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1.
J Appl Microbiol ; 123(6): 1584-1596, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28940494

RESUMO

AIMS: Test the choice of 16S rRNA gene amplicon and data analysis method on the accuracy of identification of clinically important bacteria utilizing a benchtop sequencer. METHODS AND RESULTS: Nine 16S rRNA amplicons were tested on an Ion Torrent PGM to identify 41 strains of clinical importance. The V1-V2 region identified 40 of 41 isolates to the species level. Three data analysis methods were tested, finding that the Ribosomal Database Project's SequenceMatch outperformed BLAST and the Ion Reporter Metagenomics analysis pipeline. Lastly, 16S rRNA gene sequencing mixtures of four species through a six log range of dilution showed species were identifiable even when present as 0·1% of the mixture. CONCLUSIONS: Sequencing the V1-V2 16S rRNA gene region, made possible by the increased read length Ion Torrent PGM sequencer's 400 base pair chemistry, may be a better choice over other commonly used regions for identifying clinically important bacteria. In addition, the SequenceMatch algorithm, freely available from the Ribosomal Database Project, is a good choice for matching filtered reads to organisms. Lastly, 16S rRNA gene sequencing's sensitivity to the presence of a bacterial species at 0·1% of a mixture suggests it has sufficient sensitivity for samples in which important bacteria may be rare. SIGNIFICANCE AND IMPACT OF THE STUDY: We have validated 16S rRNA gene sequencing on a benchtop sequencer including simple mixtures of organisms; however, our results highlight deficits for clinical application in place of current identification methods.


Assuntos
Bactérias/classificação , RNA Ribossômico 16S/química , Análise de Sequência de DNA/métodos , Bactérias/isolamento & purificação , Sequência de Bases , RNA Ribossômico 16S/genética , Análise de Sequência de DNA/instrumentação
2.
J Perinatol ; 35(11): 965-9, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26355942

RESUMO

OBJECTIVE: The aim of this study was to identify the best sedation/analgesia protocol for laser photocoagulation (PC) of retinopathy of prematurity (ROP). STUDY DESIGN: This multicenter observational study included five hospitals, each using a specific sedation/analgesia protocol: local anesthesia with oxybuprocaine hydrochloride (Group L); intravenous pentazocine (Group P); intravenous fentanyl (Group F); air, oxygen and sevoflurane (AOS) inhalation (Group I). The groups were compared for pain responses, vital signs and adverse events. RESULTS: Heart rates and systemic blood pressures were elevated by PC in Groups L and P and Groups L, P and F, respectively. Moreover, poor analgesic efficacy was recognized in Groups L, P and F. In contrast, Group I experienced hypothermia, enteral feeding intolerance and apnea more frequently. CONCLUSION: From the viewpoint of sedation/pain relief, AOS anesthesia should be the best protocol. However, considering all the various factors together, the most reasonable one can be varied based on the patient's condition and hospital.


Assuntos
Sedação Consciente/métodos , Recém-Nascido Prematuro , Fotocoagulação/métodos , Medição da Dor , Retinopatia da Prematuridade/cirurgia , Administração por Inalação , Estudos de Coortes , Feminino , Fentanila/administração & dosagem , Humanos , Recém-Nascido , Infusões Intravenosas , Japão , Terapia a Laser/métodos , Masculino , Éteres Metílicos/administração & dosagem , Pentazocina/administração & dosagem , Estudos Prospectivos , Retinopatia da Prematuridade/diagnóstico , Medição de Risco , Índice de Gravidade de Doença , Sevoflurano , Resultado do Tratamento
3.
J Perinatol ; 31(6): 440-5, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21164427

RESUMO

OBJECTIVE: We prospectively evaluated the rate of postnatal cytomegalovirus (CMV) transmission through breast milk in extremely premature infants to address the impact of CMV infection on preterm infants during lactation. STUDY DESIGN: A total of 25 mothers and 27 infants (two sets of twins) with birth weights <1000 g and/or gestational ages <28 weeks were enrolled in the study. They were mostly fed frozen-thawed breast milk. Breast milk, serum and urine samples were collected every 2 weeks and screened for CMV infection using the real-time polymerase chain reaction. RESULT: All of the 21 CMV-seropositive mothers had detectable CMV DNA in their breast milk, with a peak at 4 to 6 weeks postpartum. CMV infection was confirmed in only one infant (4.3%) who displayed almost no clinical symptoms. CONCLUSION: At our institutes, we mainly use frozen-thawed breast milk. We found low CMV transmission rates even in extremely premature infants, and the CMV-positive infant did not develop serious symptoms.


