bluuebunny/crossref_metadata_2025_split huggingface dataset:
The deubiquitinating enzyme Fam interacts with and stabilizes β‐catenin
Authors: Taya Shinichiro , Yamamoto Takaharu , Kanai‐Azuma Masami , Wood Stephen A. , Kaibuchi Kozo 
Published: March 2003

BackgroundIn the ubiquitin–proteasome pathway, the ubiquitinated substrates either undergo degradation by the proteasome or stabilization through the action of the deubiquitinating enzyme. We have previously found that the deubiquitinating enzyme Fam is colocalized with AF‐6, one of the effectors of the Ras small GTPase, at cell–cell contact sites in epithelial cells and interacts with AF‐6 in vivo and in vitro. Fam has deubiquitinating activity in vitro and prevents the ubiquitination of AF‐6 in intact cells. The degradation of β‐catenin, which accumulates at the cell–cell contact sites as a cadherin/catenin complex, is thought to be regulated by the ubiquitin–proteasome pathway. These observations prompted us to examine the possible Fam regulation of the stabilization of β‐catenin.ResultsWe found that Fam interacted with β‐catenin both in vivo and in vitro. The Fam‐binding site of β‐catenin mapped to the region close to the APC or Axin‐binding site of β‐catenin. Over‐expression of Fam in mouse L cells resulted in an elevation of β‐catenin levels and in an elongation of the half‐life of β‐catenin. In these L cells, Fam was colocalized with β‐catenin at the dot‐like structures in the cytoplasm.ConclusionThese results indicate that Fam interacts with and stabilizes β‐catenin in vivo, presumably through the deubiquitination of β‐catenin.
Mouse Ror2 receptor tyrosine kinase is required for the heart development and limb formation
Authors: Takeuchi Shigeto , Takeda Kiyoshi , Oishi Isao , Nomi Masashi , Ikeya Makoto , Itoh Kyoko , Tamura Shingo , Ueda Takahiro , Hatta Toshihisa , Otani Hiroki , Terashima Toshio , Takada Shinji , Yamamura Hirohei , Akira Shizuo , Minami Yasuhiro 
Published: March 2003

BackgroundsA mouse receptor tyrosine kinase (RTK), mRor2, which belongs to the Ror‐family of RTKs consisting of at least two structurally related members, is primarily expressed in the heart and nervous system during mouse development. To elucidate the function of mRor2, we generated mice with a mutated mRor2 locus.ResultsMice with a homozygous mutation in mRor2 died just after birth, exhibiting dwarfism, severe cyanosis, and short limbs and tails. Whole‐mount in situ hybridization analysis showed that mRor2 was expressed in the branchial arches, heart and limb/tailbuds, in addition to the developing nervous system. The mutants had cardiac septal defects, mainly a ventricular septal defect. In addition, an examination of the skeletal systems revealed that the mutants had shorter limbs, vertebrae and facial structure, with a particular defect in their distal portions, and that almost no calcification was observed in their distal limbs. Histological examination showed abnormalities in the chondrocytes.ConclusionsOur findings suggest that mRor2 plays essential roles in the development of the heart and in limb/tail formation, in particular cardiac septal formation and ossification of distal portions of limbs and tails.
Close kinship of human 20α‐hydroxysteroid dehydrogenase gene with three aldo‐keto reductase genes
Authors: Nishizawa Mikio , Nakajima Tatsuya , Yasuda Katsuhiko , Kanzaki Hideharu , Sasaguri Yasuyuki , Watanabe Kikuko , Ito Seiji 
Published: March 2003

