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1.
J Appl Clin Med Phys ; 24(5): e13915, 2023 May.
Artigo em Inglês | MEDLINE | ID: mdl-36934441

RESUMO

PURPOSE: We measure the dose distribution of gated delivery for different target motions and estimate the gating latency in a magnetic resonance-guided radiotherapy (MRgRT) system. METHOD: The dose distribution accuracy of the gated MRgRT system (MRIdian, Viewray) was investigated using an in-house-developed phantom that was compatible with the magnetic field and gating method. This phantom contains a simulated tumor and a radiochromic film (EBT3, Ashland, Inc.). To investigate the effect of the number of beam switching and target velocity on the dose distribution, two types of target motions were applied. One is that the target was periodically moved at a constant velocity of 5 mm/s with different pause times (0, 1, 3, 10, and 20 s) between the motions. During different pause times, different numbers of beams were switched on/off. The other one is that the target was moved at velocities of 3, 5, 8, and 10 mm/s without any pause (i.e., continuous motion). The gated method was applied to these motions at MRIdian, and the dose distributions in each condition were measured using films. To investigate the relation between target motion and dose distribution in the gating method, we compared the results of the gamma analysis of the calculated and measured dose distributions. Moreover, we analytically estimated the gating latencies from the dose distributions measured using films and the gamma analysis results. RESULTS: The gamma pass rate linearly decreased with increasing beam switching and target velocity. The overall gating latencies of beam-hold and beam-on were 0.51 ± 0.17 and 0.35 ± 0.05 s, respectively. CONCLUSIONS: Film measurements highlighted the factors affecting the treatment accuracy of the gated MRgRT system. Our analytical approach, employing gamma analysis on films, can be used to estimate the overall latency of the gated MRgRT system.


Assuntos
Planejamento da Radioterapia Assistida por Computador , Humanos , Movimento (Física) , Espectroscopia de Ressonância Magnética , Dosagem Radioterapêutica , Imagens de Fantasmas
2.
Med Phys ; 48(11): 7541-7551, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34510486

RESUMO

PURPOSE: In this study, we report on our proposed phantom based on the new end-to-end (E2E) methodology and its results. In addition, we verify whether the proposed phantom can replace conventional phantoms. METHODS: The hexagonal-shaped newly designed phantom has pockets on each side for a film dosimeter of size 80 × 90 mm2 , which is easily removable, considering the 60 Co penumbra. The new phantom comprises water, shell, and auxiliary shell phantoms. The shell and auxiliary shell materials are Solid Water HE. A mock tumor (aluminum oxide) was attached by a single prop in the water phantom and placed at the center of the new phantom. The results of a conventional E2E test were compared with those of the novel E2E test using the newly designed phantom. The irradiated film dosimeter in the novel E2E test was scanned in a flatbed scanner and analyzed using an in-house software developed with MATLAB. The irradiated field center, laser center, and mock tumor center were calculated. In the novel image-matching E2E (IM-E2E) test, image matching is performed by aligning the laser center with ruled lines. In the novel irradiation-field E2E (IF-E2E) test, the displacement of the irradiation-field center was defined as its distance from the laser center. In the composite E2E test, the overall displacement, which included the accuracy of the irradiated field and image matching, was defined as the distance between the irradiated field center and mock tumor center. In addition, using the newly designed phantom, the overall irradiation accuracy of the machine was evaluated by calculating the three-dimensional (3D) center of the irradiated field, phantom, and laser. The composite E2E test could be performed using the newly designed phantom only. RESULTS: In the IM-E2E test, the results of the conventional and novel IM-E2E tests were significantly different in each direction (left-right direction: p-value < < 0.05, anterior-posterior direction: p-value = 0.002, and superior-inferior direction: p-value = 0.002). The displacement directions were the same in both the conventional and novel IM-E2E tests. In the analysis of the IF-E2E test, no significant difference was evident between the results in each direction. Moreover, the displacement directions were the same in the conventional and novel IF-E2E tests, except for the left-right lateral direction of head three. In addition, the 3D analysis results of the novel IF-E2E test were less than 1 mm in all directions. In the analysis of the composite E2E test, the maximum displacement was 1.4 mm in all directions. In addition, almost all results of 3D analysis for the composite E2E test were less than 1 mm in all directions. CONCLUSION: The newly designed E2E phantom simplifies the E2E test for MRIdian, and is a possible alternative to the conventional E2E test. Furthermore, we can perform the previously unfeasible composite E2E tests that include the entire treatment process.


