Assuntos
Ketamina , Nefropatias , Transplante de Rim , Humanos , Imunossupressores , Ketamina/efeitos adversosRESUMO
BACKGROUND: Serrated polyps of the colorectum have distinct histological features and malignant potential. AIM: To assess the association between the presence of serrated polyps and synchronous advanced colorectal neoplasia. METHODS: Among 4989 asymptomatic Chinese individuals aged 50-70 years who underwent screening colonoscopy, 281 cases with advanced neoplasia (adenoma ≥1 cm, with tubulovillous/villous histology, with high-grade dysplasia, or invasive adenocarcinoma) were compared with 4708 controls without advanced neoplasia for age, sex, smoking history, body mass index, family history of colorectal cancer and the presence of serrated polyps. Independent predictors of advanced neoplasia were determined by multivariate logistic regression analysis. RESULTS: The prevalence of advanced neoplasia and serrated polyps (excluding small distal hyperplastic polyps) was 5.7% and 5.6%, respectively. 3.7% and 0.4% subjects had proximal and large (≥10 mm) serrated polyps, respectively. Independent predictors of synchronous advanced colorectal neoplasia were the presence of sessile serrated adenomas (OR: 4.52; 95% CI: 2.40-8.49), proximal serrated polyps (OR: 2.23, 95% CI: 1.38-3.60), large serrated polyps (OR: 59.25; 95% CI: 18.85-186.21), hyperplastic polyps (OR: 1.66; 95% CI: 1.03-2.67), three or more serrated polyps (OR: 4.86; 95% CI: 1.24-19.15) and one or more non-advanced tubular adenomas (OR: 3.58, 95% CI: 2.59-4.96). CONCLUSION: Detection of proximal, sessile and/or large serrated polyps at screening colonoscopy is independently associated with an increased risk for synchronous advanced neoplasia.
Assuntos
Adenocarcinoma/epidemiologia , Adenoma/epidemiologia , Pólipos do Colo/epidemiologia , Neoplasias Colorretais/epidemiologia , Adenocarcinoma/diagnóstico , Adenoma/diagnóstico , Fatores Etários , Idoso , Índice de Massa Corporal , China , Pólipos do Colo/diagnóstico , Colonoscopia , Neoplasias Colorretais/diagnóstico , Detecção Precoce de Câncer , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Prevalência , Fatores de Risco , Fatores Sexuais , Fumar/epidemiologiaRESUMO
BACKGROUND: The role of a faecal immunochemical test (FIT) in screening individuals with a positive family history of colorectal cancer (CRC) is not clear. AIM: To assess the diagnostic accuracy of FIT using colonoscopy findings as the gold standard in identifying colorectal neoplasms. METHODS: We analysed data from 4539 asymptomatic subjects aged 50-70 years who had both colonoscopy and FIT (Hemosure; W.H.P.M., Inc, El Monte, CA, USA) at our bowel cancer screening centre between 2008 and 2012. A total of 572 subjects (12.6%) had a family history of CRC. Our primary outcome was the sensitivity of FIT in detecting advanced neoplasms and cancers in subjects with a family history of CRC. A family history of CRC was defined as any first-degree relative with a history of CRC. RESULTS: Among 572 subjects with a family history of CRC, adenoma, advanced neoplasm and cancer were found at screening colonoscopy in 29.4%, 6.5% and 0.7% individuals, respectively. The sensitivity of FIT in detecting adenoma, advanced neoplasm and cancer was 9.5% [95% confidence interval (CI), 5.7-15.3], 35.1% (95% CI, 20.7-52.6) and 25.0% (95% CI, 1.3-78.1), respectively. Among FIT-negative subjects who have a family history of CRC, adenoma was found in 152 (29.6%), advanced neoplasm in 24 (4.7%) and cancer in 3 (0.6%) individuals. CONCLUSION: Compared with colonoscopy, FIT is more likely to miss advanced neoplasms or cancers in individuals with a family history of CRC.