Background20α‐Hydroxysteroid dehydrogenase (HSD) is a member of the aldo‐keto reductase (AKR) superfamily and catalyses the reaction of progesterone to the inactive form 20α‐hydroxyprogesterone. Progesterone plays an important role in the maintenance of pregnancy, and, in rodents, plasma progesterone levels decrease abruptly just before parturition. The induction of 20α‐HSD is thought to be responsible for the decrease in plasma progesterone at term. High homology between human 20α‐HSD [AKR 1C1] cDNA with other AKRs had caused difficulty in gene isolation and expression analysis. Thus, the metabolism of progesterone in the human reproductive system remained unclear.ResultsBy hybridization with rat 20α‐HSD [AKR 1C8] cDNA and high‐stringency polymerase chain reaction (PCR) with gene‐specific primers, we were able to isolate the human 20α‐HSD, bile acid‐binding protein (BABP) [AKR 1C2], prostaglandin F synthase (PGFS) [AKR 1C3], and dihydrodiol dehydrogenase (DD) 4 [AKR 1C4] genes. These genes had similar exon‐intron organizations and shared a high homology. The four recombinant enzymes encoded by these genes showed distinct substrate specificity. By reverse transcription‐PCR analysis, human 20α‐HSD, BABP and PGFS mRNAs were expressed ubiquitously, while DD4 mRNA was restricted to the liver. Promoter activities of the 20α‐HSD, BABP and PGFS genes were high, both in ovarian granulosa cells and hepatocytes. Radiation hybridization analysis revealed that all these genes were located close together in chromosome 10.ConclusionThe human gene encoding for the progesterone‐metabolizing enzyme 20α‐HSD in the female reproductive system was cloned, and its expression and gene localization were elucidated. BABP, PGFS and DD4 genes, which were highly homologous to the 20α‐HSD gene, were also cloned, and their structure and function were characterized.
Intronic U50 small‐nucleolar‐RNA (snoRNA) host gene of no protein‐coding potential is mapped at the chromosome breakpoint t(3;6)(q27;q15) of human B‐cell lymphoma
Authors: Tanaka Ritsuko , Satoh Hitoshi , Moriyama Masatsugu , Satoh Kasumi , Morishita Yasuyuki , Yoshida Syouko , Watanabe Toshiki , Nakamura Yoshikazu , Mori Shigeo 
Published: March 2003

BackgroundIn eukaryotic cells, nucleolar processing of preribosomal RNAs (prerRNAs) is assisted by a large number of small nucleolar RNAs (snoRNAs) that function in the 2′‐O‐methylation or the pseudouridylation of rRNAs. Most snoRNAs so far characterized are encoded and processed from introns of premRNAs.ResultsWe found a novel intronic snoRNA gene, named U50HG, located on chromosome 6q15, at the breakpoint of chromosomal translocation t(3;6)(q27;q15). The U50HG gene is composed of six exons, whose spliced transcripts have little potential for coding a protein, and its introns produce both U50 and U50‐like (U50′) snoRNAs that are localized in nucleoli. It possesses an oligopyrimidine tract that is characteristic of the 5′‐terminal oligopyrimidine (5′TOP) class of genes which have been shown to be coordinately regulated in response to cell growth.Conclusions U50HG is a member of the nonprotein‐coding multiple snoRNA host gene family, as well as of the 5′TOP gene family similar to UHG (U22 host gene), U17HG (U17 host gene), U19HG (U19 host gene) and gas5 (growth arrest‐specific transcript 5 gene). It is novel to find that the snoRNA gene is located at the breakpoint of chromosomal translocation t(3;6)(q27;q15) involved in human B‐cell lymphoma.
Rev‐dependent association of the intron‐containing HIV‐1 gag mRNA with the nuclear actin bundles and the inhibition of its nucleocytoplasmic transport by latrunculin‐B
Authors: Kimura Tominori , Hashimoto Iwao , Yamamoto Akitsugu , Nishikawa Masao , Fujisawa Jun‐ichi 
Published: March 2003

BackgroundA hallmark of HIV‐1 gene expression is that unspliced genomic RNA, which also acts as mRNA for the expression of Gag/Pol, is exported to the cytoplasm. Rev directs this transport through the nuclear export signal (NES).ResultsFluorescence in situ hybridization and immunocytochemistry demonstrated that gag mRNA, Rev, and its NES receptor, CRM1, and RanGTPase formed nuclear tracks which were congruent with underlying β‐actin bundles. Actin bundle formation was confirmed electron‐microscopically. These bundles were observed upon Rev‐containing gag RNP formation. The loss of bundles was associated with the nuclear retention of gag mRNA. Reverse transcription‐polymerase chain reaction analysis of both cytoplasmic and nuclear gag mRNAs demonstrated that disruption of nuclear actin filament formation by latrunculin‐B (LAT‐B), an F‐actin depolymerizing compound, resulted in the dose‐dependent inhibition of gag mRNA export. The differential subtyping of the mRNA‐positive cells confirmed morphologically the effect of LAT‐B treatment. The export inhibition was specific to gag mRNA and export of fully spliced HIV‐1 tat/rev mRNAs as well as cellular GAPDH mRNA was not affected by the compound.ConclusionsNuclear β‐actin bundles are suggested to be functionally involved in the Rev‐dependent nucleocytoplasmic transport of intron‐containing HIV‐1 gag mRNA.
Regulation of human myocilin/TIGR gene transcription in trabecular meshwork cells and astrocytes: role of upstream stimulatory factor
Authors: Kirstein Lucienne , Cvekl Ales , Chauhan Bharesh K. , Tamm Ernst R. 
Published: March 2003

