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Cytokines and various cellular stresses are known to activate c-Jun NH2-terminal kinase (JNK), which plays a role in conveying signals from the cytosol to the nucleus. Here we investigate the translocation and activation of JNK1 during ischemia and reperfusion in perfused rat heart. Ischemia induces the translocation of JNK1 from the cytosol fraction to the nuclear fraction in a time-dependent manner. Immunohistochemical observation also shows that JNK1 staining in the nucleus is enhanced after ischemia. During reperfusion after ischemia, further nuclear translocation of JNK1 is apparently inhibited. In contrast, JNK1 activity in the nuclear fraction does not increased during ischemia but increases significantly during reperfusion with a peak at 10 min of reperfusion. The activation of JNK1 is confirmed by the phosphorylation of endogenous c-Jun (Ser-73) with similar kinetics. The level of c-jun mRNA also increases during reperfusion but not during ischemia. Based on fractionation and immunohistochemical analyses, an upstream kinase for JNK1, SAPK/ERK kinase 1 (SEK1), is constantly present in both the nucleus and cytoplasm throughout ischemia and reperfusion, whereas an upstream kinase for mitogen-activated protein kinase, MAPK/ERK kinase 1, remains in the cytosol. Furthermore, phosphorylation at Thr-223 of SEK1, necessary for its activation, rapidly increases in the nuclear fraction during postischemic reperfusion. These findings demonstrate that JNK1 translocates to the nucleus during ischemia without activation and is then activated during reperfusion, probably by SEK1 in the nucleus.

金沢大学がん研究所がん分子細胞制御, FK506 is a powerful immunosuppressive drug currently in use that inhibits the activation of several transcription factors (nuclear factor (NF)-AT and NF-κB) critical for T cell activation. We show here that, contrary to the situation in T cells, FK506 activates transcription factor NF-κB in non-lymphoid cells such as fibroblasts and renal mesangial cells. We further show that FK506 induces NF-κB-regulated IL-6 production in vitro and in vivo, in particular in kidney. IL-6 has been shown previously to produce renal abnormalities in vivo, such as mesangioproliferative glomerulonephritis. Similar renal abnormalities were also observed in FK506-treated animals. These results thus suggest a causal relationship between FK506-induced NF-κB activation/IL-6 production and some of FK506-induced renal abnormalities.

Cdc25A, which is one of the three mammalian CDK-activating Cdc25 protein phosphatases (Cdc25A, B and C), is degraded through SCFβTrCP-mediated ubiquitylation following genomic insult; however, the regulation of the stability of the other two Cdc25 proteins is not well understood. Previously, we showed that Cdc25B is primarily degraded by cellular stresses that activate stress-activated MAPKs, such as Jun NH2-terminal kinase (JNK) and p38. Here, we report that Cdc25B was ubiquitylated by SCFβTrCP E3 ligase upon phosphorylation at two Ser residues in the βTrCP-binding-motif-like sequence D94AGLCMDSPSP104. Point mutation of these Ser residues to alanine (Ala) abolished the JNK-induced ubiquitylation by SCFβTrCP, and point mutation of DAG to AAG or DAA eradicated both βTrCP binding and ubiquitylation. Further analysis of the mode of βTrCP binding to this region revealed that the PEST-like sequence from E82SS to D94AG is crucially involved in both the βTrCP binding and ubiquitylation of Cdc25B. Furthermore, the phospho-mimetic replacement of all 10 Ser residues in the E82SS to SPSP104 region with Asp resulted in βTrCP binding. Collectively, these results indicate that stress-induced Cdc25B ubiquitylation by SCFβTrCP requires the phosphorylation of S101PS103P in the βTrCP-binding-motif-like and adjacent PEST-like sequences. © 2011. Published by The Company of Biologists Ltd.

The numb er of retrotransposon copia per genome in Drosophila melanogaster cultured cells is two to three times higher than that in D. melanogaster embryo cells. Here, we have found that the genome of the related species, Drosophila simulans , contains in cultured cells more efficiently amplified copia DNA (approximately ten fold). Furthermore, we analyzed copia virus-like particles (VLPs) prepared from D. melanogaster and D. simulans cultured cells, which contain copia RNA and reverse transcriptase (RT) activity, and thus, play a major role in copia replication. The RT activity associated with the D. simulans VLPs was 25 times higher than that associated with the D. melanogaster VLPs. Taken together with the fact that copia is believed to transpose through an RNA intermediate, these results suggest that the amplification of copia DNA should relate to copia RNA-mediated transposition, and the higher RT activity associated with the D. simulans VLPs would lead to the efficient amplification of copia DNA. In a comparison between D. melanogaster and D. simulans copia nucleotide (nt) sequences, five nt substitutions, which cause the respective amino acid changes, were found in the copia RT-coding region. Polymerase chain reaction direct sequencing showed that these five substitutions are the vast majority in each Drosophila species. The substitutions, therefore, may be responsible for the high level of the RT activity associated with the D. simulans VLPs.

