bluuebunny/crossref_metadata_2025_split huggingface dataset:
Functional sites of human PCNA which interact with p21 (Cip1/Waf1), DNA polymerase δ and replication factor C
Authors: Oku Takashi , Ikeda Soichiro , Sasaki Hisashi , Fukuda Kotaro , Morioka Hiroshi , Ohtsuka Eiko , Yoshikawa Hiroshi , Tsurimoto Toshiki 
Published: March 2003

Background:PCNA, an eukaryotic DNA sliding clamp interacts with replication factors and the cell cycle protein, p21(Cip1/Waf1) and functions as a molecular switch for DNA elongation. To understand how DNA replication is regulated through PCNA, elucidation of the precise mechanisms of these protein interactions is necessary.Results:Loop‐region mutants in which human PCNA sequences were substituted with the corresponding Saccharomyces cerevisiae PCNA regions were prepared. Analysis of their functions, along with previously prepared alanine scanning mutants, demonstrated that some loops interact with DNA polymerase δ (pol δ) and replication factor C (RFC). The p21 binding sites of PCNA, mapped by affinity measurement of the mutant forms, found to be located within a distinct structure of the PCNA monomer, overlap with RFC‐ and pol δ‐interaction sites. Competition between p21 and pol δ or RFC for binding to PCNA results in efficient inhibition of its stimulation of pol δ DNA synthesis and RFC ATPase but not of PCNA loading on DNA by RFC.Conclusions:Semi‐saturated amounts of p21 selectively block formation of the active pol δ complex but not the RFC–PCNA complex at 3′‐ends of DNA primers. This differential effect may explain the specific inhibition of DNA replication by p21.
Escherichia coli HU protein suppresses DNA‐gyrase‐mediated illegitimate recombination and SOS induction
Authors: Shanado Yuka , Kato Jun‐ichi , Ikeda Hideo 
Published: March 2003

BackgroundThe HU protein is an abundant DNA binding protein of bacteria and is a major constituent of the bacterial nucleoid. HU protein is known to be involved in several fundamental biological functions, including DNA supercoiling, DNA replication, site‐specific DNA inversion, and transposition. It is generally thought that a functional relationship exists between HU protein and DNA gyrase.ResultsWe found that an hupA hupB double mutant displays enhanced spontaneous illegitimate recombination during the formation of λbio transducing phage in Escherichia coli. Nucleotide sequence analysis of the resulting transducing phages showed that the E. coli bio and λ recombination sites did not have any homologous sequence. This mutation also enhanced the spontaneous expression of SOS functions. Furthermore, either overproduced GyrA protein or a temperature‐sensitive gyrB mutation suppressed the illegitimate recombination enhanced by the defect of HU protein.ConclusionThese results show that the defect of HU induces illegitimate recombination and SOS response, which are probably mediated by DNA gyrase, implying that HU protein plays roles in suppression of illegitimate recombination and SOS response through interaction with DNA gyrase.
Role of p300, a transcriptional coactivator, in signalling of TGF‐β
Authors: Nishihara Ayako , Hanai Jun‐ichi , Okamoto Nobuaki , Yanagisawa Jun , Kato Shigeaki , Miyazono Kohei , Kawabata Masahiro 
Published: March 2003

