On the functional ordering of biomembranes : Implications for drug binding

Cells are packed with membrane structures, defining the inside and outside, and the different subcellular compartments. These membranes consisting mainly of phospholipids have a variety of functions in addition to providing a permeability barrier for various compounds. These functions involve cellular signaling, where lipids can act as second messengers, or direct regulation of membrane associating proteins. The first part of this study focuses on relating some of the physicochemical properties of membrane lipids to the association of drug compounds to membranes. A fluorescence based method is described allowing for determination of the membrane association of drugs. This method was subsequently applied to a novel drug, siramesine, previously shown to have anti-cancer activity. Siramesine was found to associate with anionic lipids. Especially interesting is its strong affinity for a second messenger lipid phosphatidic acid. This is the first example of a small molecule drug compound specifically interacting with a cellular lipid. Phosphatidic acid in cells is required for the activation of many signaling pathways mediating growth and proliferation. This provides an intriguing possibility for a simple molecular mechanism of the observed anti-cancer activity of siramesine. In the second part the thermal behavior and self assembly of charged and uncharged membrane assemblies was studied. Strong inter-lamellar co-operativity was observed for multilamellar DPPC vesicles using fluorescence techniques together with calorimetry. The commonly used membrane models, large unilamellar vesicles (LUV) and multilamellar vesicles (MLV) were found to possess different biophysical properties as interlamellar interactions of MLVs drive segregation of a pyrene labeled lipid analogue into clusters. The effect of a counter-ion lattice on the self assembly of a cationic gemini surfactant was studied. The presence of NaCl strongly influenced the thermal phase behavior of M-1 vesicles, causing formation of giant vesicles upon exceeding a phase transition temperature, followed by a subsequent transition into a more homogenous dispersion. Understanding the underlying biophysical aspects of cellular membranes is of fundamental importance as the complex picture of the structure and function of cells is evolving. Many of the cellular reactions take place on membranes and membranes are known to regulate the activity of many peripheral and intergral membrane associating proteins. From the point of view of drug design and gene technology, membranes can provide an interesting target for future development of drugs, but also a vehicle sensitive for environmental changes allowing for encapsulating drugs and targeting them to the desired site of action.

Molecular Details of Serum Resistance of Yersinia enterocolitica

In complement activation, Factor H (FH) and C4b-binding protein (C4bp) are the key regulators that prevent the complement cascade from attacking host tissues. Some bacteria may bind and deposit these regulators on their own surfaces and thus provide themselves with an efficient means to avoid complement activation. In consequence, bacteria resist complement-mediated lysis and opsonin-dependent phagocytosis. This study has demonstrated that Y. enterocolitica, similar to many other pathogens, recruits both FH and C4bp to its surface to ensure protection against the complement-mediated killing. YadA and Ail, the most crucial serum resistance factors of Y.enterocolitica, mediate the binding of FH and C4bp. FH - YadA interaction involves multiple higher structural motifs on the YadA stalk and the short consensus repeats (SCRs) of the entire polypeptide chain of FH. The Ail binding site on FH has been located to SCRs 6 and 7. The binding site for FH on Ail, however, remains undetermined. Both YadA- and Ail-bound regulators display full cofactor activity for FI-mediated cleavage of C3b/C4b. FH/C4bp-binding characteristics do, however, differ between YadA and Ail. In addition, Ail captures the regulators only in the absence of blocking lipopolysaccharide O-antigen and outer core, whereas YadA binds FH/C4bp independent of the presence of other surface factors Independent of mode of binding, however, YadA and Ail provide Y. enterocolitica a means to avoid complement-mediated lysis, enhancing chances for the bacteria to survive in the host during various phases of infection.

