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C Melguizo

Publications and source records attributed to C Melguizo.

At least 37 records · Page 2Linked to original sources
PubMed1996

Low sample volume causes differentiation in human rhabdomyosarcoma cell line RD subjected to electroporation.

Gene transfection has been accomplished with a variety of techniques such as DEAE dextran, calcium phosphate coprecipitation, protoplast fusion, liposomes, microinjection and recombinant bacteriophages. However, transfection by electroporation, consisting of the reversible permeabilization of cell membranes after exposure to a pulsed electric field, has been shown to be the most rapid, simple and efficient method for the stable incorporation of genes in different cell lines. We studied rhabdomyosarcoma cells subjected to electroporation in two different vol. [400 microliters (group 1) and 150 microliters (group 2] of 140 mM NaCl/15 mM Hepes buffer, pH 7.2) and evaluated the effects of electroporation volume on growth and differentiation. Low sample volumes induced a terminal process of morphological and ultrastructural myogenic differentiation in rhabdomyosarcoma cells, which concluded with cell death. Our results suggest that in electroporation low sample vol. of rhabdomyosarcoma cells induced morphological and phenotypic differentiation, with increased expression of desmin, alpha-actinin and tropomyosin.

Actinin↗
PubMed1995

Differentiation of a human rhabdomyosarcoma cell line after antineoplastic drug treatment.

The feasibility of treating solid tumours with differentiation therapy using antineoplastic drugs is currently being investigated, but the emergence of multidrug resistance remains the major limitation to this therapeutic approach. A rhabdomyosarcoma cell line resistant to actinomycin D (RD-DAC) has been used as an in vitro model to investigate, with light and electron microscopy, the degree of differentiation in multidrug-resistant cells. The parental cell line (RD), derived from a human embryonic-type rhabdomyosarcoma, is undifferentiated, with no evidence of specific ultrastructural markers. Examination of resistant cells by transmission electron microscopy revealed myofilaments arranged parallel to the long axis of the cell, which was considered clear evidence of myogenic differentiation. These observations suggest that actinomycin D, the drug of choice in the treatment of rhabdomyosarcoma, induces differentiation in the cell line RD. It is postulated that multidrug resistance can interfere with cellular differentiation.

Actin Cytoskeleton↗
PubMed1995

Circulating alpha-actin in non-insulin-dependent diabetics with autonomic dysfunction.

Silent myocardial ischemia in non-insulin-dependent diabetic patients occurs frequently in association with autonomic dysfunction, suggesting that diabetic neuropathy may be involved in the development of this disorder. Repeated episodes of silent myocardial ischemia can induce myocardial necrosis. Recently, actin was detected with Western blotting in the serum of patients with acute myocardial infarction and angina pectoris. We found that a large proportion of non-insulin-dependent diabetic patients with neuropathy also have detectable circulating concentrations of alpha-actin, and therefore suggest that the determination by immunoblotting of serum alpha-actin in such patients is an effective method to detect myocardial cell suffering and to identify patients that may need special consideration.

Actins↗
PubMed1995

Swine hearts: quantitative anatomy of the right ventricle.

The right ventricle was studied in 75 anatomically normal swine hearts, using, in all, nine geometric and volumetric parameters: ventricular-wall thickness, length of the right-ventricular inflow and outflow tracts, and volume of the right-ventricular inflow and outflow tracts. The data for these parameters were compared with previously published patterns for human hearts and volumetric data were compared with patterns of normality found in human hearts. As in the human heart, the ventricular inflow tract in swine hearts was significantly shorter than the outflow tract (P < 0.0001).

Animals↗
PubMed1995

Basic fibroblast and platelet-derived growth factors as modulators of actin and alpha-actinin in chick myocardiocytes during development.

