Thermal and Structural Stability of Adsorbed Proteins

Experimental evidence suggests that proteins adsorbed to hydrophobic surfaces at low coverages are stabilized relative to the bulk. For larger coverages, proteins unfold and form β-sheets. We performed computer simulations on model proteins and found that: 1), For weakly adsorbing surfaces, unfolded conformations lose more entropy upon adsorption than folded ones. 2), The melting temperature, both in the bulk and at surfaces, decreases with increasing protein concentration because of favorable interprotein interactions. 3), Proteins in the bulk show large unfolding free energy barriers; this barrier decreases at stronger adsorbing surfaces. We conjecture that typical experimental temperatures appear to be below the bulk melting temperature for a single protein, but above the melting temperature for concentrated protein solutions. Purely thermodynamic factors then explain protein stabilization on adsorption at low concentrations. However, both thermodynamic and kinetic factors are important at higher concentrations. Thus, proteins in the bulk do not denature with increasing concentration due to large kinetic barriers, even though the aggregated state is thermodynamically preferred. However, they readily unfold upon adsorption, with the surface acting as a heterogeneous catalyst. The thermal behavior of proteins adsorbed to hydrophobic surfaces thus appears to follow behavior independent of their chemical specificity.     

Effect of Envelope Proteins on the Mechanical Properties of Influenza Virus

The envelope of the influenza virus undergoes extensive structural change during the viral life cycle. However, it is unknown how lipid and protein components of the viral envelope contribute to its mechanical properties. Using atomic force microscopy, here we show that the lipid envelope of spherical influenza virions is ∼10 times softer (∼0.05 nanonewton nm⁻¹) than a viral protein-capsid coat and sustains deformations of one-third of the virion''s diameter. Compared with phosphatidylcholine liposomes, it is twice as stiff, due to membrane-attached protein components. We found that virus indentation resulted in a biphasic force-indentation response. We propose that the first phase, including a stepwise reduction in stiffness at ∼10-nm indentation and ∼100 piconewtons of force, is due to mobilization of membrane proteins by the indenting atomic force microscope tip, consistent with the glycoprotein ectodomains protruding ∼13 nm from the bilayer surface. This phase was obliterated for bromelain-treated virions with the ectodomains removed. Following pH 5 treatment, virions were as soft as pure liposomes, consistent with reinforcing proteins detaching from the lipid bilayer. We propose that the soft, pH-dependent mechanical properties of the envelope are critical for the pH-regulated life cycle and support the persistence of the virus inside and outside the host.     

促溶剂对雷斯青霉发酵液流变特性的影响

目的:促溶剂通常用于甾体生物催化过程以提高底物溶解度,但在发酵液中添加促溶剂对菌体形态及发酵液特性的影响还少有报道。方法:利用旋转流变仪和顺磁分析仪分别对发酵液的流变特性及体积氧传递系数KLa进行监测。结果:无论是否添加促溶剂,发酵液都表现出非牛顿流体力学特性,但添加3%1,2丙二醇后同一时期发酵液稠度系数减小大约17%,而流动指数平均增加8%。结论:添加促溶剂使得发酵液表观黏度减小,体积氧传递系数增大,从而有利于甾体化合物的生物转化。     

透明质酸溶液的流变性能初步研究

透明质酸是一种酸性粘多糖,其特有的粘弹性使其广泛应用于眼科手术等医药领域。使用高级流变扩展系统研究浓度对于透明质酸溶液流变性能的影响。结果表明,透明质酸溶液的粘度随剪切速率的增加而减小。在任何剪切速率作用下,粘度总是与浓度呈正相关。而随着浓度的增加,溶液粘性与弹性频率交叉点降低。     

Effect of Substrate Concentration on Plastein Productivity and Some Rheological Properties of the Products

A bacterial arginase was purified to homogeneity from a strain of Bacillus brevis. The native enzyme, with an estimated MW of 143,000, migrated on SDS-PAGE as a single polypeptide of estimated MW of 33,000. The enzyme, highly specific to l-arginine, showed the maximum activity at pH 11.0 in the presence of Mn²⁺ ions and the pI was 4.8 by isoelectric focusing. The enzyme activity was increased significantly by the addition of Mn²⁺, Ni²⁺, or Co²⁺ ions, and inhibited potently by chemicals such as HgCl₂, N-bromosuccinimide, or glutathione. The K_ms for l-arginine and l-canavanine were 0.69 and 22.2 mm, respectively. The enzyme was inhibited competitively by γ-guanidinobutyric acid, and non-competitively by l-lysine, l-ornithine, creatine, blasticidin S, and edeine B₁ Analysis of the N-terminal amino acid sequence of the purified bacterial enzyme found 33–36% homologies with the Agrobacterium, yeast, rat, and human enzymes.     

