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1.
Future Oncol ; 20(10): 603-611, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38214131

RESUMO

Aim: To assess physician-reported treatment of metastatic bladder cancer in Japan. Methods: 76 physicians completed the CancerMPact® survey in July 2020, considering patients treated within 6 months. Results: Physicians treated a mean of 38.1 patients per month. Of cisplatin-eligible and -ineligible patients, 97.6 and 89.3%, respectively, received first-line platinum-based therapy, most commonly cisplatin plus gemcitabine (72.9%) and carboplatin plus gemcitabine (59.7%). 1.6 and 5.6% received first-line immune checkpoint inhibitors, respectively. 48.4 and 45.0%, respectively, progressed and received second-line therapy, most commonly with pembrolizumab (61.7%). Conclusion: In 2020, most patients with metastatic bladder cancer in Japan received first-line platinum-based chemotherapy; however, >50% received no subsequent treatment, highlighting the need for new treatment regimens to improve outcomes and maximize first-line treatment benefits.


In 2020, researchers surveyed 76 Japanese doctors who specialized in bladder and urinary system disorders about how they treated people with bladder cancer. Cisplatin, a type of chemotherapy drug, was the most common first treatment. For people who were unable to receive cisplatin, doctors often prescribed a similar chemotherapy drug called carboplatin. Just under half of the people received a second treatment for their cancer. New treatments are now available for bladder cancer, including the immunotherapy drug avelumab, which is given to people whose cancer stops growing or shrinks with their first chemotherapy treatment. More research is needed to better understand how bladder cancer is treated in Japan, including how new treatments are used.


Assuntos
Carcinoma de Células de Transição , Neoplasias da Bexiga Urinária , Humanos , Cisplatino , Gencitabina , Japão/epidemiologia , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/epidemiologia , Carboplatina/uso terapêutico , Desoxicitidina , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Carcinoma de Células de Transição/patologia
2.
Protein Expr Purif ; 172: 105631, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32213313

RESUMO

CD1d is a major histocompatibility complex (MHC) class I-like glycoprotein and binds to glycolipid antigens that are recognized by natural killer T (NKT) cells. To date, our understanding of the structural basis for glycolipid binding and receptor recognition of CD1d is still limited. Here, we established a preparation method for the ectodomain of human and mouse CD1d using a silkworm-baculovirus expression system. The co-expression of human and mouse CD1d and ß2-microglobulin (ß2m) in the silkworm-baculovirus system was successful, but the yield of human CD1d was low. A construct of human CD1d fused with ß2m via a flexible GS linker as a single polypeptide was prepared to improve protein yield. The production of this single-chained complex was higher (50 µg/larva) than that of the co-expression complex. Furthermore, differential scanning calorimetry revealed that the linker made the CD1d complex more stable and homogenous. These results suggest that the silkworm-baculovirus expression system is useful for structural and biophysical studies of CD1d in several aspects including low cost, easy handling, biohazard-free, rapid, and high yielding.


Assuntos
Antígenos CD1d , Baculoviridae , Expressão Gênica , Animais , Antígenos CD1d/biossíntese , Antígenos CD1d/química , Antígenos CD1d/genética , Antígenos CD1d/isolamento & purificação , Bombyx , Humanos , Camundongos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação
3.
Jpn J Clin Oncol ; 50(8): 940-947, 2020 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-32458996

RESUMO

OBJECTIVE: A prospective, observational, post-marketing surveillance was conducted to assess the safety and effectiveness of temsirolimus in patients with renal cell carcinoma in Japan. METHODS: Patients prescribed temsirolimus for advanced renal cell carcinoma were registered and received temsirolimus (25 mg weekly, intravenous infusion for 30-60 minutes) in routine clinical settings (observation period: 96 weeks). RESULTS: Among 1001 patients included in the safety analysis data set (median age, 65.0 years; men, 74.8%; Eastern Cooperative Oncology Group performance status 0 or 1, 69.6%), 778 (77.7%) reported adverse drug reactions. The most common (≥10%) all-grade adverse drug reactions were stomatitis (26.7%), interstitial lung disease (17.3%) and platelet count decreased (11.1%). The incidence rate of grade ≥3 interstitial lung disease was 4.5%. The onset of interstitial lung disease was more frequent after 4-8 weeks of treatment or in patients with lower Eastern Cooperative Oncology Group performance status (21.6% for score 0 vs 8.3% for score 4, P < 0.001). Among 654 patients in the effectiveness analysis data set, the response and clinical benefit rates were 6.7% (95% confidence interval 4.9-8.9) and 53.2% (95% confidence interval 49.3-57.1), respectively. The median progression-free survival was 18.3 weeks (95% confidence interval 16.9-21.1). CONCLUSIONS: The safety and effectiveness profile of temsirolimus observed in this study was similar to that observed in the multinational phase 3 study. The results are generalizable to the real-world scenario at the time of this research, and safety and effectiveness of temsirolimus as a subsequent anticancer therapy for renal cell carcinoma warrants further investigation. (ClinicalTrials.gov identifier NCT01210482, NCT01420601).