BackgroundSmad proteins are novel transcriptional regulators mediating the signalling of the transforming growth factor‐β (TGF‐β) superfamily. Coactivators such as p300/CBP promote transactivation by various transcription factors through a direct interaction with them. Adenoviral oncoprotein E1A, which binds p300, was shown to inhibit the signalling of TGF‐β. These findings raise the possibility that p300 may be involved in TGF‐β signalling.ResultsWe investigated whether p300 is involved in transactivation by Smads. p300 enhanced the Smad‐induced transactivation of p3TP‐Lux, a TGF‐β responsive reporter. E1A inhibited this enhancement, and the inhibition required its ability to bind p300/CBP. p300 and Smad3, as well as Smad2, interacted in vivo in a ligand‐dependent manner. The binding region in Smad3 was its C‐terminal half that was previously shown to possess an intrinsic transactivation activity. The binding region in p300 was mapped to its C‐terminal 678 amino acids. The minimal Smad2/3‐interacting region, as well as the rest of the p300, inhibited the transactivation of p3TP‐Lux in a dominant‐negative fashion.Conclusionp300 interacted with Smad2 and Smad3 in a ligand‐dependent manner, and enhanced the transactivation by Smads. Our results present the molecular basis of the transactivation by Smad proteins.
Cooperation of two actin‐binding proteins, cofilin and Aip1, in Saccharomyces cerevisiae
Authors: Iida Kazuko , Yahara Ichiro 
Published: March 2003

BackgroundCofilin is a low‐molecular weight actin‐modulating protein, and is structurally and functionally conserved among eukaryotes. Cofilin is encoded by COF1 in Saccharomyces cerevisiae, and is essential for cell viability. Cofilin binds to and severs actin filaments in vitro, and also enhances their depolymerization. A partner protein that cooperates with cofilin in vivo has not been identified.ResultsWhen COF1 was over‐expressed in yeast cells under the GAL1 promoter in a medium containing galactose as a sole carbon source, the cells did not survive. These results indicate that cells can grow only when the expression of cofilin is appropriately regulated. Several temperature sensitive (ts‐) mutants were independently created by the random mutagenesis of COF1 with hydroxylamine. Mutated amino acids in ts‐mutants were mapped in the sequences that were presumed to be involved in actin binding. A gene on a multicopy plasmid which suppresses the ts‐phenotype of cof1‐101, a typical ts‐cofilin mutant, was isolated. The suppressor gene, SCF1, was found to be identical to AIP1, a gene encoding an actin‐interacting protein. Although SCF1/AIP1 is not essential for cell viability, a combination of cof1‐101 and Δscf1/aip1 is synthetic lethal. Immunofluorescence staining of a wild‐type strain using anti‐Aip1 antibodies revealed that Aip1 was distributed in cortical actin patches where cofilin was also co‐localized. Thick and long fibres stained with anti‐cofilin antibody were detected in Δscf1/aip1 cells, but not in SCF1/AIP1 cells.ConclusionsThese results suggest the cooperative modulation of the actin cytoskeleton by cofilin and Aip1.
DCRY is a Drosophila photoreceptor protein implicated in light entrainment of circadian rhythm
Authors: Ishikawa Tomoko , Matsumoto Akira , Kato Jr Tomohisa , Togashi Shin , Ryo Haruko , Ikenaga Mituo , Todo Takeshi , Ueda Ryu , Tanimura Teiichi 
Published: March 2003

Background: Light is the major environmental signal for the entrainment of circadian rhythms. In Drosophila melanogaster, the period(per) and timeless (tim) genes are required for circadian behavioural rhythms and their expression levels undergo circadian fluctuations. Light signals can entrain these rhythms by shifting their phases. However, little is known about the molecular mechanism for the perception and transduction of the light signal. The members of the photolyase/cryptochrome family contain flavin adenine dinucleotide (FAD) as chromophore and are involved in two diverse functions, DNA repair and photoreception of environmental light signals.Results: We report the cloning of a new member of this family, dcry, from Drosophila. Northern blot analysis shows that this gene is expressed in various tissues. The dcry mRNA is expressed in a circadian manner in adult heads, while such rhythmic fluctuation is abolished in the clock‐defective per0 and tim0 mutants. The circadian expression is dampened down in constant darkness. The over‐expression of the dcry gene alters the light‐induced phase delay in the locomotor activity rhythms of flies.Conclusion: These results suggest that DCRY is a circadian photoreceptor and that its expression is regulated by circadian clock genes.
Severe osteopetrosis, defective interleukin‐1 signalling and lymph node organogenesis in TRAF6‐deficient mice
Authors: Naito Asuka , Azuma Sakura , Tanaka Sakae , Miyazaki Tsuyoshi , Takaki Satoshi , Takatsu Kiyoshi , Nakao Kazuki , Nakamura Kenji , Katsuki Motoya , Yamamoto Tadashi , Inoue Jun‐ichiro 
Published: March 2003

