RESUMO
AIM: The impact of glycaemic control on fracture risk is controversial, which may be due to the possible presence of hypoglycaemia. The aim of this study was to separately investigate the impacts of severe hypoglycaemia and poor glycaemic control on fracture risk in people with type 2 diabetes. METHODS: Overall, 4706 Japanese participants (2755 men and 1951 postmenopausal women) with type 2 diabetes (mean age 66 years) were followed prospectively (a median of 5.3 years; follow-up rate, 97.6%), and were stratified by severe hypoglycaemia status and glycaemic control. The primary outcome was fractures at any anatomic site. RESULTS: Fractures occurred in 662 participants (249 men and 413 women). The age- and sex-adjusted incidence rates (expressed per 1000 person-years) were: 71.2 (multiple episodes of severe hypoglycaemia), 43.1 (one episode), 25.2 [HbA1c < 53 mmol/mol (< 7%) without severe hypoglycaemia], 28.7 [HbA1c 53 to < 64 mmol/mol (7% to < 8%) without severe hypoglycaemia], 27.7 [HbA1c 64 to < 75 mmol/mol (8% to < 9%) without severe hypoglycaemia] and 40.5 [HbA1c ≥ 75 mmol/mol (≥ 9%) without severe hypoglycaemia]. Multivariate-adjusted hazard ratios (95% confidence intervals) for fractures were 2.24 (1.56, 3.21) in those with multiple episodes of severe hypoglycaemia, and 1.42 (1.04, 1.95) in those with HbA1c ≥ 75 mmol/mol (≥ 9%) without severe hypoglycaemia, compared with those with HbA1c < 53 mmol/mol (< 7%) without severe hypoglycaemia. CONCLUSIONS: Both severe hypoglycaemia and poor glycaemic control were significantly related to an increased risk of fracture in people with type 2 diabetes, although severe hypoglycaemia conferred a stronger risk.
Assuntos
Diabetes Mellitus Tipo 2/epidemiologia , Fraturas Ósseas/epidemiologia , Hiperglicemia/epidemiologia , Hipoglicemia/epidemiologia , Idoso , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Feminino , Controle Glicêmico , Humanos , Hipoglicemia/induzido quimicamente , Hipoglicemiantes/uso terapêutico , Japão/epidemiologia , Masculino , Pessoa de Meia-Idade , Sistema de RegistrosRESUMO
This study assessed the clinical value of CYFRA 21-1 in comparison with squamous cell carcinoma antigen (SCC-Ag), carcinoembryonic antigen (CEA), and carbohydrate antigen 19-9 (CA19-9) in patients with esophageal squamous cell carcinoma. In 112 primary cancer patients, the diagnostic sensitivity of CYFRA 21-1 (33.9%) was superior to SCC-Ag (28.6%), CEA (12.5%), and CA19-9 (6.3%). Levels of CYFRA 21-1 were closely correlated with TNM stage and were below the cutoff value in all 21 patients with stage I disease. All 38 patients with a CYFRA 21-1 level over the cutoff value among the 80 patients who underwent esophagectomy had lymph node metastases (pNl). A correlation was found between CYFRA 21-1 levels and clinical response in serial measurements of 21 patients who received chemotherapy or chemo radiotherapy. Our findings suggest that CYFRA 21-1 is not useful for diagnosis, but that it is valuable for monitoring the efficacy of therapy.
