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11.
Karina Perlaza Mary Mirvis Hiroaki Ishikawa Wallace Marshall 《Molecular biology of the cell》2022,33(2)
Length control of flagella represents a simple and tractable system to investigate the dynamics of organelle size. Models for flagellar length control in the model organism Chlamydomonas reinhardtii have focused on the length dependence of the intraflagellar transport (IFT) system, which manages the delivery and removal of axonemal subunits at the tip of the flagella. One of these cargoes, tubulin, is the major axonemal subunit, and its frequency of arrival at the tip plays a central role in size control models. However, the mechanisms determining tubulin dynamics at the tip are still poorly understood. We discovered a loss-of-function mutation that leads to shortened flagella and found that this was an allele of a previously described gene, SHF1, whose molecular identity had not been determined. We found that SHF1 encodes a Chlamydomonas orthologue of Crescerin, previously identified as a cilia-specific TOG-domain array protein that can bind tubulin via its TOG domains and increase tubulin polymerization rates. In this mutant, flagellar regeneration occurs with the same initial kinetics as in wild-type cells but plateaus at a shorter length. Using a computational model in which the flagellar microtubules are represented by a differential equation for flagellar length combined with a stochastic model for cytoplasmic microtubule dynamics, we found that our experimental results are best described by a model in which Crescerin/SHF1 binds tubulin dimers in the cytoplasm and transports them into the flagellum. We suggest that this TOG-domain protein is necessary to efficiently and preemptively increase intraflagella tubulin levels to offset decreasing IFT cargo at the tip as flagellar assembly progresses. 相似文献
12.
Expression, purification, and crystallization of endopolygalacturonase from a pathogenic fungus, Stereum purpureum, in Escherichia coli 总被引:1,自引:0,他引:1
Shimizu T Shibata H Araya T Nakatsu T Miyairi K Okuno T Kato H 《Protein expression and purification》2005,44(2):558-135
Endopolygalacturonases (EC 3.2.1.15) catalyze random hydrolysis of the alpha-1,4 glycosidic linkages in polygalacturonic acid, a component of pectin. Previously, we reported crystal structures of endogenously produced Stereum purprureum endopolygalacturonase I (endoPG I), both in its native form and complexed with its product, galacturonate. However, the substrate-binding mechanism of endoPG I is still unclear, because crystals have not yet been obtained with a substrate analog, or with mutant enzymes that can bind substrates. We describe here an expression system using Escherichia coli and a purification method to prepare functionally active endoPG I for such mutation and crystallographic studies. Expression in E. coli strain Origami (DE3) provided a soluble and active enzyme with proper disulfide bond formation, whereas the enzyme expressed in BL21 (DE3) was localized in inclusion bodies. A sufficient amount of recombinant endoPG I produced by Origami (DE3) was purified by a single-step procedure using cation exchange chromatography. The specific activity of recombinant endoPG I was equivalent to that of the enzyme produced by S. purpureum. Recombinant endoPG I was crystallized under the same conditions as those used for the native enzyme produced by S. purpureum. The crystals diffracted beyond 1.0 A resolution with synchrotron radiation. 相似文献
13.
Horiuchi T Nishimukai H Okiura T Nishimura K Nishizaka H Kojima T Tsukamoto H Hayashi K Harada M 《Biochemical and biophysical research communications》2002,298(3):450-455
Complement C7 is one of the components of membrane attack complex (MAC) generated by the terminal complement cascade. C7 protein is polymorphic and most of its polymorphisms have been identified using isoelectric focusing (IEF), which detects protein charge differences. To date, the molecular bases of the polymorphisms detected by IEF have not been determined. In this paper, we describe the structural bases of two C7 IEF-detected polymorphisms, C7*3 and C7*4, both of which are common in Asian populations. C7*3 resulted from substitution of cysteine (Cys) at amino acid residue 106 by charged arginine (Arg; C106R), while charged lysine (Lys) at amino acid residue 398 was replaced by neutral glutamine (Gln; K398Q) in C7*4. As C7*3 is hypomorphic, it is important to study its possible associations with diseases such as immunological disorders and infections. We present genetic bases for this C7 polymorphism, which we determined using polymerase chain reaction (PCR)-based genotyping, a simple and accurate method suitable for large-scale studies. 相似文献
14.
Tomomichi Chonan Takahiro Oi Daisuke Yamamoto Miyoko Yashiro Daisuke Wakasugi Hiroaki Tanaka Ayumi Ohoka-Sugita Fusayo Io Hiroko Koretsune Akira Hiratate 《Bioorganic & medicinal chemistry letters》2009,19(23):6645-6648
Acetyl-CoA carboxylases (ACCs), the rate limiting enzymes in de novo lipid synthesis, play important roles in modulating energy metabolism. The inhibition of ACC has demonstrated promising therapeutic potential for treating obesity and type 2 diabetes mellitus in transgenic mice and preclinical animal models. We describe herein the synthesis and structure–activity relationships of a series of disubstituted (4-piperidinyl)-piperazine derivatives as a new platform for ACC1/2 non-selective inhibitors. 相似文献
15.
16.
