RESUMEN
To evaluate comparative efficiency of traditional vs automated colony counting methods, cultures of Escherichia coli (ATCC 25945), Staphylococcus epidermidis (ATCC 12225), Streptococcus pyogenes (ATCC19615) and Streptococcus pneumoniae (ATCC49619) were prepared as pure cultures and mixed cultures at 0·5 McFarland standard and serial dilutions were performed. Plates were inoculated in triplicate with 50, 125, 250 and 500 colony forming units and counted by four researchers, visually and using each of the automated counters. Colony count and counting time were recorded. The pattern of efficiency for all bacterial species was similar: plates with low counts were accurate and quick to count for all methods, with an increase in time and a decrease in accuracy and precision as counts rose. Higher counts of single round colonies required less time and had greater precision with automated counters than human visual counting counts with no loss of accuracy; however, counts were reduced in accuracy and increased in time for species with less regular morphology or when plates had mixed species. Surprisingly, a free phone application was only slightly less precise and more time consuming than the high-end professional counter indicating that automation may be achievable at lower cost than expected. SIGNIFICANCE AND IMPACT OF THE STUDY: Colony quantification is essential in clinical and research settings as well as pedagogy at the college level. Human visual (HV) counting, the most common method, is time consuming and fraught with errors. The time, accuracy and precision of HV counting were compared to a high-end professional automated counter, an inexpensive phone application and a free phone application. Low cost benefits of increased speed and accuracy with automated counting are maximized when counting single round colonies; but much reduced if colonies have irregular morphology or demonstrate haemolysis.
Asunto(s)
Automatización/métodos , Escherichia coli/crecimiento & desarrollo , Staphylococcus epidermidis/crecimiento & desarrollo , Streptococcus pneumoniae/crecimiento & desarrollo , Streptococcus pyogenes/crecimiento & desarrollo , Recuento de Colonia Microbiana/métodos , Contaminación de Alimentos/análisis , Humanos , Infecciones Urinarias/diagnóstico , Infecciones Urinarias/microbiología , Contaminación del Agua/análisisRESUMEN
Various genetic conditions produce dysfunctional osteoclasts resulting in osteopetrosis or osteosclerosis. These include human pycnodysostosis, an autosomal recessive syndrome caused by cathepsin K mutation, cathepsin K-deficient mice, and mitf mutant rodent strains. Cathepsin K is a highly expressed cysteine protease in osteoclasts that plays an essential role in the degradation of protein components of bone matrix. Cathepsin K also is expressed in a significant fraction of human breast cancers where it could contribute to tumor invasiveness. Mitf is a member of a helix-loop-helix transcription factor subfamily, which contains the potential dimerization partners TFE3, TFEB, and TFEC. In mice, dominant negative, but not recessive, mutations of mitf, produce osteopetrosis, suggesting a functional requirement for other family members. Mitf also has been found-and TFE3 has been suggested-to modulate age-dependent changes in osteoclast function. This study identifies cathepsin K as a transcriptional target of Mitf and TFE3 via three consensus elements in the cathepsin K promoter. Additionally, cathepsin K mRNA and protein were found to be deficient in mitf mutant osteoclasts, and overexpression of wild-type Mitf dramatically up-regulated expression of endogenous cathepsin K in cultured human osteoclasts. Cathepsin K promoter activity was disrupted by dominant negative, but not recessive, mouse alleles of mitf in a pattern that closely matches their osteopetrotic phenotypes. This relationship between cathepsin K and the Mitf family helps explain the phenotypic overlap of their corresponding deficiencies in pycnodysostosis and osteopetrosis and identifies likely regulators of cathepsin K expression in bone homeostasis and human malignancy.
