Cationic Detergent Research Articles

Physical, chemical and kinetic factors affecting prion infectivity

ABSTRACTThe mouse-adapted scrapie prion strain RML is one of the most widely used in prion research. The introduction of a cell culture-based assay of RML prions, the scrapie cell assay (SCA) allows more rapid and precise prion titration. A semi-automated version of this assay (ASCA) was applied to explore a range of conditions that might influence the infectivity and properties of RML prions. These include resistance to freeze-thaw procedures; stability to endogenous proteases in brain homogenate despite prolonged exposure to varying temperatures; distribution of infective material between pellet and supernatant after centrifugation, the effect of reducing agents and the influence of detergent additives on the efficiency of infection. Apparent infectivity is increased significantly by interaction with cationic detergents. Importantly, we have also elucidated the relationship between the duration of exposure of cells to RML prions and the transmission of infection. We established that the infection process following contact of cells with RML prions is rapid and followed an exponential time course, implying a single rate-limiting process.

Oriented Peptide Immobilization on Microspheres.

Reproducible immobilization of peptides and proteins on microsphere surfaces is a critical factor for optimal sensitivity and selectivity in bead-based assays. However, peptides with unusually large numbers of lysine residues-whose amines are targeted in the most common microsphere immobilization chemistries-may be particularly challenging to use in bead-based arrays, as they may lose activity through multipoint attachments and incorrect presentation. For this reason, it is imperative to achieve site-directed attachment chemistry, such that a single site of attachment provides reproducibly oriented peptides on the microsphere surface. This can be achieved by inserting a unique targetable residue, such as a cysteine. Here, we present methods for attaching cysteine-containing peptides to standard carboxy-functionalized microsphere surfaces using thiol- rather than amine-directed chemistries. We show that the presence of a cationic detergent (CTAB) and a "passivating" agent such as β-mercaptoethanol facilitates improved bead recovery after peptide immobilization and may enhance functionality of the attached peptides.

Effect of surface-potential modulators on the opening of lipid pores in liposomal and mitochondrial inner membranes induced by palmitate and calcium ions

The effect of surface-potential modulators on palmitate/Ca2+-induced formation of lipid pores was studied in liposomal and inner mitochondrial membranes. Pore formation was monitored by sulforhodamine B release from liposomes and swelling of mitochondria. ζ-potential in liposomes was determined from electrophoretic mobility. Replacement of sucrose as the osmotic agent with KCl decreased negative ζ-potential in liposomes and increased resistance of both mitochondria and liposomes to the pore inducers, palmitic acid, and Ca2+. Micromolar Mg2+ also inhibited palmitate/Ca2+-induced permeabilization of liposomes. The rate of palmitate/Ca2+-induced, cyclosporin A-insensitive swelling of mitochondria increased 22% upon increasing pH from 7.0 to 7.8. At below the critical micelle concentration, the cationic detergent cetyltrimethylammonium bromide (10μM) and the anionic surfactant sodium dodecylsulfate (10–50μM) made the ζ-potential less and more negative, respectively, and inhibited and stimulated opening of mitochondrial palmitate/Ca2+-induced lipid pores. Taken together, the findings indicate that surface potential regulates palmitate/Ca2+-induced lipid pore opening.

Laboratory test system for the evaluation of nanomaterial toxicity on Dunaliella salina microalgae

We are reporting a new diagnostic test system for evaluating nanomaterial toxicity on halophile green microalgae Dunaliella salina based on colorimetric measurements enabling us to estimate cell death and growth-rate inhibition in microalgae suspension for serial dilutions of several toxicants. The principle of the method is the photodegradation of chlorophyll from damaged cells, resulting in the discoloration of the cell sus- pension, which enables us to estimate the microalgae population viability. The cell response was studied for two standard pollutants: formaldehyde and hydrogen peroxide. The toxic effect of free CTAB (cetyltrimetilammo- nium bromide, cationic detergent) water solution was compared with the effect of gold nanoparticles: nanorods, nanostars, and nanospheres, all synthesized in CTAB solutions. It was shown that toxicity is mainly caused by free (nonabsorbed on nanoparticles) surfactant molecules. The toxicity on microalgae D. salina decreases in the following order of silver-containing compounds: silver nitrate > silver proteinate >> colloidal silver in same order.

Glycosaminoglycan blotting and detection after electrophoresis separation.

