PubMed Health⌕ Search

Biomedical subjects

M Hildebrand

Publications and source records attributed to M Hildebrand.

At least 19 recordsLinked to original sources
PubMed1996

Identification and characterization of a gene cluster involved in manganese oxidation by spores of the marine Bacillus sp. strain SG-1.

The marine Bacillus sp. strain SG-1 forms spores that oxidize manganese(II) as a result of the activities of uncharacterized components of its spore coat. Nucleotide sequence analysis of chromosomal loci previously identified through insertion mutagenesis as being involved in manganese oxidation identified seven possible genes (designated mnxA to mnxG) in what appears to be an operon. A potential recognition site for the sporulation, mother-cell-specific, RNA polymerase sigma factor, sigmaK, was located just upstream of the cluster, and correspondingly, measurement of beta-galactosidase activity from a Tn917-lacZ insertion in mnxD showed expression at mid-sporulation to late sporulation (approximately stage IV to V of sporulation). Spores of nonoxidizing mutants appeared unaffected with respect to their temperature and chemical resistance properties and germination characteristics. However, transmission electron microscopy revealed alterations in the outermost spore coat. This suggests that products of these genes may be involved in the deposition of the spore coat structure and/or are spore coat proteins themselves. Regions of the deduced protein product of mnxG showed amino acid sequence similarity to the family of multicopper oxidases, a diverse group of proteins that use multiple copper ions to oxidize a variety of substrates. Similar regions included those that are involved in binding of copper, and the addition of copper at a low concentration was found to enhance manganese oxidation by the spores. This suggests that the product of this gene may function like a copper oxidase and that it may be directly responsible for the oxidation of manganese by the spores.

Amino Acid Sequence↗
PubMed1995

Stable expression of human cytochrome P450 3A4 in V79 cells and its application for metabolic profiling of ergot derivatives.

Expression of human cytochrome (CYP) in heterologous cells is a means of specifically studying the role of these enzymes in drug metabolism. The complete cDNA encoding CYP3A4 (PCN1) was inserted into an expression vector containing the strong myeloproliferative sarcoma virus promoter in combination with the enhancer of the cytomegalovirus and stably expressed in V79 Chinese hamster cells. The presence of genomically integrated CYP3A4 cDNA cell clones was confirmed by polymerase chain reaction analysis. Transcription was detected by reverse transcribed polymerase chain reaction analysis. Functional expression could be demonstrated by conversion of testosterone to the specific 6beta-hydroxylated product. In recombinant V79 cells expressing CYP3A4 about 6% of the substrate was converted to 6beta-hydroxytestosterone. The metabolism of two dopaminergic ergot derivatives was investigated in live recombinant V79 cells. Both lisuride and terguride were monodeethylated.

Animals↗
PubMed1994

Overprediction of anxiety, and disconfirmatory processes, in anxiety disorders.

The overprediction of anxiety phenomenon and its relationships with fear, dysfunctional and functional beliefs, and emotional experiences during confrontations with feared stimuli were investigated in two studies. Study 1 investigated exposure in vivo exercises executed by anxiety patients during treatment (n = 37). Study 2 investigated behavioural experiments executed by anxiety patients (n = 11) during cognitive treatment. In both studies patients rated various variables just before and immediately after their exercises. The results indicate that anxiety patients tend to overpredict the level of anxiety they are going to experience. There is no evidence that this phenomenon is a statistical artefact, caused for instance by a pre-test response style. There is also no evidence that the observed adjustment of incorrect anxiety predictions is a statistical artefact. A global negative emotional evaluation of the experience appears to have an adverse influence on the reduction of anxiety predictions and on the reduction of fear. Fear, and its reduction after exposure in vivo or after behavioural tests, appears to be influenced by both anxiety predictions and dysfunctional beliefs. Positive emotions and functional beliefs did not appear to play an important role. The interrelationships between these factors are comprised in a path-model which describes how emotional and cognitive information yielded by disconfirmatory experiences influences changes in anxiety predictions and changes in fear. Unexpectedly, change in dysfunctional belief did not relate directly to change in fear, but only indirectly, via change in anxiety predictions. Theoretical and therapeutical implications are discussed.

Adult↗
PubMed1994

Inter-species extrapolation of pharmacokinetic data of three prostacyclin-mimetics.

