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At least 937 records · Page 52Linked to original sources
PubMed1991

Two-dimensional gel electrophoresis of the membrane-bound protein complexes, including photosystem I, of thylakoid membranes in the presence of sodium oligooxyethylene alkyl ether sulfate/dimethyl dodecylamine oxide and sodium dodecyl sulfate.

Two-dimensional polyacrylamide gel electrophoresis (PAGE), using a mixture of sodium oligooxyethylene alkyl ether sulfate and dimethyl dodecylamine oxide as detergents (AES-DDAO mixture) in the first dimension and sodium dodecyl sulfate (SDS) in the second dimension, was developed and applied to an analysis of the photosystem I (PS I) complex in thylakoid membranes prepared from spinach chloroplasts. When thylakoid membranes of chloroplasts were solubilized directly in the AES-DDAO mixture and subjected to PAGE in the presence of these detergents as the first dimension, some protein complexes containing chlorophyll were observed. The protein components in these complexes separated into an array of polypeptide spots when the strip of gel after PAGE in the first dimension was subjected to PAGE in the presence of SDS as the second dimension. The main band of protein which separated in the first dimension was demonstrated to be the PS I complex. This complex retained the intrinsic photochemical activity of P700 even after it was subjected to one-dimensional PAGE. These results suggest that certain protein complexes can be separated, with the maintenance of their original structures, by electrophoresis in the presence of the AES-DDAO mixture, and this method appears to have valuable potential for analysis of the components of membrane-bound protein complexes.

Cell Membrane↗
PubMed1993

Spoilage and shelf-life of cod fillets packed in vacuum or modified atmospheres.

Microbial growth, sensory and chemical changes and composition of gas atmosphere were studied in vacuum packed (VP) and modified atmosphere packed (MAP) cod fillets stored at 0 degree C. Contrary to previous studies, coccobacilli and pleomorphic Gram-negative microorganisms (2-4 by 2-5 microns) and not Shewanella putrefaciens were found most likely to be the main spoilage organisms. These microorganisms, which may be Photobacterium phosphoreum, can explain the short shelf-life extension of VP and MAP fish products compared to meat products. It is suggested that they may inhibit the typical H2S-producing fish spoilage bacteria, S. putrefaciens, as the maximum concentration of H2S-producing bacteria found in MAP fish products is very low. Compared to VP, a shelf-life extension of 6-7 days was obtained with 48% CO2 in MAP. However, with pure CO2 the shelf life was only extended by 2-3 days. Poor texture and high drip loss indicated that the shelf life of these fillets was limited by chemical reactions and not only by microbial activity.

Animals↗
PubMed1984

Induction of tumours in rats by feeding nitrosatable amines together with sodium nitrite.

Feed containing 0.2% allantoin or diphenhydramine (as the hydrochloride) or 0.1% chlorpheniramine (as the maleate), with or without 0.2% sodium nitrite, was given ad lib. to groups of 20 or 24 male and 20 or 24 female F344 rats for 106 wk. Groups of 24 male and 24 female F344 rats were given drinking-water that contained N,N-dimethyldodecylamine-N-oxide at a concentration of 0.1%, with or without 0.2% sodium nitrite, for 93 wk. Control rats were given untreated feed or drinking-water and nitrite-treated controls were given sodium nitrite at a concentration of 0.2% in feed or drinking-water. At the end of the treatment period the rats were given untreated feed and water and observed until death. There was little or no life-shortening effect in any treatment group. None of the four amines administered alone induced an increase in the incidence of any tumour in comparison with the untreated control groups. In the male rats given diphenhydramine, chlorpheniramine or N,N-dimethyldodecylamine-N-oxide concurrently with nitrite there was a significant increase in the incidence of liver neoplasms (hepatocellular carcinomas and neoplastic nodules). In the groups given untreated feed or drinking-water there were, respectively, five and three male rats that had liver tumours. In contrast the number of male rats with liver tumours was ten in the group given dimethyldodecylamine-N-oxide plus nitrite, 11 in that given diphenhydramine plus nitrite and 14 (eight with carcinomas) in the group given chlorpheniramine plus nitrite. These results suggest that the ingestion of dimethyldodecylamine-N-oxide, diphenhydramine hydrochloride or chlorpheniramine under conditions when they could be nitrosated with nitrite in the stomach might present an increased carcinogenic risk.

Allantoin↗
PubMed1983

Cyclic amines as less mutagenic replacements for dimethyl amino (--NMe2) substituents on aromatic organic compounds: implications for carcinogenicity and toxicity.

Replacement of the dimethylamino (--NMe2) group of the rodent liver carcinogens 4-dimethylaminoazobenzene (DAB) and 6-dimethylaminophenylazobenzthiazole (6BT) with a pyrrolidinyl group leads to a marked attenuation of their mutagenicity to S. typhimurium in vitro. Replacement with the 6-membered piperidinyl group leads to a virtual loss of mutagenic activity. These results are discussed within the context of a possible, albeit limited correlation between carcinogenic potency to rodents and mutagenic potency to S. typhimurium. Based on these observations, it is suggested that replacement of the --NMe2 group of a toxic/carcinogenic/mutagenic aromatic chemical by a cyclic amine substituent may produce a less toxic (etc.) analogue with similar gross molecular properties. The significance of weak (less than 2-fold increase) mutagenic responses is discussed in relation to potential carcinogenicity.

