RESUMO
We retrospectively studied the frequency of persistent foci of fat signal on magnetic resonance (MR) imaging in osteomyelitis to assess its frequency, cause and diagnostic value. The radiographs and MR scans of 100 patients with a final diagnosis of osteomyelitis referred to a specialist orthopaedic oncology service with the presumptive diagnosis of a bone tumour were reviewed. The MR signal and morphological characteristics were recorded with particular attention to the presence of persistent fat signal within the infected area, which was classified as diffuse or focal. Seventeen cases were classified on radiographic grounds as acute, 63 as subacute and 20 as chronic osteomyelitis. In the acute group 12 (70%) showed replacement of the marrow with fluid containing residual fatty signal, diffuse in seven and focal in five cases. Two cases showed predominantly fatty marrow with very early marrow oedema and three cases (18%) showed replacement of marrow fat with fluid and no residual fatty foci. None of the subacute group showed foci of fatty signal and two cases of inactive sclerosing osyeomyelitis in the chronic group showed restoration of normal marrow. Persistent fatty signal within the bone as well as soft tissues on MR imaging is a frequent finding in acute osteomyelitis. Radiological-pathological correlation suggests that the increasing intramedullary pressure leads to septic necrosis with death of the lipocytes and release of free fatty globules. This characteristic, but not pathognomonic, MR finding supports the diagnosis of osteomyelitis and may help to exclude the presence of a tumour.
Assuntos
Tecido Adiposo/patologia , Medula Óssea/patologia , Imageamento por Ressonância Magnética , Osteomielite/patologia , Tecido Adiposo/diagnóstico por imagem , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Medula Óssea/diagnóstico por imagem , Criança , Pré-Escolar , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Masculino , Pessoa de Meia-Idade , Osteomielite/diagnóstico por imagem , Radiografia , Estudos RetrospectivosRESUMO
Peroxisome proliferators, which function as peroxisome proliferator-activated receptor alpha (PPARalpha) agonists, induce peroxisomal, microsomal, and mitochondrial fatty acid oxidation enzymes, in conjunction with peroxisome proliferation, in liver cells. Sustained activation of PPARalpha leads to the development of liver tumors in rats and mice. The assertion that synthetic PPARalpha ligands pose negligible carcinogenic risk to humans is attributable, in part, to the failure to observe peroxisome proliferation in human hepatocytes. To explore the mechanism(s) of species-specific differences in response to PPARalpha ligands, we determined the functional competency of human PPARalpha in vivo and compared its potency with that of mouse PPARalpha. Recombinant adenovirus that expresses human or mouse PPARalpha was produced and administered intravenously to PPARalpha-deficient mice. Human as well as mouse PPARalpha fully restored the development of peroxisome proliferator-induced immediate pleiotropic responses, including peroxisome proliferation and enhanced expression of genes involved in lipid metabolism as well as nonperoxisomal genes, such as CD36, Ly-6D, Rbp7, monoglyceride lipase, pyruvate dehydrogenase kinase-4, and C3f, that have been identified recently to be up-regulated in livers with peroxisome proliferation. These studies establish that human PPARalpha is functionally competent and is equally as dose-sensitive as mouse PPARalpha in inducing peroxisome proliferation within the context of mouse liver environment and that it can heterodimerize with mouse retinoid X receptor, and this human PPARalpha-mouse retinoid X receptor chimeric heterodimer transcriptionally activates mouse PPARalpha target genes in a manner qualitatively similar to that of mouse PPARalpha.
Assuntos
Fígado/metabolismo , Peroxissomos/metabolismo , Receptores Citoplasmáticos e Nucleares/fisiologia , Fatores de Transcrição/fisiologia , Animais , Divisão Celular/efeitos dos fármacos , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxirredução , Peroxissomos/efeitos dos fármacos , Pirimidinas/farmacologia , RNA Mensageiro/análiseRESUMO
Powdery mildew diseases are economically important diseases, caused by obligate biotrophic fungi of the Erysiphales. To understand the complex inheritance of resistance to the powdery mildew disease in the model plant Arabidopsis thaliana, quantitative trait loci analysis was performed using a set of recombinant inbred lines derived from a cross between the resistant accession Kashmir-1 and the susceptible accession Columbia glabrous1. We identified and mapped three independent powdery mildew quantitative disease resistance loci, which act additively to confer disease resistance. The locus with the strongest effect on resistance was mapped to a 500-kbp interval on chromosome III.
