N-trimethylammonium Bromide Research Articles

Nature of linkage between the cationic headgroup and cholesteryl skeleton controls gene transfection efficiency

Three novel cationic cholesterol derivatives with different modes of linkage between the cationic headgroup and the cholesteryl backbone have been synthesized and used as mixtures with 1,2-dioleoyl- L-α-glycero-3-phosphatidyl ethanolamine (DOPE) for liposome-mediated gene transfection. A pronounced improvement in gene transfer efficiency was observed when the cationic center was appended to the cholesteryl backbone using an ether linkage as opposed to when the linkages were based on either ester or urethane groups. Amphiphiles with ether links such as cholest-5-en-3β-oxyethane- N, N, N-trimethyl ammonium bromide ( 2) and cholest-5-en-3β-oxyethane- N, N-dimethyl- N-2-hydroxyethyl ammonium bromide ( 3) showed transfection efficiencies considerably greater than commercially available gene transfer reagents. Notably, the transfection ability of 2 with DOPE in the presence of serum was significantly greater than Lipofectamine ® and Lipofectin ®. Interestingly, 3 did not require the helper lipid DOPE for transfection. This suggests that these newly described cholesterol-based amphiphiles should be very promising in liposome-mediated gene transfection. The advantage that the ether linkage possesses would be important in the design of newer, more efficient cholesterol-based delivery reagents.

Methods for simultaneous quantitative analysis of eosinophil and neutrophil adhesion and transmigration.

Granulocytes play important roles in various inflammatory responses. The aim of this study was to develop in vitro methods to enable simultaneous analysis of eosinophil and neutrophil adhesion and transmigration in mixed granulocyte preparations. We used fibronectin-coated plates, with or without semipermeable inserts, to measure adhesion and transmigration. Granulocytes, from healthy blood donors, were stimulated with either interleukin (IL)-5 and eotaxin or N-formyl-methionyl-leucyl-phenylalanine (fMLP), during incubation in the wells. Three different detergents: n-octyl-beta-D-glucopyranoside (OG), Triton-X-100 or N-cetyl-N,N, N-trimethylammonium bromide (CTAB), were tested for their ability to cause lysis of granulocytes with minimal effect on eosinophil cationic protein (ECP) and myeloperoxidase (MPO) antigenicity. These two proteins were used for quantitative analysis of eosinophil and neutrophil adhesion/transmigration, and CTAB was the most efficient lysing reagent. Cell-recovery rates, based on ECP/MPO measurements, were > 95% in both assays. The adhesion and transmigration of eosinophils increased in a time-dependent manner upon stimulation with IL-5 and eotaxin. Eosinophil adhesion reached a plateau at 90 min of incubation and transmigration at 240 min. Neutrophils displayed a similar pattern of adhesion and transmigration upon activation with fMLP, reaching respective plateaux at 30 and 90 min. Our study shows that CTAB is an effective detergent for lysing granulocytes, yielding high and reproducible recovery rates of ECP and MPO. Measurement of ECP and MPO, as markers for cell counts, can therefore be used to quantify adhesive and transmigration properties of eosinophils and neutrophils in mixed granulocyte populations.

Development of Short‐Range Repulsive Potentials by Short‐Chain Surfactants in Aqueous Slurries

The effect of both cationic (n‐trimethylammonium bromide, CnTAB) and zwitterionic (phosphocholine, CnCnPC) surfactants on the properties of aqueous slurries of Si3N4 was studied. These surfactants were expected to adsorb to the surface of Si3N4 particles to produce short‐range repulsive interparticle potentials that might be useful for the colloidal processing of advanced ceramic powders. Electrophoretic, adsorption, and viscosity measurements showed that longer‐chain‐length surfactants (CnTAB, n≥ 12, and CnCnPC, n≥ 9) strongly adsorb. Surfactants with shorter chain lengths were highly soluble and did not adsorb. Although the CnTAB, n≥ 12, surfactants produced very weak particle networks with a low viscosity, the packing density during consolidation was very low, and the bodies were brittle (cracked before plastic deformation). The less‐soluble, longer‐chained CnCnPC, n≥ 9, surfactants did produce high particle‐packing densities but also produced brittle bodies. In all cases, it appeared that the surfactants could be pushed away from the surface during particle packing.