Assuntos
Antígenos de Neoplasias/sangue , Antígeno CA-19-9/sangue , Antígeno Carcinoembrionário/sangue , Carcinoma de Células Escamosas/sangue , Neoplasias Esofágicas/sangue , Queratina-19/sangue , Serpinas/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Escamosas/secundário , Carcinoma de Células Escamosas/terapia , Quimiorradioterapia Adjuvante , Quimioterapia Adjuvante , Neoplasias Esofágicas/patologia , Neoplasias Esofágicas/terapia , Esofagectomia , Feminino , Humanos , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Sensibilidade e Especificidade , Resultado do TratamentoRESUMO
AIMS/HYPOTHESIS: Medical nutrition therapy plays a critical role in the prevention and treatment of type 2 diabetes. However, appropriate measures of eating behaviours, such as eating rate, have not yet been clearly established. The aim of the present study was to examine the associations among eating rate, obesity and cardiovascular risk factors. METHODS: A total of 7,275 Japanese individuals aged ≥40 years who had normal fasting glucose levels, impaired fasting glucose or diabetes were divided into four groups according to self-reported eating rate: slow, medium, relatively fast and very fast. The associations between eating rate and various cardiovascular risk factors were investigated cross-sectionally. RESULTS: The proportions of participants who were obese or who had elevated waist circumference levels increased progressively with increases in eating rate (p for trend <0.001), regardless of glucose tolerance status. These associations remained significant after adjustment for potential confounders, namely, age, sex, total energy intake, dietary fibre intake, current smoking, current drinking and regular exercise (p for trend <0.001). Blood pressure and lipid levels also tended to increase in association with eating rate. HbA(1c) rose significantly as eating rate increased, even after multivariate adjustment, including BMI, in diabetic patients on insulin therapy (p = 0.02), whereas fasting plasma glucose did not increase significantly. CONCLUSIONS/INTERPRETATION: Our findings suggest that eating rate is associated with obesity and other cardiovascular risk factors and therefore may be a modifiable risk factor in the management of cardiovascular risk factors and diabetes.
Assuntos
Doenças Cardiovasculares/etiologia , Diabetes Mellitus/etiologia , Comportamento Alimentar , Intolerância à Glucose/etiologia , Obesidade/etiologia , Estado Pré-Diabético/etiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Doenças Cardiovasculares/epidemiologia , Doenças Cardiovasculares/prevenção & controle , Estudos Transversais , Diabetes Mellitus/tratamento farmacológico , Diabetes Mellitus/prevenção & controle , Feminino , Intolerância à Glucose/tratamento farmacológico , Intolerância à Glucose/prevenção & controle , Hemoglobinas Glicadas/análise , Humanos , Hipoglicemiantes/uso terapêutico , Insulina/uso terapêutico , Japão/epidemiologia , Masculino , Pessoa de Meia-Idade , Obesidade/sangue , Obesidade/epidemiologia , Obesidade/fisiopatologia , Estado Pré-Diabético/prevenção & controle , Estudos Prospectivos , Sistema de Registros , Fatores de RiscoRESUMO
BACKGROUND: The potential role of the renin-angiotensin system (RAS) in the promotion of tumour growth has been investigated, and the administration of RAS inhibitors, such as angiotensin-converting enzyme inhibitors (ACEIs) or angiotensin II receptor blockers (ARBs), may improve disease control in malignancy. We investigated the prognostic impact of RAS inhibitors by analysing data from patients with upper-tract urothelial carcinoma (UTUC). METHODS: A total of 279 patients who underwent nephroureterectomy for localised UTUC (pTa-3N0M0) were identified at our three institutions. We retrospectively investigated the prognostic outcomes following nephroureterectomy in patients administered or not administered ACEIs or ARBs. RESULTS: The median follow-up period was 3.4 years. RAS inhibitors were administered to 48 patients (17.2%). Multivariate analysis showed that the appearance of pathological T3, positive lymphovascular invasion, and no RAS inhibitor administration (P=0.027 HR=3.14) were independent risk factors for a decrease in subsequent metastasis-free survival. The 5-year metastasis-free survival rate was 93.0% in patients who administered RAS inhibitors, and 72.8% in their counterparts who did not (P=0.008). CONCLUSION: The absence of RAS inhibitor administration was an independent risk factor for subsequent tumour metastasis in patients with localised UTUC. We propose RAS inhibitors may be a potent choice as an effective treatment following nephroureterectomy.
