Influence of cholesterol on gramicidin-induced HII phase formation in phosphatidylcholine model membranes

The influence of cholesterol incorporation on gramicidin-induced hexagonal HII phase formation in different phosphatidylcholine model systems was investigated by 31P- and 2H-NMR, small-angle X-ray diffraction and differential scanning calorimetry. In liquid-crystalline distearoylphosphatidylcholine systems cholesterol inhibits gramicidin-induced HII phase formation. In dioleoylphosphatidylcholine the opposite effect is observed. Cholesterol appears to preferentially interact with gramicidin under liquid-crystalline conditions in both systems. Two phenomena that had been reported for gramicidin-treated erythrocyte membranes and derived liposomes (Tournois, H., Leunissen-Bijvelt, J., Haest, C.W.M., De Gier, J. and De Kruijff, B. (1987) Biochemistry, 26, 6613-6621) could also be observed in more simple dioleoylphosphatidylcholine-gramicidin-cholesterol systems. These are (i) an increase in tube diameter in the gramicidin-induced HII phase with increasing temperature, which is ascribed to the presence of cholesterol in this phase, and (ii) the loss of the hexagonal HII phase related 31P-NMR line shape at lower temperatures despite the presence of this phase as demonstrated with X-ray diffraction. This latter phenomenon appears to be due to restrictions in the rate of lateral diffusion of the phospholipids around the HII tubes due to the presence of gramicidin.     

Delta psi stimulates membrane translocation of the C-terminal part of a signal sequence.

For several proteins in Escherichia coli it has been shown that the protonmotive force (pmf) dependence of translocation can be varied with the signal sequence composition, suggesting an effect of the pmf on the signal sequence. To test this possibility, we analyzed the effect of the membrane potential on translocation of the signal sequence. For this purpose, a precursor peptide was used (SP+7), corresponding to the signal sequence of PhoE with the first seven amino acids of the mature part that can be processed by purified leader peptidase. Translocation was studied in pure lipid vesicles containing leader peptidase, with its active site inside the vesicles. In the presence of a positive inside Delta psi, the amount of processing of SP+7 was significantly higher than without a Delta psi, indicating that the translocation of the cleavage region is stimulated by Delta psi. Replacement of the helix-breaking glycine residue at position -10 in the signal sequence for a leucine abolished the effect of Delta psi on the translocation of the cleavage region. It is concluded that Delta psi directly acts on the wild type signal sequence by stimulating the translocation of its C terminus. We propose that Delta psi acts on the signal sequence by stretching it into a transmembrane orientation.     

Transmembrane peptides stabilize inverted cubic phases in a biphasic length-dependent manner: implications for protein-induced membrane fusion

WALP peptides consist of repeating alanine-leucine sequences of different lengths, flanked with tryptophan "anchors" at each end. They form membrane-spanning alpha-helices in lipid membranes, and mimic protein transmembrane domains. WALP peptides of increasing length, from 19 to 31 amino acids, were incorporated into N-monomethylated dioleoylphosphatidylethanolamine (DOPE-Me) at concentrations up to 0.5 mol % peptide. When pure DOPE-Me is heated slowly, the lamellar liquid crystalline (L(alpha)) phase first forms an inverted cubic (Q(II)) phase, and the inverted hexagonal (H(II)) phase at higher temperatures. Using time-resolved x-ray diffraction and slow temperature scans (1.5 degrees C/h), WALP peptides were shown to decrease the temperatures of Q(II) and H(II) phase formation (T(Q) and T(H), respectively) as a function of peptide concentration. The shortest and longest peptides reduced T(Q) the most, whereas intermediate lengths had weaker effects. These findings are relevant to membrane fusion because the first step in the L(alpha)/Q(II) phase transition is believed to be the formation of fusion pores between pure lipid membranes. These results imply that physiologically relevant concentrations of these peptides could increase the susceptibility of biomembrane lipids to fusion through an effect on lipid phase behavior, and may explain one role of the membrane-spanning domains in the proteins that mediate membrane fusion.     

