RESUMO
Objective: To investigate the rate of periprosthetic joint infection (PJI) revision surgeries and clinical information of hip-/knee- PJI cases nationwide from 2015 to 2017 in China. Methods: An epidemiological investigation. A self-designed questionnaire and convenience sampling were used to survey 41 regional joint replacement centers nationwide from November 2018 to December 2019 in China. The PJI was diagnosed according to the Musculoskeletal Infection Association criteria. Data of PJI patients were obtained by searching the inpatient database of each hospital. Questionnaire entries were extracted from the clinical records by specialist. Then the differences in rate of PJI revision surgery between hip- and knee- PJI revision cases were calculated and compared. Results: Total of 36 hospitals (87.8%) nationwide reported data on 99 791 hip and knee arthroplasties performed from 2015 to 2017, with 946 revisions due to PJI (0.96%). The overall hip-PJI revision rate was 0.99% (481/48 574), and it was 0.97% (135/13 963), 0.97% (153/15 730) and 1.07% (193/17 881) in of 2015, 2016, 2017, respectively. The overall knee-PJI revision rate was 0.91% (465/51 271), and it was 0.90% (131/14 650), 0.88% (155/17 693) and 0.94% (179/18 982) in 2015, 2016, 2017, respectively. Heilongjiang (2.2%, 40/1 805), Fujian (2.2%, 45/2 017), Jiangsu (2.1%, 85/3 899), Gansu (2.1%, 29/1 377), Chongqing (1.8%, 64/3 523) reported relatively high revision rates. Conclusions: The overall PJI revision rate in 34 hospitals nationwide from 2015 to 2017 is 0.96%. The hip-PJI revision rate is slightly higher than that in the knee-PJI. There are differences in revision rates among hospitals in different regions.
Assuntos
Artroplastia de Quadril , Artroplastia do Joelho , Infecções Relacionadas à Prótese , Humanos , Infecções Relacionadas à Prótese/epidemiologia , Infecções Relacionadas à Prótese/diagnóstico , China/epidemiologia , Hospitais , Reoperação , Estudos RetrospectivosRESUMO
Objective: To summarize the efficacy and safety of the combination of rituximab and ATG as induction therapy in highly sensitized kidney transplant recipients. Methods: Clinical data of patients who received kidney transplantation from donation after cardiac death(DCD) in Organ Transplant Center of Second Affiliated Hospital of Guangzhou Medical University from January 1st 2015 to December 31th 2016 was retrospectively analyzed. Highly sensitized patients with over 30% active panel reactive antibody (PRA>30%) received rituximab, while non-sensitized recipients as controlled group. All selected patients were observed in the renal function, urine protein, hemogram and the variation of PRA at each time point. Acute rejection, infection required hospitalization, delayed graft function(DGF), primary nonfunction (PNF), graft dysfunction, the mortality rate of patients with good allograft function and the graft survival rate were also observed. Results: 46 groups of patients were selected into highly-sensitized group and non-sensitized group. In both groups, there was no statistical difference in the renal function, urine protein and WBC (all P>0.05). Highly sensitized recipients at day 7 and day 14 following the surgery, had a significantly lower percentage of lymphocyte counts and lymphocyte proportion compared to other groups, with statistical differences(all P<0.05). Both groups had a similar incidence of DGF(2.2%) and no occurrence of PNF. 19.5% of highly sensitized recipients experienced acute rejection and 13% in control group. More specifically, no statistical difference was noted in the rate of infection required hospitalization(30.4% vs 22.2%), graft loss(2.2% vs 0) and the mortality rate of patients with good allograft function(4.3% vs 2.2%)(all P>0.05). The graft survival rate was 97.8% in the highly-sensitized group, while 100% in the control group. And the rate of patient survival in these two groups was 95.7% and 97.8%, with no statistical differences(all P>0.05). Conclusions: Immune-induction therapy that combines Rituximab with ATG can significantly inhibit lymphocyte proliferation. It is effective and safe in treating hypersensitive patients. The survival rate of human/kidney of hypersensitive patients in the short and medium term is comparable to those with low immune risk.
