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Vitamin B12 (cobalamin or Cbl) functions as a cofactor in two important enzymatic processes in human cells, and life is not sustainable without it. B12 is obtained from food and travels from the stomach, through the intestine, and into the bloodstream by three B12-transporting proteins: salivary haptocorrin (HC), gastric intrinsic factor, and transcobalamin (TC), which all bind B12 with high affinity and require proteolytic degradation to liberate Cbl. After intracellular delivery of dietary B12, Cbl in the aquo/hydroxocobalamin form can coordinate various nucleophiles, for example, GSH, giving rise to glutathionylcobalamin (GSCbl), a naturally occurring form of vitamin B12. Currently, there is no data showing whether GSCbl is recognized and transported in the human body. Our crystallographic data shows for the first time the complex between a vitamin B12 transporter and GSCbl, which compared to aquo/hydroxocobalamin, binds TC equally well. Furthermore, sequence analysis and structural comparisons show that TC recognizes and transports GSCbl and that the residues involved are conserved among TCs from different organisms. Interestingly, haptocorrin and intrinsic factor are not structurally tailored to bind GSCbl. This study provides new insights into the interactions between TC and Cbl.
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Glutatión , Ratas , Transcobalaminas , Vitamina B 12 , Animales , Cristalografía por Rayos X , Glutatión/metabolismo , Glutatión/análogos & derivados , Glutatión/química , Unión Proteica , Transcobalaminas/metabolismo , Transcobalaminas/química , Vitamina B 12/metabolismo , Vitamina B 12/análogos & derivados , Vitamina B 12/químicaRESUMEN
Despite recent advances in cryo-electron microscopy and artificial intelligence-based model predictions, a significant fraction of structure determinations by macromolecular crystallography still requires experimental phasing, usually by means of single-wavelength anomalous diffraction (SAD) techniques. Most synchrotron beamlines provide highly brilliant beams of X-rays of between 0.7 and 2 Å wavelength. Use of longer wavelengths to access the absorption edges of biologically important lighter atoms such as calcium, potassium, chlorine, sulfur and phosphorus for native-SAD phasing is attractive but technically highly challenging. The long-wavelength beamline I23 at Diamond Light Source overcomes these limitations and extends the accessible wavelength range to λ = 5.9 Å. Here we report 22 macromolecular structures solved in this extended wavelength range, using anomalous scattering from a range of elements which demonstrate the routine feasibility of lighter atom phasing. We suggest that, in light of its advantages, long-wavelength crystallography is a compelling option for experimental phasing.
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In macromolecular structure determination using X-ray diffraction from multiple crystals, the presence of different structures (structural polymorphs) necessitates the classification of the diffraction data for appropriate structural analysis. Hierarchical clustering analysis (HCA) is a promising technique that has so far been used to extract isomorphous data, mainly for single-structure determination. Although in principle the use of HCA can be extended to detect polymorphs, the absence of a reference to define the threshold used to group the isomorphous data sets (the `isomorphic threshold') poses a challenge. Here, unit-cell-based and intensity-based HCAs have been applied to data sets for apo trypsin and inhibitor-bound trypsin that were mixed post data acquisition to investigate the efficacy of HCA in classifying polymorphous data sets. Single-step intensity-based HCA successfully classified polymorphs with a certain `isomorphic threshold'. In data sets for several samples containing an unknown degree of structural heterogeneity, polymorphs could be identified by intensity-based HCA using the suggested `isomorphic threshold'. Polymorphs were also detected in single crystals using data collected using the continuous helical scheme. These findings are expected to facilitate the determination of multiple structural snapshots by exploiting automated data collection and analysis.
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Cristalografía por Rayos X , Tripsina , Difracción de Rayos X , Estructura Molecular , Análisis por ConglomeradosRESUMEN
This postmarketing surveillance study was conducted to evaluate the safety and effectiveness of onabotulinumtoxinA in Japanese patients with laryngeal dystonia (LD). Patients receiving onabotulinumtoxinA for the first time were enrolled and observed for up to 12 months following the first injection. Safety assessment included adverse drug reactions (ADRs), and effectiveness assessments included the Voice Handicap Index-10 (VHI-10) and physician's global assessment (PGA). ADRs were observed in 48 (5.8%) of 834 patients in the safety analysis set, including dysphonia in 43 (5.2%) patients and dysphagia in 7 (0.8%) patients. The change in total VHI-10 score (mean) in 790 patients included in the effectiveness analysis set showed that improvement in adductor LD peaked at 2 months after the first injection, while patients with abductor or mixed LD showed a gradual attenuation of effect 2-4 weeks post-injection. The change in total VHI-10 score in subsequent injections was generally similar to that following the first injection. The overall effectiveness rate according to the PGA was 93.4% (738/790 patients). The results demonstrate that onabotulinumtoxinA is a well-tolerated and effective treatment for LD in real-world clinical practice.
