bluuebunny/crossref_metadata_2025_split huggingface dataset:
Human homologues of the Caenorhabditis elegans cell polarity protein PAR6 as an adaptor that links the small GTPases Rac and Cdc42 to atypical protein kinase C
Authors: Noda Yukiko , Takeya Ryu , Ohno Shigeo , Naito Seiji , Ito Takashi , Sumimoto Hideki 
Published: March 2003

BackgroundAsymmetric cell division in the Caenorhabditis elegans embryos requires products of par (partitioning defective) genes 1–6 and atypical protein kinase C (aPKC), whereas Cdc42 and Rac, members of the Rho family GTPases, play an essential role in cell polarity establishment in yeast and mammalian cells. However, little is known about a link between PAR proteins and the GTPases in cell polarization.ResultsHere we have cloned cDNAs for three human homologues of PAR6, designated PAR6α, β and γ, comprising 345, 372 and 376 amino acids, respectively. The PAR6 proteins harbour a PDZ domain and a CRIB‐like motif, and directly interact with GTP‐bound Rac and Cdc42 via this motif and with the aPKC isoforms PKCι/λ and PKCζ via the N‐terminal head‐to–head association. These interactions are not mutually exclusive, thereby allowing the PAR6 proteins to form a ternary complex with the GTPases and aPKC, both in vitro and in vivo. When PAR6 and aPKC are expressed with a constitutively active form of Rac in HeLa or COS‐7 cells, these proteins co‐localize to membrane ruffles, which are known to occur at the leading edge of polarized cells during cell movement.ConclusionHuman PAR6 homologues most likely play an important role in the cell polarization of mammalian cells, by functioning as an adaptor protein that links activated Rac and Cdc42 to aPKC signalling.
Down‐regulation of α6 integrin, an anti‐oncogene product, by functional cooperation of H‐Ras and c‐Myc
Authors: Fujimoto Hiroko , Tanaka Yoshiya , Liu Zhao‐Jun , Yagita Hideo , Okumura Ko , Kosugi Atsushi , Morinobu Akio , Umehara Hisanori , Yamamura Hirohei , Minami Yasuhiro 
Published: March 2003

BackgroundThe molecular basis of cooperation of H‐Ras and c‐Myc in regulating cellular behaviour, such as cell adhesiveness, is still poorly understood. To investigate the role of H‐Ras and c‐Myc in cell adhesiveness, a constitutively active H‐RasV12 (H‐RasV12) and c‐Myc were stably expressed, singly or in combination in a haematopoietic cell line, and the expression and activity of cell adhesion molecules were monitored.ResultsWe have shown that the ectopic expression of H‐RasV12, but not c‐Myc alone, in a haematopoietic cell line, induces the activation of very late antigen‐6 (VLA‐6, α6β1) integrin. Co‐expression of H‐RasV12 and c‐Myc in the same cells further resulted in the induction of expression of vascular cell adhesion molecule‐1 (VCAM‐1) and the inhibition of expression of α6 integrin, a candidate anti‐oncogene product, leading to a loss of adhesiveness to laminin (Lm), a ligand for VLA‐6.ConclusionsCooperation of H‐Ras and c‐Myc reciprocally regulates expression of the adhesion molecules, α6 integrin and VCAM‐1. Our results represent an unprecedented account of the cooperation of the oncogene products, H‐Ras and c‐Myc, to inhibit expression of an anti‐oncogene product, α6 integrin.
Distribution of phosphorylated glial fibrillary acidic protein in the mouse central nervous system
Authors: Takemura Masaaki , Nishiyama Hiroshi , Itohara Shigeyoshi 
Published: March 2003

