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H D Fischer

Publications and source records attributed to H D Fischer.

At least 19 recordsLinked to original sources
PubMed1998

Expression of a structural domain of the beta 2 subunit essential for alpha M beta 2 ligand recognition.

The beta2 leukocyte integrins comprise a group of closely related adhesion receptors that mediate critical events during normal and inflammatory immune responses. Central to the understanding of beta2 integrin function is the basis of ligand recognition. Results from our laboratory and others indicate the presence of multiple ligand contact points in both the alpha and beta subunit. As an approach to identify and characterize regulatory domains of the beta2 subunit, we have generated two different subdomains of the beta2 subunit for expression on the surface of mammalian cells through a phosphatidyl-inositol glycan anchor. The first subdomain contains the putative beta2 MIDAS motif implicated in ligand binding [beta2(LB)], whereas the second beta2 subdomain contains the cysteine-rich region [beta2(CR)]. Cells expressing alphaM and beta2 constructs singly or cotransfected transiently in COS-7 cells were tested for the ability to bind to immobilized iC3b. Cells bearing the recombinant alphaMbeta2(LB) were capable of adhering to iC3b in a manner similar to that observed with the complete alphaMbeta2 heterodimer. In contrast, cells expressing alphaMbeta2(CR) failed to adhere to immobilized iC3b. Moreover, cells bearing singly transfected alpha or beta chains alone failed to adhere to immobilized iC3b. These results indicate that along with alphaM, the beta2(LB) subdomain contains the sufficient components within the beta2 subunit essential for ligand recognition. These findings support the hypothesis that the beta2 subunit cooperates with site(s) within the alphaM subunit in a receptor/cation/ligand complex resulting in high-affinity ligand interaction.

Animals↗
PubMed1998

A peptide inhibitor of cholesteryl ester transfer protein identified by screening a bacteriophage display library.

We screened a bacteriophage display library of random decapeptides to identify peptide inhibitors of cholesteryl ester transfer protein (CETP). After affinity selection against CETP, bacteriophage-infected Escherichia coli were plated at clonal density and 36 random clones were isolated. Analysis of the relevant portion of the bacteriophage DNA from a group of 12 clones that had a relatively high affinity for CETP revealed that the corresponding amino acid sequences of the displayed peptides exhibited an ... Xaa-Arg-Met-Arg-Tyr-Xaa ... composite motif. Based on those results, decapeptides from this group were synthesized and one of them, DP1 (NH2-VTWRMWYVPA-COOH), inhibited CETP-catalyzed transfer of cholesteryl esters and triglycerides. Amino- and carboxy-terminal truncations of DP1 demonstrated that the original decapeptide could be reduced to a pentapeptide without loss of either its ability to bind to CETP or its ability to inhibit CETP-mediated lipid transfer. That pentapeptide, NH2-WRMWY-COOH (WRMWY, PNU-107368E), binds directly to CETP and its inhibition is consistent with that of a competitive inhibitor of CETP with a Ki of 164 microM. WRMWY or modified versions of this peptide may be useful in studying the interactions between CETP and plasma lipoproteins.

Amino Acid Sequence↗
PubMed1995

Restriction and functional changes of dopamine release in rat striatum from young adult and old rats.

In order to investigate the age-related changes in dopaminergic activity in rats, we have utilized the K(+)- and veratridine-stimulated [14C]dopamine release from striatum in vitro as a functional index of responsiveness to these stimuli in aging. We found that the K(+)-stimulated dopamine release from old (12 months) rats decreased by more than 50% compared to that from young adult rats (3 months). Reserpine (5 mg/kg) led to a pronounced decrease of the K(+)-stimulated dopamine release of young adult as well as old rats. Whereas ouabain (10 mumol/l) decreased the K(+)-stimulated dopamine release from young adult rats, in old rats the K(+)-induced dopamine release was increased up to 250%. However, in old rats which were reserpine pretreated, ouabain was unable to stimulate the K(+)-induced dopamine release. In contrast, the veratridine-stimulated dopamine release of old rats was increased up to 200% compared to that of young adult rats and was highly sensitive to reserpine pretreatment but not to ouabain. However, reserpine did not alter this veratridine-stimulated dopamine release from young adult rats. The present data indicate that the age-related reduction of exocytosis-related, Ca(2+)-dependent release mechanisms (K+) are probably compensated via an increase in Ca(2+)-independent, uptake carrier-mediated release processes (veratridine).