Asunto(s)
Catepsinas/genética , Proteínas de Unión al ADN/fisiología , Regulación Enzimológica de la Expresión Génica/fisiología , Osteopetrosis/genética , Factores de Transcripción , Alelos , Animales , Secuencia de Bases , Catepsina K , Catepsinas/metabolismo , ADN , Proteínas de Unión al ADN/genética , Ratones , Ratones Endogámicos C57BL , Factor de Transcripción Asociado a Microftalmía , Datos de Secuencia Molecular , Osteopetrosis/enzimología , Regiones Promotoras Genéticas , ARN Mensajero/genética , Transcripción Genética/fisiologíaRESUMEN
We have previously shown that p38 mitogen-activated protein kinase (MAPK) inhibitors, which block the production and action of inflammatory cytokines such as tumor necrosis factor (TNF) and interleukin-1 (IL-1), are effective in models of bone and cartilage degradation. To further investigate the role of p38 MAPK, we have studied its activation in osteoblasts and chondrocytes, following treatment with a panel of proinflammatory and osteotropic agents. In osteoblasts, significant activation of p38 MAPK was observed following treatment with IL-1 and TNF, but not parathyroid hormone, transforming growth factor-beta (TGF-beta), 1,25(OH)(2)D(3), insulin-like growth factor-1 (IGF-1), or IGF-II. Similar results were obtained using primary bovine chondrocytes and an SV40-immortalized human chondrocyte cell line, T/C28A4. SB 203580, a selective inhibitor of p38 MAPK, inhibited IL-1 and TNF-induced p38 MAPK activity and IL-6 production (IC(50)s 0.3--0.5 microM) in osteoblasts and chondrocytes. In addition, IL-1 and TNF also activated p38 MAPK in fetal rat long bones and p38 MAPK inhibitors inhibited IL-1- and TNF-stimulated bone resorption in vitro in a dose-dependent manner (IC(50)s 0.3--1 microM). These data support the contention that p38 MAPK plays a central role in regulating the production of, and responsiveness to, proinflammatory cytokines in bone and cartilage. Furthermore, the strong correlation between inhibition of kinase activity and IL-1 and TNF-stimulated biological responses indicates that selective inhibition of the p38 MAPK pathway may have therapeutic utility in joint diseases such as rheumatoid arthritis (RA).
Asunto(s)
Resorción Ósea/enzimología , Interleucina-1/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Bioensayo , Radioisótopos de Calcio/análisis , Radioisótopos de Calcio/metabolismo , Bovinos , Células Cultivadas , Condrocitos/citología , Condrocitos/efectos de los fármacos , Condrocitos/enzimología , Técnicas de Cultivo , Citocinas/farmacología , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Inhibidores Enzimáticos/farmacología , Sustancias de Crecimiento/farmacología , Humanos , Interleucina-1/farmacología , Interleucina-6/biosíntesis , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Osteoblastos/enzimología , Radio (Anatomía)/citología , Radio (Anatomía)/embriología , Radio (Anatomía)/enzimología , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Factor de Necrosis Tumoral alfa/farmacología , Cúbito/citología , Cúbito/embriología , Cúbito/enzimología , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Cathepsin K is a member of the papain superfamily of cysteine proteases and has been proposed to play a pivotal role in osteoclast-mediated bone resorption. We have developed a sensitive cytochemical assay to localize and quantify osteoclast cathepsin K activity in sections of osteoclastoma and human bone. In tissue sections, osteoclasts that are distant from bone express high levels of cathepsin K messenger RNA (mRNA) and protein. However, the majority of the cathepsin K in these cells is in an inactive zymogen form, as assessed using both the cytochemical assay and specific immunostaining. In contrast, osteoclasts that are closer to bone contain high levels of immunoreactive mature cathepsin K that codistributes with enzyme activity in a polarized fashion toward the bone surface. Polarization of active enzyme was clearly evident in osteoclasts in the vicinity of bone. The osteoclasts apposed to the bone surface were almost exclusively expressing the mature form of cathepsin K. These cells showed intense enzyme activity, which was polarized at the ruffled border. These results suggest that the in vivo activation of cathepsin K occurs intracellularly, before secretion into the resorption lacunae and the onset of bone resorption. The processing of procathepsin K to mature cathepsin K occurs as the osteoclast approaches bone, suggesting that local factors may regulate this process.
Asunto(s)
Resorción Ósea/metabolismo , Catepsinas/metabolismo , Osteoclastos/metabolismo , Bioquímica/métodos , Huesos/embriología , Huesos/enzimología , Catepsina K , Catepsinas/análisis , Catepsinas/antagonistas & inhibidores , Adhesión Celular , Inhibidores de Cisteína Proteinasa/farmacología , Tumor Óseo de Células Gigantes/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Riñón/embriología , Riñón/enzimología , Leucina/análogos & derivados , Leucina/farmacología , Modelos Lineales , Oligopéptidos/farmacología , Pepstatinas/farmacología , Fluoruro de Fenilmetilsulfonilo/farmacología , Inhibidores de Proteasas/farmacología , Procesamiento Proteico-Postraduccional , Especificidad por Sustrato , Células Tumorales CultivadasRESUMEN
Cathepsin K (cat K) is the major cysteine protease expressed in osteoclasts and is thought to play a key role in matrix degradation during bone resorption. However, little is known regarding the synthesis, activation, or turnover of the endogenous enzyme in osteoclasts. In this study, we show that mature cat K protein and enzyme activity are localized within osteoclasts. Pulse-chase experiments revealed that, following the synthesis of pro cat K, intracellular conversion to the mature enzyme occurred in a time-dependent manner. Subsequently, the level of mature enzyme decreased. Little or no cat K was observed in the culture media at any timepoint. Pretreatment of osteoclasts with either chloroquine or monensin resulted in complete inhibition of the processing of newly synthesized cat K. In addition, pro cat K demonstrated susceptibility to treatment with N-glycosidase F, suggesting the presence of high-mannose-containing oligosaccharides. Treatment of osteoclasts with the PI3-kinase inhibitor, Wortmannin (WT), not only prevented the intracellular processing of cat K but also resulted in the secretion of proenzyme into the culture media. Taken together, these results suggest that the biosynthesis, processing, and turnover of cat K in human osteoclasts is constitutive and occurs in a manner similar to that of other known cysteine proteases. Furthermore, cat K is not secreted as a proenzyme, but is processed intracellularly, presumably in lysosomal compartments prior to the release of active enzyme into the resorption lacunae.
