Thermodynamics of glycophorin in phospholipid bilayer membranes

We have developed a model of glycophorin in a phospholipid bilayer membrane in order to study the thermodynamics of this system and to understand the detailed behavior of recent calorimetric data. We assume that the larger glycophorin polar group can be considered as either adopting a pancakelike conformation at the bilayer interface (D state) or be directed generally away from the interface (U state) [Ruppel, D., Kapitza, H.G., Galla, H.J., Sixl, F., & Sackmann, E. (1982) Biochim. Biophys. Acta 692, 1-17]. Lipid hydrocarbon chains are described either as excited (e state) with high energy and relatively many gauche conformers or as generally extended (g state) with low energy. We performed a Monte-Carlo simulation using the Glauber and Kawasaki procedures on a triangular lattice which represents the plane of half of the bilayer. Lattice sites can be occupied either by lipid hydrocarbon chains or by model glycophorin alpha-helical hydrophobic cores. The states D and U are represented by hexagons of different sizes in the plane of the lattice, and the hard core repulsion between two such polar groups is accounted for by forbidding hexagon-hexagon overlap. We have studied the effect of having the glycophorin polar group interact in various ways with the lipid bilayer. We find that the protein polar group in its D state interacts, either directly or indirectly, with the lipid bilayer so as to reduce the effective lateral pressure acting on the lipid hydrocarbon chains by about 3 dyn/cm. Polar groups in their U states do not reduce this lateral pressure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nonpolymorphic regions of p190, a protein of the Plasmodium falciparum erythrocytic stage, contain both T and B cell epitopes

Two conserved regions from the genetically polymorphic p190 molecule of the malaria parasite Plasmodium falciparum have previously been expressed in Escherichia coli as separate polypeptides (190.L and 190.M) or as a single fusion protein (190.N). In the present study we investigated whether human B and T lymphocytes recognize these conserved regions. The more amino-terminal region, 190.L (corresponding to residues 188-363 of the encoded protein sequence) reacted preferentially with sera from donors living in a malaria-endemic area. Also, EBV-transformed B cells, from a healthy donor living in a malaria-mesoendemic area, were fused with a human-mouse hybrid line (SPM4-0), yielding two hybridomas whose products recognized both 190.L and the fusion protein 190.N, but not the 190.M polypeptide. A large number of p190-specific T cell clones were obtained from PBMC of a noninfected donor, after in vitro stimulation with the recombinant fusion protein 190.N. The clones reacted with intact, parasite-derived p190, as well as either 190.L or 190.M. Four clones that recognized the more amino-terminal fragment also responded to infected E. According to these results the more amino-terminal conserved sequences of p190 have the requisites to be immunogenic in humans.     

Electron spin resonance and steady-state fluorescence polarization studies of lipid bilayers containing integral proteins

We derive equations that describe changes in the steady-state fluorescence polarization of the probe 1,6-diphenyl-1,3,5-hexatriene (DPH) or in the spectrum of electron spin resonance (ESR) nitroxide spin-labeled lipid probes as a function of the intrinsic molecule concentration in lipid bilayer membranes. We make use of an assumption used by us in an earlier paper. The equations are independent of any membrane model. They are valid when a DPH probe or a spin-labeled chain is equivalent to an unlabeled lipid hydrocarbon chain only as far as their general space-filling properties are concerned. We consider cases where the bilayer is either in a single homogeneous phase or in a two-phase region. We apply our equations to analyze ESR data from delipidated sarcoplasmic reticulum membranes and from egg yolk phosphatidylcholine bilayers containing Ca2+-ATPase, and DPH data from dipalmitoylphosphatidylcholine (DPPC) bilayers containing Ca2+-ATPase, both for T greater than Tc. The following conclusions were derived: (i) Ca2+-ATPase oligomers are "randomly" distributed, for the concentrations studied, in the fluid phase. (ii) There is no fixed stoichiometric ratio of "boundary" lipids and oligomers. (iii) Between 24k and 28k lipid molecules are able to surround each isolated oligomer composed of k Ca2+-ATPase monomers. Finally, we apply our equations to analyze DPH studies on DPPC bilayers containing Ca2+-ATPase for T less than Tc. We find that the results reported are in accord with the predictions of the model. In the Appendix, we show that an analytical expression for probabilities used by us is in very good agreement with the results of computer simulation.     

Interactions between two sheets of a bilayer membrane and its internal lateral pressure

We have modelled a phospholipid bilayer as two monolayer sheets which interact with each other by a coupling which depends upon the states of the lipid hydrocarbon chains in each sheet. We make use of a model (Georgallas and Pink 1982a) and its parameters, already used to study monolayer phase changes at the LC-LE transition, in order to study the lipid main transition. Although the monolayer coexistence curve can be calculated exactly, we have made use of high-temperature series expansions to calculate the critical point of the bilayer. We also present the results of computer simulations on triangular lattices for the pressure-area isotherms. We find: (i) the interaction between the sheets of a DPPC bilayer is about 1.5–2% of the maximum interaction within the plane of each sheet; (ii) the internal lateral pressure of a DPPC bilayer is about 30.5 dyne/cm; (iii) the bilayer transition enthalpy depends sensitively upon the coupling between the sheets. Should this coupling vary from sample to sample (due, possibly, to its preparation) then very different values of transition enthalpy may be measured. (iv) We present a rough rule-of-thumb for estimating the internal lateral pressure of a bilayer from a knowledge of the corresponding monolayer pressure-area isotherms.Abbreviations LC-LE liquid condensed — liquid expanded - DPPC dipalmitoylphosphatidylcholine - Q transition enthalpy Work supported in part by the Natural Sciences and Engineering Research Council of Canada     

