RESUMO
Two distinct nodules developed in a cryptorchid testis of an 8-year-old male West Highland White Terrier. One nodule was a Sertoli cell tumor. The other was a spermatocytic seminoma with focal primitive neuroectodermal differentiation: formation of Homer-Wright rosettes and perivascular pseudorosettes, with immunoreactivity for S-100 protein, neuron-specific enolase, synaptophysin, neurofilament-68 kDa, microtubule-associated protein 2, and vimentin. The dog was alive and healthy 2 years after castration.
Assuntos
Doenças do Cão/patologia , Tumores Neuroectodérmicos/veterinária , Seminoma/veterinária , Tumor de Células de Sertoli/veterinária , Neoplasias Testiculares/veterinária , Animais , Doenças do Cão/cirurgia , Cães , Imuno-Histoquímica/veterinária , Masculino , Tumores Neuroectodérmicos/patologia , Tumores Neuroectodérmicos/cirurgia , Orquiectomia/veterinária , Seminoma/patologia , Seminoma/cirurgia , Tumor de Células de Sertoli/patologia , Tumor de Células de Sertoli/cirurgia , Neoplasias Testiculares/patologia , Neoplasias Testiculares/cirurgiaRESUMO
The present study aimed to verify the changes in the expression levels of 13 candidate genes associated with chemotherapy resistance and to construct a scoring system to predict resistance to these drugs. The expression levels of the 13 candidate genes were compared between 20 dogs with lymphoma that were sensitive to drugs used in CHOP-based protocol and 16 dogs with lymphoma that were resistant to these drugs. The expression levels of six genes; ASNS, CCR3, CALCA, FCER1A, LOC448801, and EDNRB were significantly different between the two groups. A scoring system to predict resistance to cyclophosphamide, doxorubicin and vincristine, which are used in CHOP-based protocol, was constructed based on expression levels of the six genes in these 36 dogs using logistic regression models. After internal validation, sensitivity and specificity of the scoring system were 0.759 and 0.853, respectively. External validation was conducted in another cohort of 33 dogs with lymphoma, and sensitivity and specificity of the scoring system were 0.800 and 0.696, respectively. In conclusion, this study identified six genes associated with resistance to drugs used in CHOP-based protocol in canine lymphoma and proposed a novel scoring system to predict resistance to these drugs. This system might be beneficial in selecting the most appropriate chemotherapy protocol for individual dogs with lymphoma.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Doenças do Cão/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Linfoma/veterinária , Transcriptoma , Animais , Estudos de Coortes , Ciclofosfamida/uso terapêutico , Cães , Doxorrubicina/uso terapêutico , Feminino , Linfoma/tratamento farmacológico , Masculino , Prednisona/uso terapêutico , Projetos de Pesquisa , Vincristina/uso terapêuticoRESUMO
We have previously reported that a c-myc protein complex binds to the region upstream of the c-myc gene, where exist an origin of cellular DNA replication (ori) and a transcriptional enhancer. Both functions require a 21 bp long sequence, while the c-myc protein complex recognizes a 7 bp consensus therein. It was recently reported that single-stranded DNA binding proteins bound specifically to sequences that play roles in DNA replication or transcription. We examined for proteins binding to the single-stranded DNAs of the 21 bp element (myc(H-P)21). In a band shift assay with HL60 cells nuclear extract, probes of either the plus strand or the minus strand gave rise to specific signals. Mutation introduced within a short consensus (A/TCTA/TA/TT) present in both strands completely abolished binding in either case. Southwestern blotting analysis showed that proteins of molecular weight 105, 80, 50, 45, 40, 39.5 and 14 kDa bound sequence-specifically to either strand and 22 kDa to minus strand to the cognate A/TCTA/TA/TT consensus. These single-stranded DNA binding proteins were named MSSP, c-myc gene single strand binding proteins. We attempted to isolate the cDNAs encoding these proteins by screening a human cDNA library with the plus single-stranded oligonucleotide as a probe. Among several positive clones, we have characterized one, termed MSSP-1. MSSP-1 produced in E. coli as a fusion protein with GST specifically interacted with single-stranded TCTTAT (plus myc(H-P)21) and ACT-ATT (in minus myc(H-P)21), the consensus of which can be referred to as A/TCTA/TA/TT. Sequence analysis of MSSP-1 cDNA revealed that two domains thereof are homologous to the RNA binding motifs common to several ribonucleoproteins. Interestingly, the MSSP-1/GST fusion protein specifically recognized myc(H-P)21 not only in single-stranded but also in double-stranded forms. Binding properties of MSSP-1 imply its functions in DNA replication. Furthermore, when the AT stretch in the SV40 ori core was substituted by TCTTAT, MSSP-1 promoted viral DNA replication depending on the consensus sequences.