MafB is a basic leucine zipper transcription factor that plays important roles in development and differentiation processes. During osteoclastogenesis, its expression is downregulated at the transcriptional level via the JNK and p38 MAP kinase pathways. In the present study, we demonstrated that MafB protein stability is regulated by JNK and identified a phosphorylation site, Thr62. The expression of a constitutively active form of JNK (a fusion protein MKK7α1-JNK1β1) promoted the degradation of MafB in COS7 cells, and a T62A substitution significantly reduced the instability of MafB. The introduction of a four-fold (T58A/T62A/S70A/S74A) substitution in an acidic transcription-activating domain almost protected the instability resulting from the activation of JNK. Furthermore, treatment with proteasome inhibitors increased the MafB level, and a high-molecular-weight smear, characteristic of polyubiquitination, was observed in lysates from cells in which MafB, ubiquitin, and MKK7α1-JNK1β1 were co-expressed. These results suggest that phosphorylation of MafB by JNK confers susceptibility to proteasomal degradation., Article, ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS. 494(1):94-100 (2010)

金沢大学がん研究所がん分子細胞制御, Scaffold proteins for MAP kinase (MAPK) signalling modules play an important role in the specific and efficient signal transduction of the relevant MAPK cascades. Here, we investigated the function of the scaffolding protein c-Jun NH2-terminal kinase (JNK)-associated leucine zipper protein (JLP) by depleting it in cultured cells using a short hairpin RNA (shRNA) against human JLP. HeLa and DLD-1 cells stably expressing the shRNA showed a defect in cell migration. The re-expression of full-length shRNA-resistant mouse JLP rescued the impaired cell migration of the JLP-depleted HeLa cells; whereas, a C-terminal deletion mutant of mouse JLP, which failed to bind the G protein Gα13, showed little or no effect on the cell migration defect. Furthermore, although a constitutively active Gα13 enhanced the migration of control HeLa cells, the Gα13-induced cell migration was significantly suppressed in the JLP-depleted HeLa cells. Taken together, these results suggest that JLP regulates cell migration through an interaction with Gα13. © The Authors 2008. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

金沢大学がん研究所, Hepatitis C Virus (HCV) non-structural proteins are major components of replication complex that is modulated by several host factors. We previously reported that nucleolin, a representative nucleolar marker, interacts with the NS5B through two separated sequences, amino acids (aa) 208-214 and 500-506, and that W208 in the former stretch is essential for both nucleolin-binding and HCV replication. Here we evaluated the role of the latter stretch aa 500-506 of WRHRARS in nucleolin-binding and HCV replication scanned by alanine-substituted clustered mutant (cm) or point mutant (pm). One tryptophan and three arginine residues in the sequence were found to be essential both for nucleolin-binding in vivo and HCV replication detected with a HCV subgenomic replicon transfected into Huh7 cells. NS5B-binding of nucleolin was further delineated by truncation and clustered mutants of nucleolin. Arginine-glycine-glycine (RGG) repeat in the Glycine arginine rich (GAR) domain were defined to be indispensable for NS5B-binding immunologically detected in in vivo and in vitro although short internal-truncations of RGG repeat are tolerable for NS5B-binding. These results indicate that nucleolin is a critical host factor for HCV replication through the direct interaction between W208 and several residues at the sequence, aa 500-505, of NS5B, and the long-turn motif including RGG repeat at nucleolin C-terminal. © 2007 The Japanese Biochemical Society.

最近のいくつかの研究から, アルツハイマー病における痴呆の直接的原因となる選択的神経細胞死は, 細胞内情報伝達分子JNK3によって仲介されていると考えられる. 本研究では, JNK3と相互作用する蛋白質因子の同定およびその解析を試みた. 酵母 two-hybrid 法により, 新規JNK3結合蛋白 (BP#2と命名) のcDNAクローニングに成功した. BP#2は, 脊椎動物MAPキナーゼの中ではJNK3と特異的に結合し, さらにJNK活性化キナーゼSEK1との結合能も有することから, JNK3情報伝達系におけるスキャフォールド蛋白として機能していると考えられる. また, 繊維芽細胞内でBP#2を強制的に過剰発現させることによりJNK3の活性化が抑制されることを見出した. 神経細胞においても同様の現象が認められるならば, BP#2の過剰発現によりアルツハイマー病における神経細胞死を特異的に阻害することが可能となるかもしれない.

We previously reported that the level of c-Jun NH2-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1), a scaffold protein for JNK signaling, increases dramatically during nerve growth factor (NGF)-induced differentiation of PC12h cells. In the present study, we investigated the function of JSAP1 during PC12h cell differentiation by knocking down the level of JSAP1. The depletion of JSAP1 caused NGF-treated PC12h cells to form aggregates and impaired their differentiation. The aggregation was not observed in JSAP1-depleted cells that were untreated or treated with epidermal growth factor. Immunocytochemical studies indicated that N-cadherin, but not E-cadherin, was localized to sites of cell–cell contact in the aggregated cells. Furthermore, an inhibitory anti-N-cadherin antibody completely blocked the aggregation. Taken together, these results suggest that JSAP1 regulates cell–cell interactions in PC12h cells specifically in the NGF-induced signaling pathway, and does so by modulating N-cadherin.

金沢大学がん研究所がん分子細胞制御, The wild type Copia Gag precursor protein of Drosophila melanogaster expressed in Escherichia coli was shown to be processed autocatalytically to generate two daughter proteins with molecular masses of 33 and 23 kDa on SDS/PAGE. The active-site motif of aspartic proteinases, Asp-Ser-Gly, was present in the 23 kDa protein corresponding to the C-terminal half of the precursor protein. The coding region of this daughter protein (152 residues) in the copia gag gene was expressed in E. coli to produce the recombinant enzyme protein as inclusion bodies, which was then purified and refolded to create the active enzyme. Using the peptide substrate His-Gly-Ile-Ala-Phe-Met-Val-Lys-Glu-Val-Asn (cleavage site: Phe–Met) designed on the basis of the sequence of the cleavage-site region of the precursor protein, the enzymatic properties of the proteinase were investigated. The optimum pH and temperature of the proteinase toward the synthetic peptide were 4.0 and 70 °C respectively. The proteolytic activity was increased with increasing NaCl concentration in the reaction mixture, the optimum concentration being 2 M. Pepstatin A strongly inhibited the enzyme, with a Ki value of 15 nM at pH 4.0. On the other hand, the active-site residue mutant, in which the putative catalytic aspartic acid residue was mutated to an alanine residue, had no activity. These results show that the Copia proteinase belongs to the family of aspartic proteinases including HIV proteinase. The B-chain of oxidized bovine insulin was hydrolysed at the Leu15-–Tyr16 bond fairly selectively. Thus the recombinant Copia proteinase partially resembles HIV proteinase, but is significantly different from it in certain aspects.

