PubMed Health⌕ Search

Biomedical subjects

T F Taraschi

Publications and source records attributed to T F Taraschi.

At least 37 records · Page 2Linked to original sources
PubMed1996

Hydrophobic alkyl headgroups strongly promote membrane curvature and violate the headgroup volume correlation due to "headgroup" insertion.

The ability of lipid aggregates to form planar bilayers, rather than highly curved micellar or inverted structures, is dependent on the relative geometries of the headgroup and hydrocarbon regions. The headgroup volume approach to lipid structure provided a quantitative link between a lipid's headgroup size and its ability to promote curved, inverted hexagonal (H(II)) structures in a phosphatidylethanolamine (PtdEtn) matrix [Lee et al. (1993) Biophys. J. 65, 1429-1432]. Phosphatidylalkanols (PtdAlks) are shown here to promote curvature with a potency that far exceeds and a chain length dependence contrary to the expectations of the headgroup volume approach, suggestive of an atypical alkyl "headgroup" conformation. A homologous series of 3-substituted triacylglycerols (TAGs), for which 3-acyl "headgroup" insertion is established, exhibits a chain length dependence similar to the PtdAlks, evidence that the deviation is of common origin. The potency of the TAGs to promote curvature is unprecedented, and the onset of saturation, which parallels the dramatic promotion of curvature, occurs at mole fractions as low as 0.0025. The potency of the PtdAlks or TAGs to promote curvature exceeds that of all mammalian phospholipids examined. Thermodynamic analysis implicates the enthalpic curvature stress imparted upon the membrane matrix as the dominant energetic factor. The imparted stress ranges from -930 J mol(-1) for phosphatidylcholine to +7.5 kJ mol(-1) for 3-palmitoyl TAG. The results affirm the geometric considerations of membrane structure and indicate that alkyl headgroups tend to insert into the bilayer and increase the enthalpic curvature stress within the membrane.

Alcohols↗
PubMed1996

Alcohol binding to liposomes by 2H NMR and radiolabel binding assays: does partitioning describe binding?

Implicit within the concept of membrane-buffer partition coefficients of solutes is a nonspecific solvation mechanism of solute binding. However, (2)H NMR studies of the binding of (2)H(6)-ethanol and [1-(2)H(2)] n-hexanol to phosphatidylcholine vesicles have been interpreted as evidence for two distinct alcohol binding modes. One binding mode was reported to be at the membrane surface. The second mode was reported to be within the bilayer interior. An examination of the (2)H NMR binding studies, together with direct radiolabel binding assays, shows that other interpretations of the data are more plausible. The results are entirely consistent with partitioning (nonspecific binding) as the sole mode of alcohol binding to liposomes, in accord with our previous thermodynamic interpretation of alcohol action in phosphatidylcholine liposomes.

Deuterium↗
PubMed1995

Cloning the P. falciparum gene encoding PfEMP1, a malarial variant antigen and adherence receptor on the surface of parasitized human erythrocytes.

Plasmodium falciparum-infected human erythrocytes evade host immunity by expression of a cell-surface variant antigen and receptors for adherence to endothelial cells. These properties have been ascribed to P. falciparum erythrocyte membrane protein 1 (PfEMP1), an antigenically diverse malarial protein of 200-350 kDa on the surface of parasitized erythrocytes (PEs). We describe the cloning of two related PfEMP1 genes from the Malayan Camp (MC) parasite strain. Antibodies generated against recombinant protein fragments of the genes were specific for MC strain PfEMP1 protein. These antibodies reacted only with the surface of MC strain PEs and blocked adherence of these cells to CD36 but without effect on adherence to thrombospondin. Multiple forms of the PfEMP1 gene are apparent in MC parasites. The molecular basis for antigenic variation in malaria and adherence of infected erythrocytes to host cells can now be pursued.

Amino Acid Sequence↗
PubMed1994

Interdigitation-fusion: a new method for producing lipid vesicles of high internal volume.

