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1.
Front Cell Dev Biol ; 8: 580520, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33154970

RESUMO

Fibrous collagen imparts physical strength and flexibility to tissues by forming huge complexes. The density and orientation of collagen fibers must be correctly specified for the optimal physical property of the collagen complex. However, little is known about its underlying cellular mechanisms. Actinotrichia are collagen fibers aligned at the fin-tip of bony fish and are easily visible under the microscope due to their thick, linear structure. We used the actinotrichia as a model system to investigate how cells manipulate collagen fibers. The 3D image obtained by focused ion beam scanning electron microscopy (FIB-SEM) showed that the pseudopodia of mesenchymal cells encircle the multiple actinotrichia. We then co-incubated the mesenchymal cells and actinotrichia in vitro, and time-lapse analysis revealed how cells use pseudopods to align collagen fiber orientation. This in vitro behavior is dependent on actin polymerization in mesenchymal cells. Inhibition of actin polymerization in mesenchymal cells results in mis-orientation of actinotrichia in the fin. These results reveal how mesenchymal cells are involved in fin formation and have important implications for the physical interaction between cells and collagen fibers.

2.
Sci Adv ; 6(30): eaba1195, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32743070

RESUMO

Immotile cilia sense extracellular signals such as fluid flow, but whether Ca2+ plays a role in flow sensing has been unclear. Here, we examined the role of ciliary Ca2+ in the flow sensing that initiates the breaking of left-right (L-R) symmetry in the mouse embryo. Intraciliary and cytoplasmic Ca2+ transients were detected in the crown cells at the node. These Ca2+ transients showed L-R asymmetry, which was lost in the absence of fluid flow or the PKD2 channel. Further characterization allowed classification of the Ca2+ transients into two types: cilium-derived, L-R-asymmetric transients (type 1) and cilium-independent transients without an L-R bias (type 2). Type 1 intraciliary transients occurred preferentially at the left posterior region of the node, where L-R symmetry breaking takes place. Suppression of intraciliary Ca2+ transients delayed L-R symmetry breaking. Our results implicate cilium-derived Ca2+ transients in crown cells in initiation of L-R symmetry breaking in the mouse embryo.

3.
EMBO J ; 39(12): e103499, 2020 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-32368833

RESUMO

Primary cilia are antenna-like organelles on the surface of most mammalian cells that receive sonic hedgehog (Shh) signaling in embryogenesis and carcinogenesis. Cellular cholesterol functions as a direct activator of a seven-transmembrane oncoprotein called Smoothened (Smo) and thereby induces Smo accumulation on the ciliary membrane where it transduces the Shh signal. However, how cholesterol is supplied to the ciliary membrane remains unclear. Here, we report that peroxisomes are essential for the transport of cholesterol into the ciliary membrane. Zellweger syndrome (ZS) is a peroxisome-deficient hereditary disorder with several ciliopathy-related features and cells from these patients showed a reduced cholesterol level in the ciliary membrane. Reverse genetics approaches revealed that the GTP exchange factor Rabin8, the Rab GTPase Rab10, and the microtubule minus-end-directed kinesin KIFC3 form a peroxisome-associated complex to control the movement of peroxisomes along microtubules, enabling communication between peroxisomes and ciliary pocket membranes. Our findings suggest that insufficient ciliary cholesterol levels may underlie ciliopathies.

