RESUMO
BACKGROUND: P2Y10, along with GPR34 and GPR174, is a G protein-coupled receptor that is activated by an endogenous lipid mediator lysophosphatidylserine (LysoPS). Its expression pattern and its function are completely unknown. We have previously shown that P2Y10 is one of the highly up-regulated genes at the late differentiation stage during in vitro eosinophilopoiesis. OBJECTIVE: We explored the expression and functions of P2Y10 in human cord blood (CB)-derived and peripheral blood (PB) eosinophils. METHODS: Real-time PCR, FACS, Western blot, ELISA, and chemotaxis assays were performed to determine the expression and function of P2Y10. RESULTS: As CB cells differentiated towards eosinophils, P2Y10 mRNA and protein were abundantly expressed. P2Y10 was the most highly expressed in the granulocytes from PB, to a lesser extent in monocytes, and least in lymphocytes. Further fractionation of granulocytes revealed that eosinophils express P2Y10 much more strongly than do neutrophils. PB eosinophils solely expressed P2Y10 among the three LysoPS receptors, while PB neutrophils expressed the three at comparable levels. LysoPS activated both CB and PB eosinophils to induce a robust ERK phosphorylation. Importantly, LysoPS was capable of triggering degranulation of ECP in PB eosinophils. This response was significantly reduced by pharmacological inhibitors of TNF-alpha-converting enzyme (TACE), epidermal growth factor receptor (EGFR), and ERK1/2, which were known to be required in P2Y10-mediated signalling pathways. However, LysoPS had no effect on chemotaxis, differentiation, or eosinophil survival. CONCLUSIONS AND CLINICAL RELEVANCE: LysoPS provokes eosinophil degranulation through P2Y10. Therefore, P2Y10 is a potential therapeutic target to control eosinophil-associated diseases.
Assuntos
Degranulação Celular/imunologia , Eosinófilos/imunologia , Eosinófilos/metabolismo , Receptores Purinérgicos P2/metabolismo , Biomarcadores , Degranulação Celular/genética , Diferenciação Celular/imunologia , Quimiotaxia/imunologia , Suscetibilidade a Doenças , Humanos , Lisofosfolipídeos/metabolismo , Sistema de Sinalização das MAP Quinases , Receptores Acoplados a Proteínas G/metabolismo , Receptores Purinérgicos P2/genética , Transdução de SinaisRESUMO
Fusarium oxysporum, a causal agent of Fusarium wilt, is one of the most important fungal pathogens worldwide, and detection of F. oxysporum DNA at the forma specialis level is crucial for disease diagnosis and control. In this study, two novel F. oxysporum f. sp. raphani (For)-specific primer sets were designed, FOR1-F/FOR1-R and FOR2-F/FOR2-R, to target FOQG_17868 and FOQG_17869 ORFs, respectively, which were selected based on the genome comparison of other formae speciales of F. oxysporum including conglutinans, cubense, lycopersici, melonis, and pisi. The primer sets FOR1-F/FOR1-R and FOR2-F/FOR2-R that amplified a 610- and 425-bp DNA fragment, respectively, were specific to For isolates which was confirmed using a total of 40 F. oxysporum isolates. From infected plants, the FOR2-F/FOR2-R primer set directly detected the DNA fragment of For isolates even when the radish plants were collected in their early stage of disease development. Although the loci targeted by the For-specific primer sets were not likely involved in the pathogenesis, the primer set FOR2-F/FOR2-R is available for the determination of pathogenicity of radish-infecting F. oxysporum isolates. This study is the first report providing novel primer sets to detect F. oxysporum f. sp. raphani. SIGNIFICANCE AND IMPACT OF THE STUDY: Because plant pathogenic Fusarium oxysporum has been classified into special forms based on its host specificity, identification of F. oxysporum usually requires a pathogenicity assay as well as knowledge of the morphological characteristics. For rapid and reliable diagnosis, this study provides PCR primer sets that specifically detect Fusarium oxysporum f. sp. raphani (For) which is a devastating pathogen of radish plants. Because one of the primer sets directly detected the DNA fragment of For isolates from infected plants, the specific PCR method demonstrated in this study will provide a foundation for integrated disease management practices in commodity crops.