Asunto(s)
Catepsinas/biosíntesis , Osteoclastos/metabolismo , Procesamiento Proteico-Postraduccional , Androstadienos/farmacología , Anticuerpos/inmunología , Resorción Ósea , Catepsina K , Catepsinas/inmunología , Catepsinas/metabolismo , Células Cultivadas , Cloroquina/farmacología , Inhibidores Enzimáticos/farmacología , Humanos , Inmunohistoquímica , Monensina/farmacología , Osteoclastos/efectos de los fármacos , Inhibidores de las Quinasa Fosfoinosítidos-3 , WortmaninaRESUMEN
An orally active, nonpeptide Arg-Gly-Asp (RGD) mimetic alpha(v)beta3 antagonist, (S)-3-Oxo-8-[2-[6-(methylamino)-pyridin-2-yl]-1-ethoxy]-2-(2,2,2-trifluoroethyl)-2,3,4,5-tetrahydro-1H-2-benzazepine-4-acetic acid (compound 1), has been generated, which prevented net bone loss and inhibited cancellous bone turnover in vivo. The compound binds alpha(v)beta3 and the closely related integrin alpha(v)beta5 with low nanomolar affinity but binds only weakly to the related integrins alpha(IIb)beta3, and alpha5beta1. Compound 1 inhibited alpha(v)beta3-mediated cell adhesion with an IC50 = 3 nM. More importantly, the compound inhibited human osteoclast-mediated bone resorption in vitro with an IC50 = 11 nM. In vivo, compound 1 inhibited bone resorption in a dose-dependent fashion, in the acute thyroparathyroidectomized (TPTX) rat model of bone resorption with a circulating EC50 approximately 20 microM. When dosed orally at 30 mg/kg twice a day (b.i.d.) in the chronic ovariectomy (OVX)-induced rat model of osteopenia, compound 1 also prevented bone loss. At doses ranging from 3 to 30 mg/kg b.i.d., compound 1 partially prevented the OVX-induced increase in urinary deoxypyridinoline. In addition, the compound prevented the OVX-induced reduction in cancellous bone volume (BV), trabecular number (Tb.N), and trabecular thickness (Tb.Th), as assessed by quantitative microcomputerized tomography (microCT) and static histomorphometry. Furthermore, both the 10-mg/kg and 30-mg/kg doses of compound prevented the OVX-induced increase in bone turnover, as measured by percent osteoid perimeter (%O.Pm). Together, these data indicate that the alpha(v)beta3 antagonist compound 1 inhibits OVX-induced bone loss. Mechanistically, compound 1 prevents bone loss in vivo by inhibiting osteoclast-mediated bone resorption, ultimately preventing cancellous bone turnover.
Asunto(s)
Resorción Ósea/prevención & control , Osteoclastos/efectos de los fármacos , Piridinas/farmacología , Receptores de Vitronectina/antagonistas & inhibidores , Animales , Femenino , Osteoclastos/metabolismo , Ovariectomía , Piridinas/farmacocinética , Ratas , Ratas Sprague-DawleyRESUMEN
The effects of a second generation p38 mitogen-activated protein kinase (MAPK) inhibitor, SB 239063 [trans-1-(4-hydroxycyclohexyl)-4-(4-fluorophenyl)-5-(2-methoxypyridim idi n-4-yl)imidazole; IC(50) = 44 nM vs. p38 alpha], were assessed in models that represent different pathological aspects of chronic obstructive pulmonary disease (COPD) [airway neutrophilia, enhanced cytokine formation and increased matrix metalloproteinase (MMP)-9 activity] and in a model of lung fibrosis. Airway neutrophil infiltration and interleukin (IL)-6 levels, assessed by bronchoalveolar lavage 48 h after lipopolysaccharide (LPS) inhalation, were inhibited dose dependently by 3-30 mg/kg of SB 239063 given orally twice a day. In addition, SB 239063 (30 mg/kg orally) attenuated IL-6 bronchoalveolar lavage fluid concentrations (>90% inhibition) and MMP-9 activity (64% inhibition) assessed 6 h after LPS exposure. In guinea pig cultured alveolar macrophages, SB 239063 inhibited LPS-induced IL-6 production (IC(50) of 362 nM). In a bleomycin-induced pulmonary fibrosis model in rats, treatment with SB 239063 (2.4 or 4.8 mg/day via osmotic pump) significantly inhibited bleomycin-induced right ventricular hypertrophy (indicative of secondary pulmonary hypertension) and increases in lung hydroxyproline synthesis (indicative of collagen synthesis and fibrosis). Therefore, SB 239063 demonstrates activity against a range of sequelae commonly associated with COPD and fibrosis, supporting the therapeutic potential of p38 MAPK inhibitors such as SB 239063 in chronic airway disease.
