RESUMO
We successfully constructed a coupled in vitro transcription/translation (cIVTT) system based on wheat germ extract (WGE) for efficient expression from PCR-generated DNA templates in short-time (â¼3-h) batch reactions. The productivity of this system under optimized conditions was 85 µg (2.8 nmol) per 1 mL of reaction solution (corresponding to 425 µg per 1 mL of WGE), which was about 9-fold higher than that by the conventional batch method using mRNA as a template. The DNA template concentration required for efficient cIVTT was as low as 2.5 nM, which is much lower than those required for other eukaryotic cIVTT systems to maximize their productivity (30-50 nM). The productivity of the present system with a 2.5 nM template was 80-fold and 4-fold higher than that of a commercially available WGE-based cIVTT system with a 2.5 nM and a 40 nM template, respectively. In addition, the present system functioned well in a liposome (i.e., in an artificial cell) without a loss of productivity. Given that WGE-based systems have the advantage of being suitable for the expression of a broad range of proteins, the present cIVTT system is expected to be widely used in future cell-free synthetic biology.
Assuntos
Regulação da Expressão Gênica de Plantas/genética , Extratos Vegetais/genética , Reação em Cadeia da Polimerase , Transcrição Gênica/genética , Triticum/química , Estrutura Molecular , Extratos Vegetais/química , Fatores de TempoRESUMO
We here designed an in vitro selection scheme for obtaining an aptamer with which to rationally construct an artificial riboswitch as its component part. In fact, a nanosized DNA-binding aptamer obtained through this scheme allowed us to easily and successfully create eukaryotic riboswitches that upregulate internal ribosome entry site-mediated translation in response to the ligand (nanosized DNA) in wheat germ extract, a eukaryotic cell-free expression system. The induction ratio of the best riboswitch ligand-dose-dependently increased to 21 at 300 µM ligand. This switching efficiency is much higher than that of the same type of riboswitch with a widely used theophylline-binding aptamer, which was in vitro selected without considering its utility for constructing riboswitches. The selection scheme described here would facilitate obtaining various ligand/aptamer pairs suitable for constructing artificial riboswitches, which could serve as elements of synthetic gene circuits in synthetic biology.
Assuntos
Aptâmeros de Nucleotídeos/metabolismo , DNA/química , DNA/metabolismo , Extratos Vegetais/genética , Extratos Vegetais/metabolismo , Biossíntese de Proteínas/genética , Riboswitch/genética , Sistema Livre de Células/metabolismo , Células Eucarióticas/metabolismo , Expressão Gênica , Redes Reguladoras de Genes , Ligantes , Conformação de Ácido Nucleico , Ribossomos/metabolismo , Biologia Sintética/métodos , Teofilina/metabolismo , Triticum/químicaRESUMO
Wheat germ extract (WGE) is one of the most widely used eukaryotic cell-free translation systems for easy synthesis of a broad range of proteins merely by adding template mRNAs. Its productivity has thus far been improved by removing translational inhibitors from the extract and stabilizing the template with terminal protectors. Nonetheless, there remains room for increasing the yield by designing a terminally protected template with higher susceptibility to translation. Given the fact that a 5' terminal protector is a strong inhibitor of the canonical translation, we herein focused on Cripavirus internal ribosome entry sites (IRESes), which allow for a unique translation initiation from a non-AUG start codon without the help of any initiation factors. We mutated their start codons to enhance the IRES-mediated translation efficiency in WGE. One of the mutants showed considerably higher efficiency, 3-4-fold higher than that of its wild type, and also 3-4-fold higher than the canonical translation efficiency by an IRES-free mRNA having one of the most effective canonical-translation enhancers. Because this mutated IRES is compatible with different types of genes and terminal protectors, we expect it will be widely used to synthesize proteins in WGE.
