Preparation and properties of FabIgG,a chimeric univalent antibody designed to attack tumour cells

In order to promote the killing of tumour cells by antibody a derivative has been synthesized in which Fab'gamma from xenogeneic antibody is thioether-bonded to half-cystine on normal IgG of the species to be treated. The resulting entity, FabIgG, is obtained with about a 40% yield of the starting Fab'gamma. Being univalent it evades antigenic modulation. It activates complement efficiently, is minimally immunogenic, and appears to be catabolized at the slow rate characteristic of autologous IgG.     

A deuterium and phosphorus-31 nuclear magnetic resonance study of the interaction of melittin with dimyristoylphosphatidylcholine bilayers and the effects of contaminating phospholipase A2

The interaction of bee venom melittin with dimyristolphosphatidylcholine (DMPC) selectively deuteriated in the choline head group has been studied by deuterium and phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy. The action of residual phospholipase A2 in melittin samples resulted in mixtures of DMPC and its hydrolytic products that underwent reversible transitions at temperatures between 30 and 35 degrees C from extended bilayers to micellar particles which gave narrow single-line deuterium and phosphorus-31 NMR spectra. Similar transitions were observed in DMPC-myristoyllysophosphatidylcholine (lysoPC)-myristic acid mixtures containing melittin but not in melittin-free mixtures, indicating that melittin is able to stabilize extended bilayers containing DMPC and its hydrolytic products in the liquid-crystalline phase. Melittin, free of phospholipase A2 activity, and at 3-5 mol% relative to DMPC, induced reversible transitions between extended bilayers and micellar particles on passing through the liquid-crystalline to gel phase transition temperature of the lipid, effects similar to those observed in melittin-acyl chain deuterated dipalmitoylphosphatidylcholine (DPPC) mixtures [Dufourc, E. J., Smith, I. C. P., & Dufourcq, J. (1986) Biochemistry 25, 6448-6455]. LysoPC at concentrations of 20 mol% or greater relative to DMPC induced transitions between extended bilayers and micellar particles with characteristics similar to those induced by melittin. It is proposed that these melittin- and lysoPC-induced transitions share similar mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Generating sucrose gradients in three minutes by tilted tube rotation

A technique which permits rapid preparation of sucrose gradients with highly reproducible profiles is described. Tubes are filled with equal volumes of light and heavy sucrose solutions, sealed, and rotated for 3 min tilted 80 degrees from vertical. Linear 5-20% and 5-30% gradients and nonlinear but useful 5-45% gradients are obtained.     

Membrane proteins correlated with expression of the polysialic acid capsule in Escherichia coli K1.

Growth of Escherichia coli K1 strains at 15 degrees C results in a defect in the synthesis or assembly of the K1 polysialic acid capsule. Synthesis is reactivated in cells grown at 15 degrees C after upshift to 37 degrees C, and activation requires protein synthesis (Whitfield et al., J. Bacteriol. 159:321-328, 1984). Using this temperature-induced defect, we determined the molecular weights and locations of membrane proteins correlated with the expression of K1 (polysialosyl) capsular antigen. Pulse-labeling experiments demonstrated the presence of 11 proteins whose synthesis was correlated with capsule appearance at the cell surface. Using the differential solubility of inner and outer membranes in the detergent Sarkosyl, we localized five of the proteins in the outer membrane and four in the inner membrane. The subcellular location of two of the proteins was not determined. Five proteins appeared in the membrane simultaneously with the initial expression of the K1 capsule at the cell surface. One of these proteins, a 40,000-dalton protein localized in the outer membrane, was identified as porin protein K, which previously has been shown to be present in the outer membrane of encapsulated E. coli. The possible role of these proteins in the synthesis of the polysialosyl capsule is discussed.     

Effect of calmodulin on the structural state of photoreceptor membranes and rhodopsin-containing phospholipid vesicles

The effect of calmodulin on the order of lipids in rhodopsin-free and rhodopsin-containing membranes has been studied using spin-label electron spin resonance methods. Calmodulin, up to 10(-6)M, did not change the measured order of lipids in bilayer membranes containing only rhodopsin. However, for bovine rod outer segment disc membranes, which contain rhodopsin and other proteins, calmodulin induced a significant concentration and temperature dependent increase in the order of the membrane lipids. This suggests that the site of calmodulin binding is remote from rhodopsin itself, and the nature of the binding appears to be a membrane surface phenomenon.     

Deuterium and phosphorus nuclear magnetic resonance studies on the binding of polymyxin B to lipid bilayer-water interfaces

Deuterium and phosphorus NMR methods have been used to study the binding of polymyxin B to the surface of bilayers containing lipids that were deuterated at specific positions in the polar head-group region. The binding of polymyxin B to acidic dimyristoylphosphatidylglycerol (DMPG) membranes induces only small structural distortions of the glycerol head group. The deuterium spin-lattice relaxation times for the different carbon-deuterium bonds in the head group of the same phospholipid are greatly reduced on binding of polymyxin B, indicating a restriction of the motional rate of the glycerol head group. Only very weak interactions were detected between polymyxin B and bilayers of zwitterionic dimyristoylphosphatidylcholine (DMPC). In mixed bilayers of the two phospholipid types, in which either of the two phospholipids was deuterated, the presence of polymyxin B caused a lateral phase separation into DMPG-enriched phospholipid-peptide clusters and a DMPG-depleted phase. Complete phase separation did not occur: peptide-containing complexes with charged phosphatidylglycerol contained substantial amounts of zwitterionic phosphatidylcholine. Exchange of both phospholipid types between complexes and the bulk lipid matrix was shown to be fast on the NMR time scale, with a lifetime for phospholipid-peptide association of less than 1 ms.     

