP-Sia. These results demonstrated that Tyr214 and Ser216, located in the seventh transmembrane helix of the human CST, are critically important for the recognition of CMP-Sia as a transport substrate. Identification of determinants critical for the discrimination between relevant and irrelevant substrates will advance our understanding of the mechanisms of substrate recognition by nucleotide sugar transporters.

Two pathways for importing GDP-fucose into the endoplasmic reticulum lumen function redundantly in the O-fucosylation of Notch in Drosophila.

Notch is a transmembrane receptor that shares homology with proteins containing epidermal growth factor-like repeats and mediates the cell-cell interactions necessary for many cell fate decisions. In Drosophila, O-fucosyltransferase 1 catalyzes the O-fucosylation of these epidermal growth factor-like repeats. This O-fucose elongates, resulting in an O-linked tetrasaccharide that regulates the signaling activities of Notch. Fucosyltransferases utilize GDP-fucose, which is synthesized in the cytosol, but fucosylation occurs in the lumen of the endoplasmic reticulum (ER) and Golgi. Therefore, GDP-fucose uptake into the ER and Golgi is essential for fucosylation. However, although GDP-fucose biosynthesis is well understood, the mechanisms and intracellular routes of GDP-fucose transportation remain unclear. Our previous study on the Drosophila Golgi GDP-fucose transporter (Gfr), which specifically localizes to the Golgi, suggested that another GDP-fucose transporter(s) exists in Drosophila. Here, we identified Efr (ER GDP-fucose transporter), a GDP-fucose transporter that localizes specifically to the ER. Efr is a multifunctional nucleotide sugar transporter involved in the biosynthesis of heparan sulfate-glycosaminoglycan chains and the O-fucosylation of Notch. Comparison of the fucosylation defects in the N-glycans in Gfr and Efr mutants revealed that Gfr and Efr made distinct contributions to this modification; Gfr but not Efr was crucial for the fucosylation of N-glycans. We also found that Gfr and Efr function redundantly in the O-fucosylation of Notch, although they had different localizations and nucleotide sugar transportation specificities. These results indicate that two pathways for the nucleotide sugar supply, involving two nucleotide sugar transporters with distinct characteristics and distributions, contribute to the O-fucosylation of Notch.

Regulation of DNA demethylation during maturation of CD4+ naive T cells by the conserved noncoding sequence 1.

Demethylation of transcriptional regulatory elements and gene coding regions is an important step in the epigenetic regulation of gene expression. Several noncoding conserved regions are required for the efficient transcription of cytokine genes. In this paper, we show that the deletion of one such sequence, conserved noncoding sequence 1 (CNS-1), interferes with the efficient demethylation of Th2 cytokine genes but has little effect on histone modifications in the area. Th2 cells derived from CD4 single-positive (SP) mature thymocytes exhibit more rapid demethylation of CNS-1 and Th2-specific cytokine genes and produce more Th2 cytokines than do Th2 cells derived from CD4-positive peripheral naive T cells. De-repression of the Th1 cytokine IFN-gamma was also detected in Th2-primed CD4 SP thymocytes but not in naive T cells. Our results indicate that susceptibility to demethylation determines the efficiency and kinetics of cytokine gene transcription. The extrathymic maturation step undergone by naive T cells suppresses robust and rapid cytokine expression, whereas mature CD4 SP thymocytes maintain a rapid and less-specific cytokine expression profile. Finally, we detected the methyl cytosine binding protein MBD2 at CNS-1 in mature thymocytes, suggesting that this protein may regulate the demethylation of this region.

Notch deficiency implicated in the pathogenesis of congenital disorder of glycosylation IIc.