4.
Sci Rep ; 10(1): 2626, 2020 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-32060319

RESUMO

Assessing the structure and function of organelles in living organisms of the primitive unicellular red algae Cyanidioschyzon merolae on three-dimensional sequential images demands a reliable automated technique in the class imbalance among various cellular structures during mitosis. Existing classification networks with commonly used loss functions were focused on larger numbers of cellular structures that lead to the unreliability of the system. Hence, we proposed a balanced deep regularized weighted compound dice loss (RWCDL) network for better localization of cell organelles. Specifically, we introduced two new loss functions, namely compound dice (CD) and RWCD by implementing multi-class variant dice and weighting mechanism, respectively for maximizing weights of peroxisome and nucleus among five classes as the main contribution of this study. We extended the Unet-like convolution neural network (CNN) architecture for evaluating the ability of our proposed loss functions for improved segmentation. The feasibility of the proposed approach is confirmed with three different large scale mitotic cycle data set with different number of occurrences of cell organelles. In addition, we compared the training behavior of our designed architectures with the ground truth segmentation using various performance measures. The proposed balanced RWCDL network generated the highest area under the curve (AUC) value in elevating the small and obscure peroxisome and nucleus, which is 30% higher than the network with commonly used mean square error (MSE) and dice loss (DL) functions. The experimental results indicated that the proposed approach can efficiently identify the cellular structures, even when the contour between the cells is obscure and thus convinced that the balanced deep RWCDL approach is reliable and can be helpful for biologist to accurately identify the relationship between the cell behavior and structures of cell organelles during mitosis.


Assuntos
Processamento de Imagem Assistida por Computador/métodos , Redes Neurais de Computação , Rodófitas/ultraestrutura , Algoritmos , Imageamento Tridimensional/métodos , Microscopia Eletrônica de Varredura/métodos , Mitose , Organelas/ultraestrutura , Rodófitas/citologia
5.
Biophys Physicobiol ; 14: 1-11, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28409085

RESUMO

The metaphase spindle is organized for accurate chromosome segregation. One of the fundamental features of the spindle across the species is its symmetrical shape; the spindle consists of two polar arrays of microtubules at both ends. Although it has been suggested that the formation of the bipolar shape requires force balance coordination by molecular motors, i.e., kinesins and dyneins, quantitative analysis for the pole mechanics has not been conducted. Here, we demonstrate that it is not only the shape but also the stiffness and microtubule density of the pairs of pole regions are symmetrically balanced in single spindles self-assembled in Xenopus egg extracts. We found that the inhibition of dynein functions dramatically reduced the stiffness and microtubule density in the pole region. By contrast, the inhibition of one of the kinesins, Eg5, which is the antagonistic motor protein of dynein, increased the value of these parameters. Moreover, the inhibition of both dynein and Eg5 recovered these parameter values to those of non-treated spindle poles. We also found that, when one pole structure was held widened with the use of two glass microneedles, the opposite pole structure spontaneously widened, resulting in the formation of the barrel-like shaped spindle. The values of stiffness and microtubule density in the manipulated pole region decreased, following the spontaneous decrement of those in the paired unmanipulated pole region. These results suggest that the spindle possesses a mechanism to dynamically maintain its symmetry in mechanical properties.

6.
Proc Natl Acad Sci U S A ; 114(11): 2922-2927, 2017 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-28265076

RESUMO

Collective behaviors of motile units through hydrodynamic interactions induce directed fluid flow on a larger length scale than individual units. In cells, active cytoskeletal systems composed of polar filaments and molecular motors drive fluid flow, a process known as cytoplasmic streaming. The motor-driven elongation of microtubule bundles generates turbulent-like flow in purified systems; however, it remains unclear whether and how microtubule bundles induce large-scale directed flow like the cytoplasmic streaming observed in cells. Here, we adopted Xenopus egg extracts as a model system of the cytoplasm and found that microtubule bundle elongation induces directed flow for which the length scale and timescale depend on the existence of geometrical constraints. At the lower activity of dynein, kinesins bundle and slide microtubules, organizing extensile microtubule bundles. In bulk extracts, the extensile bundles connected with each other and formed a random network, and vortex flows with a length scale comparable to the bundle length continually emerged and persisted for 1 min at multiple places. When the extracts were encapsulated in droplets, the extensile bundles pushed the droplet boundary. This pushing force initiated symmetry breaking of the randomly oriented bundle network, leading to bundles aligning into a rotating vortex structure. This vortex induced rotational cytoplasmic flows on the length scale and timescale that were 10- to 100-fold longer than the vortex flows emerging in bulk extracts. Our results suggest that microtubule systems use not only hydrodynamic interactions but also mechanical interactions to induce large-scale temporally stable cytoplasmic flow.