Assuntos
Fusarium/patogenicidade , Especificidade de Hospedeiro , Doenças das Plantas/microbiologia , Raphanus/microbiologia , Primers do DNA/genética , Fusarium/genética , Fusarium/isolamento & purificação , Reação em Cadeia da PolimeraseRESUMO
Arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome is an autosomal recessive disorder caused by mutations in the VPS33B and VIPAS39. Here, we report novel mutations identified in four patients with ARC syndrome. We analyzed the entire coding regions of the VPS33B and VIPAS39 genes by direct sequencing. To detect novel splice site mutations, mRNA transcripts were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and sequencing. All four patients had compound heterozygous variants in the VPS33B gene. One patient had a previously reported splice site variant with unknown significance, c.239+5G>A, and a novel nonsense mutation, c.621G>A. The other three patients had the c.403+2T>A mutation, and each of them carried one of the splice site variants, c.239+5G>A or c.499-11G>A. c.239+5G>A and c.499-11G>A created novel splice sites which resulted in abnormal transcripts. No significant VIPAS39 mutation was detected in all patients. In patients suspected with ARC syndrome, mutation analysis of the VPS33B gene should be employed as a primary diagnostic test before performing invasive testing procedures such as organ biopsies. Performing mRNA analysis can be useful in predicting the pathogenic phenotype when the mutation seems to affect a normal splicing mechanism.
Assuntos
Artrogripose/genética , Colestase/genética , Mutação , Sítios de Splice de RNA/genética , Insuficiência Renal/genética , Proteínas de Transporte Vesicular/genética , Artrogripose/diagnóstico , Colestase/diagnóstico , Análise Mutacional de DNA , Feminino , Heterozigoto , Humanos , Lactente , Recém-Nascido , Masculino , Fenótipo , Insuficiência Renal/diagnóstico , República da CoreiaRESUMO
OBJECTIVES: Autoantibodies from the sera of Sjögren's syndrome patients (SS IgG) have been suggested to inhibit muscarinic receptor function. However, the acute nature of such an inhibitory effect remains controversial. In this study, we investigated the acute effects of SS IgG on muscarinic receptor function in human submandibular gland (HSG) cells. METHODS: The effects of autoantibodies on muscarinic receptor function were studied using microspectrofluorimetry, whole-cell patch clamp, immunofluorescence confocal microscopy, and a co-immunoprecipitation assay. RESULTS: Carbachol (CCh) was found to consistently increase intracellular calcium concentration ([Ca(2+) ](i) ) and activate K(+) current in HSG cells. However, pretreatment of the cells with SS IgG for 5 or 30 min significantly attenuated these responses, with a substantially more prominent effect after 30 min of treatment. Like CCh, adenosine 5'-triphosphate (ATP) also increased [Ca(2+) ](i) and activated K(+) currents in HSG cells, although pretreatment with SS IgG did not affect the cellular response to ATP. CCh was found to reorganize α-fodrin in HSG cells in a Ca(2+) -dependent manner. However, pretreatment with SS IgG prevented the cytoskeletal reorganization of α-fodrin induced by CCh. CONCLUSIONS: SS IgG acutely and reversibly inhibited muscarinic receptor function, thereby inhibiting the Ca(2+) mobilization necessary for the activation of K(+) currents and α-fodrin reorganization in HSG cells.
