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PubMed1987

Muscarinic receptor-detergent complexes with different biochemical properties: selective solubilization, lectin affinity chromatography and ligand binding studies.

Muscarinic receptors were solubilized by nonionic, zwitterionic and ionic detergents from porcine striatum. A mixture of digitonin and gitonin (3:2) was found to be most suitable in respect to receptor yield and stability. The solubilization of muscarinic receptors by this detergent appears to be dependent on the existence of free detergent micelles. Consequently, the receptor solubilization was studied at different protein-to-detergent ratios. Based on these experiments, a double extraction procedure was developed in which the receptor is solubilized subsequent to the solubilization of other membrane proteins. After elimination of the detergent excess, the binding of the receptor-detergent complex to six immobilized lectins was studied. In accordance with previous reports, we found a considerable portion of the digitonin/gitonin solubilized receptors (one step extraction procedure) specifically bound to wheat germ agglutinin via sialic acid residues. Muscarinic receptors solubilized by a double extraction procedure (either from porcine striatum or rat brain) did not bind to the lectin. This is not owing to selective extraction or partial denaturation, and indicates that considerable portions of the glycan residues are not covalently bound to the receptor polypeptide. A GTP-insensitive heterogenous agonist binding was found only at the non-wheat germ agglutinin binding receptors. The data analysis was performed by the affinity spectra method.

Animals↗
PubMed1992

Expression in E. coli and purification of intracellular proteins by fusion to cyclomaltodextrin glucanotransferase.

A plasmid expression vector was constructed to direct the synthesis of foreign proteins in Escherichia coli as fusions with cyclomaltodextrin glucanotransferase (CGT) with cytoplasmic location (delta ssCGT). The ability of CGT to bind to covalently immobilized cyclodextrins was utilized in purifying fused target proteins. A large proportion of the cytoplasmically synthesized delta ssCGT formed inclusion bodies which adopted the active conformation at considerably high refolding concentration (67 microM delta ssCGT solution). By lowering the cultivation temperature the proportion of the soluble delta ssCGT was slightly increased. Intracellularly expressed delta ssCGT provides a potential affinity handle which forms easily refoldable inclusion bodies increasing the yield and stability, and possibly allows the expression of lethal target proteins. Interestingly, the interaction between one model fusion protein delta ssCGT-CAT (CAT, chloramphenicol acetyltransferase) and the E. coli heat shock protein GroEL was observed.

Amino Acid Sequence↗
PubMedDate not supplied

Biochemical characterization of solubilized muscarinic acetylcholine receptors.

Several in part new methods (salt extraction, phospholipase treatment, transfer to serumlipoproteins, nonionic detergent mixtures) have been extensively examined in order to obtain a solubilized muscarinic acetylcholine receptor with properties suitable for further purification. An optimal mixture of digitonin and gitonin has been found which forms a tightly bound detergent wall around the receptor protein as indicated by ion exchange and monolayer studies and which allows to solubilize the receptor from enriched synaptosomal membranes with a high yield and stability. The specific 3H-QNB binding of this preparation is nearly unchanged between pH 7.5 and 10. The isoelectric point of the muscarinic acetylcholine receptor has been determined by isoelectric focusing to be between pH 4.5 and 4.6.

Acetylcholine↗
PubMed1985

Purification of a new restriction endonuclease, StyI, from Escherichia coli carrying the hsd+ miniplasmid.

A new restriction endonuclease, StyI, free of contaminating nuclease activities, has been isolated from Escherichia coli carrying the hsd+ miniplasmid of Salmonella typhi origin. In the presence of 10 mM Mg2+, it recognizes and cleaves a hexanucleotide sequence of 5'-C decreases C(AT)(AT)GG. The advantages of the StyI endonuclease include its stability, high yield (more than 2 X 10(3) units/g of wet cells), easy handling of producer cells, and the ability to recognize new sequences, CCAAGG and CCTTGG.

Bacteriophage lambda↗
PubMed1987

Determination of endotoxins by gas chromatography: evaluation of electron-capture and negative-ion chemical-ionization mass spectrometric detection of halogenated derivatives of beta-hydroxymyristic acid.