Assuntos
Povo Asiático , Carcinoma de Células Renais/tratamento farmacológico , Neoplasias Renais/tratamento farmacológico , Vigilância de Produtos Comercializados , Sirolimo/análogos & derivados , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/uso terapêutico , Carcinoma de Células Renais/patologia , Intervalo Livre de Doença , Feminino , Humanos , Japão , Neoplasias Renais/patologia , Doenças Pulmonares Intersticiais/induzido quimicamente , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Sirolimo/efeitos adversos , Sirolimo/uso terapêutico , Resultado do Tratamento
4.
Proc Natl Acad Sci U S A ; 113(46): 13039-13044, 2016 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-27799539

RESUMO

The 3D structure determination of biological macromolecules by X-ray crystallography suffers from a phase problem: to perform Fourier transformation to calculate real space density maps, both intensities and phases of structure factors are necessary; however, measured diffraction patterns give only intensities. Although serial femtosecond crystallography (SFX) using X-ray free electron lasers (XFELs) has been steadily developed since 2009, experimental phasing still remains challenging. Here, using 7.0-keV (1.771 Å) X-ray pulses from the SPring-8 Angstrom Compact Free Electron Laser (SACLA), iodine single-wavelength anomalous diffraction (SAD), single isomorphous replacement (SIR), and single isomorphous replacement with anomalous scattering (SIRAS) phasing were performed in an SFX regime for a model membrane protein bacteriorhodopsin (bR). The crystals grown in bicelles were derivatized with an iodine-labeled detergent heavy-atom additive 13a (HAD13a), which contains the magic triangle, I3C head group with three iodine atoms. The alkyl tail was essential for binding of the detergent to the surface of bR. Strong anomalous and isomorphous difference signals from HAD13a enabled successful phasing using reflections up to 2.1-Å resolution from only 3,000 and 4,000 indexed images from native and derivative crystals, respectively. When more images were merged, structure solution was possible with data truncated at 3.3-Å resolution, which is the lowest resolution among the reported cases of SFX phasing. Moreover, preliminary SFX experiment showed that HAD13a successfully derivatized the G protein-coupled A2a adenosine receptor crystallized in lipidic cubic phases. These results pave the way for de novo structure determination of membrane proteins, which often diffract poorly, even with the brightest XFEL beams.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Membrana/química , Cristalização , Cristalografia/métodos , Detergentes/química , Elétrons , Halobacterium , Lasers , Conformação Proteica , Ácidos Tri-Iodobenzoicos/química
5.
Nat Methods ; 12(1): 61-3, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25384243

RESUMO

Serial femtosecond X-ray crystallography (SFX) has revolutionized atomic-resolution structural investigation by expanding applicability to micrometer-sized protein crystals, even at room temperature, and by enabling dynamics studies. However, reliable crystal-carrying media for SFX are lacking. Here we introduce a grease-matrix carrier for protein microcrystals and obtain the structures of lysozyme, glucose isomerase, thaumatin and fatty acid-binding protein type 3 under ambient conditions at a resolution of or finer than 2 Å.