Molecular pathogenesis of human parechovirus 1 infection

The Parechoviruses (HPEV) belong to the family Picornaviridae of positive-stranded RNA viruses. Although the parechovirus genome shares the general properties of other picornaviruses, the genus has several unique features when compared to other family members. We found that HPEV1 attaches to αv integrins on the cell surface and is internalized through the clathrin-mediated endocytic pathway. During he course of the infection, the Golgi was found to disintegrate and the ER membranes to swell and loose their ribosomes. The replication of HPEV1 was found to take place on small clusters of vesicles which contained the trans-Golgi marker GalT as well as the viral non-structural 2C protein. 2C was additionally found on stretches of modified ER-membranes, seemingly not involved in RNA replication. The viral non-structural 2A and 2C proteins were studied in further detail and were found to display several interesting features. The 2A protein was found to be a RNA-binding protein that preferably binds to positive sense 3 UTR RNA. It was found to bind also duplex RNA containing 3 UTR(+)-3 UTR(-), but not other dsRNA molecules studied. Mutagenesis revealed that the N-terminal basic-rich region as well as the C-terminus, are important for RNA-binding. The 2C protein on the other hand, was found to have both ATP-diphosphohydrolase and AMP kinase activities. Neither dATP nor other NTP:s were suitable substrates. Furthermore, we found that as a result of theses activities the protein is autophosphorylated. The intracellular changes brought about by the individual HPEV1 non-structural proteins were studied through the expression of fusion proteins. None of the proteins expressed were able to induce membrane changes similar to those seen during HPEV1 infection. However, the 2C protein, which could be found on the surface of lipid droplets but also on diverse intracellular membranes, was partly relocated to viral replication complexes in transfected, superinfected cells. Although Golgi to ER traffic was arrested in HPEV1-infected cells, none of the individually expressed non-structural proteins had any visible effect on the anterograde membrane traffic. Our results suggest that the HPEV1 replication strategy is different from that of many other picornaviruses. Furthermore, this study shows how relatively small differences in genome sequence result in very different intracellular pathology.

Adriamycin affects plasma membrane redox functions

Adriamycin (Doxorubicin) stimulates NADH oxidase activity in liver plasma membrane, but does not cause NADH oxidase activity to appear where it is not initially present, as in erythrocyte membrane. NADH dehydrogenase from rat liver and erythrocyte plasma membranes shows similar adriamycin effects with other electron acceptors. Both NADH ferricyanide reductase and vanadate-stimulated NADH oxidation are inhibited by adriamycin, as is a cyanide insensitive ascorbate oxidase activity, whereas NADH cytochrome c reductase is not affected. The effects may contribute to the growth inhibitory (control) and/or deleterious effects of adriamycin. It is clear that adriamycin effects on the plasma membrane dehydrogenase involve more than a simple catalysis of superoxide formation.

Ionophore-mediated transmembrane movement of divalent cations in small unilamellar liposomes: An evaluation of the chlortetracycline fluorescence technique and correlations with black lipid membrane studies

Conceptual advances in the field of membrane transport have, in the main, utilized artificial membranes, both planar and vesicular. Systems of biological interest,viz., cells and organelles, resemble vesicles in size and geometry. Methods are, therefore, required to extend the results obtained with planar membranes to liposome systems. In this report we present an analysis of a fluorescence technique, using the divalent cation probe chlortetracycline, in small, unilamellar vesicles, for the study of divalent cation fluxes. An ion carrier (X537 A) and a pore former (alamethicin) have been studied. The rate of rise of fluorescence signal and the transmembrane ion gradient have been related to transmembrane current and potential, respectively. A second power dependence of ion conduction-including the electrically silent portion thereof — on X537 A concentration, has been observed. An exponential dependence of ldquocurrentrdquo on ldquotransmembrane potentialrdquo in the case of alamethicin is also confirmed. Possible errors in the technique are discussed.

