Contractile regulatory proteins tropomyosin and troponin-T as indicators of the modulatory role of retinoic acid.

Retinoic acid (RA), the active metabolite of vitamin A, plays a significant role in regulating cardiac form and function throughout the life of the organism. Both cardiac morphogenesis and myocardial differentiation are affected by alterations in RA homeostasis. In order to test the effect of all-trans RA and 13-cis RA on cardiomyocyte differentiation, we studied the level and the subcellular compartmentalization of alpha-tropomyosin and troponin-T proteins in cultures of chick embryo cardiomyocytes obtained from Hamburger and Hamilton's (HH) stage 22, 32 and 40 embryos. The retinoids increased the levels of alpha-tropomyosin and troponin-T in the cytoplasmic and cytoskeletal fractions of cells at all three stages of development. The greatest increases in alpha-tropomyosin occurred in the cytoplasmic fraction in HH22 cells cultured for 24 h with all-trans RA or 13-cis RA, whereas the greatest increases in troponin-T were found in the cytoplasmic fraction of HH32 cells exposed to retinoids for 24 h. In cultures treated for 48 h with retinoids, the levels of alpha-tropomyosin and troponin-T showed significant increases in the cytoplasmic compartment of cells treated in HH32-with respect to the control values. These findings are further evidence that RA plays a modulating role in the formation and reorganization of sarcomeric proteins during the process of cardiomyocyte maturation.

Development of chick cardiomyocytes: modulation of intermediate filaments by basic fibroblast and platelet-derived growth factors.

Recent studies suggest that peptide growth factors play a functional role in cardiac muscle. To test whether embryonic cardiac muscle is a target for regulation by basic fibroblast growth factor and platelet-derived growth factor, we analyzed the effects of these peptides on the expression of the intermediate filaments desmin and vimentin at the subcellular level during development. Sodium dodecyl sulfate-gel electrophoresis, immunoblotting and fluorescence-activated cell sorting analysis were used to study the effect of basic fibroblast growth factor and platelet-derived growth factor on cultures of chick cardiomyocytes during development. Cytoplasmic and cytoskeletal concentrations of desmin and vimentin 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 desmin expression in the cytoplasmic and cytoskeletal compartments after treatment with basic fibroblast growth factor (10 ng/ml) of chick heart cells at Hamburger and Hamilton stage 19. In more mature stages, basic fibroblast growth factor did not modify the levels of desmin expression. However, this factor led to a progressive deceleration in the rate of increase in vimentin expression. Platelet-derived growth factor increased vimentin expression in all stages studied, the greatest increases appearing in early 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, whereas platelet-derived growth factor has a dedifferentiating effect.

Modulation of myogenic differentiation in a human rhabdomyosarcoma cell line by a new derivative of 5-fluorouracil (QF-3602).

The in vitro study of mechanisms involved in drug-induced maturation has made it possible to use differentiation-based therapy in clinical practice. The goal of this new therapy is the development of specific agents to induce cancer cells to stop proliferating and express characteristics of normal cells. Recently, by structural modifications of 5-fluorouracil (5-FU), we synthesized a new pyrimidine acyclonucleoside-like compound, 1-¿[3-(3-chloro-2-hydroxypropoxy)-1-methoxy]propyl¿-5-fluorouracil (QF-3602), which showed in rhabdomyosarcoma cells a low toxicity and time-dependent growth inhibition. In this work, we compared the degree of myogenic differentiation of RD rhabdomyosarcoma (RMS) cells after treatment with QF-3602 and 5-FU. Scanning and transmission electron microscopy (SEM and TEM) and immunocytochemical analyses showed that QF-3602 induced the appearance of myofilaments along the myotube-like giant RD cells, an increase in fibronectin and a decrease in vimentin expression. In contrast, only minor changes were observed with 5-FU. Moreover, polymerase chain reaction (PCR) analyses showed that QF-3602 did not induce overexpression of the mdr 1 gene, a resistance mechanism that frequently appears in classical cytotoxic therapy in these tumors. Compounds obtained by structural modifications of 5-FU may be useful in differentiation therapy as a new approach to the treatment of RMS.