Assuntos
Arabidopsis/genética , Ascomicetos/patogenicidade , Característica Quantitativa Herdável , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/microbiologia , Arabidopsis/fisiologia , Sequência de Bases , Primers do DNA , FenótipoRESUMO
To assess the role of hepatocyte nuclear factor-3beta (HNF-3beta) in hepatocyte-specific gene transcription, we reported the characterization of the liver phenotype with transgenic mice in which the -3-kb transthyretin (TTR) promoter functioned to increase HNF-3beta expression. During breeding of the TTR-HNF-3beta transgenic mice we noticed that they displayed severe ataxia. In this study, we describe the analysis of our transgenic cerebellar phenotype and demonstrate that ectopic expression of HNF-3beta disrupted cerebellar morphogenesis and caused reduction in cerebellar size. In postnatal cerebellum, the HNF-3beta transgene expression pattern is colocalized to glial fibrillary acidic protein-positive cerebellar astrocytes and Bergmann glial cells. As a result of protracted expression, the transgenic cerebella are impaired in terms of astrocyte dispersal and formation of Bergmann glial cell processes. This caused a disruption in neuronal cell migration to the cortical laminar layers and Purkinje dendritic arbor maturation, thus leading to diminished foliation. Differential hybridization of cDNA arrays was used to identify altered expression of cerebellar genes, which is consistent with the observed defect in transgenic cerebellar morphogenesis and size as well as glial maturation. These include diminished expression of the brain lipid-binding protein, which is required for glial morphological differentiation, and the basic helix-loop-helix NeuroD/Beta2 and homeodomain Engrailed-2 transcription factors, which are required for normal cerebellar morphogenesis and foliation. Undetectable levels of ataxia telangiectasia (ATM), which is required for proper development of the Purkinje dendritic arbor, were found in postnatal transgenic cerebella. Furthermore, the transgenic cerebella displayed levels of insulin-like growth factor binding protein-1 elevated to 22 times greater than those measured for wild-type cerebella, an elevation consistent with the reduction in transgenic cerebellar size.
Assuntos
Cerebelo/crescimento & desenvolvimento , Cerebelo/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Animais , Animais Recém-Nascidos , Antígenos CD/genética , Apoptose , Astrócitos/citologia , Astrócitos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Moléculas de Adesão Celular Neuronais/genética , Diferenciação Celular , Movimento Celular , Cerebelo/anormalidades , Cerebelo/citologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Receptores ErbB/genética , Proteínas da Matriz Extracelular/genética , Expressão Gênica , Marcadores Genéticos/genética , Fator 3-beta Nuclear de Hepatócito , Hibridização In Situ , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Integrina alfa5 , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso , Receptores de Netrina , Neuroglia/citologia , Neuroglia/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/imunologia , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Células de Purkinje/citologia , Células de Purkinje/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor ErbB-4 , Receptores de Fator de Crescimento Neural/genética , Proteína Reelina , Serina Endopeptidases , Fatores de Transcrição/genéticaRESUMO
In human cells, telomerase activity is regulated by transcriptional control of the telomerase reverse transcriptase gene (hTERT) whose product is the catalytic subunit of the enzyme. The hTERT promoter is active in virtually all types of tumors and immortal cells, but is silent in most adult somatic tissues. In this study, we placed the herpes simplex virus thymidine kinase gene under the control of the hTERT promoter with the aim of restricting its expression to tumor cells. In transfection experiments, the hTERT promoter driven thymidine kinase gene (hTERTp/TK) conferred ganciclovir sensitivity to all tumor and immortal cell lines tested, whereas normal somatic cells remained largely unaffected. Human hTERTp/TK-positive cancer cells implanted in nude mice developed into tumors that could be eradicated by ganciclovir treatment. The hTERTp/TK cassette was inserted into an adenovirus vector and its efficacy in reducing tumor growth was compared with that of an adenovirus carrying the thymidine kinase gene under the control of the cytomegalovirus immediate-early promoter (CMVp/TK). In a xenograft model using the human 143B osteosarcoma cell line, a single injection of either virus resulted in equivalent tumor regression and survival upon ganciclovir treatment. In animals injected intratumorally with the CMVp/TK adenovirus, expression of the thymidine kinase gene was detected in tumors, as well as in liver samples. Expression of the suicide gene in combination with ganciclovir resulted in severe liver histopathology and in an elevation of hepatic enzymes. In sharp contrast, when the hTERT promoter controlled the thymidine kinase gene, transgene expression was observed in tumors, but not in liver samples. Normal liver function in these animals was confirmed by serum levels of hepatic enzymes that were indistinguishable from those of control healthy mice. These results indicate that by restricting thymidine kinase expression to tumor cells, the hTERT promoter allows the tumoricidal effect of the suicidal gene to be exerted without detrimental consequences on healthy tissues and vital organs. The tight specificity of expression imparted by the hTERT promoter will assist the development of novel approaches to the treatment of a broad array of cancer types.