Effect of Soy Protein Subunit Composition on the Rheological Properties of Soymilk during Acidification

The effect of soy protein subunit composition on the acid-induced aggregation of soymilk was investigated by preparing soymilk from different soybean lines lacking specific glycinin and β-conglycinin subunits. Acid gelation was induced by glucono-δ-lactone (GDL) and analysis was done using diffusing wave spectroscopy and rheology. Aggregation occurred near pH 5.8 and the increase in radius corresponded to an increase in the elastic modulus measured by small deformation rheology. Diffusing wave spectroscopy was also employed to follow acid gelation, and data indicated that particle interactions start to occur at a higher pH than the pH of onset of gelation (corresponding to the start of the rapid increase in elastic modulus). The protein subunit composition significantly affected the development of structure during acidification. The onset of aggregation occurred at a higher pH for soymilk samples containing group IIb (the acidic subunit A₃) of glycinin, than for samples prepared from Harovinton (a commercial variety containing all subunits) or from genotypes null in glycinin. The gels made from lines containing group I (A₁, A₂) and group IIb (A₃) of glycinin resulted in stiffer acid gels compared to the lines containing only β-conglycinin. These results confirmed that the ratio of glycinin/β-conglycinin has a significant effect on gel structure, with an increase in glycinin causing an increase in gel stiffness. The type of glycinin subunits also affected the aggregation behavior of soymilk.     

Oxidatively Damaged Erythrocytes Are Recognized by Membrane Proteins of Macrophages

Human erythrocytes incubated with an iron catalyst ADP-chelated Fe³⁺ undergo oxidative damage of the membrane including lipid peroxidation, protein oxidation, and protein aggregation, and become susceptible to recognition by human macrophages. In order to clarify the membrane components of macrophages responsible for the recogrution of the oxidized erythrocytes, binding of the oxidized cells to dot and Western blots of solubilized membrane of macrophages was investigated. The oxidized erythrocytes but not unoxidized cells bound to the dot blots. The binding was effectively inhibited by saccharide chains of band 3, a major glycoprotein of human erythrocytes, and lowered when the saccharide chains of band 3 were removed from the cell surface by pretreatment of the cells with endo-P-galactosidase which specifically cleaves the polylactosaminyl saccharide chains of band 3. The oxidized erythrocytes bound to the membrane proteins of macrophages with molecular mass of about 50, 80, and 120 kDa on Western blots depending on the saccharide chains of band 3 on their surface. The results suggest that the oxidatively damaged erythrocytes are specifically recognized by these proteins of macrophage membrane having saccharide binding ability.     

The Effect of Manipulating Fat Globule Size on the Stability and Rheological Properties of Dairy Creams

Recombined cream (RC, 23 % fat w/w) and standardised commercial cream (CC, 28 % fat w/w) were studied to understand the effects of manipulating fat globule size at the micron-/nano-scale on the stability and rheological properties of cream. All samples were adjusted to a fat: protein ratio of 5:1 and a fat: emulsifier (Tween 80) ratio of 30:1 to stabilize emulsion. For both CC and RC, different emulsions with droplet sizes covering micron- (3.9 μm), sub-micron (0.5 – 0.6 μm) and nano-metric scales (0.13 – 0.29 μm) were obtained using either the homogeniser (7/3 MPa) or the microfluidiser (85 MPa and 42 MPa). Fat globules from both RC and CC had high zeta potential values (-28 to -43 mV) and maintained their reduced size after 1 month of storage at 4 °C, providing evidence of emulsion stability. Droplet size had a significant effect on rheological characteristics of all creams produced. Nano-sized RC tended to have a rigid structure (solid/gel-like form) as compared to micron-sized RC (liquid-like form) as reflected by higher phase angle. Surprisingly, the rheological properties of CC exhibited an opposite tendency to that of RC. This implies that the observed rheological properties of CC and RC could not be fully explained by the discrepancy in droplet size. Differences in interfacial properties between RC and CC might also play a role in the rheological behaviour of the creams. Results indicated the stable high milk fat emulsions could be successfully created by reducing the globule size. These findings would be useful in understanding how micron-/nano-sized emulsions can be utilised in further application or processing of creams.     