Assuntos
Transplante de Rim , Soro Antilinfocitário , Rejeição de Enxerto , Sobrevivência de Enxerto , Humanos , Imunossupressores , Estudos Retrospectivos , Rituximab , Resultado do TratamentoRESUMO
To investigate the characteristics of immune cells before and after immunotherapy and their clinical significance in patients with unexplained recurrent spontaneous abortion (URSA), an analysis of 67 URSA patients, 67 sporadic spontaneous abortion (SA) patients, and 22 normal nonpregnant women (as controls) was conducted. URSA patients underwent immunotherapy using paternal lymphocytes. Peripheral blood from patients and controls was examined for lymphocytes and other markers of immune status. Before the immunotherapy, lymphocyte counts, CD4:CD8 cell ratios, and the relative proportion of natural killer (NK) cells were significantly higher in the URSA patient group than in the SA patient and control groups (P < 0.05). After the therapy, all of these three measures were decreased, whereas the percentage of T cells was increased, and statistically significant differences before and after the immunotherapy were detected (P < 0.05). Therefore, the immune system appears to be activated in the URSA patients, and the abnormal immunologic state in the URSA patients is more severe than in the SA patients. The alterations in T and NK cells may be involved in the etiopathogenesis of URSA. Lymphocyte immunotherapy appears to be an effective treatment for URSA patients.
Assuntos
Aborto Espontâneo/imunologia , Aborto Espontâneo/fisiopatologia , Células Matadoras Naturais/metabolismo , Linfócitos T/metabolismo , Aborto Espontâneo/terapia , Adulto , Relação CD4-CD8 , Estudos de Casos e Controles , Feminino , Humanos , Imunoterapia , Células Matadoras Naturais/imunologia , Gravidez , Resultado da Gravidez , Linfócitos T/imunologiaRESUMO
To study preterm birth prediction based on fetal fibronectin (fFN) in pregnant women, we randomly selected 124 patients. Vaginal posterior fornix secretions were analyzed using fFN quick test strips. Leucorrhea routine samples were collected to detect bacterial vaginosis, mycoplasma, and chlamydia. Delivery data at 7 days, 14 days, 34 weeks, and 37 weeks were documented and the sensitivity, specificity, positive predictive value, and negative predictive value were analyzed. Of the 124 cases, we found 2, 4, 10, and 18 cases of maternity within 7 days, 14 days, 34 weeks, and 37 weeks, respectively. The sensitivity, specificity, positive predictive value, and negative predictive value were as follows: 100, 77.8, 6.9, and 100% for 7 days; 75, 78.3, 10.3, and 98.9% for 14 days; 50.0, 78.9, 17.2, and 94.7% for 34 weeks; 33.3, 78.3, 20.7, and 87.4% for 37 weeks, respectively. Except for 18 preterm births, 23 cases were fFN-positive, 17 cases had lower genital tract infection. Eighty-three cases were fFN-negative, of which 18 cases had the lower genital tract infections. This difference was statistically significant (P < 0.05). Eighteen cases (14.5% of the pregnant women) had preterm birth. Ten cases delivered within 34 weeks. The negative predictive value and recent predictive value of fFN testing were higher; the positive predictive value was limited due to the impact of lower genital tract infection. The fFN-positive patients need timely clinical processing. During the pregnancy, monitoring of fFN changes and early detection of abnormalities help to reduce perinatal morbidity and mortality.