Assuntos
Infecções por Citomegalovirus/transmissão , Citomegalovirus/isolamento & purificação , Recém-Nascido de Peso Extremamente Baixo ao Nascer , Doenças do Prematuro/virologia , Leite Humano/virologia , Aleitamento Materno , Citomegalovirus/genética , Infecções por Citomegalovirus/virologia , DNA Viral/genética , Feminino , Humanos , Recém-Nascido , Doenças do Prematuro/diagnóstico , Masculino , Bancos de Leite Humano , Estudos Prospectivos , Reação em Cadeia da Polimerase Via Transcriptase Reversa
4.
Artigo em Português | LILACS | ID: lil-560261

RESUMO

A adesão ao tratamento farmacológico em doenças crônicas como a hipertensão arterial, é fundamental para o controle, prevenção de complicações e diminuição da mortalidade. Identificar os fatores que levam a não adesão ao programa de controle de hipertensão arterial, em Unidades Básicas de Saúde de Campo Grande, MS e produzir um modelo de predição desta condição foi o objetivo do presente estudo. Utilizou-se o método de caso-controle, aninhado a coorte de pacientes cadastrados no programa, no período de 2002 a 2005. Foi utilizada regressão logística tendo como variável-resposta ?adesão ao programa?. As associações significativas identificadas na análise univariada foram: características socioeconômicas, da doença, do tratamento e as relacionadas ao programa. Para prever a adesão, mantiveram-se no modelo as seguintes variáveis: dificuldade em ir ao programa, renda familiar, presença de diabetes, escolaridade e viver com companheiro. Com base no modelo, a probabilidade do paciente ser classificado corretamente como aderente, é de aproximadamente, 80% e como não aderente, 67%. O modelo identifica precocemente, pacientes vulneráveis à não adesão ao programa propiciando que este institua medidas voltadas aos prováveis, não aderentes.


Adherence to the pharmacological treatment of chronic diseases such as arterial hypertension is decisive in their control, in preventing complications, and in decreasing mortality rates. To identify factors that led patients to drop out of an arterial hypertension control program available at local district clinics of the government-run National Health Service in Campo Grande, MS, Brazil, and to design a model to predict adherence. A nested case?control study was conducted on subjects selected from within a cohort of patients enrolled in the above program, from 2002 to 2005. Binary logistic regression was used, with ?adherence to program? as the binary response variable. Data were subjected to logistic regression analysis to generate a model capable of predicting adherence. Factors identified: difficulty in going to the venue where the program was available, family income, presence of diabetes, level of education and living with a partner. When the logistic regression model was used, the probability of a patient being correctly classified as adherent and nonadherent was approximately 80% and 67%, respectively. The model enables early identification of patients prone to nonadherence to the control program, thus making it possible to implement measures directed at potentially nonadherent participants.


Assuntos
Humanos , Planos e Programas de Saúde , Hipertensão/prevenção & controle
5.
Br J Pharmacol ; 158(2): 580-7, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19663883

RESUMO

BACKGROUND AND PURPOSE: Adding spironolactone to standard therapy in heart failure reduces morbidity and mortality, but the underlying mechanisms are not fully understood. We analysed the effect of canrenone, the major active metabolite of spironolactone, on myocardial contractility and intracellular calcium homeostasis. EXPERIMENTAL APPROACH: Left ventricular papillary muscles and cardiomyocytes were isolated from male Wistar rats. Contractility of papillary muscles was assessed with force transducers, Ca(2+) transients by fluorescence and Ca(2+) fluxes by electrophysiological techniques. KEY RESULTS: Canrenone (300-600 micromol L(-1)) reduced developed tension, maximum rate of tension increase and maximum rate of tension decay of papillary muscles. In cardiomyocytes, canrenone (50 micromol L(-1)) reduced cell shortening and L-type Ca(2+) channel current, whereas steady-state activation and inactivation, and reactivation curves were unchanged. Canrenone also decreased the Ca(2+) content of the sarcoplasmic reticulum, intracellular Ca(2+) transient amplitude and intracellular diastolic Ca(2+) concentration. However, the time course of [Ca(2+)](i) decline during transients evoked by caffeine was not affected by canrenone. CONCLUSION AND IMPLICATIONS: Canrenone reduced L-type Ca(2+) channel current, amplitude of intracellular Ca(2+) transients and Ca(2+) content of sarcoplasmic reticulum in cardiomyocytes. These changes are likely to underlie the negative inotropic effect of canrenone.