Assuntos
Autoanticorpos/fisiologia , Antagonistas Muscarínicos/metabolismo , Receptores Muscarínicos/metabolismo , Síndrome de Sjogren/imunologia , Síndrome de Sjogren/metabolismo , Autoanticorpos/química , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Carbacol/farmacologia , Proteínas de Transporte/metabolismo , Células Cultivadas , Distribuição de Qui-Quadrado , Humanos , Imunoglobulina G/imunologia , Imunoprecipitação/métodos , Proteínas dos Microfilamentos/metabolismo , Microscopia Confocal/métodos , Microespectrofotometria/métodos , Técnicas de Patch-Clamp , Canais de Potássio Cálcio-Ativados/metabolismo , Glândula Submandibular/citologia , Glândula Submandibular/imunologia , Glândula Submandibular/metabolismoRESUMO
Although accurate intracranial pressure (ICP) monitoring is essential for the diagnosis and treatment of severe brain diseases, current methods are performed invasively. Therefore, a safe and less invasive ICP measurement is required. The purpose of our study was to develop a simplified cranial cavity model for a better understanding of the relationship between the ICP and the pressure measurement within the dural venous sinus (DVS) to support the validity of using sinus pressure as the surrogate of the ICP. The in-house cranial cavity model had three components: the brain part, the DVS part, and the subarachnoid space (SAS) part. Pressure in other parts was measured when the pressure in the SAS part and, separately, brain part was increased from 0 (baseline) to 50 mmHg at intervals of 10 mmHg. When the pressure in the SAS part was increased from 10 to 50 mmHg at 10 mmHg interval, pressures of both the brain and DVS parts increased without significant difference (all P > 0.05). However, pressures in both the SAS and DVS parts differed while the pressure in the brain part was increased. The pressures in both parts showed about 70% of the increase in the brain part. Nevertheless, the pressures in the SAS and DVS parts were not significantly different (P > 0.05). A simplified in-house cranial cavity model was developed consisting of three compartments to represent the actual intracranial spaces. The pressure measurement within the DVS was feasible to use as a surrogate for the ICP measurement.
RESUMO
OBJECTIVES: Recently, autoantibodies directed against muscarinic type 3 receptor (M3R) have been reported in patients with primary SS. However, the precise epitope(s) of the M3R that interacts with SS autoantibodies remains unclear. The aim of this study was to identify the functional epitope of M3R which interacts with SS immunoglobulin G (IgG). METHODS: Purified IgGs were obtained from the sera of seven SS patients (six primary and one secondary SS) and two normal persons. We examined whether SS IgG inhibits M3R function and identified the epitope using six synthetic peptides covering all the extracellular domains of M3R by microspectrofluorimetry and surface plasmon resonance-based optical biosensor system (BIAcore system). RESULTS: A volume of 0.5 mg/ml SS IgG inhibited carbachol (CCh)-induced [Ca(2+)](i) transient (CICT) in human submandibular gland (HSG) cells. However, co-incubation of SS IgG with the 6th peptide (514-527 amino acid region) corresponding to the third extracellular loop of M3R, recovered CICT. The result was further confirmed by BIAcore analysis. We found that the 6th peptide interacts with IgGs from three primary SS patients in a concentration-dependent manner. The synthetic peptide which consists of amino acids 228-237 corresponding to the COOH-terminus of the second extracellular loop of M3R also bound to SS IgG. However, normal IgGs did not interact with the 6th peptide. CONCLUSIONS: The results suggest that the third extracellular loop of M3R represents a functional epitope bound by SS IgG, and thereby partly inhibits M3R function.
