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181.
Cell division cycle 48 (CDC48), a ubiquitin-dependent molecular chaperone, is thought to mediate a variety of degradative and regulatory processes and maintain cellular homoeostasis. To investigate the protective function of CDC48 against accumulated ubiquitinated proteins during neurodevelopment, we developed an in vivo bioassay technique that detects expression and accumulation of fluorescent proteins with a polyubiquitination signal at the N terminus. When we introduced CDC48 antisense morpholino oligonucleotides into zebrafish embryos, the morphant embryos were lethal and showed defects in neuronal outgrowth and neurodegeneration, and polyubiquitinated fluorescent proteins accumulated in the inner plexiform and ganglion cell layers, as well as the diencephalon and mesencephalon, indicating that the degradation of polyubiquitinated proteins by the ubiquitin-proteasome system was blocked. These abnormal phenotypes in the morphant were rescued by CDC48 or human valosin-containing protein overexpression. Therefore, the protective function of CDC48 is essential for neurodevelopment. 相似文献
182.
Kawabata T Tanimura S Asai K Kawasaki R Matsumaru Y Kohno M 《The Journal of biological chemistry》2012,287(13):10289-10300
Blockade of the ERK signaling pathway by ERK kinase (MEK) inhibitors selectively enhances the induction of apoptosis by microtubule inhibitors in tumor cells in which this pathway is constitutively activated. We examined the mechanism by which such drug combinations induce enhanced cell death by applying time-lapse microscopy to track the fate of individual cells. MEK inhibitors did not affect the first mitosis after drug exposure, but most cells remained arrested in interphase without entering a second mitosis. Low concentrations of microtubule inhibitors induced prolonged mitotic arrest followed by exit of cells from mitosis without division, with most cells remaining viable. However, the combination of a MEK inhibitor and a microtubule inhibitor induced massive cell death during prolonged mitosis. Impairment of spindle assembly checkpoint function by RNAi-mediated depletion of Mad2 or BubR1 markedly suppressed such prolonged mitotic arrest and cell death. The cell death was accompanied by up-regulation of the pro-apoptotic protein Bim (to which MEK inhibitors contributed) and by down-regulation of the anti-apoptotic protein Mcl-1 (to which microtubule and MEK inhibitors contributed synergistically). Whereas RNAi-mediated knockdown of Bim suppressed cell death, stabilization of Mcl-1 by RNAi-mediated depletion of Mule slowed its onset. Depletion of Mcl-1 sensitized tumor cells to MEK inhibitor-induced cell death, an effect that was antagonized by knockdown of Bim. The combination of MEK and microtubule inhibitors thus targets Bim and Mcl-1 in a cooperative manner to induce massive cell death in tumor cells with aberrant ERK pathway activation. 相似文献
183.
Hua Wan Yuichi Otsuka Zeng‐Qiang Gao Yong Wei Zhen Chen Michiaki Masuda Tetsuro Yonesaki Heng Zhang Yu‐Hui Dong 《Molecular microbiology》2016,101(5):757-769
Bacteria have obtained a variety of resistance mechanisms including toxin‐antitoxin (TA) systems against bacteriophages (phages), whereas phages have also evolved to overcome bacterial anti‐phage mechanisms. Dmd from T4 phage can suppress the toxicities of homologous toxins LsoA and RnlA from Escherichia coli, representing the first example of a phage antitoxin against multiple bacterial toxins in known TA systems. Here, the crystal structure of LsoA‐Dmd complex showed Dmd is inserted into the deep groove between the N‐terminal repeated domain (NRD) and the Dmd‐binding domain (DBD) of LsoA. The NRD shifts significantly from a ‘closed’ to an ‘open’ conformation upon Dmd binding. Site‐directed mutagenesis of Dmd revealed the conserved residues (W31 and N40) are necessary for LsoA binding and the toxicity suppression as determined by pull‐down and cell toxicity assays. Further mutagenesis identified the conserved Dmd‐binding residues (R243, E246 and R305) of LsoA are vital for its toxicity, and suggested Dmd and LsoB may possess different inhibitory mechanisms against LsoA toxicity. Our structure‐function studies demonstrate Dmd can recognize LsoA and inhibit its toxicity by occupying the active site possibly via substrate mimicry. These findings have provided unique insights into the defense and counter‐defense mechanisms between bacteria and phages in their co‐evolution. 相似文献
184.