Assuntos
Canais de Cálcio Tipo L/efeitos dos fármacos , Cálcio/metabolismo , Canrenona/farmacologia , Antagonistas de Receptores de Mineralocorticoides/farmacologia , Animais , Cafeína/farmacologia , Canais de Cálcio Tipo L/metabolismo , Canrenona/administração & dosagem , Relação Dose-Resposta a Droga , Homeostase , Masculino , Antagonistas de Receptores de Mineralocorticoides/administração & dosagem , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Músculos Papilares/efeitos dos fármacos , Músculos Papilares/metabolismo , Ratos , Ratos Wistar , Retículo Sarcoplasmático/metabolismo , Espironolactona/metabolismo
6.
Braz J Med Biol Res ; 39(5): 611-4, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16648898

RESUMO

In a comparative study of erythrocyte metabolism of vertebrates, the specific activity of glucose-6-phosphate dehydrogenase (G6PD) of the Brazilian opossum Didelphis marsupialis in a hemolysate was shown to be high, 207 +/- 38 IU g-1 Hb-1 min-1 at 37 degrees C, compared to the human erythrocyte activity of 12 +/- 2 IU g-1 Hb-1 min-1 at 37 degrees C. The apparent high specific activity of the mixture led us to investigate the physicochemical properties of the opossum enzyme. We report that reduced glutathione (GSH) in the erythrocytes was only 50% higher than in human erythrocytes, a value lower than expected from the high G6PD activity since GSH is maintained in a reduced state by G6PD activity. The molecular mass, determined by G-200 Sephadex column chromatography at pH 8.0, was 265 kDa, which is essentially the same as that of human G6PD (260 kDa). The Michaelis-Menten constants (Km: 55 microM) for glucose-6-phosphate and nicotinamide adenine dinucleotide phosphate (Km: 3.3 microM) were similar to those of the human enzyme (Km: 50-70 and Km: 2.9-4.4, respectively). A 450-fold purification of the opossum enzyme was achieved and the specific activity of the purified enzyme, 90 IU/mg protein, was actually lower than the 150 IU/mg protein observed for human G6PD. We conclude that G6PD after purification from the hemolysate of D. marsupialis does not have a high specific activity. Thus, it is quite probable that the red cell hyperactivity reported may be explained by increased synthesis of G6PD molecules per unit of hemoglobin or to reduced inactivation in the RBC hemolysate.


Assuntos
Didelphis/sangue , Eritrócitos/enzimologia , Glucosefosfato Desidrogenase/sangue , Glutationa/metabolismo , Animais , Brasil , Cromatografia , Eritrócitos/química , Glucosefosfato Desidrogenase/isolamento & purificação , Oxirredução
7.
Braz. j. med. biol. res ; 39(5): 611-614, May 2006. ilus, tab
Artigo em Inglês | LILACS | ID: lil-425795

RESUMO

In a comparative study of erythrocyte metabolism of vertebrates, the specific activity of glucose-6-phosphate dehydrogenase (G6PD) of the Brazilian opossum Didelphis marsupialis in a hemolysate was shown to be high, 207 ± 38 IU g-1 Hb-1 min-1 at 37°C, compared to the human erythrocyte activity of 12 ± 2 IU g-1 Hb-1 min-1 at 37°C. The apparent high specific activity of the mixture led us to investigate the physicochemical properties of the opossum enzyme. We report that reduced glutathione (GSH) in the erythrocytes was only 50 percent higher than in human erythrocytes, a value lower than expected from the high G6PD activity since GSH is maintained in a reduced state by G6PD activity. The molecular mass, determined by G-200 Sephadex column chromatography at pH 8.0, was 265 kDa, which is essentially the same as that of human G6PD (260 kDa). The Michaelis-Menten constants (Km: 55 æM) for glucose-6-phosphate and nicotinamide adenine dinucleotide phosphate (Km: 3.3 æM) were similar to those of the human enzyme (Km: 50-70 and Km: 2.9-4.4, respectively). A 450-fold purification of the opossum enzyme was achieved and the specific activity of the purified enzyme, 90 IU/mg protein, was actually lower than the 150 IU/mg protein observed for human G6PD. We conclude that G6PD after purification from the hemolysate of D. marsupialis does not have a high specific activity. Thus, it is quite probable that the red cell hyperactivity reported may be explained by increased synthesis of G6PD molecules per unit of hemoglobin or to reduced inactivation in the RBC hemolysate.