Age Factors↗
PubMed1995

Expression and purification of recombinant cynomolgus monkey cholesteryl ester transfer protein from Chinese hamster ovary cells.

Cholesteryl ester transfer protein (CETP) mediates the transfer of cholesteryl ester from high- and low-density lipoproteins to triglyceride-rich lipoproteins, and reciprocally mediates triglyceride transfer. The gene for cynomolgus monkey CETP was expressed in serum-free CHO culture with 2 micrograms/ml insulin as its only exogenous protein supplement. Cell growth was facilitated by immobilizing the CHO cells in alginate beads. Recombinant CETP (rCETP) was purified 176-fold with a three-step protocol resulting in a 60% final yield as measured by a fluorescent CETP activity assay. Typically, 3.4 mg of rCETP was purified from 1700 ml of media by affinity-gel chromatography involving Reactive Red 120 (RR120) followed by concanavalin A Sepharose 4B and rechromatography on RR120. SDS-PAGE shows a single broad band of M(r) ranging from 68,000 to 74,000 which immunoreacts in Western blot analysis. Amino acid analysis and protein sequencing of the purified protein agree with the theoretical amino acid composition and sequence of cynomolgus CETP.

Amino Acid Sequence↗
PubMed1995

Chemical shift differences between free and Fab-bound peptide correlate with a two-stage selection of peptide sequences from a random phage display library to delineate critical and non-critical residues for antibody recognition.

Epitope libraries provide a method to identify peptide ligands for antibodies, receptors or other binding proteins. As such, they provide a powerful tool to rapidly identify lead ligands in the drug discovery process. In an attempt to correlate structural information with the results from peptide screening, we have used NMR spectroscopy of peptide/antibody complexes to demonstrate that core residues identified through a two-stage selection process undergo a larger structural change upon binding antibody than do positions in the peptide amenable to a variety of side chains. The model system used was the M2 monoclonal antibody/Flag octapeptide epitope system. We have analyzed two peptides: Ac-Asp-Tyr-Lys-Leu-Gly-Asp-Asp-Leu-NH2 (peptide 1), which contains several non-core positions randomized, and Ac-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Leu-NH2 (peptide 2), which closely corresponds to the original Flag sequence. Enrichment of the peptides with 15N facilitated the investigation by permitting spectral editing of the peptide resonances in the presence of antibody. For peptide 1 the absolute shifts for the free vs. Fab-bound peptide were found to be largest for the amide groups of Asp-1 and Asp-6, in agreement with classification of these residues as critical by the phage display library selection process. For peptide 2 the largest absolute shifts were observed for Asp-1 and Asp-4, with the other aspartic acid residues also showing significant but smaller changes.

Amino Acid Sequence↗
PubMed1994

Two-stage selection of sequences from a random phage display library delineates both core residues and permitted structural range within an epitope.

Libraries of random peptides can be screened to identify species which interact with antibodies or receptors. Similarly, maps of native molecular interactions can frequently be deduced by screening a limited set of peptide fragments derived from sequences within a native antigen or ligand. However, the existence of cross-reactive sequences that mimic original epitopes and the limited replaceability of amino acid residues suggest that the sequence space accessible by a receptor can be much broader. Definition of this space is of particular importance where structural information is required for peptidomimetic or drug design. We have used a two-stage selection scheme to expand the sequence space accessible by a phage display library and to define peptide epitopes of the anti-FLAG octapeptide monoclonal M2 antibody. Affinity selection of a primary library of 2 x 10(6) random decapeptides identified a non-contiguous core of three residues in the binding motif Tyr-Lys-Xaa-Xaa-Asp. A second stage library with 2 x 10(7) individual clones bearing the core motif but with the remaining flanking and internal residues re-randomized permitted access to a broader sequence space represented in a library equivalent to several orders of magnitude larger. Data here demonstrate that extended access to binding sequence space permitted by multi-stage screening of phage display libraries can reveal not only essential residues required for ligand binding, but also the ligand structural range permitted within the receptor binding pocket.

Amino Acid Sequence↗
PubMed1993

Biochemical diversity in a phage display library of random decapeptides.

