RESUMO
Kochi Oxydol Radiation Therapy for Unresectable Carcinoma (KORTUC) is a novel radiosensitizer invented by Professor Ogawa at Kochi University (Japan) in 2006. The current study aimed to report the experience of the present authors with the use of KORTUC treatment in combination with interstitial brachytherapy (ISBT), with or without external beam (EB) radiotherapy (RT), in patients with locally recurrent cervical cancer (LRCC), who were likely to have a high risk of poor prognosis. Between April 2012 and January 2020, 14 female patients (15 tumoral lesions) with LRCC underwent KORTUC with ISBT. Their previous treatments included surgery (n=4), radiation therapy (n=8) and surgery plus RT (n=3). The primary lesions were located in the vaginal stump (n=5), pelvic wall (n=3), cervix (n=3), vaginal wall (n=2) and lymph nodes (n=2). At 2 h before RT, KORTUC was injected intratumorally via direct colposcopy. The dose of KORTUC ranged from 4-12 ml, adjusted for the tumor size. For patients who underwent ISBT, KORTUC was administered before and after insertion of the applicator before irradiation. Intratumoral injection of KORTUC was completed without any technical or safety issues in all 15 patients; it was well tolerated with no adverse events observed. KORTUC also showed preferable efficacy; a clinical complete response was observed in 87% of patients and the initial response rate was 100%. The 2-year local control rate in patients who underwent ISBT + KORTUC was 79%, whereas it was 63% in the re-irradiation group which was significantly lower (P=0.02) than that in the non-irradiation group (100%). Based on this finding, KORTUC with external irradiation is considered to be an optimal treatment strategy for patients with newly diagnosed LRCC this disease. Additionally, KORTUC may be an effective radiation response enhancer in multiple cancer types in which locoregional control after RT alone remains poor.
RESUMO
[Ca2+]-dependent crystallization of the Ca2+-ATPase molecules in sarcoplasmic reticulum (SR) vesicles isolated from scallop striated muscle elongated the vesicles in the absence of ATP, and ATP stabilized the crystals. Here, to determine the [Ca2+]-dependence of vesicle elongation in the presence of ATP, SR vesicles in various [Ca2+] environments were imaged using negative stain electron microscopy. The images obtained revealed the following phenomena. (i) Crystal-containing elongated vesicles appeared at ≤1.4 µM Ca2+ and almost disappeared at ≥18 µM Ca2+, where ATPase activity reaches its maximum. (ii) At ≥18 µM Ca2+, almost all SR vesicles were in the round form and covered by tightly clustered ATPase crystal patches. (iii) Round vesicles dried on electron microscopy grids occasionally had cracks, probably because surface tension crushed the solid three-dimensional spheres. (iv) [Ca2+]-dependent ATPase crystallization was rapid (<1 min) and reversible. These data prompt the hypothesis that SR vesicles autonomously elongate or contract with the help of a calcium-sensitive ATPase network/endoskeleton and that ATPase crystallization may modulate physical properties of the SR architecture, including the ryanodine receptors that control muscle contraction.
Assuntos
Pectinidae , Retículo Sarcoplasmático , Animais , Retículo Sarcoplasmático/metabolismo , Adenosina Trifosfatases , ATPases Transportadoras de Cálcio/metabolismo , Contração Muscular , Pectinidae/metabolismo , Trifosfato de Adenosina , Cálcio/metabolismoRESUMO
The impact of a high-fat diet (HFD) on intestinal permeability has been well established. When bacteria and their metabolites from the intestinal tract flow into the portal vein, inflammation in the liver is triggered. However, the exact mechanism behind the development of a leaky gut caused by an HFD is unclear. In this study, we investigated the mechanism underlying the leaky gut related to an HFD. C57BL/6J mice were fed an HFD or control diet for 24 weeks, and their small intestine epithelial cells (IECs) were analyzed using deep quantitative proteomics. A significant increase in fat accumulation in the liver and a trend toward increased intestinal permeability were observed in the HFD group compared to the control group. Proteomics analysis of the upper small intestine epithelial cells identified 3684 proteins, of which 1032 were differentially expressed proteins (DEPs). Functional analysis of DEPs showed significant enrichment of proteins related to endocytosis, protein transport, and tight junctions (TJ). Expression of Cldn7 was inversely correlated with intestinal barrier function and strongly correlated with that of Epcam. This study will make important foundational contributions by providing a comprehensive depiction of protein expression in IECs affected by HFD, including an indication that the Epcam/Cldn7 complex plays a role in leaky gut.