Assuntos
Sistema Renina-Angiotensina/efeitos dos fármacos , Neoplasias Urológicas/tratamento farmacológico , Idoso , Terapia Combinada , Feminino , Humanos , Masculino , Nefrectomia , Prognóstico , Estudos Retrospectivos , Taxa de Sobrevida , Neoplasias Urológicas/patologia , Neoplasias Urológicas/cirurgiaRESUMO
BACKGROUND: Inducible activation of nuclear factor (NF)-κB is one of the principal mechanisms through which resistant prostate cancer cells are protected from radiotherapy. We hypothesised that inactivation of inducible NF-κB with a novel NF-κB inhibitor, DHMEQ, would increase the therapeutic effects of radiotherapy. METHODS: PC-3 and LNCaP cells were exposed to irradiation and/or DHMEQ. Cell viability, cell cycle analysis, western blotting assay, and NF-κB activity were measured. The antitumour effect of irradiation combined with DHMEQ in vivo was also assessed. RESULTS: The combination of DHMEQ with irradiation resulted in cell growth inhibition and G2/M arrest relative to treatment with irradiation alone. Inducible NF-κB activity by irradiation was inhibited by DHMEQ treatment. The expression of p53 and p21 in LNCaP, and of 14-3-3σ in PC-3 cells, was increased in the combination treatment. In the in vivo study, 64 days after the start of treatment, tumour size was 85.1%, 77.1%, and 64.7% smaller in the combination treatment group than that of the untreated control, DHMEQ-treated alone, and irradiation alone groups, respectively. CONCLUSION: Blockade of NF-κB activity induced by radiation with DHMEQ could overcome radio-resistant responses and may become a new therapeutic modality for treating prostate cancer.
Assuntos
Antineoplásicos/uso terapêutico , Benzamidas/farmacologia , Cicloexanonas/farmacologia , NF-kappa B/antagonistas & inibidores , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/radioterapia , Radiossensibilizantes/uso terapêutico , Animais , Antineoplásicos/farmacologia , Benzamidas/uso terapêutico , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/efeitos da radiação , Divisão Celular/efeitos dos fármacos , Divisão Celular/efeitos da radiação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Cicloexanonas/uso terapêutico , Humanos , Masculino , Camundongos , Camundongos Nus , Tolerância a Radiação/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Estimating the cost of hospital infection has become a matter of increasing interest in terms of health economics. This study aimed to assess the accuracy of economic studies on hospital infections, using surgical site infection (SSI) as an example. A search was performed for original articles reporting the cost of SSI, published in the English language between 1996 and 2005. For the critical review, the period of cost tracking, classification of costs and cost counting methods were noted. Fifteen articles met the inclusion criteria. The costs of SSI vary according to surgical procedures, country, publication year, study design and accounting method. Only two studies estimated the additional cost of SSI after discharge. All 15 studies included healthcare costs and none measured patient/family resources. In 10 studies, the costs were calculated based on accounting. Three studies used estimated costs from the ratio of costs to charges and two studies used charge data in place of cost data. It will become increasingly important for future studies to perform multi-centre prospective surveys, establish a standard method for cost accounting, include the cost of healthcare services following hospitalization and consider the morbidity cost to patients themselves from a societal perspective.
Assuntos
Infecção Hospitalar/economia , Infecção da Ferida Cirúrgica/economia , Custos de Cuidados de Saúde , HumanosRESUMO
The reaction mechanism for the formation of 2'-deoxy-oxanosine from 2'-deoxyguanosine by nitrous acid was explored using methyl derivatives of guanosine and an isolated intermediate of the reaction. When 1-methylguanosine was incubated with NaNO(2)under acidic conditions, N (5) -methyloxanosine and 1-methylxanthosine were generated, whereas the same treatment of N (2), N (2)-dimethylguanosine generated no product. In a similar experiment without NO(2)(-), participation of a Dimroth rearrangement was ruled out. In the guanosine-HNO(2)reaction system, an intermediate with a half-life of 5.6 min (pH 7.0, 20 degrees C) was isolated and tentatively identified as a diazoate derivative of guanosine. The diazoate intermediate was converted into oxanosine and xanthosine at a molar ratio (oxanosine:xanthosine) of 0.26 at pH 7.0 and 20 degrees C. The ratio was not affected by the incubation pH between 2 and 10, but increased linearly with temperature from 0.22 (0 degrees C) to 0.32 (50 degrees C). The addition of acetone also increased the ratio up to 0.85 (98% acetone). Based on these results, a con-ceivable pathway for the formation of 2'-deoxyoxanosine from 2'-deoxyguanosine by HNO(2)is proposed.
