Interaction of N-alkylanthracyclines with lipid bilayers: correlations between partition coefficients, lipid phase distributions and thermotropic behavior

The thermotropic behavior of multilamellar vesicles of dipalmitoylphosphatidylcholine (DPPC), or of DPPC in admixture with cardiolipin or cholesterol, in the presence of various N-alkyl derivatives of both adriamycin and adriamycin-14-valerate has been investigated by high sensitivity differential scanning calorimetry. The analogues, particularly the 14-valerate derivatives, which were most lipophilic as judged by their lipid/buffer, and to a lesser extent by their octanol/buffer, partition coefficients, were the most effective in depressing the tm of the investigated lipids; correlations, however, were not absolute. Other factors, such as the distribution of the drugs between the solid and liquid-crystalline phases of the bilayer, were also important to the observed membrane perturbations. With all anthracyclines, however, no major changes in the transition enthalpy were observed. In the case of vesicles prepared from pure DPPC, curve fitting analysis based on ideal solution theory (J.M. Sturtevant (1984) Proc. Natl. Acad. Sci. USA 81, 1398-1400) applied at relatively low drug concentrations where single peak transitions were produced, adequately described the differential scanning calorimetric results. At high drug concentrations, however, the presence of multi-peak transitions were indicative of non-ideality.     

A scanning calorimetric study of the interaction of anthracyclines with neutral and acidic phospholipids alone and in binary mixtures

High sensitivity differential scanning calorimetry was employed to study the thermotropic behavior of multilamellar vesicles of neutral and acidic phospholipids and binary mixtures thereof in the presence of anthracycline antibiotics. Adriamycin and its lipophilic analogue, N-trifluoroacetyladriamycin-14-valerate (AD32) were investigated and compared to chlorpromazine and quinidine with respect to their ability to affect the pretransition and the main transition of the phospholipids suspended in physiological buffer. With liposomes of neutral dipalmitoylphosphatidylcholine the observed effects paralleled to some extent the corresponding octanol/buffer partition coefficients, with adriamycin being the least effective. Calorimetric measurements on liposomes prepared from pure dipalmitoylphosphatidylglycerol or from binary mixtures of dipalmitoylphosphatidylglycerol and dipalmitoylphosphatidylcholine showed that modulation of bilayer properties by adriamycin was greatly enhanced in the presence of negatively charged lipid headgroups presumably as a result of electrostatic interactions. AD32 interacted differently from adriamycin with the acidic bilayers at low drug concentrations, in a manner similar to that of its interaction with neutral bilayers. At high drug concentrations both adriamycin and AD32 produced transitions with multiple peaks not exhibited by chlorpromazine and quinidine which may be the result of a specific association of the anthracyclines with dipalmitoylphosphatidylglycerol. All four drugs produced only minor changes in the enthalpy of the main transition of the investigated lipids. The present findings are discussed in terms of their possible physiological relevance.     

Sedimentation field flow fractionation of fused unilamellar vesicles: comparison with electron microscopy and gel filtration

Sedimentation field flow fractionation (SF3), a relatively new instrumentation methodology for separating particles according to size, has been used to monitor changes in vesicle size during the formation of fused unilamellar vesicles of dipalmitoylphosphatidylcholine. The fusion of 500-A small unilamellar vesicles to 700-A large unilamellar vesicles (LUVs) and the slower conversion to 950-A LUVs at 4 degrees C was readily monitored by SF3 over the time course of a month. Changes in the physical state of these vesicles as a result of structural reorganizations were characterized by high sensitivity differential scanning calorimetry. The advantages and limitations of SF3 are discussed and correlated with electron microscopy and gel filtration.     

Modeling, optimization, and computer control of the cephalosporin C fermentation process

In this study, a cephalosporin C producing strain, Cephalosporium acremonium (ATCC 36225), was chosen to determine the optimal conditions that maximize antibiotic production in a mixed substrate of glucose and sucrose. A model for cell growth and cephalosporin C production at different pH and temperature was developed and the associated parameters were evaluated experimentally. Pontryagin's maximum principle, in conjunction with the model, was used to predict the optimal temperature and pH control profiles to maximize the production of antibiotic.     

Micellization of fatty acyl-CoA mixtures and its relevance to the fatty acyl selectivity of acyltransferases

The critical micelle concentrations (CMCs) of palmitoyl-CoA/stearoyl-CoA and palmitoyl-CoA/oleoyl-CoA mixtures in 0.050 M KPi, pH 7.4, a buffer used in enzymatic studies, were determined by fluorescence. Mixed micelle solution theory, analogous to the thermodynamic treatment of vapor pressure, was applied to calculate monomer and micelle compositions. The behavior of the palmitoyl-CoA/stearoyl-CoA mixture is ideal, while the palmitoyl-CoA/oleoyl-CoA mixture, although not exhibiting ideal behavior, can be fitted reasonably well by nonideal theory. In both mixtures, selective micellization takes place and, unlike the case of pure fatty acyl-CoAs, above the CMC of the mixtures the concentration of molecules free in solution is strongly dependent upon total concentration. The information derived from the present physical studies becomes important in enzymatic studies with membrane-bound acyltransferases, where selectivity toward various fatty acyl donors, presented as binary mixtures, is frequently observed.     