Asunto(s)
Citocinas/biosíntesis , Inhibidores Enzimáticos/farmacología , Imidazoles/farmacología , Lipopolisacáridos/toxicidad , Enfermedades Pulmonares Obstructivas/fisiopatología , Metaloproteinasa 9 de la Matriz/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neutrófilos/fisiología , Fibrosis Pulmonar/prevención & control , Pirimidinas/farmacología , Animales , Bleomicina/toxicidad , Células Cultivadas , Citocinas/sangre , Modelos Animales de Enfermedad , Cobayas , Humanos , Hipertensión Pulmonar/inducido químicamente , Hipertensión Pulmonar/prevención & control , Inflamación/fisiopatología , Inflamación/prevención & control , Proteína Antagonista del Receptor de Interleucina 1 , Interleucina-1/sangre , Interleucina-6/sangre , Interleucina-8/sangre , Pulmón/efectos de los fármacos , Pulmón/fisiopatología , Masculino , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Neutrófilos/efectos de los fármacos , Alveolos Pulmonares/efectos de los fármacos , Alveolos Pulmonares/inmunología , Fibrosis Pulmonar/inducido químicamente , Ratas , Ratas Endogámicas Lew , Sialoglicoproteínas/sangre , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Bipotential cells in human trabecular bone explant cultures that express osteoblast characteristics are able to undergo adipogenesis in the presence of 3-isobutyl-1-methylxanthine plus dexamethasone (Nuttall et al. [1998] J Bone Miner Res 13:371-382). The initial studies of these bipotential cells in explant cultures have been extended to examine differential gene expression during osteoblast/adipocyte transdifferentiation. Using differential display, we have identified a gene expressed in trabecular bone explant cultures that is downregulated as these cells differentiate from an osteoblast to an adipocyte phenotype. Homology searching identified this gene as the human urea transporter HUT11. The expression and downregulation of HUT11 have been observed in multiple patient bone explant cultures. The size of the bone explant-derived HUT11 mRNA is approximately 4.4 kb, which is identical to the largest splice variant reported. In this article, we report the cloning and sequencing of this gene from primary human osteoblasts. In addition, we report tissue distribution for the bone explant-derived form of HUT11 mRNA and show a reciprocal relationship between the expression of HUT11 and the nuclear hormone receptor peroxisome proliferator-activated receptor gamma 2, which is a marker of adipocyte differentiation. Because the control of osteoblast/adipocyte transdifferentiation is unknown, selective downregulation of HUT11 during adipogenesis suggests that HUT11 expression may be a marker of the switch from an osteoblast to an adipocyte phenotype. Understanding the role of HUT11 in osteoblasts may provide insights into the mechanism controlling osteoblast and adipocyte differentiation.