Assuntos
Microtúbulos/química , Microtúbulos/metabolismo , Animais , Citoplasma , Citoesqueleto/química , Citoesqueleto/metabolismo , Dineínas/química , Dineínas/metabolismo , Cinesina/química , Cinesina/metabolismo , Microscopia Confocal , Xenopus laevis
7.
J Nat Prod ; 79(9): 2167-74, 2016 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-27626956

RESUMO

Seven novel spiromeroterpenoids, asnovolins A-G (1-7), one of which was shown to suppress fibronectin expression, were isolated from Aspergillus novofumigatus CBS117520 along with a known compound, novofumigatonin (8). The structures of asnovolins A-G were elucidated using MS and 2D-NMR data. Asnovolin E (5) suppressed fibronectin expression by normal human neonatal dermal fibroblast cells.


Assuntos
Compostos de Espiro/isolamento & purificação , Compostos de Espiro/farmacologia , Terpenos/isolamento & purificação , Terpenos/farmacologia , Aspergillus/química , Ensaios de Seleção de Medicamentos Antitumorais , Fibronectinas , Humanos , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Compostos de Espiro/química , Terpenos/química
8.
J Antibiot (Tokyo) ; 69(6): 451-5, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26732257

RESUMO

Growth inhibitors were isolated from an arctic strain of Trichoderma polysporum, and the structures were elucidated and the in vitro inhibitory effects of these compounds against Pythium iwayamai were investigated. Eleven compounds were isolated; four showed a concentration-dependent growth-inhibitory effect against P. iwayamai. None of these compounds have been reported previously as substances with antimicrobial activity against P. iwayamai. One of these four compounds inhibited the growth of the pathogen at 33 µg ml(-1) concentration during a 15-day incubation at 20 °C. This effect was comparable to that of chloroneb (1: 1,4-dichloro-2,5-dimethoxybenzene), a fungicide with activity against P. iwayamai. Thus, the results of the present study show that the arctic strain of T. polysporum can be an effective source of antibiotics with activity against the snow rot pathogen, P. iwayamai.


Assuntos
Antifúngicos/farmacologia , Inibidores do Crescimento/farmacologia , Pythium/efeitos dos fármacos , Trichoderma/metabolismo , Antifúngicos/isolamento & purificação , Clorobenzenos/farmacologia , Relação Dose-Resposta a Droga , Inibidores do Crescimento/química , Inibidores do Crescimento/isolamento & purificação , Pythium/crescimento & desenvolvimento , Fatores de Tempo
9.
J Antibiot (Tokyo) ; 69(2): 89-96, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26306816

RESUMO

Three new phthalide derivatives, emefuranones A1, A2 and B (1-3); six new phthalane derivatives, emefuran A, B1, B2, C1, C2 and D (4-9); three new farnesylated phthalide derivatives, farnesylemefuranones A-C (10-12); xylarinol C (13); and emericelloxide (14), along with four known compounds (dustanin, sorbicillin, aspergillodiol and xylarinol A), were isolated from the culture extracts of Emericella sp. IFM57991. Structures of 1-14 were elucidated on the basis of spectroscopic analysis and chemical evidence. Compounds 4-7 and 13 showed moderate antibacterial activities against Bacillus subtilis.