Assuntos
Dieta Hiperlipídica , Junções Íntimas , Animais , Camundongos , Dieta Hiperlipídica/efeitos adversos , Molécula de Adesão da Célula Epitelial/metabolismo , Junções Íntimas/metabolismo , Proteômica , Camundongos Endogâmicos C57BL , Mucosa Intestinal/metabolismoRESUMO
The Ca2+-ATPase is an integral transmembrane Ca2+ pump of the sarcoplasmic reticulum (SR). Crystallization of the cytoplasmic surface ATPase molecules of isolated scallop SR vesicles was studied at various calcium concentrations by negative stain electron microscopy. In the absence of ATP, round SR vesicles displaying an assembly of small crystalline patches of ATPase molecules were observed at 18 µM [Ca2+]. These partly transformed into tightly elongated vesicles containing ATPase crystalline arrays at low [Ca2+] (≤1.3 µM). The arrays were classified as ''tetramer'', "two-rail" (like a railroad) and ''monomer''. Their crystallinity was low, and they were unstable. In the presence of ATP (5 mM) at a low [Ca2+] of ~0.002 µM, "two-rail" arrays of high crystallinity appeared more frequently in the tightly elongated vesicles and the distinct tetramer arrays disappeared. During prolonged (~2.5 h) incubation, ATP was consumed and tetramer arrays reappeared. A specific ATPase inhibitor, thapsigargin, prevented both crystal formation and vesicle elongation in the presence of ATP. Together with the second part of this study, these data suggest that the ATPase forms tetramer units and longer tetramer crystalline arrays to elongate SR vesicles, and that the arrays transform into more stable "two-rail" forms in the presence of ATP at low [Ca2+].
Assuntos
Pectinidae , Retículo Sarcoplasmático , Adenosina Trifosfatases , Trifosfato de Adenosina/farmacologia , Animais , Cálcio/metabolismo , ATPases Transportadoras de Cálcio/metabolismo , Retículo Sarcoplasmático/metabolismoRESUMO
Kochi oxydol radiation therapy for unresectable carcinomas II (KORTUC II) is currently the most widely used radiosensitizer in Japan. This sensitizer is a solution consisting of 0.83% sodium hyaluronate and 0.5% hydrogen peroxide. The mixture is injected intratumorally just before radiation therapy (RT) several times. KORTUC II has the effect of neutralizing antioxidant enzymes, while increasing the oxygen tension into the tumor tissue, and achieves marked local effects without notable adverse events. The present report describes cases in which KORTUC II was used to treat patients with locally advanced breast cancer (LABC) or recurrent breast cancer (LRBC). The present study included 30 patients with LABC (n=9) or LRBC (n=21) aimed at local control of tumors, who were followed up for ≥3 months after treatment. The irradiation dose and extent fields were determined by the attending physicians considering various patient factors, such as a performance status, prognosis and presence or absence of adjuvant therapy. The median irradiation dose was 60.4 Gy3.5 (43.6-76.1 Gy3.5) based on the calculation of equivalents of 2 Gy fractions, and the median total number of sensitizer injections was 5 (2-7) times. The median maximum tumor shrinkage was 97.0% and 15 patients (50%) were assessed to have achieved a clinical complete response. The proportion with loco-regional control at 1, 2 and 3 years was 100, 94.7 and 75.4%, respectively, and progression free survival after RT at 1 and 2 years was 59.0 and 24.1%, respectively. KORTUC II exhibited high rates of local tumor control for LABC and LRBC. KORTUC II is expected to be an inexpensive and promising RT method because it is safe and has an excellent radio-sensitizing effect.
RESUMO
The Ca2+-transport ATPase of sarcoplasmic reticulum (SR) is an integral, transmembrane protein. It sequesters cytoplasmic calcium ions released from SR during muscle contraction, and causes muscle relaxation. Based on negative staining and transmission electron microscopy of SR vesicles isolated from rabbit skeletal muscle, we propose that the ATPase molecules might also be a calcium-sensitive membrane-endoskeleton. Under conditions when the ATPase molecules scarcely transport Ca2+, i.e., in the presence of ATP and ≤ 0.9 nM Ca2+, some of the ATPase particles on the SR vesicle surface gathered to form tetramers. The tetramers crystallized into a cylindrical helical array in some vesicles and probably resulted in the elongated protrusion that extended from some round SRs. As the Ca2+ concentration increased to 0.2 µM, i.e., under conditions when the transporter molecules fully carry out their activities, the ATPase crystal arrays disappeared, but the SR protrusions remained. In the absence of ATP, almost all of the SR vesicles were round and no crystal arrays were evident, independent of the calcium concentration. This suggests that ATP induced crystallization at low Ca2+ concentrations. From the observed morphological changes, the role of the proposed ATPase membrane-endoskeleton is discussed in the context of calcium regulation during muscle contraction.
