Biomedical subjects

P Volpe

Publications and source records attributed to P Volpe.

At least 145 records · Page 8Linked to original sources

PubMed1984

The effect of phenothiazines on Ca2+ fluxes in skeletal muscle sarcoplasmic reticulum.

The effect of phenothiazines (trifluoperazine, chlorpromazine, methochlorpromazine, and imipramine) on Ca2+ fluxes in light and heavy sarcoplasmic reticulum (SR) isolated from rabbit fast-twitch skeletal muscle was investigated. These drugs inhibited Ca2+ loading and (Ca2+,Mg2+)-ATPase activity, but had no effect on unidirectional Ca2+ efflux from vesicles loaded either actively or passively with Ca2+. Chlorpromazine, which is membrane permeable, and its quaternary analog, methochlorpromazine, which is membrane impermeable, gave identical results. It is concluded that (a) the enhancement of net Ca2+ release by phenothiazines is due to inhibition of Ca2+ influx mediated by the Ca2+ pump rather than to the opening of a Ca2+ channel; and (b) phenothiazines act at the outer (myoplasmic) face of the SR membrane.

Animals↗

PubMed1984

In vitro methylation of total and foldback DNAs in normal and virus-transformed cells.

The levels of the in vitro methylation of total and palindromic DNAs in nuclei isolated from normal and virus-transformed cells are compared. The methylation rate of total DNA in normal rat kidney cells is much higher than that detected in normal mouse fibroblasts. However, for both cell species, while the maximal rate of DNA methylation is observed in the mid-logarithmic phase of the cell culture growth, palindromes are always found to be more heavily methylated than total DNA. The 5-methylcytosine content of DNA, especially of palindromes, is higher in virus-transformed cells than in untransformed cells.

Animals↗

PubMed1984

Calcium-induced calcium release from purified cardiac sarcoplasmic reticulum vesicles. General characteristics.

Isolated canine cardiac sarcoplasmic reticulum exhibits Ca2+-induced Ca2+ release from both actively and passively loaded vesicles. The rate and extent of Ca2+ release depend on the extravesicular ionized Ca2+ concentration ( [Ca2+]o) at the onset of release. Maximal release following ATP-dependent, phosphate-facilitated Ca2+ loading (up to 360 nmol of Ca2+/mg of protein/min at 37 degrees C) occurs at 1.5-2 microM [Ca2+]o, with reduced release at both lower and higher Ca2+ concentrations (half-maximal Ca2+ release at approximately 0.8 and 5.5 microM [Ca2+]o). Only a portion of the accumulated Ca2+ is released and the release is followed by reuptake of Ca2+. A similar Ca2+ dependence is obtained in the absence of ATP and Pi by measuring unidirectional Ca2+ efflux from passively loaded vesicles (maximal Ca2+ efflux at 1 microM [Ca2+]o; half-maximal Ca2+-dependent efflux at approximately 0.15 and 13 microM [Ca2+]o). Although the Ca2+ release rates observed in this study are several orders of magnitude lower than the rate of Ca2+ release which occurs in muscle cells in vivo, this Ca2+ release phenomenon may be related to the Ca2+-induced Ca2+ release which has been described for skinned cardiac cells ( Fabiato , A. (1983) Am. J. Physiol. 245, C1-C14). Ca2+ release occurs in the presence of an ATP-regenerating system and is not accompanied by a reduction in ATP hydrolysis. Also, since unidirectional Ca2+ efflux (as high as 860 nmol of Ca2+/mg of protein/min at 37 degrees C) exceeds net Ca2+ release under similar conditions, Ca2+ influx proceeds during the period of net Ca2+ release. Therefore, Ca2+ release does not involve reversal or cessation of inward Ca2+ pumping. Other data indicate that Ca2+ release is not mediated through the Ca2+ pump protein, but occurs through a separate Ca2+-dependent efflux pathway, possibly a channel.

Adenosine Triphosphate↗

PubMed1984

Inhibition of calcium-induced calcium release from purified cardiac sarcoplasmic reticulum vesicles.