Assuntos
Desoxiguanosina/química , Ácido Nitroso/química , Cátions , Cromatografia Líquida de Alta Pressão , Desoxirribonucleosídeos/síntese química , Hidrólise , Espectroscopia de Ressonância MagnéticaRESUMO
Reactive oxygen species generate structurally diverse base lesions in DNA. These lesions are primarily removed by base excision repair (BER) enzymes in prokaryotic and eukaryotic cells. Biochemical properties of BER enzymes such as substrate specificity, enzymatic parameters, and action mechanisms can be best studied by employing defined oligonucleotide and DNA substrates. Currently available methods are listed to prepare defined DNA substrates containing oxidative base damage and analogs. BER enzymes for oxidative base damage are classified into two subgroups that recognize pyrimidine lesions (Endo III homologs) and purine lesions (Fpg homologs), though E. coli Fpg exhibits weak repair activity for certain pyrimidine damage. Recently, several interesting findings have been reported in relation to the substrate specificity of BER enzymes. Saccharomyces cerevisiae Endo III homologs (NTG1 and NTG2) have been shown to recognize formamidopyrimidine (Fapy) lesions that are derived from purine. Endo III and Endo VIII have a very weak activity to dihydrothymine in comparison with thymine glycol. Excision of 7,8-dihydro-8-oxoguanine by Fpg and human OGG1 is paired-base-dependent, whereas that of Fapy is essentially paired-base-independent. The repair efficiency of BER enzymes is affected by surrounding sequence contexts. In general, the sequence context effect appears to be more pronounced for Fpg homologs than Endo III homologs.
Assuntos
DNA Ligases/metabolismo , DNA/química , Desoxirribonuclease (Dímero de Pirimidina) , Proteínas de Escherichia coli , Proteínas de Bactérias/metabolismo , Sequência de Bases , DNA/genética , DNA/metabolismo , Dano ao DNA , DNA-Formamidopirimidina Glicosilase , Endodesoxirribonucleases/metabolismo , Escherichia coli/enzimologia , Proteínas Fúngicas/metabolismo , N-Glicosil Hidrolases/metabolismo , Oxidantes/toxicidade , Oxirredução , Estresse Oxidativo , Dímeros de Pirimidina/metabolismo , Espécies Reativas de Oxigênio , Saccharomyces cerevisiae/enzimologia , Especificidade da Espécie , Especificidade por SubstratoRESUMO
Bone morphogenetic proteins (BMPs) belong to the transforming growth factor-beta (TGF-beta) family and have been identified as factors that stimulate bone formation in vivo. They turned out to be multifunctional molecules regulating the growth, differentiation, and apoptosis in various target cells. Some BMPs and their receptors (BMPRs) are expressed on prostate cancer cells. We have reported previously that BMPR-IB mRNA expression is highest in the prostate, a characteristic that is not shared by the other BMPRs, BMPR-IA and BMPR-II. However, the amounts of BMPR-IB mRNA were significantly low in prostate tissues after androgen withdrawal therapy. They were also low in prostate cancer cell lines. Semiquantitative RT-PCR showed that BMPR-IB mRNA was induced by androgen in the androgen-sensitive human prostatic cancer cell line LNCaP, whereas the expression of BMPR-IA and BMPR-II mRNAs was not affected by androgen. When the recombinant human BMP-2 was added to the LNCaP cells in the presence of androgen, cell growth was inhibited. In contrast, the growth rate was increased by the addition of the same ligand when the cells were cultured in the absence of androgen; under this condition, the amounts of BMPR-IB mRNA were decreased significantly. These observations showed that the amounts of BMPR-IB, but not those of BMPR-IA, were regulated by androgen and further suggest that BMPR-IA and BMPR-IB differentially modulate prostate cancer cell growth in response to BMP under different hormonal conditions; BMPR-IA elicits growth stimulation, and BMPR-IB conveys a negative regulatory signal in response to BMP-2.