Assuntos
ATPases Transportadoras de Cálcio/metabolismo , Cálcio/farmacologia , Citoesqueleto/metabolismo , Contração Muscular/efeitos dos fármacos , Retículo Sarcoplasmático/metabolismo , Animais , Cálcio/metabolismo , Citoesqueleto/ultraestrutura , Transporte de Íons/efeitos dos fármacos , Masculino , Coelhos , Retículo Sarcoplasmático/ultraestruturaRESUMO
Chondrogenesis and angiogenesis drive endochondral ossification. Using the atmospheric scanning electron microscopy (ASEM) without decalcification and dehydration, we directly imaged angiogenesis-driven ossification at different developmental stages shortly after aldehyde fixation, using aqueous radical scavenger glucose solution to preserve water-rich structures. An embryonic day 15.5 mouse femur was fixed and stained with phosphotungstic acid (PTA), and blood vessel penetration into the hypertrophic chondrocyte zone was visualised. We observed a novel envelope between the perichondrium and proliferating chondrocytes, which was lined with spindle-shaped cells that could be borderline chondrocytes. At postnatal day (P)1, trabecular and cortical bone mineralisation was imaged without staining. Additional PTA staining visualised surrounding soft tissues; filamentous connections between osteoblast-like cells and osteocytes in cortical bone were interpreted as the osteocytic lacunar-canalicular system. By P10, resorption pits had formed on the tibial trabecular bone surface. The applicability of ASEM for pathological analysis was addressed using knockout mice of Keap1, an oxidative-stress sensor. In Keap1-/- femurs, we observed impaired calcification and angiogenesis of epiphyseal cartilage, suggesting impaired bone development. Overall, the quick ASEM method we developed revealed mineralisation and new structures in wet bone tissue at EM resolution and can be used to study mineralisation-associated phenomena of any hydrated tissue.
Assuntos
Atmosfera , Osso e Ossos/patologia , Osso e Ossos/ultraestrutura , Cartilagem/ultraestrutura , Proteína 1 Associada a ECH Semelhante a Kelch/deficiência , Microscopia Eletrônica de Varredura , Osteogênese , Osteomalacia/patologia , Animais , Osso e Ossos/diagnóstico por imagem , Calcificação Fisiológica , Cartilagem/diagnóstico por imagem , Cartilagem/patologia , Condrogênese , Osso Cortical/diagnóstico por imagem , Osso Cortical/ultraestrutura , Embrião de Mamíferos/diagnóstico por imagem , Fêmur/diagnóstico por imagem , Fêmur/ultraestrutura , Imageamento Tridimensional , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Camundongos Endogâmicos C57BL , Osteócitos/metabolismo , Fenótipo , Tíbia/diagnóstico por imagem , Tíbia/ultraestruturaRESUMO
Fluorescence microscopy (FM) has revealed vital molecular mechanisms of life. Mainly, molecules labeled by fluorescent probes are imaged. However, the diversity of labeling probes and their functions remain limited. We synthesized a pyrene-based fluorescent probe targeting SH groups, which are important for protein folding and oxidative stress sensing in cells. The labeling achieved employs thiol-ene click reactions between the probes and SH groups and is triggered by irradiation by UV light or an electron beam. When two tagged pyrene groups were close enough to be excited as a dimer (excimer), they showed red-shifted fluorescence; theoretically, the proximity of two SH residues within ~30 Å can thus be monitored. Moreover, correlative light/electron microscopy (CLEM) was achieved using our atmospheric scanning electron microscope (ASEM); radicals formed in liquid by the electron beam caused the thiol-ene click reactions, and excimer fluorescence of the labeled proteins in cells and tissues was visualized by FM. Since the fluorescent labeling is induced by a narrow electron beam, high spatial resolution labeling is expected. The method can be widely applied to biological fields, for example, to study protein dynamics with or without cysteine mutagenesis, and to beam-induced micro-fabrication and the precise post-modification of materials.
