C.M. Gupta

ABC transporters Cdr1p, Cdr2p and Cdr3p of a human pathogen Candida albicans are general phospholipid translocators.

We have used fluorescent 7‐nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl (NBD)‐tagged phospholipid analogues, NBD‐PE (phosphatidylethanolamine), NBD‐PC (phosphatidylcholine) and NBD‐PS (phosphatidylserine), to demonstrate that Cdr1p and its other homologues, Cdr2p and Cdr3p, belonging to the ATP‐binding cassette (ABC) superfamily behave as general phospholipid translocators. Interestingly, CDR1 and CDR2, whose overexpression leads to azole resistance in C. albicans, elicit in‐to‐out transbilayer phospholipid movement, while CDR3, which is not involved in drug resistance, carries out‐to‐in translocation of phospholipids between the two monolayers of plasma membrane. Cdr1p, Cdr2p and Cdr3p could be further distinguished on the basis of their sensitivities to different inhibitors. For example, the in‐to‐out activity associated with Cdr1p and Cdr2p is energy‐dependent and sensitive to sulphydryl blocking agents such as N‐ethylmaleimide (NEM) and cytoskeleton disrupting agent cytochalasin E, while Cdr3p‐associated out‐to‐in activity is energy‐dependent but insensitive to NEM and cytochalasin E. We found that certain drugs, such as fluconazole, cycloheximide and miconazole, to which Cdr1p confers resistance could also affect in‐to‐out transbilayer movement of NBD‐PE, while the same drugs had no effect on Cdr3p‐mediated out‐to‐in translocation of NBD‐PE. The ineffectiveness of these drugs to affect Cdr3p mediated out‐to‐in phospholipid translocation further confirms the inherent difference in the directionality of phospholipid translocation between these pumps. Notwithstanding the role of some of the Cdrps in drug resistance, this study clearly demonstrates that these ABC transporters of C. albicans are phospholipid translocators and this function could represent one of the physiological functions of such large family of proteins. Copyright

Asymmetric distribution of phosphatidylethanolamine in C. albicans : possible mediation by CDR1, a multidrug transporter belonging to ATP binding cassette (ABC) superfamily

By using two molecular probes, we demonstrate that only 4% of total phosphatidylethanolamine (PtdEtn) in the plasma membrane (PM) of a human pathogenic yeast, Candida albicans, is present in its external half. Evidence is presented to show that the availability of PtdEtn could be related to the expression of a multidrug transporter CDR1 of C. albicans, and the process is energy‐dependent. A homozygous CDR1 disruptant strain of C. albicans shows almost 23% reduction in the external labelling of PtdEtn. This report shows that, similar to human MDRs, yeast multidrug transporter could also be involved in aminophospholipid translocation. Copyright

Membrane phospholipid organization in calcium-loaded human erythrocytes

Intracellular Ca2+ levels in human erythrocytes were increased by incubating them with variable concentrations of Ca2+ in the presence of ionophore A23187. Experiments were done to confirm that the Ca2+ loading did induce changes in the cell shape and membrane protein composition. The effect of the increased cytoplasmic Ca2+ levels on the membrane phospholipid organization was analysed using bee venom and pancreatic phospholipases A2, Merocyanine 540 and fluorescamine as the external membrane probes. About 20% phosphatidylethanolamine (PE) and 0% phosphatidylserine (PS) were hydrolysed by the phospholipases in intact control cells, whereas in identical conditions these enzymes readily degraded, 20-30% PE and 7-30% PS, in Ca2+-loaded erythrocytes, depending on the cytoplasmic Ca2+ concentration. Also, Merocyanine 540 failed to stain the fresh or control erythrocytes, but it labeled the cells loaded with Ca2+. Furthermore, fluorescamine labeled approx. 20% PE in fresh or control erythrocytes while in identical conditions, significantly higher amounts of PE were modified in intact Ca2+-loaded cells. These results demonstrate that Ca2+ loading in human erythrocytes leads to loss of the transbilayer phospholipid asymmetry, and suggest that, together with spectrin, polypeptides 2.1 and 4.1 may also play an important role in maintaining the asymmetric distribution of various phospholipids across the erythrocyte membrane bilayer.

