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S Orlowski

Publications and source records attributed to S Orlowski.

47 records · Page 3Linked to original sources
PubMed1989

Fast kinetics of calcium release induced by myo-inositol trisphosphate in permeabilized rat hepatocytes.

We used a stopped-flow method for determining the kinetic properties (between 10 ms and 10 s) of the Ca2+ release induced by inositol 1,4,5-trisphosphate (InsP3) in saponin-treated rat hepatocytes. Preliminary experiments ensured that the indicator was able to monitor rapid changes in free Ca2+ reliably. At 20 degrees C, a maximally efficient concentration of 10 microM InsP3 released Ca2+ with a half-time of 150-300 ms, the initial rate being about 1-2 nmol of Ca2+/mg of cell protein/s. The delay between the addition of 10 microM InsP3 and the onset of Ca2+ release was shorter than 20 ms, suggesting that the opening process of Ca2+ channels after binding of InsP3 to receptors is completed within a few milliseconds. Half-maximal initial rates for Ca2+ release occurred at about 1 microM InsP3 (Hill index was 1.6). The resulting Ca2+ efflux had a moderate temperature dependence. It could not be fitted to a single exponential. After low speed centrifugation of saponin-treated cells (1000 x g for 1 min), part of the InsP3-sensitive Ca2+ pool was recovered in the cell-free supernatant fraction, suggesting that the response to InsP3 arises from a vesicular fraction which may diffuse from the saponin-treated cells into the medium.

Aminoquinolines↗
PubMed1989

Detergent structure and associated lipid as determinants in the stabilization of solubilized Ca2+-ATPase from sarcoplasmic reticulum.

The properties of detergents required to substitute the lipid environment of sarcoplasmic reticulum Ca2+-ATPase with retention of good functional properties were determined by the use of a large number of diverse detergents and delipidated enzyme. Detergents having an intermediate chain length (approximately equal to C12) and a polyoxyethylene glycol or carbohydrate polar group were optimal for Ca2+-ATPase function and stabilization, while detergents with short alkyl chain (C8) or bulky head groups and many zwitterionic detergents led to rapid inactivation. Under optimal conditions (including solubilization in the E1 state), stability of delipidated Ca2+-ATPase approximated that obtained by solubilization of Ca2+-ATPase with a layer of bound lipid. Some detergents (in particular long chain members of the Tween family) were characterized by an inadequate interaction with delipidated Ca2+-ATPase, resulting in biphasic inactivation. According to analytical ultracentrifugation and high performance liquid chromatography experiments, the rapid and slow components of biphasic inactivation were due to the formation of monomeric and oligomeric Ca2+-ATPase, respectively. It is concluded that both hydrophobic and polar interactions are important for the detergent effect and that solubilizing detergents of intermediate and short chain length may be bound as a monolayer, differently than the membrane lipid. Long chain detergents cause protein aggregation and, despite their resemblance to natural lipids, are inferior in their activity-retaining properties. The previous use of such detergents to prepare oligomeric Ca2+-ATPase with long term retention of activity (cf. Møller, J. V., Anderson, J. P., and le Maire, M. (1988) Methods Enzymol. 157, 261-270) is shown to depend on the presence of residual lipid in these preparations.

Animals↗
PubMed1989

Membrane solubilization by detergent: use of brominated phospholipids to evaluate the detergent-induced changes in Ca2+-ATPase/lipid interaction.

