Purification and partial characterization of the major outer membrane protein of Neisseria gonorrhoeae.

A procedure is described to isolate the major outer membrane protein (protein I) from Neisseria gonorrhoeae in large quantities. The method involves precipitation of protein I by hexadecyltrimethylammonium bromide (CTB) at low ionic strength. CTB is lethal for the gonococci and solubilizes most other proteins. Protein I is brought into solution by raising the ionic strength, and the nucleic acids are subsequently removed by 20% ethanol precipitation. The CTB is removed by precipitating protein I with ethanol and replaced by N-tetradecyl-N,N-dimethyl-3-ammonia-1-propanesulfonate, a dipolar ionic detergent. Further purification is accomplished by ion-exchange and molecular sieve chromatography. Two species of protein I (34,000 daltons [34K] and 32K) were purified by these methods. The purified proteins reacted with antisera prepared against the homologous organisms. The 34K proteins I generated proteolytic fragments upon treatment with trypsin and chymotrypsin similar to those generated by 34K protein in intact gonococci. The amino acid compositions of the three proteins were much like those of other major proteins of gram-negative organisms.     

Unusual complement-mediated hemolytic kinetics at low ionic strength

The dilution of human serum in relatively low ionic strength buffer (mu = 0.070) caused the spontaneous activation of C1 and a limited activation of C4 and C3 in the fluid phase. The unusual degree of activation of the complement system in the fluid phase suggested that the optimal functions of complement-regulatory systems such as C1 inhibitor might be reduced. As a function of the time of preincubation (PI) of diluted serum at 37 degrees C, under low ionic strength conditions, an unusual complement-mediated hemolytic kinetic pattern was observed upon adding sensitized erythrocytes (EA). For a 1:36 dilution of human serum, there was an initial progressive decrease in complement hemolytic activity (from 3 to 20 min PI, phase I), followed by an apparent functional reversal (increase) in hemolytic activity (20-50 min PI, phase II) and finally a gradual irreversible depletion of the hemolytic activity (after 50 min PI, phase III). This hemolytic pattern could only be adequately demonstrated using a kinetic assay which followed the course of lysis of EA in the presence of low dilutions of human serum as a complement source. Others might have missed this observation due to the use of end-point titration methods which required the use of relatively elevated serum dilutions at the time of EA addition. Mechanisms which governed the variations in hemolytic activity at low ionic strength were not clear. Speculatively, partial consumption of early complement components, generation of free C1q and generation of complement fragments might have accounted for the initial decrease in the hemolytic activity observed in phase I. The apparent functional reversal of hemolytic activity observed in phase II might have involved a critical depletion of C1 inhibitor which occurred secondary to C1 inhibitor binding to C1 (activated by low ionic strength effects) and to the C1 activated at the time of EA addition. Without sufficient regulation, a rapid unrestricted C1-mediated complement activation could have occurred, which resulted in a rapid deposition of complement on the EA. Finally, prolonged exposure of serum to low ionic strength effects appeared to induce a significant complement consumption, which caused a time-dependent irreversible depletion of complement hemolytic activity (phase III). Excess exogenous C1 inhibitor, when co-incubated with diluted serum at low ionic strength, reversed the time-dependent effects of low ionic strength and enhanced the subsequent specific complement-mediated hemolytic activity as compared to controls.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of non-lytic cytolysin-membrane intermediates

In order to understand the nature of cytolysin-membrane interactions, the characteristics of stable, non-lytic cytolysin-target cell intermediates formed at low ionic strength, neutral pH, and at physiological ionic strength, pH 6.0, were examined. Protease treatment of cytolysin-RBC intermediates formed at low ionic strength inhibited subsequent hemolysis when the intermediates were exposed to physiological ionic strength and pH. Similarly, when such intermediates were treated with anti-granule and anti-cytolysin antibodies a significant dose-dependent inhibition of hemolysis was observed. These results suggested that in this non-lytic state the cytolysin molecule was exposed on the RBC surface. If low ionic strength or pH 6.0 generated intermediates were washed in 0.5 M NaCl, hemolytic activity was greatly reduced and cytolysin activity could be recovered from the medium. In addition to RBC, both murine (Yac-1 and Lettre ascites) and human (K562) tumor targets formed cytolysin-target cell intermediates at low ionic strength and at low pH. Multilamellar vesicles composed of either phosphatidylcholine, sphingomyelin or phosphatidylserine inhibited the binding of cytolysin to RBC at both low ionic strength and pH 6.0 indicating a lack of polar head group specificity for cytolysin binding.     