Assuntos
Colo do Útero/metabolismo , Fibronectinas/análise , Nascimento Prematuro/diagnóstico , Adulto , Medicina Baseada em Evidências/métodos , Feminino , Humanos , Valor Preditivo dos Testes , Gravidez , Resultado da Gravidez , Nascimento Prematuro/epidemiologia , Sensibilidade e Especificidade , Esfregaço Vaginal/métodos , Adulto JovemRESUMO
Caffeine is a definite factor of intrauterine growth retardation (IUGR). Previously, we have confirmed that prenatal caffeine ingestion inhibits the development of hypothalamic-pituitary-adrenal (HPA) axis, and alters the glucose and lipid metabolism in IUGR fetal rats. In this study, we aimed to verify a programmed alteration of neuroendocrine metabolism in prenatal caffeine ingested-offspring rats. The results showed that prenatal caffeine (120 mg/kg.day) ingestion caused low body weight and high IUGR rate of pups; the concentrations of blood adrenocorticotropic hormone (ACTH) and corticosterone in caffeine group were significantly increased in the early postnatal period followed by falling in late stage; the level of blood glucose was unchanged, while blood total cholesterol (TCH) and triglyceride (TG) were markedly enhanced in adult. After chronic stress, the concentrations and the gain rates of blood ACTH and corticosterone were obviously increased, meanwhile, the blood glucose increased while the TCH and TG decreased in caffeine group. Further, the hippocampal mineralocorticoid receptor (MR) expression in caffeine group was initially decreased and subsequently increased after birth. After chronic stress, the 11ß-hydroxysteroid dehydrogenase-1, glucocorticoid receptor (GR), MR as well as the MR/GR ratio were all significantly decreased. These results suggested that prenatal caffeine ingestion induced the dysfunction of HPA axis and associated neuroendocrine metabolic programmed alteration in IUGR offspring rats, which might be related with the functional injury of hippocampus. These observations provide a valuable experimental basis for explaining the susceptibility of IUGR offspring to metabolic syndrome and associated diseases.
Assuntos
Cafeína/efeitos adversos , Retardo do Crescimento Fetal/induzido quimicamente , Sistema Hipotálamo-Hipofisário/efeitos dos fármacos , Sistema Hipófise-Suprarrenal/efeitos dos fármacos , Animais , Peso ao Nascer , Glicemia , Feminino , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Metabolismo dos Lipídeos , Masculino , Gravidez , Efeitos Tardios da Exposição Pré-Natal , Ratos , Estresse Fisiológico , Fatores de Tempo , Aumento de PesoRESUMO
SnO2@carbon nanostructure composites are prepared by a simple hydrothermal method. The composite exhibits unique structure, which consists of a mesoporous SnO2 core assembled of very small nanoparticles and a carbon shell with 10 nm thickness. The mesoporous SnO2@carbon core-shell nanostructures manifest superior electrochemical performance as an anode material for lithium ion batteries. The reversible specific capacity of the composite is about 908 mAh g(-1) for the first cycle and it can retain about 680 mAh g(-1) after 40 charge/discharge cycles at a current density of 0.3 C. Moreover, it shows excellent rate capability even at the high rate of 4.5 C. The enhanced performance was attributed to the mesoporous structure and a suitable carbon coating.
Assuntos
Fontes de Energia Elétrica , Lítio/química , Nanoestruturas/química , Compostos de Estanho/química , Capacitância Elétrica , Eletrodos , Nanoestruturas/ultraestrutura , PorosidadeRESUMO
We analyzed chloroplast DNA (cpDNA) polymorphism and phylogenic relationships between 6 typical indica rice, 4 japonica rice, 8 javanica rice, and 12 Asian common wild rice (Oryza rufipogon) strains collected from different latitudes in China by comparing polymorphism at 9 highly variable regions. One hundred and forty-four polymorphic bases were detected. The O. rufipogon samples had 117 polymorphic bases, showing rich genetic diversity. One hundred and thirty-one bases at 13 sites were identified with indica/japonica characteristics; they showed differences between the indica and japonica subspecies at these sites. The javanica strains and japonica shared similar bases at these 131 polymorphic sites, suggesting that javanica is closely related to japonica. On the basis of length analyses of the open reading frame (ORF)100 and (ORF)29-tRNA-Cys(GCA) (TrnC(GCA)) fragments, the O. rufipogon strains were classified into indica/japonica subgroups, which was consistent with the results of the phylogenic tree assay based on concatenated datasets. These results indicated that differences in indica and japonica also exist in the cpDNA genome of the O. rufipogon strains. However, these differences demonstrated a certain degree of primitiveness and incompleteness, as an O. rufipogon line may show different indica/ japonica attributes at different sites. Consequently, O. rufipogon cannot be simply classified into the indica/japonica types as O. sativa. Our data support the hypothesis that Asian cultivated rice, O. indica and O. japonica, separately evolved from Asian common wild rice (O. rufipogon) strains, which have different indica-japonica differentiation trends.