Assuntos
Terapia Genética/métodos , Hepatopatias/prevenção & controle , Osteossarcoma/terapia , Regiões Promotoras Genéticas , RNA , Telomerase/genética , Adenoviridae/genética , Animais , Proteínas de Ligação a DNA , Ganciclovir/uso terapêutico , Regulação Neoplásica da Expressão Gênica , Terapia Genética/efeitos adversos , Vetores Genéticos/uso terapêutico , Humanos , Hepatopatias/etiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , Osteossarcoma/genética , Osteossarcoma/patologia , RNA Mensageiro/genética , Taxa de Sobrevida , Timidina Quinase/genética , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
The cytotoxicity of high-dose methotrexate (MTX), 10 and 100 microM, and 5-fluorouracil (5-FU) combinations is independent of sequence in human MDA-MB-436 breast carcinoma cells. The growth inhibitory effects of 10 and 100 microM MTX are 22.54+/-1.56% and 16.20+/-0.74%, respectively, of the control rate. When the MTX and 5-FU concentrations are 10 microM, antiproliferative effects of MTX 2 hr before 5-FU (MTX/5-FU) and 5-FU 2 h before MTX (5-FU/MTX) are 25.17+/-1.23% and 25.60+/-1.28% of the control rate, respectively. The percentage of control rates for 5-FU alone is 94.89+/-1.35%. The growth rates of MDA-MB-436 cells in 100 microM MTX and 10 microM 5-FU are 15.19+/-0.62% (MTX/5-FU) and 16.53+/-0.85% (5-FU/MTX) of the control rate. The growth of cancer cells in the presence of 5-FU alone is 93.82+/-1.69% of the control rate. A comparison of the cell-killing effects of MTX and the nonpolyglutamable antifolate trimetrexate (TMQ) alone and in combination with 5-FU was performed to indirectly explore the role of polyglutamylation in breast cancer and bone marrow cells. The comparisons were made in equitoxic concentrations (10 microM) of MTX and TMQ and the time of exposure was the same. The inhibitory effects of TMQ, TMQ/5-FU, and 5-FU/TMQ in breast cancer cells were identical, but significantly less than MTX, MTX/5-FU, and 5-FU/MTX. The interaction between TMQ and MTX, TMQ/5-FU and MTX/5-FU, and 5-FU/TMQ and 5-FU/MTX was quantitatively similar in bone marrow. (Significant protection occurred in bone marrow cells exposed to 5-FU/TMQ and 5-FU/MTX.) Because the effects of 5-FU/MTX and 5-FU/TMQ on bone marrow were the same, it is unlikely that polyglutamylation plays a significant role in the protective effects of 5-FU. However, the greater inhibitory effect of MTX or MTX and 5-FU combinations, when compared with TMQ or TMQ and 5-FU, suggests that polyglutamylation of MTX may contribute to the cytotoxicity of this antifolate to breast cancer cells. Hence, these studies suggest that a priming and nontoxic dose of 5-FU before high-dose MTX sustains MTX cytotoxicity in breast cancer and protects against MTX toxicity to bone marrow progenitor cells.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Células da Medula Óssea/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Metotrexato/farmacologia , Antimetabólitos Antineoplásicos/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Linhagem Celular , Relação Dose-Resposta a Droga , Esquema de Medicação , Fluoruracila/administração & dosagem , Fluoruracila/farmacologia , Antagonistas do Ácido Fólico/administração & dosagem , Antagonistas do Ácido Fólico/farmacologia , Humanos , Metotrexato/administração & dosagem , Trimetrexato/administração & dosagem , Trimetrexato/farmacologia , Células Tumorais CultivadasRESUMO
Bisphosphonates are effective in the management of bone disease in patients with multiple myeloma and recent reports have suggested that they may also have an anti-tumour activity. In support of this, we have previously demonstrated that bisphosphonates can induce myeloma cell apoptosis in vitro; however, it remains unclear whether this occurs in vivo. We have therefore investigated the effect of the potent bisphosphonate ibandronate in the 5T2MM murine model of established multiple myeloma. Short-term treatment with a high dose of ibandronate had no effect on either myeloma cell number or the proportion of myeloma cells undergoing apoptosis. These observations suggest that although bisphosphonates induce apoptosis in myeloma cells in vitro, they may not have the same anti-tumour effects in vivo.