BackgroundTRAF6, a member of the tumour necrosis factor receptor‐associated factor family, was first identified as a transducer of CD40 and interleukin‐1 receptor (IL‐1R) signals based on the interaction of TRAF6 with the cytoplasmic tail of CD40 and with the IL‐1R associated kinase in vitro. However, the functions of TRAF6 in vivo remain unidentified.ResultsWe show that TRAF6−/− mice exhibit severe osteopetrosis and are defective in osteoclast formation. In vitro culture experiments revealed that osteoclast precursor cells derived from TRAF6−/− mice are unable to differentiate to functional osteoclasts in response to osteoclast differentiation factor (ODF). In bone marrow of TRAF6−/− mice, the number of sIgM+B220+ immature B cells is markedly reduced while the ratio of proB to preB cells is not affected. In contrast, development of thymocytes is not affected. Furthermore, TRAF6−/− mice are defective in lymph node organogenesis and IL‐1 signalling in thymocytes.ConclusionsThe results identify TRAF6 as an essential component of ODF signalling pathway, and also show that TRAF6 plays pivotal roles in immune and inflammatory systems in vivo.
Two separate sequences of PB2 subunit constitute the RNA cap‐binding site of influenza virus RNA polymerase
Authors: Honda Ayae , Mizumoto Kiyohiso , Ishihama Akira 
Published: March 2003

BackgroundInfluenza virus RNA polymerase with the subunit composition of PB1‐PB2‐PA is a unique multifunctional enzyme with the activities of both synthesis and cleavage of RNA, and is involved in both transcription and replication of the RNA genome. Transcription is initiated by using capped RNA fragments, which are generated after cleavage of host cell mRNA by the RNA polymerase‐associated capped RNA endonuclease. To identify the RNA cap 1‐binding site on the RNA polymerase, viral ribonucleoprotein (RNP) cores were subjected to UV‐crosslinking with RNA which was labelled with 32P only at the cap‐1 structure.ResultsAfter SDS‐PAGE of UV‐crosslinked cores, 32P was found to be associated only with the PB2 subunit (759 amino acid residues). The labelled PB2 was subjected, together with PB2 expressed in E. coli, to limited digestion with V8 protease. Analysis of the amino terminal sequences of some isolated fragments with the crosslinked cap‐1 indicated that two separate sequences within the PB2 were involved in RNA cap‐1 binding, one (N‐site) at the N‐terminal proximal region approximately between amino acid residues 242–282 downstream from the PB1 subunit‐binding site and the other (C‐site) between residues 538–577 including the cap‐binding motifs. Two lines of evidence support the prediction of the involvement of two separate PB2 sequences on the RNA cap‐binding: (i) cross‐linking of the capped RNA on to expressed and isolated PB2 fragments, each containing either the N‐site or the C‐site; and (ii) competition of capped RNA‐binding to PB2 by both of the N‐ and C‐terminal PB2 fragments. Taking together, we propose that two separate sequences within PB2 constitute the capped RNA‐binding site of the RNA polymerase.ConclusionTwo separate sequences, one N‐(242–282) and the other C‐terminal (538–577) proximal segments of PB2 subunit, constitute the RNA cap‐binding site of the influenza virus RNA polymerase.
A Hrs binding protein having a Src homology 3 domain is involved in intracellular degradation of growth factors and their receptors
Authors: Takata Hiroyuki , Kato Masaki , Denda Kimitoshi , Kitamura Naomi 
Published: March 2003