Immobilized flounder muscle glyceraldehyde 3-phosphate dehydrogenase

Summary Partially purified flounder muscle (Pseudopleuronectus americanus) glyceraldehyde 3-phosphate dehydrogenase was immobilized on cyanogen bromide-activated Sepharose. The catalytic properties of the immobilized preparation were studied to determine if immobilization alters the kinetic properties of the native holoenzyme. The results indicate that the pH activity profile of immobilized glyceraldehyde 3-phosphate dehydrogenase did not differ from that of the native enzyme. The Michaelis constants (Km) for NAD and glyceraldehyde 3-phosphate were somewhat altered. The enzyme stability toward various inactivation treatments in the presence and absence of NAD was characterized and compared to that of he native enzyme. When either form of the enzyme was incubated with urea at concentrations greater than 2m, inactivation occurred very rapidly. Incubation in 0.1% trypsin for 60 minutes decreased the activity of immobilized glyceraldehyde 3-phosphate dehydrogenase by 45% and of the native soluble enzyme by 70%. The immobilized enzyme also exhibited considerably more stability than the native soluble enzyme when exposed to a temperature of 50° or to 20 mm ATP. In all cases NAD either greatly reduced the rate of inactivation or completely protected the enzyme from inactivation.     

Detection of a novel nonsense mutation and an intragenic polymorphism in the PKD1 gene of a Cypriot family with autosomal dominant polycystic kidney disease

Mutations in the PKD1 gene on the short arm of chromosome 16 account for 85%–90% of polycystic kidney disease patients in the Caucasian population. After the recent characterization of the gene, we started a search for mutations in its 3′-end unique portion in Cypriot patients, by using the method of single-strand conformation polymorphism (SSCP). In one large family, we identified a nucleotide substitution at position 12 258 of the cDNA; this substitutes cysteine-4086 by a premature termination codon (C4086X). It has been inherited by every affected family member but not by unaffected members, nor by patients from 13 other Cypriot families. A new polymerase chain reaction (PCR) primer has been designed to engineer a novel DdeI recognition site upon PCR amplification, thereby allowing easy detection of the mutation by PCR-restriction digestion. The premature STOP codon is expected to remove 217 residues from the putative C-terminal intracellular domain of the gene product, polycystin and thus identifies this part as being critical to the production of the disease phenotype, possibly by interfering with the transmission of signals from the extracellular matrix to the cytoplasm. We also describe the identification of the first polymorphism within the encoding region of the gene. It is at alanine 4091, which is encoded by either GCA or GCG. With a heterozygosity of 35%, it should be extremely useful in informative families, especially because the gene lies in an unstable region and is prone to rearrangements. This polymorphism is readily detectable by PCR-restriction digestion with Bsp1286I. Received: 19 February 1996 / Revised: 20 April 1996     

Search for potential markers for prostate cancer diagnosis, prognosis and treatment in clinical tissue specimens using amine-specific isobaric tagging (iTRAQ) with two-dimensional liquid chromatography and tandem mass spectrometry

This study aimed to identify candidate new diagnosis and prognosis markers and medicinal targets of prostate cancer (PCa), using state of the art proteomics. A total of 20 prostate tissue specimens from 10 patients with benign prostatic hyperplasia (BPH) and 10 with PCa (Tumour Node Metastasis [TNM] stage T1-T3) were analyzed by isobaric stable isotope labeling (iTRAQ) and two-dimensional liquid chromatography-tandem mass spectrometry (2DLC-MS/MS) approaches using a hybrid quadrupole time-of-flight system (QqTOF). The study resulted in the reproducible identification of 825 nonredundant gene products (p < or = 0.05) of which 30 exhibited up-regulation (> or =2-fold) and another 35 exhibited down-regulation (< or =0.5-fold) between the BPH and PCa specimens constituting a major contribution toward their global proteomic assessment. Selected findings were confirmed by immunohistochemical analysis of prostate tissue specimens. The proteins determined support existing knowledge and uncover novel and promising PCa biomarkers. The PCa proteome found can serve as a useful aid for the identification of improved diagnostic and prognostic markers and ultimately novel chemopreventive and therapeutic targets.     

Transverse location of anthracyclines in lipid bilayers. Paramagnetic quenching studies

Quenching of anthracycline fluorescence by a series of spin-labeled fatty acids was used to probe the transverse location of the drug in phosphatidylcholine bilayers in the form of small unilamellar vesicles. Stern-Volmer plots of the quenching data indicate that the fluorophore moiety of the anthracycline is intercalated into the hydrocarbon region of the bilayer, with deeper penetration observed in fluid-phase than in solid-phase vesicles. 31P-NMR parameters (T1 and nuclear Overhauser enhancement (NOE] are unaffected by the presence of drug, consistent with a binding site removed from the interfacial region. Comparison of intensity (F0/F) plots with lifetime (tau 0/tau) data shows that the predominant mechanism of anthracycline quenching by membrane-bound nitroxides is static. Since the membrane-bound drug is also accessible to quenching by I-, the binding site in the membrane must create a channel which is accessible to solvent. Two other fluorescent probes, 12-(9-anthroyloxy)stearate (12-AS) and diphenylhexatriene (DPH), were employed to confirm the results obtained with the anthracyclines, giving quenching data representative of their location in the bilayer.