The sensitivity and selectivity of gas chromatography for analysing several halogenated ester derivatives of beta-hydroxymyristic acid were studied using both selected-ion monitoring detection with negative-ion chemical-ionization mass spectrometry and electron-capture detection. Six different derivatization methods were compared with respect to yield, chemical stability and formation of by-products. Procedures for removal of excess reagents using disposable silica columns and thin-layer chromatography were elaborated. The 3-O-pentafluorobenzoyl-methyl ester was the preferred derivative since it provided high sensitivity and had the molecular ion as the base peak in the mass spectrum. The detection limit was 0.5 pg with electron-capture detection and 0.3 pg with the mass spectrometric system. Using beta-hydroxymyristic acid as a chemical marker it was possible to detect Escherichia coli endotoxin in aqueous solution at a level of 1 ng/ml.

Chromatography, Gas↗
PubMed2005

Expression, purification, and characterization of cysteine-free mouse P-glycoprotein.

Cysteine-free mouse MDR3 P-glycoprotein (Pgp) was constructed by mutagenesis of the nine natural Cys to Ala. The Cys-free protein was expressed in Pichia pastoris and purified. Yield, purity, ATPase activity, K(m)(MgATP), and stimulation of ATPase by verapamil, were similar to wild-type mouse Ppg. Mouse Cys-free Pgp was superior in yield and stability to Cys-free human MDR1 Pgp. Mutants Y1040A and Y1040C were constructed in mouse Cys-free Pgp background. Both showed extremely low ATPase activity, strongly-impaired vanadate-trapping of ADP, and reduced photolabeling by 8-azido-ATP. The results are consistent with the conclusion that Tyr-1040 is located in the MgATP-binding site in NBD2 and is required for correct binding and/or orientation of bound MgATP substrate in Pgp as previously suggested by X-ray structures of other ABC transporters and by sequencing of photolabeled Pgp. The results also support our previous conclusion that both catalytic sites must be intact for normal function in Pgp.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed2006

Acidic cesium salts of 12-tungstophosphoric acid as catalysts for the dehydration of xylose into furfural.

Cesium salts of 12-tungstophosphoric acid, Cs(x)H(3-x)PW(12)O(40) (Cs(x)PW), in the bulk form or supported on medium-pore MCM-41 (3.7 nm) or large-pore (9.6 nm) micelle-templated silicas are active solid acid catalysts for the cyclodehydration of xylose into furfural, in a toluene/water solvent system (T/W) or in dimethyl sulfoxide (DMSO). The catalytic results are comparable to those obtained using sulfuric acid, under similar reaction conditions. The initial activities increase in the order H(3)PW(12)O(40)<Cs(2.0)PW<Cs(2.5)PW<silica-supported CsPW catalysts (15, 34wt% PW). Higher HPA loadings, larger pore diameter of the parent silica support, and higher reaction temperatures lead to higher furfural yields. The stability and reusability of the MCM-41-supported CsPW is higher in DMSO than in T/W.

Catalysis↗
PubMed2026

Towards a Robust cell-free DNA Isolation Protocol for NGS Applications in a Clinical Molecular Diagnostics Setting.

Cell-free DNA (cfDNA), released from apoptotic and necrotic cells into body fluids, is a non-invasive source of genetic information for disease prediction, diagnosis, and monitoring. However, its low abundance makes cfDNA highly susceptible to various pre-analytical influences, potentially increasing high molecular weight (HMW) or genomic DNA (gDNA) compromising downstream cfDNA analyses. This study evaluated the impact of different cfDNA-stabilizing blood collection tubes (BCT; Cell-Free DNA BCT, Streck; S-Monovette cfDNA Exact, Sarstedt) stored at room temperature for 1, 5, or 10 days, prior to plasma isolation using different isolation methods (magnetic bead-based or silica column-based) on cfDNA stability and yield. DNA quantity and quality were assessed by fluorometric quantification, automated fragment analysis, and gene-specific quantitative PCR. Streck-based workflows maintained stable cfDNA yields and characteristic mononucleosomal fragmentation profiles across all storage times. In contrast, Sarstedt tubes showed reduced cfDNA concentrations after 5 days and a pronounced increase at 10 Days, accompanied by high-molecular weight DNA patterns consistent with white-blood cells (WBC) lysis. These trends were largely independent of the extraction method. Overall, the results demonstrate that blood collection tube chemistry critically influences cfDNA integrity during delayed processing. Streck tubes, particularly when combined with silica column-based isolation method, provided the most robust and reproducible workflow for routine molecular diagnostics, whereas Sarstedt tubes produced physiologically implausible results after extended storage.

blood collection tubes↗
PubMed2006

A diffusive SI model with Allee effect and application to FIV.