Assuntos
Anti-Infecciosos/farmacologia , Bacillus subtilis/efeitos dos fármacos , Benzofuranos/farmacologia , Meios de Cultura/química , Emericella/metabolismo , Ftalimidas/farmacologia , Anti-Infecciosos/química , Anti-Infecciosos/isolamento & purificação , Benzofuranos/química , Benzofuranos/isolamento & purificação , Testes de Sensibilidade a Antimicrobianos por Disco-Difusão , Emericella/crescimento & desenvolvimento , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Estrutura Molecular , Ftalimidas/química , Ftalimidas/isolamento & purificação
10.
Sci Rep ; 5: 16611, 2015 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-26568288

RESUMO

Optical microheating is a powerful non-invasive method for manipulating biological functions such as gene expression, muscle contraction, and cell excitation. Here, we demonstrate its potential usage for regulating neurite outgrowth. We found that optical microheating with a water-absorbable 1,455-nm laser beam triggers directional and explosive neurite outgrowth and branching in rat hippocampal neurons. The focused laser beam under a microscope rapidly increases the local temperature from 36 °C to 41 °C (stabilized within 2 s), resulting in the elongation of neurites by more than 10 µm within 1 min. This high-speed, persistent elongation of neurites was suppressed by inhibitors of both microtubule and actin polymerization, indicating that the thermosensitive dynamics of these cytoskeletons play crucial roles in this heat-induced neurite outgrowth. Furthermore, we showed that microheating induced the regrowth of injured neurites and the interconnection of neurites. These results demonstrate the efficacy of optical microheating methods for the construction of arbitrary neural networks.


Assuntos
Neuritos/fisiologia , Animais , Sinalização do Cálcio , Técnicas de Cultura de Células , Crescimento Celular/efeitos da radiação , Células Cultivadas , Hipocampo/citologia , Temperatura Alta , Raios Infravermelhos , Microtúbulos/metabolismo , Ratos
11.
Biophys J ; 109(2): 355-64, 2015 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-26200871

RESUMO

Living cells sense absolute temperature and temporal changes in temperature using biological thermosensors such as ion channels. Here, we reveal, to our knowledge, a novel mechanism of sensing spatial temperature gradients within single cells. Spherical mitotic cells form directional membrane extensions (polar blebs) under sharp temperature gradients (≥∼0.065°C µm(-1); 1.3°C temperature difference within a cell), which are created by local heating with a focused 1455-nm laser beam under an optical microscope. On the other hand, multiple nondirectional blebs are formed under gradual temperature gradients or uniform heating. During heating, the distribution of actomyosin complexes becomes inhomogeneous due to a break in the symmetry of its contractile force, highlighting the role of the actomyosin complex as a sensor of local temperature gradients.


Assuntos
Forma Celular/fisiologia , Temperatura , Actomiosina/metabolismo , Cálcio/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/fisiologia , Forma Celular/efeitos dos fármacos , Células HeLa , Humanos , Raios Infravermelhos , Lasers , Gravação em Vídeo
12.
Acta Ophthalmol ; 93(3): e214-7, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25130441

RESUMO

PURPOSE: A possible association has been reported between exfoliation syndrome (EX) and various ocular and systemic vascular disorders; however, it is unclear if there is an association between EX and central retinal vein occlusion (CRVO). Because latent deposits of exfoliation materials might not be recognized during slit-lamp examination, an ocular biopsy is required to establish a precise diagnosis. We evaluated a possible association between EX and CRVO using lysyl oxidase-like 1 (LOXL1) gene variants as alternative markers for EX. METHODS: The allelic and genotypic frequencies of three LOXL1 variants (rs1048661, rs3825942, and rs2165241) were determined in 68 consecutive Japanese patients with CRVO [15 with exfoliation syndrome (EX+) and 53 without exfoliation syndrome (EX-)] and 90 control patients with cataract without EX (CT). RESULTS: The frequencies of the rs1048661 and rs3825942 variants showed borderline difference between the CRVO and CT groups (p = 0.04085 and p = 0.06088, respectively, for allelic frequencies, and p = 0.06838 and p = 0.03482, respectively, for genotypic frequencies). Compared with the CT group, subgroup analysis showed that the CRVO EX+ group had significant differences in the allelic and genotypic frequencies of rs1048661 (p = 0.0006447 and p = 0.0001392, respectively) and had borderline differences in the allelic and genotypic frequencies of rs3825942 (p = 0.03403 and p = 0.07341, respectively), while the CRVO EX- group did not (p = 0.1324-0.6306). Subgroup analysis showed that the frequencies of rs2165241 did not differ between the CRVO and CT groups. CONCLUSIONS: When the LOXL1 variants were used as disease markers for clinically undetectable EX, there was no association between CRVO and EX. The results suggested that the LOXL1 variants, which are well-established markers for EX, are not likely genetic markers for CRVO in Japanese subjects.