Assuntos
Produtos Agrícolas/genética , DNA de Cloroplastos/genética , Oryza/genética , Polimorfismo Genético , Sequência de Bases , China , Evolução Molecular , Genes de Plantas , Fases de Leitura Aberta , Filogenia , Análise de Sequência de DNARESUMO
Here, we demonstrate a previously unknown function for the 70-kDa heat-shock protein (HSP70) as a cytokine. HSP70 bound with high affinity to the plasma membrane, elicited a rapid intracellular calcium flux, activated nuclear factor (NF)-kappaB and upregulated the expression of pro-inflammatory cytokines tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-6 in human monocytes. Furthermore, two different signal transduction pathways were activated by exogenous HSP70: one dependent on CD14 and intracellular calcium, which resulted in increased IL-1beta, IL-6 and TNF-alpha; and the other independent of CD14 but dependent on intracellular calcium, which resulted in an increase in TNF-alpha but not IL-1beta or IL-6. These findings indicate that CD14 is a co-receptor for HSP70-mediated signaling in human monocytes and are indicative of an previously unrecognized function for HSP70 as an extracellular protein with regulatory effects on human monocytes, having a dual role as chaperone and cytokine.
Assuntos
Proteínas de Choque Térmico HSP70/imunologia , Proteínas I-kappa B , Interleucina-1/biossíntese , Interleucina-6/biossíntese , Fator de Necrose Tumoral alfa/biossíntese , Cálcio/imunologia , Células Cultivadas , Citocinas/imunologia , Citocinas/fisiologia , Proteínas de Ligação a DNA/imunologia , Proteínas de Ligação a DNA/metabolismo , Proteínas de Choque Térmico HSP70/farmacologia , Humanos , Receptores de Lipopolissacarídeos/imunologia , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Inibidor de NF-kappaB alfa , NF-kappa B/antagonistas & inibidores , Células Tumorais CultivadasRESUMO
PPARgamma is a nuclear receptor that has a dominant regulatory role in differentiation of cells of the adipose lineage, and has recently been shown to be expressed in the colon. We show here that PPARgamma is expressed at high levels in both well- and poorly-differentiated adenocarcinomas, in normal colonic mucosa and in human colon cancer cell lines. Ligand activation of this receptor in colon cancer cells causes a considerable reduction in linear and clonogenic growth, increased expression of carcinoembryonic antigen and the reversal of many gene expression events specifically associated with colon cancer. Transplantable tumors derived from human colon cancer cells show a significant reduction of growth when mice are treated with troglitazone, a PPARgamma ligand. These results indicate that the growth and differentiation of colon cancer cells can be modulated through PPARgamma.
Assuntos
Adenocarcinoma/fisiopatologia , Neoplasias do Colo/fisiopatologia , Receptores Citoplasmáticos e Nucleares/fisiologia , Tiazolidinedionas , Fatores de Transcrição/fisiologia , Animais , Diferenciação Celular , Divisão Celular , Cromanos/farmacologia , Expressão Gênica , Humanos , Ligantes , Camundongos , Camundongos Nus , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores Citoplasmáticos e Nucleares/genética , Tiazóis/farmacologia , Fatores de Transcrição/agonistas , Fatores de Transcrição/genética , Troglitazona , Células Tumorais CultivadasRESUMO
Previous studies have shown that integrin alpha chain tails make strong positive contributions to integrin-mediated cell adhesion. We now show here that integrin alpha4 tail deletion markedly impairs static cell adhesion by a mechanism that does not involve altered binding of soluble vascular cell adhesion molecule 1 ligand. Instead, truncation of the alpha4 cytoplasmic domain caused a severe deficiency in integrin accumulation into cell surface clusters, as induced by ligand and/ or antibodies. Furthermore, alpha4 tail deletion also significantly decreased the membrane diffusivity of alpha4beta1, as determined by a single particle tracking technique. Notably, low doses of cytochalasin D partially restored the deficiency in cell adhesion seen upon alpha4 tail deletion. Together, these results suggest that alpha4 tail deletion exposes the beta1 cytoplasmic domain, leading to cytoskeletal associations that apparently restrict integrin lateral diffusion and accumulation into clusters, thus causing reduced static cell adhesion. Our demonstration of integrin adhesive activity regulated through receptor diffusion/clustering (rather than through altered ligand binding affinity) may be highly relevant towards the understanding of inside-out signaling mechanisms for beta1 integrins.