Dielectric Properties and Ion Mobility in Erythrocytes

The impedance of erythrocytes of man, cattle, sheep, dog, cat, rabbit, and chicken was measured in the range from 0.5 to 250 Mc. The dielectric constant of the red cell interior is 50 at 250 Mc, varies but little with species, and can readily be accounted for by the cells' hemoglobin content. The electrical conductivity of the red cell interior was determined between 70 and 100 Mc. The values differ from species to species within the rather limited range from 4.4 to 5.3 mmho/cm. Removal of the cell membranes does not affect the conductivity. Hence, the cell interior behaves, from an electrical point of view, like a highly concentrated hemoglobin solution. A theoretical value for the electrical conductivity of erythrocyte interiors, which is calculated on the basis of the salt content of the cell, ion mobility, and the volume concentration of the hemoglobin, is roughly twice as large as the measured value. This discrepancy is typical not only of the red blood cell. Pertinent measurements show that it is probably caused by hydrodynamic and possibly by electrostatic effects also, which lower the mobility of the ions. From the lower electrical mobility it appears that a lowered diffusion constant of the electrolytes and nonelectrolytes within the cell is indicated.     

Rheological Properties of Blood in Athletes

Rheological properties of blood were studied in subjects with high physical activity (athletes). It was found that the whole blood viscosity decreased under conditions of relative rest. The use of the concept of hemorheological profiles made it possible to reveal different profiles in subjects exposed to different courses of training exercise. The relationship between the rheological properties of blood and the total work capacity in humans was established. The typical parameter of profiles in subjects with high physical activity was high erythrocyte deformability associated mainly with erythrocyte form and viscoelastic properties of its membrane.     

Effect of Oil Content and Composition on the Gelling Properties of Egg-SPI Proteins Stabilized Emulsion Gels

Effects of different oils on the rheological properties, textural profile, water loss (WL), oil loss (OL) and microstructure of egg-soybean protein isolate (SPI) stabilized emulsion gels were investigated at neutral pH, wherein soybean oil, olive oil and menhaden oil were used to form emulsions. The results showed that viscosity of emulsions progressively increased with the increase of oil content. Similarly, analysis of the rheological behavior of the formulated emulsion gels revealed an increase in the mechanical strength (G’) with the increase in oil concentration, indicating that oil droplets played a significant role in the formation of the gel structure. In addition, at high levels of oil, the hardness and chewiness of emulsion gels were also high, while a slight decrease in springiness and cohesiveness were observed. A linear relationship between hardness and water/oil loss was found, whereas the Pearson correlation suggested that less drainage of water may slow down the outflow of oil. The microstructural images showed a more compact network as a result of the increase of oil content in the formulation. Scarce significant differences were found among emulsion gels formulated with different oil type, suggesting oil composition played a dispensable role on the gelling properties of emulsion gels.

     

Theoretical Studies of the Electrostatic Effect on Carbocations Adsorbed over Zeolitic Sites

Theoretical studies, at MNDO level, of carbocations adsorbed over a zeolite active site were carried out, taking into account the effect of electrostatic fields. Clusters 1 and 2 were placed between two electrostatic charges of opposite sign. The distance from each charge to the center of the oxygen atom bearing the proton or alkyl group was varied in order to find the minimum in energy. The ionicity, expressed by the decrease in bond order, slightly increased with the inclusion of the charges. A more pronounced effect was observed for a positive charge of +2. The energy of the system decreased with the introduction of electrostatic charges. This fact may be partly explained by the electrostatic interaction between the opposed charges and also to an observed increase in the Si–-O–-Al bond angle, decreasing the steric repulsions between the substituents and the acid site. The bond orders remained high, indicating a predominant covalent character for the adsorption of the carbocations. The calculations are in agreement with experimental data of exchange of polyvalent cations, which pointed to an increase in the acid strength of the zeolite, due to decrease in the electron density in the aluminum atom. This revised version was published online in June 2006 with corrections to the Cover Date.     