Zinc, cadmium and lead resistance mechanisms in bacteria and their contribution to biosensing

In bacteria resistance to heavy metals is mainly achieved through active efflux, but also sequestration with proteins or as insoluble compounds is used. Although numerous studies have dealt with zinc, cadmium and lead resistance mechanisms in bacteria, it has still remained unclear how different transporters are integrated into an effective homeostasis/resistance network and whether specific mechanisms for lead sequestration exist. Furthermore, since metals are toxic not only to bacteria but to higher organisms as well, it is important to be able to estimate possible biological effects of heavy metals in the environment. This could be done by determining the bioavailable amount of the metals in the environment with bacterial bioreporters. That is, one can employ bacteria that respond to metal contamination by a measurable signal to assess the property of metals to cross biological membranes and to cause harmful effects in a possibly polluted environment. In this thesis a new lead resistance mechanism is described, interplay between CBA transporters and P-type ATPases in zinc and cadmium resistance is presented and finally the acquired knowledge is used to construct bacterial bioreporters for heavy metals with increased sensitivity and specificity. The new lead resistance model employs a P-type ATPase that removes Pb2+ ions from the cytoplasm and a phosphatase that produces inorganic phosphate for lead sequestration in the periplasm. This was the first study where the molecular mechanism of lead sequestration has been described. Characterization of two P-type ATPases and two CBA transporters showed that resistance mechanisms for Zn2+ and Cd2+ are somewhat different than for Pb2+ as these metals cannot be sequestered as insoluble compounds as easily. Resistance to Zn2+ was conferred merely by the CBA transporter that could export both cytoplasmic and periplasmic ions; whereas, full resistance to Cd2+ required interplay of a P-type ATPase that exported cytoplasmic ions to periplasm and a CBA transporter that further exported periplasmic ions to the outside. The knowledge on functionality of the transporters and metal-inducible promoters was exploited in bioreporter technology. A transporter-deficient bioreporter strain that lacked exporters for Zn2+/Cd2+/Pb2+ could detect up to 45-fold lower metal concentrations than its wild type counterpart due to the accumulation of metals in the cell. The broad specificity issue of bioreporters was overcome by using Zn-specific promoter as a sensor element, thus achieving Zn-specific bioreporter.

A vanadate-stimulated NADH oxidase in erythrocyte membrane generates hydrogen peroxide

Oxidation of NADH by rat erythrocyte plasma membrane was stimulated by about 50-fold on addition of decavanadate, but not other forms of vanadate like orthovanadate, metavanadate aad vanadyl sulphate. The vanadate-stimulated activity was observed only in phosphate buffer while other buffers like Tris, acetate, borate and Hepes were ineffective. Oxygen was consumed during the oxidation of NADH and the products were found to be NAD+ and hydrogen peroxide. The reaction had a stoichiometry of one mole of oxygen consumption and one mole of H2O2 production for every mole of NADH that was oxidized. Superoxide dismutase and manganous inhibited the activity indicating the involvement of superoxide anions. Electron spin resonance in the presence of a spin trap, 5, 5prime-dimethyl pyrroline N-oxide, indicated the presence of superoxide radicals. Electron spin resonance studies also showed the appearance of VIV species by reduction of VV of decavanadate indicating thereby participation of vanadate in the redox reaction. Under the conditions of the assay, vanadate did not stimulate lipid peroxidation in erythrocyte membranes. Extracts from lipid-free preparations of the erythrocyte membrane showed full activity. This ruled out the possibility of oxygen uptake through lipid peroxidation. The vanadate-stimulated NADH oxidation activity could be partially solubilized by treating erythrocyte membranes either with Triton X-100 or sodium cholate. Partially purified enzyme obtained by extraction with cholate and fractionation by ammonium sulphate and DEAE-Sephadex was found to be unstable.

Fluorescent alamethicin fragments A study of membrane activity and aqueous phase aggregation

The linear polypeptide antibiotic alamethicin is known to form channels in artificial lipid membranes. Synthetic 13- and 17-residue alamethicin fragments, labelled with a fluorescent dansyl group at the N-terminus, have been shown to translocate divalent cations across phospholipid membranes and to uncouple oxidative phosphorylation in rat liver mitochondria, in a manner analogous to the parent peptides. From studies of the aqueous phase aggregation behavior of the peptides, as well as their interaction with rat liver mitochondria, it is concluded that the interaction of the peptides with membranes is a complex process, probably involving both aqueous and membrane phase aggregation.