A variety of reagents (local anesthetics, phenothiazines, ruthenium red, ryanodine, dicyclohexylcarbodiimide, R 24571) inhibit Ca2+-induced Ca2+ release from purified canine cardiac sarcoplasmic reticulum (SR). Most of these compounds also increase the rate of net Ca2+ uptake by cardiac SR while moderately inhibiting Ca2+-dependent ATP hydrolysis, and together these two effects produce increased coupling ratios for ATP-dependent Ca2+ transport (Ca2+/ATP approximately equal to 2) compared to controls (Ca2+/ATP approximately equal to 1). We conclude that Ca2+ efflux normally occurs during net Ca2+ uptake by isolated cardiac SR vesicles and that this phenomenon is responsible for the low coupling ratios generally observed for cardiac SR preparations. Blockers of sarcolemmal Ca2+ channels (nitrendipine, diltiazem, methoxyverapamil, dantrolene), at concentrations much greater than those effective for sarcolemmal Ca2+ fluxes, do not affect either Ca2+ uptake or Ca2+ release by cardiac SR. Furthermore, the effects of local anesthetics and phenothiazines on Ca2+ release from cardiac SR are different from those previously reported for skeletal muscle SR. These results indicate that the Ca2+ release "channels" in cardiac SR are distinct from Ca2+ channels present in cardiac sarcolemma or in skeletal muscle SR. The common hydrophobic nature but structural dissimilarity of various inhibitors of Ca2+ release from cardiac SR suggest that in some cases a hydrophobic interaction with the membrane might be involved in blocking Ca2+ efflux. On the other hand, half-maximal inhibition by 80 nM ruthenium red is suggestive of a specific ionic interaction with some component of the Ca2+ efflux pathway.

Adenosine Triphosphate↗

PubMed1984

Virus genome integration as a function of the host cell genome replicative cycle.

Mouse 3T3 cells were grown and synchronized in monolayer with the double thymidine block. Their infection with SV40 took place continuously during the cellular cycle. However, integration of viral DNA into host cell DNA occurred preferentially during the S phase. Phase G1 appeared to be necessary for virus-cell DNA recombination in S phase. Phase G2 did not alter the stability of the integrated viral genome.

Animals↗

PubMed1984

Calcium release from sarcoplasmic reticulum of normal and dystrophic mice.

Contraction of skeletal muscle is triggered by release of calcium from the sarcoplasmic reticulum. In this study, highly purified normal and dystrophic mouse sarcoplasmic reticulum vesicles were compared with respect to calcium release characteristics. Sarcoplasmic reticulum vesicles were actively loaded with calcium in the presence of an ATP-regenerating system. Calcium fluxes were followed by dual wavelength spectrophotometry using the metallochromic indicators antipyrylazo III and arsenazo III, and by isotopic techniques. Calcium release from sarcoplasmic reticulum vesicle was elicited by (a) changing the free calcium concentration of the assay medium (calcium-induced calcium release); (b) addition of a permeant anion to the assay medium, following calcium loading in the presence of a relatively impermeant anion (depolarization-induced calcium release); (c) addition of the lipophilic anion tetraphenylboron (TPB-) to the assay medium and (d) using specific experimental conditions, i.e. high phosphate levels and low magnesium (spontaneous calcium release). Drugs known to influence Ca2+ release were shown to differentially affect the various types of calcium release. Caffeine (10 mM) was found to enhance calcium-induced calcium release from isolated sarcoplasmic reticulum. Ruthenium red (20 microM) inhibited both calcium-induced calcium release and tetraphenylboron-induced calcium release, and partially inhibited spontaneous calcium release and depolarization-induced calcium release. Local anesthetics inhibited spontaneous calcium release in a time-dependent manner, and inhibited calcium-induced calcium release instantaneously, but did not inhibit depolarization-induced calcium release. Use of pharmacological agents indicates that several types of calcium release operate in vitro. No significant differences were found between normal and dystrophic sarcoplasmic reticulum in calcium release kinetics or drug sensitivities.

Animals↗

PubMed1983

Spontaneous calcium release from sarcoplasmic reticulum. Effect of local anesthetics.