Assuntos
Códon de Iniciação/genética , Dicistroviridae/genética , Sítios Internos de Entrada Ribossomal/genética , Extratos Vegetais/genética , Biossíntese de Proteínas/genética , Triticum/genética , Relação Dose-Resposta a Droga , Estrutura Molecular , Mutação , Relação Estrutura-AtividadeRESUMO
Wheat cell-free expression systems based on wheat germ extract (WGE) enable us to briefly synthesize various types of proteins in vitro merely by exogenously adding their mRNA templates. Moreover, it is possible to produce larger amounts of protein by thoroughly removing the endosperm, which contains many translation inhibitors, including ribonucleases (RNases). However, because small amounts of RNases are also present even in an endosperm-free, high-quality WGE (hqWGE), the in-vitro transcribed mRNA is rapidly degraded. In particular, 3' exonucleases have been considered as the major RNases that degrade mRNA. We thus herein performed in vitro selection to find an effective, short 3' protector sequence from a random RNA pool. The selected sequences stabilized in vitro transcripts in the hqWGE more effectively than the previously reported, longer 3' protectors did. In addition, when one of these 3' protectors was minimized and then fused to mRNA, the translation efficiency increased 5-6-fold in the hqWGE, mainly due to the mRNA stabilization.
Assuntos
Extratos Vegetais/química , Biossíntese de Proteínas/genética , Transcrição Gênica/genética , Triticum/químicaRESUMO
We have developed a novel type of biofunction-assisted, signal-turn-on sensor for simply and homogenously detecting DNA. This sensor system is composed of two types of in vitro-transcribed label-free RNAs (a 3' premature amber suppressor tRNA probe and an amber-mutated mRNA encoding a reporter protein), RNase H, and a wheat germ extract (WGE). A target DNA induces the 3' end maturation of the tRNA probe, which is enhanced by RNase H and leads to the expression of a full-length reporter protein through amber suppression in WGE, while there is almost no expression without the target due to the inactivity of the premature probe. Therefore, the target can be readily detected with the activity of the translated reporter. The catalytic reuse of the target with the help of RNase H in addition to various bioprocesses in WGE enables this sensor system to exhibit relatively high selectivity and sensitivity.
Assuntos
DNA/análise , Sondas Moleculares/metabolismo , Extratos Vegetais/química , RNA de Transferência/metabolismo , Ribonuclease H/metabolismo , Triticum/química , Biocatálise , DNA/metabolismo , Humanos , Extratos Vegetais/metabolismo , Triticum/metabolismoRESUMO
We evaluated the efficacy of treatment using reduced cumulative doses of anthracyclines in children with acute promyelocytic leukaemia (APL) in the Japanese Paediatric Leukaemia/Lymphoma Study Group AML-P05 study. All patients received two and three subsequent courses of induction and consolidation chemotherapy respectively, consisting of all-trans retinoic acid (ATRA), cytarabine and anthracyclines, followed by maintenance therapy with ATRA. Notably, a single administration of anthracyclines was introduced in the second induction and all consolidation therapies to minimize total doses of anthracycline. The 3-year event-free (EFS) and overall survival rates for 43 eligible children were 83·6% [95% confidence interval (CI): 68·6-91·8%] and 90·7% (95% CI: 77·1-96·4%), respectively. Although two patients died of intracranial haemorrhage or infection during induction phases, no cardiac adverse events or treatment-related deaths were observed during subsequent phases. Patients not displaying M1 marrow after the first induction therapy, or those under 5 years of age at diagnosis, showed inferior outcomes (3-year EFS rate; 33·3% (95% CI: 19·3-67·6%) and 54·6% (95% CI: 22·9-78·0%), respectively). In conclusion, a single administration of anthracycline during each consolidation phase was sufficient for treating childhood APL. In younger children, however, conventional ATRA and chemotherapy may be insufficient so that alternative therapies should be considered.