Assuntos
Integrinas/genética , Integrinas/metabolismo , Mutagênese , Receptores de Retorno de Linfócitos/genética , Receptores de Retorno de Linfócitos/metabolismo , Animais , Células CHO , Adesão Celular/genética , Cricetinae , Epitopos/biossíntese , Humanos , Integrina alfa4beta1 , Integrinas/química , Leucemia Eritroblástica Aguda , Ligantes , Manganês , Ligação Proteica/genética , Conformação Proteica , Receptores de Retorno de Linfócitos/química , Receptores de Antígeno muito Tardio/efeitos dos fármacos , Deleção de Sequência , Azida Sódica , Transfecção , Células Tumorais Cultivadas , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
Epigenetic silencing of secreted frizzled-related protein (SFRP) genes, antagonists of the WNT pathway, contributes to the pathogenesis of several cancers including non-small cell lung cancer (NSCLC). We hypothesize that methylation analysis of SFRPs family could improve their use as a panel of biomarkers for diagnosing and staging of NSCLC in China. The expression of four SFRP members (SFRP1, 2, 4, and 5) in NSCLC samples was screened by RT-PCR and quantitative real-time PCR. Only SFRP1 was significantly downregulated in NSCLC, as compared to adjacent normal tissues and benign pulmonary disease tissues (P=0.006). Promoter hypermethylation of SFRP1 was found in 32.1% (25/78) NSCLC specimens and was closely correlated with loss of expression, besides SFRP1 hypermethylation was associated with lymph metastasis (P=0.039) and disease progression within one year (P=0.027). Furthermore, methylated SFRP1 was detected in 28.2% (22/78) of plasma samples from NSCLC patients while only 4% (2/50) in cancer-free controls, and the concordance of SFRP1 methylation status in tumor tissues and corresponding plasmas was satisfactory (P <0.001). In conclusion, epigenetic inactivation of SFRP1 is a common event contributing to lung carcinogenesis and maybe used as a potential biomarker for NSCLC in Chinese population.
Assuntos
Biomarcadores Tumorais , Carcinoma Pulmonar de Células não Pequenas/genética , Metilação de DNA , Peptídeos e Proteínas de Sinalização Intercelular/genética , Neoplasias Pulmonares/genética , Proteínas de Membrana/genética , Regiões Promotoras Genéticas , Ativação Transcricional , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Pulmonar de Células não Pequenas/patologia , Feminino , Humanos , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/análiseRESUMO
Cell-free fibronectin matrix (FN-matrix) isolated from chick embryo fibroblasts was used to study cell-matrix interaction. After 24 h, most fibroblastic cells, including those without cell surface fibronectin, adopted bipolar fusiform morphology. Cells grew in parallel arrays and aligned with each other apparently along FN-matrix. Since the orientation of fibronectin fibers was determined by chick embryo fibroblasts, our results suggested that intercellular organization of "matrix-using" cell type may be influenced by "matrix-producing" cell type. Whereas the elongation and alignment effects induced by FN-matrix have been detected in fibroblasts (both normal and transformed), myoblast, aortic endothelial cells, neural cell lines (B103 and RT4D1), and cardiac muscle cells, similar effects are not detected in bone marrow hemopoietic cells, circulating lymphocytic T and B cells, and sympathetic neurons. For epithelial cells, FN-matrix has varying effects. Elongation and alignment effects are detected only in transformed epithelial cells with a great reduction in keratin expression. The morphology of normal or transformed epithelial cells with abundant keratin appears unaffected by FN-matrix. FN-matrix reduced the growth of several transformed fibroblastic lines up to 25%, but did not restore the appearance of actin stress fibers and the normal migratory activities of Rous sarcoma virus-transformed rat cells.