Assuntos
Neoplasias , Radioterapia Guiada por Imagem , Humanos , Espectroscopia de Ressonância Magnética , Imagens de Fantasmas , Planejamento da Radioterapia Assistida por Computador , Software
3.
Br J Radiol ; 94(1119): 20200239, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33353402

RESUMO

OBJECTIVE: This study aimed to assess the dosimetric effect of intestinal gas of stereotactic magnetic resonance (MR)-guided adaptive radiation therapy (SMART) on target and critical organs for pancreatic cancer without online electron density correction (EDC). METHODS: Thirty pancreatic cancer patients who underwent online SMART were selected for this study. The treatment time of each stage and the total treatment time were recorded and analyzed. The concerned dose-volume parameters of target and organs-at-risk (OAR) were compared with and without an intestinal gas EDC using the Wilcoxon-signed rank test. Analysis items with p value < 0.05 were considered statistically significant. The relationships between dosimetric differences and intestinal gas volume variations were investigated using the Spearman test. RESULTS: The average treatment time was 82 min, and the average EDC time was 8 min, which accounted for 10% of the overall treatment time. There were no significant differences in CTV (GTV), PTV, bowel, stomach, duodenum, and skin (p > 0.05) with respect to dose volume parameters. For the Dmax of gastrointestinal organs (p = 0.03), the mean dose of the liver (p = 0.002) and kidneys (p = 0.03 and p = 0.04 for the left and right kidneys, respectively), there may be a risk of slight overestimation compared with EDC, and for the Dmax of the spinal cord (p = 0.02), there may be a risk of slight underestimation compared with EDC. A weak correlation for D95 in the PTV and D0.5 cc in the duodenum was observed. CONCLUSION: For patients with similar inter-fractional intestinal gas distribution, EDC had little dosimetric effects on the D0.5 cc of all GI organs and dose volume parameters of target in most plans. ADVANCES IN KNOWLEDGE: By omitting the EDC of intestinal gas, the online SMART treatment time can be shortened.


Assuntos
Imagem por Ressonância Magnética Intervencionista/métodos , Órgãos em Risco/efeitos da radiação , Neoplasias Pancreáticas/radioterapia , Radiocirurgia/métodos , Planejamento da Radioterapia Assistida por Computador/métodos , Radioterapia Guiada por Imagem/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Elétrons , Feminino , Humanos , Intestinos/diagnóstico por imagem , Masculino , Pessoa de Meia-Idade , Dosagem Radioterapêutica
4.
Psychopharmacology (Berl) ; 234(12): 1853-1869, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28332006

RESUMO

Increased risk of attention-deficit/hyperactivity disorder (AD/HD) is partly associated with the early developmental exposure to nicotine in tobacco smoke. Emerging reports link tobacco smoke exposure or prenatal nicotine exposure (PNE) with AD/HD-like behaviors in rodent models. We have previously reported that PNE induces cognitive behavioral deficits in offspring and decreases the contents of dopamine (DA) and its turnover in the prefrontal cortex (PFC) of offspring It is well known that the dysfunction of DAergic system in the brain is one of the core factors in the pathophysiology of AD/HD. Therefore, we examined whether the effects of PNE on the DAergic system underlie the AD/HD-related behavioral changes in mouse offspring. PNE reduced the release of DA in the medial PFC (mPFC) in mouse offspring. PNE reduced the number of tyrosine hydroxylase (TH)-positive varicosities in the mPFC and in the core as well as the shell of nucleus accumbens, but not in the striatum. PNE also induced behavioral deficits in cliff avoidance, object-based attention, and sensorimotor gating in offspring. These behavioral deficits were attenuated by acute treatment with atomoxetine (3 mg/kg, s.c.) or partially attenuated by acute treatment with MPH (1 mg/kg, s.c.). Taken together, our findings support the notion that PNE induces neurobehavioral abnormalities in mouse offspring by disrupting the DAergic system and improve our understanding about the incidence of AD/HD in children whose mothers were exposed to nicotine during their pregnancy.