Previously we demonstrated that fused phospholipid sheets can be formed from small unilamellar vesicles (SUVs) comprised of saturated symmetric chain lipids by exposing them to concentrations of ethanol sufficient to cause bilayer interdigitation (Boni et al. (1993) Biochim. Biophys. Acta 1146, 247-257). Here we report that these sheets spontaneously form large, predominately unilamellar vesicles, when exposed to temperatures above their main phase transition temperature (Tm). These vesicles, termed interdigitation-fusion vesicles (IFVs), have mean diameters between 1 and 6 microns, and, once produced, are stable both above and below the Tm of the lipid. The average captured volume of IFVs is dependent upon lipid chain length, the concentration of ethanol used to induce interdigitation-fusion, and size of the precursor liposomes. IFVs comprised of DPPC and DSPC had averaged captured volumes of 20-25 microliters/mumol lipid. IFVs produced from SUVs containing only DPPG or DPPC/DPPG mixtures had captured volumes equivalent to those made from pure DPPC SUVs indicating that charge can be introduced without consequence to the IFV process. Inclusion of cholesterol in precursor vesicles reduced IFV captured volume in a concentration dependent fashion by interfering with interdigitation. Cholesterol could be incorporated, however, into IFVs through admixture with the already formed phospholipid sheets producing far less comprise to captured volume. IFVs are useful as model systems or drug carriers, since their large internal volume allows for efficient encapsulation particularly with regard to compounds such as iodinated radiocontrast agents which otherwise interfere with vesicularization.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1994

PfEMP3 and HRP1: co-expressed genes localized to chromosome 2 of Plasmodium falciparum.

A malarial protein, Plasmodium falciparum erythrocyte membrane protein 3 (PfEMP3), has been recently characterized as a high-molecular-mass component (approx. 315 kDa) localized to the erythrocyte membrane of knob-bearing (K+), cytoadherent (C+) mature stages of P. falciparum-parasitized erythrocytes (PE) [Pasloske et al., Mol. Biochem. Parasitol. 59 (1993) 59-72]. Knobless (K-), non-cytoadherent (C-) parasites of the same strain were shown to lack the PfEMP3 gene. In view of the biological importance of the knobby and cytoadherent phenotypes with regard to parasite virulence, we extended the analysis of PfEMP3 and its gene product to other K+/K- and C+/C- parasites. Previously, other studies have shown that the malarial protein, knob-associated histidine-rich protein 1 (HRP1), is also strongly correlated with knob expression. Here, we show that PfEMP3 and HRP1 were absent from all the K- parasites tested, including the Palo Alto (PA) K-C+ strain. This result demonstrates that PfEMP3 and HRP1 are not essential for cytoadherence. PfEMP3 was localized to chromosome 2 of the K+ parasites, within no more than 130 kb of HRP1, between the telomere and HRP1. Stage-specific analysis of the mRNA for HRP1 and PfEMP3 indicated maximal transcription of the genes in ring-stage parasites, with little or no mRNA present during the mature parasite stages. Analysis of PfEMP3 and HRP1 by immunofluorescence assay (IFA) revealed identical staining patterns of fixed PE at all stages of the asexual life cycle. Hence, PfEMP3 and HRP1 are adjacent to each other in chromosome 2, co-expressed temporally and their gene products co-localized to the PE membrane.

Animals↗
PubMed1994

Characterization of trafficking pathways and membrane genesis in malaria-infected erythrocytes.

The origin of membraneous structures in the cytoplasm of human erythrocytes infected with the malaria parasite, Plasmodium falciparum, was determined by confocal fluorescence imaging microscopy. When infectious merozoites invaded erythrocytes labeled with the fluorescent, lipophilic, non-exchangeable molecules DiIC16 or DiOC16, a ring of fluorescence was observed surrounding the internal parasite, indicating that the parasitophorous vacuolar membrane (PVM) is formed in part from the erythrocyte membrane. As the parasites matured, fluorescent vesicles were seen to be exported into the erythrocyte cytoplasm, beginning at 6 h post-invasion. During intraerythrocytic development, these dyes were transferred from the PVM to the parasite. When fluorescently labeled merozoites were released from these cells and invaded unlabeled erythrocytes, fluorescence was confined to the parasite throughout the entire erythrocytic cycle. Taken together, these results demonstrate that all vesicles/membranous compartments in the erythrocyte cytoplasm of parasitized erythrocytes (IRBC) contain membrane derived from the PVM. Based on this information, we define pathways that the parasite utilizes to export proteins and lipids to the host cell cytoplasm and surface membrane. When IRBC were labeled post-invasion with DiIC16 or DiOC16 and the parasites allowed to mature for one life cycle, the dyes were confined to the erythrocyte membrane, demonstrating that the host cell membrane of IRBC does not endocytose and there is no membrane exchange from the erythrocyte to the parasite. This investigation helps to resolve two long-standing controversies and provides new insights into the transport pathways that malaria parasites utilize during their development within host erythrocytes.

Animals↗
PubMed1994

Taxol arrests the development of blood-stage Plasmodium falciparum in vitro and Plasmodium chabaudi adami in malaria-infected mice.