Assuntos
Difosfonatos/uso terapêutico , Mieloma Múltiplo/tratamento farmacológico , Neoplasias Experimentais/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Células da Medula Óssea/patologia , Ácido Ibandrônico , Camundongos , Camundongos Endogâmicos C57BL , Mieloma Múltiplo/patologia , Falha de Tratamento , Células Tumorais CultivadasRESUMO
Branch points and flexures in the high pressure arterial system have long been recognized as sites of unusually high turbulence and consequent stress in humans are foci for atherosclerotic lesions. We show that mice that are homozygous for a null mutation in the gene encoding an endogenous antiinflammatory cytokine, interleukin 1 receptor antagonist (IL-1ra), develop lethal arterial inflammation involving branch points and flexures of the aorta and its primary and secondary branches. We observe massive transmural infiltration of neutrophils, macrophages, and CD4(+) T cells. Animals appear to die from vessel wall collapse, stenosis, and organ infarction or from hemorrhage from ruptured aneurysms. Heterozygotes do not die from arteritis within a year of birth but do develop small lesions, which suggests that a reduced level of IL-1ra is insufficient to fully control inflammation in arteries. Our results demonstrate a surprisingly specific role for IL-1ra in the control of spontaneous inflammation in constitutively stressed artery walls, suggesting that expression of IL-1 is likely to have a significant role in signaling artery wall damage.
Assuntos
Arterite/imunologia , Receptores de Interleucina-1/antagonistas & inibidores , Sialoglicoproteínas/genética , Idade de Início , Alelos , Animais , Arterite/genética , Arterite/patologia , Linfócitos B/citologia , Linfócitos B/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Feminino , Humanos , Proteína Antagonista do Receptor de Interleucina 1 , Longevidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Interleucina-1/análise , Sialoglicoproteínas/imunologiaRESUMO
High-dose methotrexate (MTX) cytotoxicity is maintained in MCF-7 breast cancer cells but reduced in Hs824.T human bone marrow by a priming and nontoxic 5-fluorouracil (5-FU) dose. When MCF-7 breast or Hs824.T bone marrow cells are incubated with 10 microM 5-FU and 10 microM MTX for 48 h, the growth rates of breast cancer cells were 97.59 +/- 0.97% and 21.81 +/- 3.33% of the control rate, respectively, and the growth rates of bone marrow cells were 90.61 +/- 3.71% and 29.58 +/- 2.99% of the control rate. The combinations of 5-FU 2 h prior to MTX or MTX 2 h prior to 5-FU followed by a 48 h incubation, respectively, gave growth rates of 20.96 +/- 2.44% and 19.86 +/- 2.56% of the control rate for MCF-7 cells. In bone marrow cells, the combinations of 5-FU 2 h prior to MTX or MTX 2 h prior to 5-FU followed by a 48 h incubation, respectively, gave growth rates of 79.66 +/- 7.41% (protection) and 31.39 +/- 1.77% of the control rate. Similar patterns to bone marrow emerges in platelets. These studies suggest that: a) MTX and 5-FU combination on the growth of human MCF-7 breast cancer cells is independent of sequence; and b) a priming-dose of 5-FU will protect bone marrow from MTX cytotoxicity but not breast cancer cells. Therefore, a priming and non-toxic dose of 5-FU and MTX may have maximum antineoplastic activity while at the same time provide protection to the hematopoietic system.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Medula Óssea/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Fluoruracila/farmacologia , Metotrexato/farmacologia , Animais , Sinergismo Farmacológico , Feminino , Humanos , Masculino , Metotrexato/metabolismo , Metotrexato/toxicidade , CamundongosRESUMO