To test whether cardiac muscle is a target for regulation by peptide growth factors, we analyzed the effects of two growth factors on actin and alpha-actinin expression at the subcellular level. Sodium dodecyl sulfate-gel electrophoresis, immunoblotting, and fluorescense-activated cell sorter analysis were used to quantify the effects of basic fibroblast growth factor and platelet-derived growth factor on cultures of chick myocardiocytes during development. Cytoplasmic and cytoskeletal concentrations of actin and alpha-actinin were dependent on the stage of embryonic development and on the type of growth factor added to the culture. The most significant finding was the increase in actin and alpha-actinin expression in the cytoplasmic compartment after treatment with basic fibroblast growth factor of chick heart cells at Hamburger and Hamilton's stage 19. At stage 39, basic fibroblast growth factor induced less marked changes in the accumulation of actin and alpha-actinin. Platelet-derived growth factor decreased alpha-actinin expression slightly in the cytoskeletal compartment in more mature stages of heart development. Our findings support the hypothesis that basic fibroblast growth factor plays a role in cardiomyocyte differentiation during the early stages of development.

Actinin↗
PubMed1995

Modulation of cardiac intermediate filament proteins in the chick heart by fibric acid derivatives.

The effects of different fibrates (bezafibrate, fenofibrate and gemfibrozil) on intermediate filaments were studied in cultured chick embryo heart cells after treatment for 6 or 24 h. Treatment led to alterations in total protein levels, as well as changes in protein levels, in the cytoplasmic and cytoskeletal fractions of cultured cells. Desmin was increased in the cytoskeletal fraction of all cultures after 6 h of treatment regardless of the drug tested, whereas vimentin was decreased in the cytoskeletal fraction only in cells treated with fenofibrate. These findings suggest that the alterations caused by fibrates in desmin and vimentin protein content may be related with the secondary effects that these drugs have on the cardiovascular system in patients treated with fibrates.

Animals↗
PubMed1994

Influence of dimethyl sulphoxide on intermediate filament proteins in human rhabdomyosarcoma cell lines: modulation at subcellular level.

The effects of dimethyl sulphoxide have been investigated on differentiation in human rhabdomyosarcoma cell lines obtained from typically malignant, poorly differentiated tumours. The expression of cell differentiation marker proteins (desmin and vimentin) was assessed in cell lines A-204, A-673 and RD, and the modifications in expression after 3, 8 and 24 h of induction with 1.25% dimethyl sulphoxide were recorded. Protein expression in both the cytoplasm and cytoskeleton was significantly altered by treatments lasting 8 and 24 h, the most noteworthy changes being increased desmin and decreased vimentin expression. The results clearly indicate that dimethyl sulphoxide induced changes typical of differentiation in rhabdomyosarcoma cell lines A-673 and RD; less marked changes were observed in line A-204.

Blotting, Western↗
PubMed1994

Modulation of contractile proteins in embryonic and fetal chick cardiac cells by phorbol ester, gamma-interferon, 5-azacytidine and diacylglycerols.

We studied changes in the concentration of tropomyosin, actin, desmin and vimentin in cultured myocardiocytes from Hamburger and Hamilton's stages 29 and 39 chick embryos (HH29 and HH39) (1), treated with 12-o-tetradecanoyl-phorbol-13-acetate (TPA), 5-azacytidine (AZA), gamma interferon (INF) and diacylglycerols (DAG). In embryonic myocardiocytes at HH29, the first three agents modified the intracellular distribution of the thin filament proteins tropomyosin and actin, increasing their cytoplasmic concentration and decreasing their cytoskeletal concentration. The concentration of the intermediate filament proteins desmin and vimentin increased in both subcellular fractions after treatment with these drugs. In fetal myocardiocytes at HH39, total protein content decreased after treatment with these drugs. Cytoplasmic and cytoskeletal concentrations of actin and tropomyosin decreased to different degrees after treatment with TPA, AZA or DAG in HH39 myocardiocytes. TPA, AZA and DAG decreased desmin in the cytoplasmic and cytoskeletal fractions. These findings suggest that the drugs tested alter the normal protein composition in cultured myocardiocytes, and have different effects depending on the developmental stage in which the embryo is treated.

Animals↗
PubMed1994

Creatine kinase isoenzymes as markers of differentiation in rhabdomyosarcoma cells in culture.