Taxol, a natural product used to treat a variety of human cancers, is shown here to be extremely effective against chloroquine- and pyrimethamine-resistant malaria parasites. Addition of Taxol (1.0 microM) for one cycle to cultures of human erythrocytes infected with Plasmodium falciparum prevents the establishment of new infections. Blood parasitemia is eliminated in mice infected with Plasmodium chabaudi adami when they are given a single intraperitoneal injection of Taxol at 150 mg/m2. The majority of the animals treated immediately preceding parasite schizogony remain free of infection after eight replication cycles. The impressive antimalarial activity of Taxol, at a dosage that has been tolerated in humans, establishes its potential utility for treatment of severe, drug-resistant human malaria.

Animals↗
PubMed1993

Configurational entropy is the driving force of ethanol action on membrane architecture.

A colligative thermodynamic framework is developed to describe the action of ethanol on membranes. The partitioning of ethanol into a membrane structure imparts a randomness, configurational entropy, that stabilizes that structure from an energetic standpoint. When partitioning between membrane structures differs, the equilibrium between them is altered to favor the structure with the largest partition coefficient for ethanol. The action of ethanol and temperature originate in entropy and are equated through entropy. Membrane equilibria that are predicted to be most sensitive to the action of ethanol (where dilute concentrations of ethanol cause a perturbation equal to a large change in temperature) are those that exhibit a small thermal entropy change and a large difference in solute partitioning between membrane structures. Our model predicts that ethanol does not act on a single membrane structure, but on both structures in an equilibrium. The thermodynamic framework is applied to the action of ethanol on cooperative equilibria in a dipalmitoyl lecithin model membrane. Ethanol-induced perturbations are monitored by electron paramagnetic resonance (EPR) using the spin label, Tempo. The equilibrium between the gel and ripple-structures (L beta'-->P beta', pretransition) exhibits a small change in thermal entropy and, as predicted, is more sensitive to the action of ethanol than the equilibrium between the ripple and fluid bilayer-structures (P beta'-->L alpha, main transition) which exhibits a large thermal entropy change. The framework suggests that ethanol acts through entropy, as does temperature, thereby upsetting the natural thermal balance that maintains membrane architecture.

Chemical Phenomena↗
PubMed1993

Cloning and characterization of a Plasmodium falciparum gene encoding a novel high-molecular weight host membrane-associated protein, PfEMP3.

The rat monoclonal antibody, mAb 12C11, reacts with numerous proteins from mature asexual stages of Plasmodium falciparum. The largest is 315 kDa and is designated PfEMP3. A lambda gt11 expression library, generated from genomic DNA of Malayan Camp strain parasites, was screened with mAb 12C11. One positive clone, lambda 12.1.3, contained a 1.4-kb fragment in frame with the beta-galactosidase gene of lambda gt11. The deduced 455-amino acid sequence is a novel, highly charged sequence encoding two 15-amino acid repeats at the N-terminus followed by 27 repeats of 13 amino acids. The last 59 C-terminal residues are non-repetitive. Two in-frame stop codons at the 3' end of the DNA suggests that this DNA fragment encodes the C-terminus of the protein. Southern blotting with the cloned fragment identified two copies of this fragment per haploid genome in knob-positive, parasitized erythrocytes (K+PE). Both DNA fragments are absent from K - PE. Northern blotting of trophozoite-stage PE total RNA revealed mRNAs of 10, 4.4 and 2 kb in K+PE, but no hybridization with K - PE. Immune sera were elicited against the lambda 12.1.3 beta-galactosidase fusion protein and peptides generated from the predicted lambda 12.1.3 amino acid sequence. These sera and mAb 12C11 reacted specifically with PfEMP3 in Western blots of mature K+PE but not with K - PE. Rat and mouse sera against the recombinant protein produced an immunofluorescence pattern in fixed mature K+PE almost identical to the pattern produced by a monoclonal antibody against the knob-associated protein, Histidine Rich Protein 1. The same antibodies were immunofluorescence negative with fixed K - PE. Mouse antibodies against the recombinant protein reacted on immunoelectron microscopy with the erythrocyte membrane of K+PE, labeling knobs as well as the membrane between knobs. In contrast, a mAb against Histidine Rich Protein 1 reacted only under the electron dense material of knobs. We conclude that the lambda 12.1.3 clone encodes the C-terminal portion of the 315 kD PfEMP3 antigen and that PfEMP3 may be involved in knob formation or other perturbations of the erythrocyte membrane.