Analysis of parameters affecting the hemagglutination activity of Escherichia coli possessing colonization factor antigens: improved medium for observing erythrocyte agglutination.

The hemagglutination (HA) activity of two strains of Escherichia coli, each possessing different colonization factor antigens (CFA), was examined under different test conditions. The effects of ionic strength, temperature, pH, cations, and reaction surface on erythrocyte (RBC) agglutination were analyzed. Strain H-10407 (CFA/I) caused the agglutination of human, bovine, and chicken RBC, whereas strain CL-9699 (CFA/II) agglutinated only bovine and chicken RBC. The HA activity of both strains increased with decreasing ionic strength, pH, and temperature, the effects of temperature being negligible at low ionic strength. When accounting for ionic strength, the presence of Ca2+, Mg2+, Fe2+, or Fe3+ ions did not increase the HA activity of these bacteria. Optimum conditions for HA of reactive RBC by bacteria included low ionic strength (less than 50 mM) and slightly acidic pH (6.0 to 7.0). Use of a low-ionic-strength medium permitted application of microtitration methods to visualize the HA reactions. Storage of RBC in low-ionic-strength medium did not change their HA properties, and the use of this medium proved superior to saline in overcoming HA variation observed with different preparations of RBC.     

Formation of new quasi-crystalline ordered aggregates by gizzard myosin

Summary Turkey gizzard myosin was found to self-assemble into new polymorphic forms as detected by thin-section electron microscopy. In high ionic strength buffers (0.3 ihm KCl, pH 6.0), aggregates of sidepolar filaments were produced. Electron microscopy of thin sections revealed individual filaments with a 13.5 nm axial repeat. Under a number of conditions, with varying ionic strength, pH, MgCl₂ and ATP, the filaments assembled through the head region with the tail portion projecting out radially from the aggregate. The regions corresponding to heads and tails within the aggregates were established by immunoelectron microscopy using anti-Si and anti-LMM antibodies coupled to gold. These filaments often interacted to produce bilayer sheets, which, when cut perpendicular to the plane of the sheet, appeared as ladders. A hitherto unreported structure was obtained at 0.2m KCl (pH 8.0): myosin aggregated to generate a three-dimensional quasi-crystalline lattice with a 270 nm period. In these aggregates, myosin was arranged in an antiparallel fashion, stacked on one another, producing ribbon-like strips stabilized through non-covalent interactions between heads, thereby producing a crystalline lattice. Neither Mg²⁺ nor ATP were required for this form. Phosphorylation of the regulatory light chains or the cleavage of the heavy chains at a single site in the head region prevented myosin from assembling in the 3-D lattice form. Generally, unphosphorylated myosin produced periodic paracrystals at low ionic strength in the presence of 10 him MgCl₂, but as the ionic strength was increased the regular 3-D lattice became the predominant form. Some paracrystalline forms could be obtained at high ionic strength without magnesium with phosphorylated myosin.     

Effects of preparative parameters on the properties of chitosan hydrogel beads containing Candida rugosa lipase

The influences of the pH, tripolyphosphate (TPP) concentration, and ionic strength of the gelling medium on the entrapment efficiency, release, and activity of lipase in chitosan hydrogel beads were studied. A solution of Candida rugosa lipase was prepared in a 1.5% w/v chitosan and 1% (v/v) acetic acid medium, and dropped into a TPP solution. Release of lipase in pH 7.2 Tris buffer was monitored over 36 h using the micro BCA protein assay. The activity of the entrapped enzyme was assayed using the Sigma lipase activity method. Following preliminary studies, an experimental design was followed to develop mathematical models that describe bead characteristics as functions of the pH and the TPP concentration in the gelling medium. The pH and the TPP concentration each had an effect on the entrapment, retention, and activity of lipase. Entrapped lipase retained a high degree of activity in multiple reactions. The ionic strength, in the range studied, exerted a minimal effect on bead characteristics. Statistical analysis allowed optimization within the factor space with respect to maximizing the enzyme entrapment efficiency and activity, and also minimizing the amount released after 36 h in the Tris buffer.     