A genome-wide analysis of biomineralization-related proteins in the sea urchin Strongylocentrotus purpuratus

Biomineralization, the biologically controlled formation of mineral deposits, is of widespread importance in biology, medicine, and engineering. Mineralized structures are found in most metazoan phyla and often have supportive, protective, or feeding functions. Among deuterostomes, only echinoderms and vertebrates produce extensive biomineralized structures. Although skeletons appeared independently in these two groups, ancestors of the vertebrates and echinoderms may have utilized similar components of a shared genetic "toolkit" to carry out biomineralization. The present study had two goals. First, we sought to expand our understanding of the proteins involved in biomineralization in the sea urchin, a powerful model system for analyzing the basic cellular and molecular mechanisms that underlie this process. Second, we sought to shed light on the possible evolutionary relationships between biomineralization in echinoderms and vertebrates. We used several computational methods to survey the genome of the purple sea urchin Strongylocentrotus purpuratus for gene products involved in biomineralization. Our analysis has greatly expanded the collection of biomineralization-related proteins. We have found that these proteins are often members of small families encoded by genes that are clustered in the genome. Most of the proteins are sea urchin-specific; that is, they have no apparent homologues in other invertebrate deuterostomes or vertebrates. Similarly, many of the vertebrate proteins that mediate mineral deposition do not have counterparts in the S. purpuratus genome. Our findings therefore reveal substantial differences in the primary sequences of proteins that mediate biomineral formation in echinoderms and vertebrates, possibly reflecting loose constraints on the primary structures of the proteins involved. On the other hand, certain cellular and molecular processes associated with earlier events in skeletogenesis appear similar in echinoderms and vertebrates, leaving open the possibility of deeper evolutionary relationships.     

Effects of aphidicolin on premature condensation of sperm chromosomes in fertilized sea urchin eggs

Unfertilized Strongylocentrotus purpuratus eggs may be treated with ammonia to initiate maternal DNA replication and the maternal cell cycle. When these eggs are polyspermically fertilized 75 min after the beginning of ammonia treatment, the nuclei of the fertilizing spermatozoa undergo premature chromosome condensation (PCC) in an apparent attempt to conform to the advanced maternal cell cycle. PCC is inhibited if maternal DNA replication is blocked by exposing the eggs to aphidicolin but will proceed if this exposure begins after replication is complete. Additionally, PCC will proceed in ammonia-activated, polyspermically fertilized anucleate merogons in the continuous presence of aphidicolin. These results suggest that the direct inhibitory effect of aphidicolin may well be limited to the replication of DNA and that the unreplicated maternal nucleus itself exerts negative control over the development of chromosome-condensing conditions in the maternal cytoplasm.     

Probing the lipid-protein interface using model transmembrane peptides with a covalently linked acyl chain

The aim of this study was to gain insight into how interactions between proteins and lipids in membranes are sensed at the protein-lipid interface. As a probe to analyze this interface, we used deuterium-labeled acyl chains that were covalently linked to a model transmembrane peptide. First, a perdeuterated palmitoyl chain was coupled to the Trp-flanked peptide WALP23 (Ac-CGWW(LA)₈LWWA-NH₂), and the deuterium NMR spectrum was analyzed in di-C18:1-phosphatidylcholine (PC) bilayers. We found that the chain order of this peptide-linked chain is rather similar to that of a noncovalently coupled perdeuterated palmitoyl chain, except that it exhibits a slightly lower order. Similar results were obtained when site-specific deuterium labels were used and when the palmitoyl chain was attached to the more-hydrophobic model peptide WLP23 (Ac-CGWWL₁₇WWA-NH₂) or to the Lys-flanked peptide KALP23 (Ac-CGKK(LA)₈LKKA-NH₂). The experiments showed that the order of both the peptide-linked chains and the noncovalently coupled palmitoyl chains in the phospholipid bilayer increases in the order KALP23 < WALP23 < WLP23. Furthermore, changes in the bulk lipid bilayer thickness caused by varying the lipid composition from di-C14:1-PC to di-C18:1-PC or by including cholesterol were sensed rather similarly by the covalently coupled chain and the noncovalently coupled palmitoyl chains. The results indicate that the properties of lipids adjacent to transmembrane peptides mostly reflect the properties of the surrounding lipid bilayer, and hence that (at least for the single-span model peptides used in this study) annular lipids do not play a highly specific role in protein-lipid interactions.     

Estimation of individual muscle force using elastography

Background

Estimation of an individual muscle force still remains one of the main challenges in biomechanics. In this way, the present study aimed: (1) to determine whether an elastography technique called Supersonic Shear Imaging (SSI) could be used to estimate muscle force, (2) to compare this estimation to that one provided by surface electromyography (EMG), and (3) to determine the effect of the pennation of muscle fibers on the accuracy of the estimation.