Amino Acid Sequence↗
PubMed1993

Support for the shape concept of lipid structure based on a headgroup volume approach.

Headgroup volumes of seven dioleoyl lipid species, calculated from covalent radii, are shown to correlate linearly (r = 0.95) with the ability of those lipids to alter the midpoint temperature of the lamellar to inverted hexagonal phase transition (L alpha-->HII) of a 95 mole fraction percent phosphatidylethanolamine matrix. The results illustrate the utility of the shape concept and basic considerations of headgroup volume as a predictive tool for the determination of lipid structure.

Biophysical Phenomena↗
PubMed1992

Nature of alcohol and anesthetic action on cooperative membrane equilibria.

A generalized, colligative thermodynamic framework is used to treat the action of solutes on cooperative membrane equilibria. Configurational entropy, the randomness imparted by solutes through the partitioning or mixing process, is implicated as the energetic driving force for the action of anesthetics on cooperative membrane equilibria. The equilibria predicted to be most sensitive to solute action--in which the dilute solute causes a perturbation equivalent to a large change in temperature--are (1) low-enthalpy processes that coincide with (2) large partitioning differences between states. The model stresses that solutes do not act at a single site, but on both states in an equilibrium, and that the perturbation is determined by the difference in entropy. Evidence for the thermodynamic framework is obtained from the partitioning behavior of the general anesthetic 1-hexanol into a model lecithin (DMPC; 1,2-dimyristoyl-sn-glycero-3-phosphocholine) membrane as a function of temperature and alcohol concentration. The low-enthalpy equilibrium between the gel (L beta') and ripple states (P beta') (pretransition) is more sensitive to 1-hexanol than the high-enthalpy equilibrium between the ripple (P beta') and fluid bilayer states (L alpha) (main transition). The perturbations of both equilibria are accurately described by the colligative thermodynamic framework. The results suggest that alcohols and anesthetics act through entropy to upset the natural thermal balance that maintains native membrane architecture.

Anesthetics↗
PubMed1992

The characterization of two monoclonal antibodies which react with high molecular weight antigens of asexual Plasmodium falciparum.

We prepared rat monoclonal antibodies (mAb) specific for very large Plasmodium falciparum proteins to assist in their characterization. Hybridomas prepared from rats immunized with parasitized erythrocyte (PE) proteins of greater than 200 kDa exhibited two patterns of Western blot reactivity with PE SDS extracts: one represented by clone 41E11 (IgM, kappa), the other by clone 12C11 (IgM, lambda). MAb 41E11 reacted by Western blotting with at least 15 antigens, most of which comigrated with antigens identified by the 33G2 human IgM mAb. The stage specificity of mAb 41E11 reactivity and indirect immunofluorescence (IFA) pattern closely resemble those previously described for antigens that share the EEXXEE sequence motif. Unlike mAb 33G2, MAb 41E11 immunoprecipitated a biosynthetically radiolabeled protein of 320 kDa. MAb 41E11 did not immunoprecipitate any cell surface 125I proteins. MAb 12C11 reacted on Western blotting with a different group of malarial antigens of approximately 44, 95, 117, 145, and 310 kDa, as well as with some low-molecular-weight, uninfected erythrocyte antigens. MAb 12C11 did not immunoprecipitate any cell surface 125I or biosynthetically labeled proteins. The 310-kDa antigen recognized by mAb 12C11 (denoted Ag 12A) does not correspond to PfEMP2 or the 320-kDa antigen recognized by mAbs 33G2 or 41E11. With trophozoites and more mature stages, fixed IFA reactivity of mAb 12C11 was at the parasite and in antigen aggregates in the host cell cytoplasm that extended to the PE plasma membrane. Indirect results suggest that Ag 12A does not correspond to cell surface-exposed PfEMP1 and is most likely a hitherto unidentified malarial protein.

Animals↗
PubMed1992

Trafficking of malarial proteins to the host cell cytoplasm and erythrocyte surface membrane involves multiple pathways.