We isolated and sequenced caspase-10 cDNA and gene from Japanese flounder, Paralichthys olivaceus. The Japanese flounder (JF)-caspase-10 cDNA consisted of 2282 bp and encoded 495 amino acid residues. The characteristic death effector domains (DEDs) of caspases were observed in JF-caspase-10 as well as the three aspartic acid residues (D-186, -382 and -392), which are potential cleavage sites for the large and small subunit structures. The amino acid residue (His-325) and pentapeptide (QACQG), which are involved in catalytic activity, were absolutely conserved in Japanese flounder-caspase-10. JF-caspase-10 gene has a length of 6.6 kb and consists of 11 exons and 10 introns similar to that of human. The strong expression of JF-caspase-10 mRNA was detected in the gills, peripheral blood leukocytes, spleen and posterior kidney, while the weak expression was observed in the head kidney, heart, intestine, skin and stomach. The over-expression analysis of JF-caspase-10 in Japanese flounder cell line HINAE was shown to induce apoptosis 24h post-transfection using TUNEL assay. 相似文献
185.
Asano R Watanabe Y Kawaguchi H Fukazawa H Nakanishi T Umetsu M Hayashi H Katayose Y Unno M Kudo T Kumagai I 《The Journal of biological chemistry》2007,282(38):27659-27665
We previously reported the marked in vitro and in vivo antitumor activity of hEx3, a humanized diabody (small recombinant bispecific antibody) with epidermal growth factor receptor (EGFR) and CD3 retargeting. Here, we fabricated a tetravalent IgG-like bispecific antibody with two kinds of single-chain Fv (scFv), i.e. humanized anti-EGFR scFv and anti-CD3 scFv, that contains the same four variable domains as hEx3, on the platform of human IgG1 (hEx3-scFv-Fc). hEx3-scFv-Fc prepared from mammalian cells showed specific binding to both EGFR and CD3 target antigens. At one-thousandth (0.1-100 fmol/ml) of the dose of normal hEx3, hEx3-scFv-Fc showed intense cytotoxicity to an EGFR-positive cell line in a growth-inhibition assay using lymphokine-activated killer cells with the T-cell phenotype (T-LAK cells). The enhanced antitumor effect was more clearly observed when peripheral blood mononuclear cells (PBMCs) were used as effector cells, indicating the utility of IgG-like fabrication. These results suggested that the intense antitumor activity is attributable to the multivalency and the presence of the fused human Fc, a hypothesis that was supported by the results of flow cytometry, PBMC proliferation assay, and protein kinase inhibition assay. Furthermore, the growth inhibition effects of hEx3-scFv-Fc were considerably superior to those of the approved therapeutic antibody, cetuximab, which recognizes the same EGFR antigen even when using PBMCs as effector cells. The high potency of hEx3-scFv-Fc may translate into improved antitumor therapy and lower costs of production because of the smaller doses needed. 相似文献
186.