Tracking and identifying wastewater toxicants and assessing their biodegradation products

Screening of wastewater effluents from municipal and industrial wastewater treatment plants with biotests showed that the treated wastewater effluents possess only minor acute toxic properties towards whole organisms (e.g. bacteria, algae, daphnia), if any. In vitro tests (sub-mitochondrial membranes and fish hepatocytes) were generally more susceptible to the effluents. Most of the effluents indicated the presence of hormonally active compounds, as the production of vitellogenin, an egg yolk precursor protein, was induced in fish hepatocytes exposed to wastewater. In addition, indications of slight genotoxic potential was found in one effluent concentrate with a recombinant bacteria test. Reverse electron transport (RET) of mitochondrial membranes was used as a model test to conduct effluent assessment followed by toxicant characterisations and identifications. Using a modified U.S. EPA Toxicity Identification Evaluation Phase I scheme and additional case-specific methods, the main compound in a pulp and paper mill effluent causing RET inhibition was characterised to be an organic, relatively hydrophilic high molecular weight (HMW) compound. The toxicant could be verified as HMW lignin by structural analyses using nuclear magnetic resonance. In the confirmation step commercial and in-house extracted lignin products were used. The possible toxicity related structures were characterised by statistical analysis of the chemical breakdown structures of laboratory-scale pulping and bleaching effluents and the toxicities of these effluents. Finally, the biological degradation of the identified toxicant and other wastewater constituents was evaluated using bioassays in combination with chemical analyses. Biological methods have not been used routinely in establishing effluent discharge limits in Finland. However, the biological effects observed in this study could not have been predicted using only routine physical and chemical effluent monitoring parameters. Therefore chemical parameters cannot be considered to be sufficient in controlling effluent discharges especially in case of unknown, possibly bioaccumulative, compounds that may be present in small concentrations and may cause chronic effects.

Microfiltration in cheese and whey processing

Milk microfiltration (0.05-0.2 um) is a membrane separation technique which divides milk components into casein-enriched and native whey fractions. Hitherto the effect of intensive microfiltration including a diafiltration step for both cheese and whey processing has not been studied. The microfiltration performance of skimmed milk was studied with polymeric and ceramic MF membranes. The changes caused by decreased concentration of milk lactose, whey protein and ash content for cheese milk quality and ripening were studied. The effects of cheese milk modification on the milk coagulation properties, cheese recovery yield, cheese composition, ripening and sensory quality as well as on the whey recovery yield and composition by microfiltration were studied. The functional properties of whey protein concentrate from native whey were studied and the detailed composition of whey protein concentrate powders made from cheese wheys after cheese milk pretreatments such as high temperature heat treatment (HH), microfiltration (MF) and ultrafiltration (UF) were compared. The studied polymeric spiral wound microfiltration membranes had 38.5% lower energy consumption, 30.1% higher retention of whey proteins to milk retentate and 81.9% lower permeate flux values compared to ceramic membranes. All studied microfiltration membranes were able to separate main whey proteins from skimmed milk. The optimal lactose content of Emmental cheese milk exceeded 3.2% and reduction of whey proteins and ash content of cheese milk with high concentration factor (CF) values increased the rate of cheese ripening. Reduction of whey protein content in cheese milk increased the concentration of caseinomacropeptide (CMP) of total proteins in cheese whey. Reduction of milk whey protein, lactose and ash content reduces milk rennet clotting time and increased the firmness of the coagulum. Cheese yield calculated from raw milk to cheese was lower with microfiltrated milks due to native whey production. Amounts of a-lactalbumin (a-LA) and b-lactoglobulin (b-LG) were significantly higher in the reference whey, indicating that HH, MF and UF milk pretreatments decrease the amounts of these valuable whey proteins in whey. Even low CF values in milk microfiltration (CF 1.4) reduced nutritional value of cheese whey. From the point of view of utilization of milk components it would be beneficial if the amount of native whey and the CMP content of cheese whey could be maximized. Whey protein concentrate powders made of native whey had excellent functional properties and their detailed amino acid composition differed from those of cheese whey protein concentrate powders.