Tuftsin-bearing liposomes as rifampin vehicles in treatment of tuberculosis in mice.

The antitubercular activity of rifampin was considerably increased when it was encapsulated in egg phosphatidylcholine liposomes. A further increase in the activity was observed when the macrophage activator tetrapeptide tuftsin was grafted on the surface of the drug-loaded liposomes. Intermittent treatments (twice weekly) with these preparations were significantly more effective than the continuous treatments. Rifampin delivered twice weekly for 2 weeks in tuftsin-bearing liposomes was at least 2,000 times more effective than the free drug in lowering the load of lung bacilli in infected animals. However, pretreatment with drug-free tuftsin-bearing liposomes did not render the pretreated animals resistant to the Mycobacterium tuberculosis infections, neither did it appreciably increase the chemotherapeutic efficacy of the liposomized rifampin. These results clearly demonstrate that liposome targeting to macrophages could considerably increase the antitubercular activity of liposomized drugs such as rifampin. Also, it shows that immunoprophylactic treatment with macrophage activators such as tuftsin does not afford any advantage in treatment of tuberculosis infections, presumably because of inactivation of the primed macrophages by the mycobacterial sulfatides.

Membrane skeleton-bilayer interaction is not the major determinant of membrane phospholipid asymmetry in human erythrocytes☆

Transbilayer phospholipid distribution, membrane skeleton dissociation/association, and spectrin structure have been analysed in human erythrocytes after subjecting them to heating at 50 degrees C for 15 min. The membrane skeleton dissociation/association was determined by measuring the Tris-induced dissociation of Triton-insoluble membrane skeletons (Triton shells), the spectrin-actin extractability under low ionic conditions, and the binding of spectrin-actin with normal erythrocyte membrane inside-out vesicles (IOVs). The spectrin structure was ascertained by measuring the spectrin dimer-to-tetramer ratio as well as the spectrin tryptophan fluorescence. Both the Tris-induced Triton shell dissociation and the spectrin-actin extractability under low ionic conditions were considerably reduced by the heat treatment. Also, the binding of heated erythrocyte spectrin-actin to IOVs was significantly smaller than that observed with the normal cell spectrin-actin. Further, the quantity of spectrin dimers was appreciably increased in heat-treated erythrocytes as compared to the normal cells. This change in the spectrin dimer-to-tetramer ratio was accompanied by marked changes in the spectrin tryptophan fluorescence. In spite of these heat-induced alterations in structure and bilayer interactions of the membrane skeleton, the inside-outside glycerophospholipid distribution remained virtually unaffected in the heat-treated cells, as judged by employing bee venom and pancreatic phospholipase A2, fluorescamine and Merocyanine 540 as the external membrane probes. These results strongly indicate that membrane bilayer-skeleton interaction is not the major factor in determining the transbilayer phospholipid asymmetry in human erythrocyte membrane.

Actin‐depolymerizing factor, ADF/cofilin, is essentially required in assembly of Leishmania flagellum

ADF/cofilins are ubiquitous actin dynamics‐regulating proteins that have been mainly implicated in actin‐based cell motility. Trypanosomatids, e.g. Leishmania and Trypanosoma, which mediate their motility through flagellum, also contain a putative ADF/cofilin homologue, but its role in flagellar motility remains largely unexplored. We have investigated the role of this protein in assembly and motility of the Leishmania flagellum after knocking out the ADF/cofilin gene by targeted gene replacement. The resultant mutants were completely immotile, short and stumpy, and had reduced flagellar length and severely impaired beat. In addition, the assembly of the paraflagellar rod was lost, vesicle‐like structures were seen throughout the length of the flagellum and the state and distribution of actin were altered. However, episomal complementation of the gene restored normal morphology and flagellar function. These results for the first time indicate that the actin dynamics‐regulating protein ADF/cofilin plays a critical role in assembly and motility of the eukaryotic flagellum.