Assuntos
Aminoácido Oxirredutases/genética , Grupo com Ancestrais do Continente Asiático/genética , Síndrome de Exfoliação/genética , Polimorfismo de Nucleotídeo Único , Oclusão da Veia Retiniana/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Catarata/genética , Feminino , Frequência do Gene , Marcadores Genéticos , Técnicas de Genotipagem , Humanos , Japão/epidemiologia , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase
13.
J Biol Chem ; 290(5): 2784-97, 2015 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-25519903

RESUMO

Dry age-related macular degeneration (AMD) accounts for over 85% of AMD cases in the United States, whereas Japanese AMD patients predominantly progress to wet AMD or polypoidal choroidal vasculopathy. Recent genome-wide association studies have revealed a strong association between AMD and an insertion/deletion sequence between the ARMS2 (age-related maculopathy susceptibility 2) and HTRA1 (high temperature requirement A serine peptidase 1) genes. Transcription regulator activity was localized in mouse retinas using heterozygous HtrA1 knock-out mice in which HtrA1 exon 1 was replaced with ß-galactosidase cDNA, thereby resulting in dominant expression of the photoreceptors. The insertion/deletion sequence significantly induced HTRA1 transcription regulator activity in photoreceptor cell lines but not in retinal pigmented epithelium or other cell types. A deletion construct of the HTRA1 regulatory region indicated that potential transcriptional suppressors and activators surround the insertion/deletion sequence. Ten double-stranded DNA probes for this region were designed, three of which interacted with nuclear extracts from 661W cells in EMSA. Liquid chromatography-mass spectrometry (LC-MS/MS) of these EMSA bands subsequently identified a protein that bound the insertion/deletion sequence, LYRIC (lysine-rich CEACAM1 co-isolated) protein. In addition, induced pluripotent stem cells from wet AMD patients carrying the insertion/deletion sequence showed significant up-regulation of the HTRA1 transcript compared with controls. These data suggest that the insertion/deletion sequence alters the suppressor and activator cis-elements of HTRA1 and triggers sustained up-regulation of HTRA1. These results are consistent with a transgenic mouse model that ubiquitously overexpresses HtrA1 and exhibits characteristics similar to those of wet AMD patients.


Assuntos
Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas/metabolismo , Serina Endopeptidases/metabolismo , Idoso , Animais , Western Blotting , Linhagem Celular , Cromatografia Líquida , Ensaio de Desvio de Mobilidade Eletroforética , Feminino , Serina Peptidase 1 de Requerimento de Alta Temperatura A , Humanos , Imuno-Histoquímica , Hibridização In Situ , Masculino , Camundongos , Camundongos Knockout , Mutação , Ratos , Retina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas em Tandem
14.
J Ophthalmol ; 2014: 210947, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25485142