Cica-, eptalo- and iloprost are chemically and metabolically stabilized derivatives of prostacyclin which maintain the pharmacodynamic profile of the endogenous precursor. While iloprost is still subject to beta-oxidative degradation of the upper side chain, cicaprost is highly metabolically stable. Eptaloprost was synthesized to realize the pro-drug concept in PGI2-mimetics and was designed to be activated to cicaprost by single beta-oxidation. All three prostacyclin-mimetics were studied in various animal species (mouse, rat, rabbit, monkey, dog and pig) and in man to determine their pharmacokinetic profiles. Based upon this data, it was of interest whether an inter-species extrapolation of pharmacokinetic parameters can be performed to show the predictive value of animal experimentation. Allometric inter-species extrapolation is performed by modelling pharmacokinetic data (Y) as exponential functions (x) of species characteristics (e.g. body weight, W) as: Y = .aWx. For total clearance and volumes of distribution at steady state, a clear-cut correlation with x-values of 0.6-0.8 and 1.0-1.1 could be shown for all three compounds. For cicaprost, which was excreted unchanged in several species, renal and non-renal clearance was also mathematically scalable. Due to the use of different compartment models to describe plasma disposition, different sets of half-life data were obtained and could not be extrapolated reasonably. However, mean residence time showed a dependency on body weight with 0.25 as power function. In case of cicaprost, only the dog, which extensively metabolizes the compound, could not be enrolled in inter-species extrapolation. Excretion half-lives or residence times did not show a significant correlation to body weight or maximum life time potential. The present inter-species extrapolation showed a dependency from species body weight for model-independent pharmacokinetic data, e.g. clearance, volume of distribution at steady state and correspondingly mean residence time. The disposition profile of these compounds can therefore be predicted. Preliminary information on bio-degradation is an additional prerequisite for extrapolation. These data demonstrate that basic physiologically determined processes, which show some evolutionary allometric dependency, also influence the disposition of prostacyclin-mimetics. An extrapolation of data from animal to man could easily be realized giving additional justification for animal studies in pharmacology, toxicology and pharmacokinetics.

Animals↗
PubMed1994

Development and validation of a sensitive and specific radioimmunoassay for the determination of cicaprost in biological samples.

Cicaprost is a potent, chemically and metabolically stable PGI2-mimetic. Pharmacodynamic effects were observed after oral administration of approximately 10 micrograms in man when plasma levels were in the low pg-range. The present report describes the development of a selective antiserum and a tracer with high specific activity and their use for the RIA determination of Cicaprost in biological samples. Cicaprost-[3H]-methylester with a specific activity of 819 GBq/mmol was used as a tracer. RIA-analyses were carried out with 0.05-0.5 ml plasma adjusted to pH 2 with 1 N HCl and extracted with 2.5 ml diethylether. Separation of antiserum bound and unbound Cicaprost was achieved by the charcoal method. Extraction recovery of Cicaprost was approximately 90% at pH approximately 2. The detection limit of the assay was 10-20 pg/ml plasma. Coefficients of variations were 6, 3 and 9% (within-day, n = 5) and 25, 12 and 10% (day-to-day, n = 11) at 50, 100 and 200 pg/ml. HPLC-chromatograms of plasma extracts did not reveal any peak apart from Cicaprost, demonstrating the specificity of the method. The present RIA for Cicaprost exhibits high specificity and sensitivity and will be used for further bioanalyses in pharmacokinetic study.

Animals↗
PubMed1994

Coronary thrombolysis with Desmodus salivary plasminogen activator in dogs. Fast and persistent recanalization by intravenous bolus administration.