Spontaneous calcium release from purified light sarcoplasmic reticulum has been previously described (Palade, P., Mitchell, R. D., and Fleischer, S. (1983) J. Biol. Chem. 258, 8098-8107) and found to be distinct from several other forms of Ca2+ release. Ca2+ release occurs after a lag period following active Ca2+ preloading and depletion of extravesicular Ca2+. In the present study, we find that local anesthetics inhibit spontaneous Ca2+ release, in a time-dependent manner, varying considerably in the preincubation time required to exert maximal effect. At pH 7.0, hydrophilic and mostly charged local anesthetics, such as procaine, procainamide, and N-(2,6-dimethylphenyl carbamoyl methyl)triethyl ammonium bromide, inhibit Ca2+ release only after long preincubations (hours), whereas more hydrophobic local anesthetics are effective after only a short incubation (minutes) with sarcoplasmic reticulum. The more hydrophobic anesthetics take somewhat longer to reach equilibrium, as studied by inhibition of unidirectional Ca2+ efflux, and there is a direct relationship between hydrophobic partition coefficient and half-time to reach equilibrium. Agents known to inhibit permeability pathways for monovalent cations i.e. K+ channel blockers (decamethonium and n-dodecane-1, 12-N,N,N,N',N',N'-hexamethyl-bis-ammonium) or the anion blocker (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid), do not inhibit spontaneous Ca2+ release. Carbonyl cyanide m-fluorophenylhydrazone, a protonophore, and gramicidin D, a monovalent cation ionophore, have no effect on Ca2+ release whether local anesthetics are present or not, while the Ca2+ ionophore A23187 relieves inhibition of Ca2+ release by local anesthetics. Ruthenium red does not inhibit spontaneous Ca2+ release. These findings suggest that the binding site(s) for local anesthetics is located on the inner face of the sarcoplasmic reticulum membrane and that local anesthetics interact directly with a Ca2+ channel rather than with other permeability pathways which might indirectly influence Ca2+ channel gating.

Amines↗

PubMed1983

Biochemical characterization, integrity, and sidedness of purified skeletal muscle triads.

The release of Ca2+ from the terminal cisternae of sarcoplasmic reticulum in muscle fiber triggers muscle contraction. The signal for Ca2+ release is mediated via the triad junction, i.e. the junctional association of terminal cisternae and transverse tubule. Recently, highly purified morphologically intact triads were isolated from rabbit skeletal muscle (Mitchell, R. D., Palade, P., and Fleischer, S. (1983) J. Cell Biol. 96, 1008-1016). In this study, biochemical characterization of two variants of purified triad preparations (Pyrophosphate and Standard) is provided. Terminal cisternae of triads sequester Ca2+ at rates comparable to those of purified heavy sarcoplasmic reticulum which is referable to terminal cisternae (Meissner, G. (1975) Biochim. Biophys. Acta 389, 51-68). The permeability for calcium ions, as reflected by a 2-3-fold stimulation of (Ca2+, Mg2+)-ATPase activity in the presence of the Ca2+ ionophore A23187, and by the Ca2+ leak rate, is comparable in triads and heavy sarcoplasmic reticulum. Several transverse tubule characteristics are present in triads. Four of them, i.e. cholesterol content, ouabain binding, dihydroalprenolol binding (beta-adrenergic receptor), and ouabain-sensitive (Na+, K+)-ATPase activity, are comparably enriched in the Pyrophosphate triads and therefore appear to be quantitative indices of the amount of transverse tubule. Adenylate cyclase and basal ATPase are unreliable in this regard. Methodology for analyzing membrane integrity and sidedness was applied (adenylate cyclase activity) and modified (ouabain-sensitive (Na+, K+)-ATPase activity) to characterize the transverse tubule of the triad. In addition, a new method was developed making use of ouabain binding to study sidedness. These studies show that the transverse tubule is largely sealed and inside out in orientation, i.e. with the cytoplasmic face exposed. This report indicates that the t-tubule and sarcoplasmic reticulum components of the triads possess transport capability and retain permeability barriers for ions. Therefore, the isolated triads appear to be suitable for studying the physiological Ca2+ release process in vitro.