Assuntos
Proteínas Morfogenéticas Ósseas/farmacologia , Neoplasias da Próstata/patologia , Receptores de Fatores de Crescimento/metabolismo , Fator de Crescimento Transformador beta , Proteína Morfogenética Óssea 2 , Proteínas Morfogenéticas Ósseas/metabolismo , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Humanos , Masculino , Metribolona/farmacologia , Antígeno Prostático Específico/metabolismo , Neoplasias da Próstata/metabolismo , RNA Mensageiro/metabolismo , Proteínas Recombinantes/farmacologia , Congêneres da Testosterona/farmacologia , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
Bone metastasis is a common event in prostate cancer, and it is known that some of the bone morphogenetic proteins (BMPs) are expressed in prostate cancer cells, while no study on the expression of their receptors, BMPRs, has been reported. Here we report cloning and sequence analysis of the human BMPR-IB cDNA. We also analysed the expression of transcripts of three types of the BMPR genes in human tissues and prostate cancer cell lines. The BMPR-IB mRNA was present in various organs, but the highest level was found in the prostate. Moreover, the amount of BMPR-IB mRNA was significantly low in prostate cancer tissues after androgen withdrawal and was also low in prostate cancer cell lines. RT-PCR analysis showed that the BMPR-IB message was upregulated by androgen stimulation in the LNCaP cell line which expresses the androgen receptor. By contrast, the mRNA levels of BMPR-IA and BMPR-II were not significantly different among non-cancerous and cancerous prostate tissues. It was also suggested that human BMPR-IA and BMPR-IB might have different biological functions in the prostate, although their sequences were 85.3% identical in the serine-threonine kinase domain.
Assuntos
Neoplasias da Próstata/genética , Proteínas Serina-Treonina Quinases/genética , Receptores de Fatores de Crescimento/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Receptores de Proteínas Morfogenéticas Ósseas Tipo I , Clonagem Molecular , DNA Complementar , Expressão Gênica , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/genética , Homologia de Sequência de AminoácidosRESUMO
Diacylglycerol was generated in phosphatidylcholine vesicles by incubation with Clostridium welchii phospholipase C. Newly formed diacylglycerol was rapidly converted to monoacylglycerol and glycerol when rat liver cytosol fraction was present in the incubation mixture, suggesting the presence of di- and monoacylglycerol lipase activities in this subcellular fraction. On the other hand, 3H-labeled diacylglycerol co-emulsified with non-radioactive phosphatidylcholine was found to be a poor substrate for the diacylglycerol lipase. These results indicate that enzymatic generation of diacylglycerol provide a substrate having a suitable physical state for the expression of diacylglycerol lipase activity. It was also found that the rate of diacylglycerol hydrolysis was dependent upon the rate of diacylglycerol generation, but not upon the absolute concentration in the incubation mixture. When the rate of diacylglycerol hydrolysis was plotted against the rate of diacylglycerol generation, a saturation curve was obtained and the double-reciprocal plot gave a straight line. It is not known why a relationship similar to Michaelis-Menten type kinetics was obtained between the rate of diacylglycerol hydrolysis and diacylglycerol generation instead of diacylglycerol concentration, but it may be best explained by the following assumptions: (1) diacylglycerol molecules are generated at the surface of the lipid vesicles where they are readily accessible to diacylglycerol lipase; (2) soon after the generation, diacylglycerol molecules migrate into inside the vesicles where they are inaccessible to the enzyme; (3) the effective concentration of diacylglycerol, i.e., the concentration of diacylglycerol located in the surface layer of the vesicles is proportional to the rate of diacylglycerol generation.
Assuntos
Diglicerídeos/metabolismo , Glicerídeos/metabolismo , Lipase Lipoproteica/metabolismo , Fígado/enzimologia , Fosfatidilcolinas/metabolismo , Fosfolipases Tipo C/metabolismo , Animais , Citosol/enzimologia , Cinética , Masculino , Ratos , Ratos EndogâmicosRESUMO
Complementary DNAs (cDNAs) encoding alpha 1-microglobulin (alpha 1mG)/bikunin, also known as inter-alpha-inhibitor (I alpha I) light chain, were cloned from liver extracts of the Mongolian gerbil, Meriones unguiculatus, and the Syrian golden hamster, Mesocricetus auratus, by reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends methods. From the deduced amino-acid sequences of alpha 1mG/bikunin of gerbil and hamster, the basic molecular structure of the proteins seemed to be well-conserved. However, near the proposed sequence of proteinase inhibitory sites of two Kunitz domains in the bikunin part, variable regions composed of three amino acids each were observed between species, including rodents. Since the second half of bikunin is genetically identical with the mast cell proteinase inhibitor, trypstatin, the bikunin of each animal may have distinct inhibitory activity against mast cell proteinases.