Tauopathies such as Alzheimer disease (AD) probably involve a type of phosphorylation imbalance causing the accumulation of abnormally hyperphosphorylated tau in neurons and/or glias. Investigation of R406W tau mutation may provide insight into such abnormal tau hyperphosphorylation, since this mutation causes AD-like dementia and tauopathy in humans and because it has the unique ability to reduce tau phosphorylation in vitro and in cultured cells. Here we show that R406W mutation primarily disrupts tau phosphorylation at Ser404, a priming phosphorylation site of glycogen synthase kinase-3beta (GSK-3beta), thereby reducing subsequent GSK-3beta-mediated phosphorylation at the PHF-1 site (mostly Ser396). In contrast, c-jun N-terminal kinase (JNK) as activated in the mitotic phase directly hyperphosphorylates R406W tau at the PHF-1 site. This was confirmed by PHF-1 hyperphosphorylation of R406W tau in mitotic cells, its association with cytoplasmic JNK activation, and its inhibition by a JNK inhibitor, SP600125. These data unveil the unknown mechanisms of physiological tau phosphorylation at the PHF-1 site and suggest that cytoplasmic JNK activation may play an important role in the abnormal tau hyperphosphorylation associated with R406W tau mutation and in AD.

Although most somatic cells cannot proliferate, immature cells proliferate continuously to produce mature cells. Recently, we cloned mouse PSF1 from a hematopoietic stem cell specific cDNA library and reported that PSF1 is indispensable for the proliferation of immature cells. To identify the PSF1-binding protein, we used the yeast two-hybrid system with PSF1 as bait, and identified and cloned SLD5. SLD5 interacted with a central region of PSF1. Tissue distribution of SLD5 was quite similar to that of PSF1. When overexpressed, SLD5 protein was co-localized with PSF1. These data suggest that PSF1 and SLD5 may cooperate in the proliferation of immature cell populations.

c-Jun N-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1) (also termed JNK-interacting protein 3; JIP3) is a member of a family of scaffold factors for the mitogen-activated protein kinase (MAPK) cascades, and it also forms a complex with focal adhesion kinase (FAK). Here we demonstrate that JSAP1 serves as a cooperative scaffold for activation of JNK and regulation of cell migration in response to fibronectin (FN) stimulation. JSAP1 mediated an association between FAK and JNK, which was induced by either co-expression of Src or attachment of cells to FN. Complex formation of FAK with JSAP1 and p130 Crk-associated substrate (p130(Cas)) resulted in augmentation of FAK activity and phosphorylation of both JSAP1 and p130(Cas), which required p130(Cas) hyperphosphorylation and was abolished by inhibition of Src. JNK activation by FN was enhanced by JSAP1, which was suppressed by disrupting the FAK/p130(Cas) pathway by expression of a dominant-negative form of p130(Cas) or by inhibiting Src. We also documented the co-localization of JSAP1 with JNK and phosphorylated FAK at the leading edge and stimulation of cell migration by JSAP1 expression, which depended on its JNK binding domain and was suppressed by inhibition of JNK. The level of JSAP1 mRNA correlated with advanced malignancy in brain tumors, unlike other JIPs. We propose that the JSAP1.FAK complex functions cooperatively as a scaffold for the JNK signaling pathway and regulator of cell migration on FN, and we suggest that JSAP1 is also associated with malignancy in brain tumors.

Together with other Golgi matrix components, GRASP65 contributes to the stacking of Golgi cisternae in interphase cells. During mitosis, GRASP65 is heavily phosphorylated, and in turn, cisternal stacking is inhibited leading to the breakdown of the Golgi apparatus. Here we show that GRASP65 is phosphorylated on serine 277 in interphase cells, and this is strongly enhanced in response to the addition of serum or epidermal growth factor. This is directly mediated by ERK suggesting that GRASP65 has some role in growth factor signal transduction. Phosphorylation of Ser-277 is also dramatically increased during mitosis, however this is mediated by Cdk1 and not by ERK. The microinjection of recombinant GRASP65 without N-terminal myristoylation or a peptide fragment containing Ser-277 into the cytosol of normal rat kidney cells inhibits passage through mitosis. This effect is abolished when Ser-277 is replaced with alanine suggesting the phosphorylation of Ser-277 plays an important role in cell cycle regulation. The convergence of cell cycle regulation and growth factor signals on GRASP65 Ser-277 suggests that GRASP65 may function as a signal integrator controlling the cell growth.