Assuntos
Fibroblastos/ultraestrutura , Fibronectinas/análise , Animais , Adesão Celular/efeitos dos fármacos , Transformação Celular Neoplásica/efeitos dos fármacos , Embrião de Galinha , Citoesqueleto/ultraestrutura , Células Epiteliais , Fibroblastos/fisiologia , Fibronectinas/farmacologia , Crescimento , Microscopia Eletrônica de Varredura , Fatores de TempoRESUMO
We have used fluorescein isosthiocyanate-conjugated gelatin (FITC-gelatin) (1 mg/ml) to localize cell surface fibronectin in unfixed live cells in cultures. FITC-gelatin stains the fibronectin matrix on primary cultures of rat and chick embryo fibroblasts as well as untransformed, established cell lines. In live cultured cells, fibronectin in many areas of the extracellular matrix is inaccessible to antibody and cannot be visualized by immunofluorescence staining. In contrast, fibronectin in these areas is fully stainable by FITC-gelatin. At a low concentration (20 micrograms/ml), FITC-gelatin stains the fibronectin matrix of primary cultured cells but not of "untransformed" established cell lines. SEM can detect only the matrix stainable with the low concentration of FITC-gelatin, such as that expressed by primary chick embryo fibroblasts. The binding of fibronectin to the extracellular matrix is very stable and FITC-gelatin remained bound to the matrix for at least 10 d in culture. Radioiodinated gelatin has been used to quantitate the level of cell surface fibronectin in living normal and transformed cells. FITC-gelatin appears to be a useful probe for studying the fibronectin of living cells in culture.
Assuntos
Espaço Extracelular/metabolismo , Fibronectinas/metabolismo , Animais , Transformação Celular Neoplásica/patologia , Células Cultivadas , Embrião de Galinha , Fibronectinas/imunologia , Fluoresceínas , Imunofluorescência , Gelatina , RatosRESUMO
The interrelationships of the endoplasmic reticulum (ER), microtubules, and intermediate filaments were studied in the peripheral regions of thin, spread fibroblasts, epithelial, and vascular endothelial cells in culture. We combined a fluorescent dye staining technique to localize the ER with immunofluorescence to localize microtubules or intermediate filaments in the same cell. Microtubules and the ER are sparse in the lamellipodia, but intermediate filaments are usually completely absent. These relationships indicate that microtubules and the ER advance into the lamellipodia before intermediate filaments. We observed that microtubules and tubules of the ER have nearly identical distributions in lamellipodia, where new extensions of both are taking place. We perturbed microtubules by nocodazole, cold temperature, or hypotonic shock, and observed the effects on the ER distribution. On the basis of our observations in untreated cells and our experiments with microtubule perturbation, we conclude that microtubules and the ER are highly interdependent in two ways: (a) polymerization of individual microtubules and extension of individual ER tubules occur together at the level of resolution of the fluorescence microscope, and (b) depolymerization of microtubules does not disrupt the ER network in the short term (15 min), but prolonged absence of microtubules (2 h) leads to a slow retraction of the ER network towards the cell center, indicating that over longer periods of time, the extended state of the entire ER network requires the microtubule system.