AbstractBackground: Glial fibrillary acidic protein (GFAP) is the principal component of intermediate filaments (IFs) in mature astrocytes in the central nervous system (CNS). Like other IF proteins, GFAP has multiple phosphorylation sites in the N‐terminal head domain. The distribution of phospho‐GFAP in vivo has not been elucidated.Results: We generated Gfaphwt knock‐in mice, in which the coding region for the head domain of GFAP is replaced with the corresponding human sequence. In combination with a series of monoclonal antibodies (mAbs) reactive to human phospho‐GFAP, we visualized the distribution of phospho‐GFAP in vivo in mice. GFAP phosphorylated at Thr7, Ser8 and/or Ser13 increased postnatally in the CNS of these mice. Limited populations of GFAP‐positive astrocytes were labelled with anti‐phospho‐GFAP mAbs in most brain areas, whereas almost all the astrocytes in the optic nerve and spinal cord were labelled. Astrocytes in the subventricular zone and rostral migratory stream preferentially contained phospho‐GFAP. In a cold injury model of the cerebral cortex, we detected phospho‐GFAP in reactive astrocytes at 2–3 weeks after the injury.Conclusions: Phospho‐GFAP provides a molecular marker indicating the heterogeneity of astrocytes, and Gfaphwt knock‐in mice will aid in monitoring intracellular conditions of astrocytes, under various conditions. Our results suggest that the phosphorylation of GFAP plays a role in non‐dividing astrocytes in vivo.
The FYVE domain in Smad anchor for receptor activation (SARA) is sufficient for localization of SARA in early endosomes and regulates TGF‐β/Smad signalling
Authors: Itoh Fumiko , Divecha Nullin , Brocks Lenny , Oomen Lauran , Janssen Hans , Calafat Jero , Itoh Susumu , Dijke Peter ten 
Published: March 2003

Abstract Background: Transforming growth factor‐β (TGF‐β) initiates intracellular signalling by inducing the formation of a heteromeric complex between TGF‐β type I (TβR‐I) and TGF‐β type II serine/threonine kinase receptors (TβR‐II). After the activation of TβR‐I kinase by TβR‐II kinase, specific receptor‐regulated Smads (R‐Smads) are phosphorylated by TβR‐I kinase. Smad anchor for receptor activation (SARA), which contains a FYVE finger domain, regulates the subcellular localization of R‐Smads and presents them to TβR‐I. However, it is unclear where SARA is localized in the cell and which phospholipid(s) interacts with its FYVE domain.Results: Wild‐type SARA and the FYVE domain of SARA (FYVE(SARA)) reveal a punctate staining pattern and co‐localize with the early endosomal markers, early endosomal antigen‐1 (EEA1) and hepatic growth factor‐regulated tyrosine kinase substrate (Hrs). The ectopic expression of dominant negative rab5, a critical regulatory molecule in endosome function, redistributes SARA from punctate to a diffuse cytosolic staining pattern. A lipid binding assay demonstrated that the recombinant FYVE domain from SARA predominantly interacts with phosphatidylinositol 3‐phosphate (PtdIns(3)P). Consistent with this result, wortmannin, a PI3 kinase inhibitor, resulted in both a redistribution of SARA from the endosomal compartment to the cytosol and the attenuation of both TGF‐β‐induced R‐Smad phosphorylation and transcriptional activation. Ectopic expression of the FYVE domain of SARA also induced the redistribution of wild‐type SARA and inhibited TGF‐β as well as BMP/Smad‐induced transcriptional responses.Conclusion: The FYVE domain is sufficient and necessary for the early endosomal localization of SARA, probably through its interaction with PtdIns(3)P. Moreover, the localization of SARA in early endosomes is required for efficient TGF‐β/Smad signalling.
Association of frabin with specific actin and membrane structures
Authors: Kim Yongman , Ikeda Wataru , Nakanishi Hiroyuki , Tanaka Yoshinari , Takekuni Kyouji , Itoh Shinsuke , Monden Morito , Takai Yoshimi 
Published: March 2003