Methods and Results

Eleven subjects participated in two experimental sessions; one was devoted to the shear elastic modulus measurements and the other was devoted to the EMG recordings. Each session consisted in: (1) two smooth linear torque ramps from 0 to 60% and from 0 to 30% of maximal voluntary contraction, for the first dorsal interosseous and the abductor digiti minimi, respectively (referred to as “ramp contraction”); (2) two contractions done with the instruction to freely change the torque (referred to as “random changes contraction”). Multi-channel surface EMG recordings were obtained from a linear array of eight electrodes and the shear elastic modulus was measured using SSI. For ramp contractions, significant linear relationships were reported between EMG activity level and torque (R² = 0.949±0.036), and between shear elastic modulus and torque (R² = 0.982±0.013). SSI provided significant lower RMS_deviation between measured torque and estimated torque than EMG activity level for both types of contraction (1.4±0.7 vs. 2.8±1.4% of maximal voluntary contraction for “ramp contractions”, p<0.01; 4.5±2.3 vs. 7.9±5.9% of MVC for “random changes contractions”, p<0.05). No significant difference was reported between muscles.

Conclusion

The shear elastic modulus measured using SSI can provide a more accurate estimation of individual muscle force than surface EMG. In addition, pennation of muscle fibers does not influence the accuracy of the estimation.     

Mutations conferring lincomycin, spectinomycin, and streptomycin resistance in Solanum nigrum are located in three different chloroplast genes

A number of Solanum nigrum mutants resistant to the antibiotics spectinomycin, streptomycin and lincomycin have been isolated from regenerating leaf strips after mutagenesis with nitroso-methylurea. Selection of streptomycin- and spectinomycin-resistant mutants has been described earlier. Lincomycin-resistant mutants show resistance to higher levels of the antibiotic than used in the initial selection, and in the most resistant mutant (Ll7A1) maternal inheritance of the trait was demonstrated. The lincomycin-resistant mutant L17A1 and a streptomycin plus spectinomycin resistant double mutant (StSpl) were chosen for detailed molecular characterisation. Regions of the plastid DNA, within the genes encoding 16S and 23S rRNA and rps12 (3) were sequenced. For spectinomycin and lincomycin resistance, base changes identical to those in similar Nicotiana mutants were identified. Streptomycin resistance is associated with an A C change at codon 87 of rps 12 (converting a lysine into a glutamine), three codons upstream from a mutation earlier reported for Nicotiana. This site has not previously been implicated in streptomycin resistance mutations of higher plants, but has been found in Escherichia coli. The value of these mutants for studies on plastid genetics is discussed.     

Orientations of the tryptophan 9 and 11 side chains of the gramicidin channel based on deuterium nuclear magnetic resonance spectroscopy.

Deuterium nuclear magnetic resonance spectroscopy was used to investigate the orientations of the indole rings of Trp9 and Trp11 in specific indole-d5-labeled samples of gramicidin A incorporated into dimyristoyl phosphatidylcholine bilayers in the beta 6.3 channel conformation. The magnitudes and signs of the deuterium quadrupolar splittings were fit to the rings and assigned to specific ring bonds, using a full rotation search of the chi 1 and chi 2 angles of each Trp and a least-squares method. Unique assignments were obtained. The data and assignments are in close agreement with four sets of (chi 1, chi 2) angles for each Trp in which the indole N-H is oriented toward the membrane's exterior surface. (Four additional sets of (chi 1, chi 2) angles with the N-H's pointing toward the membrane interior are inconsistent with previous observations.) One of the sets of (chi 1, chi 2) angles for each Trp is consistent with the corresponding Trp orientation found by Arsen'ev et al. (1986. Biol. Membr. 3:1077-1104) for gramicidin in sodium dodecyl sulfate micelles. Together, the 1H and 2H nuclear magnetic resonance methods suggest that the Trp9 and Trp11 side chain orientations could be very similar in dimyristoyl phosphatidylcholine membranes and in sodium dodecyl sulfate micelles. The data for Trp11 could be fit using a static quadrupolar coupling constant of 180 kHz under the assumption that the ring is essentially immobile. By contrast, Trp9 could be fit only under the assumption that the quadrupolar splittings for ring 9 are reduced by approximately 14% due to motional averaging. Such a difference in motional averaging between rings 11 and 9 is also consistent with the 15N data of Hu et al. (1993. Biochemistry. 32:7035-7047).