Flotation removal of algae from water

The dispersed air flotation process was utilized to remove algae ( Scenedesmus quadricauda) from water. Three types of collector, cationic N-Cetyl- N- N- N-trimethylammonium bromide (CTAB), anionic sodium dodecylsulfate (SDS), and the nonionic Triton X-100 were used and compared. It was observed that ca. 10% of algae removal was achieved when SDS and Triton X-100 were used, respectively; and ca. 90% algae was removed when CTAB was used. Upon the addition of 10 mg l −1 of chitosan, over 90% algae was removed when SDS was used as the collector. The electrostatic interactions between collector and algae surface plays a critical role in the removal. Effects of pH, ionic strength, air flow rate, and alkalinity on flotation efficiency were also investigated.

Synthesis, characterization and sensing application of novel semiconductor oxides

Mesoporous SnO 2 with high surface areas were synthesized using a cationic surfactant ( N-cetyl- N, N, N-trimethylammonium bromide) as a synthetic template. Acidity of the starting synthesis slurry was used as one of the controlling parameters for the synthesis. After the SnO 2 was synthesized at pH 7.15, it was calcined at 723 K for 10 h in air. It had a BET surface area of 156.8 m 2 g −1 with a pore diameter of 38.4 Å. Infrared spectroscopy (FTIR) and thermal analysis techniques (thermogravimetry and differential thermal analysis) showed that the surfactant was incorporated in the mesopores of SnO 2 and calcination in air at 673–723 K was needed to remove the surfactant completely from the mesopores. The effects of SnO 2 surface area on its gas sensing properties were also investigated. It was observed that SnO 2 with higher surface areas had much higher sensitivities to hydrogen at 573 K.

Measurement of in situ halophilic glyceraldehyde-3-phosphate dehydrogenase activity from the permeabilized cells of archaebacterium Haloarcula vallismortis.

The cells of Haloarcula vallismortis, an extreme halophilic archaebacterium, were permeabilized by various chemical, physical, and biological treatments. Biological permeabilization by lysozyme and papain showed effective results as observed by studying the in situ activity of halophilic glyceraldehyde-3-phosphate dehydrogenase (hGAPDH) as the model enzyme. Detergents N-cetyl-N, N, N-trimethyl ammonium bromide (CTAB) and digitonin also showed significant results. Other strains of halobacteria could also be permeabilized by lysozyme. The cell morphology did not show any significant change after permeabilization as observed by phase contrast microscopy. The enzyme characteristics of hGAPDH were studied in situ using permeabilized H. vallismortis cells. The properties, like optimum pH, Km for GAP and NAD(+), inhibition by heavy metals, sulphydryl reagents, and other compounds, showed remarkable similarity with those studied in vitro.

Kinetic and calorimetric investigations on micelle formation of block copolymers of the poloxamer type

Block copolymers of the Poloxamer type EO x PO y EO x (where EO = ethylene oxide and PO = propylene oxide) form micelles in aqueous solution that have a hydrophobic core of PO blocks and a strongly hydrated shell of EO blocks. The influence of electrolyte and surfactant on the aggregation behavior of three Poloxamers (F127, F88 and P123) was investigated by differential scanning calorimetry (DSC) and T-jump measurements. The micelle formation is influenced both by addition of electrolyte and of surfactant. The critical micellization temperature of the Poloxamer decreases linearly with increasing weight fraction of electrolyte and the decrease is independent of the cation size (Li +, Na +, K +). The DSC peak that is due to the micelle formation of the Poloxamer disappears with addition of surfactant. The adsorption of the anionic surfactant sodium dodecyl sulfate (SDS) starts at a lower concentration than the adsorption of the cationic surfactant N-dodecyl- N, N, N-trimethyl ammonium bromide (DTAB), whereas the latter is adsorbed to a larger extent. For all investigated Poloxamers one relaxation time τ in the millisecond range is obtained. For constant temperature the relaxation time decreases with the total concentration of block copolymer. For constant concentration of block copolymer the relaxation time decreases with increasing temperature and increasing surfactant concentration. The relaxation process can be described by the Aniansson-Wall mechanism and values for the association rate constant for the poloxamers were determined. The evaluated rate constants are in the range of 0.2 × 10 7–2 × 10 7 l mol −1 s −1 and are not diffusion controlled. The micellization kinetics depends on the molecular structure of the Poloxamer, which determines the structure of the micelles. The thicker the shell of the micelle (i.e. the EO block length), the smaller the association rate constant k +. Keeping the EO block length constant and decreasing the PO block size again increases k +. Now the hydrophobic part of monomeric Poloxamer controls the penetration through the EO shell.