AbstractBackground: Frabin is an actin filament (F‐actin)‐binding protein with GDP/GTP exchange activity specific for Cdc42 small G protein. Expression of frabin forms filopodia‐like microspikes through the direct activation of Cdc42, and lamellipodia through indirect activation of Rac small G protein. Frabin consists of the F‐actin‐binding domain (FAB), the Dbl homology domain (DH), the first pleckstrin homology domain (PH1), the FYVE‐finger domain (FYVE), the second PH domain (PH2) from the N‐terminus in this order. Although DH and PH1 show exchange activity, FAB, in addition to DH and PH1, is required for the formation of microspikes, whereas FYVE and PH2, in addition to DH and PH1, are required for the formation of lamellipodia.Results: Various truncated mutants of frabin were co‐expressed with a dominant active mutant (DA) of Cdc42, Rac1DA, or full‐length frabin in L fibroblasts. FAB was recruited to the Cdc42DA‐formed filopodia‐like microspikes. FAB and a fragment containing DH, PH1, FYVE and PH2 were recruited to the Rac1DA‐formed membrane ruffles. Furthermore, each of these fragments served as a dominant negative mutant of frabin when co‐expressed with full‐length frabin, and inhibited the full‐length frabin‐formed morphological changes.Conclusion: These results suggest that frabin recognizes a specific actin structure(s) through FAB and a specific membrane structure(s) through FAB and the region containing DH, PH1, FYVE and PH2. It is likely that frabin associates with the specific actin and membrane structures and activates Cdc42 and Rac in the vicinity of these structures, eventually leading to morphological changes.
Inhibition of Nodal signalling by Lefty mediated through interaction with common receptors and efficient diffusion
Authors: Sakuma Rui , Ohnishi Yu‐ichiro , Meno Chikara , Fujii Hideta , Juan Hou , Takeuchi Jun , Ogura Toshihiko , Li En , Miyazono Kohei , Hamada Hiroshi 
Published: March 2003

AbstractBackground: Two TGFβ‐related proteins, Nodal and Lefty, are implicated in early embryonic patterning of vertebrates. Genetic data suggest that Nodal is a signalling molecule, while Lefty is an antagonist of Nodal, but their precise function remains unknown.Results: The signalling pathway of Nodal was investigated with the use of a Nodal‐responsive assay system based on frog animal caps. Expression of dominant negative mutants of various receptors indicated that ALK4, and either ActRIIA or ActRIIB, function as type I and type II receptors for Nodal, respectively. A soluble form of Cripto lacking the COOH‐terminal region interacted with Nodal but failed to mediate Nodal signalling, indicating that the native Cripto protein functions as a membrane‐bound co‐receptor for Nodal. Processed forms of Lefty proteins, both smaller and larger forms, inhibited Nodal signalling. Such Lefty‐induced inhibition was rescued by excess ActRIIA or ActRIIB, suggesting that Lefty antagonizes Nodal signalling through competitive binding to the common receptor ActRIIA or ActRIIB. This idea was supported by the demonstration of a genetic interaction between lefty2 and ActRIIB in mouse. Behaviours of GFP‐Nodal and GFP‐Lefty2 proteins were also investigated in chick embryos. Both proteins could diffuse over a long distance, but the latter diffused faster than the former.Conclusions: Efficient inhibition of Nodal signals by Lefty may involve competitive binding of Lefty to the common receptors and faster diffusion of Lefty.
ERBIN associates with p0071, an armadillo protein, at cell‐cell junctions of epithelial cells
Authors: Izawa Ichiro , Nishizawa Miwako , Tomono Yasuko , Ohtakara Kazuhiro , Takahashi Toshitada , Inagaki Masaki 
Published: March 2003