BackgroundMutations in the myocilin (MYOC)/TIGR gene are responsible for autosomal‐dominant juvenile primary open‐angle glaucoma (POAG). In patients with non‐autosomal‐dominant POAG, such mutations are rare, but the expression of MYOC/TIGR in the trabecular meshwork (TM) of the eye is considerably higher than in normals. We performed transfection, DNAse I footprinting, mutagenesis and electrophoretic mobility shift assays (EMSA) to identify elements responsible for the basal transcription of MYOC/TIGR in TM cells and astrocytes.ResultsDNAse I footprinting experiments of the human MYOC/TIGR promoter showed a major protected area between nt −106 to −77, which was not conserved in the homologous region of the mouse myoc/tigr promoter. In addition, the TATA‐box was protected, as well as at least three downstream sites, including an AP‐1‐like sequence. Deletion of the −106 to −77 region caused a substantial loss of functional promotor activity in all cell types. Site‐directed mutagenesis and EMSA experiments revealed the presence of two regulatory elements in the −106 to −77 region. Each of these cis‐elements is essential for minimal promoter activity. The 5′‐half of the region contains a sequence with similarities to NF‐κB‐related sites, however, binding of NF‐κB could not be confirmed by EMSA. The 3′‐half contains a canonical E‐box sequence. EMSA experiments showed that the upstream regulatory factor (USF) was binding to the E‐box sequence and that the binding can be supershifted by specific antibodies.ConclusionsSeveral DNA‐protein binding elements contribute to a transcription of MYOC/TIGR, and USF is critically required for its basal transcription in trabecular meshwork cells and astrocytes.
The ERK MAP kinase pathway mediates induction of SGK (serum‐ and glucocorticoid‐inducible kinase) by growth factors
Authors: Mizuno Hiroshi , Nishida Eisuke 
Published: March 2003

BackgroundThe ERK MAP kinase pathway plays a pivotal role in growth factor‐induced gene expression. However, genes whose expression is induced by the ERK pathway are not fully defined.ResultsWe have identified SGK (serum‐ and glucocorticoid‐inducible kinase) as an ERK‐inducible gene by the subtractive screening of Raf‐inducible genes. SGK is known to be similar in primary structure to AKT/PKB, PKC and PKA. Treatment of quiescent NIH‐3T3 cells with FGF, PDGF or TPA, which induced the sustained activation of ERKs, resulted in the strong induction of SGK, whereas treatment with EGF, which induced the transient activation of ERKs, did not induce a strong expression of SGK. The induction of SGK was blocked by pre‐treatment with a specific MEK inhibitor U0126, and expression of constitutively active MEK was able to induce SGK. Treatment with cycloheximide or vanadate prolonged the increased expression of SGK by FGF, concomitant with a more prolonged activation of ERKs.ConclusionGrowth factor‐induced activation of the ERK MAP kinase pathway is necessary and sufficient for the induction of SGK.
Functional divergence of the TFL1‐like gene family in Arabidopsis revealed by characterization of a novel homologue
Authors: Mimida Naozumi , Goto Koji , Kobayashi Yasushi , Araki Takashi , Ahn Ji Hoon , Weigel Detlef , Murata Minoru , Motoyoshi Fusao , Sakamoto Wataru 
Published: March 2003

BackgroundThe TERMINAL FLOWER 1 (TFL1) gene of Arabidopsis plays an important role in regulating flowering time and in maintaining the fate of inflorescence meristem (IM). TFL1 is a homologue of CENTRORADIALIS (CEN) from Antirrhinum, which is only involved in IM maintenance. Recent mutational studies and the genome project revealed that TFL1 belongs to a small gene family in Arabidopsis, in which functional divergence may have occurred among the members.ResultsWe found a new member of the TFL1 gene family, which is mapped on chromosome 2 of Arabidopsis. The predicted protein sequence encoded by this gene is more closely related to that of CEN than other Arabidopsis TFL1 homologues (and therefore named ATC for ArabidopsisthalianaCENTRORADIALIS homologue). Transgenic plants constitutively expressing the ATC gene (35S::ATC), in either wild‐type or tfl1 mutant backgrounds, showed a phenotype similar to that observed in transgenic plants constitutively expressing the TFL1 gene. However, in contrast to TFL1, the expression of ATC was only detected in the hypocotyl of young plants, and not in the IM. In addition, an atc loss‐of‐function mutant, isolated by screening a T‐DNA library, showed no phenotypes that were similar to those of tfl1 mutants.ConclusionThe phenotypes of transgenic plants over‐expressing ATC suggest that the ATC protein can functionally substitute for TFL1. However, the pattern and level of expression and the loss‐of‐function phenotype indicate that ATC does not participate in the regulation of IM identity, but rather has a role that is different from that of TFL1.
The central MHC gene, BAT1, may encode a protein that down‐regulates cytokine production
Authors: Allcock Richard J. N. , Williams James H. , Price Patricia 
Published: March 2003