Analysis of Various Very Slightly Water-Soluble Drugs in Micellar Medium. I. Potentiometric and Visual Determination of Non-Steroidal Anti-Inflammatory Therapeutic Agents

Abstract Each one of four very slightly water-soluble to practically water-insoluble, non-steroidal anti-inflammatory, antipyretic and analgesic drugs, such as indomethacin(I), aspirin(II), ibuprofen(III) and flurbiprofen(IV) were dissolved in a N-hexadecyl-N, N, N-trimethylammonium bromide (CTAB) micellar medium and the resulting solutions were titrated with a carbonate-free 2.5 mmol/L NaOH solution. The end point determined potentiometrically by the convetional manner, using the second derivative graph, while the routine analytical applications were carried out either by an automatic titration assembly or by visual titration using the selected acid-base indicator. Results for(I–IV) agreed well with those obtained by the method of the USP XXII and a statistical analysis by means of Student's t-test, as well as by the variance ratio F-test proved that between the proposed method and the official one no significant difference was observed. The method described in the present paper, is simple, precise, inexpensive and it could successfully replace the established non-aqueous techniques.

Determination of bis(amidinohydrazones) by micellar electrokinetic capillary chromatography

A micellar electrokinetic capillary chromatography method was developed for the separation and determination of aliphatic congeners of bis(amidinohydrazones) in standard solution. Eight bis(amidinohydrazones) could be determined in less than 15 min at an applied voltage of 22 kV, using 0.05 M sodium phosphate as buffer (pH 7.0) together with 1 mM N-cetyl- N, N, N-trimethylammonium bromide. Hydrostatic sample injection was employed. The method exhibited good repeatability and a linear range of 2.5–100 μg ml −1. A detection limit of 1 μg ml −1 was achieved. The method also allows the determination of bis(amidinohydrazones) in human serum samples.

Immobilization of permeabilized Escherichia coli cells with penicillin acylase activity

Escherichia coli cells with penicillin acylase activity were sequentially treated at pH 7.8 with aqueous solutions of N-cetyl-N, N, N-trimethylammonium bromide and glutaraldehyde and then immobilized within porous polyacrylamide beads. The immobilized whole cells showed enhanced hydrolysis rates in the conversion of benzylpenicillin to 6-aminopeicillannic acid (6-APA) compared to untreated cells immobilized and used under identical conditions. The immobilized system showed no apparent loss in enzyme activity when used repeatedly over 90 cycles for 6-APA production from 4% benzylpenicillin.

A multivariate calibration procedure for the tensammetric determination of detergents

A multivariate calibration procedure based on singular value decomposition (SVD) and the Ho-Kashyap algorithm is used for the tensammetric determination of the cationic detergents Hyamine 1622, benzalkonium chloride (BACl), N-cetyl- N, N, N-trimethylammonium bromide (CTABr) and mixtures of CTABr and BACl. The sensitivity and accuracy depend strongly on the nature of the detergent. Acceptable accuracy is obtained with a two-step calculation procedure in which calibration constants for the total concentration range of interest are used to guide the choice of a more specific set of calibration constants which are valid for a much smaller concentration span. For Hyamine 1622, concentrations in the range 5 × 10 −6−2 × 10 −4 M could be determined with an accuracy of ± 10 −6 M. For CTABr, these numbers were 3 × 10 −6−2 × 10 −4 M and ± 5 × 10 −7 M; for BACl, they were 2 × 10 −3−9 × 10 −2 g l −1 and ± 1 × 10 −3 g l −1. In the mixtures of CTABr and BACl, the accuracies were ± 3 × 10 −6 M and × 1 × 10 −3 g l −1, respectively.

