Toucan protein is essential for the assembly of syncytial mitotic spindles in Drosophila melanogaster.

The toc gene of Drosophila melanogaster encodes a 235-kD polypeptide with a coiled-coil domain, which is highly expressed during oogenesis (Grammont et al., 1997, 2000). We now report the localization of the Toucan protein during early embryonic development. The Toucan protein is present only during the syncytial stages and is associated with the nuclear envelope and the cytoskeletal structures of the syncytial embryo. In anaphase A, Toucan is concentrated at the spindle poles near the minus end of microtubules. This microtubule association is very dynamic during the nuclear cell cycle. Mutant embryos lacking the Toucan protein are blocked in a metaphase-like state. They display abnormal and nonfunctional spindles, characterized by broad poles, detachment of the centrosomes, and failure of migration of the chromosomes. These results strongly suggest that Toucan represents a factor essential for the assembly and the function of the syncytial mitotic spindles.

Expression and cellular localization of the Toucan protein during Drosophila oogenesis.

The toucan (toc) gene is required in the germline for somatic cell patterning during Drosophila oogenesis. To better understand the function of toc, we performed a detailed analysis of the distribution of the Toucan protein during oogenesis. Toc expression is restricted to the germline cells and shows a dynamic distribution pattern throughout follicle development. Mislocalization of the Toc protein in mutant follicles in which the microtubule network is altered indicates that microtubules play a role in Toc localization during oogenesis.

Heterogeneity of the early outward current in ventricular cells isolated from normal and hypertrophied rat hearts.

1. The nature, magnitude and kinetics of the 4-aminopyridine-sensitive early outward current (Ito) were analysed in isolated ventricular myocytes from the septum, the apex and the left ventricular free wall of rat ventricles using the whole-cell voltage clamp method. The modulatory effect of pressure overload-induced cardiac hypertrophy on the regional variations of Ito was assessed in each topographical class of cells. 2. Voltage clamp experiments were performed at room temperature (20-25 degrees C) in the absence of Na+ on both sides of the membrane and in the presence of 3 mM CoCl2. Ito was studied from a holding potential of -80 mV and determined by subtraction of total outward currents elicited by the same protocols in the presence of 3 mM 4-aminopyridine (4-AP) from those obtained in its absence. 3. In normal hearts, membrane passive properties were very similar in each topographical class of cells. Our results confirmed that the predominant early outward current in rat ventricular cells was 4-AP-sensitive, time and voltage dependent, and demonstrated that the magnitude of the current varied on a regional basis: current density of Ito in left ventricular free wall cells (30.1 +/- 9.2 pA/pF at +60 mV) was larger than in apex cells (20.2 +/- 1.7 pA/pF) or in septum cells (11.9 +/- 3.3 pA/pF). We noticed a larger variability in data from left ventricular free wall compared with other regions. 4. No shift in steady-state voltage dependence of Ito activation and inactivation was found. However, the maximal computed chord conductances were (in microS/pF): 0.18 +/- 0.07 for left ventricular free wall cells, 0.13 +/- 0.02 for apex cells, and 0.08 +/- 0.02 for septum cells. These findings might reflect a differential distribution in functional channel densities. 5. No difference in voltage-dependent Ito activation kinetics was present with respect to topography. However, inactivation time constants in septum were longer than those of both other groups. 6. Left ventricular hypertrophy was induced by abdominal aortic constriction and its effects compared to the findings from normal rats. Hypertrophied cells had similar resting potentials but higher capacitance values than normal cells. Although Ito magnitude appeared not to be modified, the current density-voltage curves were slightly shifted to more positive potentials and significantly decreased as compared to normal cells (in pA/pF, at +60 mV): 8.4 +/- 5.0 in the left free wall group, 11.6 +/- 2.0 in the apex group, and 3.8 +/- 1.5 in the septum group.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of hydroxyl radicals on ATPase and protein structure of myofibrils from rat heart.

Free oxygenated radicals frequently are involved in cardiac arrhythmias and contractility disorders during postischemic reperfusion. The aim of this study was to determine the effects of hydroxyl radicals (.OH) in vitro on myofibrillar Ca-adenosinetriphosphatase (ATPase), on the redox state of thiol groups and the electrophoretic pattern of myofibrillar proteins from rat heart. Myofibrils were treated up to 60 min by .OH generated with 0.3 mM H2O2 and 0.1 mM Fe2+. After a 60-min treatment with .OH, the measurement of thiol groups failed to show any oxidation. On the contrary, ATPase activity and electrophoretic pattern were affected dramatically by treatment with .OH. For all Ca2+ concentrations, ATPase was increased after treatment with .OH, but ATPase activation when Ca2+ rose from pCa 8 to pCa 4.5 was only 92% after 30 min of incubation rather than 226% for untreated myofibrils. The electrophoretic analysis of myofibrillar proteins showed a decrease in myosin heavy chain and formation of aggregates in treated myofibrils. All of these effects were reduced when incubation was performed in the presence of mannitol, a specific scavenger of .OH. No effect was observed with 0.1 mM Fe2+ alone or with 0.3 mM H2O2. The action of .OH was very fast to the extent that the effects were observed after only 15 s of incubation. The results reported in the present study may be related to the impaired relaxation and contracture described in vivo within the first minutes of a postischemic reperfusion and before any change in calcium homeostasis.