BackgroundBAT1 belongs to the DEAD‐box family of RNA‐binding proteins and is encoded in the central MHC. To determine whether it affects immune responses and hence diseases influenced by MHC haplotypes, U937, THP1 and Jurkat cells were stably transfected with anti‐sense DNA corresponding to exons 2–5 of BAT1 using a retroviral vector.ResultsAnti‐sense transfectants carried anti‐sense DNA and expressed anti‐sense mRNA. After mitogenic stimulation, they produced higher levels of TNFα, IL‐1 and IL‐6 than equivalent cells carrying the vector alone, suggesting that BAT1 may down‐regulate acute phase cytokine production. Polyclonal antibodies raised against a peptide in exon 8 of BAT1 recognized ≈50 kDa and ≈38 kDa proteins in all cell lines tested, including the anti‐sense transfectants. Expression was localized to the nucleolus in dividing fibroblasts. However the immunochemistry may be confounded by a recently described gene, DDXL, on chromosome 19, which shares a 89% amino acid identity with BAT1. RT‐PCR analyses established that BAT1 and DDXL mRNA are expressed in resting U937, THP1 and Jurkat cells. BAT1 and DDXL are divergent in the exons selected for the anti‐sense study.ConclusionsBAT1 is a negative regulator of inflammation. Future studies should address how its functions relate to those of DDXL.
Diverse gene expression and function of semaphorins in developing lung: positive and negative regulatory roles of semaphorins in lung branching morphogenesis
Authors: Kagoshima Masako , Ito Takaaki ,   Hitoshi Kitamura and Yoshio Goshima
Published: March 2003

Background Previously, we reported that Sema3A, one of the secreted repulsive axon guidance molecules, CRMP (collapsin response mediator protein)‐2, a putative intracellular signalling molecule for Sema3A and Sema3A receptor neuropilin‐1 are expressed in the developing lung. Sema3A inhibits branching morphogenesis of embryonic lung in organ culture.Results We examined the gene expression of Sema3A, Sema3C, Sema3F and their receptors, NP‐1, NP‐2 and plexin‐A1 by in situ hybridization. Transcripts of all six genes were detected in mouse lung from embryonic day E11.5 to E17.5, and displayed highly specific spatiotemporal distributions. The distribution of the receptor genes was detected in patterns which were consistent with known receptor usage of the semaphorins. In contrast to Sema3A, we found that the other class 3 semaphorins, Sema3C and Sema3F, stimulated branching morphogenesis. This stimulatory effect of Sema3C or Sema3F was accompanied by a moderate increase in the incorporation of bromodeoxyuridine (BrdU) into DNA in the terminal epithelial cells.Conclusion The coordinated expression patterns of different semaphorins and their receptors, together with the specific activities affecting branching morphogenesis, suggest that the semaphorins act as both positive and negative regulators of branching morphogenesis in the developing lung.
Ypel1: a novel nuclear protein that induces an epithelial‐like morphology in fibroblasts
Authors: Farlie Peter , Reid Candice , Wilcox Stephen , Peeters Justine , Reed Gullveig , Newgreen Don 
Published: March 2003

Background Embryonic development depends on the regulation of cell morphology and behaviour to carry out morphogenesis. One example of this is the development of the face, which is constructed from independent blocks of tissue that must grow in a coordinated way and then fuse to form a continuous tissue.Results We have isolated a novel gene of unknown function from mouse and quail embryos in a search for genes involved in craniofacial development. Sequence analysis of this gene, known as Ypel1, demonstrates a striking level of amino acid conservation between vertebrates and invertebrates but no significant homology with any other characterized genes. Ypel1 is expressed in the ventral half of early embryos including the branchial arches from which the face derives. Ypel1 localizes to the nucleus, and transfection into fibroblasts induces an epithelial‐like transition, which is accompanied by alterations to the cytoskeleton and cell adhesion machinery. In addition, human YPEL1 localizes to chromosome 22q11.2, a region associated with a number of syndromes involving malformation of the craniofacial complex.Conclusion These data suggest a role in regulation of cellular morphology and behaviour that is important for development of the craniofacial complex.
Cell death with predominant apoptotic features in Saccharomyces cerevisiae mediated by deletion of the histone chaperone ASF1/CIA1
Authors: Yamaki Mariko , Umehara Takashi , Chimura Takahiko , Horikoshi Masami 
Published: March 2003