Assuntos
Química Click/métodos , Cisteína/metabolismo , Corantes Fluorescentes/síntese química , Microscopia Eletrônica de Varredura/métodos , Imagem Óptica/métodos , Pirenos/química , Compostos de Sulfidrila/química , Animais , Células COS , Chlorocebus aethiops , Cisteína/química , Limite de Detecção , Masculino , Camundongos , Camundongos Endogâmicos ICR , Microscopia Eletrônica de Varredura/normas , Microscopia de Fluorescência/métodos , Microscopia de Fluorescência/normas , Imagem Óptica/normas , Conformação ProteicaRESUMO
In order to improve oncologic outcomes in radiotherapy treatments of patients with unresectable pelvic sidewall recurrences of uterine cervical cancer, we combined high-dose-rate interstitial brachytherapy (HDR-ISBT) with newly tested hypoxic radiosensitizer Kochi oxydol-radiation therapy for unresectable carcinomas (KORTUC II), an enzyme-targeting radiosensitization treatment involving intra-tumoral injection of sodium hyaluronate mixed with hydrogen peroxide. We report on a 63-year-old patient referred to our department with an extensive pelvic sidewall recurrence of uterine cervical cancer after initial hysterectomy. The tumor size was 55 × 25 × 80 mm, with a calculated volume of 89.7 cc. Whole pelvic irradiation of 50 Gy in 25 fractions was administered, combined with weekly cisplatin injections. KORTUC II injections were given two times: at day 21 (42 Gy) and at day 24 (48 Gy). After finishing whole pelvic irradiation, HDR-ISBT of 25 Gy in 5 fractions b.i.d. over 3 days was administered. KORTUC II was also injected at the time of implantation. Dose-volume histogram (DVH) values for clinical target volume were D90, D98, and D100 of 6.0, 5.0, and 3.5 Gy per fraction, respectively. D2cc values were 2.1, 4.1, 3.2, and 2.0 Gy per fraction for the bladder, rectum, sigmoid colon, and small bowel, respectively. No acute adverse events ≥ grade 3 were observed. Repeated grade 3 pyelonephritis occurred as a late complication at 11, 24, and 26 months after the treatment, and was successfully resolved with antibiotics. Moreover, grade 2 late toxicity was documented, including sciatic neuralgia, lower limb lymphedema, and urinary incontinence. At present, 32 months after HDR-ISBT, the patient remains free of disease, with no toxicity-related deterioration in physical condition.
RESUMO
Estimation of the three-dimensional (3D) structure of a protein using cryo transmission electron microscopy (cryo-TEM) is an inverse problem, which aims to estimate the parameters of a specific physical process from observations. In general, we need to model the observation process to estimate a structure. However, the inconsistency between the model and a real observation process decreases the estimation accuracy. In cryo-TEM, the flexibility of a soft protein, including the bending of a helix, can lead to inconsistencies between the observations because of the assumption that there is a consistent 3D structure behind each observed image. In this paper, we propose a 3D reconstruction algorithm for helical structures using a parametric soft-body model that can represent continuous deformation. We performed an approximate Bayesian inference for unobservable (hidden) variables, such as the deformation parameters, projection angle, and two-dimensional origin offset (shift) of each protein in the 3D structure estimation problem. Our principled approach is not only beneficial to deal with the uncertainties in the estimation, but also beneficial to make the optimization algorithm convergent and efficient. Reconstructions with artificial molecules validated the advantage of the proposed method, particularly, when deformed helices were imaged under a low signal-to-noise ratio condition. Moreover, we confirmed that the proposed method successfully reconstructed a 3D structure from cryo-TEM images of the tobacco mosaic virus.
Assuntos
Modelos Moleculares , Proteínas/química , Teorema de Bayes , Conformação Proteica em alfa-HéliceRESUMO
Melanoma is a highly aggressive cancer with a propensity for brain metastases. These can be treated by radiotherapy, but the radiation-resistant nature of melanoma makes the prognosis for melanoma patients with brain metastases poor. Previously, we demonstrated that treatment of mice with subcutaneous melanoma with 5-aminolevurinic acid (5-ALA) and X-rays in combination, ("radiodynamic therapy (RDT)"), instead of with 5-ALA and laser beams ("photodynamic therapy"), improved tumor suppression in vivo. Here, using the B16-Luc melanoma brain metastasis model, we demonstrate that 5-ALA RDT effectively treats brain metastasis. We also studied how 5-ALA RDT damages cells in vitro using a B16 melanoma culture. Cell culture preincubated with 5-ALA alone increased intracellular photosensitizer protoporphyrin IX. On X-ray irradiation, the cells enhanced their âOH radical generation, which subsequently induced γH2AX, a marker of DNA double-strand breaks in their nuclei, but decreased mitochondrial membrane potential. After two days, the cell cycle was arrested. When 5-ALA RDT was applied to the brain melanoma metastasis model in vivo, suppression of tumor growth was indicated. Therapeutic efficacy in melanoma treatment has recently been improved by molecular targeted drugs and immune checkpoint inhibitors. Treatment with these drugs is now expected to be combined with 5-ALA RDT to further improve therapeutic efficacy.