The effect of ionic strength on the kinetics of rigor development in skinned fast-twitch skeletal muscle fibres

 Recent atomic 3-D reconstructions of the acto-myosin interface suggest that electrostatic interactions are important in the initial phase of cross-bridge formation. Earlier biochemical studies had also given strong evidence for the ionic strength dependence of this step in the cross-bridge cycle. We have probed these interactions by altering the ionic strength (Γ/2) of the medium mainly with K⁺, imidazole⁺ and EGTA^2– to vary charge shielding. We examined the effect of ionic strength on the kinetics of rigor development at low Ca²⁺ (experimental temperature 18–22°C) in chemically skinned single fast-twitch fibres of mouse extensor digitorum longus (EDL) muscle. On average the delay before rigor onset was 10 times longer, the maximum rate of rigor tension development was 10 times slower, the steady-state rigor tension was 3 times lower and the in-phase stiffness was 2 times lower at high (230 mM) compared to low (60 mM) ionic strength. These results were modelled by calculating ATP depletion in the fibre due to diffusional loss of ATP and acto-myosin Mg.ATPase activity. The difference in delay before rigor onset at low and high ionic strength could be explained in our model by assuming a 15 times higher Mg.ATPase activity and a threefold increase in K _m in relaxing conditions at low ionic strength. Activation by Ca²⁺ induced at different time points before and during onset of rigor confirmed the calculated time course of ATP depletion. We have also investigated ionic strength effects on rigor development with the activated troponin/tropomyosin complex. ATP withdrawl at maximum activation by Ca²⁺ induced force transients which led into a ”high rigor” state. The peak forces of these force transients were very similar at low and high ionic strength. The subsequent decrease in tension was only 10% slower and steady-state ”high rigor” tension was reduced by only 27% at high compared to low ionic strength. Addition of 10 mM phosphate to lower cross-bridge attachment strongly suppressed the transient increases in force at high ionic strength and reduced the steady-state rigor tension by 17%. A qualitatively similar but smaller effect of phosphate was observed at low ionic strength where steady-state rigor force was reduced by 10%. The data presented in this study show a very strong effect of ionic strength on rigor development in relaxed fibres whereas the ionic strength dependence of rigor development after thin filament activation was much less. The data confirm the importance of electrostatic interactions in cross-bridge attachment and cross-bridge-attachment-induced activation of thin filaments. Received: 3 September 1997 / Received after revision and accepted: 12 December 1997     

Native protein-initiated ATRP: a viable and potentially superior alternative to PEGylation for stabilizing biologics

Comparison of in vitro serum stability and enzyme activity retention for PEGylated chymotrypsin and structurally different, biocompatible vinyl polymer grafts of chymotrypsin was performed. These polymer grafts were synthesized by atom transfer radical polymerization (ATRP) initiated by chymotrypsin covalently modified with 2-bromoisobutyric acid, the ATRP initiator. The maximum number of ATRP initiators attached to chymotrypsin was adjusted to be as close as possible to the maximum number of polyethylene glycol chains attached to chymotrypsin for better comparison and then polymerizations were conducted. In mouse serum, native and PEGylated chymotrypsin deactivated within 24h, whereas chymotrypsin-graft-poly(N-2-hydroxypropylmethacrylamide) retained >50% of its catalytic activity even after 5 days of incubation. In human serum, PEGylated chymotrypsin deactivated within 4 days of incubation, whereas native chymotrypsin and chymotrypsin-graft-poly(N-2-hydroxypropylmethacrylamide) and chymotrypsin-graft-poly(2-methacryloyloxyethyl phosphorylcholine) retained >25% catalytic activity after 5 days of incubation. Biocompatible vinyl polymer grafts of chymotrypsin synthesized by protein-initiated ATRP had higher catalytic activity retention and molecular weights and lower polydispersity than PEGylated chymotrypsin. In summary, studying the effects of structures of conjugated polymers on the stability and activity retention of modified proteins can lead to identification of a polymer-protein conjugate having superior pharmacological properties than conventionally PEGylated protein. Also, since vinyl monomers that form biocompatible polymers are easily polymerizable by ATRP, protein-initiated ATRP can become a viable and potentially superior alternative to PEGylation for stabilizing biologics.     