Assuntos
Animais , Didelphis/sangue , Eritrócitos/enzimologia , Glucosefosfato Desidrogenase/sangue , Glutationa/metabolismo , Brasil , Cromatografia , Eritrócitos/química , Glucosefosfato Desidrogenase/isolamento & purificação , Oxirredução
8.
Oncogene ; 25(39): 5405-15, 2006 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-16636674

RESUMO

Activating enhancer-binding protein 2alpha (AP-2alpha) and activating enhancer-binding protein 2gamma (AP-2gamma) are transcription factors that bind GC-rich consensus sequences and regulate the expression of many downstream genes. AP-2alpha and AP-2gamma interact with p53 both physically and functionally. Expression microarray results in human breast carcinoma cells with forced p53 expression revealed AP-2gamma as a putative transcriptional target of p53. To confirm and extend these findings we measured the effects of forced p53 expression in human breast carcinoma cells by real-time reverse transcription-PCR, Western blotting, electrophoretic gel mobility shift assays, promoter reporter, chromatin immunoprecipitation and chromatin accessibility assays. Wild-type p53 expression rapidly induced not only AP-2gamma but also AP-2alpha mRNA. The subsequent increase in these proteins led to increased AP-2 DNA-binding and transactivating activity. Candidate p53-binding sites were identified in the AP-2alpha and AP-2gamma promoters. p53 binding to these cis-elements in vivo was also observed, together with a relaxation of chromatin structure in these regions. Finally, expression of either AP-2alpha or gamma inhibited growth of human breast carcinoma cells in vitro. Taken together, our findings indicate that these AP-2 genes are targets for transcriptional activation by p53 and suggest that AP-2 proteins may mediate some of the downstream effects of p53 expression such as inhibition of proliferation.


Assuntos
Genes p53 , Fator de Transcrição AP-2/genética , Adenocarcinoma , Neoplasias da Mama/genética , Divisão Celular , Linhagem Celular Tumoral , Primers do DNA , Feminino , Genes Reporter , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Plasmídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição AP-2/metabolismo , Transcrição Gênica , Transfecção
9.
J Pharmacol Exp Ther ; 311(3): 968-81, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15302897

RESUMO

Dysregulation of epigenetic control is an important participant in carcinogenesis. The PML/RAR alpha translocation in acute promyelocytic leukemia (APL) is an example where the resultant fusion protein recruits histone deacetylase complexes to target genes resulting in their inappropriate transcriptional repression. All-trans-retinoic acid (ATRA) acts as a ligand that relieves this repression and produces an epigenetic transcriptional reprogramming of the cancer cell. CpG island microarrays were used to analyze the DNA methylation and histone acetylation state of the human APL cell line NB4 before and after differentiation with ATRA as well as normal peripheral blood mononuclear cells (PBMC). Over 70 CpG islands within 1 kb of transcription start of a known gene are aberrantly methylated in NB4 cells compared with PBMC; however, no changes in cytosine methylation were detected following ATRA-induced differentiation. With respect to histone H4 acetylation, over 100 single-copy CpG islands within 1 kb of transcription start of a known human gene became hyperacetylated following ATRA-induced differentiation. One CpG island was aberrantly methylated in NB4 cells, but became hyperacetylated and was induced following ATRA treatment and was associated with the HoxA1 gene, suggesting it may be a target gene of ATRA in APL. In addition to single-copy sequences, a selective increase in acetylation was detected in satellite DNA when compared with other high-copy sequences, such as Alu or rDNA. In summary, ATRA stimulates complex epigenomic changes during leukemic cell differentiation, and monitoring these changes may help to identify new targets of epigenetic dysfunction.


Assuntos
Ilhas de CpG/genética , Citosina/metabolismo , Histonas/metabolismo , Leucemia/genética , Leucemia/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Acetilação , Diferenciação Celular/genética , Cromatina/metabolismo , DNA/genética , DNA/isolamento & purificação , Bases de Dados Genéticas , Biblioteca Gênica , Humanos , Imunoprecipitação , Hibridização In Situ , Ceratolíticos/farmacologia , Metilação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sulfitos/química , Tretinoína/farmacologia
10.
Parasitology ; 128(Pt 5): 483-91, 2004 May.
Artigo em Inglês | MEDLINE | ID: mdl-15180316

RESUMO

The Cytochrome b (Cyt b) gene has proved to be useful for identification and classification of many mammals and plants. In order to evaluate the utility of this gene for discrimination of Leishmania parasites as well as for exploring their phylogenetic relationships, we determined the nucleotide sequences of the Cyt b gene from 13 human-infecting Leishmania species (14 strains) from the New and Old Worlds. The Cyt b genes, approximately 1080 base pairs, were found to be A/T rich, and their 5' terminal-editing regions were highly conserved. The nucleotide sequence variation among them was enough to discriminate parasite species; 245 nucleotide positions were polymorphic and 190 positions were parsimony informative. The phylogenetic relationships based on this gene, showed good agreement with the classification of Lainson & Shaw (1987) except for the inclusion of L. (L.) major in the L. (L.) tropica complex and the placement of L. tarentolae in another genus. These data show that the Cyt b gene is useful for phylogenetic study of Leishmania parasites.