BackgroundHrs (hepatocyte growth factor (HGF)‐regulated tyrosine kinase substrate) is an early endosomal protein that is rapidly tyrosine‐phosphorylated in cells stimulated with growth factors. Hrs is thought to play a regulatory role in the endocytosis of growth factor/receptor complexes through early endosomes. In this study, we searched for Hrs‐interacting molecules which may regulate the function of Hrs, using a yeast two‐hybrid system.ResultsWe isolated a cDNA clone encoding a novel Src homology 3 (SH3)‐containing protein, and named it ‘Hrs binding protein’ (Hbp). Hbp was co‐immunoprecipitated with Hrs, and its intracellular localization was similar to that of Hrs. The association between Hbp and Hrs was mediated through the coiled coil motifs in Hbp and Hrs. Deletion mutants of Hbp lacking either the SH3 domain or the Hrs binding domain showed dominantly negative effects on the intracellular degradation of a growth factor and its receptor, but not on the internalization of growth factor/receptor complexes.ConclusionsHbp is thought to be closely associated with Hrs on early endosomes. Hbp, together with Hrs may play a regulatory role in the vesicular transport of growth factor/receptor complexes through early endosomes, for their degradation.
NF‐κB activation through IKK‐i‐dependent I‐TRAF/TANK phosphorylation
Authors: Nomura  , Kawai  , Nakanishi Kenji , Akira Shizuo 
Published: March 2003

BackgroundNF‐κB is an ubiquitously expressed transcription factor that plays an important role in the immune, anti‐apoptotic and inflammatory responses. NF‐κB is normally sequestered in the cytoplasm by interacting with inhibitory IκB molecules. Upon stimulation, IκB is phosphorylated and subsequently degraded by the proteasome, allowing NF‐κB to translocate into the nucleus where they regulate target gene expression. Two kinases, IKK‐α and IKK‐β, which are responsible for IκB phosphorylation were recently identified. We have recently identified a cytokine inducible IKK‐i, a kinase related to IKK‐α and ‐β. IKK‐i significantly induced NF‐κB activation upon over‐expression, as did IKK‐α and IKK‐β. Unlike IKK‐α and IKK‐β, IKK‐i phosphorylated Ser36 but not Ser32 in vitro, suggesting that IKK‐i activates NF‐κB by distinct mechanisms from the conventional IKKs.ResultsI‐TRAF/TANK was isolated as a molecule that interacts specifically with inducible IκB kinase (IKK‐i) by the yeast two‐hybrid screening procedure. The association of IKK‐i and I‐TRAF is mediated via the interaction between the N‐terminal domain of I‐TRAF and the C‐terminal portion of IKK‐i. In vitro kinase assays demonstrate that IKK‐i phosphorylates I‐TRAF in the middle portion that associates with TRAF2. Interestingly, TRAF2 is freed from the I‐TRAF/TRAF2 complex after I‐TRAF phosphorylation. NF‐κB activation by IKK‐i is significantly blocked by coexpression of the N‐terminal domain of I‐TRAF, dominant negative TRAF2, and dominant negative NIK and IKK‐β. IKK‐i over‐expression also induced c‐Jun N‐terminal kinase. These results show that I‐TRAF is a substrate of IKK‐i. NF‐κB activation by IKK‐i may be mediated through phosphorylation of I‐TRAF by IKK‐i and subsequent liberation of TRAF2.ConclusionThese results indicate that NF‐κB activation by IKK‐i is mediated through phosphorylation of I‐TRAF/TANK by IKK‐i and subsequent liberation of TRAF2.
Identification and characterization of testis specific ornithine decarboxylase antizyme (OAZ‐t) gene: expression in haploid germ cells and polyamine‐induced frameshifting
Authors: Tosaka Yasuhiro , Tanaka Hiromitsu , Yano Yoshihisa , Masai Kumiko , Nozaki Masami , Yomogida Kentaro , Otani Shuzo , Nojima Hiroshi , Nishimune Yoshitake 
Published: March 2003