Asunto(s)
Adipocitos/metabolismo , Proteínas Portadoras/genética , Diferenciación Celular/genética , Regulación de la Expresión Génica/genética , Glicoproteínas de Membrana/genética , Proteínas de Transporte de Membrana , Osteoblastos/metabolismo , 1-Metil-3-Isobutilxantina/farmacología , Secuencia de Bases , Biomarcadores , Proteínas Portadoras/metabolismo , Células Cultivadas , Clonación Molecular , Dexametasona/farmacología , Regulación hacia Abajo/genética , Histocitoquímica , Humanos , Glicoproteínas de Membrana/metabolismo , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genética , Transportadores de UreaAsunto(s)
Benzazepinas/farmacología , Piridinas/farmacología , Receptores de Vitronectina/antagonistas & inhibidores , Animales , Benzazepinas/síntesis química , Benzazepinas/farmacocinética , Disponibilidad Biológica , Resorción Ósea/prevención & control , Adhesión Celular/efectos de los fármacos , Línea Celular , Semivida , Humanos , Imitación Molecular , Osteoclastos/efectos de los fármacos , Osteoclastos/metabolismo , Piridinas/síntesis química , Piridinas/farmacocinética , Ratas , Estereoisomerismo , Distribución TisularRESUMEN
OBJECTIVE: To evaluate the effects of SB 242235, a potent and selective inhibitor of p38 mitogen-activated protein (MAP) kinase, on joint integrity in rats with adjuvant-induced arthritis (AIA). METHODS: Male Lewis rats with AIA were orally treated either prophylactically (days 0-20) or therapeutically (days 10-20) with SB 242235. Efficacy was determined by measurements of paw inflammation, dual-energy x-ray absorptiometry for bone-mineral density (BMD), magnetic resonance imaging (MRI), microcomputed tomography (CT), and histologic evaluation. Serum tumor necrosis factor alpha (TNFalpha) in normal (non-AIA) rats and serum interleukin-6 (IL-6) levels in rats with AIA were measured as markers of the antiinflammatory effects of the compound. RESULTS: SB 242235 inhibited lipopolysaccharide-stimulated serum levels of TNFalpha in normal rats, with a median effective dose of 3.99 mg/kg. When SB 242235 was administered to AIA rats prophylactically on days 0-20, it inhibited paw edema at 30 mg/kg and 10 mg/kg per day by 56% and 33%, respectively. Therapeutic administration on days 10-20 was also effective, and inhibition of paw edema was observed at 60, 30, and 10 mg/kg (73%, 51%, and 19%, respectively). Significant improvement in joint integrity was demonstrated by showing normalization of BMD and also by MRI and micro-CT analysis. Protection of bone, cartilage, and soft tissues was also shown histologically. Serum IL-6 levels were decreased in AIA rats treated with the 60 mg/kg dose of compound. CONCLUSION: Symptoms of AIA in rats were significantly reduced by both prophylactic and therapeutic treatment with the p38 MAP kinase inhibitor, SB 242235. Results from measurements of paw inflammation, assessment of BMD, MRI, and micro-CT indicate that this compound exerts a protective effect on joint integrity, and thus appears to have disease-modifying properties.
Asunto(s)
Artritis Experimental/tratamiento farmacológico , Imidazoles/farmacología , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Piridinas/farmacología , Absorciometría de Fotón , Animales , Antiinflamatorios/farmacología , Artritis Experimental/diagnóstico por imagen , Artritis Experimental/enzimología , Artrografía , Densidad Ósea , Extremidades , Humanos , Procesamiento de Imagen Asistido por Computador , Interleucina-6/sangre , Lipopolisacáridos/farmacología , Imagen por Resonancia Magnética , Masculino , Ratas , Ratas Endogámicas Lew , Tarso Animal , Tibia , Tomografía Computarizada por Rayos X , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Idoxifene, a selective estrogen receptor modulator, was evaluated in male and female rats with adjuvant-induced arthritis (AA). AA was induced in Lewis rats with Mycobacterium butyricum in paraffin oil injected into the base of the tail, and the animals were treated with idoxifene prophylactically (days 0-21) or therapeutically (days 10-21). Efficacy was determined by measurements of paw inflammation, bone mineral content, and bone mineral density (BMD) with dual X-ray absorptiometry and by histological evaluation. Serum interleukin-6 levels were measured as a marker of the anti-inflammatory effects of the compound. Estrogen was included for comparison and was administered at 5 mg/kg, three times a week s.c. Prophylactic treatment of male AA rats with idoxifene at 10, 3, and 1 mg/kg and estrogen at 5 mg/kg significantly inhibited paw inflammation. There was improved joint integrity measured by BMD and reduced serum interleukin-6 levels in animals treated with 10 mg/kg/day idoxifene. Idoxifene and estrogen were as effective for AA in female Lewis rats as in male rats, significantly inhibiting paw inflammation and improving BMD. Histological evaluation of the tibiotarsal joints of female rats treated with 10 mg/kg showed protection of bone, cartilage, and soft tissue. Therapeutic treatment with either idoxifene or estrogen (starting on day 10 of disease) of male and female Lewis rats also was effective in reducing paw inflammation in these animals, although the effect was much less than that observed with the prophylactic dosing protocol.