Assuntos
Antraciclinas/administração & dosagem , Leucemia Promielocítica Aguda/tratamento farmacológico , Leucemia Promielocítica Aguda/mortalidade , Adolescente , Criança , Pré-Escolar , Quimioterapia de Consolidação/métodos , Citarabina/administração & dosagem , Intervalo Livre de Doença , Humanos , Quimioterapia de Indução/métodos , Lactente , Japão , Leucemia Promielocítica Aguda/complicações , Quimioterapia de Manutenção/métodos , Masculino , Estudos Prospectivos , Taxa de Sobrevida , Resultado do Tratamento , Tretinoína/administração & dosagemRESUMO
Amber suppression is a useful method of genetically incorporating a non-natural amino acid (NAA) into a protein during translation by utilizing an NAA-charged amber suppressor tRNA (sup-tRNA). A wheat germ extract (WGE) is suitable for this method by virtue of its high productivity and versatility in addition to its advantages as a cell-free translation system. However, in spite of this high potential, a genetic NAA incorporation system in WGE has not been sufficiently optimized in terms of sup-tRNAs, in contrast to that in E. coli and its cell extracts. We herein rationally optimized amber sup-tRNAs to efficiently incorporate a model NAA, p-acetyl-phenylalanine (AcPhe), into a protein in WGE, via flexizyme-based aminoacylation. The optimized sup-tRNA (named tLys-opt) that was pre-charged with AcPhe exclusively yielded up to 220 µg mL(-1) of AcPhe-incorporated protein (yellow fluorescent protein, YPet) under the optimal conditions. This high productivity is comparable to the best reported yield of a similar NAA-incorporated protein synthesized with an engineered aminoacyl-tRNA synthetase/sup-tRNA pair in WGE, despite the fact that tLys-opt that has released AcPhe was not reused at all in this study. The results clearly show both the necessity of optimizing sup-tRNAs for efficient NAA incorporation and the validity of our strategy for their optimization. Because the optimization strategy described here is expected to be applicable not only to amber sup-tRNAs for other NAAs but also to ones used in other acylation methods, it would facilitate the synthesis of large amounts of various types of NAA-incorporated proteins in WGE.
Assuntos
Fenilalanina/análogos & derivados , Extratos Vegetais/química , Biossíntese de Proteínas , Proteínas/química , RNA de Transferência/genética , Supressão Genética , Triticum/química , Estrutura Molecular , Fenilalanina/genética , Fenilalanina/metabolismo , Proteínas/metabolismoRESUMO
We have developed a novel type of biofunction-assisted aptasensor that harnesses ligand-dependent 3' processing of a premature amber suppressor tRNA and the subsequent amber suppression of a reporter gene in a wheat germ extract.
Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Extratos Vegetais/genética , RNA de Transferência/química , Teofilina/análise , Triticum/genética , Vasodilatadores/análise , Aptâmeros de Nucleotídeos/genética , Sequência de Bases , Genes Reporter , Extratos Vegetais/química , RNA de Transferência/genética , Supressão Genética , Triticum/químicaRESUMO
We investigated the end processing and degradation of premature tRNAs in wheat germ extract (WGE), which led to the discovery of end protectors useful for stabilizing an in vitro transcript against various ribonucleases and thereby enhancing its apparent activity in WGE.
Assuntos
Extratos Vegetais/farmacologia , Estabilidade de RNA/efeitos dos fármacos , RNA de Transferência/química , Triticum/química , Modelos Moleculares , Conformação de Ácido Nucleico , RNA Mensageiro/químicaRESUMO
High-quality wheat germ extract (hqWGE) is very useful for the high-yield production of various types of protein. The most important key to high productivity is the design of mRNA templates. Although the design has been refined for straightforward and efficient translation in hqWGE, there is still room for improvement in untranslated regions (UTRs), especially the 3' UTR length, because a long, cumbersome 3' UTR is commonly used for translation enhancement. Here we examined some short viral 3' cap-independent translation enhancers (3' CITEs) to identify effective ones for efficient translation in hqWGE. We then combined the most effective 3' CITE and a 5' enhancer to further increase the translation efficiency. mRNA with the optimal short 3' and 5' UTRs, both of whose length was less than 150 nt, exhibited a productivity of 1.4 mg/mL in prolonged large-scale protein synthesis in hqWGE, which was comparable to that of control mRNA with a commonly-used long 3' UTR (â¼1200 nt).