The solubilization and delipidation of sarcoplasmic reticulum Ca2+-ATPase by different nonionic detergents were measured from changes in turbidity and recovery of intrinsic fluorescence of reconstituted ATPase in which tryptophan residues had been quenched by replacement of endogenous phospholipids with brominated phospholipids. It was found that incorporation of C12E8 or dodecyl maltoside (DM) at low concentrations in the membrane, resulting in membrane "perturbation" without solubilization, displaced a few of the phospholipids in contact with the protein; perturbation was evidenced by a parallel drop in ATPase activity. As a result of further detergent addition leading to solubilization, the tendency toward delipidation of the immediate environment of the protein was stopped, and recovery of enzyme activity was observed, suggesting reorganization of phospholipid and detergent molecules in the solubilized ternary complex, as compared to the perturbed membrane. After further additions of C12E8 or DM to the already solubilized membrane, the protein again experienced progressive delipidation which was only completed at a detergent concentration about 100-fold higher than that necessary for solubilization. Delipidation was correlated with a decrease in enzyme activity toward a level similar to that observed during perturbation. On the other hand, Tween 80, Tween 20, and Lubrol WX failed to solubilize SR membranes and to induce further ATPase delipidation when added after preliminary SR solubilization by C12E8 or dodecyl maltoside. For Tween 80, this can be related to an inability to solubilize pure lipid membrane; in contrast, Tween 20 and Lubrol WX were able to solubilize liposomes but not efficiently to solubilize SR membranes. In all three cases, insertion of the detergent in SR membranes is, however, demonstrated by perturbation of enzyme activity. Correlation between detergent structure and ability to solubilize and delipidate the ATPase suggests that one parameter impeding ATPase solubilization might be the presence of a bulky detergent polar headgroup, which could not fit close to the protein surface. We also conclude that in the active protein/detergent/lipid ternary complexes, solubilized by C12E8 or dodecyl maltoside, most phospholipids remain closely associated with the ATPase hydrophobic surface as in the membranous form. Binding of only a few detergent molecules on this hydrophobic surface may be sufficient for inhibition of ATPase activity observed at high ATP concentration, both during perturbation and in the completely delipidated, solubilized protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1988

Transient electropermeabilization of cells in culture. Increase of the cytotoxicity of anticancer drugs.

The electropermeabilization (EPN) of living cells allows the uptake of non-permeant molecules and can reveal their potential activity on cells without the constraints of the plasma membrane crossing. We decided to compare the cytotoxicity of some anticancer drugs on electropermeabilized (EP) and non-permeabilized (NEP) cultured DC-3F cells exposed to the drugs for a short time. After EPN, the increase in cytotoxicity varies between 1 and more than 700 times, depending on the usual cell uptake pathway of a given drug. The most relevant increase of toxicity was observed with molecules such as netropsin (200-fold) and bleomycin (700-fold) which in ordinary conditions weakly diffuse through the plasma membrane. Only a 3-5-fold increase of the cytotoxicity was observed with lipophilic drugs able to rapidly diffuse through the plasma membrane (actinomycin D, NMHE) both in the case of drug-sensitive and resistant cell strains. This increased toxicity is clearly related to a facilitated uptake because, after electropermeabilization, the effects of melphalan (a drug which enters intact cells via leucine transporters) are not modulated by the external leucine concentration. Thus, EPN enables us to reveal the intrinsic toxicity of hydrophilic molecules which have a limited access to their intracellular targets. We propose that EPN can be used as a novel screening procedure of new cytotoxic molecules which could be modified thereafter in order to facilitate their cellular uptake.

Animals↗
PubMed1988

Phosphoenzymes formed from Mg.ATP and Ca.ATP during pre-steady state kinetics of sarcoplasmic reticulum ATPase.

We have investigated here the pre-steady state kinetics of sarcoplasmic reticulum ATPase incubated under conditions where significant amounts of Mg.ATP and Ca.ATP coexist, both of them being substrates for the ATPase. We confirmed that these two substrates are independently hydrolyzed by the ATPase, which thus apparently catalyzes Pi production by two simultaneous and separate pathways. External calcium (or the Ca2+/Mg2+ ratio) determines the extent to which Ca2+ or Mg2+ is bound at the phosphorylation site, while internal calcium controls the rate of processing of both the slow, calcium-containing and the fast, magnesium-containing phosphoenzyme. Time-dependent binding of calcium at the catalytic site is correlated with the observed burst of Pi liberation, which therefore results from reequilibration during pre-steady state of magnesium- and calcium-containing phosphoenzyme pools. Independently of direct exchange of metal at the catalytic site, ADP produced by the hydrolysis reaction contributes to reequilibration of these pools through reversal of phosphorylation by the ATP-ADP exchange pathway.

Adenosine Diphosphate↗
PubMed1988

ATP regulation of sarcoplasmic reticulum Ca2+-ATPase. Metal-free ATP and 8-bromo-ATP bind with high affinity to the catalytic site of phosphorylated ATPase and accelerate dephosphorylation.