Assuntos
Citocromos b/genética , Leishmania/enzimologia , Leishmania/genética , Sequência de Aminoácidos , Animais , Composição de Bases , Sequência de Bases , Sequência Conservada , Citocromos b/química , DNA de Protozoário/química , DNA de Protozoário/genética , Variação Genética , Humanos , Leishmania/classificação , Leishmaniose/parasitologia , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase , Alinhamento de Sequência
11.
J Fluoresc ; 14(6): 711-22, 2004 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15649023

RESUMO

Fura-2 is one of the most used fluorophore for measuring intracellular calcium concentration ([Ca2+]i). In mouse bone marrow cell suspensions ATP produces a biphasic effect: till 1 mM, ATP produces increases in [Ca2+]i; from 1 mM on an increase is observed, that is followed by the decrease in the 340/380 nm ratio (R340/380). At high ATP (4 mM) concentration fura-2 leaked from loaded bone marrow cell suspensions. We observed that ATP decreases fluorescence in the absorption and excitation spectra of fura-2, consequently the emitted one is decreased including the isobestic point (360 nm). ATP analogs: BzATP, ATPyS and UTP, but not alphabetaATP, ADP or AMP, promote decrease of fluorescence in the isobestic point of fura-2. The physical/chemical process that reduces the absorption and excitation of fura-2 by ATP is unknown. The P2X7 inhibitors, Mg2+ (5 mM), OxATP (300 microM) and Brilliant Blue (100 nM), blocked the efflux of fura-2 and ATP-induced R340/380 decrease. The J774 cell line and mononuclear cells with a higher expression of P2X7 receptors show the same decrease in R340/380 as that induced by ATP. In the HL-60 cell line, myeloid cells and erythroblasts extracted from bone marrow, such effect does not occur. It is concluded that the use of the fluorescent Ca2+ indicator fura-2 does not allow the correct measurement of [Ca2+]i in these cells in the presence of a higher concentration of ATP which activated the P2X7 receptor.


Assuntos
Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Células da Medula Óssea/metabolismo , Corantes Fluorescentes/metabolismo , Fura-2/metabolismo , Receptores Purinérgicos P2/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Células da Medula Óssea/efeitos dos fármacos , Cálcio/metabolismo , Cálcio/farmacologia , Linhagem Celular , Feminino , Células HL-60 , Humanos , Técnicas In Vitro , Magnésio/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Antagonistas do Receptor Purinérgico P2 , Receptores Purinérgicos P2X7 , Corantes de Rosanilina/farmacologia , Espectrometria de Fluorescência
13.
Leukemia ; 17(2): 451-7, 2003 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-12592346

RESUMO

Mutations of the ras gene are among the most commonly identified transforming events in human cancers, including multiple myeloma. Farnesyltransferase inhibitors (FTI) were developed to prevent Ras processing and induce cancer cell death. Several FTIs are in phase II and one is in phase III clinical trials. Preclinically, most of the focus has been on solid tumors, and the effects of FTIs in multiple myeloma have not been investigated. In this study we examined the cytotoxic activity and inhibition of Ras processing in three myeloma cell lines with differing Ras mutation status. H929 cells with activated N-Ras were more sensitive to FTI-277 treatment than 8226 and U266 cells with activated K-Ras or wild-type Ras, respectively. A combination of FTI-277 and a geranylgeranyltransferase I inhibitor (GGTI)-2166 inhibited K-Ras processing and enhanced cell death in 8226 cells. U266 cells and Bcl-x(L) transfectants were equally sensitive to FTI-277 treatment. Similarly, 8226 cells selected for resistance to various chemotherapeutic agents, which resulted in either P-glycoprotein overexpression, altered topoisomerase II activity, or elevated glutathione levels, were equally sensitive to FTI-277. These preclinical studies suggest that prenylation inhibitors may represent new therapeutic agents for the treatment of refractory or drug-resistant multiple myeloma.