Mitochondrial nucleotide translocase from skeletal muscle of halothane sensitive pigs: an electrophoretic study.

ATP translocation into mitochondria isolated from halothane-sensitive pig (HP) muscle was dramatically reduced compared with normal pigs (NP). To determine if this was due to a decreased amount of ATP translocase in the mitochondrial membranes, or a structural modification of this protein, an electrophoretic study was undertaken. Total proteins and purified translocase preparations from (NP) and (HP) mitochondria were analyzed by SDS gel electrophoresis. In the two types of mitochondria no significant differences were observed either in the amount of ATP translocase or in the molecular weight. Also, neither nonequilibrium pH gradient gel electrophoresis nor the analysis of peptides produced by limited proteolysis revealed any structural difference between the two types of protein. On the basis of these results, the depressed translocase activity observed in (HP) mitochondria cannot be explained by a reduced amount of the nucleotide translocase, nor a structural alteration of this protein. Possible inhibition of (HP) translocase activity by Ca2+ accumulation or by other mechanisms is discussed.

Photoduplication and fluorescence measurement in polyacrylamide gel electrophoresis.

A simple method of photoduplication of gel electrophoresis, visualized with fluorescent reagents, is described. The procedure is convenient and rapid and requires no camera or expensive equipment. Using electrophoresis duplicating paper (Kodak), positive prints suitable for documentation or publication may be obtained. With usual photographic paper, negative prints may be obtained, allowing reliable measurement by scanning. The technique may be applied to protein or nucleic acid electrophoresis.

[Assay of sulfhydryl groups in cardiac myofibrillar proteins: effect of oxygen radicals in vitro]. / Dosage des groupes sulfhydryles des protéines myofibrillaires cardiaques: effet des radicaux libres oxygénés in vitro.

Myofibrils from rat hearts were prepared in conditions maintaining their redox state, and their sulfhydryl groups were measured using a solution of urea and sodium dodecyl sulfate (SDS) as denaturant. The sulfhydryl content was 92 n mol/mg of protein, indicating that cysteins are in reduced form. In the presence of superoxide radicals generated in vitro with purine and xanthine oxidase, the myofibrillar sulfhydryl groups were oxidized.

Ca2+ measurement with a Ca electrode: artifacts due to some cardiotropic drugs.

In order to validate D. M. Bers' method ((1982) Amer. J. Physiol. 242, C404-C408) for the determination of free [Ca] in ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (EG-TA)-buffered Ca solution, six cardiotropic drugs were checked for their ability to interfere with Ca2+ measurements with a Ca electrode: verapamil, diltiazem, perhexiline, amrinone, bepridil, and diproteverine. Only amrinone, up to 10(-4) M, showed no effect. With the other compounds, from 5 x 10(-6) to 10(-4) M a gradual increase of the potential was observed. Negligible with 1 mM Ca2+ in the solution, the effects increased while free Ca decreased, regardless of the presence of EGTA. Similar observations were made using a K+ electrode. Relying on the results obtained by three different techniques, potentiometric study, spectrophotometric study, and binding of 45Ca on Chelex 100 resin, it was concluded that the effects of these drugs on the potential measured with an ion-specific electrode are artifacts which are not due to a release of Ca ions from the Ca-EGTA complex. When using Bers' method for the determination of free calcium, interfering drugs should be added to the solutions only after Ca2+ measurements with the Ca electrode.

Effects of bepridil on calcium release from rat heart mitochondria.

Bepridil at concentrations above 10 microM, and at pH 7.2 stimulates calcium release from rat heart mitochondria. However this action is different from that of ClCCP, an uncoupler of oxidative phosphorylations, since it is ruthenium red insensitive. At lower concentrations bepridil may inhibit the Na-induced calcium release. The effects of bepridil depend on the pH and indicate that the protonated form of the drug is more efficient on calcium release than the basic form.

Protective effects of bepridil on calcium injury of anoxic myocytes isolated from adult rat heart ventricular muscle.

Cardiac myocytes allowed to recover after the isolation procedure in a well-oxygenated medium were resistant to an extracellular Ca++ concentration of 1.5 mM. At least 90% of the isolated myocytes maintained their initial rod-shaped form after a 1-hr incubation, as well as 85% of their ATP and a low cellular content of calcium. At concentrations less than 10(-5) M, bepridil had no effect on these cells. On the other hand, when the myocytes were incubated in anaerobic conditions, 1.5 mM Ca++ was deleterious: the number of viable cells decreased by about 50%, ATP levels were lowered by 70% and the total cell calcium content increased by more than 100%. Bepridil had a biphasic effect on anaerobic cells. At concentrations below 10(7) M, the drug had a beneficial action. It restored cell viability and cellular ATP to 77 and 100% when compared to their respective level in the absence of Ca++. However, at concentrations higher than 10(-6) M, bepridil decreased the number of viable cells as well as their ATP content. At optimal concentrations, bepridil lowered the cellular calcium to its level in aerobic conditions. Without calcium addition, anoxic cells accumulated Na+ and lost K+. Calcium addition decreased Na+ accumulation by 68% and prevented the loss of K+. The Na+ and K+ content of the cells were not affected by bepridil. However, it is noteworthy that, at 10(-4) M, bepridil decreased dramatically the potassium content of the cells. In aerobic conditions, the calcium resistance of isolated ventricular myocytes may still be enhanced by Fluozol, a fluorocarbon compound which increases oxygen solubility in water.

