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Biomedical subjects

W Teschner

Publications and source records attributed to W Teschner.

At least 19 recordsLinked to original sources
PubMed2007

A new liquid, intravenous immunoglobulin product (IGIV 10%) highly purified by a state-of-the-art process.

BACKGROUND AND OBJECTIVES: The ultimate goal was to generate an industrial-scale process suitable to produce a high-yield, safe and stable immunoglobulin G (IgG) preparation for intravenous administration, which is ready to use for customer convenience. This new liquid 10% IgG preparation (IGIV 10%) was compared to Gammagard SD, a licenced lyophilized immunoglobulin in biochemical and preclinical testing. MATERIALS AND METHODS: The new process, which includes three dedicated virus clearance steps, is a streamlined combination of the currently applied and well-established manufacturing procedures. The biochemical characterization is done by standard methods focusing on purity, integrity and functionality of the preparation. Efficacy is demonstrated in vivo by mouse protection testing and in vitro by opsonization and protein A affinity chromatography. Pharmacokinetics in rats is evaluated after a single intravenous dose. The anaphylactoid potential is determined in rats and in guinea pigs, while thrombogenicity is assessed in a rabbit model. The influence of the products on vital functions is tested on dogs, while acute toxicity studies are carried out on mice and rats. RESULTS: The biochemical characterization data demonstrate the high purity of monomeric IgG in the product. The mouse protection test showed that the protective activity against systemic bacterial infections of IGIV 10% is at least as good as the reference Gammagard SD. This result is supported by the broad spectrum of antibodies in high titres against bacteria and viruses and the high functional integrity of the IgG molecule (> or = 90% functionally intact IgG) in IGIV 10%. The opsonic activity of all IGIV 10% lots is similar to the one of the reference Gammagard SD. In safety and thrombogenicity studies, no adverse effects of IGIV 10% were observed. Pharmacokinetic studies showed no statistically significant differences between the two products. In the acute toxicity animal studies, IGIV 10% compared favourably to the reference Gammagard SD. CONCLUSIONS: The new manufacturing process enables the production of a highly purified IgG preparation for intravenous administration. The product has an IgG subclass distribution similar to plasma and contains a broad spectrum of functionally intact antibodies. Preclinical studies demonstrate that the liquid IGIV 10% combines excellent qualities of efficacy, safety and tolerability.

Animals↗
PubMed2006

Fc function of a new intravenous immunoglobulin product: IGIV 10% triple virally inactivated solution.

BACKGROUND AND OBJECTIVES: Baxter AG has developed a new liquid intravenous immunoglobulin product [Immune Globulin Intravenous (IGIV) 10%] using a new manufacturing procedure. A modified Cohn fractionation and ion exchange chromatography is used to produce an IgG solution with no alterations to the Fc region. Three dedicated virus reduction steps are included: solvent-detergent treatment, nanofiltration, and incubation at low pH and elevated temperature in final formulation. We applied the reference method of the European Pharmacopoeia (EP) together with a flow-cytometric binding assay for the evaluation of the Fc function of the new product. MATERIALS AND METHODS: The EP reference method was done as described in the EP. The flow-cytometric method measured binding of IgG to Fc receptors of human monocytic THP-1 cells after exclusion of apoptotic cells. RESULTS: Sixteen lots of the new product expressed Fc functions between 84% and 110% when analysed with the EP reference method and Fc-binding activities between 82% and 121% when determined by the flow-cytometric method. CONCLUSION: All tested lots of the new product demonstrated a high level of Fc activity and met the requirements of the EP for Fc function.

Flow Cytometry↗
PubMed2005

Immunomodulatory properties of human serum immunoglobulin A: anti-inflammatory and pro-inflammatory activities in human monocytes and peripheral blood mononuclear cells.

