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831.
832.
833.
Defining Developmental Potency and Cell Lineage Trajectories by Expression Profiling of Differentiating Mouse Embryonic Stem Cells 总被引:1,自引:0,他引:1
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834.
Kazuhiro Nishimura Hiroyuki Okudaira Eriko Ochiai Kyohei Higashi Mayumi Kaneko Itsuko Ishii Tomoe Nishimura Naoshi Dohmae Keiko Kashiwagi Kazuei Igarashi 《The international journal of biochemistry & cell biology》2009,41(11):2251-2261
In Escherichia coli, several proteins whose synthesis is enhanced by polyamines at the level of translation have been identified. We looked for proteins that are similarly regulated in eukaryotes using a mouse mammary carcinoma FM3A cell culture system. Polyamine deficiency was induced by adding an inhibitor of ornithine decarboxylase, α-difluoromethylornithine, to the medium. Proteins enhanced by polyamines were determined by comparison of protein levels in control and polyamine-deficient cells using two-dimensional gel electrophoresis, and were identified by Edman degradation and/or LC/MALDI-TOF/TOF tandem mass spectrometry. Polyamine stimulation of the synthesis of these proteins at the level of translation was confirmed by measuring levels of the corresponding mRNAs and proteins, and levels of the [35S]methionine pulse-labeled proteins. The proteins identified in this way were T-complex protein 1, β subunit (Cct2); heterogenous nuclear ribonucleoprotein L (Hnrpl); and phosphoglycerate mutase 1 (Pgam1). Since Cct2 was most strongly enhanced by polyamines among three proteins, the mechanism of polyamine stimulation of Cct2 synthesis was studied using NIH3T3 cells transiently transfected with genes encoding Cct2-EGFP fusion mRNA with normal or mutated 5′-untranslated region (5′-UTR) of Cct2 mRNA. Polyamines most likely enhanced ribosome shunting on the 5′-UTR of Cct2 mRNA. 相似文献
835.
836.
Hideyuki Kawabata Taro Katsura Eiji Kondo Nobuto Kitamura Shin Miyatake Yoshie Tanabe Takao Setoguchi Setsuro Komiya Kazunori Yasuda 《Journal of biomechanics》2009,42(15):2611-2615
The effect of stress deprivation on the tendon tissue has been an important focus in the field of biomechanics. However, less is known about the in vivo effect of stress deprivation on fibroblast apoptosis as of yet. This study was conducted to test a hypothesis that complete stress deprivation of the patellar tendon induces fibroblast apoptosis in vivo with activation of Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38) within 24 h after treatment. A total of 35 mature rabbits were divided into stress-shielded (n=15), sham-operated (n=15), and control (n=5) groups. To completely shield the patellar tendon from stress, we used an established surgical method. Animals were sacrificed at 24 h, and 2, 4, 7, and 14 days after the treatment. Tendon specimens underwent TUNEL assay and immunohistological examinations of active caspase-3, JNK, and p38. Both the number and the ratio of TUNEL-positive and caspase-3-positive cells were significantly greater (p<0.0001) in the stress-shielded group than in the sham group at 24 h, 2, 4, and 7 days. Concerning JNK and p38, both the number and the ratio were significantly greater (p<0.0001) in the stress-shielded group than in the sham group at 24 h, 2, and 4 days. This study demonstrated that complete stress deprivation induces fibroblast apoptosis in vivo with activation of JNK and p38 within 24 h. This fact suggested that the fibroblast apoptosis caused by stress deprivation is induced via the mitogen-activated protein kinase signaling pathway. 相似文献
837.