Mitogen-activated protein kinase-extracellular signal-related kinase (ERK) kinase 1 (MEK1)/ERK signaling has been implicated in the regulation of tumor cell invasion and metastasis. Migration of HT1080 cells on type I collagen was suppressed by the matrix metalloproteinase (MMP) inhibitors BB94 and tissue inhibitor of metalloproteinase (TIMP)-2 but not by TIMP-1. TIMP-2-specific inhibition suggests that membrane type 1 MMP (MT1-MMP) is likely involved in this process. Activation of ERK was induced in HT1080 cells adhered on dishes coated with type I collagen, and this was inhibited by BB94. MMP-2 processing in HT1080 cells, which also was stimulated by cultivation on type I collagen, was inhibited by MEK inhibitor PD98059. Expression of a constitutively active form of MEK1 promoted MMP-2 processing concomitant with the increase of MT1-MMP levels, suggesting that MT1-MMP is regulated by MEK/ERK signaling. In addition, expression of the hemopexin-like domain of MT1-MMP in HT1080 cells interfered with MMP-2 processing, ERK activation, and cell migration, implying that the enzymatic activity of MT1-MMP is involved in collagen-induced ERK activation, which results in enhanced cell migration. Thus, adhesion of HT1080 cells to type I collagen induces MT1-MMP-dependent ERK activation, which in turn causes an increase in MT1-MMP levels and subsequent cell migration.

The c-Jun NH2-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 [JSAP1; also known as JNK-interacting protein 3 (JIP3)] has been identified as a scaffold protein for JNK mitogen-activated protein kinase signal transduction pathways and as a cargo adapter in the conventional kinesin-mediated transport system. Furthermore, a functional relationship between UNC-16, the C. elegans ortholog of JSAP1, and JNK signaling has been established genetically. In this study, we first demonstrated that the kinesin light chain is required for the targeting and localization of JSAP1 to the tips of neurites in PC12h cells. Furthermore, to understand whether JNK signaling is involved in kinesin-mediated JSAP1 trafficking, we established stable PC12h cell lines that expressed wild-type JSAP1 or its mutant lacking the JNK-binding domain (JBD). Immunocytochemical studies of the cell lines indicated that the mutant JSAP1 was localized to the growth cones of differentiating PC12h cells in a similar manner to wild-type JSAP1. Taken together, these results suggest that the proper subcellular localization of JSAP1 along microtubules probably does not require JNK signaling.

Targeted gene disruption studies have established that the c-Jun NH2-terminal kinase (JNK) is required for the stress-induced release of mitochondrial cytochrome c and apoptosis, and that the Bax subfamily of Bcl-2-related proteins is essential for JNK-dependent apoptosis. However, the mechanism by which JNK regulates Bax has remained unsolved. Here we demonstrate that activated JNK promotes Bax translocation to mitochondria through phosphorylation of 14-3-3, a cytoplasmic anchor of Bax. Phosphorylation of 14-3-3 led to dissociation of Bax from this protein. Expression of phosphorylation-defective mutants of 14-3-3 blocked JNK-induced Bax translocation to mitochondria, cytochrome c release and apoptosis. Collectively, these results have revealed a key mechanism of Bax regulation in stress-induced apoptosis.

The c-Jun N-terminal kinases (JNKs) are a subfamily of the mitogen-activated protein kinases (MAPKs). The JNKs are encoded by three separate genes (jnk1, jnk2, and jnk3), which are spliced alternatively to create 10 JNK isoforms that are either p46 or p54 in size. In this study, we found that the p52 form of JNK emerged in human leukemia MOLT-4 or U937 cells following X-irradiation or heat treatment. The accumulation of p52 coincided with the reduction of p54 JNK. On the other hand, the amounts of p46 JNK did not change by X-irradiation. Induction of the p52 form of JNK also paralleled the appearance of the active form of caspase-3 and was suppressed by a caspase-specific inhibitor, Ac-DEVD-CHO, but not by Ac-YVAD-CHO. In vitro cleavage assays indicated that recombinant human JNK1beta2 and JNK2beta2 were cleaved by caspase-3, and that the mutation of aspartic acid at position 413 of JNK1beta2 or 410 of JNK2beta2 to alanine abolished the cleavage. Altogether, our results demonstrated that p54 JNKs, at least JNK1beta2 and JNK2beta2, were new selective targets of caspases in JNK splicing variants, and suggested that the p52 form could serve as a marker of apoptosis.

Focal adhesion kinase (FAK) becomes activated and tyrosine-phosphorylated in response to cell adhesion to extracellular matrix proteins in a variety of cell types, and associates with a number of signaling molecules, structural proteins, and beta integrin cytoplasmic domains. Here we demonstrated that c-Jun N-terminal kinase (JNK)/stress activated protein kinase-associated protein 1 (JSAP1), a scaffold factor in the mitogen-activated protein kinase (MAPK) cascades, forms a complex with the N-terminus of FAK. The complex formation was further stimulated by c-Src, in which JSAP1 was tyrosine-phosphorylated and other FAK/Src signaling molecules were recruited. Fibronectin (FN) stimulation of cells expressing JSAP1 induced its tyrosine phosphorylation concomitant with association with FAK. Expression of JSAP1 in Hela cells facilitated formation of well-organized focal contacts and actin stress fibers, and promoted cell spreading onto FN. Taken together, these results suggest that JSAP1 is involved an integrin-mediated signaling pathway through FAK/Src by recruiting other signaling molecules, resulting in promotion of cell spreading onto FN.

Automated DNA sequencers draw the four-base profiles of a sample with four different colors, but it is also possible to draw the profiles of a base-specific reaction of four different samples with four colors. PCR-amplified DNAs from four individuals were subjected to a single base-specific sequencing reaction and the products were applied to a set of four lanes of an automated DNA sequencer. A base substitution in an individual was clearly identified as an individual-specific peak with a color specific for the individual. In this way, we analyzed more than 50 individuals and identified several polymorphic base substitutions. The sensitivity of this method was high enough to allow detection of the mutation/polymorphism even if samples from several individuals were applied to one lane. Thus, our method is applicable to screening of a large number of samples in an automated manner.