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Toxinas Botulínicas Tipo A , Trastornos de Deglución , Disfonía , Distonía , Humanos , Toxinas Botulínicas Tipo A/efectos adversos , Disfonía/diagnóstico , Disfonía/tratamiento farmacológico , Distonía/tratamiento farmacológico , Trastornos de Deglución/tratamiento farmacológicoRESUMEN
Ever since the historic discovery of the cooperative oxygenation of its multiple subunits, hemoglobin (Hb) has been among the most exhaustively studied allosteric proteins. However, the lack of structural information on the intermediates between oxygenated and deoxygenated forms prevents our detailed understanding of the molecular mechanism of its allostery. It has been difficult to prepare crystals of intact oxy-deoxy intermediates and to individually identify the oxygen saturation for each subunit. However, our recent crystallographic studies have demonstrated that giant Hbs from annelids are suitable for overcoming these problems and can provide abundant information on oxy-deoxy intermediate structures. Here, we report the crystal structures of oxy-deoxy intermediates of a 400 kDa Hb (V2Hb) from the annelid Lamellibrachia satsuma, following up on a series of previous studies of similar giant Hbs. Four intermediate structures had average oxygen saturations of 78%, 69%, 55%, and 26%, as determined by the occupancy refinement of the bound oxygen based on ambient temperature factors. The structures demonstrate that the cooperative oxygen dissociation is weaker, large ternary and quaternary changes are induced at a later stage of the oxygen dissociation process, and the ternary and quaternary changes are smaller with local perturbations. Nonetheless, the overall structural transition seemed to proceed in the manner of the MWC two-state model. Our crystallographic snapshots of the allosteric transition of V2Hb provide important experimental evidence for a more detailed understanding of the allostery of Hbs by extension of the Monod-Wyman-Changeux (MWC) model.
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A figure in the article by Baba et al. [(2021), J. Synchrotron Rad. 28, 1284-1295] is corrected.
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While native SAD phasing is a promising method for next-generation macromolecular crystallography, it requires the collection of high-quality diffraction data using long-wavelength X-rays. The crystal itself and the noncrystalline medium around the crystal can cause background noise during long-wavelength X-ray data collection, hampering native SAD phasing. Optimizing the crystal size and shape or removing noncrystalline sample portions have thus been considered to be effective means of improving the data quality. A crystal-processing machine that uses a deep-UV laser has been developed. The machine utilizes the pulsed UV laser soft ablation (PULSA) technique, which generates less heat than methods using infrared or visible lasers. Since protein crystals are sensitive to heat damage, PULSA is an appropriate method to process them. Integration of a high-speed Galvano scanner and a high-precision goniometer enables protein crystals to be shaped precisely and efficiently. Application of this crystal-processing machine to a long-wavelength X-ray diffraction experiment significantly improved the diffraction data quality and thereby increased the success rate in experimental phasing using anomalous diffraction from atoms.
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Cristalización/instrumentación , Cristalización/métodos , Proteínas/química , Cristalografía por Rayos X , Ferredoxina-NADP Reductasa/química , Rayos Láser , Rayos UltravioletaRESUMEN
BACKGROUND: Tenofovir disoproxil fumarate (TDF) is widely used and recommended as first-line treatment for patients infected with the hepatitis B virus (HBV). However, current data are limited regarding the efficacy and safety of switching to TDF for the treatment of chronic hepatitis B in hepatitis B e-antigen (HBeAg)-positive patients who are virologically suppressed with another nucleos(t)ide analogue. The primary objective of this study was to evaluate the hepatitis B surface antigen (HBsAg) reduction potential of switching from entecavir (ETV) to TDF at week 48 in HBeAg-positive chronic hepatitis B patients with undetectable serum HBV-DNA. METHODS: In this multicenter, single-arm, open-label, phase 4 clinical study, 75 participants currently treated with ETV 0.5 mg once daily were switched to TDF 300 mg once daily for 96 weeks. RESULTS: At week 48, 3/74 participants (4%) achieved 0.25 log10 reduction of HBsAg levels from baseline (the primary endpoint). Mean HBsAg reduction was -0.14 log10 IU/mL and 12% (9/74) achieved 0.25 log10 reduction by 96 weeks. No participants achieved HBsAg seroclearance. HBsAg reduction at weeks 48 and 96 was numerically greater in participants with higher alanine aminotransferase levels (≥ 60 U/L). Seventeen participants (25%) achieved HBeAg seroclearance up to week 96. No participants experienced viral breakthrough. All drug-related adverse events (18 participants [24%]) were mild in intensity, including an increase in urine beta-2-microglobulin (15 participants [20%]). CONCLUSIONS: In conclusion, HBsAg reduction was limited after switching from ETV to TDF in this study population. Further investigation is warranted to better understand the clinical impact of switching from ETV to TDF. ClinicalTrials.gov: NCT03258710 registered August 21, 2017. https://clinicaltrials.gov/ct2/show/NCT03258710?term=NCT03258710&draw=2&rank=1.