A minimal reaction-diffusion model for the spatiotemporal spread of an infectious disease is considered. The model is motivated by the Feline Immunodeficiency Virus (FIV) which causes AIDS in cat populations. Because the infected period is long compared with the lifespan, the model incorporates the host population growth. Two different types are considered: logistic growth and growth with a strong Allee effect. In the model with logistic growth, the introduced disease propagates in form of a travelling infection wave with a constant asymptotic rate of spread. In the model with Allee effect the spatiotemporal dynamics are more complicated and the disease has considerable impact on the host population spread. Most importantly, there are waves of extinction, which arise when the disease is introduced in the wake of the invading host population. These waves of extinction destabilize locally stable endemic coexistence states. Moreover, spatially restricted epidemics are possible as well as travelling infection pulses that correspond either to fatal epidemics with succeeding host population extinction or to epidemics with recovery of the host population. Generally, the Allee effect induces minimum viable population sizes and critical spatial lengths of the initial distribution. The local stability analysis yields bistability and the phenomenon of transient epidemics within the regime of disease-induced extinction. Sustained oscillations do not exist.

Algorithms↗
PubMedDate not supplied

Thermomechanical analysis of collagen crosslinking in the developing lamb pericardium.

In a developing lamb model, we have used hydrothermal isometric tension (HIT) techniques to assess collagen crosslink stability and its contribution to the mechanical properties of the pericardium. Strip samples of tissue were either: (i) heated to a 90 degrees C isotherm or (ii) heated in 5 degrees C increments between 60-90 degrees C and then 93 and 97 degrees C isotherms. The half-life of stress relaxation associated with peptide bond hydrolysis (t1/2) was calculated at each isotherm. The activation energy, Eact, for the hydrolysis-associated relaxation process was also calculated using the data from the stepwise HIT tests--a technical improvement which significantly reduces the experimental time required to develop statistically valid measurements. Crosslinking in the pericardium increased during development and was demonstrated both by thermoelasticity and by resistance to enzymatic solubilization. We observed greater conversation to thermally stable crosslinks upon maturation, the ratio of the NaBH4-stabilized/unstabilized half-lives peaking at 21 days postnatal. Whereas tissue from lambs (119 day fetal, and 3 day and 21 day postpartum) showed an early maximum and rapid decay of force, NaBH4 stabilization significantly increased thermal stability and yielded profiles similar to those in adult tissue.

Animals↗
PubMed1986

Monoclonal antibodies to soluble, human milk galactosyltransferase (lactose synthase A protein).

Monoclonal antibodies have been produced against soluble human milk galactosyltransferase of a blood group O donor. After initial screening by radioimmunoassay, fourteen hybridomas were further characterized by enzyme-linked immunosorbent assay, immunoblotting of purified enzyme following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, enzyme activity modification, and enzyme localization in HeLa cells by immunofluorescence. Of these fourteen clones, seven had titers between 1500 and 7800 as estimated by ELISA. In general, the titer correlated with staining intensity on immunoblots and in immunofluorescence. In the presence of monoclonal antibody, enzyme activity was usually slightly enhanced or stabilized. Subcloning yielded four monoclonal antibody preparations designated as GT2/24/108, GT2/36/118, GT2/61/14, and GT2/77/22, which belong to Ig class G2b, G3, M, and G1, respectively. They all recognized the enzyme in purified form or in defatted milk as a single, broad band on electrophoresis-immunoblotting and produced a concise juxtanuclear fluorescence typical for the Golgi apparatus in HeLa cells.

ABO Blood-Group System↗
PubMed1998

Jacalin: a jackfruit (Artocarpus heterophyllus) seed-derived lectin of versatile applications in immunobiological research.

