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PubMed1983

Determination of molecular weight of membrane proteins by the use of low-angle laser light scattering combined with high-performance gel chromatography in the presence of a non-ionic surfactant.

An assessment study was carried out to evaluate the performance of the low-angle laser light scattering technique combined with high-performance gel chromatography in the presence of a nonionic surfactant, octaethyleneglycol n-dodecyl ether, precision differential refractometry and ultraviolet photometry. It was found that the combined technique is highly promising as a method for the determination of the molecular weight of a membrane protein solubilized by the surfactant. For trial, molecular weights of the following membrane proteins of Escherichia coli, both solubilized in oligomeric forms, were measured; porin that forms the transmembrane diffusion pore in the outer membrane, and lambda-receptor protein that facilitates the diffusion of maltose-maltodextrins across the outer membrane. The result obtained indicates that both porin and lambda-receptor protein exist as trimers in the surfactant solution.

Bacterial Outer Membrane Proteins↗

PubMed2002

Thermodynamics of sodium dodecyl sulfate partitioning into lipid membranes.

The partition equilibria of sodium dodecyl sulfate (SDS) and lithium dodecyl sulfate between water and bilayer membranes were investigated with isothermal titration calorimetry and spectroscopic methods (light scattering, (31)P-nuclear magnetic resonance) in the temperature range of 28 degrees C to 56 degrees C. The partitioning of the dodecyl sulfate anion (DS(-)) into the bilayer membrane is energetically favored by an exothermic partition enthalpy of Delta H(O)(D) = -6.0 kcal/mol at 28 degrees C. This is in contrast to nonionic detergents where Delta H(O)(D) is usually positive. The partition enthalpy decreases linearly with increasing temperature and the molar heat capacity is Delta C(O)(P) = -50 +/- 3 cal mol(-1) K(-1). The partition isotherm is nonlinear if the bound detergent is plotted versus the free detergent concentration in bulk solution. This is caused by the electrostatic repulsion between the DS(-) ions inserted into the membrane and those free in solution near the membrane surface. The surface concentration of DS(-) immediately above the plane of binding was hence calculated with the Gouy-Chapman theory, and a strictly linear relationship was obtained between the surface concentration and the extent of DS(-) partitioning. The surface partition constant K describes the chemical equilibrium in the absence of electrostatic effects. For the SDS-membrane equilibrium K was found to be 1.2 x 10(4) M(-1) to 6 x 10(4) M(-1) for the various systems and conditions investigated, very similar to data available for nonionic detergents of the same chain length. The membrane-micelle phase diagram was also studied. Complete membrane solubilization requires a ratio of 2.2 mol SDS bound per mole of total lipid at 56 degrees C. The corresponding equilibrium concentration of SDS free in solution is C (sat)(D,F) approximately 1.7 mM and is slightly below the critical micelles concentration (CMC) = 2.1 mM (at 56 degrees C and 0.11 M buffer). Membrane saturation occurs at approximately 0.3 mol SDS per mol lipid and the equilibrium SDS concentration is C (sat)(D,F)approximately equal 2.2 mM +/- 0.6 mM. SDS translocation across the bilayer is slow at ambient temperature but increases at high temperatures.

Anisotropy↗

PubMed2006

Self-association and lipid binding properties of the lipoprotein initiating domain of apolipoprotein B.

The amino-terminal 20.1% of apolipoprotein B (apoB20.1; residues 1-912) is sufficient to initiate and direct the formation of nascent apoB-containing lipoprotein particles. To investigate the mechanism of initial lipid acquisition by apoB, we examined the lipid binding and interfacial properties of a carboxyl-terminal His6-tagged form of apoB20.1 (apoB20.1H). ApoB20.1H was expressed in Sf9 cells and purified by nickel affinity chromatography. ApoB20.1H was produced in a folded state as characterized by formation of intramolecular disulfide bonds and resistance to chemical reduction. Dynamic light scattering in physiological buffer indicated that purified apoB20.1H formed multimers, which were readily dissociable upon the addition of nonionic detergent (0.1% Triton X-100). ApoB20.1H was incapable of binding dimyristoylphosphatidylcholine multilamellar vesicles, unless its multimeric structure was first disrupted by guanidine hydrochloride. However, apoB20.1H multimers spontaneously dissociated and bound to the interface of naked and phospholipid-coated triolein droplets. These data reveal that the initiating domain of apoB contains solvent-accessible hydrophobic sequences, which, in the absence of a hydrophobic lipid interface or detergent, engage in self-association. The high affinity of apoB20.1H for neutral lipid is consistent with the membrane binding and desorption model of apoB-containing lipoprotein assembly.