Modulation of alpha-actin and alpha-actinin proteins in cardiomyocytes by retinoic acid during development.

Early heart development is known to be sensitive to retinoid concentrations. Although the influence of retinoids on cardiac morphogenesis has been described previously, the effect of retinoids on cardiomyocyte differentiation during development has not been characterized. We quantified the effects of the retinoic acids all-trans RA and 13-cis RA on alpha-actin and alpha-actinin at the subcellular level in cultures of chick embryo cardiomyocytes obtained from Hamburger and Hamilton's (HH) stage 22, 32 and 40 embryos. The retinoids increased the concentration of alpha-actin and alpha-actinin in the cytoplasmic and cytoskeletal fractions of cells at all three stages of development. The effect was greatest in cardiomyocytes treated for 24 h with all-trans RA and in cells from HH22 embryos. The greatest increases in alpha-actin concentration occurred in the cytoskeletal fraction of HH22 cells cultured for 24 h with all-trans or 13-cis RA, whereas the greatest increases in alpha-actinin were found in the cytoplasmic fraction of HH22 cells exposed to retinoids for 24 h. We conclude that retinoic acid plays a role in the reorganization of the pattern of sarcomeric protein expression during cardiomyocyte differentiation.

Analysis of the nucleolar organizing regions in the ant Tapinoma nigerrimum (Hymenoptera, Formicidae).

This study analyses the NORs of Tapinoma nigerrimum, a species that, as known from previous studies, has various chromosomes which carry a NOR site. The analysis was made by a combination of three methods: silver nitrate staining, in situ hybridization with fluorescein-or digoxigenin-labelled probes, and staining with the CG-specific fluorochrome chromomycin A3. The silver staining technique showed an Ag-positive region on chromosome 6 and on various other chromosomes. However, the application of in situ hybridization techniques showed only one positive signal in the proximal region of the short arm of chromosome 6 of T. nigerrimum. Similar results were observed by CMA banding. The absence of rDNA genes or the presence of only a small number of these, not detectable with the above probes, might explain the absence of hybridization signal in the remaining chromosomes.

Clinical significance of antiheart antibodies after myocardial infarction.

We used one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis of myocardial proteins followed by Western blotting to study the formation of antiheart antibodies during three months after myocardial infarction and the relationship between the appearance of antibodies and clinical and laboratory findings. Fifty-four percent of the 66 patients with infarction had different types of antiheart antibodies. The autoantibodies detected most frequently were against 35 and 42 kDa cardiac proteins. Immunoblottings with purified proteins showed that these autoantibodies reacted against myocardial tropomyosin and actin, which have been detected after acute myocardial infarction and can have immunogenetic activity through a humoral immune response. However, only the presence of autoantibody against myocardial tropomyosin correlated significantly with the presence of clinical and laboratory findings. Our results suggest that autoantibody against myocardial tropomyosin may play an immunopathogenic role in the development of symptoms in these patients.

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.

Inverse expression of mdr 1 and c-myc genes in a rhabdomyosarcoma cell line resistant to actinomycin d.

Cytotoxic agents used in cancer therapy may induce differentiation in tumour cells with no proliferative potential. However, chemotherapy can also induce multidrug resistance, a formidable obstacle to the successful treatment of tumours. Both events were recently shown to occur in a rhabdomyosarcoma cell line (RD-DAC) resistant to actinomycin D, a drug of choice in the treatment of these tumours. To analyse this connection, cell line RD cultures were investigated with progressive concentrations of actinomycin D and it was shown that a minimum dose (1.2 x 10(-6) mM) of the drug was necessary to increase mdr 1 mRNA in RD-DAC. The mechanism of mdr 1 overexpression was an increase in the number of copies of the mdr 1 gene, although the mRNA levels were not correlated with mdr 1 amplification. Drug resistance mediated by mdr 1 overexpression coincided with the development of myogenic differentiation in RD-DAC and with a decrease in c-myc mRNA levels, whereas levels of N-myc mRNA showed no modulation. These findings suggest that factors implicated in cell proliferation and differentiation, such as c-myc, may be responsible for the control of genes related to the development of multidrug resistance in rhabdomyosarcomas. Modulation of these factors may determine the sensitivity of rhabdomyosarcoma cells to drugs and may play an important role in triggering the differentiation programme found in these resistant rhabdomyosarcoma cells.

