Fusion of secretory vesicles isolated from rat liver

Secretory vesicles isolated from rat liver were found to fuse after exposure to Ca2+. Vescle fusion is characterized by the occurrence of twinned vesicles with a continuous cleavage plane between two vesicles in freeze-fracture electron microscopy. The number of fused vesicles increases with increasing Ca2+-concentrations and is half maximal around 10–6 m. Other divalent cations (Ba2+, Sr2+, and Mg2+) were ineffective. Mg2+ inhibits Ca2+-induced fusion. Therefore, the fusion of secretory vesiclesin vitro is Ca2+ specific and exhibits properties similar to the exocytotic process of various secretory cells. Various substances affecting secretionin vivo (microtubular inhibitors, local anethetics, ionophores) were tested for their effect on membrane fusion in our system. The fusion of isolated secretory vesicles from liver was found to differ from that of pure phospholipid membranes in its temperature dependence, in its much lower requirement for Ca2+, and in its Ca2+-specificity. Chemical and enzymatic modifications of the vesicle membrane indicate that glycoproteins may account for these differences

The thermodynamics of general anesthesia

It is known that the action of general anesthetics is proportional to their partition coefficient in lipid membranes (Meyer-Overton rule). This solubility is, however, directly related to the depression of the temperature of the melting transition found close to body temperature in biomembranes. We propose a thermodynamic extension of the Meyer-Overton rule which is based on free energy changes in the system and thus automatically incorporates the effects of melting point depression. This model provides a quantitative explanation of the pressure reversal of anesthesia. Further, it explains why inflammation and the addition of divalent cations reduce the effectiveness of anesthesia.Comment: 7 pages, 2 figure

The influence of anesthetics, neurotransmitters and antibiotics on the relaxation processes in lipid membranes

In the proximity of melting transitions of artificial and biological membranes fluctuations in enthalpy, area, volume and concentration are enhanced. This results in domain formation, changes of the elastic constants, changes in permeability and slowing down of relaxation processes. In this study we used pressure perturbation calorimetry to investigate the relaxation time scale after a jump into the melting transition regime of artificial lipid membranes. This time corresponds to the characteristic rate of domain growth. The studies were performed on single-component large unilamellar and multilamellar vesicle systems with and without the addition of small molecules such as general anesthetics, neurotransmitters and antibiotics. These drugs interact with membranes and affect melting points and profiles. In all systems we found that heat capacity and relaxation times are related to each other in a simple manner. The maximum relaxation time depends on the cooperativity of the heat capacity profile and decreases with a broadening of the transition. For this reason the influence of a drug on the time scale of domain formation processes can be understood on the basis of their influence on the heat capacity profile. This allows estimations of the time scale of domain formation processes in biological membranes.Comment: 12 pages, 6 figure

Phase transitions in biological membranes

Native membranes of biological cells display melting transitions of their lipids at a temperature of 10-20 degrees below body temperature. Such transitions can be observed in various bacterial cells, in nerves, in cancer cells, but also in lung surfactant. It seems as if the presence of transitions slightly below physiological temperature is a generic property of most cells. They are important because they influence many physical properties of the membranes. At the transition temperature, membranes display a larger permeability that is accompanied by ion-channel-like phenomena even in the complete absence of proteins. Membranes are softer, which implies that phenomena such as endocytosis and exocytosis are facilitated. Mechanical signal propagation phenomena related to nerve pulses are strongly enhanced. The position of transitions can be affected by changes in temperature, pressure, pH and salt concentration or by the presence of anesthetics. Thus, even at physiological temperature, these transitions are of relevance. There position and thereby the physical properties of the membrane can be controlled by changes in the intensive thermodynamic variables. Here, we review some of the experimental findings and the thermodynamics that describes the control of the membrane function.Comment: 23 pages, 15 figure

Antiproliferative effect of immunoliposomes containing 5-fluorodeoxyuridine-dipalmitate on colon cancer cells

We have investigated the antiproliferative action towards CC531 colon adenocarcinoma cells of target cell-specific immunoliposomes containing the amphiphilic dipalmitoyl derivative of 5-fluorodeoxyuridine (FUdR-dP). FUdR-dP incorporated in immunoliposomes caused a 13-fold stronger inhibition of CC531 cell growth in vitro, during a 72-h treatment, than FUdR-dP in liposomes without antibody, demonstrating that the prodrug is efficiently hydrolysed to yield the active drug, FUdR, intracellularly. The intracellular release of active FUdR was confirmed by determining the fate of H-3-labelled immunoliposomal FUdR-dP. Treatments shorter than 72 h with FUdR-dP in immunoliposomes resulted in anti-tumour activities comparable to, or even higher than, that of free FUdR. The shorter treatments reflect more closely the in vivo situation and illustrate the potential advantage of the use of immunoliposomes over non-targeted liposomal FUdR-dP or free FUdR. Association of tumour cell-specific immunoliposomes with CC531 cells was up to tenfold higher than that of liposomes without antibody or with irrelevant IgG coupled, demonstrating a specific interaction between liposomes and target cells which causes an efficient intracellular delivery of the drug. Since biochemical evidence indicates a lack of internalization or degradation of the liposomes as such; we postulate that entry of the drug most likely involves the direct transfer of the prodrug from the immunoliposome to the cell membrane during its antigen-specific interaction with the cells. followed by hydrolysis of FUdR-dP leading to relatively high intracellular FUdR-levels. In conclusion, we describe a targeted liposomal formulation for the anticancer drug FUdR, which is able to deliver the active drug to colon carcinoma cells with high efficiency, without the need for the cells to internalize the liposomes as such