Assuntos
Ácido Aminolevulínico/uso terapêutico , Neoplasias Encefálicas/secundário , Melanoma Experimental/patologia , Animais , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/radioterapia , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/radioterapia , CamundongosRESUMO
STUDY OBJECTIVE: To evaluate a new magnetic resonance imaging (MRI) grading system for preoperative differentiation between benign and variant-type uterine leiomyomas including smooth muscle tumors of uncertain malignant potential (STUMPs). DESIGN: Retrospective analysis (Canadian Task Force classification III). SETTING: Teaching hospital (Teine Keijinkai Hospital). PATIENTS: Three-hundred thirteen patient medical records were retrospectively reviewed if treated for uterine myomas and diagnosed with variant type leiomyomas or STUMPs (n = 27) or benign, typical leiomyomas (n = 286) and treated between January 2012 and December 2014. INTERVENTION: Uterine myoma classifications using MRI findings according to a 5-grade system (grades I-V) based on 3 elements. MEASUREMENTS AND MAIN RESULTS: Uterine myoma MRI classifications were based on 3 elements: T2-weighted imaging (high or low), diffusion-weighted imaging (high or low), and apparent diffusion coefficient values (high or low; apparent diffusion coefficient < 1.5 × 10-3 mm2/sec was considered low). Grades I to II were designated as typical or benign leiomyomas, grade III as degenerated leiomyomas, and grades IV to V as variant type leiomyomas or STUMPs. Accuracy levels were 98.9%, 100%, 94.3%, 58.8%, and 41.9% for grades I through V lesions, respectively. The grades were divided into 2 groups to discriminate benign leiomyomas and STUMPs (grades I-III were considered negative and grades IV-V positive). Grades IV to V scored 85.2% for sensitivity, 91.3% for specificity, 47.9% positive predictive value, 98.5% negative predictive value, a 9.745 positive likelihood ratio, and a .162 negative likelihood ratio. CONCLUSION: This novel MRI grading system for uterine myomas may be beneficial in differentiating benign leiomyomas from STUMPs or variant type leiomyomas and could be a future effective presurgical assessment tool.
Assuntos
Leiomioma/patologia , Tumor de Músculo Liso/patologia , Neoplasias Uterinas/patologia , Adulto , Imagem de Difusão por Ressonância Magnética , Feminino , Humanos , Imageamento por Ressonância Magnética/métodos , Pessoa de Meia-Idade , Gradação de Tumores/métodos , Estudos Retrospectivos , Sensibilidade e EspecificidadeRESUMO
In atmospheric scanning electron microscope (ASEM), the inverted scanning electron microscope (SEM) observes the wet sample from below, while an optical microscope observes it from above simultaneously. The ASEM sample holder has a disposable dish shape with a silicon nitride film window at the bottom. It can be coated variously for the primary-culture of substrate-sensitive cells; primary cells were cultured in a few milliliters of culture medium in a stable incubator environment. For the inverted SEM observation, cells and the excised tissue blocks were aldehyde-fixed, immersed in radical scavenger solution, and observed at minimum electron dose. Neural networking, axonal segmentation, proplatelet-formation and phagocytosis, and Fas expression in embryonic stem cells were captured by optical or fluorescence microscopy, and imaged at high resolution by gold-labeled immuno-ASEM with/without metal staining. By exploiting optical microscopy, the region of interest of organ can be found from the wide area, and the cells and organelle were successfully examined at high resolution by the following scanning electron microscopy. We successfully visualized islet of Langerhans, blood microvessels, neuronal endplate, and bacterial flora on stomach epidermal surfaces. Bacterial biofilms and the typical structural features including "leg complex" of mycoplasma were visualized by exploiting CLEM of ASEM. Based on these studies, ASEM correlative microscopy promises to allow the research of various mesoscopic-scale biological phenomena in the near future.