Assuntos
Cloridrato de Atomoxetina/toxicidade , Transtorno do Deficit de Atenção com Hiperatividade/induzido quimicamente , Dopamina/metabolismo , Nicotina/toxicidade , Córtex Pré-Frontal/metabolismo , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Inibidores da Captação Adrenérgica/toxicidade , Animais , Transtorno do Deficit de Atenção com Hiperatividade/metabolismo , Transtorno do Deficit de Atenção com Hiperatividade/psicologia , Transtornos Cognitivos/induzido quimicamente , Transtornos Cognitivos/metabolismo , Transtornos Cognitivos/psicologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Córtex Pré-Frontal/efeitos dos fármacos , Gravidez , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Efeitos Tardios da Exposição Pré-Natal/psicologia
5.
J Mol Recognit ; 24(2): 275-82, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21360613

RESUMO

Gibberellins (GAs) are phytohormones regulating various developmental processes in plants. In rice, the initial GA-signaling events involve the binding of a GA to the soluble GA receptor protein, GID1. Although X-ray structures for certain GID1/GA complexes have recently been determined, an examination of the complexes does not fully clarify how GID1s discriminate among different GAs. Herein, we present a study aimed at defining the types of forces important to binding via a combination of isothermal titration calorimetry (ITC) and computational docking studies that employed rice GID1 (OsGID1), OsGID1 mutants, which were designed to have a decreased possible number of hydrogen bonds with bound GA, and GA variants. We find that, in general, GA binding is enthalpically driven and that a hydrogen bond between the phenolic hydroxyl of OsGID1 Tyr134 and the C-3 hydroxyl of a GA is a defining structural element. A hydrogen-bond network that involves the C-6 carboxyl of a GA that directly hydrogen bonds the hydroxyl of Ser198 and indirectly, via a two-water-molecule network, the phenolic hydroxyl of Tyr329 and the NH of the amide side-chain of Asn255 is also important for GA binding. The binding of OsGID1 by GA(1) is the most enthalpically driven association found for the biologically active GAs evaluated in this study. This observation might be a consequence of a hydrogen bond formed between the hydroxyl at the C-13 position of GA(1) and the main chain carbonyl of OsGID1 Phe245. Our results demonstrate that by combining ITC experiments and computational methods much can be learned about the thermodynamics of ligand/protein binding.


Assuntos
Calorimetria/métodos , Giberelinas/metabolismo , Simulação de Dinâmica Molecular , Proteínas de Plantas/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Giberelinas/química , Ligação de Hidrogênio , Cinética , Proteínas de Plantas/química , Ligação Proteica , Termodinâmica
6.
Proc Natl Acad Sci U S A ; 107(26): 11900-5, 2010 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-20547865

RESUMO

In a previous study, we demonstrated that beta1,3-N-acetylglucosaminyltransferase 5 (B3gnt5) is a lactotriaosylceramide (Lc(3)Cer) synthase that synthesizes a precursor structure for lacto/neolacto-series glycosphingolipids (GSLs) in in vitro experiments. Here, we generated B3gnt5-deficient (B3gnt5(-/-)) mice to investigate the in vivo biological functions of lacto/neolacto-series GSLs. In biochemical analyses, lacto/neolacto-series GSLs were confirmed to be absent and no Lc(3)Cer synthase activity was detected in the tissues of these mice. These results demonstrate that beta3GnT5 is the sole enzyme synthesizing Lc(3)Cer in vivo. Ganglioside GM1, known as a glycosphingolipid-enriched microdomain (GEM) marker, was found to be up-regulated in B3gnt5(-/-) B cells by flow cytometry and fluorescence microscopy. However, no difference in the amount of GM1 was observed by TLC-immunoblotting analysis. The GEM-stained puncta on the surface of B3gnt5(-/-) resting B cells were brighter and larger than those of WT cells. These results suggest that structural alteration of GEM occurs in B3gnt5(-/-) B cells. We next examined whether BCR signaling-related proteins, such as BCR, CD19, and the signaling molecule Lyn, had moved into or out of the GEM fraction. In B3gnt5(-/-) B cells, these molecules were enriched in the GEM fraction or adjacent fraction. Moreover, B3gnt5(-/-) B cells were more sensitive to the induction of intracellular phosphorylation signals on BCR stimulation and proliferated more vigorously than WT B cells. Together, these results suggest that lacto/neolacto-series GSLs play an important role in clustering of GEMs and tether-specific proteins, such as BCR, CD19, and related signaling molecules to the GEMs.