Amino Acids↗

PubMed2006

Effect of mixing on the morphology of cylindrical micelles.

Increasing the spontaneous curvature of an amphiphile can lead to a first-order morphology transition from threadlike micelles to a branched network. The two morphologies were linked to entropy-driven topological defects; networks are dominated by Y-junctions, while linear threadlike structures are dominated by spherical end-caps. In this paper we investigate the effect of mixing on the morphological transitions in nonionic amphiphilic systems. We find that mixed equilibrium structures are obtained within seconds; these mixed cylindrical structures display comparable numbers of end-caps and branch points, resulting in a novel 'short armed' branched (SAB) morphology. Quite surprisingly, the probability of either defect (end-caps or branch points) is independent of composition, so that neither a first-order nor a second-order morphological transition is observed. A possible explanation may be local demixing of the two amphiphilic components, which adds a degree of freedom and thus enables the formation of a unique morphology that cannot be obtained in single-component systems. We further find that within a relatively large composition range phase equilibrium exists between vesicles, SAB micelles, and spherical micelles.

Butadienes↗

PubMed1996

Use of neutral surfactants for the capillary electrophoretic separation of hydrophobically modified poly(acrylic acids).

Hydrophobically modified poly(acrylic acids) (HMPAs) are random copolymers of sodium acrylate and dodecyl acrylamide, containing 0-10% mol/mol of dodecyl grafts. The hydrophobic character of different HMPAs of average molecular weight 150,000 was studied by capillary electrophoresis (CE), using neutral surfactants as buffer additives. The differentiation of the electrophoretic mobilities of HMPAs with their hydrophobicity was achieved through the use of nonionic Brij 35 and zwitterionic DAPS surfactants. A nearly baseline separation of the precursor and three HMPAs derivatives was obtained in a poly(ethylene glycol)-coated capillary with a background electrolyte containing 10 mM N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (DAPS) and 10 mM borax (pH 9.2). In addition to CE experiments, the polymer-surfactant interactions were also investigated by means of quasi-elastic light scattering (QELS) and viscosimetric measurements. According to the latter results, the separation mechanism was interpreted as an expansion of the polymer coil in the presence of micelles and subsequent change of its frictional properties. A true micellar electrokinetic capillary chromatography (MEKC) partitioning model was discarded on the basis of the relative sizes of the macromolecule and the micelles.

Acrylates↗

PubMed1997

Interaction of octyl-beta-thioglucopyranoside with lipid membranes.

Octyl-beta-thioglucopyranoside (octyl thioglucoside, OTG) is a nonionic surfactant used for the purification, reconstitution, and crystallization of membrane proteins. The thermodynamic properties of the OTG-membrane partition equilibrium are not known and have been investigated here with high-sensitivity titration calorimetry. The critical concentration for inducing the bilayer <==> micelle transition was determined as cD* = 7.3 mM by 90 degree light scattering. All thermodynamic studies were performed well below this limit. Sonified, unilamellar lipid vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) with and without cholesterol were employed in the titration calorimetry experiments, and the temperature was varied between 28 degrees C and 45 degrees C. Depending on the surfactant concentration in the membrane, the partition enthalpy was found to be exothermic or endothermic, leading to unusual titration patterns. A quantitative interpretation of all titration curves was possible with the following model: 1) The partitioning of OTG into the membrane follows a simple partition law, i.e., Xb = Kc(D,f), where Xb denotes the molar amount of detergent bound per mole of lipid and c(D,f) is the detergent concentration in bulk solution. 2) The partition enthalpy for the transfer of OTG from the aqueous phase to the membrane depends linearly on the mole fraction, R, of detergent in the membrane. All calorimetric OTG titration curves can be characterized quantitatively by using a composition-dependent partition enthalpy of the form deltaHD(R) = -0.08 + 1.7 R (kcal/mol) (at 28 degrees C). At low OTG concentrations (R < or = 0.05) the reaction enthalpy is exothermic; it becomes distinctly endothermic as more and more surfactant is incorporated into the membrane. OTG has a partition constant of 240 M(-1) and is more hydrophobic than its oxygen-containing analog, octyl-beta-D-glucopyranoside (OG). Including a third nonionic amphiphile, octa(ethyleneoxide) dodecylether (C12EO8), an empirical relation can be established between the Gibbs energies of membrane partitioning, deltaGp, and micelle formation, deltaGmic, with deltaGp = 1.398 + 0.647 deltaGmic (kcal/mol). The partition constant of OTG is practically independent of temperature and of the cholesterol content of the membrane. In contrast, the partition enthalpy shows a strong temperature dependence. The molar specific heat capacity of the transfer of OTG from the aqueous phase to the membrane is deltaCp = -98 cal/(mol x K). The OTG partition enthalpy is also dependent on the cholesterol content of the membrane. It increases by approximately 1 kcal/mol at 50 mol% cholesterol. As the partition constant remains unchanged, the increase in enthalpy is compensated for by a corresponding increase in entropy, presumably caused by a restructuring of the membrane hydration layer.