Bone is modeled during embryonic development by endochondral and membranous ossification and is continuously remodeled thereafter under the influence of local and systemic factors to provide structural support and assist in calcium homeostasis. Recent studies of knockout and transgenic mice have increased understanding of the regulation of bone modeling during development and of remodeling of mature bone and have shed new light on the pathogenesis of a number of bone disorders. For example, fibroblast growth factor receptor-3, parathyroid hormone-related protein, and tartrate-resistant acid phosphatase affect the function of chondrocytes during endochondral ossification (the latter two by regulating their life spans and thus growth plate thickness and bone length). Some ubiquitously expressed genes seem unexpectedly to have unique functions that are largely confined to bone cells: M-CSF, C-Fos, PU.1, and NF-kappaB are required for osteoclast formation, whereas c-Src and Mitf (microphthalmia transcription factor) are required for osteoclast activity after the cells have formed. Knockout of these genes results in osteopetrosis, a disorder characterized by persistence in marrow cavities of unresorbed osteocartilaginous matrix and, as in some affected humans, by increased mortality. Some proteins seem to act as negative regulators of bone cell function, for example osteoprotegerin (a soluble TNF receptor) in osteoclasts; osteocalcin, bone sialoprotein, and 5-lipoxygenase in osteoblasts. Regulation of osteoclast life span may be an important mechanism by which estrogen and bisphosphonates prevent bone loss in conditions characterized by increased bone resorption, such as postmenopausal osteoporosis. The unique requirement of bone cells for certain gene products raises the possibility that these cells may have specific responses to inhibitory or stimulatory agents, and that signaling molecules in these response pathways could be specific targets for novel therapies to treat or prevent common bone diseases.
Assuntos
Doenças Ósseas/etiologia , Osso e Ossos/fisiologia , Fisiologia/tendências , Animais , Desenvolvimento Ósseo/fisiologia , Remodelação Óssea/fisiologia , Osso e Ossos/citologia , CamundongosRESUMO
Nitrogen-containing bisphosphonates were shown to cause macrophage apoptosis by inhibiting enzymes in the biosynthetic pathway leading from mevalonate to cholesterol. This study suggests that, in osteoclasts, geranylgeranyl diphosphate, the substrate for prenylation of most GTP binding proteins, is likely to be the crucial intermediate affected by these bisphosphonates. We report that murine osteoclast formation in culture is inhibited by both lovastatin, an inhibitor of hydroxymethylglutaryl CoA reductase, and alendronate. Lovastatin effects are blocked fully by mevalonate and less effectively by geranylgeraniol whereas alendronate effects are blocked partially by mevalonate and more effectively by geranylgeraniol. Alendronate inhibition of bone resorption in mouse calvaria also is blocked by mevalonate whereas clodronate inhibition is not. Furthermore, rabbit osteoclast formation and activity also are inhibited by lovastatin and alendronate. The lovastatin effects are prevented by mevalonate or geranylgeraniol, and alendronate effects are prevented by geranylgeraniol. Farnesol and squalene are without effect. Signaling studies show that lovastatin and alendronate activate in purified osteoclasts a 34-kDa kinase. Lovastatin-mediated activation is blocked by mevalonate and geranylgeraniol whereas alendronate activation is blocked by geranylgeraniol. Together, these findings support the hypothesis that alendronate, acting directly on osteoclasts, inhibits a rate-limiting step in the cholesterol biosynthesis pathway, essential for osteoclast function. This inhibition is prevented by exogenous geranylgeraniol, probably required for prenylation of GTP binding proteins that control cytoskeletal reorganization, vesicular fusion, and apoptosis, processes involved in osteoclast activation and survival.