Assuntos
Autoanticorpos/metabolismo , Epitopos/metabolismo , Receptor Muscarínico M3/imunologia , Síndrome de Sjogren/imunologia , Adulto , Idoso , Reações Antígeno-Anticorpo/imunologia , Cálcio/metabolismo , Carbacol/antagonistas & inibidores , Carbacol/farmacologia , Células Cultivadas , Feminino , Humanos , Imunoglobulina G/metabolismo , Imunoglobulina G/farmacologia , Pessoa de Meia-Idade , Glândula Submandibular/citologia , Glândula Submandibular/efeitos dos fármacos , Glândula Submandibular/metabolismoRESUMO
Cathepsin K is an osteoclast-derived cysteine protease that has been implicated as playing a major role in bone resorption. A substantial body of evidence indicates that cathepsin K is critical in osteoclast-mediated bone resorption and suggests that its pharmacological inhibition should result in inhibition of bone resorption in vivo. Here we report the pharmacological characterization of SB-462795 (relacatib) as a potent and orally bioavailable small molecule inhibitor of cathepsin K that inhibits bone resorption both in vitro in human tissue and in vivo in cynomolgus monkeys. SB-462795 is a potent inhibitor of human cathepsins K, L, and V (K(i, app)=41, 68, and 53 pM, respectively) that exhibits 39-300-fold selectivity over other cathepsins. SB-462795 inhibited endogenous cathepsin K in situ in human osteoclasts and human osteoclast-mediated bone resorption with IC50 values of approximately 45 nM and approximately 70 nM, respectively. The anti-resorptive potential of SB-462795 was evaluated in normal as well as medically ovariectomized (Ovx) female cynomolgus monkeys. Serum levels of the C- and N-terminal telopeptides of Type I collagen (CTx and NTx, respectively) and urinary levels of NTx were monitored as biomarkers of bone resorption. Administration of SB-462795 to medically ovariectomized or normal monkeys resulted in an acute reduction in both serum and urinary markers of bone resorption within 1.5 h after dosing, and this effect lasted up to 48 h depending on the dose administered. Our data indicate that SB-462795 potently inhibits human cathepsin K in osteoclasts, resulting in a rapid inhibition of bone resorption both in vitro and in vivo in the monkey. These studies also demonstrate the therapeutic potential of relacatib in the treatment of postmenopausal osteoporosis and serves to model the planned clinical trials in human subjects.
Assuntos
Azepinas/uso terapêutico , Reabsorção Óssea/tratamento farmacológico , Catepsinas/antagonistas & inibidores , Osteoclastos/efeitos dos fármacos , Sulfonas/uso terapêutico , Administração Oral , Animais , Azepinas/administração & dosagem , Azepinas/farmacologia , Biomarcadores/sangue , Biomarcadores/urina , Catepsina K , Células Cultivadas , Colágeno Tipo I/sangue , Colágeno Tipo I/urina , Modelos Animais de Doenças , Humanos , Macaca fascicularis , Osteoclastos/enzimologia , Peptídeos/sangue , Peptídeos/urina , Sulfonas/administração & dosagem , Sulfonas/farmacologiaRESUMO
Infection of freshly isolated and cryopreserved lymphocytes with Epstein-Barr virus (EBV) leads to the establishment of human B lymphoblastoid cell lines (LCLs). Techniques for optimal infection of the lymphocytes are vital for the establishment of a human biobank. The present study found that more than half (58-86%) of such established LCLs had transport times of less than 48 h, cell densities exceeding 10(6) cells/ml and cell viabilities greater than 90%. After EBV infection, 3306 freshly isolated lymphocytes required 30.0 +/- 0.1 days to become LCLs. Conversely, 1210 cryopreserved lymphocytes required 36.2 +/- 0.4 days. Cell density and viability of the culture affected transformation time in freshly isolated lymphocytes. On the other hand, blood transport time, cryopreservation time and initial cell viability were major factors in cryopreserved specimens. These results contribute to general information concerning the establishment of a human biobank for EBV infected cells.