Assuntos
Extratos Vegetais/metabolismo , Proteínas/metabolismo , RNA Mensageiro/metabolismo , Triticum/metabolismo , Extratos Vegetais/química , Biossíntese de Proteínas , RNA Mensageiro/química , Triticum/químicaRESUMO
BACKGROUND: Traditional Japanese medicine, known as Kampo medicine, consists of mixtures of several medicinal herbs widely used to treat upper gastrointestinal disorders in Japan. Rikkunshito, one of these medicines, has not been evaluated with respect to its influence on gastrointestinal motor activity. We investigated the effect of rikkunshito on upper gastrointestinal motility and plasma ghrelin concentrations in conscious dogs. METHODS: Contractile response to intragastric administration of rikkunshito was studied via surgically implanted force transducers. A powdered extract of rikkunshito (1.3, 2.7, and 4.0 g) dissolved in water was administered into the stomachs of normal and vagotomized dogs before feeding and gastric emptying was evaluated. Several inhibitors of gastrointestinal motility (atropine, hexamethonium, and ondansetron) were injected intravenously before intragastric administration of rikkunshito. Plasma acylated ghrelin levels after intragastric administration of rikkunshito were measured. RESULTS: In a fasting state, intragastric administration of rikkunshito induced phasic contractions in the duodenum and jejunum in normal dogs. Rikkunshito-induced contractions were inhibited by atropine, hexamethonium and ondansetron. In vagotomized dogs, rikkunshito induced phasic contractions, similar to normal dogs. Gastric emptying was accelerated by intragastric administration of rikkunshito in a dose-dependent manner. The plasma acylated ghrelin level 150 min after intragastric administration of 4.0 g of rikkunshito was significantly higher than the control value. CONCLUSIONS: Intragastric administration of rikkunshito stimulates gastrointestinal contractions in the interdigestive state through cholinergic neurons and 5-HT type 3 receptors. Moreover, rikkunshito increases plasma acylated ghrelin levels. Rikkunshito may alleviate gastrointestinal disorders through its prokinetic effects.
Assuntos
Medicamentos de Ervas Chinesas/farmacologia , Motilidade Gastrointestinal/efeitos dos fármacos , Grelina/sangue , Animais , Estado de Consciência , Cães , Medicamentos de Ervas Chinesas/administração & dosagem , Feminino , Esvaziamento Gástrico/efeitos dos fármacos , Masculino , Medicina Kampo , EstômagoRESUMO
In vitro-transcribed, unmodified, and non-aminoacylated amber suppressor tRNAs that are recognized by natural aminoacyl-tRNA synthetase were improved toward higher suppression efficiency in batch-mode cell-free translation in wheat germ extract. The suppression efficiency of the suppressor obtained through four sequence optimization steps (anticodon alteration of natural tRNAs (the first generation); chimerization of the efficient suppressors in the first generation; investigation and optimization of the effective parts in the second generation; combination of the optimized parts in the third generation) and by the terminal tuning was approximately 60%, which was 2.4-fold higher than that of the best suppressor in the first generation. In addition, an eRF1 aptamer further increased the efficiency up to 85%. This highly efficient suppression system also functioned well in a dialysis-based large-scale protein synthesis.