Separation of glycosaminoglycans (GAGs) by electrophoresis and their characterization to the microgram level are integral parts of biochemical research. Their blotting on membranes after electrophoresis offers the advantage to perform further analysis on single separated species such as identification with antibodies and/or recovery of single band. A method for the blotting and immobilizing of several nonsulfated and sulfated complex GAGs on membranes made hydrophilic and positively charged by cationic detergent after their separation by conventional agarose-gel electrophoresis is illustrated. This approach to the study of these complex macromolecules utilizes the capacity of agarose-gel electrophoresis to separate single species of polysaccharides from mixtures and the membrane technology for further preparative and analytical uses. Nitrocellulose membranes are derivatized with the cationic detergent cetylpyridinium chloride (CPC) and mixtures of GAGs are capillary blotted after their separation in agarose-gel electrophoresis. Single purified species of variously sulfated polysaccharides are transferred on derivatized membranes with an efficiency of 100 % and stained with alcian blue (irreversible staining) and toluidine blue (reversible staining). This enables a lower amount limit of detection of 0.1 μg. Nonsulfated polyanions, for example hyaluronic acid (HA), may also be transferred to membranes with a limit of detection of approximately 0.1-0.5 μg after irreversible or reversible staining. The membranes may be stained with reversible staining and the same lanes used for immunological detection or other applications.

Degradation of Food industrial pollutants by photocatalysis with immobilized titanium dioxide

The paper deals with application of advanced oxidation processes in degradation of surfactants, which represent large group of pollutants in food industrial waste waters. The photocatalytic degradation of two surfactants, an anionic sodium dodecyl sulphate (SDS) and cationic N-cetyl-N,N,N-trimethylammonium bromide (CTAB), was tested in a lab-scale UV reactor (Mikropur, Czech Republic) with immobilized titanium dioxide catalyst (P25, Degussa). Various conditions were verified, e.g. initial detergent concentrations (1.2–18.7 mg/l), oxygen flow rates (0 or 72.2 ml/min) and amount of catalyst used. The degradation in a presence of catalyst, oxygen and UV irradiation was the fastest process with maximum achieved rate constants of 0.0503 min − 1 at anionic surfactant degradation and 0.342 min − 1 at cationic detergent degradation. The energy demands for SDS degradation were also calculated and possible scale-up into large industrial scale discussed. Due to the relatively high organic content, food industrial waste waters contribute to the overall pollution problem. A need for water purification at low cost and energy with limited use of chemicals is still rising. Therefore, new advanced oxidation processes (AOPs), which are capable to oxidize and mineralize almost any organic contaminant, represent rapidly developing field of recent technological research. This study deals with possible application of photocatalysis with immobilized TiO 2 in degradation of surfactants, which are quite common pollutants in food industrial waste waters, and compares this method with others. The energy balance of the process and an outline of possible industrial application were mentioned as well. • Applications of advanced oxidation processes in food waste water treatment. • Anionic and cationic surfactants decomposed by photocatalysis with immobilized TiO 2 . • The effect of UV, oxygen flow and catalyst surface density on oxidation was tested. • The energy demands for the degradation of SDS were calculated. • Photocatalysis compared with other degradation methods.

The surfactant-induced conformational and activity alterations in Rhizopus niveus lipase.

In this study, we have reported the effect of nonionic, anionic, cationic, and zwitterionic detergents on the enzymatic activity and structural stability of Rhizopus niveus lipase. Secondary structural changes were monitored by Far-UV CD which shows that surfactant induces helicity in the Rhizopus niveus lipase protein which was maximum in case of CTAB followed by SDS, CHAPS, and Brij-35. Similarly, tertiary structural changes were monitored by tryptophan fluorescence. We also carried out enzyme kinetics assays which showed that activity was enhanced by 1.5- and 1.1-fold in the presence of CHAPS and Brij-35, respectively. Furthermore, there was a decline in activity by 20 and 30% in case of SDS and CTAB, respectively. These studies may be helpful in understanding detergent-lipase interaction in greater detail as lipases are used in many industrial processes.

English

In this study, several chemical treatment techniques were verified for releasing cytoplasmic cephalosporin C deacetylase (CAH) from recombinant Escherichia coli (E. coli) cells. Among all the chemical treatment methods, using cationic detergent, organic solvents and chelating agents as permeabilizing reagents exhibited limited capacities for extraction of cytoplasmic CAH from cells. Combined use of 5.0 % (v/v) Triton X-100 and 0.3 mol L –1 NaCl achieved better effect on CAH extraction, and the release yield of CAH reached approximately 88 %. The extraction parameters such as temperature, ionic strength, Triton X-100 concentration and time were separately optimized to enhance the process of CAH release. The optimal permeabilization conditions (Triton X-100: 2.0 %, v/v; extraction temperature and time: 30 °C and 14 h) resulted in 103 % of relative CAH yield when compared with an optimized sonication process. Release yield of more than 100 % indicated that sonication could inactivate a few CAH molecules in the extraction process. This chemical treatment method could avoid the inactivation of CAH caused by sonication, and thus improve the CAH extraction process.