Adenylyl Cyclases↗

PubMed1982

Polymorphism of myosin light chains. An electrophoretic and immunological study of rabbit skeletal-muscle myosins.

Antibodies specific for rabbit fast-twitch-muscle myosin LCIF light chain were purified by affinity chromatography and characterized by both non-competitive and competitive enzyme-linked immunosorbent assay (ELISA) and a gel-electrophoresis-derived assay (GEDELISA). The antibodies did not cross-react with myosin heavy chains, and were weakly cross-reactive with the LC2F [5,5'-dithio-(2-nitrobenzoic acid)-dissociated] light chain and with all classes of dissociated light chains (LC1Sa, LC1Sb and LC2S), as well as with the whole myosin, from hind-limb slow-twitch muscle. The immunoreactivity of myosins with a truly mixed light-chain pattern (e.g. vastus lateralis and gastrocnemius) correlated with percentage content of fast-twitch-muscle-type light chains. A more extensive immunoreactivity was observed with diaphragm and masseter myosins, which were also characterized, respectively, by a relative or absolute deficiency of LC1Sa light chain. Furthermore, it was found that the LC1Sb light chain of masseter myosin is antigenically different from its slow-twitch-muscle myosin analogue, and is immunologically related to the LC1F light chain. Rabbit masseter muscle from its metabolic and physiological properties and the content, activity and immunological properties of sarcoplasmic-reticulum adenosine triphosphatase, is classified as a red, predominantly fast-twitch, muscle. Therefore our results suggest that the two antigenically different iso-forms of LC1Sb light chain are associated with the myosins of fast-twitch red and slow-twitch red fibres respectively.

Animals↗

PubMed1982

Biochemical heterogeneity of skeletal-muscle microsomal membranes. Membrane origin, membrane specificity and fibre types.

1. Microsomes were isolated from rabbit fast-twitch and slow-twitch muscle and were separated into heavy and light fractions by centrifugation in a linear (0.3-2m) sucrose density gradient. The membrane origin of microsomal vesicles was investigated by studying biochemical markers of the sarcoplasmic-reticulum membranes and of surface and T-tubular membranes, as well as their freeze-fracture properties. 2. Polyacrylamide-gel electrophoresis showed differences in the Ca(2+)-dependent ATPase/calsequestrin ratio between heavy and light fractions, which were apparently consistent with their respective origin from cisternal and longitudinal sarcoplasmic reticulum, as well as unrelated differences, such as peptides specific to slow-muscle microsomes (mol.wts. 76000, 60000, 56000 and 45000). 3. Freeze-fracture electron microscopy of muscle microsomes demonstrated that vesicles truly derived from the sarcoplasmic reticulum, with an average density of 9nm particles on the concave face of about 3000/mum(2) for both fast and slow muscle, were admixed with vesicles with particle densities below 1000/mum(2). 4. As determined in the light fractions, the sarcoplasmic-reticulum vesicles accounted for 84% and 57% of the total number of microsomal vesicles, for fast and slow muscle respectively. These values agreed closely with the percentage values of Ca(2+)-dependent ATPase protein obtained by gel densitometry. 5. The T-tubular origin of vesicles with a smooth concave fracture face in slow-muscle microsomes is supported by their relative high content in total phospholipid and cholesterol, compared with the microsomes of fast muscle, and by other correlative data, such as the presence of (Na(+)+K(+))-dependent ATPase activity and of low amounts of Na(+)-dependent membrane phosphorylation. 6. Among intrinsic sarcoplasmic-reticulum membrane proteins, a proteolipid of mol.wt. 12000 is shown to be identical in the microsomes of both fast and slow muscle and the Ca(2+)-dependent ATPase to be antigenically and catalytically different, though electrophoretically homogeneous. 7. Basal Mg(2+)-activated ATPase activity was found to be high in light microsomes from slow muscle, but its identification with an enzyme different from the Ca(2+)-dependent ATPase is still not conclusive. 8. Enzyme proteins that are suggested to be specific to slow-muscle longitudinal sarcoplasmic reticulum are the flavoprotein NADH:cytochrome b(5) reductase (mol.wt. 32000), cytochrome b(5) (mol.wt. 17000) and the stearoyl-CoA desaturase, though essentially by criteria of plausibility.