Assuntos
Genes Supressores , Extratos Vegetais/antagonistas & inibidores , RNA de Transferência/farmacologia , Supressão Genética/efeitos dos fármacos , Triticum/química , Sistema Livre de Células , Extratos Vegetais/química , Extratos Vegetais/genética , RNA de Transferência/química , RNA de Transferência/genéticaAssuntos
DNA Antissenso/química , Sondas RNA/química , Ribonuclease H/metabolismo , Sequência de Bases , Catálise , Sistema Livre de Células , DNA Antissenso/metabolismo , Corantes Fluorescentes/química , Técnicas de Amplificação de Ácido Nucleico , Extratos Vegetais/química , Extratos Vegetais/metabolismo , Biossíntese de Proteínas , Sondas RNA/metabolismo , RNA Mensageiro/metabolismo , Ribonuclease H/química , Triticum/metabolismoRESUMO
Riboswitches are RNA elements in mRNA that control gene expression in cis in response to their specific ligands. Because artificial riboswitches make it possible to regulate any gene with an arbitrary molecule, they are expected to function as biosensors, in which the output is easily detectable protein expression. I report herein a fully rational design strategy for artificially constructing novel riboswitches that work in a eukaryotic cell-free translation system (wheat germ extract). In these riboswitches, translation mediated by an internal ribosome entry site (IRES) is promoted only in the presence of a specific ligand (ON), while it is inhibited in the absence of the ligand (OFF). The first rationally designed riboswitch, which is regulated by theophylline, showed a high switching efficiency and dependency on theophylline. In addition, based on the design of the theophylline-dependent riboswitch, other three kinds of riboswitches controlled by FMN, tetracycline, and sulforhodamine B, were constructed only by calculating the ΔG value of one stem-loop structure. The rational design strategy described herein is therefore useful for easily producing various ligand-dependent riboswitches, which are available as biosensors for detecting their ligands.
Assuntos
Técnicas Biossensoriais , Desenho de Fármacos , Iniciação Traducional da Cadeia Peptídica , Extratos Vegetais/farmacologia , Ribossomos/metabolismo , Riboswitch/fisiologia , Triticum/química , Pareamento de Bases , Sequência de Bases , Sistema Livre de Células , Luciferases/metabolismo , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Extratos Vegetais/química , Ribossomos/genéticaRESUMO
BACKGROUND: The aim of this study was to investigate the effects of Rikkunshito on ghrelin secretion and on cisplatin-induced anorexia in humans. METHODS: The study was performed as a crossover design, and ten unresectable or relapsed gastric cancer patients were randomly divided into two groups. Group A (n = 5) was started on Rikkunshito (2.5 g three times daily, orally) from the first course of chemotherapy and followed by a second course without Rikkunshito. A treatment with reversed order was performed for Group B (n = 5). All patients received combined chemotherapy with S-1 plus cisplatin. The primary endpoint was the amount of oral intake, and the categories of scales of anorexia, nausea, and vomiting; secondary endpoints included the plasma concentration of acylated ghrelin. RESULTS: In the Rikkunshito-on period, no decrease of the plasma concentration of acylated ghrelin induced by cisplatin was observed. The average oral intake in the Rikkunshito-on period was significantly larger than that in the Rikkunshito-off period, and the grade of anorexia was significantly lower in the Rikkunshito-on period than in the Rikkunshito-off period. CONCLUSION: Rikkunshito appeared to prevent anorexia induced by cisplatin, resulting in effective prophylactic administration of chemotherapy with cisplatin, and patients could continue their treatments on schedule.
Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Técnicas Biossensoriais/métodos , Luciferases/genética , Extratos Vegetais/metabolismo , Biossíntese de Proteínas , RNA Catalítico/metabolismo , Triticum/química , Sistema Livre de Células/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Teofilina/metabolismoRESUMO
BACKGROUND: Besides profound hypoglycemia with hyperlacticemia, glycogen storage disease type Ia (GSD Ia) presents hypertriglyceridemia that is often resistant to dietary treatment with cornstarch. The present study aimed to evaluate the effects of medium-chain triglycerides (MCT)--which are absorbed via the portal vein without being incorporated into chylomicrons--on hypertriglyceridemia and to explore otherwise metabolic changes in children with GSD Ia. PATIENTS AND METHODS: A 13-year-old boy with GSD Ia who received a dietary treatment with MCT milk after cornstarch administration and two infants also with GSD Ia, ages 6 and 7 months, who received MCT milk after carbohydrate-rich, lipid-poor milk were enrolled. In addition to serum glucose and lactate levels, serum levels of total cholesterol, triglycerides, and high-density lipoprotein (HDL) cholesterol were serially determined. Simultaneously, serum levels of total carnitine, free carnitine, acylcarnitine, and ketone bodies were determined to evaluate fatty acid beta-oxidation. RESULTS: Mean glucose level (mmol/l) of patient 1 remained stable, the value being around 4.5, while those of patients 2 and 3 increased to this level from 4.00 and 3.72, respectively. Lactate levels were significantly decreased in all patients. Mean triglyceride levels (mM) of patient 1 decreased from 3.00 to 2.05. Also, triglyceride levels of patients 2 and 3 decreased from 2.74 and 3.15 to 2.13 and 2.70, respectively. HDL cholesterol, acylcarnitine, and ketone body levels increased in all patients after MCT administration, while total and free carnitine levels decreased. CONCLUSION: We describe here the beneficial effects on lipid and carbohydrate metabolisms in three Japanese children with GSD Ia. In light of the unfavorable influence of lipid restriction on growth and development in infancy, dietary treatment with MCT milk may be a better treatment for infants with GSD Ia. Further investigation should be required to confirm the efficacy of MCT milk in GSD Ia.