Assuntos
Cristalografia por Raios X/métodos , Lubrificantes , Proteínas/química , Aldose-Cetose Isomerases/química , Cristalização , Proteína 3 Ligante de Ácido Graxo , Proteínas de Ligação a Ácido Graxo/química , Lasers , Óleo Mineral , Muramidase/química , Proteínas de Plantas/química
6.
Chembiochem ; 18(4): 358-362, 2017 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-27905160

RESUMO

Chemical inducers that can control target-protein localization in living cells are powerful tools to investigate dynamic biological systems. We recently reported the retention using selective hook or "RUSH" system for reversible localization change of proteins of interest by addition/washout of small-molecule artificial ligands of streptavidin (ALiS). However, the utility of previously developed ALiS was restricted by limited solubility in water. Here, we overcame this problem by X-ray crystal structure-guided design of a more soluble ALiS derivative (ALiS-3), which retains sufficient streptavidin-binding affinity for use in the RUSH system. The ALiS-3-streptavidin interaction was characterized in detail. ALiS-3 is a convenient and effective tool for dynamic control of α-mannosidase II localization between ER and Golgi in living cells.


Assuntos
Ligantes , Modelos Moleculares , Ácidos Ftálicos/química , Transporte Proteico/fisiologia , Proteínas/metabolismo , Piridonas/química , Estreptavidina/química , Sulfonamidas/química , Sítios de Ligação , Cristalização , Humanos , Morfolinas/química , Morfolinas/metabolismo , Ácidos Ftálicos/farmacologia , Ligação Proteica , Proteínas/química , Piridonas/metabolismo , Piridonas/farmacologia , Siloxanas/química , Siloxanas/metabolismo , Solubilidade , Estreptavidina/metabolismo , Sulfonamidas/metabolismo
7.
J Am Chem Soc ; 137(33): 10464-7, 2015 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-26261872

RESUMO

Artificial ligands of streptavidin (ALiS) with association constants of ∼10(6) M(-1) were discovered by high-throughput screening of our chemical library, and their binding characteristics, including X-ray crystal structure of the streptavidin complex, were determined. Unlike biotin and its derivatives, ALiS exhibits fast dissociation kinetics and excellent cell permeability. The streptavidin-ALiS system provides a novel, practical compound-dependent methodology for repeated reversible cycling of protein localization between intracellular organella.


Assuntos
Espaço Intracelular/metabolismo , Estreptavidina/metabolismo , Avaliação Pré-Clínica de Medicamentos , Cinética , Ligantes , Modelos Moleculares , Permeabilidade , Conformação Proteica , Transporte Proteico , Estreptavidina/química
8.
Chem Rec ; 15(4): 675-90, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26123293

RESUMO

Determining the bioactive structure of membrane lipids is a new concept, which aims to examine the functions of lipids with respect to their three-dimensional structures. As lipids are dynamic by nature, their "structure" does not refer solely to a static picture but also to the local and global motions of the lipid molecules. We consider that interactions with lipids, which are completely defined by their structures, are controlled by the chemical, functional, and conformational matching between lipids and between lipid and protein. In this review, we describe recent advances in understanding the bioactive structures of membrane lipids bound to proteins and related molecules, including some of our recent results. By examining recent works on lipid-raft-related molecules, lipid-protein interactions, and membrane-active natural products, we discuss current perspectives on membrane structural biology.


Assuntos
Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Microdomínios da Membrana/química , Microdomínios da Membrana/metabolismo , Ressonância Magnética Nuclear Biomolecular/métodos , Animais , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Modelos Moleculares
9.
Appl Opt ; 54(16): 5193-202, 2015 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-26192683

RESUMO

An immersion grating composed of a transmissive material with a high refractive index (n>2) is a powerful device for high-resolution spectroscopy in the infrared region. Although the original idea is attributed to Fraunhofer about 200 years ago, an immersion grating with high diffraction efficiency has never been realized due to the difficulty in processing infrared crystals that are mostly brittle. While anisotropic etching is one successful method for fabricating a fine groove pattern on Si crystal, machining is necessary for realizing the ideal groove shape on any kind of infrared crystal. In this paper, we report the realization of the first, to the best of our knowledge, machined immersion grating made of single-crystal CdZnTe with a high diffraction efficiency that is almost identical to that theoretically predicted by rigorous coupled-wave analysis.