Assuntos
Adenoma/diagnóstico , Colonoscopia/métodos , Neoplasias Colorretais/diagnóstico , Programas de Rastreamento/métodos , Adenoma/patologia , Idoso , Neoplasias Colorretais/patologia , Intervalos de Confiança , Detecção Precoce de Câncer/métodos , Fezes/química , Feminino , Humanos , Imunoquímica/métodos , Masculino , Pessoa de Meia-IdadeRESUMO
In this study, we evaluated the pharmacological effects of Ganoderma lucidum (G. lucidum) (water-extract) (0.003, 0.03 and 0.3g/kg, 4-week oral gavage) consumption using the lean (+db/+m) and the obese/diabetic (+db/+db) mice. Different physiological parameters (plasma glucose and insulin levels, lipoproteins-cholesterol levels, phosphoenolpyruvate carboxykinase (PEPCK), 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA reductase) and isolated aorta relaxation of both species were measured and compared. G. lucidum (0.03 and 0.3g/kg) lowered the serum glucose level in +db/+db mice after the first week of treatment whereas a reduction was observed in +db/+m mice only fed with 0.3g/kg of G. lucidum at the fourth week. A higher hepatic PEPCK gene expression was found in +db/+db mice. G. lucidum (0.03 and 0.3g/kg) markedly reduced the PEPCK expression in +db/+db mice whereas the expression of PEPCK was attenuated in +db/+m mice (0.3g/kg G. lucidum). HMG CoA reductase protein expression (in both hepatic and extra-hepatic organs) and the serum insulin level were not altered by G. lucidum. These data demonstrate that G. lucidum consumption can provide beneficial effects in treating type 2 diabetes mellitus (T2DM) by lowering the serum glucose levels through the suppression of the hepatic PEPCK gene expression.
Assuntos
Glicemia/metabolismo , Diabetes Mellitus Experimental/tratamento farmacológico , Hipoglicemiantes/uso terapêutico , Fitoterapia , Extratos Vegetais/uso terapêutico , Proteínas Serina-Treonina Quinases/metabolismo , Reishi , Gordura Abdominal/efeitos dos fármacos , Animais , Antioxidantes/farmacologia , Antioxidantes/uso terapêutico , Peso Corporal/efeitos dos fármacos , Colesterol/sangue , Diabetes Mellitus Experimental/sangue , Ingestão de Energia/efeitos dos fármacos , Hidroximetilglutaril-CoA Redutases/metabolismo , Hipoglicemiantes/farmacologia , Insulina/sangue , Lipólise/efeitos dos fármacos , Lipoproteínas/sangue , Camundongos , Obesidade/tratamento farmacológico , Extratos Vegetais/farmacologia , Vasodilatação/efeitos dos fármacosRESUMO
BACKGROUND AND PURPOSE: Statins (3-hydroxy-3-methyl-glutaryl coenzyme A (HMG CoA) reductase inhibitors) have been demonstrated to reduce cardiovascular mortality. It is unclear how the expression level of HMG CoA reductase in cardiovascular tissues compares with that in cells derived from the liver. We hypothesized that this enzyme exists in different cardiovascular tissues, and simvastatin modulates the vascular iberiotoxin-sensitive Ca2+-activated K(+) (BK(Ca)) channels. EXPERIMENTAL APPROACHES: Expression of HMG CoA reductase in different cardiovascular preparations was measured. Effects of simvastatin on BK(Ca) channel gatings of porcine coronary artery smooth muscle cells were evaluated. KEY RESULTS: Western immunoblots revealed the biochemical existence of HMG CoA reductase in human cardiovascular tissues and porcine coronary artery. In porcine coronary artery smooth muscle cells, extracellular simvastatin (1, 3 and 10 microM) (hydrophobic), but not simvastatin Na+ (hydrophilic), inhibited the BK(Ca) channels with a minimal recovery upon washout. Isopimaric acid (10 microM)-mediated enhancement of the BK(Ca) amplitude was reversed by external simvastatin. Simvastatin Na+ (10 microM, applied internally), markedly attenuated isopimaric acid (10 microM)-induced enhancement of the BK(Ca) amplitude. Reduced glutathione (5 mM; in the pipette solution) abolished simvastatin -elicited inhibition. Mevalonolactone (500 microM) and geranylgeranyl pyrophosphate (20 microM) only prevented simvastatin (1 and 3 microM)-induced responses. simvastatin (10 microM ) caused a rottlerin (1 microM)-sensitive (cycloheximide (10 microM)-insensitive) increase of PKC-delta protein expression. CONCLUSIONS AND IMPLICATIONS: Our results demonstrated the biochemical presence of HMG CoA reductase in different cardiovascular tissues, and that simvastatin inhibited the BK(Ca) channels of the arterial smooth muscle cells through multiple intracellular pathways.