Assuntos
Alquil e Aril Transferases/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Metionina/análogos & derivados , Metionina/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Farnesiltranstransferase , Genes ras/efeitos dos fármacos , Humanos , Mieloma Múltiplo/genética , Células Tumorais Cultivadas , Ensaio Tumoral de Célula-Tronco
14.
Clin Cancer Res ; 7(12): 4262-71, 2001 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11751528

RESUMO

Our previous work demonstrated that the Janus kinase (JAK)-Stat3 pathway regulates expression of Bcl-x(L) in the U266 human multiple myeloma cell line and prevents Fas-mediated apoptosis. Inhibition of this pathway by the JAK selective kinase inhibitor AG490 or dominant-negative Stat3 protein results in down-regulation of Bcl-x(L) expression and enhanced sensitivity to Fas-mediated apoptosis. Because Bcl-x(L) has also been implicated in resistance to chemotherapeutic drugs, we investigated whether inhibition of the JAK-Stat3 pathway and subsequent reduction in Bcl-x(L) expression would also enhance cytotoxic drug activity. Contrary to this prediction, pretreatment of U266 myeloma cells with AG490, followed by exposure to topoisomerase II- inhibiting agents, antagonized drug-induced apoptosis. This effect correlated with reduced cyclin D1 expression and cell cycle arrest. The cell cycle arrest following AG490 pretreatment further correlated with reduced mitoxantrone-induced DNA double-strand breaks and reduced cell death, findings consistent with the critical requirement of DNA damage for drug cytotoxicity. These studies demonstrate that inhibition of the JAK-Stat3 pathway can result in paradoxical effects relative to cytotoxic drug response. These paradoxical responses may be explained by the findings that JAK-Stat3 signaling regulates the expression of multiple genes involved in controlling cell proliferation and apoptosis. Thus, understanding the cellular context of inhibiting signal transduction pathways is essential for the design of novel combination therapies for cancer.


Assuntos
Apoptose/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Proteínas Proto-Oncogênicas , Inibidores da Topoisomerase II , Receptor fas/fisiologia , Antineoplásicos/farmacologia , Regulação Neoplásica da Expressão Gênica , Humanos , Janus Quinase 1 , Janus Quinase 2 , Janus Quinase 3 , Mieloma Múltiplo/patologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Recombinantes/antagonistas & inibidores , Transfecção , Células Tumorais Cultivadas , Tirfostinas/farmacologia , Proteína bcl-X
15.
J Dermatol ; 28(9): 475-80, 2001 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-11603387

RESUMO

In this study, an attempt was made to identify different Leishmania species by polymerase chain reaction (PCR). Fourteen Leishmania strains from stock were tested by PCR and Southern blotting. A pair of primers were employed that anneal to the kinetoplast DNA sequence conserved among subgenus Leishmania. Of the 14 Leishmania strains used in this study, six showed strong bands of approximately 170 bp, and all the positive strains belonged to the species of the subgenus Leishmania viz., Leishmania (Leishmania) garnhami, L. (L.) amazonensis, L. (L.) pifanoi, L. (L.) mexicana, L. (L.) chagasi, and L. (L.) major All the species belonging to the subgenus Viannia used in this study were negative by PCR. These results suggest that the primer pair may be useful for identification of the species belonging to the subgenus Leishmania of the New World as well as to distinguish subgenus Leishmania from subgenus Viannia.


Assuntos
Southern Blotting/métodos , Leishmania/classificação , Leishmania/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Animais , Sequência de Bases , Humanos , Leishmaniose Cutânea/diagnóstico , Dados de Sequência Molecular , Sensibilidade e Especificidade
16.
Histochem Cell Biol ; 115(5): 373-80, 2001 May.
Artigo em Inglês | MEDLINE | ID: mdl-11449885

RESUMO

Heparan sulfate proteoglycans (HS-PGs) are associated with important cell functions, for example, cell motility, cell adhesion, and oncogenesis. We examined the localization of HS-PGs in normal and carcinoma tissues of the gastrointestinal tract to help elucidate their roles in these organs. Fresh surgical materials from 134 patients with carcinoma of the stomach or large intestine and 26 patients with various diseases of the small intestine were immunostained after fixation with 10E4 (an antibody against the HS of HS-PG) as a primary antibody. Immunoelectron microscopy (immunogold method) was also performed. The basolateral surfaces of normal tissues of the large and small intestines were strongly stained with antibody confirmed by electron microscopy. In the stomach, lesions with intestinal metaplasia showed the same staining as the intestines, although normal gastric tissue showed staining only in some parts of the basal layer of fundic and pyloric glands. Carcinoma tissues in all cases examined showed staining with antibody. Better results were obtained after fixation in acetic alcohol or zinc-containing solutions than in ordinary formalin. These characteristic localizations of HS-PG in intestines and stomachs suggest that this kind of HS-PG staining could be a hallmark characteristic of the intestine.