Asunto(s)
Artritis Experimental/tratamiento farmacológico , Moduladores de los Receptores de Estrógeno/farmacología , Tamoxifeno/análogos & derivados , Animales , Artritis Experimental/metabolismo , Artritis Experimental/patología , Densidad Ósea/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Estrógenos/farmacología , Femenino , Pie/patología , Interleucina-6/metabolismo , Masculino , Ratas , Ratas Endogámicas Lew , Tamoxifeno/farmacologíaRESUMEN
The Arg-Gly-Asp (RGD)-binding integrin alpha(V)beta(3) is highly expressed on osteoclasts and has been proposed to mediate cell-matrix adhesion required for osteoclast-mediated bone resorption. Antagonism of this receptor should prevent stable osteoclast adhesion and thereby inhibit bone resorption. We have generated an orally bioavailable, nonpeptide RGD mimetic alpha(v)beta(3) antagonist, SB 265123, which prevents bone loss in vivo when dosed by oral administration. SB 265123 binds alpha(v)beta(3) and the closely related integrin alpha(v)beta(5) with high affinity (K(i) = 3.5 and 1.3 nM, respectively), but binds only weakly to the related RGD-binding integrins alpha(IIb)beta(3) (K(i) >1 microM) and alpha(5)beta(1) (K(i) >1 microM). The compound inhibits alpha(v)beta(3)-mediated cell adhesion with an IC(50) = 60 nM and more importantly, inhibits human osteoclast-mediated bone resorption in vitro with an IC(50) = 48 nM. In vivo, SB 265123 completely blocks bone resorption in a thyroparathyroidectomized rat model of acute bone resorption when dosed at 2.5 mg/kg/h by continuous i.v. infusion. When dosed orally with 3 to 30 mg/kg b.i.d. , in the ovariectomy-induced rat model of osteoporosis, SB 265123 prevents bone resorption in a dose-dependent fashion. This is the first report of an orally active alpha(v)beta(3) antagonist that is effective at inhibiting bone resorption when dosed in a pharmaceutically acceptable fashion. Such a molecule may provide a novel therapeutic agent for the treatment of postmenopausal osteoporosis.
Asunto(s)
Acetatos/farmacología , Aminopiridinas/farmacología , Resorción Ósea/prevención & control , Adhesión Celular/efectos de los fármacos , Agregación Plaquetaria/efectos de los fármacos , Receptores de Vitronectina/antagonistas & inhibidores , Acetatos/síntesis química , Acetatos/farmacocinética , Administración Oral , Aminopiridinas/síntesis química , Aminopiridinas/farmacocinética , Animales , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Técnicas In Vitro , Infusiones Intravenosas , Integrinas/metabolismo , Osteoporosis/prevención & control , Ovariectomía , Paratiroidectomía , Unión Proteica , Ratas , Tiroidectomía , Factores de TiempoRESUMEN
A new series of potent nonpeptide vitronectin receptor antagonists, based on a novel carbocyclic Gly-Asp mimetic, has been discovered. A representative of this series, SB 265123 (4), has 100% oral bioavailability in rats, and is orally active in vivo in the ovariectomized rat model of osteoporosis.
Asunto(s)
Acetatos/síntesis química , Acetatos/farmacocinética , Aminopiridinas/síntesis química , Aminopiridinas/farmacocinética , Osteoporosis/tratamiento farmacológico , Receptores de Vitronectina/antagonistas & inhibidores , Administración Oral , Animales , Benzodiazepinonas/síntesis química , Benzodiazepinonas/farmacocinética , Disponibilidad Biológica , Modelos Animales de Enfermedad , Cinética , Ratas , Factores de TiempoRESUMEN
We have identified and cloned a novel connective tissue growth factor-like (CTGF-L) cDNA from primary human osteoblast cells encoding a 250-amino acid single chain polypeptide. Murine CTGF-L cDNA, encoding a polypeptide of 251 amino acids, was obtained from a murine lung cDNA library. CTGF-L protein bears significant identity ( approximately 60%) to the CCN (CTGF, Cef10/Cyr61, Nov) family of proteins. CTGF-L is composed of three distinct domains, an insulin-like growth factor binding domain, a von Willebrand Factor type C motif, and a thrombospondin type I repeat. However, unlike CTGF, CTGF-L lacks the C-terminal domain implicated in dimerization and heparin binding. CTGF-L mRNA ( approximately 1.3 kilobases) is expressed in primary human osteoblasts, fibroblasts, ovary, testes, and heart, and a approximately 26-kDa protein is secreted from primary human osteoblasts and fibroblasts. In situ hybridization indicates high expression in osteoblasts forming bone, discrete alkaline phosphatase positive bone marrow cells, and chondrocytes. Specific binding of 125I-labeled insulin-like growth factors to CTGF-L was demonstrated by ligand Western blotting and cross-linking experiments. Recombinant human CTGF-L promotes the adhesion of osteoblast cells and inhibits the binding of fibrinogen to integrin receptors. In addition, recombinant human CTGF-L inhibits osteocalcin production in rat osteoblast-like Ros 17/2.8 cells. Taken together, these results suggest that CTGF-L may play an important role in modulating bone turnover.