Our study investigated the immunomodulatory activities of human plasma-derived serum immunoglobulin (Ig)A. Previous findings seem contradictory indicating either pro- or anti-inflammatory activities. We used serum IgA purified from large plasma pools and studied the modulation of the release of cytokines and chemokines from resting and lipopolysaccharide (LPS, endotoxin)-stimulated human adherent monocytes and human peripheral blood mononuclear cells (PBMC). Our results indicate that IgA down-modulates the release of the pro-inflammatory chemokines monocyte chemoattractant protein (MCP) 1, macrophage inflammatory protein (MIP) 1alpha and MIP1beta from LPS-stimulated PBMC and the release of MCP1, MIP1alpha and MIP1beta from LPS-stimulated monocytes. Furthermore, we confirmed previous reports that plasma-derived serum IgA down-modulates the release of the pro-inflammatory cytokines, interleukin (IL)-6 and tumour necrosis factor (TNF)-alpha, from LPS-stimulated monocytes and PBMC, and up-regulates the release of IL-1 receptor antagonist (IL-1RA) from resting and LPS-stimulated monocytes and resting PBMC. This IgA-mediated up-regulation of IL-1RA is independent of the simultaneous up-regulation of IL-1beta release, as shown by blocking the biological activity of IL-1beta with a neutralizing antibody. On the other hand, we also found an IgA-induced pro-inflammatory activity, namely IgA-mediated up-regulation of the release of pro-inflammatory IL-1beta as well as down-regulation of the anti-inflammatory cytokines IL-10 and IL-12p40 from LPS-stimulated monocytes and PBMC and a down-regulation of transforming growth factor (TGF)-beta from resting and LPS-stimulated PBMC. We conclude that human serum IgA has both an anti-inflammatory and a pro-inflammatory capacity and this dual capacity might contribute to the feedback mechanisms maintaining a balance between pro-inflammatory and anti-inflammatory activities.

Cells, Cultured↗
PubMed1990

Specific suppression of heterotropic interactions in phosphofructokinase by the mutation of leucine 178 into tryptophan.

The leucine residue at position 178 in the allosteric phosphofructokinase from Escherichia coli has been changed into a tryptophan residue by oligonucleotide-directed mutagenesis. The modified enzyme has been purified to homogeneity, and its enzymatic properties show that this single mutation suppresses the heterotropic interactions without affecting the homotropic ones. The mutant has the same saturation curve by fructose 6-phosphate as the wild type, showing that its active site binds this substrate with the same affinity and cooperativity. The regulatory site of the mutant enzyme can bind the effectors, the activator GDP, or the inhibitor phosphoenolpyruvate, as measured by protection against irreversible thermal denaturation. However, the binding of either effector does no longer influence the activity. This specific suppression of the coupling between the regulatory and active sites is not predicted by the concerted model which postulates that the same structural transition between two states R and T is responsible for both homotropic and heterotropic interactions. Leu-178 belongs to neither the active nor the regulatory site but appears as an important residue in the conformational change(s) involved in the regulation by allosteric effectors.

Allosteric Regulation↗
PubMed1990

Fructose-6-phosphate modifies the pathway of the urea-induced dissociation of the allosteric phosphofructokinase from Escherichia coli.

Phosphofructokinase from Escherichia coli binds fructose-6-phosphate with the sugar moiety of the substrate interacting with one subunit and the phosphate group with another one, so that bound fructose-6-phosphate lies across the interface between the subunits [(1988) J. Mol. Biol. 204, 973-994]. When this interface is 'cross-linked' by fructose-6-phosphate, it becomes more stable because of the extra interactions between subunits: inactivation upon dissociation occurs only above 5 M urea, instead of 1 M urea for the free protein. At saturation in fructose-6-phosphate, this interface is no longer the first to dissociate as in the free protein [(1989) Biochemistry 28, 6836-6841]: instead, the addition of urea to phosphofructokinase in the presence of fructose-6-phosphate induces a conformational change within the tetramer which alters the environment of Trp-311 and distorts the regulatory site.

Allosteric Regulation↗
PubMed1990

Enzymatic properties, renaturation and metabolic role of mannitol-1-phosphate dehydrogenase from Escherichia coli.