Assuntos
Músculo Liso Vascular/efeitos dos fármacos , Peptídeos/farmacologia , Canais de Potássio Cálcio-Ativados/antagonistas & inibidores , Sinvastatina/farmacologia , Adulto , Idoso , Animais , Western Blotting , Caveolina 1/biossíntese , Linhagem Celular , Linhagem Celular Tumoral , Vasos Coronários/citologia , Vasos Coronários/efeitos dos fármacos , Vasos Coronários/fisiologia , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Feminino , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Imidazóis/farmacologia , Técnicas In Vitro , Masculino , Potenciais da Membrana/efeitos dos fármacos , Pessoa de Meia-Idade , Músculo Liso Vascular/citologia , Músculo Liso Vascular/fisiologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/fisiologia , Ésteres de Forbol/farmacologia , Canais de Potássio Cálcio-Ativados/metabolismo , Canais de Potássio Cálcio-Ativados/fisiologia , Proteína Quinase C-delta/metabolismo , Piridinas/farmacologia , Sinvastatina/química , SuínosRESUMO
INTRODUCTION: Haemorrhoidectomy is traditionally an inpatient procedure. With many benefits, the day-surgery arrangement is an attractive alternative. The feasibility of day-surgery haemorrhoidectomy was explored and the hospital days were calculated in a case controlled design. METHODS: A single surgeon's experience of day-surgery haemorrhoidectomy between 1 July 1999 and 31 March 2000 was compared with inpatient haemorrhoidectomy during the same period. The operations were performed at United Christian Hospital Department of Surgery, Hong Kong (a government-funded public hospital). Statistical tests were applied where appropriate. RESULTS: There were 30 day-surgery and 15 inpatient haemorrhoidectomies. The groups were comparable in terms of age, gender, severity of haemorrhoids, method and duration of haemorrhoidectomy, blood loss, residual haemorrhoids, duration of follow up and unplanned readmission rate. Significantly more day-patients received general than spinal anaesthesia. Twenty-six of 30 (87%) patients were successfully discharged after day surgery. Two were admitted for transient fever (< 24 h), one for micturition syncope and one for acute urinary retention. There were four unplanned readmissions after day surgery: one for pain and three for secondary bleeding. All stopped spontaneously. All three unplanned readmissions after inpatient surgery were for secondary bleeding. All stopped spontaneously. Patient stay was significantly shorter for day surgery (1 +/- 1 day) than for the inpatient arrangement (4 +/- 1.6 days). CONCLUSION: Day-surgery haemorrhoidectomy is feasible. The significantly shorter hospital stay implies savings in public medical expenses.
Assuntos
Procedimentos Cirúrgicos Ambulatórios , Hemorroidas/cirurgia , Procedimentos Cirúrgicos Ambulatórios/estatística & dados numéricos , Estudos de Casos e Controles , Estudos de Viabilidade , Feminino , Humanos , Tempo de Internação/estatística & dados numéricos , Masculino , Pessoa de Meia-Idade , Readmissão do Paciente/estatística & dados numéricosRESUMO
BACKGROUND AND PURPOSE: One advantage of minimal-access surgery is that it produces less pain. A radially expanding trocar has been claimed to reduce pain further. We aimed to evaluate this claim. PATIENTS AND METHODS: This was a randomized controlled single-blind clinical trial. Fifty-four patients who underwent laparoscopic cholecystectomy at the Department of Surgery, United Christian Hospital, Hong Kong, between July 1997 and September 1998 were randomized into either the study group or the control group. The radially expanding 10-mm trocar was used for the epigastric port in the study group. The conventional 10-mm metal trocar was used similarly in the control group. The operation was otherwise performed with a standardized technique. Another conventional 10-mm metal trocar was used for the subumbilical port for all patients. Pain was measured using a visual analog scale. Pain scores for the epigastric port and subumbilical port were documented for 3 days after the surgery. RESULTS: There was no difference in age, sex, diagnoses, operating time, or conversion rate. There was consistently no difference in the pain experienced in the subumbilical wound, whereas pain at the epigastric wound was consistently less with the radially expanding trocar (p < 0.05). CONCLUSION: The radially expanding trocar produces less early postoperative pain than the conventional metal trocar.
