Molecular weights of three mouse milk caseins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and kappa-like characteristics of a fourth casein

Caseins of mouse milk are phosphoproteins which precipitate at pH 4.6, stain blue with "Stains-all," and stain red with "Stains-all" following alkaline phosphatase digestion. Four caseins were separated electrophoretically in sodium dodecyl sulfate-polyacrylamide gels varying from 8.5 to 15% acrylamide. Molecular weights for three of these proteins were 43,200, 27,700, and 25,900. The molecular weights determined for bovine alphas1 and beta caseins by this method were similar to those previously obtained by other methods. A fourth mouse casein contained carbohydrate, phosphorus, and sialic acid. This protein was rennin-sensitive and behaved anomalously on sodium dodecyl sulfate polyacrylamide gels, as did bovine kappa-casein. Because of similarities with bovine kappa-casein, it was designated with "kappa-casein" of mouse milk.     

Effect of the A and B variants of both alpha s1- and kappa-casein on bovine casein micelle solvation and kappa-casein content

Casein micelle solvation, a micelle characteristic that is sensitive to many factors, has been measured by a centrifugation technique at 30 degrees C for a series of uncooled fresh skim milks at pH 6.3, 6.6, 6.9 and 7.1. The relative alpha s-(alpha s1-plus alpha s2-), beta- and kappa-casein contents of all centrifuge pellets and supernatants were determined by a standardized electrophoretic method. The calcium and phosphate contents of a number of the pellets and milk samples were also determined. Solvation of micelles from milks with various genetic variants of beta-lactoglobulin (A and B), alpha s1-casein (A and B) and kappa-casein (A and B) was often found to be lower for milks containing either the B variant of alpha s1-casein or the A variant of kappa-casein. It was also found that these two variant caseins were associated with a lower kappa-casein. It was also found that these two variant caseins were associated with a lower kappa-casein content of the milks and the micelles, which is consistent with the lower solvation as kappa-casein is associated with smaller micelle size and greater solvation. The solvations also seemed to increase during the lactation period. It is possible that some of the other features of milk and its products that have been ascribed to the differences in functional character between the A and B variants of alpha s1-casein may be partly caused by the increased level of kappa-casein. The reason for the association of the A variant of alpha s1-casein with higher concentrations of kappa-casein (and micelle solvation) is not obvious but possibly the haplotype alpha s1-casein A, beta-casein A1, kappa-casein A contains a controlling sequence in the chromosomal DNA that enhances expression of the kappa-casein gene.     

Genetic analysis of receptor-Galphaq coupling selectivity

Many different G protein-linked receptors are preferentially coupled to G proteins of the Gq/11 family. To elucidate the molecular basis underlying this selectivity, different Gq/11-coupled receptors (m3 muscarinic, V1a vasopressin, and gastrin-releasing peptide receptor) were coexpressed (in COS-7 cells) with mutant alphas subunits in which residues present at the C terminus of alphas were replaced with the corresponding alphaq/11 residues. Remarkably, whereas none of the receptors was able to interact with wild type alphas to a significant extent, all three receptors gained the ability to productively couple to a mutant alphas subunit containing a single Glu --> Asn point mutation at position -3. Moreover, the m3 muscarinic and the V1a vasopressin receptors but not the GRP receptor also gained the ability to interact with a mutant alphas subunit containing a single Gln --> Glu point mutation at position -5, indicating that the alphaq/11 residues present in these mutant G protein constructs play key roles in determining the selectivity of receptor recognition. To identify the site(s) on Gq/11-coupled receptors that can functionally interact with the C terminus of alphaq/11 subunits, we next analyzed the ability of a series of hybrid m2/m3 muscarinic receptors to interact with a mutant alphas subunit (sq5) in which the last five amino acids of alphas were replaced with the corresponding alphaq/11 sequence. Similar to the wild type m2 and m3 muscarinic receptors, none of the investigated hybrid receptors was able to efficiently interact with wild type alphas. Interestingly, however, three mutant m2 receptors in which different segments of the second and third intracellular loops were replaced with the corresponding m3 receptor sequences were identified, which, in contrast to the Gi/o-coupled wild type m2 receptor, gained the ability to efficiently activate the sq5 subunit. This observation suggests that multiple intracellular receptor domains form a binding pocket for the C terminus of G protein alphaq/11 subunits.     