金沢大学がん研究所, Human Rad9 is a key cell-cycle checkpoint protein that is postulated to function in the early phase of cell-cycle checkpoint control through complex formation with Rad1 and Hus1. Rad9 is also thought to be involved in controlling apoptosis through its interaction with Bcl-2. To explore the biochemical functions of Rad9 in these cellular control mechanisms, we performed two-hybrid screening and identified Tetratricopeptide repeat protein 2 (Tpr2) as a novel Rad9-binding protein. We found that Tpr2 binds not only to Rad9, but also to Radl and Hus1, through its N-terminal tetratricopeptide repeat region, as assessed by in vivo and in vitro binding assays. However, the in vivo and in vitro interactions of Tpr2 with Rad9 were greatly enhanced by the deletion of its C-terminal J domain or by a point mutation in the conserved HPD motif in the J domain, though the binding of Tpr2 to Rad1 and Hus1 was not influenced by these J-domain mutations. We further found: (1) Rad9 transiently dissociates from Tpr2 following heat-shock or UV treatments, but the mutation of the J domain abrogates this transient dissociation of the Tpr2/Rad9 complex; and (2) the J domain of Tpr2 modulates the cellular localization of both Tpr2 itself and Rad9. These results indicate that the J domain of Tpr2 plays a criticai role in the regulation of both physical and functional interactions between Tpr2 and Rad9. © 2001 Academic Press.

Drosophila retrotransposon copia produces virus-like particles (VLPs) in the nuclei of cultured Drosophila cells. The VLPs contain copia RNA and reverse transcriptase activity, and thus, play a major role in copia replication. Here we have expressed the copia gag polyprotein precursor in yeast. The precursor, which includes copia protease itself, showed correct autoprocessing to produce a unique multi-lamella structure in the nuclei of the yeast cells. This expression system should be useful for the analysis of nuclear localization ot'the major copia VLP protein, and Furthermore, would provide important information concerning the mechanism of copia VLPs formation.

p53 and NF-kappaB RelA are activated by various genotoxic agents and mutually suppress each other's ability to activate transcription, most likely through competition for transcriptional coactivators such as CBP or p300. However, we found that the inhibition by RelA of p53 transcriptional activity is not completely restored by CBP/p300 overexpression and that a p53 mutant can not suppress RelA activity despite of its ability to bind CBP/p300. In the present study, we further present evidence that these two transcriptional factors directly interact both in vivo and in vitro. These results therefore indicate that the cross transcriptional interference between p53 and RelA is partly caused by the direct interaction between these two transcription factors which is mediated by their dimerization/tetramerization domains and results in inhibition of each other's transcriptional activity. Finally, cells derived from RelA knockout mice showed enhanced p53 transcriptional activity, suggesting that this cross transcriptional interference is physiologically important in cellular response to genotoxic stress.

We have generated a mouse model for hepatic ischemia in which surgical subcutaneous transposition of the spleen allows hepatic ischemia to be applied without affecting other tissues. Using this mouse model we investigated the relationship between the length of ischemic periods in the liver and subsequent liver function; furthermore, we assayed the activation of c-Jun N-terminal kinase (JNK) during ischemia and reperfusion. Although prior to this study only the activated form of JNK was known to be translocated to the nucleus, we found that JNK translocates to the nucleus during ischemia without activation and is then activated during reperfusion. These results suggest a novel mechanism of JNK activation.

Compared with the knowledge of maternal care, much less is known about the factors required for paternal parental care. Here we report that new sires of laboratory mice, though not spontaneously parental, can be induced to show maternal-like parental care (pup retrieval) using signals from dams separated from their pups. During this interaction, the maternal mates emit 38-kHz ultrasonic vocalizations to their male partners, which are equivalent to vocalizations that occur following pheromone stimulation. Without these signals or in the absence of maternal mates, the sires do not retrieve their pups within 5 min. These results show that, in mice, the maternal parent communicates to the paternal parent to encourage pup care. This new paradigm may be useful in the analysis of the parental brain during paternal care induced by interactive communication., Parental responsibilities in mice are usually carried out by the mother of the pups. In this study, the authors show that when mothers are separated from their mouse pups, they emit ultrasonic vocalizations to their male partners, who respond by administering paternal care to the pups.

Induction of phase 2 detoxification enzymes by phenolic antioxidants can account for prevention of tumor initiation but cannot explain why these compounds inhibit tumor promotion. Phase 2 genes are induced through an antioxidant response element (ARE). Although the ARE resembles an AP-1 binding site, we show that the major ARE binding and activating protein is not AP-1. Interestingly, AP-1 DNA binding activity was induced by the phenolic antioxidant tert-butylhydroquinone (BHQ), but the induction of AP-1 transcriptional activity by the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) was inhibited by this compound. BHQ induced expression of c-jun, junB, fra-1, and fra-2, which encode AP-1 components, but was a poor inducer of c-fos and had no effect on fosB. Like c-Fos and FosB, the Fra proteins heterodimerize with Jun proteins to form stable AP-1 complexes. However, Fra-containing AP-1 complexes have low transactivation potential. Furthermore, Fra-1 repressed AP-1 activity induced by either TPA or expression of c-Jun and c-Fos. We therefore conclude that inhibitory AP-1 complexes composed of Jun-Fra heterodimers, induced by BHQ, antagonize the transcriptional effects of the tumor promoter TPA, which are mediated by Jun-Fos heterodimers. Since AP-1 is an important mediator of tumor promoter action, these findings may explain the anti-tumor-promoting activity of phenolic antioxidants.