Enzymatic properties, renaturation and metabolic role of mannitol-1-phosphate dehydrogenase from Escherichia coli. D-mannitol-1-phosphate dehydrogenase was purified to homogeneity from Escherichia coli, and its physicochemical and enzymatic properties were investigated. The molecular weight of the polypeptide chain is 45,000 as determined by polyacrylamide gel electrophoresis in denaturing conditions. High performance size exclusion chromatography gives an apparent molecular weight of 47,000 for the native enzyme, showing that D-mannitol-1-phosphate dehydrogenase is a monomeric NAD-dependent dehydrogenase. D-mannitol-1-phosphate dehydrogenase is rapidly denatured by 6 M guanidine hydrochloride. Non-superimposable transition curves for the loss of activity and the changes in fluorescence suggest the existence of a partially folded inactive intermediate. The protein can be fully renatured after complete unfolding, and the regain of both native fluorescence and activity occurs rapidly within a few seconds at pH 7.5 and 20 degrees C. Such a high rate of reactivation is unusual for a protein of this size. D-mannitol-1-phosphate dehydrogenase is specific for mannitol-1-phosphate (or fructose-6-phosphate) as a substrate and NAD+ (or NADH) as a cofactor. Zinc is not required for the activity. The affinity of D-mannitol-1-phosphate dehydrogenase for the reduced or oxidized form of its substrate or cofactor remains constant with pH. The affinity for NADH is 20-fold higher than for NAD+. The forward and reverse catalytic rate constants of the reaction: mannitol-1-phosphate + NAD+ in equilibrium fructose-6-phosphate + NADH have different pH dependences. The oxidation of mannitol-1-phosphate has an optimum pH of 9.5, while the reduction of fructose-6-phosphate has its maximum rate at pH 7.0. At pH values around neutrality the maximum rate of reduction of fructose-6-phosphate is much higher than that of oxidation of mannitol-1-phosphate. The enzymatic properties of isolated D-mannitol-1-phosphate dehydrogenase are discussed in relation to the role of this enzyme in the intracellular metabolism.

Amino Acid Sequence↗
PubMed1989

Urea-induced inactivation, dissociation, and unfolding of the allosteric phosphofructokinase from Escherichia coli.

The influence of urea on the allosteric phosphofructokinase from Escherichia coli has been studied by measuring the changes in enzymatic activity, protein fluorescence, circular dichroism, and retention in size-exclusion chromatography. Tetrameric, dimeric, and monomeric forms of the protein can be discriminated by their elution from a high-performance liquid chromatography gel filtration column. Three successive steps can be detected during the urea-induced denaturation of phosphofructokinase: (i) the dissociation of the native tetramer into dimers which abolishes the activity; (ii) the dissociation of dimers into monomers which exposes the unique tryptophan, Trp-311, to the aqueous solvent; (iii) the unfolding of the monomers which disrupts most of the secondary structure. This pathway involves the ordered dissociation of the interfaces between subunits and supports a previous hypothesis (Deville-Bonne et al., 1989). Phosphofructokinase can be quantitatively renatured from urea solutions, provided that precautions are taken to avoid the aggregation of one insoluble monomeric state. The renaturation of phosphofructokinase from urea implies three steps: an initial folding reaction within the monomeric state is followed by two successive association steps. The faster association step restores the native fluorescence, and the slower regenerates the active enzyme. The renaturation and denaturation of phosphofructokinase correspond to the complex pathway: tetramer in equilibrium dimer in equilibrium folded monomer in equilibrium unfolded monomer. It is found that the subunit interface which forms the regulatory site is more stable and associates 40 times more rapidly than the subunit interface which forms the active site.

Allosteric Regulation↗
PubMed1989

A carboxypeptidase Y pulse method to study the accessibility of the C-terminal end during the refolding of ribonuclease A.

Carboxypeptidase Y pulses, applied after various times of refolding, were employed to probe the accessibility of the C-terminus of RNAase A during the refolding process. The increase in resistance against proteolytic cleavage was measured by determination of the amount of liberated C-terminal amino acids and by activity assays. The results indicate that the C-terminus of RNAase becomes inaccessible early in the course of refolding, if folding is carried out at low temperatures under conditions that effectively stabilize the native state. At higher temperatures (25 degrees C) or under conditions of marginal stability, intermediates are not populated and protection against proteolytic cleavage is not detectable before the formation of the native state. The method described may be used to monitor the accessibility of the C-terminus of various proteins during refolding. However, intermediates on the folding pathway can only be observed if the native state is stable against carboxypeptidase attack.