AbstractWorking model for the ERBIN–p0071 interaction at cell‐cell adhesions. ERBIN is associated with p0071 at adherens junctions and desmosomes. p0071 is thought to interact with classic cadherins or desmosomal cadherins (Hatzfeld 1999). The subcellular localization of ERBIN may be regulated by the Rho family and other signals controlling cell polarity.imageBackground: ERBIN, an ErbB2 receptor‐interacting protein, belongs to a recently described family of proteins termed the LAP [leucine‐rich repeats and PSD‐95/dLg‐A/ZO‐1 (PDZ) domains] family which has essential roles in establishment of cell polarity.Results: To identify new ERBIN‐binding proteins, we screened a yeast two‐hybrid library, using the carboxyl‐terminal fragment of ERBIN containing PDZ domain as the bait, and we isolated p0071 (also called plakophilin‐4) as an ERBIN‐interacting protein. p0071 is a member of the p120 catenin family, which are defined as proteins with 10 armadillo repeats, and localizes along the cell‐cell border. The ERBIN PDZ domain binds the COOH‐terminus of p0071 containing the PDZ domain‐binding sequence. Endogenous ERBIN was co‐immunoprecipitated with p0071. In fully polarized Madin–Darby canine kidney (MDCK) cells, ERBIN co‐localized largely with β‐catenin and partly with desmoplakin along the lateral plasma membrane domain. At these cell‐cell contact regions, ERBIN co‐localizes with p0071. Over‐expression of the dominant active forms of Cdc42, Rac1 or RhoA, Rho family small GTPases, resulted in a marked accumulation of ERBIN at the cell‐cell contacts of MDCK and HeLa cells.Conclusion: These results show that ERBIN interacts in vivo with p0071 and that it may be involved in the organization of adherens junctions and the desmosomes of epithelia. In addition, we demonstrated that the subcellular localization of ERBIN might be regulated by Rho family small GTPases.
Yeast Whi2 and Psr1‐phosphatase form a complex and regulate STRE‐mediated gene expression
Authors: Kaida Daisuke , Yashiroda Hideki , Toh‐e Akio , Kikuchi Yoshiko 
Published: March 2003

AbstractBackground:  In response to various stressful situations, including diauxic conditions, the Msn2 and Msn4 transcription factors induce STRE‐mediated gene expression of many stress responsive genes in Saccharomyces cerevisiae. This is called the general stress response. The whi2 cells in the stationary phase are smaller than wild‐type cells.Results:  Here we demonstrate that STRE‐mediated gene expression in whi2 cells is reduced to half of that in the wild‐type cells under various stress conditions. It is also delayed for several hours when the mutant cells enter the stationary phase. Using the two‐hybrid system, we isolated a WHI2‐interacting gene, PSR1, which is one of the redundant genes encoding plasma membrane phosphatases. whi2 and psr1 psr2 mutants had similar phenotypes, including reduced STRE‐mediated gene expression, higher sensitivity to sodium ions and heat shock, and hyper‐phosphorylation of Msn2. The phosphatase activity of Psr1 was necessary for the full activation of STRE‐mediated gene expression. Furthermore, both Psr1 and Msn2 were co‐immunoprecipitated with Whi2.Conclusions:  Thus, Whi2 and its binding partner, Psr1‐phosphatase, are required for a full activation of the general stress response, possibly through the dephosphorylation of Msn2. These results may explain why stationary phase whi2 cells are small.
MBLR, a new RING finger protein resembling mammalian Polycomb gene products, is regulated by cell cycle‐dependent phosphorylation
Authors: Akasaka Takeshi , Takahashi Naomi , Suzuki Maki , Koseki Haruhiko , Bodmer Rolf , Koga Hisashi 
Published: March 2003