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Antígenos e de la Hepatitis B , Hepatitis B Crónica , Guanina/análogos & derivados , Hepatitis B Crónica/tratamiento farmacológico , Humanos , Estudios Prospectivos , Tenofovir/uso terapéuticoRESUMEN
Cooperative oxygen binding of hemoglobin (Hb) has been studied for over half a century as a representative example of the allostericity of proteins. The most important problem remaining to be solved is the lack of structural information on the intermediates between the oxygenated and deoxygenated forms. In order to characterize the intermediate structures, it is necessary to obtain intermediate-state crystals, determine their oxygen saturations and then determine the oxygen saturations of each of their constituent subunits, all of which are challenging issues even now. Here, intermediate forms of the 400â kDa giant Hb from the tubeworm Oligobrachia mashikoi are reported. To overcome the above problems without any artificial modifications to the protein or prosthetic groups, intermediate crystals of the giant Hb were prepared from fully oxygenated crystals by a soaking method. The oxygen saturation of the crystals was measured by in situ observation with a microspectrophotometer using thin plate crystals processed by an ultraviolet laser to avoid saturation of absorption. The oxygen saturation of each subunit was determined by occupancy refinement of the bound oxygen based on ambient temperature factors. The obtained structures reveal the detailed relationship between the structural transition and oxygen dissociation. The dimer subassembly of the giant Hb shows strong correlation with the local structural changes at the heme pockets. Although some local ternary-structural changes occur in the early stages of the structural transition, the associated global ternary-structural and quaternary-structural changes might arise at about 50% oxygen saturation. The models based on coarse snapshots of the allosteric transition support the conventional two-state model of Hbs and provide the missing pieces of the intermediate structures that are required for full understanding of the allosteric nature of Hbs in detail.
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Intense micro-focus X-ray beamlines available at synchrotron facilities have achieved high-quality data collection even from the microcrystals of membrane proteins. The automatic data collection system developed at SPring-8, named ZOO, has contributed to many structure determinations of membrane proteins using small-wedge synchrotron crystallography (SWSX) datasets. The `small-wedge' (5-20°) datasets are collected from multiple crystals and then merged to obtain the final structure factors. To our knowledge, no systematic investigation on the dose dependence of data accuracy has so far been reported for SWSX, which is between `serial crystallography' and `rotation crystallography'. Thus, herein, we investigated the optimal dose conditions for experimental phasing with SWSX. Phase determination using anomalous scattering signals was found to be more difficult at higher doses. Furthermore, merging more homogeneous datasets grouped by hierarchical clustering with controlled doses mildly reduced the negative factors in data collection, such as `lack of signal' and `radiation damage'. In turn, as more datasets were merged, more probable phases could be obtained across a wider range of doses. Therefore, our findings show that it is essential to choose a lower dose than 10â MGy for de novo structure determination by SWSX. In particular, data collection using a dose of 5â MGy proved to be optimal in balancing the amount of signal available while reducing the amount of damage as much as possible.