Assuntos
Alendronato/farmacologia , Reabsorção Óssea/metabolismo , Diterpenos/farmacologia , Osteoclastos/efeitos dos fármacos , Proteínas Quinases/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Colesterol/biossíntese , Ácido Clodrônico/farmacologia , Ativação Enzimática/efeitos dos fármacos , Farneseno Álcool/farmacologia , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Lovastatina/farmacologia , Ácido Mevalônico/metabolismo , Ácido Mevalônico/farmacologia , Camundongos , Osteoclastos/citologia , Coelhos , Crânio/metabolismo , Esqualeno/farmacologiaRESUMO
The growth inhibitory effect of trimetrexate (TMQ) is maintained in MCF-7 breast cancer but is decreased in Hs 824.T human bone marrow cells by a priming- and non-toxic 5-fluorouracil (5-FU) dose. Incubation of MCF-7 breast cells with 10 microM TMQ alone or in combination with 10 M 5-FU (TMQ 2 h prior to 5-FU [TMQ/5-FU] or 5-FU 2 h prior to TMQ[5-FU/TMQ]) resulted in similar inhibitory effects but dissimilar effects occurred in Hs 824.T bone marrow. In breast cancer, the percentage differences among TMQ and TMQ/5-FU, TMQ and 5-FU/TMQ, and TMQ/5-FU and 5-FU/TMQ on growth rates, respectively, were 3.56%, 2.35%, and 1.68%. The percentage differences on growth rates of TMQ and TMQ/5-FU, TMQ and 5-FU/TMQ, and TMQ/5-FU and 5-FU/TMQ in bone marrow, respectively, were 5.76%, 30.03% (significant protection by 5-FU, i.e. the inhibitory effect of 5-FU/TMQ < or = TMQ), and 35.78% (sequence dependent). The growth rates of breast cancer and bone marrow cells in the presence of 5-FU were 96.03 +/- 1.17% and 94.59 +/- 1.15%, respectively, of control rates. These studies suggest that (a) TMQ and 5-FU combinations on the growth of MCF-7 breast cancer cells are independent of sequence of administration and best related to TMQ and (b) a priming- and non-toxic 5-FU dose protects against TMQ toxicity in human bone marrow while not affecting the maximum inhibitory effect of TMQ in breast cancer.
Assuntos
Antimetabólitos Antineoplásicos/uso terapêutico , Medula Óssea/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Fluoruracila/uso terapêutico , Trimetrexato/uso terapêutico , Antimetabólitos Antineoplásicos/administração & dosagem , Antimetabólitos Antineoplásicos/toxicidade , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Esquema de Medicação , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Fluoruracila/administração & dosagem , Fluoruracila/toxicidade , Humanos , Trimetrexato/administração & dosagem , Células Tumorais CultivadasRESUMO
Bisphosphonates are currently the most important class of antiresorptive drugs used for the treatment of metabolic bone diseases. Although the molecular targets of bisphosphonates have not been identified, these compounds inhibit bone resorption by mechanisms that can lead to osteoclast apoptosis. Bisphosphonates also induce apoptosis in mouse J774 macrophages in vitro, probably by the same mechanisms that lead to osteoclast apoptosis. We have found that, in J774 macrophages, nitrogen-containing bisphosphonates (such as alendronate, ibandronate, and risedronate) inhibit post-translational modification (prenylation) of proteins, including the GTP-binding protein Ras, with farnesyl or geranylgeranyl isoprenoid groups. Clodronate did not inhibit protein prenylation. Mevastatin, an inhibitor of 3-hydroxy-3-methylglutatyl (HMG)-CoA reductase and hence the biosynthetic pathway required for the production of farnesyl pyrophosphate and geranylgeranyl pyrophosphate, also caused apoptosis in J774 macrophages and murine osteoclasts in vitro. Furthermore, alendronate-induced apoptosis, like mevastatin-induced apoptosis, could be suppressed in J774 cells by the addition of farnesyl pyrophosphate or geranylgeranyl pyrophosphate, while the effect of alendronate on osteoclast number and bone resorption in murine calvariae in vitro could be overcome by the addition of mevalonic acid. These observations suggest that nitrogen-containing bisphosphonate drugs cause apoptosis following inhibition of post-translational prenylation of proteins such as Ras. It is likely that these potent antiresorptive bisphosphonates also inhibit bone resorption by preventing protein prenylation in osteoclasts and that enzymes of the mevalonate pathway or prenyl protein transferases are the molecular targets of the nitrogen-containing bisphosphonates. Furthermore, the data support the view that clodronate acts by a different mechanism.