Detailed knowledge and understanding of the structure and function of biologically important macromolecules is frequently insufficient to permit rational, de novo design of recognition molecules and therapeutics. Traditional drug discovery has, thus, focused on screening and identifying native molecules as drugs or as templates for genetic engineering or organic synthesis. The number and novelty of lead compounds for drug discovery might be expanded significantly, however, by the ability to express and screen large libraries of peptide structures with phage display technologies [Scott and Smith, Science 249 (1990) 386-390]. The significance of such libraries as sources of novel biological ligands will depend in part on the depth and degree of biochemical diversity they comprise. We have prepared a phage display library of greater than 2 x 10(6) individual decapeptides produced as N-terminal fusions to the pIII surface protein of fd filamentous phage. The decapeptides were expressed from a degenerate DNA insert sequence that was chemically synthesized with an equal mixture of all four nucleotide bases at the three positions in each of the ten codons. Fifty-two clones were picked randomly and without prior selection from the population and the sequences of their peptide inserts were determined. Our results confirm and document the broad representation at the primary amino acid sequence level that is expected in a library expressed from random DNA inserts. More significantly, biochemical characterization shows these insert sequences correspond to structures comprising a wide range and combination of isoelectric, hydropathic, and biochemical properties necessary in drug discovery to access a significant percentage of the repertoire of possible peptide structures by affinity or activity screening.

Amino Acid Sequence↗
PubMed1993

Long-term effects of postnatal hypoxia and flunarizine on the dopaminergic system.

Long-term changes of learning behavior and of the striatal dopaminergic system were observed in a rat model of early postnatal hypoxia. Striatal dopamine (DA) concentration, K(+)-stimulated DA release from slices, and DA uptake into crude synaptosomal preparations (S1 fractions) were used as markers of the striatal DAergic system. Active avoidance learning was tested as behavioral criterion. Cyclodextrin and flunarizine were found to produce long-term effects on the DAergic system in control animals. While cyclodextrin normalized hypoxia-induced effects in DA release, flunarizine prevented those in DA uptake and improved avoidance learning.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1993

Effects of potassium channel openers and their antagonists on rat locus coeruleus neurones.

1. Intracellular recordings were obtained from a pontine slice preparation of the rat brain containing the locus coeruleus (LC). Two openers of ATP-sensitive potassium (K(ATP)) channels, RO 31-6930 (10 microM) and cromakalim (100 microM) decreased the spontaneous discharge of action potentials without altering their amplitude or duration. Neither compound changed the resting membrane potential. 2. Of two K(ATP) channel blockers, tolbutamide (300 microM) increased the firing rate, while glibenclamide (3 microM) only tended to do so. In addition, both compounds antagonized the effect of RO 31-6930 (10 microM). Neither glibenclamide (3 microM) nor tolbutamide (300 microM) altered the resting membrane potential. 3. Tetrodotoxin (0.5 microM) depressed the firing, but did not influence the inhibitory action of RO 31-6930 (10 microM). The excitatory amino acid antagonist, kynurenic acid (500 microM), did not change the spontaneous discharge of action potentials. 4. Small shifts (2-4 mV) of the membrane potential by hyper- or depolarizing current injections markedly decreased and increased the firing rate, respectively. 5. Noradrenaline (100 microM) hyperpolarized the cells and decreased their input resistance. This effect was not antagonized by glibenclamide (3 microM) or tolbutamide (300 microM). Ba2+ (2 mM), a blocker of both ATP-sensitive and inwardly rectifying potassium channels, abolished the effects of RO 31-6930 (10 microM) and noradrenaline (100 microM). 6. These data suggest that K(ATP) channels are present on the noradrenergic LC neurones, but are not coupled to alpha 2-adrenoceptors.

Action Potentials↗
PubMed1992

Stable production of an analog of human tissue plasminogen activator from cultured Drosophila cells.