Assuntos
DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/química , Genes myc/genética , Proteínas de Ligação a RNA , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Ciclo Celular , Clonagem Molecular , Sequência Consenso , Replicação do DNA , DNA Complementar , Escherichia coli/genética , Glutationa Transferase/genética , Humanos , Dados de Sequência Molecular , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Células Tumorais CultivadasRESUMO
The sequence motif GGAGGC (Alu core) is present in the Alu family repeats, where it is required for RNA polymerase III promoter function. This motif is also found in the SV40 origin (ori) of replication. Here, an oligonucleotide containing the Alu sequence was inserted into pSV2CAT, a plasmid composed of the SV40 enhancer/promoter/ori linked to the bacterial chloramphenicol acetyltransferase gene (CAT), to see the effect of the Alu sequence on SV40 DNA replication and transcription. Results of transfection experiments in human HeLa cells showed that the Alu sequence stimulated sequence-specifically replication and transcription in the SV40 system. Stimulation effects on DNA replication were observed when the Alu sequence was placed upstream of enhancer/promoter/ori in either orientation, while effects on transcription were detected only when it was inserted in the normal orientation. These effects correlate with sequence-specific binding of two proteins (40 kDa and 120 kDa) to this motif. In fact, binding was abolished by a mutation in the cognate sequence that disrupted stimulation of replication and transcription. Both proteins bind duplex DNA, while the 40 kDa one also binds the minus strand with high affinity.
Assuntos
DNA Viral/genética , Regulação Viral da Expressão Gênica , Sequências Repetitivas de Ácido Nucleico , Vírus 40 dos Símios/genética , Transcrição Gênica , Sequência de Bases , Proteínas de Transporte/análise , Análise Mutacional de DNA , Replicação do DNA , Elementos Facilitadores Genéticos , Células HeLa , Humanos , Dados de Sequência MolecularRESUMO
The GC-rich segment containing GGAGGC (Alu core) is conserved within the RNA polymerase III (pol III) promoters of Alu family sequences. We have shown that the GGAGGC motif functions as a modulator of DNA replication as well as of transcription, and identified the proteins binding to the motif in human HeLa cells. In this study, the Alu core binding proteins were partially purified from human Raji cells by using an Alu core DNA affinity column. Both the proteins thus purified were implied to be subunits of Ku antigen based on the following criteria: The molecular weights of the proteins estimated on gel electrophoreses were 70 and 85 kDa, respectively, under denaturing conditions, while under non-denaturing conditions only one band was observed for the same sample at 150 kDa, probably representing hetero-dimer formed between the 70 and 85 kDa proteins. The sizes and the hetero-dimer formation are reminiscent of the 70 and 80 kDa subunits of Ku antigen (Ku-p70 and Ku-p80). Moreover, the purified proteins were immunoreactive with anti-Ku antibodies, and the specific DNA-protein complex on the Alu core element was cancelled by the anti-Ku antibodies. The nucleoprotein complex showed the same clipping patterns as those of the complex between the Alu core element and an authentically purified Ku antigen after proteolytic cleavage with trypsin and chymotrypsin.
Assuntos
Antígenos Nucleares , DNA Helicases , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Proteínas Nucleares/metabolismo , Sequências Repetitivas de Ácido Nucleico , Linhagem Celular , Cromatografia de Afinidade , Metilação de DNA , Proteínas de Ligação a DNA/isolamento & purificação , Humanos , Autoantígeno Ku , Proteínas Nucleares/isolamento & purificaçãoRESUMO
A new lipophilic platinum (Pt) compound, trans-bis(n-valerato)(1R,2R-cyclohexanediamine)(oxalato)Pt(lV) (C5-OHP), was compared with cisplatin (CDDP) by intraperitoneal injection for antitumor activities against 3 different mouse models, L1210 leukemia, LMFS sarcoma with high metastatic potential and spontaneous mammary tumor. C5-OHP cured both CDDP-sensitive and -resistant L1210 leukemia frequently. CDDP did not cure the leukemia, although it prolonged the survival of diseased mice significantly. C5-OHP cured early and advanced LMFS sarcomas more frequently than CDDP. Cured mice had acquired antitumor immunity, indicating the pivotal role of the immune system in induction of the cure of tumors. It is likely tllat C5-OHP can eradicate tumor cells more effectively than CDDP by virtue of its weaker suppressive effects on the immune system. C5-OHP but not CDDP could cure mammary tumors. C5-OHP manifested a curative effect against LMFS tumors by oral route. These results indicate that C5-OHP is a promising anticancer agent worthy of clinical trial.