RESUMO

Purpose. To investigate genetic and clinical features of patients with rhodopsin (RHO) mutations in two Japanese families with autosomal dominant retinitis pigmentosa (adRP). Methods. Whole-exome sequence analysis was performed in ten adRP families. Identified RHO mutations for the cosegregation analysis were confirmed by Sanger sequencing. Ophthalmic examinations were performed to evaluate the RP phenotypes. The impact of the RHO mutation on the rhodopsin conformation was examined by molecular modeling analysis. Results. In two adRP families, we identified two RHO mutations (c.377G>T (p.W126L) and c.1036G>C (p.A346P)), one of which was novel. Complete cosegregation was confirmed for each mutation exhibiting the RP phenotype in both families. Molecular modeling predicted that the novel mutation (p.W126L) might impair rhodopsin function by affecting its conformational transition in the light-adapted form. Clinical phenotypes showed that patients with p.W126L exhibited sector RP, whereas patients with p.A346P exhibited classic RP. Conclusions. Our findings demonstrated that the novel mutation (p.W126L) may be associated with the phenotype of sector RP. Identification of RHO mutations is a very useful tool for predicting disease severity and providing precise genetic counseling.

16.
Biophys J ; 106(3): 735-40, 2014 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-24507614

RESUMO

The meiotic spindle is a bipolar molecular machine that is designed to segregate duplicated chromosomes toward the opposite poles of the cell. The size and shape of the spindle are considered to be maintained by a balance of forces produced by molecular motors and microtubule assembly dynamics. Several studies have probed how mechanical perturbations of the force balance affect the spindle structure. However, the spindle's response to a stretching force acting at the spindle pole and along its long axis, i.e., the direction in which chromosomes are segregated, has not been examined. Here, we describe a method to apply a stretching force to the metaphase spindle assembled in Xenopus egg extracts and measure the relationship between the force and the three-dimensional deformation of the spindle. We found that the spindle behaves as a Zener-type viscoelastic body when forces are applied at the spindle pole, generating a restoring force for several minutes. In addition, both the volume of the spindle and the tubulin density are conserved under the stretching force. These results provide insight into how the spindle size is maintained at metaphase.


Assuntos
Elasticidade , Fuso Acromático/metabolismo , Animais , Oócitos/metabolismo , Fuso Acromático/química , Estresse Mecânico , Xenopus
17.
Clin Ophthalmol ; 8: 143-8, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24403817

RESUMO

BACKGROUND: The purpose of this study was to investigate the association between ARMS2/HTRA1, CFH, and C3 gene polymorphisms and retinal angiomatous proliferation (RAP), an infrequent and severe form of exudative age-related macular degeneration, which is characterized by intraretinal neovascularization. METHODS: Diagnosis of RAP was based on fundus photographs, images of fluorescein and indocyanine green angiographies, and optical coherence tomography findings. Six single nucleotide polymorphisms (SNPs), A69S (rs10490924) in ARMS2, rs11200638 in HTRA1, I62V (rs800292) in CFH, Y402H (rs1061170) in CFH, R80G (rs2230199) in C3, and rs2241394 in C3, were genotyped in eight Japanese patients with RAP. RESULTS: The two SNPs in the ARMS2/HTRA1 were in complete linkage disequilibrium. The frequency of the risk T allele in ARMS2 (the risk A allele in HTRA1) was 93.8% in the RAP patients. The frequency of homozygosity for the risk genotype TT of ARMS2 (the risk genotype AA of HTRA1) was 87.5%. The frequency of the non-risk allele (A) of I62V was 100%. The frequencies of risk alleles of Y402H, R80G, and rs2241394 were 12.5%, 0%, and 18.8%, respectively. CONCLUSION: Our results suggest that the risk alleles of the ARMS2/HTRA1 SNPs may be associated with development of RAP and play a major role in the pathogenesis of intraretinal angiogenesis.

18.
Cell Rep ; 5(1): 44-50, 2013 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-24120869

RESUMO

The polymerization/depolymerization dynamics of microtubules (MTs) have been reported to contribute to control of the size and shape of spindles, but quantitative analysis of how the size and shape correlate with the amount and density of MTs in the spindle remains incomplete. Here, we measured these parameters using 3D microscopy of meiotic spindles that self-organized in Xenopus egg extracts and presented a simple equation describing the relationship among these parameters. To examine the validity of the equation, we cut the spindle into two fragments along the pole-to-pole axis by micromanipulation techniques that rapidly decrease the amount of MTs. The spheroidal shape spontaneously recovered within 5 min, but the size of each fragment remained small. The equation we obtained quantitatively describes how the spindle size correlates with the amount of MTs while maintaining the shape and the MT density.