Assuntos
Microscopia Eletrônica de Varredura/métodos , Animais , Axônios/metabolismo , Biofilmes , Neoplasias da Mama/patologia , Neoplasias da Mama/ultraestrutura , Células COS , Cálcio/metabolismo , Chlorocebus aethiops , Drosophila melanogaster/ultraestrutura , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Feminino , Humanos , Neoplasias Pulmonares/secundário , Neoplasias Pulmonares/ultraestrutura , Mycoplasma/ultraestrutura , Especificidade de Órgãos , Staphylococcus aureus/fisiologia , Staphylococcus aureus/ultraestrutura , Molécula 1 de Interação Estromal/metabolismoRESUMO
T antigen, mucin-type core 1 O-glycan, is highly expressed in the embryonic central nervous system (CNS) and co-localizes with a Drosophila CNS marker, BP102 antigen. BP102 antigen and Derailed, an axon guidance receptor, are localized specifically in the proximal axon segment of isolated primary cultured neurons, and their mobility is restricted at the intra-axonal boundary by a diffusion barrier. However, the preferred trafficking mechanism remains unknown. In this study, the major O-glycan T antigen was found to localize within the proximal compartments of primary cultured Drosophila neurons, whereas the N-glycan HRP antigen was not. Ultrastructural analysis by atmospheric scanning electron microscopy revealed that microtubule bundles cross one another at the intra-axonal boundary, and that T antigens form circular pattern before the boundary. We then identified Short stop (Shot), a crosslinker protein between F-actin and microtubules, as a mediator for the proximal localization of T antigens; null mutation of shot cancelled preferential localization of T antigens. Moreover, F-actin binding domain of Shot was required for their proximal localization. Together, our results allow us to propose a novel trafficking pathway where Shot crosslinks F-actin and microtubules around the intra-axonal boundary, directing T antigen-carrying vesicles toward the proximal plasma membrane.
Assuntos
Axônios/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas dos Microfilamentos/metabolismo , Mucinas/metabolismo , Polissacarídeos/metabolismo , Actinas/metabolismo , Animais , Antígenos Virais de Tumores , Axônios/ultraestrutura , Células Cultivadas , Proteínas de Drosophila/química , Proteínas dos Microfilamentos/química , Modelos Biológicos , Mutação/genética , Neurônios/metabolismo , Ligação Proteica , Domínios Proteicos , Tubulina (Proteína)/metabolismoRESUMO
T antigen (Galß1-3GalNAcα1-Ser/Thr), a core 1 mucin-type O-glycan structure, is synthesized by Drosophila core 1 ß1,3-galactosyltrasferase 1 (dC1GalT1) and is expressed in various tissues. We previously reported that dC1GalT1 synthesizes T antigen expressed in hemocytes, lymph glands, and the central nervous system (CNS) and that dC1GalT1 mutant larvae display decreased numbers of circulating hemocytes and excessive differentiation of hematopoietic stem cells in lymph glands. dC1GalT1 mutant larvae have also been shown to have morphological defects in the CNS. However, the functions of T antigen in other tissues remain largely unknown. In this study, we found that glycans contributed to the localization of neuromuscular junction (NMJ) boutons. In dC1GalT1 mutant larvae, NMJs were ectopically formed in the cleft between muscles 6 and 7 and connected with these two muscles. dC1GalT1 synthesized T antigen, which was expressed at NMJs. In addition, we determined the function of mucin-type O-glycans in muscle cells. In dC1GalT1 mutant muscles, myofibers and basement membranes were disorganized. Moreover, ultrastructural defects in NMJs and accumulation of large endosome-like structures within both NMJ boutons and muscle cells were observed in dC1GalT1 mutants. Taken together, these results demonstrated that mucin-type O-glycans synthesized by dC1GalT1 were involved in the localization of NMJ boutons, synaptogenesis of NMJs, establishment of muscle cell architecture, and endocytosis.