Upasana Shukla Tomoko Hatani Kenji Nakashima Kazuhiro Ogi Kiyonao Sada 《The Journal of biological chemistry》2009,284(49):33719-33728
Adaptor protein c-Abl SH3 domain-binding protein-2 (3BP2, also referred to SH3BP2) regulates immune receptor-mediated signal transduction. In this report we focused on the molecular mechanism of 3BP2 function in B cell receptor (BCR) signaling. Engagement of BCR induces tyrosine phosphorylation of 3BP2. Genetic analysis demonstrated that Syk is critical for BCR-mediated tyrosine phosphorylation of 3BP2. Mutational analysis of 3BP2 revealed that both Tyr183 and Src homology 2 (SH2) domain are necessary for 3BP2-mediated BCR-induced activation of nuclear factor of activated T cells (NFAT). Point mutation of Tyr183 or Arg486 in the SH2 domain of 3BP2 diminished BCR-mediated tyrosine phosphorylation of 3BP2. Endogenous 3BP2 forms a complex with tyrosine-phosphorylated cellular signaling molecules. Peptide binding experiments demonstrated that only phosphorylated Tyr183 in 3BP2 could form a complex with the SH2 domain(s) of phospholipase Cγ2 and Vav1 from B cell lysates. These interactions were represented by using bacterial glutathione S-transferase-phospholipase Cγ2 or -Vav1 SH2 domain. Furthermore, pulldown and Far Western experiments showed that the 3BP2-SH2 domain directly binds to B cell linker protein (BLNK) after BCR stimulation. These results demonstrated that 3BP2 induces the protein complex with cellular signaling molecules through phosphorylation of Tyr183 and SH2 domain leading to the activation of NFAT in B cells. 相似文献
838.
Yuusuke Maruyama Toshihiko Ogura Kazuhiro Mio Kenta Kato Takeshi Kaneko Shigeki Kiyonaka Yasuo Mori Chikara Sato 《The Journal of biological chemistry》2009,284(20):13676-13685
The Ca2+ release-activated Ca2+ channel is a
principal regulator of intracellular Ca2+ rise, which conducts
various biological functions, including immune responses. This channel,
involved in store-operated Ca2+ influx, is believed to be composed
of at least two major components. Orai1 has a putative channel pore and
locates in the plasma membrane, and STIM1 is a sensor for luminal
Ca2+ store depletion in the endoplasmic reticulum membrane. Here we
have purified the FLAG-fused Orai1 protein, determined its tetrameric
stoichiometry, and reconstructed its three-dimensional structure at 21-Å
resolution from 3681 automatically selected particle images, taken with an
electron microscope. This first structural depiction of a member of the Orai
family shows an elongated teardrop-shape 150Å in height and 95Å in
width. Antibody decoration and volume estimation from the amino acid sequence
indicate that the widest transmembrane domain is located between the round
extracellular domain and the tapered cytoplasmic domain. The cytoplasmic
length of 100Å is sufficient for direct association with STIM1. Orifices
close to the extracellular and intracellular membrane surfaces of Orai1 seem
to connect outside the molecule to large internal cavities.Ca2+ is an intracellular second messenger that plays important
roles in various physiological functions such as immune response, muscle
contraction, neurotransmitter release, and cell proliferation. Intracellular
Ca2+ is mainly stored in the endoplasmic reticulum
(ER).2 This ER system
is distributed through the cytoplasm from around the nucleus to the cell
periphery close to the plasma membrane. In non-excitable cells, the ER
releases Ca2+ through the inositol 1,4,5-trisphosphate
(IP3) receptor channel in response to various signals, and the
Ca2+ store is depleted. Depletion of Ca2+ then induces
Ca2+ influx from outside the cell to help in refilling the
Ca2+ stores and to continue Ca2+ rise for several
minutes in the cytoplasm (1,
2). This Ca2+ influx
was first proposed by Putney
(3) and was named
store-operated Ca2+ influx. In the immune system, store-operated
Ca2+ influx is mainly mediated by the Ca2+
release-activated Ca2+ (CRAC) current, which is a highly
Ca2+-selective inwardly rectified current with low conductance
(4,
5). Pathologically, the loss of
CRAC current in T cells causes severe combined immunodeficiency
(6) where many Ca2+
signal-dependent gene expressions, including cytokines, are interrupted
(7). Therefore, CRAC current is
necessary for T cell functions.Recently, Orai1 (also called CRACM1) and STIM1 have been physiologically
characterized as essential components of the CRAC channel
(8–12).