Abstract 221 A functional variation in the NKG2D gene regulates NKG2D receptor expression and is associated with better transplant outcomes after fully-HLA-matched unrelated bone marrow transplantation NKG2D, an activating receptor expressed by NK cells and some T cells, plays important roles in both immunity to microbial infections and cancer immune surveillance. A single nucleotide polymorphism (SNP rs1049174) in the 3′-untranslated region (3′UTR) of the NKG2D gene determines two genotypes, HNK1 genotype with high NK cytotoxicity, and the LNK1 genotype with low NK cytotoxicity. Individuals with the HNK1 genotype have reduced risk of cancer (Cancer Res 2006; 66(1):63-70). In the present study, we analyzed the impact of SNP rs1049174 on transplant outcomes after HLA-matched unrelated bone marrow transplantation. The NKG2D genotypes were retrospectively analyzed in a cohort of 360 pairs of patients and their unrelated donors transplanted through the Japan Marrow Donor Program. The donor HNK1 genotype was associated with lower transplant related mortality (TRM) in patients with standard risk (hazard ratio [HR], 0.58; 95% confidence interval [CI], 0.35–0.97; P=0.04) (Fig.1). The donor HNK1 genotype was also associated with a trend toward better OS (HR, 0.69; 95% CI, 0.46–1.04; P=0.08). The host NKG2D genotypes did not significantly influence the transplant outcomes. NK cells from HNK1 donors expressed higher levels of NKG2D on the cell surface, and also expressed higher levels of NKG2D mRNA. Luciferase reporter gene constructs transfected into NK cells showed that the LNK1 construct more effectively attenuated gene expression compared with the HNK1 construct (60% vs. 35%). Bioinformatics tools predicted an interaction between the rs1049174 SNP motif with the micro-RNA-1245 (miR1245). Next, in an mRNA/miRNA binding assay, the LNK allele showed higher affinity to the miR1245 than the HNK1 allele. Moreover transfection of NK cells possessing the LNK1 allele with a miR1245 mimic resulted in 25 fold decrease in mRNA NKG2D level versus only 2 fold decrease in mRNA NKG2D expression in HNK1 allele derived NK cells, thus suggesting that the 3′UTR SNP may regulate the expression of NKG2D through its binding to the putative repressor miR1245. These findings provide evidence that the NKG2D 3′UTR polymorphism is functional, and that the NK cells from donors with the HNK1 genotype exert higher cytotoxicity through their increased expression of NKG2D, thereby resulting in an improved outcome for bone marrow transplantation. Disclosures: No relevant conflicts of interest to declare.

We examined the ontogenesis of a subtype of metabotropic glutamate receptors, termed mGluR1, which is linked to phosphoinositide metabolism, in various regions of rat brain during neonatal development. Northern blot analyses of mGluR1 mRNA indicated that mRNA increased monotonously or remained at plateau levels during the first 5 weeks after birth. In situ hybridization analyses supported this conclusion. The result is in contrast with the reported development of the activity in excitatory amino acid-stimulated phosphoinositide turnover during the same period. The latter increases during the first few weeks and then decreases sharply.

We describe a rapid and sensitive DNA sequencing method for an automated fluorescent DNA sequencer (AFDS) and its application for detection of point mutations. The method is based on an improved cycle sequencing procedure in which only 10–50 fmol of template DNA is required. Furthermore, it is able to use crude DNA preparation as a template as well as the purified one. Thus, the improved method provided a simplified procedure for sequencing of various types of DNA, including cosmid DNA, in which purification steps were unnecessary. We also developed a novel system for detection of point mutations using AFDS. A set of four lanes is used for the parallel analysis of single-base profiles of four different samples, instead of for the four-base profile of a sample. The AFDS exhibits the base profiles of the samples with four different colors in the analyzed data, which enables us to identify a mutation as an additional peak with a color specific for the lane. The feasibility of our system was tested by analyzing polymerase chain reaction (PCR)-amplified genomic DNAs from four individuals including a carrier of a mutation of C to T. The mutation was clearly identified as an additional “T” peak of a color specific for the carrier. The mutation was also detectable even if 16 individuals including the carrier were simultaneously analyzed on a set of four lanes (four individual samples for each lane). Thus, the novel system is useful for simultaneous detection of mutations in a large number of individual samples.

Alzheimer's disease (AD) is a devastating neurological disorder and the leading cause of dementia among aged individuals. The human amyloid beta protein, which is a cleavage product of amyloid precursor protein (APP), is a major component of the amyloid deposited in the brain of patients with AD. By using PCR direct sequencing of exon 17 (encoding part of the beta protein) of the APP gene, we have found that a Japanese AD patient harbours a C to T substitution, responsible for a valine to isoleucine change at position 717, heterogeneously. The mutation is exactly the same as that found in a Caucasian AD family by Goate et al. (1). Furthermore, the mutation was shown to co-segregate with AD in his family. These results suggest that the Val----Ile change in the APP causes AD, regardless of ethnic background.