Assuntos
Antineoplásicos/uso terapêutico , Compostos Organoplatínicos/uso terapêutico , Animais , Cisplatino/uso terapêutico , Feminino , Leucemia L1210/tratamento farmacológico , Fígado/patologia , Neoplasias Mamárias Experimentais/tratamento farmacológico , Camundongos , Camundongos Endogâmicos BALB C , Sarcoma Experimental/tratamento farmacológico , Sarcoma Experimental/imunologia , Sarcoma Experimental/patologia , Baço/citologia , Baço/transplanteRESUMO
Nitric oxide (NO) is generated from L-arginine by NO synthase. NO has been reported to be produced by a variety of cell types such as vascular endothelial cells, macrophages, neutrophils and articular chondrocytes. A recent report demonstrated that NO inhibits osteoclast (OC) function and, in this way, is critically associated with bone metabolism. In the present study we have studied NO synthesis by osteoblasts (OBs). OB cell line, MC3T3-E1, was cultured with the various cytokines for 72 hrs. Nitrite, a stable endproduct of cell-generated NO, in the culture supernatant was then determined by using a spectrophotometric method based on Griess reaction. IL-1 alpha increased nitrite release in a dose-dependent fashion and a significant enhancement (p < 0.01) was attained at 10 U/ml. OBs released 14.2 nmol/4.0 x 10(4) cells of nitrite after 72 hrs stimulation by 100 U/ml IL-1 alpha. In contrast IL-1 beta, TNF-alpha and INF-gamma failed to affect NO synthesis by MC3T3-E1. The results suggest that OBs produce NO in response to IL-1 alpha and OB-induced NO may play a role in OB-OC interaction in the inflammatory process.
Assuntos
Citocinas/farmacologia , Lipopolissacarídeos/farmacologia , Óxido Nítrico/biossíntese , Osteoblastos/metabolismo , Animais , Arginina/análogos & derivados , Arginina/farmacologia , Glucocorticoides/farmacologia , Camundongos , Óxido Nítrico/antagonistas & inibidores , Nitroarginina , Células Tumorais CultivadasRESUMO
Many patients undergo operations of the hip joint for avascular necrosis of the femoral head (ANF). Abnormal lipid metabolism in thought to play a certain role in the pathogenesis of ANF. In the present study we have detected the abnormal lipid from the necrotic area of the femoral heads suffering from ANF by gas chromatography-mass spectrometry (GC/MS). This abnormal lipid was supposed to be cholesterol epoxide, because its peak was observed at one oxygen molecular weight larger position than that of cholesterol. However, this abnormal lipid was not found in the femoral heads with osteoarthritis (OA). We also investigated the effect of the lipids (cholesterol and cholesterol epoxide) and corticosteroid (prednisolone) on proliferation of osteoblastic cell line (MC3T3-E1) cells and vascular endothelial cells (EC) derived from human umbilical vein using 3H-thymidine (3H-TdR) incorporation. Cholesterol has no effect on osteoblast proliferation up to 100 micrograms/ml, while cholesterol epoxide inhibited the 3H-TdR incorporation in a dose dependent fashion and a significant suppression was attained at the concentration of 10 micrograms/ml. In contrast, both cholesterol and cholesterol epoxide had suppressive effect on the EC proliferation. DNA synthesis of the osteoblasts was suppressed by prednisolone and additive inhibitory effect was observed in the combination of cholesterol epoxide and prednisolone. None of the free radical scavengers such as mannitol, dimethylsulfoxide (DMSO), super oxide dismutase (SOD) and catalase had significant recovery effect on the suppressed 3H-TdR incorporation of both MC3T3-E1 cells and EC. Only alpha-tocopherol restored the suppressed incorporation of 3H-TdR. These results suggest that lipid peroxide has an important role in the progression of ANF by suppressing the proliferation of osteoblasts and EC.