Mono-N-acyl-2,6-diaminopimelic acid derivatives: Analysis by electromigration and spectroscopic methods and examination of enzyme inhibitory activity

Thirteen mono-N-acyl derivatives of 2,6-diaminopimelic acid (DAP)—new potential inhibitors of the dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE; EC 3.5.1.18)—were analyzed and characterized by infrared (IR) and nuclear magnetic resonance (NMR) spectroscopies and two capillary electromigration methods: capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC). Structural features of DAP derivatives were characterized by IR and NMR spectroscopies, whereas CZE and MEKC were applied to evaluate their purity and to investigate their electromigration properties. Effective electrophoretic mobilities of these compounds were determined by CZE in acidic and alkaline background electrolytes (BGEs) and by MEKC in acidic and alkaline BGEs containing a pseudostationary phase of anionic detergent sodium dodecyl sulfate (SDS) or cationic detergent cetyltrimethylammonium bromide (CTAB). The best separation of DAP derivatives, including diastereomers of some of them, was achieved by MEKC in an acidic BGE (500mM acetic acid [pH 2.54] and 60mM SDS). All DAP derivatives were examined for their ability to inhibit catalytic activity of DapE from Haemophilus influenzae (HiDapE) and ArgE from Escherichia coli (EcArgE). None of these DAP derivatives worked as an effective inhibitor of HiDapE, but one derivative—N-fumaryl, Me-ester-DAP—was found to be a moderate inhibitor of EcArgE, thereby providing a promising lead structure for further studies on ArgE inhibitors.

Spectrophotometric, spectrofluorimetric, and potentiometric assays of cetyltrimethylammonium bromide in industrial wastewater samples.

This work deals with spectrophotometric, spectrofluorimetric, and potentiometric analyses of cetyltrimethylammonium bromide (CTAB) cationic detergent. The spectrophotometric procedure depends on measuring the absorbance of its binary complex with eosin yellow in Britton-Robinson buffer (pH 4) at Lambda max 547 nm in the range of 2.0-14.0 microg/mL with an accuracy of 100.15 +/- 0.54%. The spectrofluorimetric procedure depends on determining the quenching of the fluorescence intensity of fluorescein dye by CTAB in the presence of borate buffer at Lambda em = 500 nm, Lambda ex = 304 nm, in the range of 2.90-14.50 microg/mL with an accuracy of 99.81 + 0.33%. The electrochemical procedure describes an ionophore-based technique using a graphite sensor to measure 0.036 microg/mL and showed an accuracy of 100.11 +/- 0.61%. The experimental conditions affecting each of the three suggested procedures were studied and optimized. All the developed procedures were validated and satisfactorily applied for the determination of CTAB in industrial wastewater samples.

Functional states of antioxidant enzymatic complex of tissues of Mytillus galloprovincialis Lam. under conditions of oxidative stress

There are generalized materials on reaction of enzymatic antioxidant (AO) complex of tissues of molluscs of the genus Mytilus to oxidative loading of different intensity. It is shown that with increase of level of oxidative stress, a rise of activity of glutathione peroxidase (GP) and glutathione reductase (GR) is observed, as well as the rate of glutathione (GSH) in tissues also increases. Then the toxic loading is compensated by a rise of activities of enzymes of low affinity to hydroperoxides, specifically of catalase. In the glutathione system, it is possible to identify several relatively steady states: of low, moderate, and highly intensive processes. In several tissues, functioning of the AO system seems to be directed not to rendering harmless, but to generation of reactive oxygen species (O2-, superoxide dismutase), which is likely to be determined by specificity of their running processes (destruction of damaged byssus theads). The molluscan AO complex is characterized by high lability and sensitivity both to physiological states (spawning) and to action of factors of anthropogenic nature (cationic detergents). Reactions of AO complex of the mussel to the natural states and to toxic action are comparable between each other, which is necessary to be taken into account at diagnostics of the aquatic medium.