Carbamyl analogs of phosphatidylcholines: Synthesis, interaction with phospholipases and permeability behavior of their liposomes

A novel class of phospholipase-resisting phosphatidylcholine analogs, in which the C-2 ester group or both C-1 and C-2 ester groups have been replaced by carbomyloxy functions (Formula--see text), have been synthesized. These lipids were not degraded by phospholipase A2, while complete hydrolysis occurred with phospholipase C. Ultrasonic irradiation of the aqueous dispersions of the phospholipids in the presence as well as in the absence of cholesterol resulted in the formation of closed bilayer structures as evidenced by negative staining electron microscopy and also by their ability to entrap [14C]glucose. The leakage rates of glucose at 37 degrees C from liposomes of these compounds have also been measured. Liposomes consisting of 1,2-dipentadecanylcarbamyloxy-sn-glycero-3-phosphorylcholine were found to be more leaky (2.1%/h) as compared to the liposomes of 1-palmitoyl-2-pentadecanylcarbamyloxy-sn-glycero-3-phosphorylcholine (0.5%/h). Moreover, inclusion of cholesterol (33 mol%) into the bilayers of the former phospholipid had no effect on the leakage rate (2.4%/h) while it effectively reduced permeability of the latter (0.22%/h). These phosphatidylcholines are useful for studying the possible role of phospholipases in the capture and lysis of liposomes in vivo.

Superior Chemotherapeutic Efficacy of Amphotericin B in Tuftsin-bearing Liposomes against Leishmania Donovani Infection in Hamsters

Chemotherapeutic efficacy of the amphotericin B (Amp B), which is the drug of choice for treatment of the leishmanial infections (kala-azar) that become resistant to the conventional chemotherapy using antimonials, has been examined in the Leishmania donovani infected hamsters after encapsulating the drug in tuftsin-free as well as tuftsin-bearing liposomes. The activity was significantly increased (p <0.05) by delivering Amp B in tuftsin-free liposomes. This antileishmanial effect of the liposomized Amp B was further increased (p <0.05) by grafting the natural macrophage-activator tetrapeptide, tuftsin (Thr-Lys-Pro-Arg), on the liposomes surface. This could possibly be attributed to both the enhanced drug tolerance after liposomization as well as to the increased uptake of tuftsin-bearing Amp B-laden liposomes by the macrophages. In addition to the increased efficacy, encapsulation of Amp B in the tuftsin-bearing liposomes also enhanced the drug accessibility to areas (e.g. bone marrow) that are otherwise inaccessible to the free drug. These results further demonstrate the usefulness of tuftsin-bearing liposomes as drug vehicles in treatment of the macrophage-based infections that have been reviewed recently (Agrawal, A.K. and Gupta, C.M. (2000). Tuftsin-bearing liposomes in treatment of macrophage-based infections, Adv. Drug Deliv. Rev., 41, 135-146).

Specific interactions of liposomes with PMN leukocytes upon incorporating tuftsin in their bilayers

Incorporation of tuftsin derivatives, Thr‐Lys‐Pro‐Arg‐NH‐C18H37 or Thr‐Lys‐Pro‐Arg‐NH‐(CH2)2‐NH‐COC15H31, into an egg phosphatidylcholine/cholesterol liposome bilayer led to significantly enhanced binding of the liposomes to PMN leukocytes at 37°C but not at 0°C. Under identical conditions, no such enhanced binding of the liposomes was observed with erythrocytes and lymphocytes. These results demon‐strate that grafting of tuftsin on the liposome bilayer enables the liposome to recognize specifically the PMN leukocytes and to deliver its contents to these cells.

Influence of the phospholipid structure on the stability of liposomes in serum

The effect of serum on the structural integrity of liposomes consisting of ether and/or carbamyl analogs of 1,2-diester phosphatidylcholine (PC) has been evaluated by measuring both the efflux of the entrapped 6-carboxyfluorescein and the lipid transfer to serum proteins, and the results have been compared with the egg PC liposomes. Replacement of the C-1 ester bond in PC by an ether linkage did not significantly enhance the liposome stability, but it was markedly increased upon introducing further structural changes in the C-2 ester region of the resulting 1-ether-2-ester PC. However, the stability was not influenced by altering the steric configuration of the latter phospholipid. These results strongly suggest that lysis of liposomes in serum can be prevented by structurally modifying the ester bond(s) in the phospholipid component of liposomes.