Asunto(s)
Huesos/metabolismo , Sustancias de Crecimiento/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Proteínas de Neoplasias , Osteoblastos/metabolismo , Factores de Transcripción , Secuencia de Aminoácidos , Animales , Proteínas CCN de Señalización Intercelular , Adhesión Celular , Clonación Molecular , ADN Complementario/genética , Fibrinógeno/metabolismo , Sustancias de Crecimiento/genética , Humanos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Factor II del Crecimiento Similar a la Insulina/metabolismo , Ratones , Datos de Secuencia Molecular , Familia de Multigenes , Osteocalcina/biosíntesis , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Unión Proteica , Ratas , Receptores de Vitronectina/metabolismo , Proteínas Represoras , Homología de Secuencia de Aminoácido , Distribución TisularRESUMEN
A novel, immortalized, human bone marrow stroma-derived cell line TF274 is described which has the ability to form bone both in vitro and in vivo. Under basal conditions these cells expressed alkaline phosphatase (ALP) and type I collagen genes which are characteristic of the osteoblast phenotype. ALP levels were upregulated in the presence of osteotropic agents such as parathyroid hormone (PTH), transforming growth factor beta (TGF-beta), and BMP-2. In addition, PTH also increased cAMP levels in these cells. The capacity of these cells to form bone in vitro was evaluated by culturing them in the presence of L-ascorbic acid and beta-glycerophosphate. Matrix mineralization in these cultures was assessed by Alizarin Red staining and increased 45Ca uptake. Under these conditions mineralized nodule formation was observed in less than 2 weeks. Northern analysis of TF274 cells at various times during the mineralization process indicated a temporal expression of the osteocalcin gene that is typically associated with differentiating osteoblasts. The osteogenic nature of TF274 cells was confirmed in vivo using the severe combined immunodeficient (SCID) mouse model. Antibodies to human leukocyte antigens (HLA), class I antigens, and human OKa blood group antigen were used to demonstrate that the lesions formed were of human origin. By 21 days, the lesion consisted of a homogeneous focus of ALP-positive cells containing areas of mineralized bone lined with tartarate-resistant acid phosphatase (TRAP) positive osteoclasts. Thus, the TF274 cells exhibit osteogenic potential both in vitro and in vivo. This immortalized cell line represents a consistent source of cells that can be used to study human osteoblast differentiation both in vitro and in vivo.
Asunto(s)
Osteoblastos/citología , Osteogénesis , 1-Metil-3-Isobutilxantina/farmacología , Adulto , Fosfatasa Alcalina/metabolismo , Animales , Northern Blotting , Calcificación Fisiológica , Línea Celular , Colágeno/metabolismo , AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Masculino , Ratones , Ratones SCID , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Osteoblastos/trasplante , Hormona Paratiroidea/farmacología , ARN Mensajero/análisisRESUMEN
In a 3-oxo-1,4-benzodiazepine-2-acetic acid series of vitronectin receptor (alpha v beta 3) antagonists containing a benzimidazole as a novel arginine mimetic, we examined the effects of benzimidazole modifications and amide substitutions on both activity and pharmacokinetics.
Asunto(s)
Bencimidazoles/farmacología , Benzodiazepinas/farmacología , Receptores de Vitronectina/antagonistas & inhibidores , Arginina , Relación Estructura-ActividadRESUMEN
In the 3-oxo-1,4-benzodiazepine-2-acetic acid series of vitronectin receptor (alpha v beta 3) antagonists, a compound containing an imidazopyridine arginine mimetic was discovered which had sufficient potency and i.v. pharmacokinetics for demonstration of efficacy in a rat restenosis model.