Forward osmosis as a pre-treatment for treating coal seam gas associated water: Flux and fouling behaviours

In this study, a bench scale forward osmosis (FO) process was operated using two commonly available FO membranes in different orientations in order to examine the removal of foulants in the coal seam gas (CSG) associated water, the water flux and fouling behaviours of the process were also investigated. After 48 h of fouling simulation experiment, the water flux declined by approximately 55 and 35% of its initial level in the TFC-PRO and CTA-PRO modes (support layer facing the feed), respectively, while the flux decline in the TFC-FO and CTA-FO modes (active layer facing the feed) was insignificant. The flux decline in PRO modes was caused by the compounding effects of internal concentration polarisation and membrane fouling. However, the declined flux was completely recovered to its initial level following the hydraulic cleaning using deionised water. Dissolved organic carbon (DOC), adenosine tri-phosphate (ATP) and major inorganic scalants (Ca, Mg and silica) in the CSG feed were effectively removed by using the FO process. The results of this study suggest that the FO process shows promising potential to be employed as an effective pre-treatment for membrane purification of CSG associated water.

Monitoring of Fungal Growth and Degradation of Wood

Valko- ja ruskolahosienet tunnetaan luonnossa tehokkaimpina puun ja karikkeen lignoselluloosan lahottajina. Valkolahosienet pystyvät hajottamaan kaikkia puun osia: ligniiniä, selluloosaa ja hemiselluloosaa. Selektiivisesti ligniiniä hajottavat sienet lahottavat puusta suhteessa enemmän vaikeasti hajoavaa ligniiniä kuin selluloosaa tai hemiselluloosaa, jolloin jäljelle jää valkoista ja miltei puhdasta selluloosaa. Bioteknisissä sovelluksissa juuri selektiviiviset valkolahottajat ovat kiinnostavia. Niiden avulla voidaan puuhaketta esikäsitellä esimerkiksi paperinvalmistuksessa haitallisen ligniinin poistamiseksi. Ruskolahosienet ovat huomattavia puun, puutavaran ja puisten rakenteiden lahottajia, kuten tässä työssä käytetty Gloeophyllum trabeum (saunasieni ) ja Poria (Postia) placenta (istukkakääpä). Ruskolahosienet hajottavat puusta hemiselluloosan lisäksi selluloosaa, jolloin jää jäljelle ruskea ja jauhomaiseksi mureneva ligniini. Ruskolahosienet muovaavat ligniiniä jonkin verran. Kahden ruskolahosienen G. trabeumin ja P. placentan lisäksi tutkittiin valkolahosieniä, joista Ceriporiopsis subvermispora (karstakääpä) ja harvinainen Physisporinus rivulosus -sieni (talikääpä) hajottavat ligniiniä erittäin selektiivisesti. Phanerochaete chrysosporium on kaikkialla paljon tutkittu sieni, ja Phlebia radiata valkolahosientä (rusorypykkä) on tutkittu paljon mikrobiologian osastolla. Lisäksi tutkittiin Phlebia tremellosa -sienten (hytyrypykkä) ligninolyyttisten entsyymien tuottoa ja 14C-leimatun synteettisen ligniinin (DHP) hajotusta. P. radiata ja P. tremellosa -sienten on todettu aiemmin hajottavan ligniiniä selektiivisesti. Työssä selvitettiin miten sienten kasvua voi mitata, miten vertailukelpoisia eri mittaamismenetelmillä saadut tulokset ovat ja ilmenevätkö sienten aktiivisimmat kasvuvaiheet samaan aikaan eri menetelmillä mitattuna. Tärkeimmät tulokset olivat seuraavat havainnot: (i) P. radiata ja P. tremellosa -sienikannat tuottivat ligniini- ja mangaaniperoksidaasientsyymejä (LiP ja MnP) sekä lakkaasia, ja sienistä puhdistettiin 2-3 LiP- ja P. radiatasta yksi MnP-entsyymi; (ii) P. tremellosa -sienet hajottivat leimattua synteettistä ligniiniä (DHP) yhtä hyvin kuin paljon tutkitut P. chrysosporium ja P. radiata -sienet; (iii) puu, sienen luonnollinen kasvualusta, lisäsi valkolaho- ja ruskolahosienten demetoksylaatiota [O14CH3]-leimatusta ligniinin malliyhdisteestä 14CO2:ksi ilman puuta olleeseen alustaan verrattuna; (iv) demetoksylaatio (14CO2:n tuotto) oli normaalissa ilma-atmosfäärissä useimmiten parempi happeen verrattuna; (v) hapessa paras 14CO2:n tuotto saatiin puupalakasvatuksissa, joihin oli lisätty ravinnetyppeä tai typen lisäksi glukoosia sekä valkolaho- että ruskolahosienillä; (vi) ilmassa 14CO2:n tuotto oli puulla voimakkainta valkolahosienillä ilman lisäravinteita, kun taas G. trabeum -sienellä se oli yhtä hyvä eri alustoissa; (vii) biomassan muodostuminen rihmastojen ergosterolipitoisuuksista mitattuna oli ruskolahosienillä parempi kuin valkolahosienillä; (viii) ja biomassojen huippupitoisuudet olivat 6:lla sienellä eri suuruisia ja niiden maksimimäärien ajankohdat vaihtelivat viiden viikon kasvatusten kuluessa. Mikrobiologian osastolla Viikissä eristetty ja paljon tutkittu P. radiata -valkolahosieni oli mukana kaikissa tehdyissä kokeissa. Sienen LiP-aktiivisuus ja 14CO2:n tuotto 14C-rengas-leimatusta synteettisestä ligniinistä (DHP) korreloivat erittäin hyvin. Biomassan muodostuminen ergosterolilla määritettynä tuki hyvin entsyymiaktiivisuusmittauksilla ja isotooppikasvatuksilla saatuja tuloksia.

Food and Indoor Air Isolated Bacillus Non-Protein Toxins: : Structures, Physico-Chemical Properties and Mechanisms of Effects on Eukaryotic Cells