To localize and characterize the regulatory nucleotide site of skeletal muscle sarcoplasmic reticulum Ca2+-ATPase, we have investigated the effects of ADP, ATP, and analogues of these nucleotides on the rate of dephosphorylation of both native ATPase and ATPase modified with fluorescein 5'-isothiocyanate (FITC), a reagent which hinders access of nucleotides to the ATPase catalytic site without affecting phosphorylation from Pi. Dephosphorylation of the phosphoenzyme formed from Pi was monitored by rapid filtration or stopped-flow fluorescence, mostly at 20 degrees C, pH 6.0, and in the absence of potassium. Fluorescence measurements were made possible through the use of 8-bromo-ATP, which selectively quenched certain tryptophan residues of the ATPase, thereby allowing the intrinsic fluorescence changes associated with dephosphorylation to be measured in the presence of bound nucleotide. ATP, 8-bromo-ATP, and trinitrophenyladenosine diand triphosphate, but not ADP, enhanced the rate of dephosphorylation of native ATPase 2-3-fold when added in the absence of divalent cations. Millimolar concentrations of Mg2+ eliminated the accelerating effects. Acceleration in the absence of Mg2+ was observed at relatively low concentrations of ATP and 8-bromo-ATP (0.01-0.1 mM) and binding of metal-free ATP and ADP, but not Mg.ATP, to the phosphoenzyme in this concentration range was demonstrated directly. Modification of the ATPase with FITC blocked nucleotide binding in the submillimolar concentration range and eliminated the nucleotide-induced acceleration of dephosphorylation. These results show that dephosphorylation, under these conditions, is regulated by ATP but not by Mg.ATP or ADP, and that the catalytic site is the locus of this "regulatory" ATP binding site.

Adenosine Diphosphate↗
PubMed1976

Stability characteristics of freeze-dried human live virus vaccines.

Freeze-drying (lyophilization) of biologically active materials such as human live virus vaccines is an accepted method of preserving potency. Since freeze-drying does not assure total stability, a formal system of monitoring stability, i.e., potency, has been established. Two experimental desings are utilized: (1) Long term studies to duplicate normal use storage at -20 degrees C and 2-8 degrees C and (2) Accelerated studies which predict long term stability through regression analysis using the Arrhenius equation. Data summarizing stability experience over a long period of study will be presented.

Drug Stability↗
PubMedDate not supplied

Multiple recognition of various amphiphilic molecules by the multidrug resistance P-glycoprotein: molecular mechanisms and pharmacological consequences coming from functional interactions between various drugs.

P-glycoprotein (P-gp) is an active, ATP-dependent plasma membrane transporter which is responsible for the expulsion of various cytotoxic drugs with different chemical structures out of resistant (MDR) cells. It is also capable of transporting a number of other amphiphilic molecules, the so-called MDR-reversing agents, which belong to a very broad variety of chemical families. Moreover, P-gp can also play a role in steroid secretion and cellular detoxification by transporting various other substrates. In this review, we address the problem of the multiple recognition by P-gp of such a large number of amphiphilic molecules. This is both (i) from a basic viewpoint in order to discuss the underlying molecular mechanisms explaining how the general rule of substrate-enzyme specificity can be violated, and (ii) from a more applied pharmacological viewpoint to show in detail how the interaction of various drugs with P-gp leads to important consequences in terms of the relative effects of these drugs in the anticancer chemotherapy context, as well as for their pharmacokinetic distributions in the whole organism, rationalizing possible adverse drug reactions. In particular, we will present evidence that, independently of the technique used, the mutual interactions between P-gp transport substrates cannot always be reduced to simple competitive effects.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMedDate not supplied

Electrochemotherapy tumor treatment is improved by interleukin-2 stimulation of the host's defenses.

We have recently described a new antitumor treatment, electrochemotherapy (ECT), based on the large local potentiation of the effects of the chemotherapeutic agent bleomycin (BLM) by electric pulses (EP) delivered at the tumor site. We demonstrate here that the host's immune response participates in cure achievement. We obtain an increase of the rate of completely cured animals by injecting mice with interleukin-2 (IL-2).

Animals↗