Assuntos
alfa-Globulinas/genética , DNA Complementar/química , Glicoproteínas/genética , Glicoproteínas de Membrana , Inibidores de Proteases/química , Proteínas , Inibidor da Tripsina de Soja de Kunitz , alfa-Globulinas/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Cricetinae , Gerbillinae , Humanos , Fígado/química , Mesocricetus , Dados de Sequência Molecular , Inibidores de Serina Proteinase/genética , Especificidade da EspécieRESUMO
Diacylglycerol kinase activity is found in both adult and fetal lung. Approximately 27 and 52% of the total activity is found in microsomes and cytosol, respectively. The activity is maximal at pH 7.4. The apparent Km for ATP is 0.11 mM and 0.21 mM for cytosol and microsomes, respectively. The apparent Km for dioleoylglycerol is 0.05 mM for cytosol and 0.14 for microsomes. Maximal activity in cytosol and microsomes is obtained with 2.0 mM dexoycholate. Other detergents cannot substitute for deoxycholate. Phosphatidylglycerol stimulates activity in the absence and in the presence of deoxycholate. Phosphatidylserine also stimulates activity, whereas phosphatidylethanolamine was inactive and phosphatidylcholine inhibited the reaction. Linoleic acid produced inhibition. The general properties of the enzyme were similar for fetal and adult lung. Diacylglycerol kinase from microsomes and cytosol fraction from both fetal and adult lung was most active with dioleoylglycerol and diacylglycerol from egg phosphatidylcholine. Significantly lower activity was obtained with dipalmitoylglycerol. Phosphatidylglycerol did not alter the relative substrate preferences. The activity in microsomes increased with development from 19 days gestation to a maximal activity at 21 days gestation. Maximal activity was about 2-fold higher than the adult. The activity dropped rapidly reaching adult values prior to birth (22 days gestation). The activity in cytosol fractions increased gradually from 19 days gestation, reaching adult values by 22 days gestation.
Assuntos
Pulmão/enzimologia , Fosfotransferases/metabolismo , Animais , Citosol/enzimologia , Diacilglicerol Quinase , Feminino , Feto , Cinética , Pulmão/embriologia , Microssomos/enzimologia , Gravidez , Ratos , Especificidade por SubstratoRESUMO
Ethanolaminephosphotransferase (EC 2.7.8.1) activity was determined in lung microsomes using diacylglycerols generated endogenously from [14C]glycerol 3-phosphate and different mixtures of fatty acids. Ethanolaminephosphotransferase used endogenously generated dipalmitoylglycerol better than dioleoylglycerol. The apparent Km and the reaction rates for four different endogenously generated mixtures were the same (16 nmol/mg microsomal proteins). The apparent Km values for CDP-ethanolamine were the same (0.26 mm) for endogenously generated dipalmitoylglycerol and dioleoylglycerol. The amount of diacylglycerol generated in microsomes was 2-3-times the apparent Km for diacylglycerol. Dipalmitoylglycerol, supplied exogenously as a Tween 20/phosphatidylglycerol emulsion, was nearly twice as active as dioleoylglycerol. Both dipalmitoylglycerol and dioleoylglycerol were more active as substrates when emulsions were made with phosphatidylglycerol/Tween 20 than with Tween 20 alone. The results suggest that ethanolaminephosphotransferase in lung is relatively nonselective for molecular species of diacylglycerol. In addition, the results suggest that the concentration of diacylglycerol and the physical state in which it is presented to the enzyme can affect the apparent selectivity of ethanolaminephosphotransferase for diacylglycerols.
