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At least 775 records · Page 43Linked to original sources
PubMed1996

Peptide-mass profiles of polyvinylidene difluoride-bound proteins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in the presence of nonionic detergents.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), in conjunction with enzymatic digestion of proteins and molecular weight search of peptide-mass database is a powerful technique for peptide/protein identification. Ideally, peptide mixtures should be compatible with both MALDI-TOF and microsequencing. In our laboratory, enzymatic digestion and extraction of peptides from polyvinylidene difluoride (PVDF)-bound proteins is performed in the presence of nonionic detergents. However, nonionic detergents have been shown to cause signal suppression in MALDI-TOF analysis. This study demonstrates that by using a modified matrix solution, peptide-mass fingerprinting of PVDF-bound proteins by MALDI-TOF can be obtained in the presence of nonionic detergents such as hydrogenated Triton X-100 (RTX-100), octylglucopyranoside, and Tween 20.

Amino Acid Sequence↗
PubMed1997

BIACORE analysis of histidine-tagged proteins using a chelating NTA sensor chip.

While BIACORE instruments are routinely used for kinetic measurements and for the determination of binding constants, the immobilization of a ligand onto the sensor chip surface has to be individually optimized for every system. We show here that the histidine (His) tag, routinely used in protein purification and in detection is an ideal tag for immobilization, despite the intrinsically low affinity between an immobilized metal ion and the His tag. This is due to strong rebinding effects caused by the high surface density of immobilized Ni2+-nitrilotriacetic acid (NTA) on the chips used here. The immobilization of the ligand can be adjusted to a low level using the same chip, such that mass transport limitation and rebinding of the analyte to the immobilized ligand is minimal. Nine different proteins with different numbers of His tags were tested for stable binding to the Ni2+-NTA surface. Most proteins with one His tag dissociate very rapidly from the Ni2+-NTA surface, and the KD for the interaction between His tag and Ni2+-NTA was estimated to about 10(-6) m at neutral pH. In contrast, two His tags are usually found to be sufficient for stable binding. The kinetics of the chaperonin system of Escherichia coli GroEL and GroES were analyzed as a model using this system and found to be very similar to those obtained with covalently immobilized ligands. The sensor chip can be reused many times, because of the powerful regeneration methods. The ligand can be freshly immobilized after each cycle, thus eliminating potential denaturation upon regeneration as a source of error.

Adenosine Triphosphate↗
PubMed2000

Quantitation of the blocking effect of tween 20 and bovine serum albumin in ELISA microwells.

ELISA provides a highly sensitive procedure for quantitating antigens and antibodies. In that assay, microwells are coated initially with a specific ligand and then saturated with inert molecules to minimize nonspecific background. Coating can be improved by pretreating the microwells with poly-l-lysine (PLL). Proteins and Tween 20 are most often used to block vacant binding sites in enzyme-linked immunosorbent assay (ELISA). In the present study the blocking effects of Tween 20 and bovine serum albumin (BSA) were estimated using an original novel approach. In the assay the magnitude of saturation of the microwells was quantitated by measuring the enzymatic activity of alkaline phosphatase adsorbed to residual vacant sites in the microwell. Tween 20 completely saturated ELISA microwells at concentrations higher than 2 microg/ml. If the microwells were pretreated with PLL, even high concentrations of the detergent did not completely saturate the wells. In contrast, BSA completely saturated both PLL-treated and nontreated microwells at 5 microg/ml. Complementation of Tween 20-induced saturation of PLL-treated microwells was achieved only by addition of BSA at concentration required for BSA alone to reach complete saturation. This approach is applicable for assessing binding to ELISA microwells of any reagent of choice either as a ligand or as a blocking reagent.

Alkaline Phosphatase↗
PubMed2000

Optimization of detergents for the assay of cathepsins B, L, S, and K.