Assuntos
Drosophila/metabolismo , Mucinas/metabolismo , Músculos/metabolismo , Junção Neuromuscular/metabolismo , Polissacarídeos/metabolismo , Animais , Microscopia Eletrônica de VarreduraRESUMO
The molecular motor kinesin moves along microtubules using energy from ATP hydrolysis in an initial step coupled with ADP release. In neurons, kinesin-1/KIF5C preferentially binds to the GTP-state microtubules over GDP-state microtubules to selectively enter an axon among many processes; however, because the atomic structure of nucleotide-free KIF5C is unavailable, its molecular mechanism remains unresolved. Here, the crystal structure of nucleotide-free KIF5C and the cryo-electron microscopic structure of nucleotide-free KIF5C complexed with the GTP-state microtubule are presented. The structures illustrate mutual conformational changes induced by interaction between the GTP-state microtubule and KIF5C. KIF5C acquires the 'rigor conformation', where mobile switches I and II are stabilized through L11 and the initial portion of the neck-linker, facilitating effective ADP release and the weak-to-strong transition of KIF5C microtubule affinity. Conformational changes to tubulin strengthen the longitudinal contacts of the GTP-state microtubule in a similar manner to GDP-taxol microtubules. These results and functional analyses provide the molecular mechanism of the preferential binding of KIF5C to GTP-state microtubules.
Assuntos
Cinesinas/química , Cinesinas/metabolismo , Microtúbulos/química , Microtúbulos/metabolismo , Difosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Microscopia Crioeletrônica/métodos , Cristalografia por Raios X , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Cinesinas/genética , Camundongos , Microtúbulos/ultraestrutura , Modelos Moleculares , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Conformação ProteicaRESUMO
In the atmospheric scanning electron microscope (ASEM), a 2- to 3-µm layer of the sample resting on a silicon nitride-film window in the base of an open sample dish is imaged, in liquid, at atmospheric pressure, from below by an inverted SEM. Thus, the time-consuming pretreatments generally required for biological samples to withstand the vacuum of a standard electron microscope are avoided. In the present study, various mouse tissues (brain, spinal cord, muscle, heart, lung, liver, kidney, spleen and stomach) were fixed, stained with heavy metals, and visualized in radical scavenger D-glucose solution using the ASEM. While some stains made the nuclei of cells very prominent (platinum-blue, phosphotungstic acid), others also emphasized cell organelles and membranous structures (uranium acetate or the NCMIR method). Notably, symbiotic bacteria were sometimes observed on stomach mucosa. Furthermore, kidney tissue could be stained and successfully imaged in <30 min. Lung and spinal cord tissue from normal mice and mice metastasized with breast cancer cells was also examined. Cancer cells present in lung alveoli and in parts of the spine tissue clearly had larger nuclei than normal cells. The results indicate that the ASEM has the potential to accelerate intraoperative cancer diagnosis, the diagnosis of kidney diseases and pathogen detection. Importantly, in the course of the present study it was possible to increase the observable tissue area by using a new multi-windowed ASEM sample dish and sliding the tissue across its eight windows.
Assuntos
Microscopia Eletrônica de Varredura/métodos , Neoplasias/diagnóstico , Animais , Pressão Atmosférica , Feminino , Cuidados Intraoperatórios , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos ICR , Neoplasias/cirurgia , Coloração e Rotulagem/métodosRESUMO
Proteins play important roles in cell functions such as enzymes, cell trafficking, neurotransmission, muscle contraction and hormone secretion. However, some proteins are very difficult to be crystallized and their structures are undetermined. Several techniques have been developed to elucidate the structure of macromolecules; X-ray or electron crystallography, nuclear magnetic resonance spectroscopy, and high-resolution electron microscopy. Among them, electron microscopy based single particle reconstruction (SPA) technique is a computer-aided structure determination method. This method reconstructs the 3D structure from projection images of dispersed protein. A large number of two-dimensional particle images are picked up from EM films, aligned and classified to generate 2D averages, and used to reconstruct the 3D structure by assigning the Euler angle of each 2D average. Due to the necessity of elaborate collaboration between the classical biology and the innovative information technology including parallel computing, scientists needed to break unseen barriers to get a start of this analysis. However, recent progresses in electron microscopes, mathematical algorithms, and computational abilities greatly reduced the height of barriers and expanded targets that are considered to be primarily addressable using single particle analysis. Membrane proteins are one of these targets to which the single particle analysis is successfully applied for the understanding of their 3D structures. For this purpose, we have developed various SPA methods [1-5] and applied them to different proteins [6-8].Here, we introduce reconstructed proteins, and discuss the availability of this technique. The