Assuntos
Diglicerídeos/metabolismo , Etanolaminofosfotransferase/metabolismo , Glicerídeos/metabolismo , Pulmão/enzimologia , Fosfotransferases/metabolismo , Animais , Relação Dose-Resposta a Droga , Ácidos Graxos/metabolismo , Masculino , Microssomos/enzimologia , Fosfatidiletanolaminas/biossíntese , Ratos , Ratos Endogâmicos , Especificidade por SubstratoRESUMO
The influence of sequence context on the ability of DNA polymerase to bypass sites of base loss was addressed using an in vitro selection system. Oligonucleotides containing either an aldehydic abasic site or tetrahydrofuran surrounded by four randomized bases on both the 5' and 3' sides were used as templates for synthesis by phage T4 DNA polymerase holoenzyme proficient or deficient in the 3'-->5' proofreading exonuclease activity. Successful bypass products were purified, subcloned and the sequences of approximately 100 subclones were determined for each of the four polymerase/lesion combinations tested. Between 7 and 19 % of the bypass products contained deletions of one to three nucleotides in the randomized region. In bypass products not containing deletions, biases for and against certain nucleotides were readily noticeable across the entire randomized region. Template strands from successful bypass products of abasic sites had a high frequency of T in most of the randomized positions, while those from bypass products of tetrahydrofuran had a high frequency of G at the positions immediately to the 3' and 5' side of the lesion. Consensus sequences were shared by successful bypass products of the same lesion but not between bypass products of the two lesions. The consensus sequence for efficient bypass of tetrahydrofuran was over-represented in several frames relative to the lesion. T4 DNA polymerase inserted A opposite abasic sites 63 % of the time in the presence of proofreading and 79 % of the time in its absence, followed by G>T>C, while the insertion of A opposite tetrahydrofuran ranged between 93 % and 100 % in the presence and absence of proofreading, respectively. Finally, sequence context influenced the choice of nucleotide inserted opposite abasic sites and consensus sequences which favored the incorporation of nucleotides other than A were defined.
Assuntos
Replicação do DNA , DNA Polimerase Dirigida por DNA , DNA/metabolismo , Furanos/metabolismo , Holoenzimas/metabolismo , Proteínas Virais/metabolismo , Bacteriófago T4/enzimologia , Bacteriófago T4/metabolismo , Sequência de Bases , DNA/biossíntese , DNA/química , Dano ao DNA , DNA Viral/biossíntese , Dados de Sequência MolecularRESUMO
Endonuclease III (endoIII; nth gene product) of Escherichia coli is known to be a DNA repair enzyme having a relatively broad specificity for damaged pyrimidine bases of DNA. Here, we describe the cloning and characterization of the cDNA and the gene for a mouse homologue (mNthl1/mNth1) of endoIII. The cDNA was cloned from a mouse T-cell cDNA library with a probe prepared by PCR using the library and specific PCR primers synthesized based on the reported information of partial amino acid sequences of bovine NTHL1/NTH1 and of EST Data Bases. The cDNA is 1025 nucleotides long and encodes a protein consisting of 300 amino acids with a predicted molecular mass of 33.6 kDa. The amino acid sequence exhibits significant homologies to those of endoIII and its prokaryotic and eukaryotic homologues. The recombinant mNthl1 with a hexahistidine tag was overexpressed in a nth::cmr nei::Kmr double mutant of E. coli, and purified to apparent homogeneity. The enzyme showed thymine glycol DNA glycosylase, urea DNA glycosylase and AP lyase activities. Northern blot analysis indicated that mNthl1 mRNA is about 1 kb and is expressed ubiquitously. A 15 kb DNA fragment containing the mNthl1 gene was cloned from a mouse genomic library and sequenced. The gene consists of six exons and five introns spanning 6.09 kb. The sequenced 5' flanking region lacks a typical TATA box, but contains a CAAT box and putative binding sites for several transcription factors such as Ets, Sp1, AP-1 and AP-2. The mNthl1 gene was shown to lie immediately adjacent to the tuberous sclerosis 2 (Tsc2) gene in a 5'-to-5' orientation by sequence analysis and was assigned to chromosome 17A3 by in situ hybridization.