The Effect of Ionic Strength on the Haemolytic Activity of Complement

The haemolytic activity of guinea-pig complement has been measured in isotonic solutions of various ionic strengths in the range 0.034–0.28 and shown to be maximum at an ionic strength close to 0.08. Haemolytic activity was virtually abolished at ionic strength 0.034, while at 0.28, the complement titre was only about 20 per cent of the value found at the physiological ionic strength 0.155. NaCl, KCl, LiBr and K₂SO₄ were the electrolytes used to provide ionic strength, and sucrose, mannitol and inositol the non-electrolytes used to maintain isotonicity. Nine permutations of the four electrolytes with the three non-electrolytes were tested and gave similar results. Human and rabbit complements also showed optimum haemolytic activity at ionic strength 0.08–0.10.     

Drug release from a porous ion-exchange membrane in vitro.

The effect of environmental ionic strength on the rate of drug release from a cation exchange membrane was evaluated. Cationic propranolol-HCl, timolol, sotalol-HCl, atenolol and dexmedetomidine-HCl and neutral diazepam were adsorbed onto a porous poly(vinylidene fluoride) (PVDF) membrane that was grafted with bioadhesive poly(acrylic acid) chains (PAA-PVDF). Despite its porosity, the PAA-PVDF membrane acted as a cation exchange membrane. The release of adsorbed drug from the PAA-PVDF membrane was investigated by using a USP rotating basket apparatus. Adsorption of cationic drugs onto the PAA-PVDF membrane tended to increase with increasing lipophilicity of the drug. A decrease in the ionic strength of the adsorption medium increased the amount of the cationic drugs adsorbed onto the membrane, but had no effect on diazepam adsorption. The release of cationic drugs from the PAA-PVDF membrane was greatly affected by the ionic strength of both the adsorption medium and the dissolution medium, while ionic strengths did not affect diazepam release. Our results suggest that the ionic strength of both the adsorption and dissolution media substantially affects the release rate of a drug that has been adsorbed onto the ion exchange membrane, primarily via electrostatic interactions, while ionic strength has no effect on the release of a drug which has been adsorbed onto the membrane via non-electrostatic forces.     

Binding of complement component C1q by rat adipocyte membranes

Human C1q was found to bind to rat adipocyte membranes with an affinity comparable to that for aggregated immunoglobulin. The binding was ionic strength dependent, and modification of arginyl and histidyl residues in C1q abrogated its binding activity. Treatment of the adipocyte membranes with either high ionic strength buffers, EDTA or trypsin had little effect on their C1q-binding activity.     

Self-interaction of dynein fromTetrahymena cilia

Summary The molecular mass (M_r) and enzymic activity of the larger dynein species fromTetrahymena thermophila has been studied in the high (600mm) to low (40mm) ionic strength range. The apparent M_r is found to vary with both ionic strength (by sedimentation velocity and quasi elastic light scattering analysis) and with protein concentration at low ionic strength (by sedimentation equilibrium analysis). These data indicate a strong self-interaction, resulting in dimer formation under low salt conditions. There is no evidence for the formation of species of higher than dimeric mass. A molecular mass for the dynein monomer of 1.64×10⁶ daltons has been determined, a value rather lower than previous published estimates.The ATPase activity of dynein increases with increasing ionic strength. The possible relationship between this effect and the self-association phenomenon is discussed.     

Porous akermanite scaffolds for bone tissue engineering: preparation, characterization, and in vitro studies