10.
Chem Soc Rev ; 43(7): 2147-58, 2014 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-24252936

RESUMO

With the recent development in pulsed lasers with ultrashort pulse widths or wavelengths, spatially precise, low-damage processing by femtosecond or deep-UV laser ablation has shown promise for the production of protein single crystals suitable for X-ray crystallography. Femtosecond laser processing of supersaturated solutions can shorten the protein nucleation period or can induce nucleation at low supersaturation, which improves the crystal quality of various proteins including membrane proteins and supra-complexes. In addition to nucleation, processing of protein crystals by femtosecond or deep-UV laser ablation can produce single crystalline micro- or macro-seeds without deterioration of crystal quality. This tutorial review gives an overview of the successful application of laser ablation techniques to nucleation and seeding for the production of protein single crystals, and also describes the advantages from a physico-chemical perspective.


Assuntos
Lasers , Proteínas/química , Cristalização
11.
Angew Chem Int Ed Engl ; 54(5): 1508-11, 2015 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-25491543

RESUMO

Long-chain fatty acids (FAs) with low water solubility require fatty-acid-binding proteins (FABPs) to transport them from cytoplasm to the mitochondria for energy production. However, the precise mechanism by which these proteins recognize the various lengths of simple alkyl chains of FAs with similar high affinity remains unknown. To address this question, we employed a newly developed calorimetric method for comprehensively evaluating the affinity of FAs, sub-Angstrom X-ray crystallography to accurately determine their 3D structure, and energy calculations of the coexisting water molecules using the computer program WaterMap. Our results clearly showed that the heart-type FABP (FABP3) preferentially incorporates a U-shaped FA of C10-C18 using a lipid-compatible water cluster, and excludes longer FAs using a chain-length-limiting water cluster. These mechanisms could help us gain a general understanding of how proteins recognize diverse lipids with different chain lengths.


Assuntos
Proteínas de Ligação a Ácido Graxo/metabolismo , Miocárdio/metabolismo , Água/metabolismo , Sítios de Ligação , Calorimetria , Cristalografia por Raios X , Proteína 3 Ligante de Ácido Graxo , Proteínas de Ligação a Ácido Graxo/química , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Humanos , Simulação de Dinâmica Molecular , Estrutura Terciária de Proteína , Termodinâmica , Água/química
12.
Bioorg Med Chem ; 22(6): 1804-8, 2014 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-24581547

RESUMO

The role of heart-type fatty acid-binding protein (FABP3) in human physiology as an intracellular carrier of fatty acids (FAs) has been well-documented. In this study, we aimed to develop an analytical method to study real-time interaction kinetics between FABP3 immobilized on the sensor surface and unsaturated C18 FAs using surface plasmon resonance (SPR). To establish the conditions for SPR experiments, we used an FABP3-selective inhibitor 4-(2-(1-(4-bromophenyl)-5-phenyl-1H-pyrazol-3-yl)-phenoxy)-butyric acid. The affinity index thus obtained was comparable to that reported previously, further supporting the usefulness of the SPR-based approach for evaluating interactions between FABPs and hydrophobic ligands. A pseudo-first-order affinity of FABP3 to K(+) petroselinate (C18:1 Δ6 cis), K(+) elaidate (C18:1 Δ9 trans), and K(+) oleate (C18:1 Δ9 cis) was characterized by the dissociation constant (K(d)) near micromolar ranges, whereas K(+) linoleate (C18:2 Δ9,12 cis/cis) and K(+) α-linolenate (C18:3 Δ9,12,15 cis/cis/cis) showed a higher affinity to FABP3 with Kd around 1 × 10(-6)M. Interactions between FAPB3 and C18 FAs incorporated in large unilamellar vesicles consisting of 1,2-dimyristoyl-sn-glycero-3-phosphocholine and FAs (5:1 molar ratio) were also analysed. Control DMPC liposomes without FA showed only marginal binding to FABP3 immobilized on a sensor chip while liposome-incorporated FA revealed significant responses in sensorgrams, demonstrating that the affinity of FAs to FABP3 could be evaluated by using the liposome-incorporated analytes. Significant affinity to FABP3 was observed for monounsaturated fatty acids (K(d) in the range of 1 × 10(-7)M). These experiments demonstrated that highly hydrophobic compounds in a liposome-incorporated form could be subjected to SPR experiments for kinetic analysis.