Assuntos
Linfócitos B/citologia , Linfócitos B/virologia , Transformação Celular Viral , Herpesvirus Humano 4 , Animais , Callithrix , Contagem de Células , Linhagem Celular Transformada/citologia , Linhagem Celular Transformada/virologia , Separação Celular , Sobrevivência Celular , Criopreservação , HumanosRESUMO
The yeast Rox1 hypoxic transcriptional repressor protein binds to and bends a specific DNA sequence through an HMG domain located at the N-terminus. To better understand the structure of Rox1 and how it interacts with DNA, 38 missense mutations in the HMG domain were isolated through a combination of random and site-directed mutageneses, the latter directed to two Ile residues that play an important role in DNA recognition and bending by HMG domains. The mutants were characterized in terms of their ability to repress the hypoxic gene ANB1 and the auto-repressed ROX1 gene in vivo. The mutant HMG domains were fused to maltose binding protein and expressed in and purified from Escherichia coli and their relative affinities for DNA and ability to bend DNA were determined. A model of the structure of the Rox1 HMG domain was derived using sequence similarities between Rox1 and the human protein SRY, the structure of which has been determined. The results of the mutational analysis are interpreted in terms of the model structure of Rox1.
Assuntos
Proteínas de Ligação a DNA/química , DNA/metabolismo , Proteínas Nucleares , Proteínas Repressoras/química , Fatores de Transcrição , Sequência de Aminoácidos , Sequência de Bases , Simulação por Computador , Análise Mutacional de DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Relação Dose-Resposta a Droga , Escherichia coli/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , Plasmídeos/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/fisiologia , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae , Homologia de Sequência de Aminoácidos , Proteína da Região Y Determinante do SexoRESUMO
Synaptosomes isolated from adult or newborn rat cerebrum take up L-lysine by two saturable systems, one with a high affinity low capacity and the other with a low affinity high capacity. Initial rate of uptake for low lysine concentrations is mort tissue. Analysis of kinetic data indicates that synaptosomes of the newborn have a higher Vmax than those of the adult for high affinity system but adult for high affinity system but adult synaptosomes have a higher Vmax than newborn for low affinity system. At a physiological lysine concentration of 0.5 mM, the calculated contributions of two systems indicate that the adult uptake occurs for about 71% by low affinity system but the newborn utilizes both systems to the same extent. The uptake is sodium independent but pH dependent. Lysine uptake is inhibited by other dibasic amino acids, arginine and ornithine but not cystine. Kinetic analysis indicates that arginine specifically inhibits the high affinity, low Km system for lysine uptake.
Assuntos
Encéfalo/metabolismo , Lisina/metabolismo , Sinaptossomos/metabolismo , Envelhecimento , Animais , Animais Recém-Nascidos , Transporte Biológico , Encéfalo/crescimento & desenvolvimento , Cinética , Masculino , Ratos , Sódio/farmacologiaRESUMO
ALPHA-Methyl-D-glucoside has been used to study the invitro mechanism of the effect of maleic acid on sugar transport, using isolated rat renal tubule fragments. 6 mM maleate maximally inhibits the ability of the tubule to establish a concentration gradient for this model sugar with no evidence of ultrastructural changes. This inhibition is due to a 100% increase in efflux, as well as to a 50% decrease in influx with more prolonged incubation. The data presented here are consistent with those of other workers, but their work does not explain our results, which therefore deserve further investigation by other techniques.
Assuntos
Túbulos Renais/metabolismo , Maleatos/farmacologia , Metilglucosídeos/metabolismo , Metilglicosídeos/metabolismo , Animais , Transporte Biológico Ativo , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/ultraestrutura , Cinética , Masculino , Microscopia Eletrônica , Ratos , Fatores de TempoRESUMO
Isolated renal cortical tubule fragments from rats ranging in age from less than 48 h to 15 weeks were used to examine the pattern of cystine uptake with development. Immature tubules took up cystine with a faster initial rate than mature tubules and did not reach a steady state by 60 min. By eight weeks of age, the timed uptake of cystine began to approach a steady state and between 8 and 11 weeks the uptake pattern achieved its adult form of reaching a steady state by 30 min of incubation. Analysis of the intracellular metabolism of the cystine taken up by the newborn tubules revealed that the majority had been reduced to cysteine with the formation of small amounts of reduced glutathione. Cystine entered the renal cortical tubule cell from the newborn via two saturable transport systems similar to the mature animal. The kinetic parameters of initial uptake of these two transport systems were similar in the mature and newborn animal except for a higher maximum transport velocity for the low Km, low capacity system in the newborn. Lysine inhibited cystine uptake by newborn tubules and this inhibition appeared to occur on the low Km, low capacity transport system similar to the adult. Cystine uptake was sodium dependent with an apparent affinity for sodium of 36 mequiv./l. From this data, the physiologic cystinuria of the immature animal does not appear to be refeable to a lower rate of influx as previously observed with the cortical slice. Other mechanisms should be sought to explain this phenomenon of immaturity.