Detection of circulating alpha-actinin in non-insulin-dependent diabetics with autonomic dysfunction.

The presence of cardiac proteins in serum has been related to ischemic heart diseases such as acute myocardial infarction and angina pectoris, which are more frequent in non-insulin-dependent diabetic patients. Silent myocardial ischemia, which is also more frequent in these patients, occurs in association with autonomic dysfunction. We used Western blot analysis to search the myocardial protein alpha-actinin in sera from non-insulin-dependent diabetic patients with or without autonomic dysfunction. Of the 24 diabetic patients with neuropathy, 18 were positive for circulating alpha-actinin; this protein was found in only 1 of the 22 diabetic patients without neuropathy. Our results showed a significant correlation between non-insulin-dependent diabetic patients with neuropathy and detectable circulating alpha-actinin in serum, and suggest that the determination by immunoblotting of serum alpha-actinin in these patients may be an effective method to detect myocardial cell impairment, and to identify diabetic patients that may need special consideration.

Low concentrations of actinomycin D potentially cause therapeutic differentiation in human rhabdomyosarcoma cell line RD.

Neoplastic transformation may be an alteration in the process of cell maturation that leads to an infinite capacity for proliferation. Because the cytodestruction caused by most drugs available for cancer chemotherapy is often accompanied by significant morbidity and poor response, the induction of differentiation has been proposed as an alternative approach to conventional anticancer therapy. We used human rhabdomyosarcoma cell line RD to analyze the differentiation process induced by actinomycin D, a drug of choice in the conventional treatment of rhabdomyosarcomas. Low concentrations of actinomycin D induced a terminal process of morphological and ultrastructural myogenic differentiation in rhabdomyosarcoma cells, which concluded with cell death. However, this potential therapeutic effect cannot be considered complete because of the presence of tumoral cells that are heterogeneous with respect to actinomycin D chemosensitivity. This heterogeneity led to the appearance of foci of resistant cells which, despite their greater degree of differentiation in comparison with the parental cell line, escaped from terminal myogenic differentiation. This subgroup of tumoral cells may be responsible for the failure of cytotoxic treatment.

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.

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.

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.

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).

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.

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.

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.

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.

[The swine heart: the papillo-tendino-valvular system of the right ventricle]. / Corazón cerdo: sistema papilo-tendino-valvular del ventrículo derecho.

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.

Actin, tropomyosin and alpha-actinin as markers of differentiation in human rhabdomyosarcoma cell lines induced with dimethyl sulfoxide.

Most rhabdomyosarcomas are poorly differentiated malignant tumors. Dimethyl sulfoxide has been shown to modulate cell differentiation in cultured human cells. We induced differentiation in human rhabdomyosarcoma cell lines A-673, RD and A-204 with 1.25% dimethyl sulfoxide, and used desmin, the protein most frequently used as a marker of muscle cell differentiation, to trace this process. As alternative markers of the degree of differentiation, we quantified the expression of the proteins actin, tropomyosin and alpha-actinin in these cell lines, and followed the changes in expression of these proteins after induction for 8, 12, 24, 48 and 72 hrs. In the process of differentiation, protein expression in both the cytoplasm and cytoskeleton was significantly increased by treatments lasting 12 hrs. (alpha-actinin) and 24 hrs. (actin). On the basis of our results, alpha-actinin can be considered as an earlier marker of differentiation than actin in human rhabdomyosarcoma cell lines. However, the earliest indication of differentiation was a modification in desmin expression (8 hrs.). Because changes in tropomyosin expression were less marked, we consider this protein as a poor marker of rhabdomyosarcoma cell differentiation.