Assuntos
Proteínas de Ligação a Ácido Graxo/química , Ácidos Graxos Insaturados/química , Lipossomos/química , Ressonância de Plasmônio de Superfície , Proteína 3 Ligante de Ácido Graxo , Proteínas de Ligação a Ácido Graxo/genética , Humanos , Cinética , Lipossomos/síntese química
13.
J Synchrotron Radiat ; 20(Pt 6): 923-8, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24121341

RESUMO

Heart-type fatty-acid-binding protein (FABP3), which is a cytosolic protein abundantly found in cardiomyocytes, plays a role in trafficking fatty acids throughout cellular compartments by reversibly binding intracellular fatty acids with relatively high affinity. The fluorescent probe 1-anilinonaphthalene-8-sulfonate (ANS) is extensively utilized for examining the interaction of ligands with fatty-acid-binding proteins. The X-ray structure of FABP3 was determined in the presence of ANS and revealed the detailed ANS-binding mechanism. Furthermore, four water molecules were clearly identified in the binding cavity. Through these water molecules, the bound ANS molecule forms indirect hydrogen-bond interactions with FABP3. The adipocyte-type fatty-acid-binding protein (FABP4) exhibits 67% sequence identity with FABP3 and its crystal structure is almost the same as that of FABP3. However, FABP4 can bind with a higher affinity to ANS than FABP3. To understand the difference in their ligand specificities, a structural comparison was performed between FABP3-ANS and FABP4-ANS complexes. The result revealed that the orientation of ANS binding to FABP3 is completely opposite to that of ANS binding to FABP4, and the substitution of valine in FABP4 to leucine in FABP3 may result in greater steric hindrance between the side-chain of Leu115 and the aniline ring of ANS.


Assuntos
Naftalenossulfonato de Anilina/química , Proteínas de Ligação a Ácido Graxo/química , Corantes Fluorescentes/química , Proteína 3 Ligante de Ácido Graxo , Humanos , Conformação Proteica
14.
J Virol ; 86(14): 7565-76, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22573863

RESUMO

The genomes of the Tomato mosaic virus and many other plant and animal positive-strand RNA viruses of agronomic and medical importance encode superfamily 1 helicases. Although helicases play important roles in viral replication, the crystal structures of viral superfamily 1 helicases have not been determined. Here, we report the crystal structure of a fragment (S666 to Q1116) of the replication protein from Tomato mosaic virus. The structure reveals a novel N-terminal domain tightly associated with a helicase core. The helicase core contains two RecA-like α/ß domains without any of the accessory domain insertions that are found in other superfamily 1 helicases. The N-terminal domain contains a flexible loop, a long α-helix, and an antiparallel six-stranded ß-sheet. On the basis of the structure, we constructed deletion mutants of the S666-to-Q1116 fragment and performed split-ubiquitin-based interaction assays in Saccharomyces cerevisiae with TOM1 and ARL8, host proteins that are essential for tomato mosaic virus RNA replication. The results suggested that both TOM1 and ARL8 interact with the long α-helix in the N-terminal domain and that TOM1 also interacts with the helicase core. Prediction of secondary structures in other viral superfamily 1 helicases and comparison of those structures with the S666-to-Q1116 structure suggested that these helicases have a similar fold. Our results provide a structural basis of viral superfamily 1 helicases.


Assuntos
RNA Helicases/química , Tobamovirus/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , GTP Fosfo-Hidrolases/química , Modelos Moleculares , Mutação , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , RNA Helicases/genética , RNA Helicases/metabolismo , Saccharomyces cerevisiae/virologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Deleção de Sequência , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo
15.
Artigo em Inglês | MEDLINE | ID: mdl-23908034

RESUMO

The spermidine acetyltransferase (SAT) from Escherichia coli catalyses the transfer of acetyl groups from acetyl-CoA to spermidine. SAT has been expressed and purified from E. coli. SAT was crystallized by the sitting-drop vapour-diffusion method to obtain a more detailed insight into the molecular mechanism. Preliminary X-ray diffraction studies revealed that the crystals diffracted to 2.5 Å resolution and belonged to the cubic space group P23, with unit-cell parameters a = b = c = 148.7 Å. They contained four molecules per asymmetric unit.