The differential effects of representative, commonly available ionic (SDS), nonionic (Brij 35, Tween 20, and Triton X-100), and zwitterionic (Chaps) detergents on the catalytic activity and properties of human cathepsins B, L, S, and K were examined. The presence of detergents in the assay buffer affected the activity of cathepsins to variable extents; Chaps enhanced the activity of all the enzymes while SDS was most detrimental. Tween 20 lowered cathepsin S activity, while it slightly enhanced that of all other cathepsins studied. The presence of detergents in the activation buffer was clearly beneficial to both cathepsins L and K, possibly by favoring the release of the enzyme from the walls of the incubation vessel. Overall, the results indicate that Chaps is the optimal detergent for use with this family of enzymes.

Cathepsin B↗
PubMed1993

Inhibition kinetics of chloramphenicol acetyltransferase by selected detergents.

Kinetic analyses indicate that the inhibitory effects of the nonionic detergents Triton X-100 and Nonidet P-40 on chloramphenicol acetyltransferase are exerted by a competitive and a non-competitive mechanism with respect to the substrates chloramphenicol and acetyl-CoA, respectively. Comparison with nonionic detergents without an aromatic moiety like that present in Triton X-100 and Nonidet P-40 suggests that the aromatic groups in these two detergents may compete with chloramphenicol for binding to the hydrophobic, active site in the chloramphenicol acetyltransferase.

Acetyl Coenzyme A↗
PubMed1995

The identification and partial characterisation of a novel inducible extracellular thermostable esterase from the archaeon Sulfolobus shibatae.

Extracellular esterases have so far only been reported in eubacteria, here we report the first identification and partial characterisation of a novel inducible extracellular esterase from the thermoacidophilic archaeon Sulfolobus shibatae. This esterase exhibits remarkable stability to both acid and heat. Esterase activity is induced by growth on a range of polyoxyethylenesorbitan (Tween) compounds as sole carbon source. Activity occurs over a wide temperature (25-99 degrees C) and pH (pH4.0-9.0) range and is optimal at 90 degrees C and pH6.0. It exhibits high thermal stability, with a half-life of 20 min at 120 degrees C, and shows a transient thermal activation of 60% at 90 degrees C. The thermal inactivation of function occurs by first order kinetics, and after 120 min incubation at 120 degrees C 50% of activity still remains. It is able to hydrolyse mono- and diglycerides, but is unable to hydrolyse the triglycerides olive oil and triolein, which is indicative of an esterase and not a lipase.

Enzyme Induction↗
PubMed1996

A novel ligand blot assay detects different hyaluronan-binding proteins in rat liver hepatocytes and sinusoidal endothelial cells.

We have developed a sensitive ligand blot assay to detect hyaluronan (HA) binding proteins in cell extracts using 125I-HA. Samples to be tested are electrophoresed using standard, nonreducing SDS-PAGE conditions, electro-transferred to nitrocellulose then blocked in buffer containing Tween 20. After incubation with 125I-HA the nitrocellulose is washed and HA-binding proteins are detected by autoradiography. This method was used to detect different HA-binding proteins in isolated rat liver cell preparations. Two HA-binding bands of 175 kD and 350 kD were detected in sinusoidal endothelial cells. Both bands were competed virtually 100% with nonlabeled HA. Using rat hepatocytes, the assay detected major bands at 85 kD and 180 kD. In addition, histones present in both cell types were readily detected in the low MW region. Thus, two different liver cell types show different HA-binding patterns. The blocking procedure is critical for successful renaturation of HA-binding activity, since substitution of BSA for Tween 20 did not result in detectable 125I-HA-binding. This ligand blot assay will be a powerful tool to detect HA-binding proteins in various other tissues and cell types.

Animals↗
PubMed1999

Glutathione-dependent oxidative modification of protoporphyrin and other dicarboxylic porphyrins by mammalian and plant peroxidases.