BACKGROUND: DSPA (Desmodus salivary plasminogen activator) is a new thrombolytic agent corresponding to a natural plasminogen activator discovered in the saliva of the vampire bat Desmodus rotundus. Compared with tissue plasminogen activator (TPA), DSPA, produced in a recombinant cell line, is more fibrin cofactor dependent than TPA. METHODS AND RESULTS: The thrombolytic properties of DSPA and TPA were compared in a canine model of copper coil-induced coronary thrombosis. All dogs received heparin 200 IU/kg IV and SC. Whereas controls did not reperfuse within 180 minutes (none of six), intravenous bolus administration of DSPA at 25, 50, and 100 micrograms/kg resulted in a 100% incidence (6 of 6) of recanalization within 37, 23, and 18 minutes, respectively. TPA at 63 and 125 micrograms/kg reopened the coronaries in 33% (two of six) and 50% (three of six) of cases within 40 minutes. Eighty-three percent (5 of 6) of the arteries were still patent 3 hours after 50 and 100 micrograms/kg DSPA, whereas only 20% (one of five) of all coronaries originally recanalized with both doses of TPA were still open at 3 hours. Plasma levels of alpha 2-antiplasmin decreased significantly only with 125 micrograms/kg TPA. The clearance of DSPA (2.3 to 3.5 mL.min-1.kg-1) was lower compared with TPA (11.4 to 20 mL.min-1.kg-1) due to a prolonged terminal half-life. CONCLUSIONS: In a canine coronary thrombosis model, DSPA exhibited higher potency and recanalized coronary arteries faster and with a lower incidence of reocclusion than TPA. Its properties may translate into a higher efficacy in patients compared with available thrombolytic agents. The long half-life of DSPA may allow for single bolus administration in the treatment of acute myocardial infarction.

Animals↗
PubMed1993

Silicon-responsive cDNA clones isolated from the marine diatom Cylindrotheca fusiformis.

In organisms ranging from single-celled algae to mammals, including humans, silicon is essential for, and actively participates in, a variety of life processes. It has become clear that silicon (i) acts as a metabolite affecting a variety of cellular processes, and (ii) regulates gene expression. However, the mechanisms by which silicon (i.e., Na2SiO3.9H2O, in the present study) acts are not clear, due to inherent methodological difficulties. As part of our program to understand how silicon acts in biological systems, we present the first isolation of cDNA clones derived from silicon-responsive mRNAs, from the marine diatom Cylindrotheca fusiformis. We distinguish between clones responding only to silicon starvation and replenishment, and those also responding to other cellular conditions. Some of the clones can be identified by similarity to other genes, and should be useful as probes to isolate genes from other organisms. Isolation of these clones provides the means to (i) identify metabolic pathways affected by silicon, and (ii) investigate the mechanism(s) of silicon-regulated gene expression.

Cloning, Molecular↗
PubMed1993

Bioactivation of eptaloprost in animals and man.

Eptaloprost is a novel concept PGI2-mimetic, which is designed to be activated to the pharmacologically potent cicaprost via beta-oxidation. By pro-drug formation advantages in terms of sustained delivery of prostacyclin-mimetic activity were envisaged. The active metabolite is known to be metabolically stable and highly pharmacologically potent. In the present set of experiments the pharmacokinetics of eptaloprost was studied in rat, monkey and man by i.v. and ig administration of tritiated compound. Eptaloprost was completely and rapidly absorbed in all three species. Peak plasma levels of the parent compound were observed within 30 min postdose. Total clearance of the pro-drug accounted for 170, 62 and 66 ml/min/kg in rat, monkey and man. Disposition of eptaloprost exhibited half-lives of 0.1 to 0.5 h and mean residence times accounted for 0.15, 0.4 and 0.6 h in the three species. The active metabolite cicaprost was present in the central compartment with a slight delay as compared to eptaloprost. Its peak plasma levels were found within 0.25 to 0.5 h postdose. Disposition of radiolabel in plasma and 3H-excretion with the urine and feces was determined by the pharmacokinetic behaviour of cicaprost. In rats excretion was mainly biliary while monkeys and man excreted almost unchanged cicaprost in equal portions with urine and feces. Half-lives of renal excretion were in the range of terminal half-lives in the central compartment. Neither in animals nor in man eptaloprost administration resulted in an advantageous systemic profile of cicaprost. On the contrary the bioavailable dose fraction of cicaprost was lower as compared to cicaprost administration. A delay or an extension of cicaprost plasma levels was not observed. The present pharmacokinetic data of eptaloprost studied in three species demonstrated that a pro-drug concept based on simple beta-oxidative bioactivation could be successfully realized for a special PGI2-mimetic. An advantage resulting from oral pro-drug administration as compared to direct treatment with the active metabolite could not be shown. For long-lasting plasma levels of cicaprost a chemically determined retardation might require a more sophisticated pro-drug concept or alternatively pharmaceutical technology is required.

Absorption↗
PubMed1992

Nucleotide sequence of diatom plasmids: identification of open reading frames with similarity to site-specific recombinases.