BackgroundPolyamines are known to play important roles in the proliferation and differentiation of many types of cells. However, in the testis, where polyamines such as spermidine and spermine exist in high concentrations, their roles still remains to be elucidated.ResultsWe have cloned a testis‐specific gene encoding an ornithine decarboxylase antizyme known to control intracellular concentrations of polyamines in a feedback manner. The mRNA encoding the protein named ornithine decarboxylase antizyme in testis (OAZ‐t) was specifically expressed in haploid germ cells. In contrast, the mRNA level of somatic ornithine decarboxylase antizyme 1 (OAZ1) decreased markedly at the late stages of haploid germ cell differentiation. OAZ‐t mRNA was first observed in 23‐day‐old mice, whereas the OAZ‐t protein was detected much later, at 35 days after birth. Further experiments on OAZ‐t revealed that polyamines were capable of inducing a frameshifting at the frameshift sequence of OAZ‐t mRNA, resulting in the translation of OAZ‐t, as was the case with the somatic OAZ1. Transfection of OAZ‐t cDNA inactivated the ornithine decarboxylase activity in the HEK293 cells.ConclusionsResults indicate that the expression of OAZ‐t is controlled at both transcriptional and translational levels, and that OAZ‐t likely plays a key role in spermatogenesis by regulating the intracellular concentration of polyamines in haploid germ cells.
MST1‐JNK promotes apoptosis via caspase‐dependent and independent pathways
Authors: Ura Seiji , Masuyama Norihisa , Graves Jonathan D. , Gotoh Yukiko 
Published: March 2003

Background MST1 is an upstream kinase of the JNK and p38 MAPK pathways whose expression induces apoptotic morphological changes such as nuclear condensation. During apoptosis, caspase cleavage of MST1 removes a C‐terminal regulatory domain, increasing the kinase activity of the MST1 N‐terminal domain. Downstream pathways of MST1 in the induction of apoptosis remain to be clarified.
Results
In this study, we found that the expression of MST1 resulted in caspase‐3 activation. Therefore, MST1 is not only a target of caspases but also an activator of caspases. This caspase activation and apoptotic changes occur through JNK, since the co‐expression of a dominant‐negative mutant of JNK inhibited MST1‐induced morphological changes as well as caspase activation. In contrast, neither a dominant‐negative p38 nor the p38 inhibitor SB203580 inhibited them. MST1 induced nucleosomal DNA fragmentation, which was suppressed by caspase inhibitors or ICAD (Inhibitor of Caspase‐Activated DNase). Surprisingly, however, other changes such as membrane blebbing and chromatin condensation were not inhibited by caspase inhibitors.
Conclusion
These results suggest that MST1 most likely promotes two events through JNK activation; first, MST1 induces the activation of caspases, resulting in CAD‐mediated DNA fragmentation, and second, MST1 induces chromatin condensation and membrane blebbing without utilizing downstream caspases.
Bacterial SsrA system plays a role in coping with unwanted translational readthrough caused by suppressor tRNAs
Authors: Ueda Koji , Yamamoto Yasufumi , Ogawa Kazuko , Abo Tatsuhiko , Inokuchi Hachiro , Aiba Hiroji 
Published: March 2003

AbstractBackgrounds: Bacterial SsrA RNA (also known as tmRNA or 10Sa RNA) mediates the addition of a short peptide tag to the C‐terminus of the nascent polypeptide when a ribosome is stalled at the 3′ end of an mRNA lacking a stop codon. This process, called trans‐translation, rescues the stalled ribosome and ensures degradation of tagged polypeptides by ATP‐dependent proteases. To fully understand the physiological roles of SsrA RNA, it is essential to know how endogenous mRNA targets for the SsrA system are generated in cells. The aim of the present study is to examine how translational readthrough by suppressor tRNAs affects trans‐translation in Escherichia coli.Results: We demonstrated that SsrA tagging of bulk cellular proteins was significantly enhanced by an ochre or an amber suppressor tRNA. Western blot analysis of proteins produced from specific genes possessing a Rho‐independent terminator revealed that readthrough at the normal stop codon leads to an efficient tagging and proteolysis of the extended proteins. Size analyses of both protein and mRNA suggested that tagging of extended proteins occurs because ribosome passing through the normal stop codon presumably reach the 3′ end of mRNA defined by the transcription terminator hairpin. The inhibitory effect of ssrA mutation on cell growth was markedly amplified in cells with an ochre suppressor tRNA.Conclusion: The present finding suggests that the SsrA system contributes to scavenge errors and/or problems caused by translational readthrough that occurs typically in the presence of a suppressor tRNA.
The CLN3/SWI6/CLN2 pathway and SNF1 act sequentially to regulate meiotic initiation in Saccharomyces cerevisiae
Authors: Purnapatre Kedar , Piccirillo Sarah , Schneider Brandt L. , Honigberg Saul M. 
Published: March 2003