Rhabdomyosarcomas have been classified on the basis of their degree of differentiation, a feature closely related with their response to chemotherapy. We recently reported the role of creatine kinase isoenzymes as tumoral markers in the diagnosis of rhabdomyosarcoma. The present study was designed to determine whether these isoenzymes are also good markers of the degree of differentiation of these neoplasm. Dimethyl sulfoxide, a well-known differentiating agent, was used to induce myogenic differentiation. This agent increased CK-MM and/or CK-MB fractions, and decreased CK-BB isoenzyme in rhabdomyosarcoma cell lines. The variable behavior of MCK-1 macromolecule was unrelated to the degree of differentiation. Our findings suggest that CK-MM, CK-MB and CK-BB isoenzymes are good markers of the degree of differentiation in rhabdomyosarcomas and could be used as specific markers of prognostic and diagnostic value.

Biomarkers, Tumor↗
PubMed1994

Modulation of contractile protein troponin-T in chick myocardial cells by basic fibroblast growth factor and platelet-derived growth factor during development.

We quantified the effect of basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) on the contractile protein troponin-T (TnT) at the cellular and subcellular level in cultures of chick embryo cardiomyocytes obtained from Hamburger and Hamilton's (HH) stage 19, 29, and 39 embryos. Because expression of thin-filament molecules is considered a good marker of differentiation in muscle cell cultures, we analyzed the modifications these growth factors induced in the transcription of the gene for chick cardiac TnT. Sodium dodecyl sulfate-polyacrylamate gel electrophoresis (SDS-PAGE) and immunoblotting showed that cytoplasmic and cytoskeletal concentrations of TnT are dependent on the stage of embryonic development analyzed and on the type of growth factors added to the culture. The most significant finding was the increase in TnT expression in the cytoplasmic compartment (p < 0.001), accompanied by a slight increase in TnT mRNA, after treatment with bFGF of chick heart cells obtained at HH stage 19. At HH stage 39, bFGF induced less marked changes in the accumulation of TnT in comparison with untreated cardiomyocytes. Our findings support the hypothesis that bFGF plays a role in cardiomyocyte differentiation during early stages of development.

Animals↗
PubMed1994

Modulation of contractile protein troponin-T in chick myocardial cells by catecholamines during development.

In the present study we quantified the contractile protein troponin-T at the cellular and subcellular level in chick embryo cardiomyocytes to investigate the modulation of cardiac development by catecholamines. We analyzed the effects of these drugs on cultures of chick cardiomyocytes obtained from Hamburger and Hamilton's (HH) stage 21, HH stage 29 and HH stage 40 embryos; cardiomyocytes are considered to be mature at HH stage 40. We analyzed the modifications these drugs induced in the transcription of the gene for chick cardiac troponin-T. Sodium dodecyl sulfate-gel electrophoresis and immunobloting showed that cytoplasmic and cytoskeletal concentrations of troponin-T are dependent on the stage of embryonic development analyzed, and on the type of catecholamine added to the culture. The most significant finding was the increase in troponin-T mRNA in the chick heart at HH stage 40, accompanied by an increase in the increase in the expression of this protein in the cytoskeletal compartment after treatment with norepinephrine. At HH stage 21, norepinephrine induced less marked changes in the accumulation of troponin-T in comparison with untreated cardiomyocytes.

Animals↗
PubMed1994

Production and characterization of a new monoclonal antibody, GR-ICOR-2, recognizing sarcomeric actin: analysis of the expression in the developing chick heart.

We produced and characterized a specific monoclonal antibody (mAB) designated GR-ICOR-2. This mAb recognizes sarcomeric actin molecules (43 kDa) and was used in an immunohistochemical analysis of staining patterns in Hamburger and Hamilton's stages 18, 22 and 25 (HH 18, 22 and 25) embryonic chick hearts. Staining showed a mainly cytoplasmic distrubition in three regions: the atrioventricular (AV) canal cushion tissue, the primitive ventricle, and conal crests. In addition, this mAb-cross-reacted with rabbit and human cardiac and skeletal muscle tissue; but not with smooth muscle tissue.

Actins↗
PubMed1994

Actinomycin D causes multidrug resistance and differentiation in a human rhabdomyosarcoma cell line.