Assuntos
Desoxirribonuclease (Dímero de Pirimidina) , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Proteínas de Escherichia coli , Escherichia coli/enzimologia , Sequência de Aminoácidos , Animais , Southern Blotting , Carbono-Oxigênio Liases/metabolismo , Clonagem Molecular , DNA Glicosilases , DNA Liase (Sítios Apurínicos ou Apirimidínicos) , Desoxirribonuclease IV (Fago T4-Induzido) , Endodesoxirribonucleases/química , Endodesoxirribonucleases/isolamento & purificação , Escherichia coli/genética , Éxons/genética , Etiquetas de Sequências Expressas , Humanos , Íntrons/genética , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , N-Glicosil Hidrolases/metabolismo , Fases de Leitura Aberta/genética , Mapeamento Físico do Cromossomo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/genética , Elementos de Resposta/genética , Homologia de Sequência de Aminoácidos , Timina/análogos & derivados , Timina/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor , Ureia/metabolismoRESUMO
1H-Nuclear magnetic resonance (NMR) microscopy was used to study the freezing behavior of wintering buds of full-moon maple (Acer japonicum Thunb.). The images obtained predominantly reflected the density of mobile (i.e. non-ice) protons from unfrozen water. A comparison of NMR images taken at different subfreezing temperatures revealed which tissues produced high- and low-temperature exotherms in differential thermal analyses. In leaf and lower buds of A. japonicum, the scales and stem bark tissues were already frozen by -7[deg]C, but the primordial inflorescence and terminal primordial shoots remained supercooled at -14[deg]C, and the lateral primordial shoots were unfrozen even at -21[deg]C. The freezing of these supercooled tissues was associated with their loss of viability. The size of the supercooled primordial shoots and inflorescences was gradually reduced with decreasing temperature, indicating extraorgan freezing in these tissues. During this process the formation of dark regions beneath the primordia and subsequent gradual darkening in the basal part of supercooled primordia were visible. As the lateral shoot primordia were cooled, the unfrozen area was considerably reduced. Since the lateral primordia remained viable down to -40[deg]C, with no detectable low-temperature exotherms, they probably underwent type I extraorgan freezing. Deep supercooling in the xylem was clearly imaged. NMR microscopy is a powerful tool for noninvasively visualizing harmonized freezing behaviors in complex plant organs.
RESUMO
To examine differences in the surface properties of limb bud cells, we mixed cells from the progress zone at different stages or from different positions along proximodistal axis of chick and quail wing limb buds. To identify the origin of cells, a chick-specific antibody was used in a mixed culture of chick and quail cells, or cells from one of the stages were labeled with a fluorescent dye, PKH-26. Within 18 hours in mixed culture, cells segregated from each other and formed patches of various sizes. The process zone cells at early developmental stages mixed homogeneously with the cells from proximal region of old limb buds and the progress zone cells at late stages mixed homogeneously with the cells from distal region of the old limb buds. These results suggest that surface properties of cells in progress zone change during limb bud development and vary along the proximodistal and anteroposterior axes of the limb bud and that these differences in surface property may correspond to the positional values for limb pattern formation.
Assuntos
Asas de Animais/embriologia , Animais , Anticorpos Monoclonais , Cartilagem/citologia , Cartilagem/embriologia , Separação Celular , Células Cultivadas , Embrião de Galinha , Codorniz , Especificidade da Espécie , Asas de Animais/citologiaRESUMO
The apical mesodermal region of chick limb buds (progress zone, PZ) which is essential for limb pattern formation contains uncommitted cells that change their positional values instructed by the apical ectodermal ridge (AER). Reciprically, the PZ cells maintain the AER activity. FGF-2 and FGF-4 can substitute for the AER to maintain normal outgrowth and gene expression in the limb bud. We examined the effects of FGF on the maintenance of PZ cells characteristics in culture by making recombinant limbs with anterior PZ cells that were pre-cultured in the presence of FGF-2 or FGF-4 and analyzed their morphogenic potency and responsiveness to positional cues arising from the zone of polarizing activity (ZPA). The limb buds expressed distal Hox genes and could form a segmented digit. Recombinant limb buds consisting of anterior PZ cells cultured without FGF failed to express Hox genes and formed instead a small cartilage nodule. These results indicate that addition of FGF-2 or FGF-4 to cultured PZ cells maintains their competence for Hox gene expression and digit formation, but not their responsiveness to positional cues from the ZPA. We also found that when anterior PZ cells which had been pre-cultured with FGF-2 or FGF-4 were implanted underneath the AER, they could maintain Fgf-8 expression in the AER, whereas this expression was not detected in the AER on the grafted anterior PZ cells that had been pre-cultured without FGF, indicating that FGF maintains AER-maintenance activity of PZ cells in culture.