Azo Compounds↗
PubMed2004

Synthesis, in vitro transfection activity and physicochemical characterization of novel N,N'-diacyl-1,2-diaminopropyl-3-carbamoyl-(dimethylaminoethane) amphiphilic derivatives.

A novel series of N,N'-diacyl-1,2-diaminopropyl-3-carbamoyl-(dimethylaminoethane) cationic derivatives was synthesized and screened for in vitro transfection activity at different charge ratios in the presence and absence of the helper lipids DOPE and cholesterol. Physicochemical properties of lipid-DNA complexes were studied by gel electrophoresis, fluorescence spectroscopy and dynamic light scattering. The interfacial properties of the lipids in isolation were studied using the Langmuir film balance technique at 23 degrees C. It was found that only lipoplexes formulated with the dioleoyl derivative, 1,2lmt[5], mediated significant in vitro transfection activity. Optimum activity was obtained with 1,2lmt[5]/DOPE mixture at a +/-charge ratio of 2. In agreement with the transfection results, 1,2lmt[5] was the only lipid found to complex and retard DNA migration as verified by gel electrophoresis. Despite the efficient complexation, no significant condensation of plasmid DNA was observed as indicated by fluorescence spectroscopy measurements. Monolayer studies showed that the dioleoyl derivative 1,2lmt[5] was the only lipid that existed in an all liquid-expanded state with a collapse area and collapse pressure of 59.5 A2 and 38.7 mN/m, respectively. This lipid was also found to have the highest elasticity with a compressibility modulus at monolayer collapse of 80.4 mN/m. In conclusion, increased acyl chain fluidity and high molecular elasticity of cationic lipids were found to correlate with improved transfection activity.

Cations↗
PubMed2004

Validated HPLC determination of 2-[(dimethylamino)methyl]cyclohexanone, an impurity in tramadol, using a precolumn derivatisation reaction with 2,4-dinitrophenylhydrazine.

A new method for the determination of 2-[(dimethylamino)methyl]cyclohexanone (DAMC) in Tramadol (as active substance or active ingredient in pharmaceutical formulations) is described. The method is based on the derivatisation of 2-[(dimethylamino)methyl]cyclohexanone with 2,4-dinitrophenylhydrazine (2,4-DNPH) in acidic conditions followed by a reversed-phase liquid chromatographic separation with UV detection. The method is simple, selective, quantitative and allows the determination of 2-[(dimethylamino)methyl]cyclohexanone at the low ppm level. The proposed method was validated with respect to selectivity, precision, linearity, accuracy and robustness.

Chromatography, High Pressure Liquid↗
PubMed2005

Temperature-induced sedimentation to dispersion of ionic vesicles.

We present a temperature-induced sedimentation/dispersion transition of ionic vesicles in the system of alkyldimethylamine oxide hemihydrochloride (CnDMAO.1/2HCl) with a hydrocarbon chain length of 12-16 (n = 12, 14, and 16) and sodium 2-naphthalenesulfonate (NaNphS). The temperature-sensitive sedimentation/dispersion of ionic vesicles took place around a temperature of 50 degrees C, which was weakly dependent on the alkyl chain length. The combined effect of the thermally induced dissociation of the counterions from the vesicle and a hydrogen bonding between the nonionic and the cationic head groups is likely to be responsible for this unique behavior.

Chemical Precipitation↗
PubMed2005

Hydrogen ion titration of alkyldimethylamine oxides by 13C and 1H NMR and conventional methods.

In the hydrogen ion titration of micelles, the degree of ionization of the micelle at a given pH has to be evaluated to obtain a pKa value of micelles (Ka being the proton dissociation constant) at the pH. We compared the degree of ionization obtained from 13C and 1H NMR spectra with that obtained from the stoichiometric method. We used dodecyldimethylamine oxide (C12DMAO) and hexyldimethylamine oxide (C6DMAO) to examine the titration behavior of micelles and monomers, respectively. We determined pKa values of amine oxides both in H2O and D2O. As to the monomer (C6DMAO), the degree of ionization from NMR, alpha(NMR), coincided with that from the conventional stoichiometric method alpha. The difference of pK1 of amine oxide monomer between D2O and H2O was about 0.5: pK1(D) approximately pK1(H) + 0.5. The difference was about the same as that for carboxylic acids. As to the C12DMAO micelle, alphaNMR did not coincide with alpha over a considerable range of alpha. The NMR chemical shift might be influenced by micellar structure changes induced by the ionization, such as the sphere-to-rod transition. The intrinsic logarithmic dissociation constants of the micelle were 5.9+/-0.1 for H2O, and 6.5+/-0.1 for D2O.

Carbon Isotopes↗
PubMed2005

A sensitive flow cytometry-based cytotoxic T-lymphocyte assay through detection of cleaved caspase 3 in target cells.