The aim of this study was to develop a bioactive, degradable, and cytocompatible akermanite (Ca2MgSi2O7) scaffold with high porosity and pore interconnectivity. In brief, porous akermanite scaffolds were prepared using polymer sponge method. The porosity and corresponding compressive strength were evaluated. The in vitro degradability was investigated by soaking the scaffolds in Ringer's solution. Hydroxyapatite (HAp)-formation ability of akermantite scaffolds in simulated body fluid (SBF) and the effect of ionic products from the scaffolds dissolution on osteoblasts were investigated. In addition, bone marrow stromal cells (BMSC) adhesion and proliferation on the scaffolds were evaluated. Differentiation of the cells was assessed by measuring alkaline phosphatase (ALP) activity. The results showed that akermanite scaffolds possessed 63.5-90.3% of porosity, with a corresponding compressive strength between 1130 and 530 kPa. The weight loss of the scaffolds and ionic content of the Ringer's solution increased with the increase in soaking time, indicating the degradability of scaffolds. HAp was formed on the scaffolds in SBF and the ionic products from akermanite scaffolds dissolution stimulated osteoblasts proliferation, indicating good in vitro bioactivity. Furthermore, BMSC adhered and spread well on akermanite scaffolds and proliferated with the increase in the culture time, and the differentiation rate of osteoblasts on scaffolds was comparable to that on blank culture plate control. Our results suggested that akermanite scaffolds were bioactive, degradable, and cytocompatible, and might be used as bone tissue engineering materials.     

Diffusion of lysozyme in gels and liquids. A general approach for the determination of diffusion coefficients using holographic laser interferometry

A study on diffusion measurements of the protein lysozyme in liquids and agarose gels, at different pH and ionic strengths, has been performed using holographic laser interferometry. The measurements showed that the diffusive flux was very dependent on pH and ionic strength when the protein was not at its isoelectric point or when the charge of the lysozyme molecules was not screened by ions in the solution. Evaluation of the experimental data with Fick's law, resulted in diffusion coefficients for lysozyme that are strongly dependent on pH and ionic strength. Evaluation of the experimental data using a more general transport model, based on chemical potential gradients instead of concentration gradients resulted in lysozyme diffusion coefficients that are independent of pH and ionic strength. The chemical potential was estimated by using the Poisson-Boltzmann equation.     

Surface characterization and on-line activity measurements of microorganisms by capillary zone electrophoresis

Capillary zone electrophoresis (CZE) was applied to the electrophoretic characterization for microorganisms. The electrophoretic peaks detected using light scattering phenomena were characteristic of the microorganisms used. The electrophoretic mobility (mu) evaluated by CZE was in good agreement with that obtained by classical electrophoresis of microorganisms. The migration time was reproducible and depended on the ionic strength (I). Analysis of the mu vs. I relationship provided information regarding the charge density and the hardness of the microbial cell surface. The redox enzymatic activity of microorganisms was also evaluated by CZE using a running buffer containing a corresponding substrate and an appropriate exogenous electron acceptor. A decrease in the concentration of the electron acceptor due to microbial activity can be simultaneously monitored during the electrophoretic process without significant modification of the CZE instrument. Effects of some chemical treatments of microbial cells were also studied using this technique.     

A novel pH- and ionic-strength-sensitive carboxy methyl dextran hydrogel

A fast and simple method for the preparation of pH-sensitive hydrogel membranes for drug delivery and tissue engineering applications has been developed using carbodiimide chemistry. The hydrogels were formed by the intermolecular cross-linking of carboxymethyl dextran (CM-dextran) using 1-ethyl-(3-3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). Infrared spectra of the hydrogels suggest the formation of ester bonds between the hydroxyl and carboxyl groups in the CM-dextran. The porosity of the hydrogels produced, as shown by protein diffusion, increases in response to changes in the pH and the ionic strength of the external medium. The results show pH-dependent swelling behaviour arising from the acidic pedant groups in the polymer network. The diffusion of the protein lysozyme through the hydrogel membranes increased with increases in both pH (5.0-9.0) and ionic strength. The effect of changes of pH and ionic strength on the hydrogel's permeability was shown to be reversible. Scanning electron microscopy of these hydrogels showed that pH-dependent changes in permeability are mirrored by morphological changes in gel structure.     