intramembrane-cleaving proteases (I-CLiPs) that sever the transmembrane domains of their substrates have been identified in a range of organisms and play a variety of roles in biological conditions. I-CLiPs have been classified into three groups: serine-, aspartyl- and metalloprotease-type. Signal peptide peptidase (SPP) is an atypical aspartic protease that hydrolyzes peptide bonds within the transmembrane domain of substrates and is implicated in several biological and pathological functions. The structure of human SPP was determined by SPA at a resolution of 22 Å [8]. SPP forms a slender, bullet-shaped homotetramer with dimensions of 85 x 85 x 130 Å. The SPP complex has four concaves on the rhombus-like sides, connected to a large chamber inside the molecule. For the tetrameric assembly, the N-terminal region of SPP was found to be sufficient. Moreover, when N-terminal region was overexpressed, the formation of the endogenous SPP tetramer was inhibited, which suppressed the proteolytic activity within cells. From these data, the N-terminal region is considered to work as the structural scaffold.Transmembrane (TM) translocation of newly synthesized secretion proteins and membrane proteins are carried out by a Sec translocon protein complex. The polypeptide-conducting pore is formed by the SecYEG-SecA complex in bacteria, and the membrane protein SecDF is necessary for the efficient transport of proteins. However the molecular mechanism how SecDF realized efficient transport is not clear. A previous X-ray structural study of the whole protein and subdomain suggest that SecDF has at least two conformational variants, which could reflect molecular dynamics of this protein. To confirm this hypothesis, we analyzed the 3D structure of SecDF using dark field STEM electron tomography and single particle reconstruction. We determined two different whole SecDF protein structures which well explains the X-ray data. From these data, we would like to propose the possible molecular mechanism of SecDF during polypeptide translocation.
RESUMO
The Sec translocon facilitates transportation of newly synthesized polypeptides from the cytoplasm to the lumen/periplasm across the phospholipid membrane. Although the polypeptide-conducting machinery is formed by the SecYEG-SecA complex in bacteria, its transportation efficiency is markedly enhanced by SecDF. A previous study suggested that SecDF assumes at least two conformations differing by a 120° rotation in the spatial orientation of the P1 head subdomain to the rigid base, and that the conformational dynamics plays a critical role in polypeptide translocation. Here we addressed this hypothesis by analyzing the 3D structure of SecDF using electron tomography and single particle reconstruction. Reconstruction of wt SecDF showed two major conformations; one resembles the crystal structure of full-length SecDF (F-form structure), while the other is similar to the hypothetical structural variant based on the crystal structure of the isolated P1 domain (I-form structure). The transmembrane domain of the I-form structure has a scissor like cleft open to the periplasmic side. We also report the structure of a double cysteine mutant designed to constrain SecDF to the I-form. This reconstruction has a protrusion at the periplasmic end that nicely fits the orientation of P1 in the I-from. These results provide firm evidence for the occurrence of the I-form in solution and support the proposed F- to I-transition of wt SecDF during polypeptide translocation.
Assuntos
Proteínas de Bactérias/ultraestrutura , Proteínas de Membrana/ultraestrutura , Proteínas de Membrana Transportadoras/ultraestrutura , Thermus thermophilus/genética , Proteínas de Bactérias/genética , Cristalografia por Raios X , Tomografia com Microscopia Eletrônica , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras/genética , Mutação , Estrutura Terciária de Proteína , Transporte ProteicoRESUMO
Optical microscopy is generally the first choice to observe microbes and cells. However, its resolution is not always sufficient to reveal specific target structures, such as flagella and pili, which are only nanometers wide. ASEM is an attractive higher resolution alternative, as the sample is observed in aqueous solution at atmospheric pressure. Sample pretreatment for ASEM only comprises simple tasks including fixation, gold labeling, and reagent exchange, taking less than 1 h in total. The lengthy sample pretreatments often required for more classical electron microscopies, such as embedding and dehydration, are unnecessary, and native morphology is preserved. In this study, positively charged nanogold particles were used to label the surfaces of bacteria and cultured animal cells, exploiting their net negative surface charge. After gold enhancement to increase the size of the nanogold particles, ASEM imaging of the bacteria in aqueous solution revealed pili and delicate spiral flagella. This natural shape contrasts starkly with images of dried flagella recorded by standard SEM. Positively charged nanogold labeled the plasma membrane of cultured COS7 cells, and after enhancement allowed filopodia as thin as 100 nm in diameter to be clearly visualized. Based on these studies, ASEM combined with positively charged nanogold labeling promises to become an important tool for the study of cell morphology and dynamics in the near future.