Preparation and characterization of biodegradable nanospheres composed of methoxy poly(ethylene glycol) and DL-lactide block copolymer as novel drug carriers

We synthesized amphiphilic diblock copolymer based on methoxy poly(ethylene glycol) (MePEG) and dl-lactide with different molar composition in bulk without catalyst. Using the resulting amphiphilic diblock copolymers, we prepared drug-loaded polymeric nanospheres by micelle formation through solution behavior of amphiphilic copolymer in selective solvents. The structure of MePEG/dl-lactide diblock copolymers was identified by IR, WAXD, GPC, 1H-NMR. The size of nanosphere measured by dynamic light scattering showed a narrow monodisperse size distribution and average diameter less than 200 nm. From the surface chemical composition of nanosphere by ESCA, the presence of MePEG chains on the nanosphere layers was confirmed. The critical micelle concentration of ML50 sample investigated by fluorescence spectroscopy was 1.44x10-7 mol/l which is lower than common low molecular weight surfactants. In addition, we could obtain nanospheres having a relatively high drug-loading of about 33.0% when the feed weight ratio of indomethacin to polymer was 1:1. In vitro release experiments of the indomethacin-loaded MePEG/dl-lactide nanospheres exhibited sustained release behavior without any burst effects. The results of cytotoxicity tests showed that the MePEG/dl-lactide nanospheres didn't induce any relevant cell damage.     

alpha-Crystallin polymers and polymerization: the view from down under

Several models have been proposed for the quaternary structure of alpha-crystallin. Some suggest the subunits are arranged in concentric shells. Others propose that the subunits are in a micelle-like arrangement. However, none is able to satisfactorily account for all observations on the protein and the quaternary structure of alpha-crystallin remains to be established. In this review, factors contributing to the assembly and polymerization are examined in order to evaluate the different models. Consideration of the variations in particle size and molecular weight under different conditions leads to the conclusion that alpha-crystallin cannot be a micelle or a layered structure. Instead, it is suggested that the protein may be assembled from a 'monomeric' unit comprising eight subunits arranged in two tetramers with cyclic symmetry. The octameric unit is proposed to be disc-like particle with a diameter of 9.5 nm and a height of 3 nm. The larger particles, chains and sheet-like structures commonly observed are assembled from the octamers. Structural predictions indicate that the polypeptide may be folded into three independent domains which have different roles in the structural organization and functions of the protein. It is suggested that the tetramers are stabilized through interactions involving the second domain (residues 64-104) while assembly into the octamers and higher polymers requires hydrophobic interactions involving the N-terminal domain. Deletion of parts of this domain by site directed mutagenesis revealed that residues 46-63 play a critical role in the assembly. Current research aims to identify the specific amino acids involved.     

The interfacial pressure is an important parameter for the rate of phospholipase D catalyzed reactions in emulsion systems

Phospholipase D (PLD) is widely used for the transformation of phospholipids, which is preferably performed in aqueous-organic emulsion systems. The influence of the organic solvent on the reaction rates has been studied on the hydrolysis of phosphatidylcholine (PC) and its transesterification with glycerol by two types of PLD (cabbage and Streptomyces sp.). The initial rates determined by quantitative HPTLC show great differences in dependence on the solvent used with a similar tendency for both reactions and both PLDs. Since the polymorphism of the PC aggregates was assumed to be responsible for these effects, the critical concentration of micelle formation, the size of the aggregates, the water content of the organic phase, and the interfacial tension were determined in the different reaction systems. As result the interfacial pressure in the reaction systems influencing the package density of the PC aggregates is suggested to regulate the enzymatic activity.     