Doi Y Kubo M Yonemoto K Ninomiya T Iwase M Tanizaki Y Shikata K Iida M Kiyohara Y 《Obesity (Silver Spring, Md.)》2007,15(7):1841-1850
Objective: We studied the relationship between liver enzymes and the development of diabetes in a general Japanese population. Research Methods and Procedures: A total of 1804 non‐diabetic subjects 40 to 79 years of age were followed‐up prospectively for a mean of 9.0 years. Results: During the follow‐up, 135 subjects developed diabetes. In both sexes, the age‐adjusted cumulative incidence of diabetes increased significantly with elevating quartiles of serum γ‐glutamyltransferase (GGT) and alanine aminotransferase (ALT) levels. This pattern was also observed in aspartate aminotransferase (AST) quartiles for men but not for women. In multivariate analyses after adjusting for comprehensive risk factors and other liver enzymes, the risk of developing diabetes was significantly higher in the highest GGT quartile than in the lowest quartile [odds ratio (OR), 2.54; 95% confidence interval (CI), 1.03 to 6.26 for men; OR, 5.73; 95% CI, 1.62 to 20.19 for women]. Similar results were observed in ALT quartiles (OR, 2.32; 95% CI, 0.91 to 5.92 for men; OR, 4.40; 95% CI, 1.38 to 14.06 for women) but not in AST quartiles in either sex. Significant positive associations of GGT and ALT with diabetes were seen within each stratified category of risk factors, namely fasting insulin, BMI, waist‐to‐hip ratio, high‐sensitivity C‐reactive protein, and alcohol consumption. In receiver operating characteristic analyses, the areas under the receiver operating characteristic curve of GGT and ALT were significantly larger than that of AST, fasting insulin, waist‐to‐hip ratio, or C‐reactive protein. Discussion: Our findings suggest that serum GGT and ALT concentrations are strong predictors of diabetes in the general population, independent of known risk factors. 相似文献
187.
Cancer prevention by carotenoids 总被引:1,自引:0,他引:1
Nishino H Murakoshi M Tokuda H Satomi Y 《Archives of biochemistry and biophysics》2009,483(2):165-526
Various natural carotenoids seem to be valuable for cancer prevention, and these carotenoids may be more suitable in combinational use, rather than in single use. In fact, we have proven that combinational use of natural carotenoids resulted in significant suppression of liver cancer. Patients of viral hepatitis with cirrhosis were administered with β-cryptoxanthin-enriched Mandarin orange juice, in addition to capsules of carotenoids mixture. Cumulative incidence of hepatocellular carcinoma development was compared with that in the group treated with carotenoids mixture capsules alone, or in the group without treatment (control group). In the data analysis at year 2.5, cumulative incidence of liver cancer in β-cryptoxanthin-enriched orange juice with carotenoids mixture capsules-treated group was lower than that in the control group (p = 0.05). Cumulative incidence of liver cancer in the group treated with carotenoids mixture capsules alone was also lower than that in the control group, but not statistically significant. 相似文献
188.
Eli Kakiashvili Pam Speight Faiza Waheed Romy Seth Monika Lodyga Susumu Tanimura Michiaki Kohno Ori D. Rotstein Andr��s Kapus Katalin Sz��szi 《The Journal of biological chemistry》2009,284(17):11454-11466
Tumor necrosis factor-α (TNF-α), an inflammatory cytokine, has
been shown to activate the small GTPase Rho, but the underlying signaling
mechanisms remained undefined. This general problem is particularly important
in the kidney, because TNF-α, a major mediator of kidney injury, is
known to increase paracellular permeability in tubular epithelia. Here we
aimed to determine the effect of TNF-α on the Rho pathway in tubular
cells (LLC-PK1 and Madin-Darby canine kidney), define the upstream
signaling, and investigate the role of the Rho pathway in the
TNF-α-induced alterations of paracellular permeability. We show that
TNF-α induced a rapid and sustained RhoA activation that led to stress
fiber formation and Rho kinase-dependent myosin light chain (MLC)
phosphorylation. To identify new regulators connecting the TNF receptor to Rho
signaling, we applied an affinity precipitation assay with a Rho mutant
(RhoG17A), which captures activated GDP-GTP exchange factors (GEFs). Mass
spectrometry analysis of the RhoG17A-precipitated proteins identified GEF-H1
as a TNF-α-activated Rho GEF. Consistent with a central role of GEF-H1,
its down-regulation by small interfering RNA prevented the activation of the
Rho pathway. Moreover GEF-H1 and Rho activation are downstream of ERK
signaling as the MEK1/2 inhibitor PD98059 mitigated TNF-α-induced
activation of these proteins. Importantly TNF-α enhanced the ERK
pathway-dependent phosphorylation of Thr-678 of GEF-H1 that was key for
activation. Finally the TNF-α-induced paracellular permeability increase
was absent in LLC-PK1 cells stably expressing a
non-phosphorylatable, dominant negative MLC. In summary, we have identified
the ERK/GEF-H1/Rho/Rho kinase/phospho-MLC pathway as the mechanism mediating
TNF-α-induced elevation of tubular epithelial permeability, which in
turn might contribute to kidney injury.Tumor necrosis factor-α
(TNF-α)2 is a
pleiotropic proinflammatory cytokine that is synthesized as a membrane protein
in response to inflammation, infection, and injury
(1). Subsequently it is cleaved
by the metalloprotease TNF-α convertase enzyme to release a 17-kDa
soluble peptide (for a review, see Ref.