Assuntos
Laparoscópios/efeitos adversos , Laparoscopia/efeitos adversos , Dor Pós-Operatória/prevenção & controle , Instrumentos Cirúrgicos/efeitos adversos , Desenho de Equipamento , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Método Simples-CegoRESUMO
BACKGROUND: The technique of laparoscopic-assisted resection of colorectal carcinoma has been established. However, whether such a procedure is beneficial to patients is uncertain. OBJECTIVE: To review the immediate and medium-term results of laparoscopic-assisted resection in patients with rectosigmoid carcinoma. PATIENTS AND INTERVENTIONS: We attempted laparoscopic-assisted sigmoid colectomy or anterior resection in 50 patients with rectosigmoid carcinoma (ie, the study group). The results were compared with those of 50 matched patients who underwent conventional open resection in the immediate prelaparoscopic era (ie, the control group). RESULTS: The median follow-up times for the study and control groups were 32.8 and 39.1 months, respectively. The operating time was significantly longer (P < .001, Student t test), while the analgesic requirement was significantly less (P < .001, Mann-Whitney U test) and the duration of hospitalization was significantly shorter (P = .001, Mann-Whitney U test), in the study group than in the control group. The oncological clearance (ie, the number of lymph nodes removed and the distal resection margin), the complication rate, the disease-free rate, and the survival rate were comparable in the 2 groups. CONCLUSION: The immediate and medium-term results of laparoscopic-assisted resection of rectosigmoid carcinoma are promising.
Assuntos
Laparoscopia , Neoplasias Retais/cirurgia , Neoplasias do Colo Sigmoide/cirurgia , Idoso , Analgésicos/uso terapêutico , Estudos de Casos e Controles , Dieta , Intervalo Livre de Doença , Feminino , Humanos , Tempo de Internação , Masculino , Análise por Pareamento , Pessoa de Meia-Idade , Dor Pós-Operatória/tratamento farmacológico , Neoplasias Retais/patologia , Neoplasias do Colo Sigmoide/patologia , Análise de Sobrevida , Fatores de Tempo , Resultado do TratamentoRESUMO
Extracellular cAMP serves as a chemoattractant as well as a signal which regulates gene expression during development of Dictyostelium discoideum. The cell adhesion molecule gp80 is expressed at the aggregation stage, between 6 and 10 h of development, and is known to be under cAMP regulation. Transcription of the gp80 gene is first turned on at a low, basal level at the preaggregation stage and is then greatly augmented by pulses of low levels of cAMP at the aggregation stage. Using cloned cDNA sequences, we have isolated genomic DNA fragments encompassing the gp80 gene. The gp80 gene has a single open reading frame, with multiple transcription start sites located downstream from a putative TATA box. Several short, repeated sequences in the upstream sequence have also been identified. The cloned 1.3-kilobase upstream DNA was sufficient to confer proper temporal and cAMP regulation on a gp80 minigene reporter in Dictyostelium cells. Deletional analysis of this 5'-flanking DNA led to the mapping of a cAMP-response element (CRE) in the gp80 gene to sequences between -306 and -289 base pairs upstream of the translational start site. Present within this region is a sequence we refer to as box 1 (TGGTGTG). The gp80 box 1-CRE binds specifically to a protein present in nuclear extracts, but binding is abolished when mutations are introduced in the box 1 sequence. The gp80 box 1-CRE shows little sequence homology to CREs of late developmental genes and the expression of gp80 may involve a distinct signal transduction pathway.