Assuntos
Reabsorção Óssea/tratamento farmacológico , Difosfonatos/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas ras/metabolismo , Alendronato/antagonistas & inibidores , Alendronato/farmacologia , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Ácido Clodrônico/farmacologia , Inibidores Enzimáticos/farmacologia , Ácido Etidrônico/análogos & derivados , Ácido Etidrônico/farmacologia , Hidroximetilglutaril-CoA Redutases/metabolismo , Hidroximetilglutaril-CoA-Redutases NADP-Dependentes , Ácido Ibandrônico , Lovastatina/análogos & derivados , Lovastatina/farmacologia , Macrófagos/efeitos dos fármacos , Ácido Mevalônico/metabolismo , Camundongos , Osteoclastos/efeitos dos fármacos , Prenilação de Proteína/efeitos dos fármacos , Ácido Risedrônico , Relação Estrutura-Atividade , Proteínas ras/químicaRESUMO
The potential of a new microanalytical method using magnetic beads (MBs) and commercial capillary electrophoresis (CE) instrumentation for performing enzymatic and inhibition assays, as well as for analysis of biological molecules such as antigens, substrates, etc., has been explored. A small quantity of magnetic beads containing immobilized biomolecules was injected into a neutral hydrophilic-coated fused-silica capillary. The short plug (2-3 mm) of beads was held fixed by a magnet placed in the cartridge of the CE system, without the use of frits. The beads could be replaced after each run, eliminating the need to regenerate the solid support. Two protocols were used for analysis: sequential injection (SI) and SI followed by isotachophoretic (ITP) focusing. Alkaline phosphatase (AP) and HIV-protease were used to demonstrate the SI procedure for enzymatic and inhibition assays. The second protocol, SI/ITP, was employed to quantitate an antigen (mouse mAB) using antibodies (sheep IgG towards mouse AB) immobilized on the beads. The MB-CE method, requiring only femtomole (fmol) quantities of material, can potentially be employed in diagnostic and forensic assays, kinetic studies and searching for inhibitors, ligands, receptors, etc.
Assuntos
Fosfatase Alcalina/análise , Eletroforese Capilar/métodos , Enzimas Imobilizadas/análise , Protease de HIV/análise , Fosfatase Alcalina/química , Fosfatase Alcalina/imunologia , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Enzimas Imobilizadas/química , Enzimas Imobilizadas/imunologia , Protease de HIV/química , Inibidores da Protease de HIV/química , Magnetismo , Camundongos , Microesferas , Concentração Osmolar , Pepstatinas/química , Espectrometria de Fluorescência , Fatores de TempoRESUMO
Within the pH range 2-12, the monoclonal chimeric antibody BR96 can be separated into one to five isoforms by micellar electrokinetic capillary chromatography (MECC). The distribution of the immunoglobulin between these isoforms is pH dependent and apparently reversible. Some of the changes in the electrophoretic profile are represented by alterations in the immunoglobulin secondary structure. MECC and CD data demonstrate that, in other cases, differences in electrophoretic mobilities of the intact and acid-stressed antibody molecules were not due to differences in the ionization of the protein functional groups or changes in secondary structure, but rather resulted from differences in the exposure of the molecule's structural elements to the solvent. The results indicate that the interaction of the isoforms with sodium dodecyl sulfate micelles plays a crucial role in MECC isoform separations. The formation of analyte-micelle complexes was postulated to make electrophoretic mobilities, especially of large protein molecules, susceptible to subtle conformational changes that are not detectable by other methods.
Assuntos
Anticorpos Monoclonais/análise , Varredura Diferencial de Calorimetria , Dicroísmo Circular , Eletroforese Capilar , Concentração de Íons de Hidrogênio , IsomerismoRESUMO
Assays for detecting and measuring antinuclear antibodies (ANA) in dogs were compared. They included the indirect immunofluorescence test, using rat liver as substrate, and ELISAs for three nuclear antigens: double-stranded DNA, single-stranded DNA, and histone. There was no correlation between the ANA titre and antibodies to the three nuclear antigens. Analysis of ANA in different arthropathies showed no specific disease association. HEP-2 cells showed no fluorescence reaction with either ANA-positive or ANA-negative dog sera. Western blotting produced too complex a pattern to identify specific antigens. The antigens that reacted with ANA in dogs were not identified; there is either a broad range of reactivities or non-specific binding of immunoglobulins.
Assuntos
Anticorpos Antinucleares/sangue , Cães/imunologia , Ensaio de Imunoadsorção Enzimática/veterinária , Técnica Indireta de Fluorescência para Anticorpo/veterinária , Animais , Western Blotting/métodos , Western Blotting/veterinária , DNA/sangue , DNA de Cadeia Simples/sangue , Cães/sangue , Ensaio de Imunoadsorção Enzimática/métodos , Técnica Indireta de Fluorescência para Anticorpo/métodos , Histonas/sangue , RatosRESUMO
Map positions have been determined for 42 non-redundant Arabidopsis expressed sequence tags (ESTs) showing similarity to disease resistance genes (R-ESTs), and for three Pto-like sequences that were amplified with degenerate primers. Employing a PCR-based strategy, yeast artificial chromosome (YAC) clones containing the EST sequences were identified. Since many YACs have been mapped, the locations of the R-ESTs could be inferred from the map positions of the YACs. R-EST clones that exhibited ambiguous map positions were mapped as either cleavable amplifiable polymorphic sequence (CAPS) or restriction fragment length polymorphism (RFLP) markers using F8 (Ler x Col-0) recombinant inbred (RI) lines. In all cases but two, the R-ESTs and Pto-like sequences mapped to single, unique locations. One R-EST and one Pto-like sequence each mapped to two locations. Thus, a total of 47 loci were identified in this study. Several R-ESTs occur in clusters suggesting that they may have arisen via gene duplication events. Interestingly, several R-ESTs map to regions containing genetically defined disease resistance genes. Thus, this collection of mapped R-ESTs may expedite the isolation of disease resistance genes. As the cDNA sequencing projects have identified an estimated 63% of Arabidopsis genes, a very large number of R-ESTs (approximately 95), and by inference disease resistance genes of the leucine-rich repeat-class probably occur in the Arabidopsis genome.