Identification of common and distinct residues involved in the interaction of alphai2 and alphas with adenylyl cyclase

The G protein alpha subunits, alphas and alphai2, have stimulatory and inhibitory effects, respectively, on a common effector protein, adenylyl cyclase. These effects require a GTP-dependent conformational change that involves three alpha subunit regions (Switches I-III). alphas residues in three adjacent loops, including Switch II, specify activation of adenylyl cyclase. The adenylyl cyclase-specifying region of alphai2 is located within a 78-residue segment that includes two of these loops but none of the conformational switch regions. We have used an alanine-scanning mutagenesis approach within Switches I-III and the 78-residue segment of alphai2 to identify residues required for inhibition of adenylyl cyclase. We found a cluster of conserved residues in Switch II in which substitutions cause major losses in the abilities of both alphai2 and alphas to modulate adenylyl cyclase activity but do not affect alpha subunit expression or the GTP-induced conformational change. We also found two regions within the 78-residue segment of alphai2 in which substitutions reduce the ability of alphai2 to inhibit adenylyl cyclase, one of which corresponds to an effector-activating region of alphas. Thus, both alphai2 and alphas interact with adenylyl cyclase using: 1) conserved Switch II residues that communicate the conformational state of the alpha subunit and 2) divergent residues that specify particular effectors and the nature of their modulation.     

Amphiphilic poly(Ala)-b-poly(Sar) microspheres loaded with hydrophobic drug

Amphiphilic block polypeptides, (Ala)m(Sar)n, were synthesized. Transmission electron microscopy showed that three kinds of block polypeptides, (Ala)42(Sar)21, (Ala)34(Sar)22, and (Ala)34(Sar)27, formed spherical aggregates in water. Dynamic light scattering measurement revealed that the average diameters of (Ala)42(Sar)21 and (Ala)34(Sar)22 aggregates were in the range of 80-90 nm, whilst that of (Ala)34(Sar)27 was about 30 nm. The polypeptides in aqueous solution took a beta-sheet structure, while they took an alpha-helical conformation in trifluoroethanol. These polypeptide aggregates took up 8-anilinonaphthalene-1-sulfonate (ANS), and the capacity of the aggregates for ANS decreased in the order of (Ala)42(Sar)21 > (Ala)34(Sar)22 > (Ala)34(Sar)27. Sumithion, which is a commercial agricultural insecticide, was also taken up by the polypeptide aggregates. When increasing amounts of Sumithion were introduced, (Ala)42(Sar)21 aggregates kept their shape, but (Ala)34(Sar)27 aggregate increased in size. These different behaviors of the polypeptide aggregates were discussed in terms of different structure of aggregates.     

In vitro assembly of virus-like particles with Rous sarcoma virus Gag deletion mutants: identification of the p10 domain as a morphological determinant in the formation of spherical particles

Retroviruses are unusual in that expression of a single protein, Gag, leads to budding of virus-like particles into the extracellular space. We have developed conditions under which virus-like particles are formed spontaneously in vitro from fragments of Rous sarcoma virus (RSV) Gag protein purified after expression in Escherichia coli. The CA-NC fragment of Gag was shown previously to assemble into hollow cylinders (S. Campbell and V. M. Vogt, J. Virol. 69:6487-6497, 1995). We have now extended these studies to larger Gag proteins. In every case examined, assembly into regular structures required RNA. A nearly full-length Gag missing only the C-terminal PR domain, as well as similar proteins missing in addition the N-terminal half of MA, the C-terminal half of MA, the entire MA sequence, or the entire p2 sequence, all assembled into spherical particles resembling RSV in size. By contrast, proteins missing p10 assembled into cylindrical particles like those formed by CA-NC alone. Thin section electron microscopy showed that each of these Gag proteins formed in the expressing E. coli cells particles similar in shape to those seen in vitro. We conclude from these results that neither the sequences required for membrane binding in vivo, near the N terminus of Gag, nor the sequences required for a late step in budding, in the p2 portion of Gag, are essential for formation of virus-like particles in this system. Furthermore, we postulate the existence of a shape-determining sequence in p10, which provides or facilitates interactions required for the growing particle to be constrained to a spherical shape.     

Experimental evaluation of three single-particle dissolution models

The dissolution of the 60-85-mesh fraction os recording, flow-through dissolution apparatus equipped with a dissolution cell; it was particularly suitable for kinetic analysis of multiparticulate systems. By using a time-scaling approach, experimental data are compared with theoretical calculations to evaluate, quantitatively, which of three single-particle dissolution models best describes the data and how well the multiparticulate kinetics can be explained mathematically. The nonspherical tolbutamide particles are replaced in the calculations by a hypothetical system of spherical particles that appears to be log-normally distributed. This procedure permits the calculation of the intrinsic dissolution profile, considering both size distribution and particle shape effects.     