RETRACTED: Calcium-dependent effect of the thymic polypeptide thymopoietin on the desensitization of the nicotinic acetylcholine receptor.

The effects of the thymic polypeptide thymopoietin (Tpo) on the properties of the nicotinic acetylcholine receptor (AcChoR) were investigated by patch clamp techniques on mouse C2 myotubes and by biochemical assays on AcChoR-rich membrane fragments purified from the Torpedo marmorata electric organ. At high concentrations (greater than 100 nM), Tpo inhibits the binding of cholinergic agonists to the AcChoR in a Ca2+-insensitive manner. At lower concentrations (2 nM), Tpo applied on C2 myotubes simultaneously with nondesensitizing concentrations of acetylcholine results in the appearance of long closed times separating groups of openings. This effect depends on the presence of Ca2+ in the external medium. Outside-out recordings, performed with various concentrations of EGTA in the intracellular medium, suggest that Ca2+ acts on the cytoplasmic face of the membrane after entry through acetylcholine-activated channels. Parallel studies with T. marmorata AcChoR-rich membranes show that in the presence of Ca2+ Tpo causes a decrease in the apparent equilibrium dissociation constant of the noncompetitive blocker [3H]phencyclidine, enhances, at low concentrations, the binding of [3H]acetylcholine, and also alters the binding kinetics of the fluorescent agonist 6-(5-dimethylamino-1-naphthalenesulfonamido)-n-hexanoic acid beta-(N-trimethylammonium bromide) ethyl ester to the AcChoR. It was concluded that, in the presence of Ca2+, Tpo displaces the conformational equilibrium of the AcChoR towards a high-affinity desensitized state and increases the transition rate towards the same state.

Occurrence of short-sized oligo(A) fragments during course of cell cycle and ageing

Affinity chromatography of nucleic acids precipitated by N-cetyl- N, N, N-trimethyl ammonium bromide on poly(U)-Sepharose has proved to be a suitable method for a nearly quantitative isolation of oligo(A) sequences down to a chain length of 4 nucleotide units. Analysis of short oligo(A) fragments in synchronized L5178y mouse lymphoma cells after labeling with [ 3H]Ado revealed that the percentage of A 2–6 sequences on the total radioactivity amounted in S-phase cells to 1.6%, while the value obtained for the stationary L-cell system was 8.0%. The alterations of occurrence and chain length distribution of short oligo(A) fragments during ageing were studied in two age groups of female quails: mature (250–320 days old) and senescent animals (3–3.5 yr old). It was found that the amount of low molecular weight oligo(A) fragments gradually decreases during ageing of the animals; the amount in the mature animal group was significantly higher (6-fold) than in the old animal group. The decreased amounts of oligo(A) during S phase and ageing could in part be due to posttranslational modification of enzymes involved in poly(A) metabolism. It could be demonstrated that both homogeneous poly(A) anabolic poly(A) polymerase and homogeneous poly(A) catabolic endoribonuclease IV are phosphorylated by nuclear protein kinase NI.

Fast Kinetic Studies on the Interaction of a Fluorescent Agonist with the Membrane‐Bound Acetylcholine Receptor from Torpedo marmorata