Calorimetry↗

PubMedDate not supplied

[Effect of water on DNA structure in a model microemulsion system].

The physical state of water in a system of reversed micelles at different molar rations of H2O and a nonionic surfactant (tetraethylene glycol monododecyl ether, Brij 30) was studied by NMR spectroscopy. In addition, the effect of water on the structure of the intramicellar DNA was investigated using the intercalator ethidium bromide. It was shown by NMR diffusometry that the state of water inside micelles can be considered as supercooled. It was found that the condensation of DNA inside micelles takes place only at definite ratios of water and Brij 30 (from 1.26 to 5). If free water is absent and if the percentage of free water inside in reversed micelles is more than 50%, the process of DNA condensation is disturbed. It is assumed that the molecular memory inherent in the supercooled state of water affects the formation and stabilization of the condensed state of DNA.

DNA↗

PubMed1985

Phase distribution studies on an oil-water emulsion based on a eutectic mixture of lidocaine and prilocaine as the dispersed phase.

The distribution conditions in oil-water emulsions prepared by emulsifying a 1:1 eutectic mixture of lidocaine and prilocaine with a nonionic surfactant in water were studied by membrane and gel filtration methods. In this system, the local anesthetics are considered to be freely dissolved, surfactant solubilized, and emulsified in three separate phases. The dispersity of the oil phase was investigated by light microscopy and light-scatter spectroscopy. The majority of drops in the lidocaine-prilocaine emulsions were less than 1 micron in size. The concentration of freely dissolved drug in the aqueous phase of the emulsions was equal to the aqueous solubility of lidocaine-prilocaine in a 1:1 ratio. At constant lidocaine/prilocaine/surfactant ratio, increasing the total drug concentration in the emulsion resulted in an increase of the emulsified fraction of lidocaine-prilocaine, whereas the surfactant-solubilized fraction remained constant.

Chemistry, Pharmaceutical↗

PubMed2000

Analysis of aliphatic alcohol ethoxylates in terms of alkyl and ethylene oxide chain lengths by reversed-phase liquid chromatography with evaporative light scattering detection.

Aliphatic alcohol ethoxylates are nonionic surfactants which are incorporated in many industrial formulations as complex mixtures of alkyl homologs and ethylene oxide oligomers. Determination of both the homolog and oligomer distributions is required for product control. The proposed method consisted of three reversed-phase liquid chromatographic separation steps carried out on the same C18-bonded silica column. The first step was a preparative one: the sample mixture was fractionated according to the alkyl chain length without discrimination between ethylene oxide oligomers by using methanol-water eluent. The even homologs (EH) were collected together in a single fraction, the odd homologs (OH) in another. In the second and third steps, respectively, EH and OH fractions were separated according to the alkyl chain length and the number of ethylene oxide units simultaneously by changing the mobile phase composition to acetonitrile-water and by using evaporative light scattering detection.

Alcohols↗

PubMed2005

Barriers to folding of the transmembrane domain of the Escherichia coli autotransporter adhesin involved in diffuse adherence.