Gsalpha contains an unidentified covalent modification that increases its affinity for adenylyl cyclase

Many G protein alpha subunits are dually acylated with myristate and palmitate or are palmitoylated on more than one cysteine residue near their N termini. The Galpha protein that activates adenylyl cyclase, alphas, is not myristoylated but can be reversibly palmitoylated. It appears that alphas contains another, as-yet-unidentified covalent modification that decreases its apparent dissociation constant for adenylyl cyclase from 50 nM to <0. 5 nM. This modification is at or near the N terminus of the protein and is hydrophobic. Palmitoylation of native alphas does not account for its high affinity for adenylyl cyclase.     

Ag对抗菌不锈钢组织性能的影响及其析出行为

在304L不锈钢的成分基础上,添加不同质量分数(0.072%~0.132%)的Ag,并对其热轧板进行固溶处理和冷轧,发现材料的显微组织均为均匀的奥氏体组织,且随着Ag含量的增加,屈服强度及伸长率变化均不明显。利用OM、SEM和TEM观察不同变形条件下材料中Ag颗粒变化,发现Ag颗粒主要为材料冶炼时未熔入到基体内的Ag,其在基体中弥散分布,呈椭球形或球形;Ag颗粒有2种存在形式,即几纳米到几十纳米的富Ag相、由多个纳米级Ag相团簇聚集在一起形成的几百纳米到几微米的Ag颗粒聚集物。另外,热轧后,富Ag颗粒聚集物沿轧制方向变成带状、棒状,部分分裂为椭球状或球状;进一步冷轧变形后,富Ag颗粒的尺寸减小,且变形量越大,Ag颗粒尺寸越小。     

Ag对抗菌不锈钢组织性能的影响及其析出行为

在304L不锈钢的成分基础上,添加不同质量分数(0.072%~0.132%)的Ag,并对其热轧板进行固溶处理和冷轧,发现材料的显微组织均为均匀的奥氏体组织,且随着Ag含量的增加,屈服强度及伸长率变化均不明显。利用OM、SEM和TEM观察不同变形条件下材料中Ag颗粒变化,发现Ag颗粒主要为材料冶炼时未熔入到基体内的Ag,其在基体中弥散分布,呈椭球形或球形;Ag颗粒有2种存在形式,即几纳米到几十纳米的富Ag相、由多个纳米级Ag相团簇聚集在一起形成的几百纳米到几微米的Ag颗粒聚集物。另外,热轧后,富Ag颗粒聚集物沿轧制方向变成带状、棒状,部分分裂为椭球状或球状;进一步冷轧变形后,富Ag颗粒的尺寸减小,且变形量越大,Ag颗粒尺寸越小。     

Investigation of the morphology and particle size of silicon carbide nanopowders using electron microscopy

Using transmission and scanning electron microscopy, the shape and particle size of nanopowders (NP) of silicon carbide and pyrolytic carbon are determined. The results are compared with other methods of analyzing the dimensional characteristics of NPs, and their tendency to aggregation is analyzed. It is established that silicon carbide NPs in the state of delivery are presented by globular aggregates from 0.1 to 1 μm in size which are formed by faced nanoparticles that are preferentially 50–70 nm in size. The latter are characterized by octahedron-, cube-, and intermediate-shaped facing; i.e., the tendency for rounded edges and vertexes and the formation of convex faces is observed. Nanoparticles of pyrolytic graphite form aggregates up to 150–200 nm in size containing globular particles 30–40 nm in size.     

Intestinal permeability in patients with Crohn''s disease and ulcerative colitis and their first degree relatives

A method has been devised for predicting the ability of drugs to cross the blood-brain barrier. The criteria depend on the amphiphilic properties of a drug as reflected in its surface activity. The assessment was made with various drugs that either penetrate or do not penetrate the blood-brain barrier. The surface activity of these drugs was quantified by their Gibbs adsorption isotherms in terms of three parameters: (i) the onset of surface activity, (ii) the critical micelle concentration, and (iii) the surface area requirement of the drug at the air/water interface. A calibration diagram is proposed in which the critical micelle concentration is plotted against the concentration required for the onset of surface activity. Three different regions are easily distinguished in this diagram: a region of very hydrophobic drugs which fail to enter the central nervous system because they remain adsorbed to the membrane, a central area of less hydrophobic drugs which can cross the blood-brain barrier, and a region of relatively hydrophilic drugs which do not cross the blood-brain barrier unless applied at high concentrations. This diagram can be used to predict reliably the central nervous system permeability of an unknown compound from a simple measurement of its Gibbs adsorption isotherm.     