Assuntos
Retículo Endoplasmático/fisiologia , Microtúbulos/fisiologia , Animais , Linhagem Celular , Movimento Celular , Chlorocebus aethiops , Retículo Endoplasmático/ultraestrutura , Fibroblastos/fisiologia , Fibroblastos/ultraestrutura , Imunofluorescência , Filamentos Intermediários/fisiologia , Filamentos Intermediários/ultraestrutura , Rim , Macropodidae , Microtúbulos/ultraestrutura , Mitocôndrias/ultraestruturaRESUMO
To study the construction of the ER, we used the microtubule-disrupting drug nocodazole to induce the complete breakdown of ER structure in living cells followed by recovery in drug-free medium, which regenerates the ER network within 15 min. Using the fluorescent dye 3,3'-dihexyloxacarbocyanine iodide to visualize the ER, we have directly observed the network construction process in living cells. In these experiments, the ER network was constructed through an iterative process of extension, branching, and intersection of new ER tubules driven by the ER motility previously described as tubule branching. We have tested the cytoskeletal requirements of this process. We find that newly formed ER tubules are aligned with single microtubules but not actin fibers or vimentin intermediate filaments. Microtubule polymerization preceded the extension of ER tubules and, in experiments with a variety of different drugs, appeared to be a necessary condition for the ER network formation. Furthermore, perturbations of the pattern of microtubule polymerization with microtubule-specific drugs caused exactly correlated perturbations of the pattern of ER construction. Induction of abnormally short, nonintersecting microtubules with 20 microM taxol prevented the ER network formation; ER tubules only extended along the few microtubules contacting the aggregated ER membranes. This requirement for a continuous network of intersecting microtubules indicates that ER network formation takes place through the branching and movement of ER membranes along microtubules. Cytochalasin B had no apparent effect on the construction of the ER network during recovery, despite apparently complete disruption of actin fibers as stained by phalloidin. Blockage of protein synthesis and disorganization of intermediate filaments with cycloheximide pretreatment also failed to perturb ER construction.
Assuntos
Retículo Endoplasmático/ultraestrutura , Actinas/análise , Alcaloides/farmacologia , Animais , Carbocianinas , Linhagem Celular , Cicloeximida/farmacologia , Citocalasina B/farmacologia , Retículo Endoplasmático/efeitos dos fármacos , Imunofluorescência , Corantes Fluorescentes , Filamentos Intermediários/ultraestrutura , Microtúbulos/efeitos dos fármacos , Microtúbulos/ultraestrutura , Nocodazol/farmacologia , Paclitaxel , Coloração e RotulagemRESUMO
Tensin is a focal adhesion phosphoprotein that binds to F-actin and contains a functional Src homology 2 domain. To explore the biological functions of tensin, we cloned the mouse tensin gene, determined its program of expression, and used gene targeting to generate mice lacking tensin. Even though tensin is expressed in many different tissues during embryogenesis, tensin null mice developed normally and appeared healthy postnatally for at least several months. Over time, -/- mice became frail because of abnormalities in their kidneys, an organ that expresses high levels of tensin. Mice with overt signs of weakness exhibited signs of renal failure and possessed multiple large cysts in the proximal kidney tubules, but even in tensin null mice with normal blood analysis, cysts were prevalent. Ultrastructurally, noncystic areas showed typical cell-matrix junctions that readily labeled with antibodies against other focal adhesion molecules. In abnormal regions, cell-matrix junctions were disrupted and tubule cells lacked polarity. Taken together, our data imply that, in the kidney, loss of tensin leads to a weakening, rather than a severing, of focal adhesion. All other tissues appeared normal, suggesting that, in most cases, tensin's diverse functions are redundant and may be compensated for by other focal adhesion proteins.
Assuntos
Falência Renal Crônica/patologia , Rim/anormalidades , Proteínas dos Microfilamentos/deficiência , Doenças Renais Policísticas/patologia , Animais , Adesão Celular , Polaridade Celular , Matriz Extracelular/metabolismo , Feminino , Morte Fetal/genética , Junções Intercelulares , Rim/embriologia , Rim/crescimento & desenvolvimento , Rim/metabolismo , Falência Renal Crônica/genética , Pelve Renal/anormalidades , Túbulos Renais Proximais/patologia , Tamanho da Ninhada de Vivíparos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas dos Microfilamentos/fisiologia , Especificidade de Órgãos , Doenças Renais Policísticas/genética , Gravidez , TensinasRESUMO
Tensin, a 200-kD phosphoprotein of focal contacts, contains sequence homologies to Src (SH2 domain), and several actin-binding proteins. These features suggest that tensin may link the cell membrane to the cytoskeleton and respond directly to tyrosine kinase signalling pathways. Here we identify three distinct actin-binding domains within tensin. Recombinant tensin purified after overexpression by a baculovirus system binds to actin filaments with Kd = 0.1 microM, cross-links actin filaments at a molar ratio of 1:10 (tensin/actin), and retards actin assembly by barbed end capping with Kd = 20 nM. Tensin fragments were constructed and expressed as fusion proteins to map domains having these activities. Three regions from tensin interact with actin: two regions composed of amino acids 1 to 263 and 263 to 463, cosediment with F-actin but do not alter the kinetics of actin assembly; a region composed of amino acids 888-989, with sequence homology to insertin, retards actin polymerization. A claw-shaped tensin dimer would have six potential actin-binding sites and could embrace the ends of two actin filaments at focal contacts.