2). TNF-α has two
receptors, the constitutively expressed, ubiquitous TNF receptor 1 and the
inducible TNF receptor 2.An increasing body of evidence supports a key role for TNF-α in both
acute renal injury and chronic kidney diseases (for reviews, see Refs.
3 and
4). Although TNF-α is
almost undetectable in normal kidneys, elevated intrarenal, serum, or urine
concentrations have been reported in various pathological states including
ischemia-reperfusion, endotoxinemia, and early diabetic nephropathy
(5–8).
Moreover kidney injury in various pathological states was prevented or
mitigated by inhibition of TNF-α production, by addition of neutralizing
antibodies, or in TNF receptor knock-out mice (for a review, see Ref.
3). The central role of
TNF-α in mediating kidney injury is therefore well established.
Importantly TNF-α can be produced in the kidney not only by infiltrating
macrophages and lymphocytes but by resident cells including the tubular
epithelium. For example, in reperfusion injury TNF-α expression precedes
macrophage infiltration and localizes mostly to the tubules
(3,
7). Tubular TNF-α
production is also enhanced by endotoxin and hypoxia
(9–12).
Although effects of locally released TNF-α on the tubular epithelium
could contribute to its deleterious actions, the underlying mechanisms have
been incompletely explored.Although a large number of studies have focused on the inflammatory and
apoptotic signaling initiated by TNF-α in various cells, its
cytoskeletal effects remain much less explored. In recent years Rho and its
effector, Rho kinase (ROK), key regulators of both the actin cytoskeleton and
myosin phosphorylation (13),
have emerged as important mediators of TNF-α effects in endothelial
cells
(14–18).
Similar effects in the tubular epithelium, however, have not been established.
Even more importantly, the upstream signaling that connects the TNF receptor
to activation of the Rho pathway remains completely unknown. Like other small
GTPases, Rho cycles between an inactive (GDP-bound) and active (GTP-bound)
form (13). The exchange of GDP
to GTP during activation is stimulated by GDP-GTP exchange factors (GEFs). The
diverse family of Rho GEFs contains >70 members in humans
(19), making it challenging to
identify the specific factors involved in mediating Rho activation through
receptor-mediated stimuli. In the case of TNF-α, neither the particular
Rho GEF involved nor the mechanism of its regulation has been identified in
any of the cell systems studied.A rise in epithelial paracellular permeability through the intercellular
junctions is a prominent event during inflammation (“leaky
epithelium”) (for reviews, see Refs.
20 and
21). In addition, the
junctions maintain the polarized phenotype of epithelial cells that is
necessary for directional transport processes and constitute an important
signaling platform that transmits environmental cues to the cells. Therefore,
the consequences of junction disruption during inflammation might go beyond
the compromised barrier functions. Interestingly TNF-α has been reported
to affect the permeability of the tubular epithelium. Mullin et al.
(22) have reported that in a
tubular cell line TNF-α induced a temporary elevation in transepithelial
resistance followed by a drop in transepithelial resistance and increased
paracellular permeability. The transepithelial resistance decrease was blocked
by genistein, a general tyrosine kinase inhibitor; however, the exact
mechanism underlying the observed permeability changes remained incompletely
explored.The actin cytoskeleton and especially phosphorylation of myosin light chain
(MLC) was shown to be essential for the permeability increase caused by
pathogens, cytokines, and growth factors in various epithelial and endothelial
systems (for reviews, see Refs.