We have determined the nucleotide sequence of two small circular DNA plasmids, pCf1 and pCf2 [22], from the marine diatom Cylindrotheca fusiformis. pCf1 is 4273 bp, and pCf2 is 4079 bp in size. In each plasmid, all of the major open reading frames (ORFs) are encoded on the same DNA strand. Two ORFs are similar, comparing the two plasmids. ORF218 (pCf1) and ORF217 (pCf2) share 80% amino acid identity and ORF482 (pCf1) and ORF484 (pCf2) share 54% amino acid identity. ORF218/217 shows significant similarity (28-31% amino acid identity) to the Tn3 class of resolvases. Resolvases are most commonly found in bacterial transposons. However, two other features found in the Tn3 class of transposon are missing in the plasmids; an ORF encoding a transposase and terminal inverted repeat sequences. This, and data mapping the portions of the plasmids that hybridize to genomic chloroplast DNA, suggest that the plasmids do not contain active transposons. By analogy with the R46 plasmid from Enterobacter [5, 6], another potential role for the resolvases encoded by pCf1 and pCf2 is the conversion of multimeric forms of the plasmid to monomers. The similarity of ORF218/217 to resolvases documents the first identification of a potential coding function in an algal plasmid.

Amino Acid Sequence↗
PubMed1992

Characterization of two circular plasmids from the marine diatom Cylindrotheca fusiformis: plasmids hybridize to chloroplast and nuclear DNA.

This paper reports the discovery and initial characterization of two small plasmids, pCf1 and pCf2, in the marine diatom Cylindrotheca fusiformis. Extracted diatom DNA separates into two bands in CsCl-Hoechst 33258 dye gradients. Upon agarose gel electrophoresis of a sample of the upper band of the gradient we observed, in addition to high molecular weight (genomic) chloroplast and mitochondrial DNA, pairs of lower molecular weight bands. These bands contained two species of circular plasmid DNA molecules, as shown by electron microscopy. The nucleotide composition of the plasmids, and chloroplast and mitochondrial DNAs is similar, as indicated by their co-banding in the gradients. They were cloned, and their restriction maps determined, showing that pCf1 is 4.27 and pCf2 4.08 kb in size. By hybridization analysis, we showed that pCf1 and pCf2 share regions of similarity, but not identity. Neither plasmid hybridizes with mitochondrial DNA. Both plasmids hybridize with chloroplast DNA, and pCf2 also hybridizes with nuclear DNA.

Cell Nucleus↗
PubMed1992

Somatic symptoms in social phobia: a treatment method based on rational emotive therapy and paradoxical interventions.

Social phobia is often accompanied by somatic symptoms such as trembling, blushing, and sweating. In cases where these symptoms are predominant and, rather than the social situation, represent the feared stimulus, their unpredictable occurrence may reduce the effectiveness of an otherwise successful treatment, such as exposure in vivo. In this study, a paradoxical treatment approach, combined with rational emotive therapy, is employed with three social phobic patients with fears of blushing, trembling, and sweating, respectively. Results indicate that a paradoxical treatment may be an effective strategy in reducing somatic symptoms in social phobia.

Adult↗
PubMed1992

Pharmacokinetics of iloprost and cicaprost in mice.