Assuntos
Endotélio Vascular/efeitos dos fármacos , Peróxidos Lipídicos/farmacologia , Osteoblastos/efeitos dos fármacos , Progressão da Doença , Endotélio Vascular/patologia , Necrose da Cabeça do Fêmur/patologia , Humanos , Osteonecrose/patologia , Traumatismo por Reperfusão/patologiaRESUMO
A permanent cell line, termed LMFS (liver metastasis from sarcoma) was established in vivo and in vitro from a spontaneously occurring murine retroperitoneal tumor of BALB/c mouse origin. After a subcutaneous injection of more than 1 x 10(5) cells in the dorsal side of mice, the LMFS cells proliferated at the inoculation site (100% take) and induced metastatic nodules spontaneously in the liver but not in the lung. The antitumor effects of 5'-DFUR against the LM FS tumor were examined. Mice with the LMFS tumor were given 5'-DFUR (0, 0.75, 1.5, 3.0 mM/kg) per os for 7 days from day 21 through day 27. Prolonging of the survival period was observed by the 5'-DFUR administration and the increase in the liver weight, which reflects tumor growth in the liver, was suppressed. Mice with the LMFS tumor were given 5'-DFUR (0, 0.75, 3.0 mM/kg) every day without Saturday and Sunday from day 21. Mean survival time of the control group was 57.8 +/- 4.9 days. And that of 5'-DFUR groups (0.75, 3.0 mM/kg) was 72.1 +/- 16.0 day (p < 0.05), and 80.5 +/- 9.5 days (p < 0.001), respectively. These results reveal that 5'-DFUR is effective for not only the primary site but also the liver metastasis.
Assuntos
Antineoplásicos/uso terapêutico , Floxuridina/uso terapêutico , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Neoplasias Hepáticas Experimentais/secundário , Neoplasias Retroperitoneais/patologia , Sarcoma Experimental/tratamento farmacológico , Sarcoma Experimental/secundário , Animais , Antineoplásicos/administração & dosagem , Feminino , Floxuridina/administração & dosagem , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias Retroperitoneais/tratamento farmacológicoRESUMO
BACKGROUND: Physical or psychological stress causes functional disorders in the upper gastrointestinal tract. This study aims to elucidate the ameliorating effect of exogenous acylated ghrelin or rikkunshito, a Kampo medicine which acts as a ghrelin enhancer, on gastric dysfunction during acute restraint stress in mice. METHODS: Fasted and postprandial motor function of the gastric antrum was wirelessly measured using a strain gauge force transducer and solid gastric emptying was detected in mice exposed to restraint stress. Plasma corticosterone and ghrelin levels were also measured. To clarify the role of ghrelin on gastrointestinal dysfunction in mice exposed to stress, exogenous acylated ghrelin or rikkunshito was administered, then the mice were subjected to restraint stress. KEY RESULTS: Mice exposed to restraint stress for 60 min exhibited delayed gastric emptying and increased plasma corticosterone levels. Gastric motility was decreased in mice exposed to restraint stress in both fasting and postprandial states. Restraint stress did not cause any change in plasma acylated ghrelin levels, but it significantly increased the plasma des-acyl ghrelin levels. Administration of acylated ghrelin or rikkunshito improved the restraint stress-induced delayed gastric emptying and decreased antral motility. Ameliorating effects of rikkunshito on stress-induced gastric dysfunction were abolished by simultaneous administration of a ghrelin receptor antagonist. CONCLUSIONS & INFERENCES: Plasma acylated/des-acyl ghrelin imbalance was observed in acute restraint stress. Supplementation of exogenous acylated ghrelin or enhancement of endogenous ghrelin signaling may be useful in the treatment of decreased gastric function caused by stress.