Adhesin involved in diffuse adherence (AIDA) is an autotransporter protein that confers the diffuse adherence phenotype to certain diarrheagenic Escherichia coli strains. It consists of a 49 amino acid signal peptide, a 797 amino acid passenger domain, and a 440 amino acid beta-domain integrated in the outer membrane. The beta-domain consists of two parts: the beta(1)-domain, which is predicted to form two beta-strands on the bacterial cell surface, and the beta(2)-domain, which constitutes the transmembrane domain. We here present a detailed biophysical analysis of the AIDA beta-domain addressing its refolding properties and its different conformational states and their stability. We find that the beta(2)-domain in solution can fold only when the beta(1)-domain is present and only with 50% efficiency. However, 100% refolding of the beta(2)-domain, with or without the beta(1)-domain, can be achieved in the presence of a solid support. Folding can only take place above the cmc of the detergent used, but the refolded state is retained if diluted below the cmc, revealing a kinetic barrier to dissociation of the detergent molecules from the folded protein. Refolding attempts of the beta(2)-domain in the absence of a solid support result in the formation of an oligomeric misfolded state both in the absence and in the presence of detergent. Despite being misfolded, these states unfold cooperatively with a T(m) approximately 70 degrees C. The refolded protein in the nonionic detergent octylpolyoxyethylene (oPOE) can only be thermally unfolded in the presence of SDS. The linear relationship between SDS mole fraction and unfolding temperature, T(m), predicts a T(m) of 112.9 +/- 1.2 degrees C for the beta(2)-domain and 132.7 +/- 12.2 degrees C for the entire beta-domain in pure oPOE. Thus, the beta(1)-domain also stabilizes the beta(2)-domain. In conclusion, our data show that the in vitro refolding of the AIDA beta-domain is critically dependent on a solid support, suggesting that in vivo specific biological factors may assist in folding the protein correctly into the outer membrane to avoid the formation of stably misfolded conformations.

Adhesins, Escherichia coli↗

PubMedDate not supplied

[An evaluation of orthotopic ileal neobladders by dynamic digital urography].

PURPOSE: To report on the diagnostic capabilities of dynamic digital urography in the evaluation of orthotopic ileal neobladders. MATERIAL AND METHODS: Ten male patients (aged 61.3 +/- 14.7 years) with orthotopic ileal neobladder (4 Studer, 4 Camey type II with spiralized ileal segment and 2 classic Camey type II) underwent dynamic digital urography using an angiographic unit (Philips DVI/ARC-A). Eight patients were asymptomatic and two presented mild nycturia. All patients should have undergone follow-up conventional urography, which was replaced, for the purposes of our study, by dynamic digital urography. The intestinal cleansing regimen usual for the double contrast barium enema was used in all the patients. Ninety mL of a nonionic iodinated contrast agent were administered i.v. with an automatic injector. After precontrast mask images, two postcontrast sequences were acquired 15 s and 30 min after the injection (each made of 20 images acquired every 10 seconds). Postprocessing consisted of digital image subtraction and videorecording. RESULTS: Renal pelvis and calyceal systems were well visualized in 18/20 excretory systems. Eighteen of 20 ureters were visualized completely up to the ureteral jet, and two were only partially visualized. Calyceal, pyelic and ureteral enlargement with ureteral kinking were observed in a patient with classic Camey type II. High motility was seen in 11 ureters, moderate in 5 and mild in 4. The orthotopic ileal neobladder was well opacified in 8/10 patients. Effective peristalsis of the afferent ileal segment was seen in all the Studer type neobladders. Ureteral reflux was not observed in any patient. DISCUSSION: After orthotopic ileal neobladder reconstruction, the most frequent complications include urinary leakage, intestinal obstruction, venous thrombosis, stenosis of the neobladder anastomoses, incontinence, cancer recurrence, stone formation. In the follow-up, many diagnostic tools are used: intravenous urography, retrograde cystography, urodynamic studies, transabdominal and transrectal ultrasonography. With dynamic digital urography the nephrographic evaluation was possible in all patients, as well as the evaluation of calyceal, pyelic and ureteral opacification and even ureteral peristalsis. Moreover, this diagnostic tool allows the morphofunctional dynamic study of the ureteral-neobladder anastomoses and of the orthotopic ileal neobladder, even evaluating the residual peristalsis of the detubularized ileal segment. A major drawback of the new method is the relatively high radiation dose given to the patient. A limitation of the study is the selection of a population of completely or nearly asymptomatic patients. CONCLUSIONS: Dynamic digital urography provides useful morphologic and functional information in the follow-up of patients with orthotopic ileal neobladder and could replace conventional urography in symptomatic patients.

Aged↗

PubMed1994

The chemistry of iomeprol and physico-chemical properties of its aqueous solutions and pharmaceutical formulations.