Congenital disorder of glycosylation IIc (CDG IIc), also termed leukocyte adhesion deficiency II, is a recessive syndrome characterized by slowed growth, mental retardation, and severe immunodeficiency. Recently, the gene responsible for CDG IIc was found to encode a GDP-fucose transporter. Here, we investigated the possible cause of the developmental defects in CDG IIc patients by using a Drosophila model. Biochemically, we demonstrated that a Drosophila homolog of the GDP-fucose transporter, the Golgi GDP-fucose transporter (Gfr), specifically transports GDP-fucose in vitro. To understand the function of the Gfr gene, we generated null mutants of Gfr in Drosophila. The phenotypes of the Drosophila Gfr mutants were rescued by the human GDP-fucose transporter transgene. Our phenotype analyses revealed that Notch (N) signaling was deficient in these Gfr mutants. GDP-fucose is known to be essential for the fucosylation of N-linked glycans and for O-fucosylation, and both fucose modifications are present on N. Our results suggest that Gfr is involved in the fucosylation of N-linked glycans on N and its O-fucosylation, as well as those of bulk proteins. However, despite the essential role of N O-fucosylation during development, the Gfr homozygote was viable. Thus, our results also indicate that the Drosophila genome encodes at least another GDP-fucose transporter that is involved in the O-fucosylation of N. Finally, we found that mammalian Gfr is required for N signaling in mammalian cultured cells. Therefore, our results implicate reduced N signaling in the pathology of CDG IIc.

Identification and characterization of human Golgi nucleotide sugar transporter SLC35D2, a novel member of the SLC35 nucleotide sugar transporter family.

We report the molecular cloning of SLC35D2, a novel member of the SLC35 nucleotide sugar transporter family. The gene SLC35D2 maps to chromosome 9q22.33. SLC35D2 cDNA codes for a hydrophobic protein consisting of 337 amino acid residues with 10 putative transmembrane helices. Northern blot analysis revealed the SLC35D2 mRNA as a single major band corresponding to 2.0 kb in length. SLC35D2 was localized in the Golgi membrane and exhibited around 50% similarity with three nucleotide sugar transporters: human SLC35D1 (UDP-glucuronic acid/UDP-N-acetylgalactosamine transporter), fruitfly fringe connection (frc) transporter, and nematode SQV-7 transporter, the latter two being involved in developmental and organogenetic processes. Heterologous expression of SLC35D2 protein in yeast indicated that UDP-N-acetylglucosamine is a candidate for the substrate(s) of the transporter. The sequence similarity, subcellular localization, and transporting substrate suggest that SLC35D2 is a good candidate for the ortholog of frc transporter, which is involved in the Notch signaling system by providing the fringe N-acetylglucosaminyltransferase with the substrate. We also describe the identification and categorization of the human SLC35 gene family.

Substrate recognition by nucleotide sugar transporters: further characterization of substrate recognition regions by analyses of UDP-galactose/CMP-sialic acid transporter chimeras and biochemical analysis of the substrate specificity of parental and chimeric transporters.

Human UDP-Gal transporter 1 (hUGT1) and the human CMP-Sia transporter (hCST) are similar in structure, with amino acid sequences that are 43% identical, but they have quite distinct transport substrates. To define their substrate recognition regions, we constructed various chimeras between the two transporters and demonstrated that distinct submolecular regions of the transporter molecules are involved in the specific recognition of UDP-Gal and CMP-Sia (Aoki, K., Ishida, N., and Kawakita, M. (2001) J. Biol. Chem. 276, 21555-21561). In a further attempt to define the minimum submolecular regions required for the recognition of specific substrates, we found that substitution of helix 7 of hCST into the corresponding part of hUGT1 was necessary and sufficient for a chimera to show CST activity. Additional replacement of helix 2 or 3 of hUGT1 with the corresponding hCST sequence markedly increased the efficiency of CMP-Sia transport. For UGT activity, helices 1 and 8 of hUGT1 were necessary (but not sufficient), and helices 9 and 10 or helices 2, 3, and 7 derived from hUGT1 were also required to render the chimera competent for UDP-Gal transport. The in vitro analyses of a chimera with dual specificity indicated that it transported both UMP and CMP and mediated exchange reactions between these nucleotides and nucleotide sugars that are recognized specifically by either of the parental transporters.