Lipid-protein interactions in frog rod outer segment disc membranes. Characterization by spin labels

Freely-diffusing phospholipid spin labels have been employed to study rhodopsin-lipid interactions in frog rod outer segment disc membranes. Examination of the ESR spectra leads us to the conclusion that there are two motionally distinguishable populations of lipid existing in frog rod outer segment membranes over a wide physiological temperature range. Each of the spin probes used shows a two-component electron spin resonance (ESR) spectrum, one component of which is motionally restricted on the ESR timescale, and represents between 33 and 40% of the total integrated spectral intensity. The second spectral component which accounts for the remainder of the spectral intensity possesses a lineshape characteristic of anisotropic motion in a lipid bilayer, very similar in shape to that observed from the same spin labels in dispersions of whole extracted frog rod outer segment lipid. The motionally restricted spectral component is attributed to those spin labels in contact with the surface of rhodospin, while the major component is believed to originate from spin labels in the fluid lipid bilayer region of the membranes. Calculations indicate that the motionally restricted lipid is sufficient to cover the protein surface. This population of lipids is shown here and elsewhere (Watts, A., Volotovski, I.D. and Marsh, D. (1979) Biochemistry 18, 5006-5013) to be by no means rigidly immobilized, having motion in the 20 ns time regime as opposed to motions in the one nanosecond time regime found in the fluid bilayer. Little selectivity for the motionally restricted population is observed between the different spin-labelled phospholipid classes nor with a spin-labelled fatty acid or sterol.     

Cooperativity of the phase transition in single- and multibilayer lipid vesicles.

The effect of membrane morphology on the cooperativity of the ordered-fluid, lipid phase transition has been investigated by comparing the transition widths in extended, multibilayer dispersons of dimyristoyl phosphatidyl-choline, and also of dipalmitoyl phosphatidylcholine, with those in the small, single-bilayer vesicles obtained by sonication. The electron spin resonance spectra of three different spin-labelled probes, 2,2,6,6-tetramethylpiperdine-N-oxyl, phosphatidylcholine and stearic acid, and also 90 degrees light scattering and optical turbidity measurements were used as indicators of the phase transition. In all cases the transition was broader in the single-bilayer vesicles than in the multibilayer dispersions, corresponding to a decreased cooperativity on going to the small vesicles. Comparison of the light scattering properties of centrifuged and uncentrifuged, sonicated vesicles suggests that these are particularly sensitive to the presence of intermediate-size particles, and thus the spin label measurements are likely to give a more reliable measure of the degree of cooperativity of the small, single-bilayer vesicles. Application of the Zimm and Bragg theory ((1959) J. Chem. Phys. 31, 526-535) of cooperative transitions to the two-dimensional bilayer system shows that the size of the cooperative unit, 1/square root sigma, is a measure of the mean number of molecules per perimeter molecule, in a given region of ordered or fluid lipid at the centre of the transition. From this result it is found that it is the vesicle size which limits the cooperativity of the transition in the small, single-bilayer vesicles. The implications for the effect of membrane structure and morphology on the cooperativity of phase transitions in biological membranes, and for the possibility of achieving lateral communication in the plane of the membrane, are discussed.     

SUMO modification is involved in the maintenance of heterochromatin stability in fission yeast

Several studies have suggested that SUMO may participate in the regulation of heterochromatin, but direct evidence is lacking. Here, we present a direct link between sumoylation and heterochromatin stability. SUMO deletion impaired silencing at heterochromatic regions and induced histone H3 Lys4 methylation, a hallmark of active chromatin in fission yeast. Our findings showed that the SUMO-conjugating enzyme Hus5/Ubc9 interacted with the conserved heterochromatin proteins Swi6, Chp2 (a paralog of Swi6), and Clr4 (H3 Lys9 methyltransferase). Moreover, chromatin immunoprecipitation (ChIP) revealed that Hus5 was highly enriched in heterochromatic regions in a heterochromatin-dependent manner, suggesting a direct role of Hus5 in heterochromatin formation. We also found that Swi6, Chp2, and Clr4 themselves can be sumoylated in vivo and defective sumoylation of Swi6 or Chp2 compromised silencing. These results indicate that Hus5 associates with heterochromatin through interactions with heterochromatin proteins and modifies substrates whose sumoylations are required for heterochromatin stability, including heterochromatin proteins themselves.     

Activation of a novel PKC isoform synergistically enhances D2L dopamine receptor-mediated sensitization of adenylate cyclase type 6

Despite acutely inhibiting adenylate cyclase, prolonged activation of Galpha(i/o)-coupled receptors leads to a subsequent heterologous sensitization of adenylate cyclase responsiveness. Recently, protein kinase signaling and phosphorylation have been implicated in the sensitization of adenylate cyclase type 6 (AC6). To examine the sensitization specifically of AC6, we constructed human embryonic kidney cells (HEK293) cells stably expressing AC6 and the Galpha(i/o)-coupled D2L dopamine receptor. In contrast to observations in delta-opioid-expressing Chinese hamster ovary (CHO) cells that express endogenous AC6 and AC7, neither protein kinase C (PKC) nor tyrosine kinase inhibitors attenuated D2L receptor-mediated sensitization of AC6. Inhibition of Raf1 modestly inhibited the magnitude of D2L receptor-induced sensitization of AC6; however, activation of PKC robustly enhanced D2L receptor-mediated AC6 sensitization in a Raf1-dependent manner. These data indicate that, although PKC and Raf1 are not required for sensitization, activation of the PKC-Raf1 pathway robustly potentiated D2L receptor-mediated sensitization of AC6.