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Cristalografía por Rayos X/métodos , Proteínas de la Membrana/química , Proteínas de la Membrana/efectos de la radiación , Muramidasa/química , Muramidasa/efectos de la radiación , Modelos Moleculares , Dosis de Radiación , Traumatismos por Radiación , Dispersión de Radiación , SincrotronesRESUMEN
OBJECTIVES: Most dentists maintain awkward and restricted postures for prolonged periods that stress the body while providing direct patient care. Information about working posture must be analysed in a more systematic manner to provide a deeper understanding of the relationship between working posture and work-related musculoskeletal disorders (MSD). Therefore, the present study aimed to clarify the effects of different lines of vision during tooth preparation, such as the direct and the mirror view technique, on-body tilt (angle), muscle activity, and sitting balance, which may correlate with the reduction or prevention of MSD. METHODS: A mannequin head with a maxillary right first molar embedded in a model was attached to the dental chair headrest. Two different techniques for tooth preparation were selected: direct view and mirror view. Muscle activity, body tilt (angle), and sitting balance were analysed as independent parameters. RESULTS: Different tooth preparation techniques had a distinct influence on body tilt (angle), muscle activity, and sitting balance. The direct view technique resulted in significantly larger values for all parameters, except for the activity of the spinal column erector muscles than the mirror view technique. CONCLUSION: Based on these results, the direct view technique for tooth preparation, which is used by most dentists in practice, imposes a burden on the lower back, shoulders, and neck of the dentist.
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Postura , Sedestación , Electromiografía , Humanos , Músculos , Preparación del DienteRESUMEN
The human adiponectin receptors, AdipoR1 and AdipoR2, are key anti-diabetic molecules. We previously reported the crystal structures of human AdipoR1 and AdipoR2, revealing that their seven transmembrane helices form an internal closed cavity (the closed form). In this study, we determined the crystal structure of the D208A variant AdipoR1, which is fully active with respect to the major downstream signaling. Among the three molecules in the asymmetric unit, two assume the closed form, and the other adopts the open form with large openings in the internal cavity. Between the closed- and open-form structures, helices IV and V are tilted with their intracellular ends shifted by about 4 and 11 Å, respectively. Furthermore, we reanalyzed our previous wild-type AdipoR1 diffraction data, and determined a 44:56 mixture of the closed and open forms, respectively. Thus, we have clarified the closed-open interconversion of AdipoR1, which may be relevant to its functional mechanism(s).
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Receptores de Adiponectina/química , Receptores de Adiponectina/metabolismo , Secuencia de Aminoácidos , Cristalografía por Rayos X , Células HEK293 , Humanos , Modelos Moleculares , Conformación ProteicaRESUMEN
Plant-type ferredoxin (Fd) is an electron transfer protein in chloroplast. Redox-dependent structural change of Fd controls its association with and dissociation from Fd-dependent enzymes. Among many X-ray structures of oxidized Fd have been reported so far, very likely a given number of them was partially reduced by strong X-ray. To understand the precise structural change between reduced and oxidized Fd, it is important to know whether the crystals of oxidized Fd may or may not be reduced during the X-ray experiment. We prepared the thin plate-shaped Fd crystals from Chlamydomonas reinhardtii and monitored its absorption spectra during experiment. Absorption spectra of oxidized Fd crystals were clearly changed to that of reduced form in an X-ray dose-dependent manner. In another independent experiment, the X-ray diffraction images obtained from different parts of one single crystal were sorted and merged to form two datasets with low and high X-ray doses. An Fo-Fo map calculated from the two datasets showed that X-ray reduction causes a small displacement of the iron atoms in the [2Fe-2S] cluster. Both our spectroscopic and crystallographic studies confirm X-ray dose-dependent reduction of Fd, and suggest a structural basis for its initial reduction step especially in the core of the cluster.
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Chlamydomonas reinhardtii/metabolismo , Ferredoxinas/química , Ferredoxinas/efectos de la radiación , Cristalización , Cristalografía por Rayos X , Ferredoxinas/metabolismo , Modelos Moleculares , Oxidación-Reducción , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/efectos de la radiación , Espectroscopía de Absorción de Rayos X , Rayos XRESUMEN
Changes in NIRS signals are related to changes in local cerebral blood flow or oxy-Hb concentration. On the other hand, recent studies have revealed the effect of chewing gum on cognitive performance, stress control etc. which accompanied brain activity in the prefrontal cortex (PFC). However, these relationships are still controversial. To evaluate the chewing effect on PFC, NIRS seems to be a suitable method of imaging such results. When measuring NIRS on PFC, blood volume in superficial tissues (scalp, skin, muscle) might have some affect. The aim of the present study was to clarify the effect of the anterior temporal muscle on NIRS signals during gum chewing. Eight healthy volunteers participated. Two-channel NIRS (HOT-1000, NeU, Japan), which can distinguish total-Hb concentrations in deep tissue and superficial tissue layers, was used. In addition to a conventional optode separation distance of 3.0 cm, Hot 1000 has a short distance of 1.0 cm (NEAR channel) to measure NIRS signals that originate exclusively from surface tissues. NIRS probes were placed at Fp1 and Fp2 in the normal probe setting. The headset was displaced to the left in order to allow the left probe to be placed over the left anterior temporal muscle. In the normal setting, the superficial signal curve shows no notable change; however, the neural (calculated and defined in HOT-1000) and deep curves show an increase during the gum chewing task. At the deviated setting, all three signals show marked changes during the task. Total-Hb concentration in the deviated probe setting is significantly large (p < 0.05) than that of in the normal probe setting. When using gum chewing as a task, it would be better to consider a probe position carefully so that the influence of muscle activity on NIRS signal can be distinguished.