Fluorescence studies on new potential antitumoral benzothienopyran-1-ones in solution and in liposomes

Fluorescence properties of four new potential antitumoral compounds, 3-arylbenzothieno[2,3-c]pyran-1-ones, were studied in solution and in lipid membranes of dipalmitoyl phosphatidylcholine (DPPC), egg yolk phosphatidylcholine (Egg-PC) and dioctadecyldimethylammonium bromide (DODAB). The 3-(4-methoxyphenyl)benzothieno[2,3-c]pyran-1-one (1c) exhibits the higher fluorescence quantum yields in all solvents studied. All compounds present a solvent sensitive emission, with significant red shifts in polar solvents for the methoxylated compounds. The results point to an ICT character of the excited state, more pronounced for compound 1c. Fluorescence (steady-state) anisotropy measurements of the compounds incorporated in liposomes of DPPC, DODAB and Egg-PC indicate that all compounds have two different locations, one due to a deep penetration in the lipid membrane and another corresponding to a more hydrated environment. In general, the methoxylated compounds prefer hydrated environments inside the liposomes. The 3-(4- fluorophenyl)benzothieno[2,3-c]pyran-1-one (1a) clearly prefers a hydrated environment, with some molecules located at the outer part of the liposome interface. On the contrary, the preferential location of 3-(2-fluorophenyl)benzothieno[2,3-c]pyran-1-one (1b) is in the region of lipid hydrophobic tails. Compounds with a planar geometry (1a and 1c) have higher mobility in the lipid membranes when phase transition occurs.Portugal and FEDER (Fundo Europeu de Desenvolvimento Regional), for financial support through Centro de Física (CFUM) and Centro de Química (CQ-UM) of University of Minho and through the Project PTDC/QUI/81238/2006. M.S.D. Carvalho and R.C. Calhelha acknowledge FCT for their PhD grants SFRH/BD/47052/2008 and SFRH/BD/29274/2006, respectively.Fundação para a Ciência e a Tecnologia (FCT

New potential antitumoral fluorescent tetracyclic thieno[3,2-b]pyridine derivatives: interaction with DNA and nanosized liposomes

Fluorescence properties of two new potential antitumoral tetracyclic thieno[3,2-b]pyridine derivatives were studied in solution and in liposomes of DPPC (dipalmitoyl phosphatidylcholine), egg lecithin (phosphatidylcholine from egg yolk; Egg-PC) and DODAB (dioctadecyldimethylammonium bromide). Compound 1, pyrido[2',3':3,2]thieno[4,5-d]pyrido[1,2-a]pyrimidin-6-one, exhibits reasonably high fluorescence quantum yields in all solvents studied (0.20 ≤ ΦF ≤ 0.30), while for compound 2, 3-[(p-methoxyphenyl)ethynyl]pyrido[2',3':3,2]thieno[4,5-d]pyrido[1,2-a]pyrimidin-6-one, the values are much lower (0.01 ≤ ΦF ≤ 0.05). The interaction of these compounds with salmon sperm DNA was studied using spectroscopic methods, allowing the determination of intrinsic binding constants, Ki = (8.7 ± 0.9) × 103 M-1 for compound 1 and Ki = (5.9 ± 0.6) × 103 M-1 for 2, and binding site sizes of n = 11 ± 3 and n = 7 ± 2 base pairs, respectively. Compound 2 is the most intercalative compound in salmon sperm DNA (35%), while for compound 1 only 11% of the molecules are intercalated. Studies of incorporation of both compounds in liposomes of DPPC, Egg-PC and DODAB revealed that compound 2 is mainly located in the hydrophobic region of the lipid bilayer, while compound 1 prefers a hydrated and fluid environment

Thyroid control over biomembranes: VI. Lipids in liver mitochondria and microsomes of hypothyroid rats

The lipids of liver mitochondria prepared from normal rats and from rats made hypothyroid by thyroidectomy and injection with131INa contained similar amounts, per mg protein, of total lipids, phospholipids, neutral lipids and lipid phosphorus. Hypothyroidism caused a doubling of the relative amounts of mitochondrial cardiolipins (CL; to 20.5% of the phospholipid P) and an accompanying trend (although statistically not significant) toward decreased amounts of both phosphatidylcholines (PC) and phosphatidylserines (PS), with phosphatidylethanolamines (PE) remaining unchanged. The pattern of elevated 18∶2 fatty acyl content and depleted 20∶4 acyl groups of the mitochondrial phospholipids of hypothyroid preparations was reflected to varying degrees in the resolved phospholipids, with PC showing greater degrees of abnormality than PE, and CL showing none. Hypothyroidism produced the same abnormal pattern of fatty acyl distributions in liver microsomal total lipids as was found in the mitochondria. Hypothyroid rats, when killed 6 hr after injection of [1‐14C] labeled linoleate, showed the following abnormalities: the liver incorporated less label into lipids, and converted 18∶2 not exclusively to 20∶4 (as normals do) but instead incorporated the label mainly into saturated fatty acids. These data, together with the known decrease in β‐oxidation, suggest that hypothyroidism involves possible defective step(s) in the conversion of 18∶2 to 20∶4.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/142296/1/lipd0328.pd