Alterations in the Immunogenic Properties of Sheep Erythrocytes by Sonic Disruption

A solubilized sheep red blood cell (SRBC) antigen (supernatant fraction obtained by centrifuging 10^7-2 × 10⁸ sonicated SRBC at 6 × 10⁴ g for 30 min [Sup-SRBC]), whose ability to inhibit anti-SRBC plaque formation was 70% of that of the original sonicated SRBC, was unable to elicit a detectable antibody response in either unprimed or SRBC-primed mice. However, Sup-SRBC as well as intact SRBC antigens generated memory for the secondary response, which was transferable to irradiated syngeneic recipients by injection of immune spleen cells. The memory generated by Sup-SRBC involved helper memory for anti-trinitrophenyl group (TNP) response to challenge with TNP-conjugated SRBC. Increase in the helper T cell memory in the spleens of Sup-SRBC-primed mice was also demonstrated by an in vitro culture experiment and by an adoptive cell transfer experiment. In contrast, no detectable B cell memory was generated by Sup-SRBC. Repeated stimulation with Sup-SRBC never induced significant antibody response but reduced the level of memory. A single injection of a low dose (10⁶) of SRBC also failed to induce a definite primary antibody response generating memory for the secondary response. However, repeated stimulation with this dose of SRBC induced a high antibody response and generated good memory. From these results it is suggested that the intact structure of SRBC is required for the activation of B cells, but is not necessary for the stimulation of T cells.     

Effects of Lipids on the Rheological Properties of Aqueous Soybean Protein Dispersions

The flow properties of soybean protein–lipid–water suspension systems and coagulated gels were related to a protein–lipid interaction. For powdered soybean lecithin-added soybean protein suspension systems and their heat-induced gels, the yield stress (σ_y) and the consistency index (K) increased with increasing amounts of added lipid, but the flow behavior index (n) and the thixotropy index (TI) decreased. On the other hand, there were only small changes in the magnitudes of the thixotropic parameters and the viscometric parameters (σ_y, K and n) after adding soybean oil at various concentrations to soybean protein dispersions. These facts suggested that the formation of a protein–phospholipid complex increased the effective particle size, and that the intermolecular entanglements and linkages among the protein molecules or among the protein-phospholipid complexes were weakened by the addition of polar lipids. The thixotropy index defined in this study is available for characterizing the stress decay that occurs within soybean protein dispersions and heat-induced gels as they are sheared.     

Physicochemical and Rheological Properties of Romanian Honeys

The purpose of this work was to investigate the physicochemical and rheological properties of Romanian honey, and particularly to analyze the influence of moisture content, °Brix concentration and temperature. All the samples were characterized to determine their physicochemical (moisture, °Brix, pH, ash, conductivity, colour, 5-hydroxymethylfurfural content, sugar content, diastase activity) and bioactive (antioxidant activity, total phenols content and total flavonoids content) parameters. All the investigated honeys displayed Newtonian behaviour at all temperatures. Four experimental viscosity models were checked using the experimental data to correlate the influence of temperature upon honey viscosity; The William-Landel-Ferry model was much suitable than Power Law, Arrhenius and Vogel-Taumman-Fulcher models for temperature-viscosity modelling. By using the polynomial modeling method, it was possible to develop a mathematical model to describe honey viscosity in the function of temperature and chemical composition. The model achieved, developed from the viscosity, temperature and chemical composition allows predicting the evolution of the dynamic viscosity.     

凉粉草胶的初级结构与流变性质的研究

凉粉草胶经过DEAE-Sepharose FF分离可以得到中性糖和酸性糖两种组分,其中中性糖的尖峰分子量Mp为5227,酸性糖的尖峰分子量为6566,前者的单糖组成(以摩尔百分比计)为半乳糖:葡萄糖:甘露糖:木糖:阿拉伯糖:鼠李糖=9.9∶15.3∶4.31∶1.48∶11.6∶1,后者的单糖组成为半乳糖:葡萄糖:甘露糖:木糖:阿拉伯糖:半乳糖醛酸:鼠李糖=2.66∶1∶0.37∶2.29∶12.5∶23.5∶5.99。两者再经Sephadex G-100凝胶色谱分离,均为单一对称峰,酸性糖经琼脂糖凝胶电泳,甲苯胺蓝显色为均一斑点,表明所分离得到的两种组分都为均一组分。紫外光谱分析表明两种糖都不含蛋白质或肽段,红外光谱分析表明NMBG和AMBG都具有多糖特征吸收峰,NMBG仅在872 cm-1附近有吸收峰,表明其结构中只有β-糖苷键,AMBG在896和858 cm-1附近有吸收峰,表明其结构中既有β-糖苷键,又有α-糖苷键。流变学实验表明在1%~5%(w/w)内中性糖不具有流变学性质,但酸性糖却表现出明显地流变学性质。     

Effects of the Binding of Imipramine to Erythrocytes and Plasma Proteins on Its Transport Through the Rat Blood-Brain Barrier

