Chromatin binding of RCC1 during mitosis is important for its nuclear localization in interphase

RCC1, a guanine nucleotide exchange factor of the small GTPase Ran, plays various roles throughout the cell cycle. However, the functions of RCC1 in biological processes in vivo are still unclear. In particular, although RCC1 has multifunctional domains, the biological significance of each domain is unclear. To examine each domain of RCC1, we established an RCC1 conditional knockout chicken DT40 cell line and introduced various RCC1 mutants into the knockout cells. We found that nuclear reformation did not occur properly in RCC1-deficient cells and examined whether specific RCC1 mutants could rescue this phenotype. Surprisingly, we found that neither the nuclear localization signal nor the chromatin-binding domain of RCC1 is essential for its function. However, codisruption of these domains resulted in defective nuclear reformation, which was rescued by artificial nuclear localization of RCC1. Our data indicate that chromatin association of RCC1 during mitosis is crucial for its proper nuclear localization in the next interphase. Moreover, proper nuclear localization of RCC1 in interphase is essential for its function through its nucleotide exchange activity.     

Microarray analysis of the developing rat mandible

To analyze the molecular events that occur in the developing mandible, we examined the expression of 8803 genes from samples taken at different time points during rat postnatal mandible development. Total RNA was extracted from the mandibles of 1-day-old, 1-week-old, and 2-week-old rats. Complementary RNA (cRNA) was synthesized from cDNA and biotinylated. Fragmented cRNA was hybridized to RGU34A GeneChip arrays. Among the 8803 genes tested, 4344 were detectable. We identified 148 genes with significantly increased expression, and 19 genes with significantly decreased expression. A comprehensive analysis appears to be an effective method of studying the complex process of development.     

Expression of long-form N-acetylglucosamine-6-O-sulfotransferase 1 in human high endothelial venules

Two members of the N-acetylglucosamine-6-O-sulfotransferase (GlcNAc6ST) family, GlcNAc6ST-1 and GlcNAc6ST-2, function in the biosynthesis of 6-sulfo sialyl Lewis X-capped glycoproteins expressed on high endothelial venules (HEVs) in secondary lymphoid organs. Thus, both enzymes play a critical role in L-selectin-expressing lymphocyte homing. Human GlcNAc6ST-1 is encoded by a 1593-bp open reading frame exhibiting two 5' in-frame methionine codons spaced 141 bp apart. Both resemble the consensus sequence for translation initiation. Thus, it has been hypothesized that both long and short forms of GlcNAc6ST-1 may be present, although endogenous expression of either form has not been confirmed in humans. Here, the authors developed an antibody recognizing amino acid residues between the first two human GlcNAc6ST-1 methionines. This antibody specifically recognizes the long form of the enzyme, a finding validated by Western blot analysis and immunofluorescence cytochemistry of HeLa cells misexpressing long and/or short forms of human GlcNAc6ST-1. Using this antibody, the authors carried out immunofluorescence histochemistry of human lymph node tissue sections and found endogenous expression of the long form of the enzyme in human tissue, predominantly in the trans-Golgi network of endothelial cells that form HEVs.     

Involvement of the cannabinoid CB2 receptor and its endogenous ligand 2-arachidonoylglycerol in oxazolone-induced contact dermatitis in mice

The possible involvement of 2-arachidonoylglycerol (2-AG), an endogenous ligand for the cannabinoid receptors (CB1 and CB2), in contact dermatitis in mouse ear was investigated. We found that the level of 2-AG was markedly elevated in the ear following a challenge with oxazolone in sensitized mice. Of note, the swelling following the challenge was suppressed by either the administration of SR144528, a CB2 receptor antagonist, immediately after sensitization, or the administration of SR144528 upon the challenge. The effect of AM251, a CB1 receptor antagonist, was marginal in either case. It seems apparent, therefore, that the CB2 receptor and its endogenous ligand 2-AG are closely involved in both the sensitization phase and the elicitation phase of oxazolone-induced contact dermatitis. In line with this, we found that Langerhans cells (MHC class II(+)) contain a substantial amount of CB2 receptor mRNA, whereas keratinocytes (MHC class II(-)) do not. We also obtained evidence that the expression of mRNAs for proinflammatory cytokines following a challenge with oxazolone was markedly suppressed by treatment with SR144528. We next examined whether the CB2 receptor and 2-AG participate in chronic contact dermatitis accompanied by the infiltration of tissues by eosinophils. The amount of 2-AG in mouse ear dramatically increased following repeated challenge with oxazolone. Importantly, treatment with SR144528 attenuated both the recruitment of eosinophils and ear swelling in chronic contact dermatitis induced by repeated challenge with oxazolone. These results strongly suggest that the CB2 receptor and 2-AG play important stimulative roles in the sensitization, elicitation, and exacerbation of allergic inflammation.     