Animals↗

PubMed1982

Transitions in membrane composition during postnatal development of rabbit fast muscle.

Early postnatal changes (4-5 days to 15 days after birth) in the biochemical composition of microsomes were investigated in rabbit skeletal muscles destined to become fast-twitch muscles. During this period, a steady decrease in the microsomal content of cholesterol and of ouabain-sensitive Na + /K + -ATPase activity, as well as a decrease in protein electrophoretic components in the 80 000-70 000 molecular weight range, were observed. These changes are probably due to a diminishing yield of microsomal membranes derived from T-tubules, as the age of the animals increases, and are indicated from a knowledge of the mixed composition of muscle microsomes and previous biochemical data on isolated T-tubules. The content of cytochrome b5, which was found to be high in muscle microsomes of newborn animals, decreased strikingly as the amount of membrane-bound Ca2 + -ATPase protein increased, with a crossing-over point at about 7-10 days after birth. These changes, possibly corresponding to a transition from precursor sarcoplasmic reticulum (SR) to mature SR, were found to be temporally correlated with changes in [3H] alpha-tocopherol binding ability of the microsomes and in the mitochondrial content of glycerol phosphate dehydrogenase. At the same critical periods, coincident with the onset of motile activity, the immunological cross-reactivity of the Ca2 + -ATPase protein of microsomal vesicles, with antibody specific for the Ca2 + -ATPase of adult fast SR, was found to increase markedly, as tested by competitive enzyme-linked immunosorbent assay (ELISA). The immunological data are consistent with data in the literature demonstrating an increase in the concentration of Ca2 + -ATPase molecules in the SR membranes during ontogenic development. Both these data and catalytic data, however, suggest that the Ca2 + -ATPase protein is present in the same form in the SR of immature and of adult fast muscle and, in an antigenically different form, in slow muscle SR.

Animals↗

PubMed1982

Fast to slow change of myosin in nemaline myopathy: electrophoretic and immunologic evidence.

Muscle biopsies from two familial and one sporadic case with congenital nemaline myopathy and seven healthy family members were examined for myosin composition. Myosin was characterized with respect to light chain (LC) composition by one-dimensional and two-dimensional electrophoresis, and by immunologic methods (enzyme-linked immunosorbent assay [ELISA]), using specific antibody for rabbit fast myosin LCl (LC1F). Type I fiber predominance was associated with the substitution of a hybrid, predominantly "slow" to a virtually pure "slow" myosin LC pattern for the "mixed" pattern found with myosin of normal muscle. Muscle myosin from the relatives had apparently normal light chain composition.

Adult↗

PubMed1981

Polymorphism of sarcoplasmic-reticulum adenosine triphosphatase of rabbit skeletal muscle.

Antibody was raised in chickens against purified sarcoplasmic-reticulum Ca2+-activated ATPase (Ca2+-ATPase). The immunological relationship between the Ca2+-ATPase of fast-muscle and slow-muscle sarcoplasmic reticulum was investigated by a one-step and a two-step competitive enzyme-linked immunosorbent assay (ELISA). The results show marked antigenic differences between the membrane-bound Ca2+-ATPase of the sarcoplasmic-reticulum vesicles from fast muscle and slow muscle, beside differences in the membrane content of ATPase protein.

Animals↗

PubMed1981

Characterization of human muscle myosins with respect to the light chains.

Isolated myosins from human predominantly fast and slow muscles, human neonatal and foetal muscle were examined for light chain composition by one- and two-dimensional electrophoresis. The LC1F, LC2F and LC3F light chains were identical with their counterparts from rabbit fast myosin. Human LC1S was identified by correlative criteria as a single component having a molecular weight slightly lower than, but an electric charge similar to, that of rabbit LC1Sb. Consequently, human LC1S appears to be much less heterogeneous relative to LC1F than is the case with other mammalian species. A high immunological cross-reactivity was likewise observed, with antibody specific to rabbit LC1F, between the isolated myosins from several human mixed muscles and rabbit fast myosin, though reactivity was highest with foetal myosin (having a pure-fast-light-chain pattern).