The fluorescent cholinergic effector, [1‐(5‐dimethylamino naphthalene)sulfonamido] n‐hexanoic acid β‐(N‐trimethylammonium bromide) ethyl ester (Dns‐C6‐Cho) acts as a potent depolarizing agent or agonist on Electrophorus electricus electroplaque (maximal depolarization 65–70 mV). In vitro, it increases the efflux of 22Na+ from receptor‐rich membrane fragments prepared from Torpedo marmorata electric organ. The apparent dissociation constant determined with the two systems is close to 1 μM. Preincubation of the receptor‐rich microsacs with Dns‐C6‐Cho results in a decrease of the amplitude of the permeability response to carbamylcholine. Like the physiological transmitter, acetylcholine, Dns‐C6‐Cho triggers the two characteristic reactions of ‘activation’ and ‘desensitization’.The equilibrium dissociation constant of Dns‐C6‐Cho measured by following the decrease of the initial velocity of binding of Naja nigricollisα‐[3H]toxin to receptor‐rich microsacs is close to 0.01 μM after prolonged incubation. Using the same method, it is found that Dns‐C6‐Cho triggers, in the range of a few minutes, a time‐dependent increased of affinity.The interaction of Dns‐C6‐Cho with the receptor‐rich membranes can be monitored under conditions of energy transfer from proteins (λex= 290 nm) and results in an approximately 10‐fold increase of fluorescence intensity at the maximal emission wavelength (λem= 557 nm). Rapid mixing of a suspension of membrane fragments with Dns‐C6‐Cho gives rise to changes of fluorescence intensity which can be recorded in the time range of seconds to milliseconds. The signals disappear after pre‐incubation of the membrane fragments with saturating levels of N. nigricollisα‐toxin and therefore are due to the interaction of Dns‐C6‐Cho with the receptor site. Three major relaxation processes are observed. (a) A ‘rapid’ one, in the millisecond range, is characterized by a linear dependence of its apparent rate constant with Dns‐C6‐Cho concentration. It is analysed in terms of Dns‐C6‐Cho binding to receptor sites spontaneously present in a high‐affinity state for agonists (Kd∼ 3 nM). These sites represent approximately 20% of the total population of receptor sites and exist prior to agonist binding in the membrane at rest. (b) An ‘intermediate’ relaxation process, in the second–millisecond range, shows a linear dependence of the apparent rate constant with Dns‐C6‐Cho concentration and is accounted for by a bimolecular binding reaction between Dns‐C6‐Cho and acetylcholine receptor sites in a state (or states) of lower affinity (∼ 1 μM). (c) Finally, a ‘slow’ relaxation process, in the seconds range, is characterised by an apparent rate of an interconversion between low‐affinity and high‐affinity states of the receptor.A model with two pre‐existing and interconvertible affinity states for the receptor qualitatively and quantitatively accounts for the data, and the corresponding rate constants have been determined; the model also accounts for the complex kinetics monitored upon addition of a non‐fluorescent effector to a suspension of receptor‐rich membrane fragments equilibrated with Dns‐C6‐Cho; the possible occurrence of a third state with intermediate affinity is discussed.The slow interconversion from low‐affinity to high‐affinity states of the receptor is correlated with the physiological process of ‘desensitization’ on the basis of its dependence on agonist concentration and temperature and of the effect of local anesthetics; the features of the intermediate relaxation process and its possible correlation with the ‘activation’ reaction are discussed.

Procedure for the simultaneous large‐scale isolation of pullulanase and 1,4‐α‐glucan phosphorylase from Klebsiella pneumoniae involving liquid–liquid separations

A procedure for the simultaneous large-scale isolation of pullulanase and 1,4-alpha-glucan phosphorylase from Klebsiella pneumoniae is described. The pullulanase is solubilized from the cell wall by cholate treatment; cells and cell debris are removed by partition in a poly(ethylene glycol) (PEG)-dextran two-phase system and from the upper (PEG) phase of this system the pullulanase is isolated by ultrafiltration and precipitation with N-cetyl,N-,N-,N-trimethyl ammonium bromide to a purity of about 80% with a yield of 70%. The preparations are free of alpha-amylase activity. The cell containing dextran-rich phase is passed through a Manton-Gaulin homogenizer. Then the phosphorylase is separated from the cell debris by partition in a second PEG-dextran system. From the top phase of this system the phosphorylase is isolated by distribution in a PEG-salt two-phase system followed by batch adsorption on carboxymethyl-Sephadex in a yield of 55%, a purity of around 90%, and nearly free of glycosyltransferase activity. All steps in the isolation of the two enzymes can be performed easily in a large scale.