Effects of BTS (N-benzyl-p-toluene sulphonamide), an inhibitor for myosin-actin interaction, on myofibrillogenesis in skeletal muscle cells in culture

Actin filaments align around myosin filaments in the correct polarity and in a hexagonal arrangement to form cross-striated structures. It has been postulated that this myosin-actin interaction is important in the initial phase of myofibrillogenesis. It was previously demonstrated that an inhibitor of actin-myosin interaction, BDM (2,3-butanedione monoxime), suppresses myofibril formation in muscle cells in culture. However, further study showed that BDM also exerts several additional effects on living cells. In this study, we further examined the role of actin-myosin interaction in myofibril assembly in primary cultures of chick embryonic skeletal muscle by applying a more specific inhibitor, BTS (N-benzyl-p-toluene sulphonamide), of myosin ATPase and actin-myosin interaction. The assembly of sarcomeric structures from myofibrillar proteins was examined by immunocytochemical methods with the application of BTS to myotubes just after fusion. Addition of BTS (10-50 microM) significantly suppressed the organization of actin and myosin into cross-striated structures. BTS also interfered in the organization of alpha-actinin, C-protein (or MyBP-C), and connectin (or titin) into ordered striated structures, though the sensitivity was less. Moreover, when myotubes cultured in the presence of BTS were transferred to a control medium, sarcomeric structures were formed in 2-3 days, indicating that the inhibitory effect of BTS on myotubes is reversible. These results show that actin-myosin interaction plays a critical role in the process of myofibrillogenesis.     

Ars insulator identified in sea urchin possesses an activity to ensure the transgene expression in mouse cells

Sea urchin arylsulfatase (Ars) gene locus has features of an insulator, i.e., blocking of enhancer and promoter interaction, and protection of a transgene against positional effects [Akasaka et al. (1999) Cell. Mol. Biol. 45, 555-565]. To examine the effect of Ars insulator on long-term expression of a transgene, the insulator was inserted into LTR of retrovirus vector harboring hrGFP gene as a reporter, and then introduced into mouse myoblast cells. The isolated clones transduced with the reporter gene with or without Ars insulator were cultured for more than 20 wk in the absence of a selection reagent, and the expression of hrGFP was periodically determined. Expression of hrGFP in four clones transduced with the reporter gene without Ars insulator was completely silenced after 20 wk of culture. On the other hand, hrGFP was expressed in all clones with Ars insulator inserted in one of the two different orientations. Histone H3 deacetylation and DNA methylation of the 5'LTR promoter region, signs for heterochromatin and silencing, were suppressed in the clones that were expressing hrGFP. Ars insulator is effective in maintaining a transgene in mouse cells in an orientation-dependent manner, and will be a useful tool to ensure stable expression of a transgene.     

Three enhancer regions regulate gbx2 gene expression in the isthmic region during zebrafish development

In vertebrate embryos, positioning of the boundary between the midbrain and hindbrain (MHB) and subsequent isthmus formation are dependent upon the interaction between the Otx2 and Gbx genes. In zebrafish, sequential expression of gbx1 and gbx2 in the anterior hindbrain contributes to this process, whereas in mouse embryos, a single Gbx gene (Gbx2) is responsible for MHB development. In the present study, to investigate the regulatory mechanism of gbx2 in the MHB/isthmic region of zebrafish embryos, we cloned the gene and showed that its organization is conserved among different vertebrates. Promoter analyses revealed three enhancers that direct reporter gene expression after the end of epiboly in the anterior-most hindbrain, which is a feature of the zebrafish gbx2 gene. One of the enhancers is located upstream of gbx2 (AMH1), while the other two enhancers are located downstream of gbx2 (AMH2 and AMH3). Detailed analysis of the AMH1 enhancer showed that it directs expression in the rhombomere 1 (r1) region and the dorsal thalamus, as has been shown for gbx2, whereas no expression was induced by the AMH1 enhancer in other embryonic regions in which gbx2 is expressed. The AMH1 enhancer is composed of multiple regulatory subregions that share the same spatial specificity. The most active of the regulatory subregions is a 291-bp region that contains at least two Pax2-binding sites, both of which are necessary for the function of the main component (PB1-A region) of the AMH1 enhancer. In accordance with these results, enhancer activity in the PB1-A region, as well as gbx2 expression in r1, was missing in no isthmus mutant embryos that lacked functional pax2a. In addition, we identified an upstream conserved sequence of 227bp that suppresses the enhancer activity of AMH1. Taken together, these findings suggest that gbx2 expression during the somitogenesis stage in zebrafish is regulated by a complex mechanism involving Pax2 as well as activators and suppressors in the regions flanking the gene.     

Shisa2 promotes the maturation of somitic precursors and transition to the segmental fate in Xenopus embryos

In vertebrate somitogenesis, FGF and Wnt signals constitute a morphogenetic gradient that controls the maturation of the presomitic mesoderm (PSM) as well as the transition to segmental units. It remains unclear, however, whether there is a regulatory mechanism that promotes the transition by a direct regulation of FGF and Wnt signaling in the PSM. Here we show that Shisa2, a member of a novel Shisa gene family, plays an essential role in segmental patterning during Xenopus somitogenesis. Shisa2 encodes an endoplasmic reticulum (ER) protein that cell-autonomously inhibits FGF and Wnt signaling by preventing the maturation and the cell-surface expression of their receptors. Shisa2 is expressed in the PSM and its knockdown caused a reduction in somite number by the delayed maturation of PSM and anterior shift of the transition; however, the phase of the segmental clock remained intact. These phenotypes were abolished by the inhibition of both FGF and Wnt signals, but by neither alone. We therefore propose that the individual inhibition of both types of signaling by the regulation of receptor maturation in the ER plays an essential role in the establishment of proper segmental patterning.