21,
23, and
24). Interestingly although
myosin phosphorylation mediates the TNF-α-elicited permeability changes
in intestinal cells (25,
26), phospho-MLC was reported
not to be involved in the TNF-α-induced permeability rise in endothelial
cells (17). The possible role
of the Rho pathway and myosin phosphorylation in the TNF-α-induced
permeability changes in the tubular epithelium therefore remains to be
established.The aim of this study was to explore the signaling pathways through which
TNF-α causes cytoskeleton remodeling and elevates paracellular
permeability in kidney tubular cells. Our findings show that TNF-α
induces rapid activation of RhoA that leads to Rho/Rho kinase-dependent actin
remodeling and myosin phosphorylation. Using an affinity precipitation assay
followed by mass spectrometry, we identified GEF-H1 as a TNF-α-activated
GEF. We showed that GEF-H1 mediates the TNF-α-induced stimulation of Rho
and its effectors. In addition, activation of the GEF-H1/Rho pathway by
TNF-α was downstream of ERK signaling and required GEF-H1
phosphorylation on Thr-678. Finally using a dominant negative MLC mutant, we
showed that myosin phosphorylation is essential for the TNF-α-induced
elevation in paracellular permeability. 相似文献
189.
Toshihiro Horii Hiroki Shirai Li Jie Ken J. Ishii Nirianne Q. Palacpac Takahiro Tougan Mariko Hato Nobuo Ohta Albino Bobogare Nana Arakaki Yoshitsugu Matsumoto Junko Namazue Toyokazu Ishikawa Shigeharu Ueda Michiaki Takahashi 《Parasitology international》2010,59(3):380-386
An effective malaria vaccine is a public health priority. Proteins expressed during the blood-stage of the parasite life cycle have been proposed as good vaccine candidates. No such blood-stage vaccine, however, is available against Plasmodium falciparum, the deadliest Plasmodium species. We show here that P. falciparum serine repeat antigen 5 (SERA5) is a potential vaccine immunogen. We have constructed a new recombinant molecule of SERA5, namely SE36, based on previously reported SE47′ molecule by removing the serine repeats. Epidemiological study in the holo-endemic population of Solomon Islands shows highly significant correlation of sero-conversion and malaria protective immunity against this antigen. Animal experiments using non-human primates, and a human phase 1a clinical trial assessed SE36 vaccine immunogenicity. Vaccination of squirrel monkeys with SE36 protein and aluminum hydroxyl gel (SE36/AHG) conferred protection against high parasitemia and boosted serum anti-SE36 IgG after P. falciparum parasite challenge. SE36/AHG was highly immunogenic in chimpanzees, where serum anti-SE36 IgG titers last more than one year. Phase 1a clinical trial (current controlled trials, ISRCTN78679862) demonstrated the safety and immunogenicity of SE36/AHG with 30 healthy adults and 10 placebo controls. Three subcutaneous administrations of 50 and 100 μg dose of SE36/AHG were well-tolerated, with no severe adverse events; and resulted in 100% sero-conversion in both dose arms. The current research results for SE36/AHG provide initial clinical validation for future trials and suggest clues/strategies for further vaccine development. 相似文献
190.
Masahiro Nakajima Atsushi Takahashi Ikuyo Kou Cristina Rodriguez-Fontenla Juan J. Gomez-Reino Tatsuya Furuichi Jin Dai Akihiro Sudo Atsumasa Uchida Naoshi Fukui Michiaki Kubo Naoyuki Kamatani Tatsuhiko Tsunoda Konstantinos N. Malizos Aspasia Tsezou Antonio Gonzalez Yusuke Nakamura Shiro Ikegawa 《PloS one》2010,5(3)
Osteoarthritis (OA) is a common disease that has a definite genetic component. Only a few OA susceptibility genes that have definite functional evidence and replication of association have been reported, however. Through a genome-wide association study and a replication using a total of ∼4,800 Japanese subjects, we identified two single nucleotide polymorphisms (SNPs) (rs7775228 and rs10947262) associated with susceptibility to knee OA. The two SNPs were in a region containing HLA class II/III genes and their association reached genome-wide significance (combined P = 2.43×10−8 for rs7775228 and 6.73×10−8 for rs10947262). Our results suggest that immunologic mechanism is implicated in the etiology of OA. 相似文献