We report here the structures and properties of heat-stable, non-protein, and mammalian cell-toxic compounds produced by spore-forming bacilli isolated from indoor air of buildings and from food. Little information is available on the effects and occurrence of heat-stable non-protein toxins produced by bacilli in moisture-damaged buildings. Bacilli emit spores that move in the air and can serve as the carriers of toxins, in a manner similar to that of the spores of toxic fungi found in contaminated indoor air. Bacillus spores in food cause problems because they tolerate the temperatures applied in food manufacture and the spores later initiate growth when food storage conditions are more favorable. Detection of the toxic compounds in Bacillus is based on using the change in mobility of boar spermatozoa as an indicator of toxic exposure. GC, LC, MS, and nuclear magnetic resonance NMR spectroscopy were used for purification, detection, quantitation, and analysis of the properties and structures of the compounds. Toxicity and the mechanisms of toxicity of the compounds were studied using boar spermatozoa, feline lung cells, human neural cells, and mitochondria isolated from rat liver. The ionophoric properties were studied using the BLM (black-lipid membrane) method. One novel toxin, forming ion channels permeant to K+ > Na+ > Ca2+, was found and named amylosin. It is produced by B. amyloliquefaciens isolated from indoor air of moisture-damaged buildings. Amylosin was purified with an RP-HPLC and a monoisotopic mass of 1197 Da was determined with ESI-IT-MS. Furthermore, acid hydrolysis of amylosin followed by analysis of the amino acids with the GS-MS showed that it was a peptide. The presence of a chromophoric polyene group was found using a NMR spectroscopy. The quantification method developed for amylosin based on RP-HPLC-UV, using the macrolactone polyene, amphotericin B (MW 924), as a reference compound. The B. licheniformis strains isolated from a food poisoning case produced a lipopeptide, lichenysin A, that ruptured mammalian cell membranes and was purified with a LC. Lichenysin A was identified by its protonated molecules and sodium- and potassium- cationized molecules with MALDI-TOF-MS. Its protonated forms were observed at m/z 1007, 1021 and 1035. The amino acids of lichenysin A were analyzed with ESI-TQ-MS/MS and, after acid hydrolysis, the stereoisomeric forms of the amino acids with RP-HPLC. The indoor air isolates of the strain of B. amyloliquefaciens produced not only amylosin but also lipopeptides: the cell membrane-damaging surfactin and the fungicidal fengycin. They were identified with ESI-IT-MS observing their protonated molecules, the sodium- and potassium-cationized molecules and analysing the MS/MS spectra. The protonated molecules of surfactin and fengycin showed m/z values of 1009, 1023, and 1037 and 1450, 1463, 1493, and 1506, respectively. Cereulide (MW 1152) was purified with RP-HPLC from a food poisoning strain of B. cereus. Cereulide was identified with ESI-TQ-MS according to the protonated molecule observed at m/z 1154 and the ammonium-, sodium- and potassium-cationized molecules observed at m/z 1171, 1176, and 1192, respectively. The fragment ions of the MS/MS spectrum obtained from the protonated molecule of cereulide at m/z 1154 were also interpreted. We developed a quantification method for cereulide, using RP-HPLC-UV and valinomycin (MW 1110, which structurally resembles cereulide) as the reference compound. Furthermore, we showed empirically, using the BLM method, that the emetic toxin cereulide is a specific and effective potassium ionophore of whose toxicity target is especially the mitochondria.

Paramagnetic probes in membrane biophysics

The use of paramagnetic probes in membrane research is reviewed. Electron paramagnetic resonance studies on model and biological membranes doped with covalent and non-covalent spin-labels have been discussed with special emphasis on the methodology and the type of information obtainable on several important phenomena like membrane fluidity, lipid flip-flop, lateral diffusion of lipids, lipid phase separation, lipid bilayer phase transitions, lipid-protein interactions and membrane permeability. Nuclear magnetic resonance spectroscopy has also been effectively used to study the conformations of cation mediators across membranes and to analyse in detail the transmembrane ionic motions at the mechanistic level.

Studies on the synthesis of cytochrome P-450 and cytochrome P-448 in rat liver

The synthesis of cytochrome P-450 (phenobarbital inducible) and cytochrome P-448 (3-methylcholanthrene inducible) have been studied in rat liver in vivo and in the wheat germ cell-free system using anti- cytochrome P-450 and anti-cytochrome P-448 antibodies. The major mature forms synthesized in vivo correspond to a molecular weight of 47,000 for cytochrome P-450 and 53,000 for cytochrome P-448. Translation of poly(A)-containing RNA from phenobarbital-treated rats in the wheat germ cell-free system reveals that the cell-free product immunoprecipitated with anti-cytochrome P-450 antibody has a molecular weight close to 47,000. In the case of 3-methylcholanthrene, the cell- free product immunoprecipitated with anti-cytochrome P-448 antibody shows a molecular weight around 59,000. Significant conversion of the 59,000 species to the 53,000 species can be demonstrated when the translation is carried out in the presence of microsomal membranes isolated from rat liver. Phenobarbital and 3-methylcholanthrene enhance the translatable messenger.