Jacalin, the major protein from the jackfruit (Artocarpus heterophyllus) seeds, is a tetrameric two-chain lectin (molecular mass 65 kDa) combining a heavy alpha chain of 133 amino acid residues with a light beta chain of 20-21 amino acid residues. It is highly specific for the alpha-O-glycoside of the disaccharide Thomsen-Friedenreich antigen (Gal beta1-3GalNAc), even in its sialylated form. This property has made jacalin suitable for studying various O-linked glycoproteins, particularly human IgA1. Jacalin's uniqueness in being strongly mitogenic for human CD4+ T lymphocytes has made it a useful tool for the evaluation of the immune status of patients infected with human immunodeficiency virus (HIV)-1. The abundance of source material for the production of jacalin, its ease of purification, yield and stability have made it an attractive cost-effective lectin. It has found applications in diverse areas such as the isolation of human plasma glycoproteins (IgA1, C1-inhibitor, hemopexin, alpha2-HSG), the investigation of IgA-nephropathy, the analysis of O-linked glycoproteins and the detection of tumours.

Allergy and Immunology↗
PubMed2006

An evaluation of surgical approaches for posterior urethral distraction defects in boys.

PURPOSE: We present our preliminary experience with operative approaches in the treatment of complex posterior urethral distraction defects in children. MATERIALS AND METHODS: A total of 24 boys 7 to 14 years old with obliterative complex posterior urethral distraction defects were treated with 1 of 3 surgical approaches between January 1998 and December 2002. A perineal approach was used in 5 cases, a transperineal-inferior pubic approach including distal urethral mobilization, corporeal body separation and inferior pubectomy in 10, and a combined transpubic-perineal approach in 9. RESULTS: Patients were followed 1 to 5 years postoperatively. A successful result of no recurrence of stricture was achieved in 19 of 24 patients (79.2%), including 3 of 5 (60%) treated with a perineal approach, 9 of 10 (90%) with a transperineal-inferior pubic approach and 7 of 9 (77.8%) with a transpubic-perineal approach. Complications included 1 combined false passage, which was resolved using a transperineal-inferior pubic approach, 2 urethrocutaneous fistulas, which were resolved using a perineal approach in 1 and a transpubic-perineal approach in 1, and 3 rectourethral fistulas, which were resolved using a transpubic-perineal approach in 1 and a transperineal-inferior pubic approach in 1. Of the 9 patients treated with the transpubic-perineal approach 3 walked with an abnormal gait postoperatively. CONCLUSIONS: The transperineal-inferior pubic approach fully exposes the space behind the pubis, does not damage pelvic stability and yields better results compared to the perineal and transpubic-perineal approaches. We recommend it as first line operative treatment for complex posterior urethral distraction defects in boys.

Adolescent↗
PubMed2005

Dynamics and pathology of dendritic spines.

Dendritic spines are key players in information processing in the brain. Changes in spine shape and wholesale spine turnover provide mechanisms for modifying existing synaptic connections and altering neuronal connectivity. Although neuronal cell death in acute and chronic neurodegenerative diseases is clearly an important factor in decline of cognitive or motor function, loss of dendritic spines, in the absence of cell death, may also contribute to impaired brain function in these diseases, as well as in psychiatric disorders and aging. Because spines can function in neuroprotection in vitro, advances toward a molecular understanding of spine maintenance might one day aid in the design of therapies to minimize neurological damage following excitotoxic injury. In addition, progress in defining the biochemical basis of spine development and stabilization may yield insights into mental retardation and psychiatric disorders.

Animals↗
PubMedDate not supplied

A directional process of gene conversion is expected to yield dynamic polymorphism associated with stability of alternative alleles in class I histocompatibility antigens gene family.

This work is a contribution to the discussion of the polymorphism of class I histocompatibility antigens (H-2 antigens in the mouse). Specially, the involvement of gene conversion in H-2 polymorphism has been explored through the use of a simplified computer model. It thus appears that in a population of limited size, gene conversion can promote allelic polymorphism while homogenizing the DNA sequences in a multigene family, only if it acts as a directional process. The DNA sequences of the preferential targets are then polymorphic and evolutionary versatile, while other genes of the family mostly behave as sequence donors, have more stable DNA sequences and are much less polymorphic. The characteristics of such a polymorphism generator are discussed with respect to their functional and evolutionary implications.