Reduction of ionic strength activates single volume-regulated anion channels (VRAC) in endothelial cells

We have previously shown that a reduction of intracellular ionic strength is involved in the activation of volume-regulated anion channels (VRAC). Here we show in a single-channel study that VRAC can be activated in a cell-attached patch when the cell interior is dialyzed with a solution of decreased ionic strength. For this purpose, bovine pulmonary endothelial (CPAE) cells) were permeabilized with alpha-staphylotoxin (alphaST) which has a molecular weight cut-off size of 2 kDa. If the ionic strength in the bath solution is reduced from 160 mM to 95 mM, single-channel activity is initiated in cell-attached patches sealed before permeabilization. Conductance is outwardly rectifying with approximately 17 pS at negative and 57 pS at positive potentials. Single-channel currents reverse near the calculated equilibrium potential for Cl-. The averaged current shows inactivation at positive potentials. The current is blocked by 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB). An increase in ionic strength reversibly inhibits current activation. It is concluded that a decrease in ionic strength activates single-channel currents through VRAC rather than shifting the set point of a hypothetical volume sensor.     

Controlled release of doxorubicin from pH-responsive microgels

Stimuli-responsive hydrogels have enormous potential in drug delivery applications. They can be used for site-specific drug delivery due to environmental variables in the body such as pH and temperature. In this study, we have developed pH-responsive microgels for the delivery of doxorubicin (DOX) in order to optimize its anti-tumor activity while minimizing its systemic toxicity. We used a copolymer of oligo(polyethylene glycol) fumarate (OPF) and sodium methacrylate (SMA) to fabricate the pH-responsive microgels. We demonstrated that the microgels were negatively charged, and the amounts of charge on the microgels were correlated with the SMA concentration in their formulation. The resulting microgels exhibited sensitivity to the pH and ionic strength of the surrounding environment. We demonstrated that DOX was efficiently loaded into the microgels and released in a controlled fashion via an ion-exchange mechanism. Our data revealed that the DOX release was influenced by the pH and ionic strength of the solution. Moreover, we designed a phenomenological mathematical model, based on a stretched exponential function, to quantitatively analyze the cumulative release of DOX. We found a linear correlation between the maximum release of DOX calculated from the model and the SMA concentration in the microgel formulation. The anti-tumor activity of the released DOX was assessed using a human chordoma cell line. Our data revealed that OPF–SMA microgels prolonged the cell killing effect of DOX.     

Chemical Characterization of Macrophage Cytotoxicity Factor,Macrophage Migration Inhibitory Factor,T-Helper Cell-Replacing Factor and Colony-Stimulating Factor from Culture Supernatants of Concanavalin A-Stimulated Murine Spleen Cells

Supernatants from Concanavalin A-stimulated murine spleen cells were subjected to hydrophobic interaction chromatography on phenyl-Sepharose. Macrophage cytotoxicity factor (MCF), macrophage migration inhibitory factor (MIF), T-helper cell-replacing factor (TRF) and colony-stimulating factor (CSF) were bound at high ionic strength and were released stepwise at low ionic strength. CSF thus could be separated from MCF, MIF and TRF and the bulk of other proteins. Chromatography of pools containing MCF, MIF and TRF on Sephadex did not lead to a separation of the three activities which were all found in a molecular weight range of 25.000-55.000. Isoelectric focusing of these pools in pH range from 4 to 9 gave two peaks for MCF at pH 8.2 and 7.2, whereas MIF activity focused from pH 4.5 to 5.5. TRF activity was found in a single sharp peak at pH 5.3. The results demonstrate that the four biological activities can be distinguished on a chemical basis and are accessible for purification and chemical characterization.     

Chicken (Gallus gallus) hemolytic complement: optimal conditions for its titration.

The effect of pH, ionic strength, cation concentration, ethylenediaminetetraacetic acid trisodium salt (Na3EDTA), time and temperature were studied to determine the optimal conditions for titrating the hemolytic complement (C) activity in sera of chicken (Gallus gallus). Swine erythrocytes (E) sensitized with rabbit antibodies were the most sensitive, while chicken serum had a smaller amount of "natural" antibody against them than against red blood cells from other five species tested. The highest titers of chicken C, when tested with swine sensitized E, were detected when isotonic NaCl-barbital buffer was used as diluent, having the ionic strength of 0.15, conductance of 11 millimhos/cm at 20 degrees C. However, maximal chicken C titers detected with sensitized rabbit E were obtained at ionic strength of 0.07 to 0.11 depending on pH. A final concentration of 1 X 10(-3) M of Mg2+ and 3 X 10(-4) M of Ca2+ and pH 8 were optimal in both cases. The temperature of 30 degrees C and time of 60 minutes were appropriate to reveal the maximal titers..