We describe a highly sensitive flow cytometry-based CTL assay using the cleavage of caspase 3 in target cells as a readout. The assay involved labeling of cells with a cell tracker dye and staining permeabilized cells with an antibody recognizing cleaved caspase 3. The assay proved to be robust and reliable in measuring antigen-specific CTL activity in a number of human and murine systems, including MLR, human peptide-specific T-cell responses induced in vitro, and CTL responses following immunization of mice with viral and peptide vaccines. The assay was found to yield comparable results as 51Cr-release, but with markedly higher sensitivity. When compared to detection of antigen-specific T cells via HLA tetramer/pentamer-based methods of T-cell staining in HIV gag peptide-specific human T cell lines the caspase 3 cleavage readout assay exhibited a comparable level of sensitivity with detection of CTL function at antigen-specific T-cell frequencies of 1:15,000 or lower. A similar level of sensitivity was obtained when murine CTL assays were performed with MLR in which effector cells were highly diluted with naïve syngeneic spleen cells. Our results indicate that the caspase 3 cleavage assay may be a powerful tool to measure antigen-specific CTL responses in human vaccine trials and in pre-clinical animal models of CTL function at both high and low effector cell frequencies.

Animals↗
PubMed2005

Purification, crystallization, and properties of F1-ATPase complexes from the thermoalkaliphilic Bacillus sp. strain TA2.A1.

Recently, we reported the cloning of the atp operon encoding for the F(1)F(0)-ATP synthase from the extremely thermoalkaliphilic bacterium Bacillus sp. strain TA2.A1. In this study, the genes encoding the F(1) moiety of the enzyme complex were cloned from the atp operon into the vector pTrc99A and expressed in Escherichia coli in two variant complexes, F(1)-wt consisting of subunits alpha(3)beta(3)gammadeltaepsilon and F(1)Deltadelta lacking the entire delta-subunit as a prerequisite for overproduction and crystallization trials. Both F(1)-wt and F(1)Deltadelta were successfully overproduced in E. coli and purified in high yield and purity. F(1)Deltadelta was crystallized by micro-batch screening yielding three-dimensional crystals that diffracted to a resolution of 3.1A using a synchrotron radiation source. After establishing cryo and dehydrating conditions, a complete set of diffraction data was collected from a single crystal. No crystals were obtained with F(1)-wt. Data processing of diffraction patterns showed that F(1)Deltadelta crystals belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell parameters of a=121.70, b=174.80, and c=223.50A, alpha, beta, gamma=90.000. The asymmetric unit contained one molecule of bacterial F(1)Deltadelta with a corresponding volume per protein weight (V(M)) of 3.25A(3) Da(-1) and a solvent content of 62.1%. Silver staining of single crystals of F(1)Deltadelta analyzed by SDS-PAGE revealed four bands alpha, beta, gamma, and epsilon with identical M(r)-values as those found in the native F(1)F(0)-ATP synthase isolated from strain TA2.A1 membranes. ATPase assays of F(1)Deltadelta crystals exhibited latent ATP hydrolytic activity that was highly stimulated by lauryldimethylamine oxide, a hallmark of the native enzyme.

Bacillus↗
PubMed2006

Role of PKA and PKC in histamine H1 receptor-mediated activation of catecholamine neurotransmitter synthesis.

Activation of the histamine H1 receptor stimulates tyrosine hydroxylase (TH) to increase catecholamine neurotransmitter synthesis in mammalian brain and adrenal tissues. Histamine non-selectively activates both H1-linked phospholipase (PL) C/inositol phosphates (IP)/diacylglycerol (DAG) signaling and adenylyl cyclase (AC)/adenosine 3',5'-cyclic monophosphate (cAMP) signaling, confounding determination of signaling events involved in H(1)-mediated TH activation. This research uses two new functionally-selective H1 agonists, cis-PAB and trans-PAT, that selectively activate H1/PLC/IP/DAG and H1/AC/cAMP signaling, respectively, to characterize H(1)-mediated activation of TH in rat striatum and bovine adrenal chromaffin (BAC) cells. Histamine, cis-PAB, and trans-PAT produced a two-fold maximal TH activation by an H1 receptor mechanism in rat striatum and BAC cells. Histamine is more potent and efficacious in BAC cells (EC50 approximately 0.2 microM, Emax approximately 200% basal) versus rat striatum (EC50 approximately 0.4 microM; Emax approximately 150%). Cis-PAB and trans-PAT are more potent in rat striatum (EC50 approximately 0.1 microM for both agonists) versus BAC cells (EC50 approximately 1.0 microM for both), with similar efficacy in both preparations (Emax approximately 160% for both agonists). Signaling studies in BAC cells revealed that protein kinase (PK) A but not PKC is involved in H1 -mediated TH activation by trans-PAT and histamine, while, both PKA and PKC are involved for cis-PAB. Results for cis-PAB suggest H1/PLC/IP/DAG/PKC signaling activates PKA, downstream of cAMP formation, indicating apparent direct activation of PKA by PKC.

Adenylyl Cyclases↗