Assuntos
Arabidopsis/genética , Mapeamento Cromossômico , Genes de Plantas , Doenças das Plantas/genética , Sequência de Bases , Mapeamento Cromossômico/métodos , Cromossomos Artificiais de Levedura , Cruzamentos Genéticos , Marcadores Genéticos , Imunidade Inata , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Recombinação Genética , Sitios de Sequências RotuladasRESUMO
The kinetics of inhibition of metastasis by the immunomodulator swainsonine (SW) is effective 1 to 3 days after administration. It is likely that SW's prolonged antimetastatic effect is due to its mitogenic property (spleenocytes isolated from animals treated with SW for 42-72 hours stimulated DNA synthesis that remained elevated for up to 3 days after removal of the drug from the drinking water). An analysis of SW in lymphoid (spleen and thymus) and highly perfused tissues was undertaken to determine if SW's sustained antimetastatic effect could be correlated to its retention. C57BL/6 mice received [3H]SW in drinking water for 24-72 hours and thereafter, received SW-free drinking for 24, 48, and 72 hours. Lymphoid and highly perfused tissues were analyzed for [3H]SW. At 24, 48, and 72 hours, spleen SW levels are, respectively, at least 2.33, 2.25, and 2.00 times greater than the perfused tissue; and thymus are, respectively, 1.44, 1.50, and 1.77 as great as the perfused tissue (kidney) with the highest SW level. These studies suggest that SW is predominantly retained for at least 72 hours, in lymphoid tissue. The targeting and retention of SW for lymphoid tissue days after removal of SW from animal drinking water is consistent with a) the immunomodulatory/mitogenic property and b) the sustained antimetastatic effect attributed to SW.
Assuntos
Baço/metabolismo , Swainsonina/farmacologia , Swainsonina/farmacocinética , Timo/metabolismo , Adjuvantes Imunológicos/farmacocinética , Adjuvantes Imunológicos/farmacologia , Animais , Antineoplásicos Fitogênicos/farmacocinética , Antineoplásicos Fitogênicos/farmacologia , DNA/biossíntese , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Metástase Neoplásica/prevenção & controle , Distribuição Tecidual , TrítioRESUMO
Postmenopausal osteoporosis, the most common bone disease in the developed world, is associated with estrogen deficiency. This deficiency induces increased generation and activity of osteoclasts, which perforate bone trabeculae, thus reducing their strength and increasing fracture risk. Estrogen replacement prevents these effects, indicating that estrogen negatively regulates osteoclast formation and function, but how it does this is unclear. Because functional osteoclast life span and thus the amount of bone that osteoclasts resorb could also be enhanced following estrogen deficiency, and since sex steroids regulate apoptosis in other target tissues, we investigated whether estrogen may affect osteoclast function by promoting apoptosis. 17 beta-Estradiol promoted apoptosis of murine osteoclasts in vitro and in vivo by two- to threefold. Tamoxifen, which has estrogenic effects on bone resorption, and transforming growth factor-beta 1 (TGF-beta), whose production by osteoblasts is increased by estrogen, had similar effects in vitro. Anti-TGF-beta antibody inhibited TGF-beta-, estrogen- and tamoxifen-induced osteoclast apoptosis, indicating that TGF-beta might mediate this effect. These findings suggest that estrogen may prevent excessive bone loss before and after the menopause by limiting osteoclast life span through promotion of apoptosis. The development of analogues to promote this mechanism specifically could be a useful and novel therapeutic approach to prevent postmenopausal osteoporosis.