Assuntos
Sistema Digestório/química , Heparina/análogos & derivados , Heparina/metabolismo , Proteoglicanas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais , Colo/química , Colo/citologia , Sistema Digestório/citologia , Feminino , Heparina/imunologia , Humanos , Imuno-Histoquímica , Neoplasias Intestinais/metabolismo , Neoplasias Intestinais/patologia , Intestino Delgado/química , Intestino Delgado/citologia , Masculino , Microscopia Imunoeletrônica , Pessoa de Meia-Idade , Proteoglicanas/imunologia , Coloração e Rotulagem , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/secundário , Distribuição Tecidual
17.
Blood ; 98(3): 805-13, 2001 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-11468182

RESUMO

Multiple myeloma (MM) is a clonal B-cell malignancy characterized by slow-growing plasma cells in the bone marrow (BM). Patients with MM typically respond to initial chemotherapies; however, essentially all progress to a chemoresistant state. Factors that contribute to the chemorefractory phenotype include modulation of free radical scavenging, increased expression of drug efflux pumps, and changes in gene expression that allow escape from apoptotic signaling. Recent data indicate that arsenic trioxide (As(2)O(3)) induces remission of refractory acute promyelocytic leukemia and apoptosis of cell lines overexpressing Bcl-2 family members; therefore, it was hypothesized that chemorefractory MM cells would be sensitive to As(2)O(3). As(2)O(3) induced apoptosis in 4 human MM cell lines: 8226/S, 8226/Dox40, U266, and U266/Bcl-x(L). The addition of interleukin-6 had no effect on cell death. Glutathione (GSH) has been implicated as an inhibitor of As(2)O(3)-induced cell death either through conjugating As(2)O(3) or by sequestering reactive oxygen induced by As(2)O(3). Consistent with this possibility, increasing GSH levels with N-acetylcysteine attenuated As(2)O(3) cytotoxicity. Decreases in GSH have been associated with ascorbic acid (AA) metabolism. Clinically relevant doses of AA decreased GSH levels and potentiated As(2)O(3)-mediated cell death of all 4 MM cell lines. Similar results were obtained in freshly isolated human MM cells. In contrast, normal BM cells displayed little sensitivity to As(2)O(3) alone or in combination with AA. Together, these data suggest that As(2)O(3) and AA may be effective antineoplastic agents in refractory MM and that AA might be a useful adjuvant in GSH-sensitive therapies. (Blood. 2001;98:805-813)


Assuntos
Arsenicais/farmacologia , Ácido Ascórbico/farmacologia , Mieloma Múltiplo/patologia , Óxidos/farmacologia , Acetilcisteína/farmacologia , Antineoplásicos/farmacologia , Antioxidantes/farmacologia , Trióxido de Arsênio , Morte Celular/efeitos dos fármacos , Sinergismo Farmacológico , Glutationa/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/metabolismo , Plasmócitos/efeitos dos fármacos , Superóxidos/metabolismo , Células Tumorais Cultivadas/efeitos dos fármacos
18.
J Clin Invest ; 107(3): 351-62, 2001 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-11160159

RESUMO

Large granular lymphocyte (LGL) leukemia is characterized by the expansion of antigen-activated cytotoxic T lymphocytes. These leukemic cells are resistant to Fas-mediated apoptosis despite expressing high levels of Fas. We found that leukemic LGL from 19 patients displayed high levels of activated STAT3. Treatment of leukemic LGL with the JAK-selective tyrosine kinase inhibitor AG-490 induced apoptosis with a corresponding decrease in STAT-DNA binding activity. Moreover, using an antisense oligonucleotide approach to diminish STAT3 expression, we found that Fas sensitivity was restored in leukemic LGL. AG-490-induced apoptosis in leukemic LGL was independent of Bcl-xL or Bcl-2 expression. However, we found that the Bcl-2-family protein Mcl-1 was significantly reduced by AG-490 treatment. Activated STAT3 was shown to bind an SIE-related element in the murine mcl-1 promoter. Using a luciferase reporter assay, we demonstrated that v-src overexpression in NIH3T3 induced STAT3-dependent transcriptional activity from the mcl-1 promoter and increased endogenous Mcl-1 protein levels. We conclude that STAT3 activation contributed to accumulation of the leukemic LGL clones. These findings suggest that investigation should focus on novel strategies targeting STAT3 in the treatment of LGL leukemia.