Silver staining of proteins in polyacrylamide gels: increased sensitivity by a blue toning.

An image amplification of silver-stained proteins in polyacrylamide gels by a blue toning has been developed. Following the silver staining, this technique is fast, simple, requires only stable and inexpensive reagents, and its sensitivity is three- to sevenfold higher than the silver staining alone, and 300- to 700-fold higher than the Coomassie blue. When combined with a silver staining utilizing dithiothreitol (J. H. Morrissey, (1981) Anal. Biochem. 117, 307-310) this method is shown to be uniform in sensitivity for five standard proteins. The blue toning does not affect the linearity of the staining and allows the quantitation of proteins by densitometric evaluation.

The incorporation of radioactive lysine or tyrosine into cardiac and skeletal myofibrillar and non-myofibrillar contractile proteins.

The labelled amino acids incorporation into cardiac and skeletal contractile proteins has been compared after a single injection of 3H lysine, repeated injections of 3H lysine during 1 or 6 hours or after a continuous infusion of both 3H-lysine and 14C-tyrosine. The myofibrillar incorporation was higher in the heart than in the skeletal muscle. Myosin heavy chains and actin have been prepared using gel filtration. The incorporation was again higher in the heart for both these proteins but the labelling of actin in both the muscles reaches rapidly a plateau in contrast with myosin, suggesting that these two proteins possess a different precursor pool. Myosin heavy chains prepared from the supernatant obtained after a relaxing treatment were more labelled than those extracted from myofibrils. These heavy chains from the supernatant were presumably newly synthetized and not yet incorporated into myofibrils. They also were more labelled in the heart than in the skeletal muscle which means that the myosin synthesis itself was different and not the process of assembly of the myofibrils.

A comparative study of the cardiac troponin inhibitory factor (TNI) from mammalians.

Troponin inhibitory factor, TNI, was prepared by affinity chromatography from different mammalian hearts. (i) Structure. These different TNI have the same M.W. (28000), which is higher than that found in rabbit skeletal muscle (23000). Nevertheless they differ with respect of their charge as shown by alkaline urea polyacrylamide gel electrophoresis using cardiac TNI which has previously been bound to an excess of skeletal troponin Ca2+-binding factor. These changes do not correlate with the PO4 content of TNI. They are associated with structural differences demonstrated by peptide mapping of the unfolded molecule after papain treatment. The structure of cardiac TNI from rat and rabbit differs clearly from that of crow and pig. (ii) Biological activity. These different TNI have the same inhibitory effect on skeletal actomyosin. ATPase, the same content of PO4 and the same ability to be phosphorylated in-vitro by a bovine heart c-AMP-dependent protein kinase.

Heart contractile proteins.

That several proteins of the sarcomere differ from one muscle to the next is well documented, and it is becoming evident that homogeneous muscles, like the heart, are also species specific. 1) Clear-cut evidence is available concerning myosin, and, to date, several types of molecules have been described. a) The myosins of white skeletal, heart, and smooth muscle differ in the activity of their Ca2+ and K+ATPases, as also in the structure of their light subunits. b) The Ca2+ATPases of the various cardiac myosins have been shown to exhibit species differences and correlate with the speed of shortening of the muscle. 2) The structures of tropomyosin, some troponin components, and alpha actinin (but not actin) appear to be unlike in the different types of muscle. 3) These phylogenic modifications may be related to the changes characteristic of the particular muscles under pathological conditions, which are accompanied by substantial increase in protein synthesis.

Cardiac myosin: preparation, ATPase in chronic heart hypertrophy.

A low myofibrillar ATPase seems to be established definitely in several experimental models of chronic heart hypertrophy as well as in humans, but the biochemical pathogenesis of this defect is still unclear. Three different preparations of myosin were studied. Their purity was estimated by measuring MgATPase or by polyacrylamide gel electrophoresis. The first preparation was highly contaminated by actin and tropomyosin; the second was rather pure, and the third (chromatography on DEAE-Sephadex) was pure but slightly denaturated. Heart myosin CaATPase (ionic strength 0.6 or 0.06) was decreased in chronic aortic insufficiency in the rabbits (CAI) when all three preparations were tested. Two (molecular weight 18,000 and 26,000), sometimes three light subunits were found in heart myosin. Their charge and molecular weight are normal in CAI. The third subunit (molecular weight 15,500) was found in control as well as in CAI. Search for an inhibitor was unsuccessful since the two myosin ATPases are additive. The nucleoprotein peak separated from myosin during chromatography was identical in control and CAI. Therefore, myosin seems to be abnormal in CAI.