Brain extraction of a tricyclic antidepressant, imipramine, was investigated using the carotid injection technique in the rat. The extent to which drug binding to plasma proteins and erythrocytes could inhibit the brain extraction was measured. Equilibrium dialysis showed that imipramine is highly bound to human serum albumin (HSA), alpha 1-acid glycoprotein (AAG), lipoproteins, and erythrocytes. The free dialyzable drug fraction was inversely related to the protein concentration. Despite this degree of binding, no significant reduction in the brain extraction of the drug was observed in the presence of HSA, lipoprotein, or erythrocytes. Only AAG reduced the brain transport of this drug in a ratio related to the protein concentration. However, the rat brain extraction was higher than expected from the in vitro measurement of the dialyzable fraction. These data indicate that the amount of circulating imipramine available for penetration in brain exceeds widely the dialyzable fraction of the drug as measured in vitro.     

The Effect of UV-C Pasteurization on Bacteriostatic Properties and Immunological Proteins of Donor Human Milk

Background

Human milk possesses bacteriostatic properties, largely due to the presence of immunological proteins. Heat treatments such as Holder pasteurization reduce the concentration of immunological proteins in human milk and consequently increase the bacterial growth rate. This study investigated the bacterial growth rate and the immunological protein concentration of ultraviolet (UV-C) irradiated, Holder pasteurized and untreated human milk.

Methods

Samples (n=10) of untreated, Holder pasteurized and UV-C irradiated human milk were inoculated with E. coli and S. aureus and the growth rate over 2 hours incubation time at 37°C was observed. Additionally, the concentration of sIgA, lactoferrin and lysozyme of untreated and treated human milk was analyzed.

Results

The bacterial growth rate of untreated and UV-C irradiated human milk was not significantly different. The bacterial growth rate of Holder pasteurized human milk was double compared to untreated human milk (p<0.001). The retention of sIgA, lactoferrin and lysozyme after UV-C irradiation was 89%, 87%, and 75% respectively, which were higher than Holder treated with 49%, 9%, and 41% respectively.

Conclusion

UV-C irradiation of human milk preserves significantly higher levels of immunological proteins than Holder pasteurization, resulting in bacteriostatic properties similar to those of untreated human milk.     

The Effect of Shear on the Desorption of Liposomes Adsorbed to Bacterial Biofilms

Abstract

With the aid of a flow cell assembly the desorption of cationic liposomes prepared from mixtures of dipalmitoylphoshatidylcholine (DDPC), cholesterol, and either dimethyldioctadecylammonium bromide (DDAB) or 3,β[N-(N¹,N-dimethylethylenediamine)-carbamoyl]cholesterol (DC-chol) from immoblized biofilms of Staphylococcus aureus has been studied as a function of shear stress by confocal microscopy. A shear stress theory has been adapted from fluid mechanics of laminar flow between parallel plates and used to determine the critical shear stress for liposome desorption. The critical shear stress for both DDAB and DC-chol liposomes has been determined as a function of cationic lipid content and hence surface charge as reflected in their zeta potentials. The critical shear stress has been used to obtain the potential energy of liposome–biofilm interaction which together with the electrostatic interaction energy has enabled estimates of the London-Hamaker constants to be made. The values of the London-Hamaker constants at small liposome-bacterial cell separation were found to be independent of liposome composition.     

Effect of Cholesterol Content on Gramicidin S-Induced Hemolysis of Erythrocytes

Gramicidin S (GS) is a cyclo-decapeptide antibiotic with wide Gram+ and Gram− antimicrobial spectrum. However, its therapeutic application is very limited due to hemolytic activity of GS. The presence of cholesterol defines one of the most significant differences between eukaryotic plasma membranes and bacterial inner membranes. To find out the cholesterol effect on the GS hemolytic efficiency we compared GS-induced hemolysis of erythrocytes extracted from the blood of healthy donors against donors with atherosclerosis, “naturally” enriched with cholesterol. Our results show that increased cholesterol levels significantly attenuates yet does not abolishes the GS hemolytic activity. High levels of cholesterol content in erythrocyte membranes results in a decrease in the membrane fluidity and deformability leading to a decrease in the rate of GS interaction with membranes. The results obtained confirm that hydrophobic as well as electrostatic interactions must be involved in the binding of GS to cell membranes. Lipid peroxidation occurring within atherosclerotic erythrocytes leads to considerable decrease in the degree of GS-induced erythrocyte hemolysis in vitro. These results can be applied to the rational design of GS analogs with increased antibacterial efficiency but reduced hemolytic activity.