Assuntos
Actinas/metabolismo , Proteínas dos Microfilamentos/metabolismo , Citoesqueleto de Actina/ultraestrutura , Actinas/ultraestrutura , Animais , Sítios de Ligação , Galinhas , Contração Muscular , Ligação Proteica , Proteínas Recombinantes de Fusão , TensinasRESUMO
Permeant cationic fluorescent probes are shown to be selectively accumulated by the mitochondria of living cells. Mitochondria-specific interaction of such molecules is apparently dependent on the high trans-membrane potential (inside negative) maintained by functional mitochondria. Dissipation of the mitochondrial trans-membrane and potential by ionophores or inhibitors of electron transport eliminates the selective mitochondrial association of these compounds. The application of such potential-dependent probes in conjunction with fluorescence microscopy allows the monitoring of mitochondrial membrane potential in individual living cells. Marked elevations in mitochondria-associated probe fluorescence have been observed in cells engaged in active movement. This approach to the analysis of mitochondrial membrane potential should be of value in future investigations of the control of energy metabolism and energy requirements of specific biological functions at the cellular level.
Assuntos
Membranas Intracelulares/fisiologia , Mitocôndrias/fisiologia , Animais , Linhagem Celular , Movimento Celular , Sobrevivência Celular , Cricetinae , Transporte de Elétrons , Metabolismo Energético , Gerbillinae , Haplorrinos , Potenciais da Membrana , Camundongos , Microscopia de Fluorescência , Vison , RatosRESUMO
Rabbit bladder epithelium, grown on collagen gels and exposed to the chemical carcinogen benzo[a]pyrene, produced nontumorigenic altered foci as well as tumorigenic epithelial cell lines during 120-180 d in culture. Immunofluorescence studies revealed extensive keratin filaments in both primary epithelial cells and benzo[a]pyrene-induced altered epithelial foci but showed no detectable vimentin filaments. The absence of vimentin expression in these cells was confirmed by two-dimensional gel electrophoresis. In contrast, immunofluorescence staining of the cloned benzo[a]pyrene-transformed rabbit bladder epithelial cell line, RBC-1, revealed a reduction in filamentous keratin concomitant with the expression of vimentin filaments. The epithelial nature of this cell line was established by the observation that cells injected into nude mice formed well-differentiated adenocarcinomas. Frozen sections of such tumors showed strong staining with antikeratins antibodies, but no detectable staining with antivimentin antibodies. These results demonstrated a differential expression of intermediate filament type in cells at different stages of neoplastic progression and in cells maintained in different growth environments. It is apparent that the expression of intermediate filaments throughout neoplastic progression is best studied by use of an in vivo model system in parallel with culture studies.
Assuntos
Transformação Celular Neoplásica , Citoesqueleto/análise , Queratinas/análise , Proteínas Musculares/análise , Animais , Benzo(a)pireno , Benzopirenos/farmacologia , Linhagem Celular , Células Cultivadas , Epitélio , Imunofluorescência , Coelhos , Bexiga Urinária , VimentinaRESUMO
Rhodamine-123, a cationic laser dye, markedly reduced the clonal growth of carcinoma cells but had little effect on nontumorigenic epithelial cells in vitro. This selective inhibitory effect of Rhodamine-123 on some carcinomas is unusual since known anticancer drugs, such as arabinosyl cytosine and methotrexate, have not been shown to exhibit such selectivity in vitro.