AbstractBackground: The RING finger proteins function in a variety of fundamental cellular processes. The products of some members of the Polycomb group (PcG) bear ring finger domains and are defined as a subclass of RING finger proteins. Among them are Drosophila posterior sex combs and suppressor 2 of zeste, whose RING fingers are conserved in vertebrate PcG proteins Mel18 and Bmi1.Results: We have identified a new mammalian RING finger protein, termed MBLR due to its structural similarity to Mel18 and Bmi1 (Mel18 and Bmi1‐like RING finger protein). MBLR interacts with some PcG proteins: in vitro biochemical data support the idea of a direct interaction of MBLR’s RING finger domain with Ring1B, which is highly homologous to one of the mammalian PcG genes, Ring1A. We also show that MBLR acts as a transcriptional repressor in transiently transfected cells, as is the case for other PcG proteins. Immunocytochemical analysis reveals that MBLR protein is localized in a fine‐grained distribution throughout the nucleoplasm in interphase cultured cells and in a fainter diffuse cytoplasmic distribution in mitotic cells. In addition, we find that serine 32 of MBLR is specifically phosphorylated during mitosis, most likely by CDK7, a component of the basal transcriptional machinery.Conclusion: Similarities to previously defined PcG proteins suggest that MBLR should be included in the same subclass of RING finger proteins as Mel18 and Bmi1. Although the biological relevance of the cell cycle‐related phosphorylation remains to be demonstrated, serine 32 phosphorylation could nevertheless be functionally important.
Cytoskeletal reorganization induced by insulin: involvement of Grb2/Ash, Ras and phosphatidylinositol 3‐kinase signalling
Authors: Tobe Kazuyuki , Asai Shohji , Matuoka Koozi , Yamamoto Tadashi , Chida Kazuhiro , Kaburagi Yasushi , Akanuma Yasuo , Kuroki Toshio , Takenawa Tadaomi , Kimura Satoshi , Nagai Ryozo , Kadowaki Takashi 
Published: March 2003

AbstractBackground:  Cytoskeletal reorganization is important for a wide variety of insulin‐mediated biological actions, including cell growth, migration and metabolism, but the intracellular signalling pathways leading to insulin‐induced cytoskeletal reorganization have largely been unknown. We therefore investigated the involvement of Grb2/Ash‐Ras and phosphatidylinositol (PI) 3‐kinase in the insulin‐induced morphological changes in fibroblasts over‐expressing human insulin receptors (HIRcB cells).Results:  I nsulin, as well as 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) and 8‐bromo‐cAMP, induced a unique morphological change associated with actin cytoskeletal reorganization characterized by the disruption of actin stress fibres and thicker actin bundle formation. Microinjection of an anti‐Grb2/Ash antibody, but not control IgG, inhibited the insulin‐induced actin reorganization, whereas the TPA‐ and 8‐bromo‐cAMP‐induced morphological changes were not inhibited by microinjection of the anti‐Grb2/Ash antibody. In addition, microinjection of dominant negative ras p21 protein, but not the heat‐treated protein, inhibited insulin‐induced cytoskeletal reorganization. Microinjection of activated p21ras protein resulted in very similar cytoskeletal reorganization with actin bundle formation in the cytoplasm. The PI3‐kinase inhibitor wortmannin inhibited insulin‐induced cytoskeletal reorganization, but not the TPA‐ nor 8‐bromo‐cAMP‐induced reorganization. Interestingly, wortmannin also inhibited the activated p21ras‐induced morphological change.Conclusions:  We concluded that Grb2/Ash‐Ras activation and probably Ras‐associated PI3‐kinase activation are involved in the insulin‐induced morphological change.
Regulation of c‐Fos and Fra‐1 by the MEK5‐ERK5 pathway
Authors: Terasawa Kazuya , Okazaki Kenji , Nishida Eisuke 
Published: March 2003