Assuntos
Moléculas de Adesão Celular/genética , AMP Cíclico/fisiologia , Dictyostelium/genética , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Regiões Promotoras Genéticas , Sequências Reguladoras de Ácido Nucleico , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Análise Mutacional de DNA , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/química , Sequências Repetitivas de Ácido Nucleico , Mapeamento por Restrição , Homologia de Sequência do Ácido NucleicoRESUMO
cDNAs encoding the largest subunit of RNA polymerase II were isolated from a Dictyostelium cDNA library. A total of 2.9 kilobases (kb) of cDNA was sequenced and the amino acid sequence of the carboxyl-terminal half of the protein was deduced. Similar to other eukaryotic RNA polymerases II, the largest subunit of Dictyostelium RNA polymerase II contains a unique repetitive tail domain at its carboxyl-terminal region. It consists of 24 highly conserved heptapeptide repeats, with a consensus sequence of Tyr-Ser-Pro-Thr-Ser-Pro-Ser. In addition to the tail domain, five segments of the deduced primary structure show > 50% sequence identity with either yeast or mouse protein. RNA blots show that cDNA probes hybridized with a single mRNA species of approximately 6 kb and immunoblots using a monoclonal antibody raised against the tail domain lighted up a single protein band of 200 kilodaltons. Interestingly, expression of the largest subunit of RNA polymerase II appears to be under developmental regulation. The accumulation of its mRNA showed a 60% increase during the first 3 h of development, followed by a steady decrease during the next 6 h. Cells began to accumulate a higher level of the RNA polymerase II mRNA after 9 h of development. When cells were treated with low concentrations of cAMP pulses to stimulate the developmental process, the pattern of mRNA accumulation moved 3 h ahead, but otherwise remained similar to that of control cells.
Assuntos
Dictyostelium/enzimologia , Regulação Fúngica da Expressão Gênica/fisiologia , Estrutura Terciária de Proteína , RNA Polimerase II/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Immunoblotting , Dados de Sequência Molecular , RNA Polimerase II/genética , Homologia de Sequência de AminoácidosRESUMO
We previously provided in vitro evidence that the cell surface glycoprotein of Mr80,000 (gp80) of Dictyostelium discoideum is capable of mediating EDTA-resistant cell-cell binding. Expression of gp80 is specific for the aggregation stage when cells form tight aggregates. To investigate the physiological role of gp80, Dictyostelium cells were transformed with a vector containing gp80 cDNA fused to an actin promoter. gp80 transcripts were detected in transformed cells in their vegetative growth phase. Transformants at this stage also exhibited EDTA-resistant cell cohesion, thus providing direct in vivo evidence that gp80 mediates cell-cell binding via homophilic interaction. While aggregates of the parental strain KAX3 had the tendency to break up to form small slugs, transformants expressing an increased amount of gp80 were able to maintain the integrity of aggregates, giving rise to larger slugs, resulting in the formation of bigger fruiting bodies. To further demonstrate that the increase in slug size could be correlated with the expression of gp80, cells of the parental strain were treated with exogenous cAMP pulses to stimulate an over-expression of gp80. The treated cells also gave rise to larger slugs, consistent with the notion that slug size is influenced by intercellular adhesiveness during development.
Assuntos
Moléculas de Adesão Celular/fisiologia , Dictyostelium/fisiologia , Proteínas de Protozoários , Adesão Celular , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/isolamento & purificação , AMP Cíclico/farmacologia , Dictyostelium/efeitos dos fármacos , Dictyostelium/genética , Ácido Edético/farmacologia , Imunofluorescência , Regulação Fúngica da Expressão Gênica , Vetores Genéticos , RNA Fúngico/genética , RNA Fúngico/isolamento & purificação , Transformação GenéticaRESUMO
At the aggregation stage of Dictyostelium discoideum development, a cell surface glycoprotein of Mr 80,000 (gp80) has been found to mediate the EDTA-resistant type of cell-cell adhesion via homophilic interaction (Siu, C.-H., A. Cho, and A. H. C. Choi. 1987. J. Cell Biol. 105:2523-2533). To investigate the structure-function relationships of gp80, we have isolated full length cDNA clones for gp80 and determined the DNA sequence. The deduced structure of gp80 showed three major domains. An amino-terminal globular domain composed of the bulk of the protein is supported by a short stalk region, which is followed by a membrane anchor at the carboxy terminus. Structural analysis suggested that the cell-binding domain of gp80 resides within the globular domain near the amino terminus. To investigate the relationship of the cell-binding activity to this region of the polypeptide, three protein A/gp80 (PA80) gene fusions were constructed using the expression vector pRIT2T. These PA80 fusion proteins were assayed for their ability to bind to aggregation stage cells. Binding of 125I-labeled fusion proteins PA80I (containing the Val123 to Ile514 fragment of gp80) and PA80II (Val123 to Ala258) was dosage dependent and could be inhibited by precoating cells with the cell cohesion-blocking mAb 80L5C4. On the other hand, there was no appreciable binding of PA80III (Ile174 to Ile514) to cells. Reassociation of cells was significantly inhibited in the presence of PA80I or PA80II. In addition, 125I-labeled PA80II exhibited homophilic interaction with immobilized PA80I, PA80II, or gp80. The results of these studies lead to the mapping of a cell-binding domain in the region between Val123 and Leu173 of gp80 and provide direct evidence that the cell-binding activity of gp80 resides in the protein moiety.