AbstractBackground: IME1, which is required for the initiation of meiosis, is regulated by Cln3:Cdc28 kinase, which activates the G1‐to‐S transition, and Snf1 kinase, which mediates glucose repression. Here we examine the pathway by which Cln3:Cdc28p represses IME1 and the relationship between Cln3:Cdc28p and Snf1p in this regulation.Results: When wild‐type yeast cease growth, they express IME1 to moderate levels, intermediate between the low levels expressed during growth and the high levels expressed during sporulation. Moderate IME1 expression occurred in cln3Δ, cln1Δ cln2Δ, cdc28‐4 and swi6Δ mutants, even during growth. These mutants also induced IME1 expression more rapidly than the wild‐type. CLN3 required SWI6 and CLN2 to repress IME1 and IME2, but CLN1 was much less active than CLN2 in this repression. The phenotype of the cln3Δ snf1Δ double mutant indicated that Cln3:Cdc28p regulates IME1 independently of SNF1.Conclusion: Entry into meiosis involves two independent but sequential controls, which regulate IME1 via a three position switch: (i) during growth IME1 is repressed by the CLN3/SWI6/CLN2 pathway, (ii) once growth ceases, this repression is released and IME1 is expressed at moderate levels, and (iii) subsequently, nutritional conditions that activate Snf1p allow high IME1 expression.
A role of the C‐terminus of aquaporin 4 in its membrane expression in cultured astrocytes
Authors: Nakahama Ken‐ichi , Fujioka Atsuko , Nagano Mamoru , Satoh Shinsuke , Furukawa Kei‐ichi , Sasaki Hiroshi , Shigeyoshi Yasufumi 
Published: March 2003

AbstractBackground: Aquaporin 4 (AQP4) is a predominant water channel protein in mammalian brains, which is localized in the astrocyte plasma membrane. Membrane targeting of AQP4 is essential to perform its function. The mechanism(s) of membrane targeting is not clear in astrocytes.Results: We investigated the role of the C‐terminus of AQP4 (short isoform) in its membrane targeting by an expression study of C‐terminal mutants of AQP4 in cultured astrocytes. The deletion of 26 C‐terminal residues of AQP4 (AQP4Δ276–301aa) results in the intracellular localization of the protein. However, smaller deletions than 21 C‐terminal residues did not alter its plasma membrane localization. These results suggest that C‐terminal residues between Val276 and Ile280 play an important role in the expression of AQP4 in the plasma membrane. However, the plasma membrane localization of the AQP4(A276AAAA280) mutant (alanine substitution of Val276‐Ile280 of AQP4) suggests that another signal for membrane targeting exists in the C‐terminus of AQP4. The deletion or point mutations of the PDZ binding motif of the AQP4(A276AAAA280) mutant resulted in the intracellular localization of the proteins. These results suggest that the PDZ binding motif may also be involved in the membrane targeting of AQP4.Conclusions: We found that the C‐terminal sequence of AQP4 contains two important signals for membrane expression of AQP4 in cultured astrocytes. One is a hydrophobic domain and the other is a PDZ binding motif that exists in the C‐terminus.