Reaction principle of the turbidimetric method for urine protein using cationic detergent

Reaction principle of the turbidimetric method for urine protein using cationic detergent

A Case of Fatal Ingestion of a 10% Benzalkonium Chloride Solution

A Case of Fatal Ingestion of a 10% Benzalkonium Chloride Solution Benzalkonium chloride (BAC) is a mixture of quaternary ammonium compounds of alkyl-dimethylbenzylammonium chlorides. It has wide ranging use as a disinfectant, germicidal cleaner, algaecide, cationic detergent and topical dermal antiseptic. Ingestion of BAC can cause local caustic effects and systemic effects. The present report involved a 78 year old male with a history of severe dementia who ingested up to 240ml of a germicidal disinfectant containing 10% benzalkonium chloride. Shortly after, he complained of oral pain and nausea, and experienced one episode of vomiting. Upon arrival to the emergency department approximately 45 minutes post-ingestion the patient’s complaints included oral pain, sialorrhea, drooling, spitting, nausea, and coughing, but no increase in work of breathing. Initial evaluation of the oral cavity and oropharynx showed mild erythema and swelling, but without lesions or erosions.

Orientation of lamellar phases of lyotropic multicomponent mixtures, based on cetyltrimethylammonium bromide cationic detergent, in magnetic field

The orientation of the lamellar phases in lyotropic systems based on cetyltrimethylammonium bromide (CTAB) detergent has been studied by polarization optical microscopy and 2H-NMR methods. The lamellar lyotropics studied are shown to align under a strong magnetic field of 11.7 T. According to 2H-NMR data, structural transformations of the lamellar phases may occur during orientation when the sample temperature increases.

A novel method for purification of Vi capsular polysaccharide produced by Salmonella enterica subspecies enterica serovar Typhi

Vi capsular polysaccharide is the major component of Vi polysaccharide typhoid vaccines. Vi is synthesized during growth of Salmonella enterica subspecies enterica serovar Typhi and is released into the fermentation broth in large quantities. Along with the Vi considerable amounts of impurities consisting of bacterial protein, nucleic acid and lipopolysaccharide (LPS) as well as media components contaminate the fermentation broth. A purification method based on selective precipitation of Vi using the cationic detergent cetavlon was developed to separate impurities from Vi. A novel method for handling the Vi precipitate using 0.2μm sterilizing grade filters to trap and wash the Vi and then, after re-solubilization, allow the Vi to pass through the filter was developed. Cetavlon selectively precipitates Vi and is the major purification step in the process, however, the conditions must be carefully controlled otherwise LPS will co-precipitate in large quantities. Various diafiltration steps help to remove contaminating protein, nucleic acid and fermentation media components as well as chemicals added during the process to induce precipitation of either Vi or contaminants. The final yield of purified Vi was approximately 45% and the bulk concentrate complied with the specifications defined in the WHO recommendations for Vi polysaccharide vaccine. Analysis of the Vi by size exclusion chromatography revealed a uniform peak with a narrow size distribution. The Nuclear Magnetic Resonance spectrum was similar to Vi produced by other methods. The method developed produces large quantities of Vi using low cost production methods translating into Vi based vaccines that can be produced at affordable prices for use in developing countries.

Denaturation of bovine serum albumin under the action of cetyltrimethylammonium bromide, according to data from fluorescence analysis

The tryptophan fluorescence of bovine serum albumin (BSA) in solutions with different concentrations of cationic detergent cetyltrimethylammonium bromide (CTAB) at different pH is investigated, providing information on BSA denaturation under the action of CTAB. It is found that BSA denaturation under the action of CTAB at all of the investigated pH values (3.5–8.0) is a single-stage process, as determined by BSA tryptophan fluorescence quenching, by an increased degree of the BSA tryptophan fluorescence polarization, and by the values of the parameters for the rotational diffusion of BSA molecules in CTAB solutions. It is shown that the cationic detergent CTAB is more efficient for BSA denaturation at pH values higher than the BSA isoelectric point (4.9).

Non-activated T and B lymphocytes become morphologically distinguishable after detergent treatment

T and B lymphocytes are difficult to distinguish morphologically even with electron microscopy, and antibodies are generally used to make the distinction. A specific reagent, consisting of nonionic and cationic detergents, is used for leukocyte differentiation using the Sysmex automated blood analyzer. This reagent increases cell membrane porosity and enables the introduction of fluorescent dye into leukocytes. In this study, we investigated the effect of this specific detergent on the morphology of T and B lymphocytes. T and B lymphocytes were obtained by density gradient centrifugation and magnetic cell sorting, with a minimum of 90% isolation efficiency. T and B lymphocytes were then treated with the specific detergent and fluorescent dye, and their distribution was analyzed based on side scatter and fluorescence intensity using general-purpose flow cytometry (FCM). Fluorescent images were observed using a confocal laser scanning microscope (CLSM), cellular inner structures using a transmission electron microscopy (TEM), and cell surfaces using a scanning electron microscope (SEM). The ratio of cholesterol to total lipid in cell membranes of B and T lymphocytes was measured using a fluorescent assay kit. The distribution of fluorescence intensity was different between T and B lymphocyte clusters, according to the FCM analysis. CLSM observations revealed that the fluorescent dye mainly stained cytoplasmic organelles. FCM, TEM, and SEM observations revealed that B lymphocytes are more likely to lose surface antigens and intracellular organelles than T lymphocytes, which allows the visual distinction between T and B lymphocytes. The ratio of cholesterol to total lipid in T lymphocyte membranes had tendency higher than that in B lymphocyte membranes. In this study, we demonstrate that cells with differences in cell membrane cholesterol amounts, such as B and T lymphocytes could be identified using an inexpensive detergent, as an alternative to costly antibodies.