Animals↗

PubMed1981

Electron spin resonance of growing normal and virus-transformed cells.

A g = 2.003 ESR signal, attributed to a free radical localized in HeLa cell nuclei and mitochondria but absent in membranes and cytoplasm, has been studied as a function of the culture growth cycle in normal (NRK and 3T3) and virus transformed (NRK/RSV and 3T3/SV40) cells. For both these cell pairs, the signal is higher during the "lag" stage and lower during the "growth" stage. The average specific intensity of the signal in normal cells is about twice that in virus-transformed cells. However, the maximal point of resonance during the lag state is higher in transformed cells than in normal ones. The lag stage in NRK and NRK/RSV cells is much longer than in 3T3 and 3T3/SV40 cells, while the maximal value of the g = 2.003 ESR signal occurs, early in the lag stage of 3T3 and 3T3/SV40 cells and late in the lag stage of NRK and NRK/RSV cells.

Animals↗

PubMed1980

Organization, replication and modification of the human genome: synthesis and methylation of palindromic, repeated and unique HeLa nDNA sequences during the S-phase.

At least five supermethylated nDNA families have been found in HeLa cells. The concentration of 5-methylcytosine increases on palindromes from 1 to 5 hours during the nDNA duplicative phase, decreasing again at the 6th hour of S. The early S-phase involves the accumulation of 5-methylcytosines on the nDNA sequences reassociating near a Cot = 3 x 10(-1). Late S involves increment of this base on the sequences which reassociate near a Cot = 9 x 10(-1) and Cot = 7 x 10(1). The unique sequences show a moderate methylation from 3 to 6 hours of S. This information shows that genes are methylated with an order during the S-phase in a system in which nDNA organization, modification and replication appear to be severely subordinated one to another. The possible role of methylation of foldback nDNA in regulation of transcription during the eukaryotic cell life cycle is discussed.

5-Methylcytosine↗

PubMed1979

Organization, replication and modification of the human genome: temporal order of synthesis and methylation of two classes of HeLa nDNA separated in Ag+--Cs2-SO4 gradients.

During the HeLa S-phase, DNA was methylated, at 1-hr intervals in isolated nuclei and fractionated in Ag+-Cs2SO4 gradients providing a heavy GC-rich peak and a main light AT-rich peak. Both size and specific methylation of these peaks changed during the nDNA duplicative phase. Replication of the heavy GC-rich nDNA fraction, which contains genes for ribosomal RNA, occurred in early S; in contrast, replication of the main AT-rich nDNA fraction was maximal in late S. Concomitantly, specific methylation of the GC-rich nDNA was maximal in the first part of S, while that of the AT-rich nDNA was maximal in the second part of S. This suggested that genes are replicated and methylated with order during the S-phase.

Cell Cycle↗

PubMed1978

Organization, replication and modification of the human genome: differential methylation of two classes of HeLa nuclear DNA separated on Ag+--Cs2SO4 gradients.

HeLa nuclear DNA sediments as a single peak, in neutral CsCl, while it is separated in a heavier and a lighter components, in alkaline Ag+--Cs2SO4. The heavy fraction, on the average, represents about 20% of the total DNA. CsCl analytical ultracentrifugation shows that heavy DNA bands at 1.715 g/cm3 and contains 53% GC (10% of the total GC), whereas light DNA bands at 1.703 g/cm3 and contains 40% GC (32% of the total GC). Coherently, Tm values in 0.1 x SSC are 82.5 degrees C, for heavy DNA, and 72.5 degrees C, for light DNA. After treatment with [3H-methyl-S-adenosyl-L-methionine in isolated nuclei, the concentration of labelled 5-methylcytosine was found to be highest in the more dense regions of the heavy peak and in the less dense regions of the light peak. Exposure to ultrasound modifies the quantitative relationship of the two peaks and improves the separation of supermethylated AT- and GC-rich DNAs. Four possible triplets as sites for DNA-methylase recognition are discussed.

Cell Nucleus↗