Peripheral tolerance and the qualitative characteristics of autoreactive T cell clones in primary biliary cirrhosis 总被引:1,自引:0,他引:1
Kawano A Shimoda S Kamihira T Ishikawa F Niiro H Soejima Y Taketomi A Maehara Y Nakamura M Komori A Migita K Ishibashi H Azuma M Gershwin ME Harada M 《Journal of immunology (Baltimore, Md. : 1950)》2007,179(5):3315-3324
Primary biliary cirrhosis is characterized by autoreactive T cells specific for the mitochondrial Ag PDC-E2(163-176). We studied the ability of eight T cell clones (TCC) specific for PDC-E2(163-176) to proliferate or become anergic in the presence of costimulation signals. TCC were stimulated with either human PDC-E2(163-176), an Escherichia coli 2-oxoglutarate dehydrogenase mimic (OGDC-E2(34-47)), or analogs with amino acid substitutions using HLA-matched allogeneic PBMC or mouse L-DR53 fibroblasts as APC. Based on their differential responses to these peptides (human PDC-E2(163-176), E. coli OGDC-E2(34-47)) in the different APC systems, TCC were classified as costimulation dependent or independent. Only costimulation-dependent TCC could become anergic. TCC with costimulation-dependent responses to OGDC-E2 become anergic to PDC-E2 when preincubated with mimic, even if costimulation is independent for PDC-E2(163-176). Anergic TCC produced IL-10. One selected TCC could not become anergic after preincubation with PDC-E2(163-176)-pulsed L-DR53 but became anergic using L-DR53 pulsed with PDC-E2 peptide analogs with a substitution at a critical TCR binding site. TCC that only respond to peptide-pulsed PBMC, but not L-DR53, proliferate with peptide-pulsed CD80/CD86-transfected L-DR53; however, anergy was not induced with peptide-pulsed L-DR53 transfected with only CD80 or CD86. These data highlight that costimulation plays a dominant role in maintaining peripheral tolerance to PBC-specific Ags. They further suggest that, under specific circumstances, molecular mimicry of an autoantigen may restore rather than break peripheral tolerance. 相似文献
17.
Kazushi Yukiiri Naohisa Hosomi Takayuki Naya Tsutomu Takahashi Hiroyuki Ohkita Mao Mukai Hisashi Masugata Koji Murao Masaki Ueno Takehiro Nakamura Hiroaki Dobashi Takanori Miki Yasuhiro Kuroda Masakazu Kohno 《BMC neurology》2008,8(1):1-8
Background
Cardioembolic stroke generally results in more severe disability, since it typically has a larger ischemic area than the other types of ischemic stroke. However, it is difficult to differentiate cardioembolic stroke from non-cardioembolic stroke (atherothrombotic stroke and lacunar stroke). In this study, we evaluated the levels of plasma brain natriuretic peptide in acute ischemic stroke patients with cardioembolic stroke or non-cardioembolic stroke, and assessed the prediction factors of plasma brain natriuretic peptide and whether we could differentiate between stroke subtypes on the basis of plasma brain natriuretic peptide concentrations in addition to patient's clinical variables.Methods
Our patient cohort consisted of 131 consecutive patients with acute cerebral infarction who were admitted to Kagawa University School of Medicine Hospital from January 1, 2005 to December 31, 2007. The mean age of patients (43 females, 88 males) was 69.6 ± 10.1 years. Sixty-two patients had cardioembolic stroke; the remaining 69 patients had non-cardioembolic stroke (including atherothrombotic stroke, lacunar stroke, or the other). Clinical variables and the plasma brain natriuretic peptide were evaluated in all patients.Results
Plasma brain natriuretic peptide was linearly associated with atrial fibrillation, heart failure, chronic renal failure, and left atrial diameter, independently (F4,126 = 27.6, p < 0.0001; adjusted R2 = 0.45). Furthermore, atrial fibrillation, mitral regurgitation, plasma brain natriuretic peptide (> 77 pg/ml), and left atrial diameter (> 36 mm) were statistically significant independent predictors of cardioembolic stroke in the multivariable setting (Χ2 = 127.5, p < 0.001).Conclusion
It was suggested that cardioembolic stroke was strongly predicted with atrial fibrillation and plasma brain natriuretic peptide. Plasma brain natriuretic peptide can be a surrogate marker for cardioembolic stroke. 相似文献18.
19.
20.
Junichi Ushiyama Kei Masani Motoki Kouzaki Hiroaki Kanehisa Tetsuo Fukunaga 《Journal of applied physiology》2005,98(4):1427-1433
It has been suggested that a suppression of maximal voluntary contraction (MVC) induced by prolonged vibration is due to an attenuation of Ia afferent activity. The purpose of the present study was to test the hypothesis that aftereffects following prolonged vibration on muscle activity during MVC differ among plantar flexor synergists owing to a supposed difference in muscle fiber composition. The plantar flexion MVC torque and surface electromyogram (EMG) of the medial head of gastrocnemius (MG), the lateral head of gastrocnemius (LG), and the soleus (Sol) were recorded in 13 subjects before and after prolonged vibration applied to the Achilles tendon at 100 Hz for 30 min. The maximal H reflexes and M waves were also determined from the three muscles, and the ratio between H reflexes and M waves (H/Mmax) was calculated before and after the vibration. The MVC torque was decreased by 16.6 +/- 3.7% after the vibration (P < 0.05; ANOVA). The H/Mmax also decreased for all three muscles, indicating that Ia afferent activity was successfully attenuated by the vibration in all plantar flexors. However, a reduction of EMG during MVC was observed only in MG (12.7 +/- 4.0%) and LG (11.4 +/- 3.9%) (P < 0.05; ANOVA), not in Sol (3.4 +/- 3.0%). These results demonstrated that prolonged vibration-induced MVC suppression was attributable mainly to the reduction of muscle activity in MG and LG, both of which have a larger proportion of fast-twitch muscle fibers than Sol. This finding suggests that Ia-afferent activity that reinforces the recruitment of high-threshold motor units is necessary to enhance force exertion during MVC. 相似文献