Carboxypeptidases↗
PubMed1989

Intermediates on the reassociation pathway of phosphofructokinase I from Escherichia coli.

The folding and association pathway of the allosteric phosphofructokinase from Escherichia coli has been investigated after complete denaturation of the protein in guanidine hydrochloride by spectroscopical methods, fluorescence and circular dichroism. Three successive processes can be observed during the renaturation of this protein. First, a fast reaction, detected by fluorescence, results in the formation of a (partially) structured monomer. Second, two monomers associate into a dimeric species. This step involves the shielding of the unique tryptophan residue, Trp 311, from the aqueous solvent, and it corresponds to the formation of the interface containing the effector binding site. The presence of ATP during renaturation increases the rate of formation of this dimeric species. The other ligands of the enzyme have no effect on this reaction as well as on the whole reactivation. Finally, the enzymatic activity is regained during the third slowest step. This last reaction is due to the association of two dimers into the native tetrameric structure. The presence of fructose 6-phosphate does not increase the rate of reactivation, even though this ligand strongly stabilizes the native enzyme against denaturation by bridging the interface corresponding to the active site. The self-assembly of phosphofructokinase from E. coli from its unfolded and separated chains follows a specific order in the formation of the interactions between subunits and involves a dimeric intermediate with a defined geometry.

Allosteric Regulation↗
PubMed1989

Ordered disruption of subunit interfaces during the stepwise reversible dissociation of Escherichia coli phosphofructokinase with KSCN.

The reversible inactivation and dissociation of the allosteric phosphofructokinase from Escherichia coli has been studied in relatively mild conditions, i.e., in the presence of the chaotropic agent KSCN. At moderate KSCN concentration, the loss of enzymatic activity involves two separated phases: first, a rapid dissociation of part of the tetramer into dimers, second, a slower displacement of the dimer-tetramer equilibrium upon further dissociation of the dimer into monomers. These two reactions can no longer be distinguished above 0.3 M KSCN since complete inactivation occurs in a single reaction. Different changes are observed for the fluorescence and the activity of the enzyme in KSCN: the fluorescence is not affected by the dissociation into dimers which is responsible for inactivation. The decrease in fluorescence reflects the change in environment of the unique tryptophan residue, Trp 311, during the dimer to monomer dissociation. This residue belongs to the interface containing the regulatory site, and its native fluorescence indicates that this interface is still present in the dimer. The substrate fructose 6-phosphate protects phosphofructokinase from inactivation by binding to the tetramer and prevents its dissociation into dimers. The presence of phosphoenolpyruvate prevents the slow dissociation of the dimer into monomers, which shows the ability of the dimer to bind the inhibitor. Two successive processes can be observed during reassociation of the protein upon KSCN dilution. First, a fast reaction (k1 = 2 x 10(5) M-1.s-1) is accompanied by a fluorescence increase and results in the formation of the dimeric species.(ABSTRACT TRUNCATED AT 250 WORDS)

Allosteric Regulation↗
PubMed1984

Isolation, physicochemical properties, and folding of octopine dehydrogenase from Pecten jacobaeus.