The emergence of drug-resistant tumor cells remains a major problem in cancer chemotherapy. Resistance to multiple unrelated antineoplastic drugs may be related, in part, to expression of the P-glycoprotein. The cell line RD, derived from an embryonic rhabdomyosarcoma tumor, was used as an in vitro model to examine the development of drug resistance. A cell line resistant to actinomycin D (RD-DAC) was developed by growing RD in increasing concentrations of the drug. The ID50 (concentration of drug needed to induce a 50% reduction in cell growth) of the resultant line to actinomycin D was more than 15 times that of the parental line. The resistant line was cross-resistant to vincristine and doxorubicin. Resistance to actinomycin D resulted in increased P-glycoprotein expression, which was associated with a change in desmin and vimentin expression. These results suggest that exposure to chemotherapeutic drugs can induce not only classical multidrug resistance, but also a process of cellular differentiation in rhabdomyosarcoma cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Expression of epidermal growth factor receptor in chick embryo myocardiocytes: relation with desmin expression during cardiac development.

The epidermal growth factor receptor is related with processes of cell replication and differentiation. We used the intermediate filament protein desmin as a marker to study the relation between muscle cell differentiation and modifications in the expression of this receptor during heart development in the chick embryo. Epidermal growth factor receptor was expressed as early as Hamburger and Hamilton's stage 17, when myocardiocytes are still poorly differentiated and desmin-negative. Expression became steadily weaker as the heart matured, and decreased after Hamburger and Hamilton's stage 25, a key stage in heart maturation characterized by a sharp increase in desmin expression. Our findings suggest that in the chick embryo, the expression of epidermal growth factor receptor becomes steadily weaker as myocardiocyte differentiation progresses.

Animals↗
PubMed1993

Circulating alpha-actin in angina pectoris.

We used Western blot, a highly sensitive technique that detects amounts of protein as low as 0.1 to 1.0 ng, to investigate the possible presence in the blood stream of the contractile protein alpha-actin in 29 patients diagnosed with angina pectoris (Braunwald's classification). Circulating protein was identified with a monoclonal antibody specific for cardiac alpha-actin. Of the 20 control samples of blood, the immunoblot results were negative for alpha-actin in 19. Of the 30 patients with skeletal muscle damage caused by surgery, 27 were negative for circulating alpha-actin. Of the 29 patients with angina pectoris, circulating alpha-actin was found in 19 as a 43 kDa band in immunoblots. Of the four patients with anterior acute myocardial infarction, mean concentration of circulating alpha-actin was 58 mg/l. Among the patients with angina pectoris, the highest circulating concentrations (mean 40 mg/l) was found in those with prolonged angina (class III B, according to Braunwald's classification). In the entire group of individuals with angina pectoris alpha-actin was detectable in serum for up to 175 h after the onset of pain, and showed two peaks, one at 1 h (112 mg/l) and one at 50 h (82 mg/l) after the onset of pain. These findings reinforce the notion that unstable angina should be considered a serious condition.

Actins↗
PubMed1993

Changes in subcellular accumulation of contractile proteins in myocardiocyte cultures: effects of fibric acid derivatives.

We analyzed the influence of 6 and 24 h of treatment with the fibric acid derivatives bezafibrate (10 micrograms/ml), gemfibrozil (23 micrograms/ml), and fenofibrate (30 micrograms/ml) on alpha-actinin, troponin-T, and tropomyosin proteins in the cytoplasmic and cytoskeletal fractions of cultured chick myocardiocytes. The findings with sodium dodecyl sulfate-gel electrophoresis and immunoblotting showed that all three drugs modified cellular and subcellular protein levels in different ways: bezafibrate and fenofibrate produced the most significant alterations in both fractions, modifying alpha-actinin, troponin T, and tropomyosin compartmentalization in myocardiocytes, whereas gemfibrozil altered these proteins less notably. Given the role of these proteins in heart muscle contraction, fibric acid derivative-induced changes may be related with the secondary effects of these drugs on heart rhythmicity.

Actinin↗
PubMed1993

[The swine heart: the papillo-tendino-valvular system of the right ventricle].

The right ventricle was studied in 75 anatomically normal swine hearts. Nine parameters in the papillo-tendino valvular system and three corresponding to the tricuspid orifice, pulmonary orifice and length of the inflow tract were measured. Correlations were established between the parameters and heart weight in grams, between the different parameters themselves, and between heart weight and body weight. The results were compared with similar data from human hearts, and were considered of use to researchers planning to use the swine heart as an experimental model to study congenital or induced heart diseases, or as a reference for the clinical interpretation of spontaneous cardiac, anomalies in swine.

Animals↗