The synthesis and the chemical characterization of iomeprol, a new triiodinated nonionic radiographic contrast agent, are reported. The physico-chemical properties both of aqueous solutions of the pure compound and of its pharmaceutical formulations are presented and these last data are compared with those of other contrast media. The pharmaceutical development of the product is described. Iomeprol is obtained via a synthesis which is particularly friendly to the environment. The compound shows an unusually high solubility which allows the formulation of contrast media with the lowest osmolalities and viscosities as compared with corresponding contrast media of the same category. In view of these favourable characteristics and of the remarkably high stability of its solutions, iomeprol for injection is formulated also at 400 mgI/ml, the highest concentration so far available on the market for non-ionic contrast media.

Anilides↗

PubMed1992

Effects of the aggregation state of amphotericin B on its toxicity to mice.

Amphotericin B (AmB) is a very effective antifungal agent for most systemic fungal infections. However, the relatively high toxicity of this drug imposes limits on its clinical usefulness. Most of the current work in this field is devoted to the search for less-toxic formulations of the drug. Here we describe the effects of three surfactants, one anionic and the other two nonionic, on the aggregation state of AmB in solutions which were injected intravenously into mice. The degree of aggregation of AmB was monitored spectroscopically and by light scattering. The toxicity was expressed as percentage of survivors. These results were compared with those obtained with doses of AmB the same as those present in a commercial formulation of AmB, Fungizone. Two surfactants, lauryl sucrose and sodium deoxycholate, used at concentrations which induced monomerization of AmB, substantially decreased the acute toxicity of AmB to mice. Conversely, the third surfactant, Tween 80, showed a synergistic potentiation of the toxicity of the antibiotic. A good correlation was found between the in vivo toxicity and the aggregation state of AmB in injected solutions. Solutions in which AmB was almost entirely monomeric were half as toxic after 24 h and about six times less toxic after 1 week than the corresponding solutions of Fungizone.

Amphotericin B↗

PubMedDate not supplied

Enhancing effect of nonionic surfactant on the inactivation of lipopolysaccharide by steam-heat treatment II.

n-alkylpolyoxyethylene surfactants (CnH2n+1O(CH2CH2O)mH; CnEm) showed a strong enhancing effect on the inactivation of lipopolysaccharide (LPS) by heat treatment over a wide range of temperatures. The effect of CnE8 (n = 10-16) was observed above the critical micelle concentration (CMC) and above the cloud point, and was influenced by the length of the alkyl chains. The efficacy of the surfactants was in the order C10E8 < C12E8, C16E8 < C14E8. However, the hydrophilic moiety seemed to have no influence. An 80-95% solution of n-butanol showed a similar effect, indicating that LPS was more effectively inactivated in the oily phase of the surfactants than in water. The effect of surfactant on the hydrodynamic diameter of LPS was the same before and after steam-heat treatment for 20 minutes at 121 degrees C. Each surfactant disaggregated LPS without alteration of the activity of LPS before the heat treatment. We consider that the surfactants interact with LPS in the region of lipid A in a manner that favors loss of the activities of LPS during heating.

Chemical Phenomena↗

PubMed2002

How to control the molecular architecture of a monolayer of proteins supported by a lipid bilayer.

In this work, we report the spontaneous formation of a new structure composed of two lipid layers surrounding a dense monolayer of soluble proteins (lysozyme). We extend a process, initially discovered with nonionic surfactants to phospholipids (DMPC and DOPC). The motor of the protein insertion process is the difference between the protein chemical potential in the solution and in the freshly formed Newton black film (NBF). This process is completely controlled by adjusting the protein chemical potential in the solution. By means of x-ray reflectivity, we follow the evolution of the freestanding sandwich structure until a stable equilibrium state is reached. Depending on the lipid concentration with respect to the protein concentration, we observe two different behaviors of the film leading to the formation of such unique structure: at the highest lipid concentration, the usual protein diffusion into the NBF, and, at the lowest lipid concentration, the spontaneous formation of a sandwich structure immediately obtained after the drainage. Finally, we show that the insertion process is reversible, because, if the lipid concentration varies in the bulk solution, a "deswelling" of the film can be observed.

Dimyristoylphosphatidylcholine↗

PubMed2001

Maltodextrin-based proniosomes.