Background Although no potential homologues of multicellular apoptotic genes (e.g. Bax, Bak, Bcl‐2, caspases and p53) have been identified in a unicellular eukaryote, previous reports contain several implications of the apoptotic behaviour of yeasts (i.e. Saccharomyces cerevisiae and Schizosaccharomyces pombe). Therefore, whether or not yeast undergoes apoptosis has been a topic of some debate. hCCG1, which is the largest subunit of TFIID and a histone acetyltransferase, appears to be involved in the regulation of apoptosis. The factor hCIA interacts with hCCG1 and functions as a histone chaperone in mammalian cells; its homologue in yeast is Asf1p/Cia1p. Therefore, we anticipated that a yeast mutant in Asf1p/Cia1p would be a valuable tool for studying apoptosis in yeast.Results We established a strain of S. cerevisiae lacking the histone chaperone ASF1/CIA1. This disruptant, asf1/cia1, arrested preferentially at the G2/M‐phase and died. We systematically analysed the phenotype associated with the death of this mutant yeast and identified many changes, such as fragmentation of the nuclei, condensation and fragmentation of chromatin, reduction of the mitochondrial membrane‐potential, dysfunction of the mitochondrial proton pump, and a discernible release of cytochrome c to cytoplasm that resembles those in apoptotic multicellular organisms. Other changes potentially associated with the death in our mutant included a reduction in the vacuolar membrane potential, dysfunction of the vacuolar proton pump, reduction of endocytosis, and the presence of many autophagic bodies. However, these mutant yeast cells also showed cellular enlargement, which is characteristic of necrosis.Conclusions Cell death in S. cerevisiae occurs with a phenotype that largely resembles apoptosis in multicellular organisms, but that has some features of necrosis. Therefore, we indicate that yeast undergoes a ‘prototypal active cell death’ that retains some characteristics of passive cell death (necrosis). In addition, we think that active cell death is ubiquitously the essential attribute of life. Although such an active cell death system in yeast remains open to confirmation, we speculate that deletion of the histone chaperone Asf1p/Cia1p inhibits the normal assembly/disassembly of nucleosomes in yeast and thereby initiates the active cell death system.
The interaction between σS, the stationary phase σ factor, and the core enzyme of Escherichia coli RNA polymerase
Authors: Colland Frédéric , Fujita Nobuyuki , Ishihama Akira , Kolb Annie 
Published: March 2003

AbstractBackground: The RNA polymerase holoenzyme of Escherichia coli is composed of a core enzyme (subunit structure α2ββ′) associated with one of the σ subunits, required for promoter recognition. Different σ factors compete for core binding. Among the seven σ factors present in E. coli, σ70 controls gene transcription during the exponential phase, whereas σS regulates the transcription of genes in the stationary phase or in response to different stresses. Using labelled σS and σ70, we compared the affinities of both σ factors for core binding and investigated the structural changes in the different subunits involved in the formation of the holoenzymes.Results: Using native polyacrylamide gel electrophoresis, we demonstrate that σS binds to the core enzyme with fivefold reduced affinity compared to σ70. Using iron chelate protein footprinting, we show that the core enzyme significantly reduces polypeptide backbone solvent accessibility in regions 1.1, 2.5, 3.1 and 3.2 of σS, while increasing the accessibility in region 4.1 of σS. We have also analysed the positioning of σS on the holoenzyme by the proximity‐dependent protein cleavage method using σS derivatives in which FeBABE was tethered to single cysteine residues at nine different positions. Protein cutting patterns are observed on the β and β′ subunits, but not α. Regions 2.5, 3.1 and 3.2 of σS are close to both β and β′ subunits, in agreement with iron chelate protein footprinting data.Conclusions: A comparison between these results using σS and previous data from σ70 indicates similar contact patterns on the core subunits and similar characteristic changes associated with holoenzyme formation, despite striking differences in the accessibility of regions 4.1 and 4.2.