Assuntos
Linfócitos B/imunologia , Linfócitos B/metabolismo , Glicoesfingolipídeos/imunologia , Glicoesfingolipídeos/metabolismo , Microdomínios da Membrana/imunologia , Microdomínios da Membrana/metabolismo , Animais , Sequência de Bases , Primers do DNA/genética , Feminino , Gangliosídeo G(M1)/imunologia , Gangliosídeo G(M1)/metabolismo , Técnicas In Vitro , Lactosilceramidas/metabolismo , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , N-Acetilglucosaminiltransferases/deficiência , N-Acetilglucosaminiltransferases/genética
7.
Chem Pharm Bull (Tokyo) ; 56(8): 1138-42, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18670114

RESUMO

From the whole plants and the roots of Gueldenstaedtia multiflora, which has been used in traditional Chinese medicine, five new oleanane glycosides and one lupane glycoside were isolated together with eight known oleanane glycosides and a medicarpin derivative. These structures were determined based on MS and 2D-NMR spectra.


Assuntos
Ácido Oleanólico/análogos & derivados , Raízes de Plantas/química , Plantas Medicinais/química , Triterpenos/isolamento & purificação , Espectrometria de Massas , Medicina Tradicional Chinesa , Ácido Oleanólico/química , Ácido Oleanólico/isolamento & purificação , Pterocarpanos/isolamento & purificação , Pterocarpanos/farmacologia , Triterpenos/química
8.
Proc Natl Acad Sci U S A ; 104(40): 15829-34, 2007 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-17890318

RESUMO

beta1,3-N-acetylglucosaminyltransferase 2 (beta3GnT2) is a polylactosamine synthase that synthesizes a backbone structure of carbohydrate structures onto glycoproteins. Here we generated beta3GnT2-deficient (beta3GnT2(-/-)) mice and showed that polylactosamine on N-glycans was markedly reduced in their immunological tissues. In WT mice, polylactosamine was present on CD28 and CD19, both known immune costimulatory molecules. However, polylactosamine levels on these molecules were reduced in beta3GnT2(-/-) mice. beta3GnT2(-/-) T cells lacking polylactosamine were more sensitive to the induction of intracellular calcium flux on stimulation with anti-CD3epsilon/CD28 and proliferated more strongly than T cells from WT mice. beta3GnT2(-/-) B cells also showed hyperproliferation on BCR stimulation. Macrophages from beta3GnT2(-/-) mice had higher cell surface CD14 levels and enhanced responses to endotoxin. These results indicate that polylactosamine on N-glycans is a putative immune regulatory factor presumably suppressing excessive responses during immune reactions.


Assuntos
Ativação Linfocitária/genética , Ativação de Macrófagos/genética , N-Acetilglucosaminiltransferases/deficiência , Linfócitos T/imunologia , Amino Açúcares/deficiência , Animais , Antígenos CD19/imunologia , Antígenos CD28/imunologia , Solanum lycopersicum , Camundongos , Camundongos Knockout , Lectinas de Plantas/imunologia , Polissacarídeos/deficiência , Receptores de Antígenos de Linfócitos T/imunologia
9.
J Biol Chem ; 280(49): 41015-24, 2005 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-16186120

RESUMO

EL5 is a rice ubiquitin-protein isopeptide ligase (E3) containing a RING-H2 finger domain that interacts with Oryza sativa (Os) UBC5b, a rice ubiquitin carrier protein. We introduced point mutations into the EL5 RING-H2 finger so that residues that functionally interact with OsUBC5b could be identified when assayed for ubiquitination activity in vitro. The residue positions were selected based on the results of an EL5 RING-H2 finger/OsUBC5b NMR titration experiment. These RING-H2 finger residues form or are adjacent to a shallow groove that is recognized by OsUBC5b. The E3 activity of EL5 is shown to be dependent on a Trp located at the center of the groove. We classified rice RING fingers according to the type of metal-chelating motif, i.e. RING-H2 or RING-HC, and according to the presence or absence of a conserved EL5-like Trp. We discuss the probable relationship between E3 activity and the conserved Trp.


Assuntos
Oryza/enzimologia , Triptofano , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Dedos de Zinco , Sequência de Aminoácidos , Sítios de Ligação , Clonagem Molecular , Sequência Conservada , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oryza/química , Mutação Puntual , Proteínas Recombinantes de Fusão , Alinhamento de Sequência , Relação Estrutura-Atividade , Especificidade por Substrato , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética
10.
Protein Expr Purif ; 42(2): 268-77, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15914031