Assuntos
Medicamentos de Ervas Chinesas/farmacologia , Gastroenteropatias/tratamento farmacológico , Motilidade Gastrointestinal/efeitos dos fármacos , Grelina/farmacologia , Período Pós-Prandial/efeitos dos fármacos , Estresse Psicológico/complicações , Animais , Modelos Animais de Doenças , Medicamentos de Ervas Chinesas/administração & dosagem , Gastroenteropatias/sangue , Gastroenteropatias/etiologia , Grelina/administração & dosagem , Masculino , Camundongos , Camundongos Endogâmicos ICR , Estresse Psicológico/sangueAssuntos
Glândulas Endócrinas/anatomia & histologia , Hipófise/citologia , Gravidez , Adolescente , Glândulas Suprarrenais/anatomia & histologia , Adulto , Povo Asiático , Feminino , Humanos , Japão , Fígado/anatomia & histologia , Tamanho do Órgão , Pâncreas/anatomia & histologia , Baço/anatomia & histologia , Timo/anatomia & histologia , Glândula Tireoide/anatomia & histologiaAssuntos
Afogamento , Animais , Encéfalo/patologia , Eletroencefalografia , Humanos , Hipóxia/etiologia , Mudanças Depois da Morte , Baço/patologiaRESUMO
We examined the effect of low concentrations of gold compounds on the proliferation of human fibroblasts. Gold sodium thiomalate (GST) inhibited both basal and interleukin-1-induced tritiated thymidine incorporation into fibroblasts in a dose- and time-dependent manner. Significant inhibition was observed at the level of 5 micrograms/ml GST, and greater than 50% inhibition was attained at 10 micrograms/ml. These concentrations are attainable in the serum of treated patients. Similar inhibition was observed when less than 1 micrograms/ml auranofin, which is also within a serum-attainable range, was added. Low concentrations of GST (0-10 micrograms/ml) did not affect interleukin-1 secretion from lipopolysaccharide-stimulated human mononuclear phagocytes (M phi) when assessed by both human fibroblast and C3H/HeJ mouse thymocyte proliferation assays. When M phi precultured for 48 hours with GST (0-10 micrograms/ml) were added to the fibroblast culture in the presence or absence of lipopolysaccharide, there was no significant inhibition of M phi-induced DNA synthesis of fibroblasts. In contrast, when fibroblasts were precultured with GST (0-10 micrograms/ml) for 48 hours and freshly separated M phi were added, significant inhibition was observed in M phi-induced fibroblast proliferation at 5 micrograms/ml. These results suggest that low concentrations of GST directly cause a reduction of fibroblast proliferation, but do not affect the capability of M phi for induction of fibroblast proliferation. Therefore, gold compounds may play a role in the inhibition of the growth of rheumatoid pannus by direct inhibition of fibroblast proliferation.
Assuntos
Auranofina/farmacologia , Tiomalato Sódico de Ouro/farmacologia , Interleucina-1/metabolismo , Macrófagos/metabolismo , Pele/citologia , Divisão Celular/efeitos dos fármacos , DNA/antagonistas & inibidores , DNA/biossíntese , Relação Dose-Resposta a Droga , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Interleucina-1/farmacologia , Masculino , Monócitos/metabolismo , Monócitos/fisiologia , Pele/metabolismo , Membrana Sinovial/citologia , Membrana Sinovial/metabolismo , Linfócitos T/metabolismoRESUMO
The common occurrence of fibrin deposits in chronic inflammatory lesions suggests a possible role for thrombin in the mobilization of mononuclear cell infiltrates. For this reason, the effect of thrombin on the binding of mononuclear cells to endothelial cells (EC) was investigated. Incubation of confluent monolayers of human umbilical vein endothelial cells with thrombin markedly enhanced EC adhesiveness for both T lymphocytes and U937 cells (a monocyte-like cell line) in a time- and dose-dependent fashion. This effect was EC specific: 1) treatment of the T cells or the U937 cells with thrombin did not stimulate their adherence to EC, and 2) treatment of human foreskin fibroblasts with thrombin did not stimulate their inherently low adhesiveness for T cells. Fixation of EC monolayers with paraformaldehyde after pre-incubation with thrombin did not affect the increased adhesiveness for T cells. mAb against the LFA-1 antigen (mAb 60.3 (anti-CD18) or mAb TS1/22 (anti-CD11a), which inhibit the binding of T cells to unstimulated EC, failed to block the increased adhesion induced by thrombin, indicating that the increased binding induced by thrombin is similar to that induced by IL-1 and TNF, which showed similar resistance. These results suggest that thrombin may have a role in the extravascular emigration of mononuclear cells from post-capillary venules by virtue of its ability to stimulate the adhesiveness of EC for both lymphocytes and monocytes.