Assuntos
Linfócitos T CD8-Positivos/fisiologia , Proteínas de Ligação a DNA/antagonistas & inibidores , Leucemia/fisiopatologia , Proteínas de Neoplasias/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2 , Transativadores/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Western Blotting , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Dimerização , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica , Humanos , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteínas de Neoplasias/genética , Proteína Oncogênica pp60(v-src) , Fosforilação , Fator de Transcrição STAT3 , Transdução de Sinais , Transativadores/metabolismo , Células Tumorais Cultivadas , Tirfostinas/farmacologia
19.
Br J Ophthalmol ; 85(1): 21-3, 2001 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11133706

RESUMO

AIM: To investigate change in the area of anterior capsular opening (ACO) after cataract surgery and its relation to the degree of postoperative anterior inflammation in patients with diabetes mellitus (DM). METHODS: 31 eyes of 31 patients with DM and 30 eyes of 30 normal controls scheduled to undergo cataract surgery were examined prospectively. The area of ACO was measured with an anterior eye segment analysis system (EAS-1000) on the day following surgery and 3, 6, and 12 months after surgery. Comparative analyses were made on the area of ACO relative to the presence of DM and diabetic retinopathy (DR). The percentage reduction of area of ACO was calculated from values 1 day and 12 months after surgery, and multiple regression analysis was performed on the presence of DM, patient age, ACO area on the first postoperative day, and aqueous flare intensity 1 day and 12 months after surgery. RESULTS: The area was significantly smaller in the DM group at 3 (p=0.015, Student's t test), 6 (p=0.011), and 12 (p=0.010) months postoperatively. Patients having DR showed significantly smaller ACO area than the non-DR group 3 (p=0.039), 6 (p=0.033), and 12 (p=0.028) months after surgery. Multiple regression analysis revealed that presence of DM (p=0.003) and aqueous flare intensity 12 months after surgery (p=0.039) significantly correlated with the percentage reduction of area of ACO. Age, ACO area at 1 day postoperatively, and aqueous flare intensity immediately after surgery were not relevant to ACO contraction. CONCLUSIONS: Anterior capsular contraction after cataract surgery was greater in eyes of DM patients, especially in those with DR and increased permeability of the blood-aqueous barrier.


Assuntos
Diabetes Mellitus/patologia , Cápsula do Cristalino/patologia , Facoemulsificação , Idoso , Humor Aquoso , Retinopatia Diabética/patologia , Endoftalmite/patologia , Seguimentos , Humanos , Pessoa de Meia-Idade , Complicações Pós-Operatórias/patologia , Período Pós-Operatório , Estudos Prospectivos , Análise de Regressão
20.
Acta Paediatr ; 90(11): 1283-7, 2001 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11808900

RESUMO

UNLABELLED: To discover the relationship between antioxidant enzyme activities and trace elements in low-birthweight infants during the early postnatal period, we analysed catalase (CAT), CuZn-superoxide dismutase (CuZn-SOD) and glutathione peroxidase (GSH-Px) activities in erythrocytes. and compared them with Fe, Cu. Zn and Se levels in plasma, and Cu, Zn and Se levels in erythrocytes until 16 wk after birth. Thirteen low-birthweight infants whose mean birthweight and gestation were 1520 +/- 293 g and 32.0 +/- 2.8 wk were enrolled in this study. All infants were without chronic complications, well nourished, and predominantly fed standard formula or preterm formula based on cow's milk, commercially available in Japan. Cu and Zn levels in erythrocytes did not decline after birth, in contrast to a temporal decrease in plasma Zn. Erythrocyte CAT activity was significantly higher at 16 wk than that at birth or 4 wk of age. Erythrocyte CuZn-SOD activity did not change throughout the study period. Only Se in plasma and erythrocytes decreased remarkably after birth, which resulted in a significant decline in erythrocyte GSH-Px activity at 8 and 16 wk (11.2 +/- 2.0 and 11.0 +/- 1.1 U/g Hb) compared to that at birth (12.4 +/- 2.1 U/g Hb). CONCLUSION: During the early postnatal period. erythrocyte CAT and CuZn-SOD activities did not decrease in formula-fed, low-birthweight infants. Japanese commercial formula not supplemented with Se, however, caused postnatal plasma and erythrocyte Se deficiency and a concomitant decline in erythrocyte GSH-Px activity in the same subjects.


Assuntos
Recém-Nascido de Baixo Peso/sangue , Oxirredutases/sangue , Oligoelementos/sangue , Catalase/sangue , Eritrócitos/química , Feminino , Glutationa Peroxidase/sangue , Humanos , Alimentos Infantis , Recém-Nascido , Unidades de Terapia Intensiva Neonatal , Japão , Modelos Lineares , Masculino , Selênio/sangue , Estatísticas não Paramétricas , Superóxido Dismutase/sangue
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