Assuntos
Fuso Acromático/ultraestrutura , Animais , Extratos Celulares/química , Humanos , Imageamento Tridimensional/métodos , Micromanipulação/métodos , Microtúbulos/metabolismo , Óvulo/metabolismo , Óvulo/ultraestrutura , Fuso Acromático/metabolismo , Xenopus
19.
Sci Rep ; 3: 2808, 2013 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-24077015

RESUMO

The chromosome alignment is mediated by polar ejection and poleward forces acting on the chromosome arm and kinetochores, respectively. Although components of the motile machinery such as chromokinesin have been characterized, their dynamics within the spindle is poorly understood. Here we show that a quantum dot (Qdot) binding up to four Xenopus chromokinesin (Xkid) molecules behaved like a nanosize chromosome arm in the meiotic spindle, which is self-organized in cytoplasmic egg extracts. Xkid-Qdots travelled long distances along microtubules by changing several tracks, resulting in their accumulation toward and distribution around the metaphase plate. The analysis indicated that the direction of motion and velocity depend on the distribution of microtubule polarity within the spindle. Thus, this mechanism is governed by chromokinesin motors, which is dependent on symmetrical microtubule orientation that may allow chromosomes to maintain their position around the spindle equator until correct microtubule-kinetochore attachment is established.


Assuntos
Cromossomos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Cinesina/metabolismo , Microtúbulos/metabolismo , Fuso Acromático/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Animais , Movimento , Pontos Quânticos
20.
Mol Vis ; 19: 1580-90, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23885164

RESUMO

PURPOSE: To describe the molecular characteristics of four Japanese patients with cone dystrophy with supernormal rod responses (CDSRR). METHODS: Four individuals with a clinical and electrophysiological diagnosis of CDSRR were ascertained. The pathognomonic findings of the full-field electroretinograms (ERGs) included a decrease in the rod responses, a square-shaped a-wave, an excessive increase in the b-wave in the bright flash responses, and decreased cone-derived responses. Mutational screening of the coding regions and flanking intronic sequences of the potassium channel, subfamily V, member 2 (KCNV2) gene was performed with bidirectional sequencing. The segregation of each allele was confirmed by screening other family members. Subsequent in silico analyses of the mutational consequences for protein function were performed. RESULTS: There were two siblings from one family and one case in each of the two families. One family had a consanguineous marriage. Mutational screening revealed compound heterozygosity for the two alleles, p.C177R and p.G461R, in three patients, and homozygosity for complex alleles, p.R27H and p.R206P, in one patient from the consanguineous family. There were three putative novel variants, p.R27H, p.C177R, and p.R206P. The four variants in the families with KCNV2 were highly conserved in other species. In silico analyses predicted that all of the missense variants would alter protein function. CONCLUSIONS: Biallelic disease-causing variants were identified in four Japanese patients with CDSRR suggesting that the pathognomonic electrophysiological features are helpful in making a molecular diagnosis of KCNV2. Three novel variants were identified, and we conclude that there may be a distinct spectrum of KCNV2 alleles in the Japanese population.


Assuntos
Grupo com Ancestrais do Continente Asiático/genética , Predisposição Genética para Doença , Mutação/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Doenças Retinianas/genética , Adolescente , Sequência de Aminoácidos , Criança , Pré-Escolar , Demografia , Eletrorretinografia , Família , Feminino , Humanos , Japão , Masculino , Dados de Sequência Molecular , Linhagem , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Doenças Retinianas/fisiopatologia , Alinhamento de Sequência , Adulto Jovem
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