Assuntos
Adesão Celular/efeitos dos fármacos , Endotélio Vascular/fisiologia , Leucócitos Mononucleares/fisiologia , Trombina/farmacologia , Anticorpos Monoclonais/fisiologia , Antígenos de Diferenciação de Linfócitos T/imunologia , Fatores Biológicos/farmacologia , Células Cultivadas , Cicloeximida , Citocinas , Endotélio Vascular/metabolismo , Endotoxinas/farmacologia , Fixadores , Humanos , Interleucina-1/fisiologia , Leucócitos Mononucleares/imunologia , Linfócitos T/imunologia , Linfócitos T/fisiologiaRESUMO
Lymphokines and monokines have been reported to affect endothelial cell (EC) morphology and function. In experiments here described, we have demonstrated that recombinant tumor necrosis factor (TNF) stimulates the adhesion of T lymphocytes to confluent monolayers of human umbilical vein EC. The increase in adhesion induced by TNF was EC-specific inasmuch as preincubation of the lymphocytes with TNF did not alter binding, and preincubation of human dermal fibroblasts with TNF did not increase their inherently low adhesiveness for lymphocytes. Stimulation of T-EC binding occurred after treatment of the EC with as little as 0.01 U/ml (1 pg/ml) of TNF. In kinetic experiments, preincubation of EC with TNF for 4 hr resulted in optimal adhesion. TNF-treated EC retained their increased adhesiveness after fixation with paraformaldehyde, suggesting that TNF stimulated binding by increasing the expression or accessibility of EC surface receptors for lymphocytes. Although antibodies to the lymphocyte function-associated antigen 1 alpha- or beta-chains on the T cell markedly inhibited unstimulated T-EC binding, such antibodies had no effect on the increase in EC adhesiveness induced by TNF, indicating that the increased binding resulted from the generation of an alternate binding receptor on the EC membrane. These findings provide additional evidence that cytokines participate in the mobilization of mononuclear cells in the chronic inflammatory reaction by stimulation of the adhesiveness of endothelium for circulating lymphocytes.
Assuntos
Endotélio/citologia , Glicoproteínas/farmacologia , Linfócitos T/citologia , Anticorpos Monoclonais , Antígenos de Superfície/imunologia , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Técnicas In Vitro , Interleucina-1/farmacologia , Ativação de Macrófagos , Fator de Necrose Tumoral alfaRESUMO
The purpose of this study was to improve our previously described enzymatic fluorometric assay of the adenylate cyclase activity. Using physicochemical characteristics of NADPH, of which a 0.1 mmol/L solution would have an optical density of 0.627, we measured the adenylate cyclase activity by the spectral absorption of NADPH. The assay consists of two parts: pharmacological modulation of adenylate cyclase and measurement of newly synthesized cyclic AMP. The latter part involves four steps: enzymatic destruction of noncyclic adenine nucleotides and phosphorylated metabolites, conversion of cyclic AMP to ATP, amplification of ATP by enzymatic cycling, and measurement of NADPH with spectral absorption, which is generated in proportion to initial cyclic AMP levels. This new assay was tested in membrane preparations made from rat hearts in comparison with the previously described fluorometric assay. We obtained identical results by spectrophotometry and fluorometry with high reproducibility. Because the fluorometric assay possesses a high sensitivity while the spectrophotometric method is advantageous because of its wide analytical range of cyclic AMP measurement, combination of fluorometric and spectrophotometric methods may offer a convenient way to measure the adenylate cyclase activities in various samples.
Assuntos
Adenilil Ciclases/metabolismo , Ensaios Enzimáticos Clínicos/normas , Espectrometria de Fluorescência/normas , Adenilil Ciclases/análise , Animais , Membrana Celular/enzimologia , Química Clínica/métodos , Química Clínica/normas , Ensaios Enzimáticos Clínicos/métodos , AMP Cíclico/análise , Masculino , Miocárdio/enzimologia , NADP/análise , Ratos , Ratos Sprague-Dawley , Valores de Referência , Espectrometria de Fluorescência/métodosRESUMO
Neovascularization, a common occurrence in chronic inflammatory lesions, requires endothelial cell (EC) proliferation. Because this form of inflammation is often mediated by immunologically generated cytokines, the effects of such cytokines on human umbilical vein EC proliferation in vitro were investigated. Low concentrations of recombinant interferon gamma (rIFN-gamma) (10-100 U/ml), but not a higher concentration (1,000 U/ml), enhanced both basal and endothelial cell growth factor (ECGF)-stimulated EC proliferation. Recombinant interleukin 1 (rIL-1) and recombinant tumor necrosis factor-alpha (rTNF) had minor effects on basal EC proliferation, but significant inhibition was observed in the presence of ECGF. A combination of rIFN-gamma and rTNF induced marked suppression of EC proliferation, which appeared to be due to a cytotoxic effect on the EC, as demonstrated by 51Cr release. In contrast, the combination of rIFN-gamma and rIL-1 had only an additive effect on EC proliferation, with no evidence of cytotoxicity. These results suggest that cytokines have important regulatory roles in local vascular proliferation. These effects varied not only with the individual cytokine, but also with the combination of cytokines used. The most striking effects were 1) the stimulation of proliferation by IFN-gamma at a low concentration and 2) the inhibition by both rIL-1 and rTNF of ECGF-stimulated proliferation.