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Masticación , Corteza Prefrontal , Espectroscopía Infrarroja Corta , Adulto , Hemoglobinas/metabolismo , Humanos , Japón , Proyectos Piloto , Corteza Prefrontal/fisiología , Adulto JovenRESUMEN
AIM: To evaluate the effects of early (≤6 months after starting any medical treatment [baseline] for benign prostatic hyperplasia [BPH]), intermediate (between >6 months and 2 years from baseline) and late (2 years after baseline) initiation of add-on dutasteride therapy on the incidence of acute urinary retention (AUR) and BPH-related surgery in Japanese patients with moderate-to-severe BPH. METHODS: This multicentre, observational, retrospective chart review study used anonymised data from Japanese medical records. Eligible patients (≥50 years) were followed from baseline until first AUR, BPH-related surgery or Year 4. RESULTS: Overall, 1206 patients were included (early initiation: n = 793; intermediate: n = 233; late: n = 180). Early dutasteride initiation was not superior to late initiation in reducing the risk of first AUR or BPH-related surgery from baseline (hazard ratio [HR] 0.733; 95% confidence interval [CI] 0.468-1.150) but was superior in reducing the risk of first AUR alone (HR 3.449; 95% CI 1.796-6.623). One year after initiation, the cumulative incidence of first AUR rose rapidly in the late vs early and intermediate initiation groups. Incidences of all parameters (first AUR/BPH-related surgery, first AUR alone and BPH-related surgery alone) in patients undergoing BPH-related surgery in low incidence sites (ie clinical sites with ≤ 16% incidence of first AUR or BPH-related surgery) were significantly lower in the early vs late initiation groups. CONCLUSION: Early dutasteride initiation reduced the risk of AUR in a Japanese real-world setting. A randomised controlled trial is warranted to evaluate the benefit of early initiation in preventing BPH-related surgery in Japanese patients.
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Native single-wavelength anomalous dispersion (SAD) is an attractive experimental phasing technique as it exploits weak anomalous signals from intrinsic light scatterers (Z < 20). The anomalous signal of sulfur in particular, is enhanced at long wavelengths, however the absorption of diffracted X-rays owing to the crystal, the sample support and air affects the recorded intensities. Thereby, the optimal measurable anomalous signals primarily depend on the counterplay of the absorption and the anomalous scattering factor at a given X-ray wavelength. Here, the benefit of using a wavelength of 2.7 over 1.9â Å is demonstrated for native-SAD phasing on a 266â kDa multiprotein-ligand tubulin complex (T2R-TTL) and is applied in the structure determination of an 86â kDa helicase Sen1 protein at beamline BL-1A of the KEK Photon Factory, Japan. Furthermore, X-ray absorption at long wavelengths was controlled by shaping a lysozyme crystal into spheres of defined thicknesses using a deep-UV laser, and a systematic comparison between wavelengths of 2.7 and 3.3â Å is reported for native SAD. The potential of laser-shaping technology and other challenges for an optimized native-SAD experiment at wavelengths >3â Å are discussed.