They are separately located in the plasma membrane and in the ER membrane;
co-expression of these proteins presents heterologous CRAC-like currents in
various types of cells (10,
13–15).
Both of them are shown to be expressed ubiquitously in various tissues
(16–18).
STIM1 senses Ca2+ depletion in the ER through its EF hand motif
(19) and transmits a signal to
Orai1 in the plasma membrane. Although Orai1 is proposed as a regulatory
component for some transient receptor potential canonical channels
(20,
21), it is believed from the
mutation analyses to be the pore-forming subunit of the CRAC channel
(8,
22–24).
In the steady state, both Orai1 and STIM1 molecules are dispersed in each
membrane. When store depletion occurs, STIM1 proteins gather into clusters to
form puncta in the ER membrane near the plasma membrane
(11,
19). These clusters then
trigger the clustering of Orai1 in the plasma membrane sites opposite the
puncta (25,
26), and CRAC channels are
activated (27).Orai1 has two homologous genes, Orai2 and Orai3
(8). They form the Orai family
and have in common the four transmembrane (TM) segments with relatively large
N and C termini. These termini are demonstrated to be in the cytoplasm,
because both N- and C-terminally introduced tags are immunologically detected
only in the membrane-permeabilized cells
(8,
9). The subunit stoichiometry
of Orai1 is as yet controversial: it is believed to be an oligomer, presumably
a dimer or tetramer even in the steady state
(16,
28–30).Despite the accumulation of biochemical and electrophysiological data,
structural information about Orai1 is limited due to difficulties in
purification and crystallization. In this study, we have purified Orai1 in its
tetrameric form and have reconstructed the three-dimensional structure from
negatively stained electron microscopic (EM) images. 相似文献
839.
Developmental Change of Sialidase Neu4 Expression in Murine Brain and Its Involvement in the Regulation of Neuronal Cell Differentiation 总被引:1,自引:0,他引:1
Kazuhiro Shiozaki Koichi Koseki Kazunori Yamaguchi Momo Shiozaki Hisashi Narimatsu Taeko Miyagi 《The Journal of biological chemistry》2009,284(32):21157-21164
Sialidase Neu4 is reported to be dominantly expressed in the mouse brain, but its functional significance is not fully understood. We previously demonstrated that sialidase Neu3, also rich in mouse brain, is up-regulated during neuronal differentiation with involvement in acceleration of neurite formation. To elucidate physiological functions of Neu4, as well as Neu3, we determined expression during mouse brain development by quantitative RT-PCR. Expression was relatively low in the embryonic stage and then rapidly increased at 3–14 days after birth, whereas Neu3 demonstrated high levels in the embryonic stage and down-regulation after birth. Murine Neu4 was found to possess two isoforms differing in expression levels, developmental pattern, and enzymatic character. Distinct from the human isoforms, the murine forms, to a different extent, both catalyzed the removal of sialic acid from gangliosides as well as glycoproteins, and one isoform seemed to act on polysialylated NCAM efficiently, despite the low activity toward ordinary substrates. In situ hybridization demonstrated Neu4 mRNA to be present mainly in the hippocampus in which NCAM is rich and decreases after birth. During retinoic acid-induced differentiation, Neu4 expression was down-regulated in Neuro2a cells. Overexpression of Neu4 resulted in suppression of neurite formation, and its knockdown showed the acceleration. Thin layer chromatography of the glycolipids from Neu4-transfected cells showed ganglioside compositions to be only slightly affected, although lectin blot analysis revealed increased binding to Ricinus communis agglutinin (RCA) lectin of a ∼95-kDa glycoprotein, which decreased with cell differentiation. These results suggest that mouse Neu4 plays an important regulatory role in neurite formation, possibly through desialylation of glycoproteins.Sialidases catalyze the removal of sialic acid from non-reducing ends of glycoproteins and glycolipids. In mammals, four types of sialidases have so far been cloned, classified according to their