Assuntos
Cistina/metabolismo , Córtex Renal/crescimento & desenvolvimento , Túbulos Renais/crescimento & desenvolvimento , Envelhecimento , Animais , Transporte Biológico/efeitos dos fármacos , Feminino , Córtex Renal/metabolismo , Túbulos Renais/metabolismo , Cinética , Lisina/farmacologia , Masculino , Ratos , Ratos EndogâmicosRESUMO
The initial uptake of glycine by renal cortical slices from newborn Sprague-Dawley and Long-Evans rats is the same as that observed in adult tissues. Both newborn and adult tissue possess similar high and low affinity glycine transport systems which require an examination of velocity measurements over a wide range of concentration (0.02--50.0 mM) for their discernment. Initial rates of glycine uptake by isolated renal tubule fragments from newborn and adults are similar at a physiological substrate concentration but at high glycine levels there appears to be a decrease in velocity of uptake (V) associated with the high Km system in the young. Whatever preparation of renal cortex is studied, there is a consistent finding that immature tissue is able to accumulate much higher intracellular levels of glycine than the adult, a finding consistent with slower efflux from the cell. An interpretation of the etiology of physiologic aminoaciduria in young animals should take this into account.
Assuntos
Glicina/metabolismo , Córtex Renal/crescimento & desenvolvimento , Envelhecimento , Animais , Animais Recém-Nascidos , Transporte Biológico , Feminino , Humanos , Córtex Renal/metabolismo , Túbulos Renais/metabolismo , Cinética , RatosRESUMO
The uptake of L-lysine was examined in isolated renal cortical tubule fragments from adult and 1-week-old dogs. Lysine uptake by adult tubules was initially more rapid than that by the immature tubules. This uptake by mature tubules reached a steady state after 30 min of incubation, while the newborn tubules still had not reached a steady state by 90 min of incubation. Because a steady state of lysine uptake was not attained with the immature tubules, their uptake of lysine exceeded that of the adult after 60 min of incubation. Kinetic studies revealed that lysine was taken up by one saturable transport system with a Km of 0.56 mM and Vmax of 6.18 mmol/liter intercellular fluid per 5 min in the adult and one saturable transport system in the 1-week-old with a Km of 0.38 mM and Vmax of 3.66 mmol/l intracellular fluid per 5 min. Lysine also entered the renal tubule cells in both age groups via a diffusional pathway with a kd of 0.35 min-1 in the adult and 0.30 min-1 in the newborn. Cystine competitively inhibited lysine uptake by adult dog tubules with a Ki of 0.61 mM. The other dibasic amino acids, ornithine and arginine, also inhibited lysine uptake in both the adult and the newborn.