Two types (isoenzymes) of octopine dehydrogenase (A and B) from Pecten jacobaeus adductor muscle were purified to homogeneity, applying affinity chromatography as an efficient final step of purification. Both forms of the enzyme differ in their electrophoretic mobility. All other physico-chemical and enzymatic properties, as well as the folding behaviour were found to be identical. Interconversion of one form into the other was not detectable. Sedimentation equilibrium, gel permeation chromatography, and NaDodSO4/polyacrylamide gel electrophoresis yield a relative molecular mass of 45000 +/- 1500 for both native and denatured enzyme. The unfolding transition at varying guanidine X HCl concentrations is characterized by a two-step profile: at 0.4-0.8 M, partial unfolding is parallelled by inactivation; at 2.0-2.4 M the residual structure is destroyed in a second unfolding step. Beyond 2.8 M no further changes in fluorescence emission and dichroic absorption are observed. At 0.4-1.8 M guanidine X HCl, partial unfolding is superimposed by aggregation. The emission maximum of the intrinsic protein fluorescence at 327 nm is shifted to 352 nm upon denaturation in 6 M guanidine X HCl. Changes in the far-ultraviolet circular dichroism indicate complete loss of the overall backbone structure in this denaturant, including the native helix content of about 33%. Denaturation in 6 M guanidine X HCl, as monitored by the decrease of protein fluorescence, is fast (less than 8s). Upon reactivation after short denaturation, about 25% of the activity is recovered in a fast initial phase (less than 20s). The product of this phase has a similar stability towards destabilizing additives or proteases as the native enzyme. The slow phase of reactivation, which predominates after long-term denaturation, is determined by a single first-order reaction characterized by tau = 29 +/- 3 min (20 degrees C). This reaction must be a relatively late event on the folding pathway, preceded by the fast formation of a structured intermediate, as indicated by the immediate recovery of the native fluorescence. The structural rearrangements, which are rate-limiting for reactivation after long-term denaturation, are characterized by a high energy of activation (112 +/- 8 kJ/mol). The slow reactivation step is compatible in rate with the first-order folding reactions involved in the reconstitution of several oligomeric dehydrogenases [c.f. R. Jaenicke and R. Rudolph (1983) Colloq. Ges. Biol. Chem. Mosbach 34, 62-90].

Amino Acid Oxidoreductases↗
PubMed1979

DIADEM--a system for the interactive data acquisition and processing in an analytical laboratory.

A conversational program for the acquisition of experimental data in a multi-user, multi-instrument computer system is described. It assists the researcher when recording on-time data. Due to the simple structure of the dialogue, no special knowledge of computer handling is required by the experimenter. Whereas the experimental methods are versatile, a uniform concept of the dialogue and the file structure is realized.

Chemistry Techniques, Analytical↗
PubMedDate not supplied

Introduction by site-directed mutagenesis of a tryptophan residue as a fluorescent probe for the folding of Escherichia coli phosphofructokinase.

The leucine residue at position 178 in the major allosteric phosphofructokinase from Escherichia coli has been replaced by a tryptophan using site-directed mutagenesis. Transformation by the mutated gene of pfk- bacteria results into the expression of a pfk+ phenotype and the production of an active enzyme. The modified protein has been purified and its fluorescence properties show that it contains 2 tryptophan residues, the original Trp 311 and the new Trp 178. During unfolding of the protein by guanidine hydrochloride, the changes in the fluorescence of these 2 residues take place at different steps: Trp 311 becomes exposed to solvent when the dimeric form dissociates into monomers, while Trp 178 is exposed only when a folded chain loses its tertiary structure. The mutant enzyme is stabilized by its substrate fructose-6-phosphate against denaturation induced by heat or guanidine hydrochloride.

Base Sequence↗
PubMedDate not supplied

[Shear fracture stress of the spongiosa and pressure fracture stress of cortical bone of cadaveric vertebrae].

By biomechanic examinations of the vertebrae of human dead bodies the ultimate stress due to shearing (Schubbruchspannung) of the spongy substance could be found out with 1.80 N/mm2 and the ultimate stress due to compression (Druckbruchspannung) of the cortical-belonging to the thick of cortical--with 280 N/mm2. Both measuring results are important for the ultimate strength of the connection (Verankerungsfestigkeit) of screws in vertebae.

Adolescent↗
PubMedDate not supplied

[Anchoring experiments on human cadaver vertebrae using various types of screws].

The firmness of the bone structure of vertebrae of human cadavers was investigated by turning screws of four different types into vertebrae T6 to L5 and subjecting them to axial tension up to the point of extraction. It was found that the cancellous bone screws (rather than the Zielke screw) had the best anchoring characteristics in bone tissue. The shearing fracture stress of the vertebral bone tissue was found to be approx. 1.80 N/mm2; the relation between the extraction forces and the individual vertebrae was also examined. In addition, the extraction forces were determined for screws inserted into vertebrae T6 to L4, as performed in Zielke's ventral spondylodesis procedure, as well as the lower tolerance limit for each vertebra in this segment of the vertebral column.

Adult↗