Determination of radioactivity of proline and hydroxyproline in tissue hydrolysates after the elimination of interference by primary amino acids by deamination

A simple method for the determination of radioactivity of proline and hydroxyproline, particularly of small amounts, in hydrolysates of tissues is described. Specificity is assured by eliminating primary amino acids from the hydrolysates by deamination and then extraction before separation of proline from hydroxyproline by paper chromatography. Six to eight tissue samples may be compared simultaneously. The efficiency and reproducibility are good, as indicated by the use of labeled l-proline, labeled dl-hydroxyproline, a hydrolysate of a protein in which the amino acids (and proline) were labeled, and hydrolysates of tissues cultured in media containing radioactive l-proline. The method is particularly useful when ion-exchange column chromatography of amino acids is not in routine use.     

Application of an optimized marker amplification protocol in immunohistochemistry — a valuable tool for visualizing antigenic sites of low signal strength or sparse distribution

Amplification of immunohistochemical markers received considerable attention during the 1980s and 1990s. The amplification approach was largely abandoned following the development of antigen retrieval and reporter amplification techniques, because the latter were incorporated more easily into high throughput automated procedures in industrial and diagnostic laboratories. There remain, however, a number of instances where marker amplification still has much to offer. Consequently, we examined experimentally the utility of an optimized marker amplification technique in diagnostically relevant tissue where either the original signal strength was low or positive sites were visible, but sparsely distributed. Marker amplification in the former case not only improved the visibility of existing positive sites, but also revealed additional sites that previously were undetectable. In the latter case, positive sites were rendered more intense and therefore more easily seen during low magnification examination of large areas of tissue.     

EFFECT OF VITAMIN E SUPPLEMENTATION AND PARTIAL SUBSTITUTION OF POLY- WITH MONO-UNSATURATED FATTY ACIDS IN PIG DIETS ON MUSCLE,AND MICROSOME EXTRACT a-TOCOPHEROL CONCENTRATION AND LIPID OXIDATION

The experiment was organized in a 3×2 factorial arrangement with three dietary fat blends and a basal (20 mg kg^?1 diet) or supplemented (220 mg kg^?1) level of α-tocopheryl acetate. Dietary vitamin E and monounsaturated to polyunsaturated fatty acid ratio (dietary MUFA/PUFA) affected muscle α-tocopherol concentration (α-tocopherol [log μg g^?1]=0.18 (±0.105)+0.0034 (±0.0003)·dietary α-tocopherol [mg kg^?1 diet] (P<0.0001)+0.39 (±0.122)·dietary MUFA/PUFA (P<0.0036)). An interaction between dietary α-tocopherol and dietary MUFA/PUFA exists for microsome α-tocopherol concentration (α-tocopherol [log μg g^?1]=1.14 (±0.169) (P<0.0001)+0.0056 (±0.00099)·dietary α-tocopherol [mg kg^?1 diet] (P<0.0001)+0.54 (±0.206)·dietary MUFA/PUFA (P<0.0131)?0.0033 (±0.0011)·dietary α-tocopherol [mg kg^?1)]×dietary MUFA/PUFA (P<0.0067)), and hexanal concentration in meat (hexanal [ng·g^?1]=14807.9 (±1489.8)?28.8 (±10.6) dietary α-tocopherol [mg·kg^?1] (P<0.01)?8436.6 (±1701.6)·dietary MUFA/PUFA (P<0.001)+24.0 (±11.22)·dietary α-tocopherol·dietary MUFA/PUFA (P<0.0416)). It is concluded that partial substitution of dietary PUFA with MUFA lead to an increase in the concentration of α-tocopherol in muscle and microsome extracts. An interaction between dietary α-tocopherol and fatty acids exists, in which at low level of dietary vitamin E inclusion, a low MUFA/PUFA ratio leads to a reduction in the concentration of α-tocopherol in microsome extracts and a concentration of hexanal in meat above the expected values.     

Quirks of dye nomenclature. 5. Rhodamines

Rhodamines were first produced in the late 19^th century, when they constituted a new class of synthetic dyes. These compounds since have been used to color many things including cosmetics, inks, textiles, and in some countries, food products. Certain rhodamine dyes also have been used to stain biological specimens and currently are widely used as fluorescent probes for mitochondria in living cells. The early history and current biological applications are sketched briefly and an account of the ambiguities, complications and confusions concerning dye identification and nomenclature are discussed.     

Quirks of dye nomenclature. 6. Malachite green

Malachite green was discovered independently by two researchers in Germany in the 19^th century and found immediate employment as a dye and a pigment. Subsequently, other uses, such as staining biological specimens, emerged. A much later application was the control of fungal and protozoan infections in fish, for which the dye remains popular, although illegal in many countries owing to a variety of toxicity problems. In solution, malachite green can exist as five different species depending on the pH. The location of the positive charge of the colored cation on a carbon atom or a nitrogen atom is still debated. The original names of this dye, and their origins, are briefly surveyed.