Protoporphyrin, an intermediate in heme and chlorophyll biosynthesis, can accumulate in human and plant tissues under certain pathological conditions and is a photosensitizer used in cancer phototherapy. We previously showed that protoporphyrin and the related non-natural dicarboxylic porphyrin deuteroporphyrin are rapidly oxidized by horseradish peroxidase in the presence of some thiols, especially glutathione. This study reports that bovine lactoperoxidase, but not leucocyte myeloperoxidase, can also catalyze this reaction and that Tween and ascorbic acid are inhibitors. Exogenous hydrogen peroxide is not required and cannot replace glutathione. Deuteroporphyrin was oxidized to a unique green chlorin product with two oxygen functions added directly to the characteristic reduced pyrrole ring of the chlorin. Spectroscopic and chromatographic results suggest that protoporphyrin was oxidized not to a green chlorin, but to a much more polar red porphyrin modified by oxidative addition to the two vinyl side chains. Two related nonnatural dicarboxylic porphyrins, with ethyl or hydroxyethyl instead of vinyl side chains, are not substrates or products for this enzymatic conversion.

Animals↗
PubMed1998

Studies on the loss of infectivity of live type 3 poliovaccine on storage.

The Children's Vaccine Initiative established a Product Development Group in 1991 to help in the development of oral poliovaccines of improved thermostability. Under the auspices of this group, the effect of different stabilizers on the loss of infectivity of commercial monovalent type 3 live poliovaccine bulks at 37 degrees C, 42 degrees C and 45 degrees C has been examined. The only effective conditions were the maintenance of the pH at 6.5, and the use of deuterium oxide at a concentration of 90%. All other additives, including detergents, albumin, antiviral compounds, sucrose and magnesium chloride, alone or in combination either had no effect or increased the rate of loss of infectivity.

Antiviral Agents↗
PubMed1998

Dendritic cells internalize vaccine adjuvant after intramuscular injection.

Vaccine adjuvants help antigens elicit rapid, potent, and long-lasting immune responses. The lack of understanding of the immunological mechanism of action of adjuvants has limited the rational development of vaccines for human use. In particular, little is known about how the immune system processes adjuvants. The goal of the present study was to determine the fate of the vaccine adjuvant MF59, labeled with the fluorescent dye Dil, after injection with fluorescein-labeled gD2 antigen from type 2 herpes simplex virus. At 3 h after intramuscular injection into BALB/c mice, most of the MF59 was still in the form of extracellular droplets in the muscle, but a detectable fraction of the MF59 was in cells in the subcapsular sinus of draining inguinal lymph nodes. At 48 h, most of the MF59 at the site of injection was inside cells that were immunoreactive for the dendritic cell markers DEC-205 and MHC class II molecules, reflecting the interaction of MF59 with antigen presenting cells. At this time, intracellular MF59 was also abundant in the paracortical (T cell) region of lymph nodes. The gD2 antigen was also intracellular in muscle and colocalized MF59 at 48 h, and the presence of MF59 increased the amount of intracellular antigen. Similarly, serological antibody titers to gD2 were 207-fold higher after two injections when MF59 was administered with the antigen. These findings suggest that MF59 interacts with antigen presenting cells at the site of injection and then moves to the draining lymph nodes, where it increases the efficiency of antigen presentation to T cells.

Adjuvants, Immunologic↗
PubMed1997

Studies on the mutagenicity of mild gasification products of coal and their subfractions by the Salmonella/microsomal assay.

Mild gasification of coal is a technology being developed in the United States in order to upgrade lower rank coals and facilitate their use in coal-burning electric generation plants. Thirteen coal-derived mild gasification products from different coal sources and processing conditions have been examined for their potential biohazards. The mutagenicity of these samples was tested with the Ames Salmonella/microsomal assay. Two solvents, dimethyl sulfoxide (DMSO) and polyoxyethylene-sorbitan monooleate (Tween 80), were used to dissolve samples in a manner to facilitate their interaction with the test organisms. The results showed that 9 of the 13 samples displayed mutagenic activity in test strains TA98 and/or TA100 with or without metabolic activation, whether dissolved in Tween 80 or DMSO. Five mutagenic and two nonmutagenic samples were class-fractionated into basic, acidic, nonpolar, and polar neutral subfractions to examine their class-related mutagenic activities. Results of the testing of subfractions of the five mutagenic and one nonmutagenic samples showed mutagenic activity in at least the nonpolar neutral fraction. The subfractions of the another nonmutagenic sample did not display any mutagenic activity. Chemical characterization of the subfractions revealed the existence of aromatic hydrocarbons in certain subfractions, which may be responsible for the mutagenic activity of the coal-derived mild gasification products.