RESUMO

For proteins of higher eukaryotes, such as plants, which have large genomes, recombinant protein expression and purification are often difficult. Expression levels tend to be low and the expressed proteins tend to misfold and aggregate. We tested seven different expression vectors in Escherichia coli for rapid subcloning of rice genes and for protein expression and solubility levels. Each expressed gene product has an N-terminal fusion protein and/or tag, and an engineered protease site upstream of the mature rice protein. Several different fusion proteins/tags and protease sites were tested. We found that the fusion proteins and the protease sites have significant and varying effects on expression and solubility levels. The expression vector with the most favorable characteristics is pDEST-trx. The vector, which is a modified version of the commercially available expression vector, pET-32a, contains an N-terminal thioredoxin fusion protein and a hexahistidine tag, and is adapted to the Gateway expression system. However, addition of an engineered protease site could drastically change the expression and solubility properties. We selected 135 genes corresponding to potentially interesting rice proteins, transferred the genes from cDNAs to expression vectors, and engineered in suitable protease sites N-terminal to the mature proteins. Of 135 genes, 131 (97.0%) could be expressed and 72 (53.3%) were soluble when the fusion proteins/tags were present. Thirty-eight mature-length rice proteins and domains (28.1%) are suitable for NMR solution structure studies and/or X-ray crystallography. Our expression systems are useful for the production of soluble plant proteins in E. coli to be used for structural genomics studies.


Assuntos
Clonagem Molecular , Oryza/genética , Proteínas de Plantas/biossíntese , Proteínas de Plantas/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Escherichia coli/genética , Vetores Genéticos , Oryza/química , Proteínas de Plantas/química , Proteínas Recombinantes de Fusão/química , Solubilidade
11.
Science ; 301(5631): 376-9, 2003 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-12869764

RESUMO

We collected and completely sequenced 28,469 full-length complementary DNA clones from Oryza sativa L. ssp. japonica cv. Nipponbare. Through homology searches of publicly available sequence data, we assigned tentative protein functions to 21,596 clones (75.86%). Mapping of the cDNA clones to genomic DNA revealed that there are 19,000 to 20,500 transcription units in the rice genome. Protein informatics analysis against the InterPro database revealed the existence of proteins presented in rice but not in Arabidopsis. Sixty-four percent of our cDNAs are homologous to Arabidopsis proteins.


Assuntos
Genoma de Planta , Oryza/genética , Análise de Sequência de DNA , Processamento Alternativo , Sequência de Aminoácidos , Clonagem Molecular , Biologia Computacional , DNA Complementar , Bases de Dados de Ácidos Nucleicos , Bases de Dados de Proteínas , Genes de Plantas , Dados de Sequência Molecular , Fases de Leitura Aberta , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/fisiologia , Estrutura Terciária de Proteína , RNA Antissenso/genética , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Fatores de Transcrição/química , Fatores de Transcrição/genética , Transcrição Gênica
12.
J Biol Chem ; 278(17): 15341-8, 2003 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-12588869

RESUMO

EL5, a RING-H2 finger protein, is rapidly induced by N-acetylchitooligosaccharides in rice cell. We expressed the EL5 RING-H2 finger domain in Escherichia coli and determined its structure in solution by NMR spectroscopy. The EL5 RING-H2 finger domain consists of two-stranded beta-sheets (beta1, Ala(147)-Phe(149); beta2, Gly(156)-His(158)), one alpha-helix (Cys(161)-Leu(166)), and two large N- and C-terminal loops. It is stabilized by two tetrahedrally coordinated zinc ions. This structure is similar to that of other RING finger domains of proteins of known function. From structural analogies, we inferred that the EL5 RING-H2 finger is a binding domain for ubiquitin-conjugating enzyme (E2). The binding site is probably formed by solvent-exposed hydrophobic residues of the N- and C-terminal loops and the alpha-helix. We demonstrated that the fusion protein with EL5-(96-181) and maltose-binding protein (MBP) was polyubiquitinated by incubation with ubiquitin, ubiquitin-activating enzyme (E1), and a rice E2 protein, OsUBC5b. This supported the idea that the EL5 RING finger domain is essential for ubiquitin-ligase activity of EL5. By NMR titration experiments, we identified residues that are critical for the interaction between the EL5 RING-H2 finger and OsUBC5b. We conclude that the RING-H2 finger domain of EL5 is the E2 binding site of EL5.


Assuntos
Ligases/química , Oryza/química , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Ligases/metabolismo , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Proteínas de Plantas/química , Polissacarídeos Bacterianos/farmacologia , Conformação Proteica , Ubiquitina/metabolismo , Enzimas Ativadoras de Ubiquitina , Enzimas de Conjugação de Ubiquitina , Ubiquitina-Proteína Ligases , Dedos de Zinco
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