Niosomes are nonionic surfactant vesicles that have potential applications in the delivery of hydrophobic or amphiphilic drugs. Our lab developed proniosomes, a dry formulation using a sorbitol carrier coated with nonionic surfactant, which can be used to produce niosomes within minutes by the addition of hot water followed by agitation. The sorbitol carrier in the original proniosomes was soluble in the solvent used to deposit surfactant, so preparation was tedious and the dissolved sorbitol interfered with the encapsulation of one model drug. A novel method is reported here for rapid preparation of proniosomes with a wide range of surfactant loading. A slurry method has been developed to produce proniosomes using maltodextrin as the carrier. The time required to produce proniosomes by this simple method is independent of the ratio of surfactant solution to carrier material and appears to be scalable. The flexibility of the proniosome preparation method would allow for the optimization of drug encapsulation in the final formulation based on the type and amount of maltodextrin. This formulation of proniosomes is a practical and simple method of producing niosomes at the point of use for drug delivery.

Alprenolol↗

PubMed1996

Preparation and characterization of polyethyl-2-cyanoacrylate nanocapsules containing antiepileptic drugs.

Biocompatible and biodegradable colloidal drug delivery systems can be obtained by means of in situ polymerization of alkylcyanoacrylate. In particular, nanocapsules of polyethylcyanoacrylate (PECA) were prepared by adding the monomer to an organic phase, consisting of Miglyol 812 and an organic solvent (ethanol, acetone or acetonitrile), and subsequently mixing the organic phase with an aqueous phase containing Pluronic F68 at different concentrations. The possible mechanism of formation and the influence of preparation conditions on the quality of nanocapsule formulations were investigated by freeze-fracture electron microscopy and laser light scattering using both the inverse Laplace transform and the standard cumulant analysis for data fitting. High-quality nanocapsule systems were obtained using an aprotic fully water-miscible organic solvent such as acetone. The presence of ethanol led to the formation of both nanospheres and nanocapsules. The concentrations of nonionic surfactant in the aqueous phase of monomer in the organic phase did not influence the kind of colloidal suspension obtained. The oil simply plays the role of monomer support. The diameter of PECA nanoparticles (nanospheres and nanocapsules) ranged from 100 to 400 nm. Three antiepileptic drugs (Ethosuximide, 5,5-diphenyl hydantoin and carbamazepine) were entrapped in PECA nanocapsules. The loading capacity of PECA nanocapsules, prepared using acetone as organic solvent, varied from 1% to 11% (drug/dried material) as a function of the solubility (affinity) of the different drugs with the oil core. This parameter also influenced the release from PECA nanocapsules, which was slower for drugs with a higher affinity for Miglyol 812. By encapsulating the three antiepileptic drugs in the PECA nanocapsules, it was possible to achieve controlled drug release. The mechanism of drug release from PECA nanocapsules was mainly diffusion from the oil core through the intact polymer barrier.

Acetone↗

PubMed1990

An ultraviolet-sensitive maternal mRNA encoding a cytoskeletal protein may be involved in axis formation in the ascidian embryo.

Ultraviolet (uv) irradiation of the vegetal hemisphere of fertilized eggs during ooplasmic segregation inhibits subsequent gastrulation and axis formation in ascidian embryos. The molecular basis of this phenomenon was investigated in by comparing in vivo protein synthesis and in vitro mRNA translation in normal and uv-irradiated embryos of the ascidian Styela clava. Analysis of protein synthesis by [35S]methionine incorporation, two-dimensional (2D) gel electrophoresis, and autoradiography showed that only 21 (or about 5%) of 433 labeled polypeptides were missing or decreased in labeling intensity in uv-irradiated embryos. The most prominent of these was a 30,000 molecular weight (pI 6.0) polypeptide (p30). Extraction of gastrulae with the nonionic detergent Triton X-100 showed that p30 is retained in the detergent insoluble residue, suggesting that it is associated with the cytoskeleton. Several lines of evidence suggest that p30 may be involved in axis formation. First, p30 labeling peaks during gastrulation, when the embryonic axis is being established. Second, axis formation and p30 labeling are abolished by the same threshold uv dose, which is distinct from that required to inactivate muscle cell development. Third, the uv sensitivity period for abolishing p30 labeling and axis formation are both restricted to ooplasmic segregation. In vitro translation of egg RNA followed by 2D gel electrophoresis and autoradiography of the protein products showed that p30 is encoded by a maternal mRNA. The translation of p30 mRNA was abolished by uv irradiation of fertilized eggs during ooplasmic segregation suggesting that this message is a uv-sensitive target. The results are consistent with the hypothesis that uv irradiation blocks gastrulation and axis formation by inhibiting the translation of maternal mRNA localized in the vegetal hemisphere of the fertilized egg.

Acetylcholinesterase↗