Assuntos
Doença de Depósito de Glicogênio Tipo I/dietoterapia , Hipertrigliceridemia/prevenção & controle , Lactatos/sangue , Ácido Láctico/sangue , Leite , Triglicerídeos/administração & dosagem , Adolescente , Animais , Biomarcadores/sangue , Pré-Escolar , Colesterol/sangue , Carboidratos da Dieta/metabolismo , Suplementos Nutricionais , Doença de Depósito de Glicogênio Tipo I/sangue , Doença de Depósito de Glicogênio Tipo I/complicações , Crescimento , Humanos , Japão , Lipoproteínas/metabolismo , Masculino , Triglicerídeos/sangueRESUMO
A pool of 84-nt RNAs containing a randomized sequence of 50 nt was selected against gel-immobilized Escherichia coli release factor 1 (RF-1) responsible for translation termination at amber (UAG) stop codon. The strongest aptamer (class II-1) obtained from 43 clones bound to RF-1, but not to UAA/UGA-targeting RF-2, with Kd = 30+/-6 nM (SPR). A couple of unpaired hairpin domains in the aptamer were suggested as the sites of attachment of RF-1. By binding to and hence inhibiting the action of RF-1 specifically or bio-orthogonally, aptamer class II-1 enhanced the amber suppression efficiency in the presence of an anticodon-adjusted (CUA) suppressor tRNA without practically damaging the protein translation machinery of the cell-free extract of E. coli, as confirmed by the translation of amber-mutated (gfp(amber141) or gfp(amber178)) and wild-type (gfp(wild)) genes of GFP.
Assuntos
Aptâmeros de Nucleotídeos/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Proteínas de Escherichia coli/antagonistas & inibidores , Fatores de Terminação de Peptídeos/antagonistas & inibidores , Sítios de Ligação , Códon de Terminação , Genes Supressores , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
BACKGROUND: The aim of this study was to investigate the effects of arginine on nutrition, growth and urea cycle function in boys with late-onset ornithine transcarbamylase deficiency (OTCD). Seven Japanese boys with late-onset OTCD enrolled in this study resumed arginine treatment after the cessation of this therapy for a few years. Clinical presentations such as vomiting and unconsciousness, plasma amino acids and urinary orotate excretion were followed chronologically to evaluate urea cycle function and protein synthesis with and without this therapy. In addition to height and body weight, blood levels of proteins, lipids, growth hormone (GH), insulin-like growth factor-I (IGF-I) and IGF-binding protein -3 (IGFBP-3) were monitored. RESULTS: The frequency of hyperammonemic attacks and urinary orotate excretion decreased significantly following the resumption of arginine treatment. Despite showing no marked change in body weight, height increased gradually. Extremely low plasma arginine increased to normal levels, while plasma glutamine and alanine levels decreased considerably. Except for a slight increase in high-density lipoprotein cholesterol level, blood levels of markers for nutrition did not change. In contrast, low serum IGF-I and IGFBP-3 levels increased to age-matched control levels, and normal urinary GH secretion became greater than the level observed in the controls. CONCLUSION: Arginine treatment is able to reduces attacks of hyperammonemia in boys with late-onset OTCD and to increase their growth.