The Drosophila melanogaster transposable element copia is usually 5 kb long with long terminal repeats (LTRs), and its major transcripts are a full-length 5-kb RNA and a 2-kb RNA. We have previously shown that the 2-kb RNA is generated through splicing. Here, we have cloned a genomic intronless copia using an oligodeoxyribonucleotide probe which is specific for the junction of the two exons. The unusual copia is bounded by two LTRs and lacks precisely the intron of the 2-kb copia RNA. Identification of genomic intronless copia strongly suggests that copia transposes through an RNA intermediate. Moreover, we have found that copia virus-like particles (VLPs), in which reverse transcription of copia RNA seems likely to occur, packages the spliced copia RNA much less efficiently than the full-length copia RNA. This result leads to the suggestion that much lower copy number of genomic intronless copia, as compared with that of 'normal' copia, may be responsible for the inefficient packaging of the spliced copia RNA into the VLP.

Northern blot and nucleotide sequence analyses of copia RNA from a transfectant made by introducing a genomic copia into copia-free cells showed that the 2 kb RNA, one of the major transcripts of copia, is generated through splicing. Using the polymerase chain reaction (PCR), we have also found that the position of the splice sites used in Drosophila larvae and cultured cells originally containing copia is the same as that used in the transfectant. To investigate the function of the 2 kb RNA, we constructed mutant copias which harboured a single point mutation at the splice site or approximately 3 kb deletion of the internal region corresponding to the spliced out sequence. Analyses of transfectants made by introducing these mutant copias into copia-free cells demonstrated that the spliced 2 kb RNA contains sufficient information to make copia virus-like particles (VLPs). Furthermore, when copia RNA corresponding to the spliced RNA was translated in vitro, the major VLP protein was found to be released autocatalytically from its own precursor. A single amino acid substitution at the putative protease active site in the precursor prevented the processing, and resulted in accumulation of the mutant precursor in vitro. From these results, we conclude that copia VLPs are produced through autocatalytic processing of the precursor polyprotein encoded by the spliced copia RNA.

The cerebellum is essential for the motor control of movement and posture. Due to its apparent simplicity and geometrical arrangement, the cerebellum provides an excellent model for studying the mechanisms that control development of the central nervous system (CNS). The cerebellar cortex is formed from two distinct proliferative zones: one ventricular and one superficial called the external granule layer (EGL). Massive clonal expansion of granule cell precursors (GCPs) occur in the EGL, and ultimately generates by far the most abundant neuronal population in the CNS. In this review, I describe recent advances in understanding the control of GCP proliferation in the developing cerebellum. I also briefty review the uncontrolled GCP proliferation associated with medulloblastoma, the most common brain malignancy in children. Biomedical Reviews 2005; 16: 35-41.

The genetic structure of the 42.84-43.6 F (BamHI-PstI) segment of the F plasmid, which contains all the F DNA sequences necessary for coupling cell division of F+ bacteria with plasmid DNA replication, was analyzed by isolating a series of amber mutants. Two cistrons were found in this region and they were designated letA and letD (an abbreviation for lethal mutation). The letA and letD cistrons were mapped on the 42.84-43.35 F (BamHI- XmaI ) segment and the 43.07-43.6 F (HincII-PstI) segment, respectively, and are presumed to correspond to the first (43.04-43.26 F) and second (43.26-43.57 F) open reading frames, respectively, which were found in this region by nucleotide sequencing. The letD gene product acts to inhibit cell division of the host bacteria and to induce prophages in lysogenic bacteria, whereas the letA gene product acts to suppress the activity of the letD gene product. Taking into consideration the fact that the 42.84-43.6 F segment carries all the F plasmid genes necessary for coupling cell division with plasmid DNA replication, and that the expression of the genes is likely to be controlled by plasmid DNA replication, we constructed the following hypothesis. Before completion of plasmid DNA replication, LetD protein acts to prevent cell division of the host bacteria. When plasmid DNA replication is completed, synthesis of LetA protein (and also LetD protein) takes place and the LetA protein synthesized acts to suppress the activity of LetD protein and make the cell ready for cell division. Actual cell division will take place when replication of both chromosomal and plasmid DNA is completed and the termination protein of the chromosome and the LetA protein of F plasmid are both synthesized. When cell division takes place LetA protein is consumed, and as a result LetD protein becomes active and prevents cell division until the next round of DNA replication is completed.

The assignment of the human prealbumin (PALB) gene to chromosome region 18q11-q12.1 has been achieved using a human genomic probe in the study of human-mouse somatic cell hybrids and by in situ hybridization. Because familial amyloidotic polyneuropathy was reported previously to be due to a mutation in prealbumin, it can be inferred that the gene for this disorder also maps to 18q11.2-q12.1.

We examined the DNA analysis of familial amyloid polyneuropathy (FAP) patients and their families from Nagano and Hiroshima prefectures in Japan using recombinant DNA techniques and compared the results with the clinical features. This study indicated that the valine-methionine change prealbumin gene was closely related to the clinical features of type 1 FAP. The DNA analysis was valuable for the definite diagnosis of type 1 FAP even in sporadic and asymptomatic cases. FAP patients from Iiyama city and Ogawa village area in the Nagano prefecture had the same mutation despite differences in clinical features. The onset of the sporadic FAP cases was later than that of the FAP patients who had family histories.

金沢大学がん研究所がん分子細胞制御, A Japanese family with atypical type I familial amyloidotic polyneuropathy (FAP) in Iiyama, Japan, was studied. Most of the family members have dysfunctions of the central nervous system, in addition to typical symptoms of type I FAP. The transthyretin (TTR, also called prealbumin) gene of the atypical FAP (FAP-IY) was analyzed with recombinant DNA techniques and a RIA method. FAP-IY was found to have the mutation responsible for the methionine-for-valine substitution at position 30 of TTR, as in the case of typical type I FAP. However, analysis of DNA polymorphisms in the TTR locus showed that FAP-IY has a genetic background differing from that of the typical type I FAP. These observations lead to the consideration that a genetic factor(s) involved in the dysfunction of the central nervous system may locate in a chromosome region in close proximity to the TTR gene.