Asunto(s)
Angioplastia Coronaria con Balón/efectos adversos , Benzodiazepinas/uso terapéutico , Enfermedad Coronaria/tratamiento farmacológico , Imidazoles/uso terapéutico , Piridinas/uso terapéutico , Receptores de Vitronectina/antagonistas & inhibidores , Animales , Modelos Animales de Enfermedad , RatasRESUMEN
We have identified a novel integrin beta3 subunit, termed beta3C, from a human osteoclast cDNA library. The COOH-terminal sequence and 3'-untranslated region of the beta3C subunit differs from the previously reported beta3A (platelet) and beta3B (placenta) sequences, while the regions coding for the transmembrane and extracellular domains are identical. The beta3C cytoplasmic domain contains 37 amino acids, the last 17 of which are encoded by a novel exon located about 6 kilobase pairs downstream of exon 14 of the beta3A gene. HEK 293 cells were stably co-transfected with alphaV and either beta3C (HEKbeta3C) or beta3A (HEKbeta3A). The viability of HEKbeta3C cells was lower than that of HEKbeta3A cells, and HEKbeta3C cells in culture grew as clusters rather than as a monolayer. The novel cytoplasmic domain did not affect receptor binding affinity; both alphaVbeta3A and alphaVbeta3C isoforms exhibited high affinity binding to 125I-echistatin and cyclic and linear RGD peptides. However, in contrast to HEKbeta3A, HEKbeta3C cells failed to adhere to osteopontin, an alphaVbeta3 matrix protein. The data provide further support for the key role of the cytoplasmic domain of the beta3 integrin in cell adhesion and suggest a potential role for the beta3C integrin subunit in modulating cell-matrix interactions.
Asunto(s)
Antígenos CD/genética , Glicoproteínas de Membrana Plaquetaria/genética , Secuencia de Aminoácidos , Animales , Antígenos CD/química , Antígenos CD/fisiología , Secuencia de Bases , Northern Blotting , Adhesión Celular , Clonación Molecular , Humanos , Inmunohistoquímica , Integrina beta3 , Datos de Secuencia Molecular , Glicoproteínas de Membrana Plaquetaria/química , Glicoproteínas de Membrana Plaquetaria/fisiología , Ratas , TransfecciónRESUMEN
Random high throughput sequencing of a human osteoclast cDNA library was employed to identify novel osteoclast-expressed genes. Of the 5475 ESTs obtained, approximately 4% encoded cathepsin K, a novel cysteine protease homologous to cathepsins S and L; ESTs for other cathepsins were rare. In addition, ESTs for cathepsin K were absent or at low frequency in cDNA libraries from numerous other tissues and cells. In situ hybridization in osteoclastoma and osteophyte confirmed that cathepsin K mRNA was highly expressed selecively in osteoclasts; cathepsins S, L, and B were not detectable. Cathepsin K was not detected by in situ hybridization in a panel of other tissues. Western blot of human osteoclastoma or fetal rat humerus demonstrated bands of 38 and 27 kDa, consistent with sizes predicted for pro- and mature cathepsin K. Immunolocalization in osteoclastoma and osteophyte showed intense punctate staining of cathepsin K exclusively in osteoclasts, with a polar distribution that was more intense at the bone surface. The abundant expression of cathepsin K selectively in osteoclasts strongly suggests that it plays a specialized role in bone resorption. Furthermore, the data suggest that random sequencing of ESTs from cDNA libraries is a valuable approach for identifying novel cell-selective genes.
Asunto(s)
Catepsinas/genética , Endopeptidasas , Osteoclastos/metabolismo , Secuencia de Aminoácidos , Animales , Resorción Ósea/enzimología , Catepsina B/genética , Catepsina K , Catepsina L , Cisteína Endopeptidasas , ADN Complementario , Humanos , Hibridación in Situ , Datos de Secuencia Molecular , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Lugares Marcados de SecuenciaRESUMEN
OBJECTIVE: To evaluate the effect of SK&F 106615 on joint integrity in rats with adjuvant-induced arthritis (AIA). METHODS: AIA was induced in Lewis rats on day 0, and the animals were treated either prophylactically (days 0-16 or days 0-23) or therapeutically (days 10-23) with SK&F 106615. Efficacy was determined by measurements of paw inflammation, bone mineral density (BMD) using dual x-ray absorptiometry, and magnetic resonance imaging (MRI). Joint integrity was also determined histologically, and serum interleukin-6 (IL-6) levels were measured as a marker of the antiinflammatory effects of the compound. RESULTS: Prophylactic treatment (days 0-16) of AIA rats with SK&F 106615 significantly inhibited paw volume at doses of 545 mg/kg/day given orally on 5 days each week. Extensive evaluation of joint integrity in rats treated with SK&F 106615 20 mg/kg/day orally for 23 days showed inhibition of paw volume, normalization of BMD, and significant improvement in disease by MRI and histologic assessment compared with the AIA controls. Elevated levels of serum IL-6 in AIA rats were reduced dramatically by SK&F 106615. Therapeutic treatment (days 10-23) resulted in similar protective effects measured by paw inflammation, BMD, and MRI. In the therapeutic protocol, serum IL-6 appeared to be a more sensitive marker of antiinflammatory activity than paw edema. CONCLUSION: Symptoms of AIA in rats are significantly reduced by prophylactic and therapeutic treatment with SK&F 106615. Of particular note, this compound appears to exert a protective effect on joint integrity and to have disease-modifying properties.