Assuntos
Acetiltransferases/biossíntese , Acetiltransferases/isolamento & purificação , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/isolamento & purificação , Escherichia coli/enzimologia , Regulação Bacteriana da Expressão Gênica , Acetiltransferases/química , Cristalização , Cristalografia por Raios X , Proteínas de Escherichia coli/química
16.
Biosci Biotechnol Biochem ; 77(3): 612-7, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23470768

RESUMO

Remarkable progress has been made in genome science during the past decade, but understanding of genomes of eukaryotes is far from complete. We have created DNA flexibility maps of the human, mouse, fruit fly, and nematode chromosomes. The maps revealed that all of these chromosomes have markedly flexible DNA regions (We named them SPIKEs). SPIKEs occur more frequently in the human chromosomes than in the mouse, fruit fly, and nematode chromosomes. Markedly rigid DNA regions (rSPIKEs) are also present in these chromosomes. The ratio of the number of SPIKEs to the total number of SPIKEs and rSPIKEs correlated positively with evolutionary stage among the organisms. Repetitive DNA sequences with flexible and rigid properties contribute to the formation of SPIKEs and rSPIKEs respectively. However, non-repetitive flexible and rigid sequences appear to play a major role in SPIKE and rSPIKE formation respectively. They might be involved in the genome-folding mechanism of eukaryotes.


Assuntos
DNA/genética , Genoma Humano/genética , Animais , Sequência de Bases , Caenorhabditis elegans/genética , Cromossomos Humanos/genética , Biologia Computacional , Drosophila melanogaster/genética , Evolução Molecular , Humanos , Camundongos , Fenômenos Físicos
17.
Gut ; 61(4): 554-61, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21836027

RESUMO

BACKGROUND AND AIMS: The mechanisms of cancer cell growth and metastasis are still not entirely understood, especially from the viewpoint of chemical reactions in tumours. Glycolytic metabolism is markedly accelerated in cancer cells, causing the accumulation of glucose (a reducing sugar) and methionine (an amino acid), which can non-enzymatically react and form carcinogenic substances. There is speculation that this reaction produces gaseous sulfur-containing compounds in tumour tissue. The aims of this study were to clarify the products in tumour and to investigate their effect on tumour proliferation. METHODS: Products formed in the reaction between glucose and methionine or its metabolites were analysed in vitro using gas chromatography. Flatus samples from patients with colon cancer and exhaled air samples from patients with lung cancer were analysed using near-edge x-ray fine adsorption structure spectroscopy and compared with those from healthy individuals. The tumour proliferation rates of mice into which HT29 human colon cancer cells had been implanted were compared with those of mice in which the cancer cells were surrounded by sodium hyaluronate gel to prevent diffusion of gaseous material into the healthy cells. RESULTS: Gaseous sulfur-containing compounds such as methanethiol and hydrogen sulfide were produced when glucose was allowed to react with methionine or its metabolites homocysteine or cysteine. Near-edge x-ray fine adsorption structure spectroscopy showed that the concentrations of sulfur-containing compounds in the samples of flatus from patients with colon cancer and in the samples of exhaled air from patients with lung cancer were significantly higher than in those from healthy individuals. Animal experiments showed that preventing the diffusion of sulfur-containing compounds had a pronounced antitumour effect. CONCLUSIONS: Gaseous sulfur-containing compounds are the main products in tumours and preventing the diffusion of these compounds reduces the tumour proliferation rate, which suggests the possibility of a new approach to cancer treatment.


Assuntos
Antineoplásicos/uso terapêutico , Neoplasias do Colo/metabolismo , Gases/metabolismo , Compostos de Enxofre/metabolismo , Animais , Antineoplásicos/farmacologia , Testes Respiratórios/métodos , Proliferação de Células , Cromatografia Gasosa , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/patologia , Difusão/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Feminino , Flatulência/metabolismo , Glucose/metabolismo , Humanos , Ácido Hialurônico/farmacologia , Ácido Hialurônico/uso terapêutico , Sulfeto de Hidrogênio/metabolismo , Neoplasias Pulmonares/metabolismo , Reação de Maillard , Metionina/metabolismo , Camundongos , Camundongos Nus , Transplante de Neoplasias , Compostos de Sulfidrila/metabolismo , Transplante Heterólogo , Espectroscopia por Absorção de Raios X/métodos
18.
Biochemistry ; 51(1): 32-42, 2012 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-22133006