Familial amyloidotic polyneuropathy (FAP) is an autosomal dominant genetic disease characterized by systemic accumulation of amyloid fibrils. A major component of FAP amyloid has been identified as variant transthyretin (TTR, also called prealbumin). In particular, a variant with the substitution 30Val----Met has been commonly found in FAP of various ethnic groups. To understand the origin and spread of the Val----Met mutation, we analyzed DNA polymorphisms associated with the TTR gene in six Japanese FAP families and several Portuguese FAP patients. Three distinct haplotypes associated with the Val----Met mutation were identified in Japanese FAP families, one of which was also found in Portuguese patients. On the other hand, it was found that the Val----Met mutation can be explained by a C-T transition at the CpG dinucleotide sequence of a mutation hot spot. Thus, our findings indicate that the Val----Met mutation has probably recurred in the human population, to generate FAP families of independent origin.

Drosophila copia protease is likely to be encoded in the gag gene. We have expressed copia gag polyprotein precursor in E. coli. The gag precursor was correctly processed to generate a unique laminate structure in E. coli. The processing was almost completely blocked by a mutation at the putative active site of copia protease, and resulted in accumulation of the precursor. Furthermore, the laminate structure was not found in E. coli expressing the mutant precursor. These results indicate that the protease is involved in cleaving the gag precursor itself. Also, the assembly of copia gag protein should correlate to the autoprocessing of copia gag polyprotein precursor.

The largest group of transposable elements in Drosophila melanogaster, copia-like elements1,2, share some important structural features with and are intimately related in evolution to vertebrate retroviruses2–12. To further clarify the relationship between retroviruses and copia-like transposable elements, we set out to determine the complete nucleotide sequence of the genome of 17.6, which has long terminal repeats homologous in nucleotide sequence to those of avian leukaemia–sarcoma virus12. We report here that 17.6 contains three long open reading frames comparable with gag, pol and env genes in retrovirus. At the level of amino acid sequence, the longest open reading frame of 17.6 includes a coding sequence similar to that for reverse transcriptase, suggesting a role for this enzyme in the life cycle of some Drosophila copia-like elements, analogous to the situation in retrovirus.

Familial amyloidotic polyneuropathy (FAP) is an autosomal dominant genetic disease, and the major component of the amyloid fibrils from FAP patients was shown to be variants of transthyretin (TTR) with single amino acid substitutions. The nucleotide sequence analysis of the TTR cDNA and its corresponding gene enabled us to detect the base substitutions responsible for the variant TTR by conventional Southern blotting or polymerase chain reaction (PCR) method and allowed us to screen a number of FAP families in Japan. Among seven TTR variants related to FAP, all the FAP patients tested in Japan had the particular 30Val----Met mutation. Haplotype analysis revealed that the Val----Met mutation has recurred frequently in the population to generate the FAP families of independent origins. Although the primary cause of FAP has become clear, extensive screening of FAP families revealed that there existed late onset cases and also patients with atypical symptoms in FAP families with the Val----Met mutation, suggesting that the expression of FAP is a complex process and affected by some (unknown) factors other than TTR.

The major components of the mitogen-activated protein kinase (MAPK) cascades are MAPK, MAPK kinase (MAPKK), and MAPKK kinase (MAPKKK). Recent rapid progress in identifying members of MAPK cascades suggests that a number of such signaling pathways exist in cells. To date, however, how the specificity and efficiency of the MAPK cascades is maintained is poorly understood. Here, we have identified a novel mouse protein, termed Jun N-terminal protein kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1), by a yeast two-hybrid screen, using JNK3 MAPK as the bait. Of the mammalian MAPKs tested (JNK1, JNK2, JNK3, ERK2, and p38alpha), JSAP1 preferentially coprecipitated with the JNKs in cotransfected COS-7 cells. JNK3 showed a higher binding affinity for JSAP1, compared with JNK1 and JNK2. In similar cotransfection studies, JSAP1 also interacted with SEK1 MAPKK and MEKK1 MAPKKK, which are involved in the JNK cascades. The regions of JSAP1 that bound JNK, SEK1, and MEKK1 were distinct from one another. JNK and MEKK1 also bound JSAP1 in vitro, suggesting that these interactions are direct. In contrast, only the activated form of SEK1 associated with JSAP1 in cotransfected COS-7 cells. The unstimulated SEK1 bound to MEKK1; thus, SEK1 might indirectly associate with JSAP1 through MEKK1. Although JSAP1 coprecipitated with MEK1 MAPKK and Raf-1 MAPKKK, and not MKK6 or MKK7 MAPKK, in cotransfected COS-7 cells, MEK1 and Raf-1 do not interfere with the binding of SEK1 and MEKK1 to JSAP1, respectively. Overexpression of full-length JSAP1 in COS-7 cells led to a considerable enhancement of JNK3 activation, and modest enhancement of JNK1 and JNK2 activation, by the MEKK1-SEK1 pathway. Deletion of the JNK- or MEKK1-binding regions resulted in a significant reduction in the enhancement of the JNK3 activation in COS-7 cells. These results suggest that JSAP1 functions as a scaffold protein in the JNK3 cascade. We also discuss a scaffolding role for JSAP1 in the JNK1 and JNK2 cascades.