We have studied the expression of an analog of human tissue plasminogen activator, FK2P, in Drosophila Schneider 2 cells. A number of promoters were tested, including the Drosophila metallothionein promoter (MTd), baculovirus immediate early promoter (IE), Drosophila copia promoter, mouse metallothionein promoter, cytomegalovirus immediate early promoter with or without intron, SV40 immediate early promoter, and human elongation factor 1 alpha promoter. Two of these promoters drove significant expression of FK2P. The MTd promoter is tightly regulated and upon induction with copper or cadmium expression of FK2P increases as much as 180-fold, accumulating in the culture medium to about 7 micrograms FK2P/10(6) cells/day as determined by ELISA. The IE promoter can direct the constitutive expression to yield about 0.4 microgram FK2P/10(6) cells/day. The production of FK2P in these cell lines remains at about the same level after repeated passages, even in the absence of selective pressure. The FK2P accumulated in the culture medium is fully active in an assay using a chromogenic substrate for serine proteases. Western immunoblot analysis shows that the product remains predominantly as single-chain molecules in serum-free medium, while in serum-containing medium two-chain material occurs as expected due to the presence of plasmin in serum. Judged from the size in Western immunoblots, the FK2P produced is glycosylated.

Amino Acid Sequence↗
PubMed1990

Serum activity that confers acid lability to alpha-interferon in systemic lupus erythematosus: its association with disease activity and its independence from circulating alpha-interferon.

We conducted a longitudinal evaluation of a patient with systemic lupus erythematosus who constitutively exhibited elevated levels of circulating alpha-interferon (alpha-IFN). This study demonstrated that the serum levels of an activity that renders the endogenous alpha-IFN acid labile are positively correlated with disease activity. This IFN acid lability-inducing activity can also be found in the sera of systemic lupus erythematosus patients who have active disease but who do not have circulating alpha-IFN.

Acids↗
PubMed1990

Effect of 2-mercapto-ethanol on some brain biochemical characteristics and behavioural changes in the ageing CBA/Ca mice.

Male CBA/Ca inbred mice were treated with a dose of 8 micrograms 2-mercapto-ethanol per animal per day in the drinking water from the age of 5 months onwards up to the age of 24 months. Dopamine release was greatly decreased in old animals in contrast to the elevation of dopamine release in the treated mice. Similarly, an elevated malondialdehyde content in brain homogenates was also observed in the aged treated animals compared with their controls. No essential differences were observed in locomotor activity and learning between treated an control mice.

Aging↗
PubMed1990

Effect of 2-mercaptoethanol on posthypoxic and age-related biochemical and behavioural changes in mice and rats.

2-Mercaptoethanol (2-ME) has a beneficial effect on the mean life span of laboratory rodents. This paper deals with the effects of 2-ME on changes of dopamine release from brain slices of old aged or hypoxia exposed mice and rats. The results were compared with data which reflect spontaneous peroxidation of brain lipid constituents. In addition, adequate behavioural properties were studied. Long-term 2-ME treatment for months abolishes the age-related decrease of transmitter release and prevents changes in malondialdehyde formation. If age-dependent release failure is already established, then neither single high doses of 2-ME nor repeated treatment for 3 weeks are effective. Posthypoxic release inhibition is prevented by a 2-ME pretreatment for 3 weeks but not by an acute single application even at high dosages. The preventive effect of 2-ME is a mediated one rather than an immediate direct action. Age-related behavioural deficits, such as locomotor activity, habituation performance and learning ability, do not reflect any effect of 2-ME long-term treatment.

Aging↗
PubMed1990

Influence of nootropic drugs on the age-dependent potassium-coupling of transmitter release.

The potassium-induced dopamine release from rat striatum slices shows an age-dependent decline comparable to observations after hypoxia. Pretreatment of aged animals with antihypoxically active nootropic drugs for three weeks results in an improvement of the impaired transmitter release. Simultaneously the slope of the stimulus-release relation is increased and an age-related 50% decrease of the high affinity Ca(++)-ATPase activity (brain P2 fraction) is partially compensated. Like the antihypoxic effect, the effectiveness of nootropic drugs regarding age-dependent changes of neuronal functions probably will consist, above all, in vascular influences of the microcirculation, repair of phospholipids damaged by free radical triggered peroxidation and improvement of stimulus-release coupling.

Aging↗
PubMed1990

[Characterization and pharmacological effect on brain damage as a result of cold injury].

Freezing lesion of brain damages the blood-brain-barrier and induces edema. Both injuries are partially separate processes with different progress and diverse response to pharmacological treatment. A very weak lesion fails in to induce edema despite of an enhanced permeation of Evans blue. The injured permeability responds to treatment with dexamethasone or piracetam whereas the edema does not.

Animals↗