RESUMO

Molecular recognition such as antigen-antibody interaction is characterized by the parameters of kinetics and the energy landscape. Examinations of molecules involved in the interaction at different temperatures using atomic force microscopy (AFM) can provide information on not only the effects of temperature on the unbinding force between a molecule of interest and a complementary molecule but also the parameters of kinetics and the energy landscape for dissociation of the molecular complex. We investigated the effect of temperature on the dissociation process of the complex of ß-lactoglobulin and anti-bovine ß-lactoglobulin IgG polyclonal antibody using AFM. Measurements of the unbinding forces between ß-lactoglobulin and the antibody were performed at 25, 35, and 45 °C. The following results were obtained in our present study: (i) The unbinding forces decreased as temperature increased, suggesting that the binding force between ß-lactoglobulin and the antibody includes the force originating from temperature-dependent interactions (e.g., hydrogen bonding). (ii) At each temperature, the unbinding force exhibited two linear regimes in the force spectra, indicating that the dissociation process of the ß-lactoglobulin-antibody complex passes at least two energy barriers from the bound state to the dissociated state. (iii) The dissociation rates at zero force and the position of energy barriers increased as temperature increased. (iv) The heights of the two energy barriers in the reaction coordinates were 49.7 k(B)T and 14.5 k(B)T. (v) The values of roughness of the barriers were ca. 6.1 k(B)T and 3.2 k(B)T. Overall, the present study using AFM revealed more information about the ß-lactoglobulin-antibody interaction than studies using conventional bulk measurement such as surface plasmon resonance.


Assuntos
Anticorpos Imobilizados/química , Afinidade de Anticorpos , Lactoglobulinas/química , Lactoglobulinas/imunologia , Microscopia de Força Atômica , Temperatura , Alérgenos/química , Alérgenos/imunologia , Alérgenos/farmacocinética , Animais , Anticorpos Imobilizados/metabolismo , Bovinos , Metabolismo Energético , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Lactoglobulinas/farmacocinética , Microscopia de Força Atômica/métodos , Ressonância de Plasmônio de Superfície
19.
J Am Chem Soc ; 134(13): 5786-9, 2012 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-22435400

RESUMO

High-throughput protein X-ray crystallography offers a significant opportunity to facilitate drug discovery. The most reliable approach is to determine the three-dimensional structure of the protein-ligand complex by soaking the ligand in apo crystals. However, protein apo crystals produced by conventional crystallization in a solution are fatally damaged by osmotic shock during soaking. To overcome this difficulty, we present a novel technique for growing protein crystals in a high-concentration hydrogel that is completely gellified and exhibits high strength. This technique allowed us essentially to increase the mechanical stability of the crystals, preventing serious damage to the crystals caused by osmotic shock. Thus, this method may accelerate structure-based drug discoveries.


Assuntos
Cristalização/métodos , Hidrogéis/química , Muramidase/química , Fenômenos Biomecânicos , Dureza , Pressão Osmótica , Estabilidade Proteica
20.
Nucleic Acids Res ; 38(21): 7822-9, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20675355

RESUMO

Aptamers are short single-stranded nucleic acids with high affinity to target molecules and are applicable to therapeutics and diagnostics. Regardless of an increasing number of reported aptamers, the structural basis of the interaction of RNA aptamer with proteins is poorly understood. Here, we determined the 2.15 Å crystal structure of the Fc fragment of human IgG1 (hFc1) complexed with an anti-Fc RNA aptamer. The aptamer adopts a characteristic structure fit to hFc1 that is stabilized by a calcium ion, and the binding activity of the aptamer can be controlled many times by calcium chelation and addition. Importantly, the aptamer-hFc1 interaction involves mainly van der Waals contacts and hydrogen bonds rather than electrostatic forces, in contrast to other known aptamer-protein complexes. Moreover, the aptamer-hFc1 interaction involves human IgG-specific amino acids, rendering the aptamer specific to human IgGs, and not crossreactive to other species IgGs. Hence, the aptamer is a potent alternative for protein A affinity purification of Fc-fusion proteins and therapeutic antibodies. These results demonstrate, from a structural viewpoint, that conformational plasticity and selectivity of an RNA aptamer is achieved by multiple interactions other than electrostatic forces, which is applicable to many protein targets of low or no affinity to nucleic acids.


Assuntos
Aptâmeros de Nucleotídeos/química , Fragmentos Fc das Imunoglobulinas/química , Imunoglobulina G/química , Cátions Bivalentes/química , Cristalografia , Humanos , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , RNA/química
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