Experimental developments in movement disorders: update on proposed free radical mechanisms

A possible physical explanation of the echinocyte -spheroechinocyte red blood cell (RBC) shape transformation induced by the intercalation of amphiphilic molecules into the outer layer of the RBC plasma membrane bilayer is given. The stable RBC shape is determined by the minimization of the membrane elastic energy, consisting of the bilayer bending energy, the bilayer relative stretching energy and the skeleton shear elastic energy. It is shown that for a given relative cell volume the calculated number of echinocyte spicula increases while their size decreases as the number of the intercalated amphiphilic molecules in the outer layer of the cell membrane bilayer is increased, which is in agreement with experimental observations. Further, it is show that the equilibrium difference between the outer and the inner membrane leaflet areas of the stable RBC shapes increases if the amount of the intercalated amphiphiles is increased, thereby verifying theoretically the original bilayer couple hypothesis of Sheetz and Singer (1974) and Evans (1974).     

Intact photoreceptor membrane from bovine rod outer segment: size and shape in bleached state

Photoreceptor disk membranes isolated from bovine rod outer segments are suspended in dilute aqueous sucrose (4.36 X 10(-3%)) and bleached, and their size and shape are determined with quasielastic and elastic light scattering. They are found to be extremely homogeneous spherical vesicles with a radius of 0.49 +/- 0.07 mum which appear to have derived from the native disk shape by osmotic swelling.     

Deformation of Pores in Viscoplastic Soil Material

Changes in soil pore volume and shape in response to internal and external mechanical stresses alter key soil hydrologic and transport properties. The extent of these changes is dependent on details of pore shape and size evolution. We present a model for quantifying rates of deformation and shape evolution of idealized spheroidal pores as functions of macroscopic stresses and soil rheological properties. Previous solutions for shrinkage of spherical pores embedded in a viscoplastic matrix under isotropic stress were extended to spheroidal pore shapes and biaxial stresses using Eshelby’s classical theory. Bulk soil behavior was obtained from upscaling of detailed single pore deformation. Results show that pore closure rates increase with decreasing initial aspect ratio (i.e., oblate pores close faster than spherical pores), and with higher deviatoric stress. Incomplete pore closure is attributed to soil hardening due to pore shape accommodation under biaxial stresses. The model provides a means for approximating pore deformation as input to predictive models for soil hydraulic properties.     

In-place leaching of primary sulfide ores: Laboratory leaching data and kinetics model

Experimental results obtained in laboratory leaching studies of primary copper sulfide ore in sulfuric acid systems pressurized with oxygen are interpreted by a computerized geometric model involving the movement of a reaction zone through the ore fragments. Physical properties of the ore, including size, shape, and mineral content, are considered. The leaching mechanism involves mixed kinetics and includes a surface reaction within a moving reaction zone plus pore diffusion of dissolved oxygen through the reacted portion of the ore fragment to the reaction zone. The results are applicable to conditions that would exist innuclear solution mining or similar processes in which the ore is converted into rubble and then inundated by a leach solution adequately supplied with oxidants. Experimental results at 90°C are correlated with the model, which includes temperature-dependent parameters.     

粉末烧结法制备开孔泡沫铝压缩性能的研究

采用粉末烧结工艺制备开孔泡沫铝并研究了其压缩性能，不同形态的尿素和氯化钠颗粒作为造孔剂使泡沫铝的孔隙度控制在70％。结果表明：粉末烧结法制备的泡沫铝呵以容易地控制孔隙度及孔径的大小，并且孔结构很好地保持了造孔剂的形状。不同的孔结构对泡沫铝的压缩性能具有显著影响，球形孔结构得到了最佳的压缩效果。