Alleles↗
PubMed2000

Effective onion vinegar production by a two-step fermentation system.

A two-step fermentation system combining a repeated batch process using a flocculating yeast with a charcoal pellet bioreactor was developed for onion vinegar production. Juice from the red onion R-3, which contained 67.3 g/l total sugar, was smoothly converted to onion alcohol containing 30.6 g/l ethanol by repeated batch operation using the flocculating yeast Saccharomyces cerevisiae strain IR-2. Stable operation was possible and the maximum productivity was about 8.0 g/l/h. A packed bed bioreactor containing charcoal pellets produced from waste mushroom medium was then applied to continuous onion vinegar production from the onion alcohol. Onion vinegar was successfully produced, with a maximum productivity and acetic acid concentration of about 3.3 g/l/h and 37.9 g/l, respectively. The total acetic acid yield calculated from the amount of sugar consumed was 0.86. The two-step system was operated for 50 d and proved to be competitive with other systems in terms of its high productivity, high acetic acid yield, operational stability and low production costs.

Journal Article↗
PubMed2005

Electrospray diagnostics by Fourier analysis of current oscillations and fast imaging.

The different spraying modes in electrospray ionization sources exhibit large variations in their ion yield and stability. To achieve consistently optimal ion production, active control of the spray parameters is desirable. To diagnose the changes in spraying mode, the spray current and its Fourier spectrum were monitored under a wide range of conditions, that is, as a function of the spray voltage, liquid flow rate, and composition. Most Fourier spectra indicated a strong dc component, a low-frequency branch at low flow rates and applied voltages, and a high-frequency branch and their harmonics. Changing of these parameters resulted in several spraying mode changes that were reflected in the Fourier spectra of the spray current. Significant mode changes and the malformation of the Taylor cone were detected as peak shifts or sudden changes in the spectrum quality. This was confirmed by fast imaging that showed a reduction in the size of the Taylor cone under hydrophobic tip conditions and rapid periodic ejection of filaments and droplets for high conductivity solutions. Comparing the oscillation frequencies of Taylor cones of different sizes, good correlation was found with the frequencies of capillary waves on comparablly sized liquid spheres. Spray stability was also linked with the positional stability of the contact line between the liquid meniscus and the capillary tip.

Electrochemistry↗
PubMed2004

Kinetic mechanism and quaternary structure of Aminobacter aminovorans NADH:flavin oxidoreductase: an unusual flavin reductase with bound flavin.

The homodimeric NADH:flavin oxidoreductase from Aminobacter aminovorans is an NADH-specific flavin reductase herein designated FRD(Aa). FRD(Aa) was characterized with respect to purification yields, thermal stability, isoelectric point, molar absorption coefficient, and effects of phosphate buffer strength and pH on activity. Evidence from this work favors the classification of FRD(Aa) as a flavin cofactor-utilizing class I flavin reductase. The isolated native FRD(Aa) contained about 0.5 bound riboflavin-5'-phosphate (FMN) per enzyme monomer, but one bound flavin cofactor per monomer was obtainable in the presence of excess FMN or riboflavin. In addition, FRD(Aa) holoenzyme also utilized FMN, riboflavin, or FAD as a substrate. Steady-state kinetic results of substrate titrations, dead-end inhibition by AMP and lumichrome, and product inhibition by NAD(+) indicated an ordered sequential mechanism with NADH as the first binding substrate and reduced FMN as the first leaving product. This is contrary to the ping-pong mechanism shown by other class I flavin reductases. The FMN bound to the native FRD(Aa) can be fully reduced by NADH and subsequently reoxidized by oxygen. No NADH binding was detected using 90 microM FRD(Aa) apoenzyme and 300 microM NADH. All results favor the interpretation that the bound FMN was a cofactor rather than a substrate. It is highly unusual that a flavin reductase using a sequential mechanism would require a flavin cofactor to facilitate redox exchange between NADH and a flavin substrate. FRD(Aa) exhibited a monomer-dimer equilibrium with a K(d) of 2.7 microM. Similarities and differences between FRD(Aa) and certain flavin reductases are discussed.

Alphaproteobacteria↗