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PURPOSE: To compare the efficacy, safety, and tolerability profiles of pegylated liposomal doxorubicin and carboplatin (PLDC) with those of gemcitabine and carboplatin (GC) for the treatment of patients with platinum-sensitive recurrent ovarian cancer. METHODS: Ovarian cancer patients with recurrence > 6 months after first-line platinum and taxane-based therapies were randomly assigned to PLDC [pegylated liposomal doxorubicin 30 mg/m2 plus carboplatin area under the curve (AUC) 5 mg/mL/min on day 1] every 4 weeks or GC (gemcitabine 1000 mg/m2 on days 1 and 8 plus carboplatin AUC 4 mg/mL/min on day 1) every 3 weeks for at least 6 cycles. The primary endpoint was progression-free survival, and overall response rate, overall survival, toxicity, and dose administration were secondary endpoints. RESULTS: One-hundred patients (49 PLDC; 51 GC) were randomly assigned. Over a median follow-up of 24 months, the median progression-free survival was 12.0 months (95% CI 9.2-15.0) for PLDC and 9.8 months (8.9-12.3) for GC [HR 0.69 (0.455-1.047)] with a difference of 2.2 months. The response rate was 57.1% (41.0-72.3) for PLDC and 56.4% (39.6-72.2) for GC. No obvious differences in toxicity (G3/4) were noted between arms. The median relative dose intensity of planned dose per week was 88.9% for pegylated liposomal doxorubicin and 53.1% for gemcitabine (p < 0.0001). CONCLUSIONS: PLDC and GC are both good treatment candidates for platinum-sensitive recurrent ovarian cancer patients; however, the dose intensity was lower for GC than for PLDC. PLDC had a more favorable risk-benefit profile than that of GC for patients.
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Adenocarcinoma de Células Claras/tratamiento farmacológico , Adenocarcinoma Mucinoso/tratamiento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Recurrencia Local de Neoplasia/tratamiento farmacológico , Neoplasias Ováricas/tratamiento farmacológico , Adenocarcinoma de Células Claras/patología , Adenocarcinoma Mucinoso/patología , Adulto , Anciano , Anciano de 80 o más Años , Carboplatino/administración & dosificación , Desoxicitidina/administración & dosificación , Desoxicitidina/análogos & derivados , Doxorrubicina/administración & dosificación , Doxorrubicina/análogos & derivados , Femenino , Humanos , Persona de Mediana Edad , Recurrencia Local de Neoplasia/patología , Neoplasias Ováricas/patología , Polietilenglicoles/administración & dosificación , Pronóstico , Tasa de Supervivencia , Adulto Joven , GemcitabinaRESUMEN
Recent research on the structure and mechanism of DNA polymerases has continued to generate fundamentally important features, including a noncanonical pathway involving "prebinding" of metal-bound dNTP (MdNTP) in the absence of DNA. While this noncanonical mechanism was shown to be a possible subset for African swine fever DNA polymerase X (Pol X) and human Pol λ, it remains unknown whether it could be the primary pathway for a DNA polymerase. Pol µ is a unique member of the X-family with multiple functions and with unusual Mn2+ preference. Here we report that Pol µ not only prebinds MdNTP in a catalytically active conformation but also exerts a Mn2+ over Mg2+ preference at this early stage of catalysis, for various functions: incorporation of dNTP into a single nucleotide gapped DNA, incorporation of rNTP in the nonhomologous end joining (NHEJ) repair, incorporation of dNTP to an ssDNA, and incorporation of an 8-oxo-dGTP opposite template dA (mismatched) or dC (matched). The structural basis of this noncanonical mechanism and Mn2+ over Mg2+ preference in these functions was analyzed by solving 19 structures of prebinding binary complexes, precatalytic ternary complexes, and product complexes. The results suggest that the noncanonical pathway is functionally relevant for the multiple functions of Pol µ. Overall, this work provides the structural and mechanistic basis for the long-standing puzzle in the Mn2+ preference of Pol µ and expands the landscape of the possible mechanisms of DNA polymerases to include both mechanistic pathways.
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ADN Polimerasa Dirigida por ADN/metabolismo , Manganeso/metabolismo , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/aislamiento & purificación , Humanos , Manganeso/química , Modelos MolecularesRESUMEN
Owing to the development of brilliant microfocus beamlines, rapid-readout detectors and sample changers, protein microcrystallography is rapidly becoming a popular technique for accessing structural information from complex biological samples. However, the method is time-consuming and labor-intensive and requires technical expertise to obtain high-resolution protein crystal structures. At SPring-8, an automated data-collection system named ZOO has been developed. This system enables faster data collection, facilitates advanced data-collection and data-processing techniques, and permits the collection of higher quality data. In this paper, the key features of the functionality put in place on the SPring-8 microbeam beamline BL32XU are described and the major advantages of this system are outlined. The ZOO system will be a major driving force in the evolution of the macromolecular crystallography beamlines at SPring-8.