Assuntos
Córtex Renal/metabolismo , Túbulos Renais/metabolismo , Lisina/metabolismo , Fatores Etários , Animais , Animais Recém-Nascidos/metabolismo , Cistina/farmacologia , Cães , Relação Dose-Resposta a Droga , Feminino , Técnicas In Vitro , Cinética , MasculinoRESUMO
Aerobic repression of the hypoxic genes of Saccharomyces cerevisiae is mediated by the DNA-binding protein Rox1 and the Tup1/Ssn6 general repression complex. To determine the DNA sequence requirements for repression, we carried out a mutational analysis of the consensus Rox1-binding site and an analysis of the arrangement of the Rox1 sites into operators in the hypoxic ANB1 gene. We found that single base pair substitutions in the consensus sequence resulted in lower affinities for Rox1, and the decreased affinity of Rox1 for mutant sites correlated with the ability of these sites to repress expression of the hypoxic ANB1 gene. In addition, there was a general but not complete correlation between the strength of repression of a given hypoxic gene and the compliance of the Rox1 sites in that gene to the consensus sequence. An analysis of the ANB1 operators revealed that the two Rox1 sites within an operator acted synergistically in vivo, but that Rox1 did not bind cooperatively in vitro, suggesting the presence of a higher order repression complex in the cell. In addition, the spacing or helical phasing of the Rox1 sites was not important in repression. The differential repression by the two operators of the ANB1 gene was found to be due partly to the location of the operators and partly to the sequences between the two Rox1-binding sites in each. Finally, while Rox1 repression requires the Tup1/Ssn6 general repression complex and this complex has been proposed to require the aminoterminal regions of histones H3 and H4 for full repression of a number of genes, we found that these regions were dispensable for ANB1 repression and the repression of two other hypoxic genes.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Regiões Operadoras Genéticas , Proteínas Repressoras/metabolismo , Saccharomyces cerevisiae/genética , Sequência Consenso , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Histonas/fisiologia , Mutagênese , Oxigênio/metabolismo , Mutação Puntual , Proteínas Repressoras/genética , Proteínas de Saccharomyces cerevisiaeRESUMO
LSTRA is a mouse lymphoma cell line overexpressing the src-related oncogene product and T cell marker p56lck. We have discovered that LSTRA cells, like HL60 and U937 promyelocytic leukemia cells, can be induced to differentiate toward macrophages by the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) or granulocytes by the cyclic nucleotide analogue dibutyryl cAMP (dbcAMP). One property of mature macrophages and granulocytes is the ability to produce nitric oxide, a highly reactive free radical that is important in non-specific host defense. Nitric oxide production by mature macrophages is stimulated by inflammatory mediators via an inducible form of the enzyme, nitric oxide synthase. We report that LSTRA cells acquire the ability to produce nitric oxide following differentiation induced by TPA and, to a lesser extent, by dbcAMP. Nitric oxide production by the cells is dependent on L-arginine and blocked by several inhibitors of nitric oxide synthase, including N-monomethyl-L-arginine, L-canavanine, L-arginine benzyl ester, and L-arginine methyl ester. In macrophage-differentiated LSTRA cells, the inflammatory cytokine interferon-gamma was found to be a potent inducer of nitric oxide production. This was correlated with a marked increase in nitric oxide synthase activity in the cells that was due to interferon-gamma-induced expression of the macrophage-inducible form of nitric oxide synthase protein as well as mRNA. Differentiated LSTRA cells also expressed increased amounts of a constitutive form of nitric oxide synthase that was also present in undifferentiated cells. Taken together with previous findings, these results support the model that LSTRA cells have the capacity to differentiate toward mature macrophages.
Assuntos
Aminoácido Oxirredutases/metabolismo , Macrófagos/fisiologia , Óxido Nítrico/metabolismo , Aminoácido Oxirredutases/genética , Animais , Arginina/análogos & derivados , Arginina/farmacologia , Bucladesina/farmacologia , Canavanina/farmacologia , Diferenciação Celular/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Camundongos , Óxido Nítrico Sintase , RNA Mensageiro/genética , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais Cultivadas , ômega-N-MetilargininaRESUMO
We present an unusual case of metastatic renal cell carcinoma (RCC) mimicking diverticulitis in a 76-year-old man with a 16-year history of chronic lymphocytic leukaemia (CLL) and a 2â cm left renal mass. The patient presented with severe abdominal pain and lower gastrointestinal bleeding with anticoagulation from recent pulmonary embolism. His clinical course was troubled by recurrent hospitalisations and complications that delayed investigations and potential treatments. Radiographic findings revealed stable CLL, mild sigmoid diverticulitis and a small renal mass. Small renal masses (less than 4â cm) are considered low risk for metastasising and are, thus, often observed or ablated, rather than resected. Furthermore, gastrointestinal metastases from RCC are rare. This case adds new perspective to the unpredictable nature of RCC and how synchronous malignancies may be masked in patients with long-standing CLL.