Assuntos
Antígenos de Superfície/metabolismo , Moléculas de Adesão Celular , Dictyostelium/metabolismo , Proteínas de Protozoários , Sequência de Aminoácidos , Bacteriófagos/genética , Sequência de Bases , Ligação Competitiva , Metabolismo dos Carboidratos , Adesão Celular , Clonagem Molecular , DNA Fúngico , Eletroforese em Gel de Poliacrilamida , Vetores Genéticos , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/metabolismo , Mapeamento por Restrição , Relação Estrutura-AtividadeRESUMO
During development of the cellular slime mold Dictyostelium discoideum, cells migrate in response to cAMP to form aggregates, which give rise to fruiting bodies consisting of two major cell types: spores and stalk cells. Multicellularity is achieved by the expression of two types of cell-cell adhesion sites. The EDTA-sensitive binding sites are expressed at the initial stage of development. At the aggregation stage, cells acquire EDTA-resistant binding sites, which are mediated by a cell-surface glycoprotein of Mr80,000 (gp80). gp80 is preferentially associated with cell surface filopodia, which are probably involved in the initiation of contact formation between cells. Covaspheres conjugated with gp80 bind specifically to aggregation-stage cells. The binding can be inhibited by precoating cells with an anti-gp80 monoclonal antibody, thus suggesting that gp80 mediates cell-cell binding via homophilic interaction. The structure of gp80 predicted from its cDNA sequence can be divided into three major domains: a membrane anchor, a hinge, and a globular region. An analysis of fusion proteins containing different gp80 segments shows that the cell-binding activity resides in the globular region. In the postaggregation stages, gp80 is replaced by other surface glycoproteins in maintaining cell-cell adhesion. One of them has a Mr of 150,000 (gp150). Anti-gp150 antibodies have no effect on aggregation-stage cells, but they disrupt cell-cell adhesion at subsequent stages. It becomes evident that the complex phenomena of cell adhesion and tissue organization involve the participation of a number of surface glycoproteins.
Assuntos
Dictyostelium/citologia , Sequência de Aminoácidos , Sequência de Bases , Carboidratos/fisiologia , Adesão Celular , Comunicação Celular , Dados de Sequência MolecularRESUMO
In Dictyostelium discoideum, a surface glycoprotein with Mr 80,000 (gp80) has been found to mediate the EDTA-resistant contact sites A at the aggregation stage of development. To evaluate the role of the carbohydrate moiety in cell-cell adhesion, we have examined the accumulation and activity of an altered gp80 molecule in two glycosylation (modB) mutants. Both mutants synthesize an altered gp80 of lower molecular size. This modB-gp80 can be detected by the monoclonal antibody 80L5C4, which is capable of blocking cell-cell adhesion (C. -H. Siu, T. Y. Lam, and A. Choi, (1985) J. Biol. Chem. 260, 16,030-16,036). The mutant cells exhibit both EDTA-sensitive and EDTA-resistant types of cell-cell binding, though to a lesser extent than that of the parental strain, and the EDTA-resistant binding sites are blocked in the presence of 80L5C4 Fab. Mutant cells can also bind Covaspheres conjugated with gp80. These results suggest that the modB-gp80 protein still retains the domain essential for its cell binding activity and the carbohydrate moiety affected by the modB mutation is not directly involved in cell-cell adhesion.