Mechanism of Coomassie Brilliant Blue G-250 binding to cetyltrimethylammonium bromide: An interference with the Bradford assay

The Bradford protein assay is a popular method because of its rapidity, sensitivity, and relative specificity. This method is subject to some interference by nonprotein compounds. In this study, we describe the interference of cetyltrimethylammonium bromide (CTAB) with the Bradford assay. This interference is based on the interaction of Coomassie Brilliant Blue G-250 (CBB) with this cationic detergent. This study suggests that both electrostatic and hydrophobic interactions are involved in the interaction of CTAB and CBB. The anionic and neutral forms of CBB bind to CTAB by electrostatic attraction, which accelerates hydrophobic interactions of these CBB forms and the hydrophobic tail of CTAB. Consequently, the hydrophobic regions of the dominant free cationic form of CBB dye compete for the tail of CTAB with two other forms of the dye and gradually displace the primary hydrophobic interactions and rearrange the primary CBB–CTAB complex. This interaction of CTAB and CBB dye produces a primary 650-nm-absorbing complex that then gradually rearranges to a complex that shows an absorbance shoulder at 800–950nm. This study conclusively shows a strong response of CBB to CTAB that causes a time-dependent and nearly additive interference with the Bradford assay. This study also may promote an application of CBB for CTAB quantification.

Rapid isolation of high molecular weight DNA from single dry preserved adult beetle of Cryptolaemus montrouzieri for polymerase chain reaction (PCR) amplification

For studying genetic diversity in populations of predatory coccinellid, Cryptolaemus montrouzieri Mulsant (Coccinellidae: Coleoptera), our attempts to isolate high quality DNA from individual adult beetle using several previously reported protocols and even modifications were quite unsuccessful as the insect size was small and was preserved at -20°C as dry specimen. Here we describe a simple, rapid and efficient method of isolating high-quality intact genomic DNA with reduced protein contamination for polymerase chain reaction (PCR) amplification from a single, dry preserved specimen of adult Cryptolaemus. The procedure features macerating and mixing the single adult specimen of Cryptoalemus with cationic detergent cetyltrimethylammonium bromide (CTAB) in the homogenization buffer, two chloroform-isoamylalcohol extractions and an alcohol precipitation. RNA contamination was eliminated with RNAse treatment. The purity of DNA was high since the A260/A280 ratio ranged from 1.78 to 1.97. The isolated DNA was used as template for PCR, and the results were evaluated by comparing with different preserved samples.

Spontaneous vesicles, disks, threadlike and spherical micelles found in the solubilization of DMPC liposomes by the detergent DTAC

The spontaneous colloidal nanostructures formed in water by the zwitterionic phospholipid DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) with the cationic detergent DTAC (n-dodecyltrimethylammonium chloride) were investigated at a fixed DMPC concentration and variable detergent:lipid total molar ratios (D:L). Apparent (neutral-sphere-equivalent) hydrodynamic diameters (Φe) of liposomes and micelles were obtained by dynamic light scattering (DLS). Fluorescence lifetime imaging microscopy (FLIM), using chlorophyll-a as a probe, showed the morphology of giant vesicles and threadlike micelles. Micro-differential scanning calorimetry (micro-DSC) detected the presence of bilayers, in vesicles and discoidal micelles (disks). Pure DMPC liposomes are multilamellar and polydisperse (Φe≈100–10,000nm). As D:L increased, smaller vesicles were found, due to the bigger spontaneous curvature of the bilayer: at D:L=1, ULVs (unilamellar vesicles; Φe≈100nm) appeared and, at D:L=2–10, ULVs coexisted with disks (Φe≈30nm). Bilayers totally disappeared at D:L⩾15, giving rise to spheroidal (Φe≈2–16nm) and threadlike (Φe≈100–10,000nm) micelles. A quasi-equilibrium structural diagram for the DMPC–DTAC–water system shows equivalent diameters of the scattering nanoparticles as a function of D:L. The results obtained herein for the system DMPC–DTAC show the role of electrostatic interactions in the formation of the mixed structures.