Assuntos
Carcinoma de Células Renais/secundário , Diverticulite/diagnóstico , Hemorragia Gastrointestinal/etiologia , Neoplasias Renais/patologia , Leucemia Linfocítica Crônica de Células B/complicações , Doenças do Colo Sigmoide/diagnóstico , Dor Abdominal/etiologia , Idoso , Carcinoma de Células Renais/complicações , Carcinoma de Células Renais/diagnóstico , Diagnóstico Diferencial , Evolução Fatal , Humanos , Neoplasias Renais/complicações , Neoplasias Renais/diagnóstico , Masculino , Doenças do Colo Sigmoide/etiologiaRESUMO
A human in vitro resorption assay has been developed using osteoclastoma-derived osteoclasts and used to evaluate novel antiresorptive agents including antagonists of the alphavbeta3 integrin, and inhibitors of cathepsin K and the osteoclast ATPase. The potency of novel compounds in the in vitro resorption assay correlates with functional assays for each class of inhibitor: the human alphavbeta3-mediated cell adhesion assay for the vitronectin receptor antagonists (r2 = 0.82), the chick osteoclast vacuolar ATPase enzyme assay for the H+-ATPase inhibitors (r2 = 0.77) and the recombinant human cathepsin K enzyme assay for the cathepsin K inhibitors (r2 = 0.80). Cell suspensions, rich in osteoclasts, are prepared by collagenase digestion of the tumor tissue. These cells can be stored long-term in liquid nitrogen and upon thawing maintain their bone-resorbing phenotype. The cryopreserved cells can be cultured on bovine cortical bone for 24-48 h and resorption can be measured by either confocal microscopy or biochemical assays. The resorptive activity of osteoclasts derived from a number of tumors can be inhibited reproducibly using a number of mechanistically unique antiresorptive compounds. In addition, the measurement of resorption pits by laser confocal microscopy correlates with the release of type I collagen C-telopeptides or N-telopeptides, as measured by enzyme-linked immunosorbent assay. Resorption can be measured reproducibly using a 48-h incubation of osteoclasts on bone slices, or a 24-h incubation with bone particles. This in vitro human osteoclast resorption assay provides a robust system for the evaluation of inhibitors of osteoclastic function that may be developed for the treatment of metabolic bone diseases such as osteoporosis.
Assuntos
Biomarcadores Tumorais/análise , Neoplasias Ósseas/química , Reabsorção Óssea , Tumor de Células Gigantes do Osso/química , Adenosina Trifosfatases/metabolismo , Animais , Catepsina K , Catepsinas/metabolismo , Bovinos , Adesão Celular , Separação Celular , Ensaio de Imunoadsorção Enzimática , Congelamento , Humanos , Técnicas In Vitro , Fenótipo , Reprodutibilidade dos TestesRESUMO
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.
Assuntos
Reabsorção Óssea/metabolismo , Catepsinas/metabolismo , Osteoclastos/metabolismo , Bioquímica/métodos , Osso e Ossos/embriologia , Osso e Ossos/enzimologia , Catepsina K , Catepsinas/análise , Catepsinas/antagonistas & inibidores , Adesão Celular , Inibidores de Cisteína Proteinase/farmacologia , Tumor de Células Gigantes do Osso/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Rim/embriologia , Rim/enzimologia , Leucina/análogos & derivados , Leucina/farmacologia , Modelos Lineares , Oligopeptídeos/farmacologia , Pepstatinas/farmacologia , Fluoreto de Fenilmetilsulfonil/farmacologia , Inibidores de Proteases/farmacologia , Processamento de Proteína Pós-Traducional , Especificidade por Substrato , Células Tumorais CultivadasRESUMO
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.