AbstractBackground: ERK5 is the newest subfamily member of the mitogen‐activated protein kinase (MAPK) family, and is activated by various extracellular signals including growth factors. MEK5 is a specific activator of ERK5. c‐Fos and Fra‐1, well‐known immediate early gene products, are members of the AP‐1 family. We previously reported that activation of the MEK5‐ERK5 pathway is able to induce expression of c‐Fos.Results: We have found that activation of the MEK5‐ERK5 pathway causes the phosphorylation and stabilization of c‐Fos and Fra‐1. Phosphorylation of c‐Fos appears to be mediated by ERK5 and a kinase(s) lying downstream of ERK5, and the MEK5‐ERK5 pathway‐dependent phosphorylation sites on c‐Fos are different from the ERK1/2 pathway‐dependent ones. Interestingly, activation of the MEK5‐ERK5 pathway, but not that of the ERK1/2 pathway, is found to markedly increase the transactivation activity of c‐Fos. Furthermore, our results show that the C‐terminal half of ERK5 is necessary for the maximal activation of the transactivation activity of c‐Fos and Fra‐1.Conclusion: These results reveal a role of the MEK5‐ERK5 pathway in modulating the function of the Fos family proteins which is different from the role of the ERK1/2 pathway.
Geophysical inversion with a neighbourhood algorithm—II. Appraising the ensemble
Authors: Sambridge Malcolm 
Published: March 2003

SummaryMonte Carlo direct search methods, such as genetic algorithms, simulated annealing, etc., are often used to explore a finite-dimensional parameter space. They require the solving of the forward problem many times, that is, making predictions of observables from an earth model. The resulting ensemble of earth models represents all ‘information’ collected in the search process. Search techniques have been the subject of much study in geophysics; less attention is given to the appraisal of the ensemble. Often inferences are based on only a small subset of the ensemble, and sometimes a single member.This paper presents a new approach to the appraisal problem. To our knowledge this is the first time the general case has been addressed, that is, how to infer information from a complete ensemble, previously generated by any search method. The essence of the new approach is to use the information in the available ensemble to guide a resampling of the parameter space. This requires no further solving of the forward problem, but from the new ‘resampled’ ensemble we are able to obtain measures of resolution and trade-off in the model parameters, or any combinations of them.The new ensemble inference algorithm is illustrated on a highly non-linear wave-form inversion problem. It is shown how the computation time and memory requirements scale with the dimension of the parameter space and size of the ensemble. The method is highly parallel, and may easily be distributed across several computers. Since little is assumed about the initial ensemble of earth models, the technique is applicable to a wide variety of situations. For example, it may be applied to perform ‘error analysis’ using the ensemble generated by a genetic algorithm, or any other direct search method.
Measurement of magnetic properties of steel sheets
Authors: Eskola  , Puranen  , Soininen  
Published: March 2003

Magnetic methods are used in detection of environmental, engineering and military objects fabricated of thin ferromagnetic sheets having volume susceptibilities higher than 100 SI units. Magnetic modelling of such objects would be advantageous, but it requires knowledge of the susceptibility and remanence values of sheet materials, which is scarce. We introduce a magnetometer method for the determination of susceptibility and remanence on thin steel samples. The area of the sample must be so large that its within‐sheet magnetization remains below the saturation state. The measurements are made in normal office surroundings in the Earth's magnetic field with an ordinary fluxgate magnetometer.The square‐shaped sheet samples measured in this work have an edge length of 17.5 cm and a thickness in the range 0.5–1.0 mm. During the measuring procedure the sample is placed in four positions on a subvertical measurement board. For each position, the magnetic field in the dip direction of the board plane is measured on the opposite sides of the sample. The secondary field values are averaged for each sample position in order to reduce the effect of sample inhomogeneities. With these data, the susceptibility and remanence of the sample in its edge directions are then determined, based on a model curve which is calculated numerically using thin‐sheet integral equations.The susceptibilities measured for different steel types (cold rolled and hot‐dip zinc‐coated steel sheets) varied in the range 200–500 SI units, and the remanence varied in the range 1000–20 000 A/m. No systematic differences were observed between the magnetic properties of various steel types. The repeatability of the susceptibility measurements was good (variations < 5%) but the remanence could be changed by 50% between repeated determinations.The measured susceptibility range signifies that pieces of steel with a typical thickness of 0.5 mm remain below magnetic saturation when their edge dimension is larger than 5 cm. Therefore magnetic modelling of larger steel pieces must be made using the thin‐sheet theory with known magnetic properties, whereas smaller saturated pieces can be alternatively modelled as an equipotential system.