Animals↗
PubMed2002

Multiple emulsion stability: pressure balance and interfacial film strength.

The objective of this study was to investigate the significance of inner and outer phase pressure, as well as interfacial film strength on W/O/W multiple emulsion stability using microscopy and long-term stability tests. It was observed that immediately upon applying a coverslip to samples the multiple droplets deformed and there was coalescence of the inner aqueous droplets. Under certain conditions (such as lipophilic surfactant concentration and internal phase osmotic pressure) the destabilized multiple emulsions formed unique metastable structures that had a "dimpled" appearance. The formation of these metastable structures correlated with the real-time instability of the W/O/W multiple emulsions investigated. Multiple emulsion stability also correlated with the interfacial film strength (measured by interfacial elasticity) of the hydrophobic surfactant at the mineral oil/external continuous aqueous phase interface. The formation of the metastable dimpled structures and the long-term stability of the multiple emulsions were dependent on the osmotic pressure of the inner droplets and the Laplace curvature pressure as described by the Walstra Equation (P. Walstra, "Encyclopedia of Emulsion Technology" (P. Becher, Ed.), Vol. 4. Dekker, New York, 1996). It appears that the effect of coverslip pressure on multiple emulsions may be useful as an accelerated stability testing method or for initial formulation screening.

Drug Stability↗
PubMed1998

Arachidonic acid metabolism in primary irritant dermatitis produced by patch testing of human skin with surfactants.

A clinical study was performed to determine the effects of patch testing human skin with four industrially used surfactants on erythema formation, transepidermal water loss, and the contents in suction blister fluids of primary proinflammatory mediators including arachidonic acid, eicosanoids, and IL-1 alpha, which were analyzed by quantitative gas chromatography/negative ion chemical ionization mass spectrometry and by an enzyme-immunoassay, respectively. Benzalkonium chloride (BKCI) and sodium lauryl sulfate (SLS) elicited erythema and caused increased transepidermal water loss, indicating a disturbance of the epidermal barrier. Triethanolamine (TEA) and Tween 80 did not evoke these gross symptoms of inflammation. Suction blister fluids collected after a 24-h application of BKCl, SLS, and Tween 80 contained significantly increased amounts of individual eicosanoids whereas TEA induced no response. The induced eicosanoid profile was characteristic for each compound, pointing to different cell types of skin to be involved in their production. The elevation of prostaglandin and LTB4 contents correlated with the induction of erythema and the impairment of the epidermal barrier as shown for BKCl and SLS and preceded the maximum of erythema formation. IL-1 alpha contents did not correlate with these gross symptoms of inflammation. The results of this in vivo study support those of a previous study using human keratinocytes in culture indicating the release of arachidonic acid and prostaglandins to be an early event involved in the interaction of keratinocytes with surfactants. Moreover, the in vivo data with human skin underscore the mechanistic relationship to the in vitro model and support the concept that arachidonic acid and eicosanoid release from keratinocytes can be used as a marker of primary skin irritation.

Adult↗
PubMed1995

MF59. Design and evaluation of a safe and potent adjuvant for human vaccines.

MF59 is a safe, practical, and potent adjuvant for use with human vaccines. The formulation is easily manufactured, may be sterilized by filtration, and is both compatible and efficacious with all antigens tested to date. MF59 has been shown to be a potent stimulator of cellular and humoral responses to subunit antigens in both animal models and clinical studies. Toxicology studies in animal models and Phase I-III studies in humans have demonstrated the safety of MF59 with HSV, HIV, and influenza vaccines.

Adjuvants, Immunologic↗