subcellular localization and enzymatic properties (abbreviated to Neu1, Neu2, Neu3, and Neu4) (1–3). Studies have provided strong evidence that these sialidases play crucial roles in various physiological functions such as cell differentiation, cell growth, and malignant transformation. Among these sialidases, Neu4 is unique in its tissue expression pattern and enzymatic properties. In the mouse, it is dominantly expressed in brain, but its sialidase activity is very weak compared with other mouse sialidases (4). In contrast, human NEU4 is expressed not only in brain, but also in liver, kidney, and colon (5–7). We have demonstrated that NEU4 has two isoforms, differing in the N-terminal 12-amino acid residues that act as a mitochondrial-targeting sequence (7). Except for the subcellular localization, enzymatic properties are very similar. The short form of NEU4 (NEU4S) suppresses malignancy in colon cancer cells, mainly through desialylation of some glycoproteins, whereas the long form of NEU4 (NEU4L) may be involved in apoptosis with hydrolysis of ganglioside GD3 in mitochondria (8). Recently, Neu4 knockout mice (Neu4−/−) were generated for pathological analysis (9). Neu4−/− grew normally with a normal lifespan and proved fertile, but vacuolization of the lung and spleen was observed with a lysosomal storage phenotype, and the GM1/GD1a ratio was decreased in the brain. The observations on Neu4−/− are very interesting, but there is some ambiguity in the available previous reports, because, as mentioned above, mouse Neu4 has been reported to have weak sialidase activity in vitro, and its expression is restricted in brain. To clarify this ambiguity and further understand the physiological functions of Neu4, we examined expression in the mouse brain and observed a possible involvement in neural differentiation in connection with another sialidase, Neu3, which greatly increases during differentiation of neuroblastoma cells (10, 11) and causes acceleration of neurite formation (10–13).In the GenBankTM data base, nucleotide sequences of mouse Neu4 have been submitted as and AY258421. The former contains a complete coding sequence of 1506 bp, with two ATGs at positions 1 and 70, and AK034236 encodes only the second ATG ( AK0342364). The gene from has been reported to encode Neu4, showing weak sialidase activity, but there is no information on whether the gene based on AY258421 encodes Neu4 with sialidase activity toward natural substrates. We have now extended our studies to the existence of different mouse Neu4 isoforms, focusing on their significance in neuronal cells by measuring expression levels during cell differentiation. We present, here, evidence that two murine Neu4 isoforms contribute to neurite formation. AK034236相似文献
840.
Yoshihiro Ishikawa Jackie Wirz Janice A. Vranka Kazuhiro Nagata Hans Peter B?chinger 《The Journal of biological chemistry》2009,284(26):17641-17647
The rough endoplasmic reticulum-resident protein complex consisting of prolyl 3-hydroxylase 1 (P3H1), cartilage-associated protein (CRTAP), and cyclophilin B (CypB) can be isolated from chick embryos on a gelatin-Sepharose column, indicating some involvement in the biosynthesis of procollagens. Prolyl 3-hydroxylase 1 modifies a single proline residue in the α chains of type I, II, and III collagens to (3S)-hydroxyproline. The peptidyl-prolyl cis-trans isomerase activity of cyclophilin B was shown previously to catalyze the rate of triple helix formation. Here we show that cyclophilin B in the complex shows peptidyl-prolyl cis-trans isomerase activity and that the P3H1·CRTAP·CypB complex has another important function: it acts as a chaperone molecule when tested with two classical chaperone assays. The P3H1·CRTAP·CypB complex inhibited the thermal aggregation of citrate synthase and was active in the denatured rhodanese refolding and aggregation assay. The chaperone activity of the complex was higher than that of protein-disulfide isomerase, a well characterized chaperone. The P3H1·CRTAP·CypB complex also delayed the in vitro fibril formation of type I collagen, indicating that this complex is also able to interact with triple helical collagen and acts as a collagen chaperone. 相似文献