During the asexual stage of malaria infection, the intracellular parasite exports membranes into the erythrocyte cytoplasm and lipids and proteins to the host cell membrane, essentially "transforming" the erythrocyte. To investigate lipid and protein trafficking pathways within Plasmodium falciparum-infected erythrocytes, synchronous cultures are temporally analyzed by confocal fluorescence imaging microscopy for the production, location and morphology of exported membranes (vesicles) and parasite proteins. Highly mobile vesicles are observed as early as 4 h postinvasion in the erythrocyte cytoplasm of infected erythrocytes incubated in vitro with C6-NBD-labeled phospholipids. These vesicles are most prevalent in the trophozoite stage. An immunofluorescence technique is developed to simultaneously determine the morphology and distribution of the fluorescent membranes and a number of parasite proteins within a single parasitized erythrocyte. Parasite proteins are visualized with FITC- or Texas red-labeled monoclonal antibodies. Double-label immunofluorescence reveals that of the five parasite antigens examined, only one was predominantly associated with membranes in the erythrocyte cytoplasm. Two other parasite antigens localized only in part to these vesicles, with the majority of the exported antigens present in lipid-free aggregates in the host cell cytoplasm. Another parasite antigen transported into the erythrocyte cytoplasm is localized exclusively in lipid-free aggregates. A parasite plasma membrane (PPM) and/or parasitophorous vacuolar membrane (PVM) antigen which is not exported always colocalizes with fluorescent lipids in the PPM/PVM. Visualization of two parasite proteins simultaneously using FITC- and Texas red-labeled 2 degrees antibodies reveals that some parasite proteins are constitutively transported in the same vesicles, whereas other are segregated before export. Of the four exported antigens, only one appears to cross the barriers of the PPM and PVM through membrane-mediated events, whereas the others are exported across the PPM/PVM to the host cell cytoplasm and surface membrane through lipid (vesicle)-independent pathways.

4-Chloro-7-nitrobenzofurazan↗
PubMed1991

Direct access to serum macromolecules by intraerythrocytic malaria parasites.

Trafficking pathways in malaria-infected erythrocytes are complex because the internal parasite is separated from the serum by the erythrocyte and parasitophorous vacuolar membranes. Intraerythrocytic Plasmodium falciparum parasites can endocytose dextrans, protein A and an IgG2a antibody. Here we show that these macromolecules do not cross the erythrocyte or parasitophorous vacuolar membranes, but rather gain direct access to the aqueous space surrounding the parasite through a parasitophorous duct. Evidence for this structure includes visualization of membranes that are continuous between the parasitophorous vacuolar and erythrocyte membranes, and surface labelling of the parasite with fluorescent macromolecules under conditions that block endocytosis. The parasite can internalize by fluid-phase endocytosis macromolecules from the aqueous compartment surrounding it. Thus, surface antigens on trophozoites and schizonts should be considered as targets for antibody-directed parasiticidal agents.

Animals↗
PubMed1991

Maintenance of structural and functional characteristics of skeletal-muscle mitochondria and sarcoplasmic-reticular membranes after chronic ethanol treatment.

The effect of long-term ethanol intake on the structural and functional characteristics of rat skeletal-muscle mitochondria and sarcoplasmic reticulum was investigated. Functionally, skeletal-muscle mitochondria were characterized by a high respiratory control index and ADP/O ratio and a high State-3 respiration rate with different substrates. These parameters were not significantly different in preparations from control and ethanol-fed rats, except for a small increase in the rate of oxidation of alpha-oxoglutarate/malate in the latter. In submitochondrial particles from the two groups of animals there was no significant difference in cytochrome content, ATPase activity or the activity of respiratory-chain complexes. Mitochondrial membranes from untreated and ethanol-fed rats showed no difference in the baseline e.s.r. order parameter, and both preparations were equally sensitive to disordering by ethanol in vitro. Similarly, sarcoplasmic-reticulum preparations were not significantly affected by long-term ethanol feeding with respect to Ca2(+)-ATPase activity or in baseline order parameter and susceptibility to membrane disordering by ethanol in vitro. These membranes were also equally sensitive to degradation by exogenous phospholipase A2. Ethanol feeding did not alter the class composition of mitochondrial or sarcoplasmic-reticulum membrane phospholipids, nor the acyl composition of individual phospholipid classes. Specifically, the changes in acyl composition that characteristically occur in liver microsomal phosphatidylinositol and liver mitochondrial cardiolipin were not observed in the corresponding phospholipids from skeletal-muscle membranes. In experiments where membrane preparations from liver and skeletal muscle from the same ethanol-fed animals were compared, the liver membranes developed membrane tolerance, with the muscle membranes retaining normal sensitivity to disordering effects by ethanol. It is concluded that: (a) different tissues from the same animals differ in their susceptibility to ethanol; (b) the tissue-specific lack of development of membrane tolerance correlates with a lack of chemical changes in the phospholipids and with a retention of normal function of mitochondria and sarcoplasmic reticulum; (c) effects of chronic ethanol intake on muscle function are not due to a defect in the mitochondrial energy supply.

Adenosine Triphosphatases↗