Assuntos
Arginina/uso terapêutico , Crescimento/efeitos dos fármacos , Fenômenos Fisiológicos da Nutrição/efeitos dos fármacos , Doença da Deficiência de Ornitina Carbomoiltransferase/tratamento farmacológico , Doença da Deficiência de Ornitina Carbomoiltransferase/fisiopatologia , Ureia/metabolismo , Idade de Início , Aminoácidos/sangue , Aminoácidos/efeitos dos fármacos , Amônia/sangue , Análise de Variância , Arginina/sangue , Biomarcadores/sangue , Biomarcadores/urina , Proteínas Sanguíneas/efeitos dos fármacos , Proteínas Sanguíneas/metabolismo , Estatura/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Pré-Escolar , HDL-Colesterol/sangue , LDL-Colesterol/sangue , Dieta com Restrição de Proteínas , Hormônio do Crescimento/sangue , Hormônio do Crescimento/efeitos dos fármacos , Hormônio do Crescimento/urina , Humanos , Hiperamonemia/dietoterapia , Hiperamonemia/tratamento farmacológico , Hiperamonemia/etiologia , Hiperamonemia/metabolismo , Hiperamonemia/fisiopatologia , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/sangue , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/efeitos dos fármacos , Fator de Crescimento Insulin-Like I/efeitos dos fármacos , Fator de Crescimento Insulin-Like I/metabolismo , Japão , Masculino , Doença da Deficiência de Ornitina Carbomoiltransferase/complicações , Doença da Deficiência de Ornitina Carbomoiltransferase/dietoterapia , Doença da Deficiência de Ornitina Carbomoiltransferase/metabolismo , Ácido Orótico/urina , Tireotropina/sangue , Fatores de Tempo , Resultado do Tratamento , Triglicerídeos/sangueRESUMO
Local hyperthermia of living tissue can cause significant increases in blood flow and oxygenation depending on time-temperature history. Increases in perfusion of the abnormal and insufficient vasculature found in solid tumors may increase tumor oxygenation, thereby increasing the radiation sensitivity of the tumor. We hypothesized that local heating of tumor would increase the oxygenation of the tumor tissue and allow other oxygenating agents to further modify tumor oxygenation and radiation response. In the present study the effect of moderate temperature hyperthermia (MTH) at 41.5-42.5 degrees C for 30-60 min, 250 mg/kg nicotinamide, or carbogen breathing (95% O2/5% CO2) on the radiation sensitivity of FSaII murine fibrosarcomas or R3230 AC rat adenocarcinomas was studied. Individually, these treatments increased the tumor cell sensitivity to single dose 10-15 Gy X-irradiation by 1-5 fold on average, as measured by the in vivo/in vitro tumor excision assay. The combination of tumor MTH with nicotinamide or carbogen breathing increased the radiation sensitivity by 3-5 fold in FSaII tumors and 10-30 fold in R3230 tumors with varying levels of statistical significance. Finally, the triple combination of adjuvant MTH, nicotinamide and carbogen breathing increased the radiation-induced cell death in FSaII tumors to a similar extent as the dual combinations of MTH, nicotinamide or heat, carbogen breathing. However, in R3230 AC tumors the triple adjuvant combination significantly increased radiation-induced cell killing compared to all other dual adjuvant treatments (p < 0.04). To interrogate the mechanism by which heating alters tumor physiology, nitric oxide production in tumor and endothelial cells in culture and tumor tissue after heating was studied. Heating caused an increase in nitric oxide production over a 24 h period after treatment. Subsequently, inhibiting the enzymatic production of NO with L-NAME was found to increase heat-induced growth delay of FSaII tumors. The cause and effect of increased nitric oxide production and the response of the tumor vasculature to heat are discussed in the context of the tumor radiosensitization achieved by heating, carbogen breathing and nicotinamide.