Assuntos
Adesão Celular/efeitos dos fármacos , Dictyostelium/genética , Glicoproteínas de Membrana/farmacologia , Mutação , Carboidratos/análise , Linhagem Celular , Ácido Edético/farmacologia , Peso MolecularRESUMO
We have previously reported that a monoclonal antibody directed specifically against a surface glycoprotein of Mr 80,000 (gp80) inhibits the EDTA-resistant contact sites A of Dictyostelium discoideum (Siu, C.-H., and Choi, A.H.C. (1985) J. Biol. Chem. 260, 16030-16036). In this report, we describe an assay using this monoclonal antibody to quantitate the amount of gp80 expressed at different developmental stages. Under normal conditions, gp80 is detectable after 6 h of development and it rapidly accumulates between 6 and 10 h, corresponding to the time when cells acquire their EDTA-resistant binding sites. At the peak level, there are 1.5.10(5) gp80 molecules per cell. More than 90% of the cellular gp80 is located on the cell surface. When cells are given exogenous pulses of cAMP, a precocious and enhanced expression of gp80 is induced. At the peak level, the cAMP-pulsed cells accumulate five times more gp80 than the non-pulsed cells. This is preceeded by an equally rapid accumulation of gp80 transcripts, suggesting that cAMP regulates gp80 synthesis at the transcriptional level.
Assuntos
Antígenos de Superfície/biossíntese , Moléculas de Adesão Celular , Dictyostelium/metabolismo , Glicoproteínas de Membrana/biossíntese , Proteínas de Protozoários , Anticorpos Monoclonais , Antígenos de Superfície/análise , Antígenos de Superfície/imunologia , Comunicação Celular , Membrana Celular/análise , Membrana Celular/metabolismo , AMP Cíclico/farmacologia , Dictyostelium/análise , Dictyostelium/genética , Dictyostelium/ultraestrutura , Glicoproteínas de Membrana/análise , Glicoproteínas de Membrana/imunologia , Hibridização de Ácido Nucleico , RNA Fúngico/análise , Transcrição GênicaRESUMO
Monoclonal antibodies were prepared against a putative cell-cell adhesion molecule, a surface glycoprotein with an apparent Mr of 80,000 (gp80), from Dictyostelium discoideum. Seven monoclonal antibodies directed against gp80 were characterized and found to fall into three distinct classes. Class I consisted of one monoclonal antibody, is monospecific for gp80, and probably recognizes the peptide portion of the molecule. This class was capable of blocking the EDTA-resistant contact sites effectively. Class II recognized the carbohydrate moiety of gp80 and cross-reacted with a large number of glycoproteins. These monoclonal antibodies partially inhibited cell reassociation. Class III recognized gp80 and one other glycoprotein of Mr 95,000. This class had no effect on cell-cell binding. The class I monoclonal antibody was most potent in inhibiting cell reassociation at the aggregation stage of development. Its effect decreased drastically as development progressed and became negligible by the culmination stage. These observations are consistent with a direct role of gp80 in cell-cell binding and suggest a transient function for gp80 at the aggregation stage.
Assuntos
Anticorpos Monoclonais , Dictyostelium/citologia , Glicoproteínas/imunologia , Adesividade , Aglutinação , Animais , Ácido Edético/farmacologia , Técnicas de Imunoadsorção , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Contraste de Fase , Peso MolecularRESUMO
Cell sorting among prespore cells and prestalk cells in Dictyostelium discoideum was studied by using fluorochrome-labeled cells in an in vitro assay. Labeled prestalk cells first formed randomly mixed aggregates with unlabeled prespore cells. Then cells began to sort out from each other. About 3-4 hr later, prestalk cells became clustered at one pole of the aggregate. Aggregates deposited on an agar surface underwent morphogenesis and formed migrating slugs within 3 hr. The addition of Fab fragments directed against a cell-surface glycoprotein of Mr 150,000 (gp 150) to the cell mixture completely inhibited the cell-sorting phenomenon. Morphogenesis of such aggregates on agar was also delayed by 5 hr. However, inclusion of Fab fragments directed against the endogenous lectins, the contact site A glycoprotein, or vegetative cells had no detectable effect on cell sorting or morphogenesis of these reconstituted aggregates.