Iloprost and cicaprost are two PGI2-mimetics, which are chemically stable and highly pharmacologically potent. Both compounds differ by their susceptibility to metabolic degradation. While iloprost contains a pentanoic acid upper side chain, which is subject to beta-oxidative degradation, cicaprost is metabolically stabilized by the introduction of an oxygen atom at position 3 of the pentanoic acid chain, preventing beta-oxidation. Both compounds have been characterized concerning their pharmacological and pharmacokinetic profile in a number of animal species and in man. In the present set of experiments both drugs were characterized in terms of pharmacokinetics in mice, an animal species quite routinely used in long-term toxicity studies on cancerogenicity, by iv and ig administration of 0.2 mg/kg (iloprost) and 0.01 mg/kg (cicaprost) using tritiated substances. Iloprost was rapidly inactivated after iv dosing with plasma levels declining from 247 to 0.27 ng/ml within 60 min. Disposition half-lives were 3 and 14 min. Total clerance accounted for 152 ml/min/kg. Total radiolabel exhibited a clearance of 35 ml/min/kg, its AUC in plasma was 146 ng-equiv.h/ml. After ig administration Iloprost peak plasma levels of 9.2 ng/ml occurred after 5 min. Bioavailability was 10%. AUC of total radiolabel was 152 ng-equiv.h/ml, showing complete absorption. Excretion of 3H-label was 41%/57% of dose (iv) and 36%/47% o.d. (ig) with the urine and 32%/18% o.d. (iv) and 36%/25% o.d. (ig) in male/female animals and proceeded for > 90% of dose fraction recovered with half-lives of 0.2-0.3 d. Metabolic patterns revealed the known profile consisting of unchanged drug, dinor- and tetranor-metabolites in plasma and mainly, tetranor-products in urine and feces. After iv dosing of cicaprost total radiolabel plasma levels declined biphasically with half-lives of approx. 0.05 h and 0.31 h. Extrapolated AUC was 1.6 ng-equiv. h/ml and total clearance accounted for 108 ml/min/kg. After ig treatment peak radioactivity plasma levels of 0.7 and 1 ng-equiv./ml were observed at 0.16 and 1 h postdose, probably due to differences between animal groups. Extrapolated AUC was 1 ng-equiv.h/ml. Excretion of 3H-label was mainly biliary: With the feces 83%/89% o.d. (iv) and 93%/92% o.d. (ig) were excreted by male/female animals, while 8.3%/5.7% o.d. (iv) and 2.6%/5.5% were recovered in the urine. More than 90% of the excreted radiolabel was found in samples collected up to 24 h postdose. Metabolic patterns in plasma revealed that after both routes of administration 3H-cicaprost was the dominant radiolabel fraction accounting for up to 90% of total radiolabel chromatographed.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Comparison of bioanalytical determinations of Iloprost, a chemically stable PGI2 mimetic, by conventional radioimmunoassay (RIA) and scintillation proximity assay (SPA).

The scintillation proximity assay is a novel variant of classical radioimmunoassay. It can be performed as a single tube measurement because the separation of bound and unbound tracer fraction is avoided. In principle, microbeads are coated with anti-species antibodies that can couple with the respective antiserum used for RIA. By means of special cores, light emission takes place if labelled, antiserum-bound tracer is coupled to the anti-species antibody on the fluomicrosphere surface. In the present report, the novel assay was compared to a validated RIA for the bioanalysis of the PGI2 mimetic, Iloprost. Extraction recovery of Iloprost was approximately 90% at pH less than or equal to 4. The detection limit of the novel assay was 2-4 pg/sample, corresponding to 10-20 pg/ml plasma (if 0.2 ml plasma was used). Coefficients of variations were 9, 7 and 6% (within-day, n = 5) and 30, 11 and 10% (day-to-day, n = 10) at 50, 100 and 200 pg/ml. RIA and SPA levels of Iloprost measured in human plasma samples (n = 428) were similar. The SPA method exhibits both a similar specificity and detection limit to RIA and will be used for further analyses.

Humans↗
PubMed1991

Plasmids in diatom species.

We have discovered plasmids in 5 of 18 diatom species surveyed. In several species, more than one type of plasmid is present. Several of the plasmids show similarity by hybridization previously characterized plasmids in Cylindrotheca fusiformis (J. D. Jacobs et al., unpublished data). Additionally, there is similarity between the plasmids found in C. fusiformis and chloroplast DNA in three diatom species. These results add to the evidence that the plasmids have features of mobile genetic elements.

Cloning, Molecular↗
PubMed1991

Oral iloprost in healthy volunteers.

Iloprost is a potent chemically stable PGI2-mimetic. Therapeutic efficacy was shown after i.v. infusion treatment in several states of peripheral vascular disease. For out-patient therapy an oral dosage form should be developed. Based upon dissolution profiles and in vivo data of a pig model, three different film-coated pellet formulations were selected for pharmacokinetic characterization in nine healthy volunteers. In the first part of the study groups of three test subjects were treated with increasing dosages (150-300 micrograms) of iloprost. At 300 micrograms flush and headache led to the discontinuation of those titration. All formulations exhibited dose-dependent serum level profiles. The cross-over characterization in all test subjects showed that one formulation, which exhibited a modified in vitro dissolution of 60% of the dose within 1 h in pH 7.4 phosphate buffer, was optimal from the pharmacokinetic profile. After oral administration of this formulation the bioavailable dose fraction was highest and half-maximal serum levels lasted for 2.4 h (mean); therapeutic serum levels were maintained for 2.1-5.0 h. This formulation was chosen for further investigation to imitate therapeutic serum level profiles as obtained after i.v. infusion for 4-6 h with a once-a-day dosage form.

Administration, Oral↗