Biomedical subjects

P Franke

Publications and source records attributed to P Franke.

At least 73 records · Page 4Linked to original sources

PubMed1996

The multiple carrier model of nonribosomal peptide biosynthesis at modular multienzymatic templates.

Gramicidin S synthetase 1 and 2 were affinity-labeled at their thiolation centers either by thioesterification with the amino acid substrate or by specific alkylation with the thiol reagent N-ethylmaleimide in combination with a substrate protection technique. The labeled proteins were digested either chemically by cyanogen bromide or by proteases. An efficient multistep high pressure liquid chromatography methodology was developed and used to isolate the active site peptide fragments of all five thiolation centers of gramicidin S synthetase in pure form. The structures of these fragments are investigated by N-terminal sequencing, mass spectrometry, and amino acid analysis. Each of the active site peptide fragments contains the consensus motif LGG(H/D)S(L/I), which is specific for thioester formation in nonribosomal peptide biosynthesis. It was demonstrated that a 4'-phosphopantetheine cofactor is attached to the central serine of the thiolation motif in each amino acid-activating module of the gramicidin S synthetase multienzyme system forming the thioester binding sites for the amino acid substrates and catalyzing the elongation process. Our data are strong support for a "multiple carrier model" of nonribosomal peptide biosynthesis at multifunctional templates, which is discussed in detail.

Amino Acid Isomerases↗

PubMed1996

Force magnitude applied by orthodontists. An inter- and intra-individual study.

The aim of this investigation was to study the magnitude and variation of forces routinely applied by different orthodontists for buccal tipping of premolars and canines in the maxilla, and also to compare these forces with the individually considered ideal force. Nineteen clinically experienced orthodontists were asked to bend and activate sectional arch wires on a plastic model with bands on the first molars. Round 0.016 Australian wires were used on one side and square 0.016 x 0.016 Blue Elgiloy wires on the other. The tests were repeated a second time 3-4 weeks later. The applied mean force magnitude was 48.4 cN (g) for premolars and 40.0 cN for canines, with a considerable intra-individual variation (range 25-75 cN, and 21-62 cN) using the round Australian wire. In general, the activation of the square wire resulted in a higher force, mean difference 29 per cent (11.4 cN) for canines and 23 per cent (11.1 cN) for premolars when compared with the round wire. On each side, the shorter wire for premolars was activated with a higher force when compared with the longer canine wire, mean 16 per cent (8.0 cN) for the square wire and 21 per cent (8. cN) for the round wire. Sex, age and clinical experience had no major influence on the applied force magnitude. On average, the orthodontists considered an ideal force for tipping of canines and premolars to be mean 62.5 cN (range 30-100 cN) and mean 56.1 cN (range 30-100 cN) respectively. They also reported that a strain gauge was not often used in everyday clinical practice. This study showed substantial differences between applied forces and considered ideal forces. It is suggested that regular checks of the force magnitude should be performed in situations where a certain force is considered important.

Adult↗

PubMed1996

[Biotransformation of the lipoxygenase inhibitor 2-hydroxy-5-methyl-laurophenone-oxime (FLM 5011)].

2-Hydroxy-5-methyl-laurophenone-oxime (FLM 5011, 1) is an inhibitor of the lipoxygenase with antiinflammatory and antiallergic actions. The studies on the biotransformation using in vivo investigations and in vitro test systems resulted in finding of at least eight metabolites. Four of these compounds have been detected and identified in urine and faeces after p.o. administration in male Wistar rats. By means of cultures of hepatocytes, lymphocytes and myeloma cells additional metabolites were found and the main pathways of metabolism could be suggested. Furthermore it was possible to confirm the sequence of the metabolic reactions. First of all, 1 is hydroxylated in the omega-position of the lauryl side chain by the cytochrome P-450 system. The further oxidation to the carboxylated compound is followed by the stepwise degradation of the side chain by beta-oxidation similarly to the pathways of fatty acid metabolism. Simultaneously the oxime group is converted to the keto group. The metabolites and 1 partly occur as sulfate or glucuronide conjugates. Additionally all compounds produced by beta-oxidation are conjugated with other partners, probably amino acids. By omega-oxidation, compounds with higher inhibitory potency on the lipoxygenase than the parent compound are formed. These results suggest that the activity of 1 is partly caused by the initial metabolites.

Animals↗

PubMed1995

Gramicidin S synthetase 1 (phenylalanine racemase), a prototype of amino acid racemases containing the cofactor 4'-phosphopantetheine.

The biosynthesis of the decapeptide antibiotic gramicidin S in Bacillus brevis ATCC 9999 is catalyzed by a multienzyme system consisting of two multifunctional proteins, gramicidin S synthetase 1 and 2, encoded by the grsA and grsB genes, respectively. Gramicidin S synthetase 1 (phenylalanine racemase, EC 5.1.1.11, GS1) racemizes phenylalanine in the thioester-bound stage. The amount of 4'-phosphopantetheine liberated from highly purified GS1 was determined microbiologically using Lacto-bacillus plantarum as the test organism. It matches exactly with the amount of L-[14C]phenylalanine covalently incorporated by GS1 as thioester. The reaction center of GS1 for L-phenylalanine thiolation and racemization was labeled with [3H]iodoacetic acid. After tryptic fragmentation of the 3H-carboxymethylated enzyme, the active site peptide for thioester binding and racemization of phenylalanine was isolated in pure form by multistep methodology and investigated by sequence, amino acid, and mass spectrometric analysis. A 4'-phosphopantetheine carrier was found to be attached to the active site serine of the consensus motif LGGDSI forming the thiolation site of phenylalanine. These specific properties establish GS1 as a prototype of amino acid racemases using 4'-phosphopantetheine as a cofactor and yield further evidence that multiple Pan carriers are involved in gramicidin S formation. Our results are strong evidence for the "multiple carrier model" as a new concept of nonribosomal peptide biosynthesis at protein templates as recently proposed [Stein, T., et al. (1994) FEBS Lett. 340, 39-44].

Amino Acid Isomerases↗

PubMed1995

A new class of photoactivatable and cleavable derivatives of neurotoxin II from Naja naja oxiana. Synthesis, characterisation, and application for affinity labelling of the nicotinic acetylcholine receptor from Torpedo californica.

A new series of photoactivatable and cleavable derivatives of neurotoxin II from the cobra Naja naja oxiana is investigated which can be used for mapping the surface topology of the nicotinic acetylcholine receptor from Torpedo electric tissue. The preparation and characterisation of five toxin derivatives, each with a radioactive 125I-azidosalicylamidoethyl-1,3'-dithiopropyl group in a defined position within the primary structure, are described. The photoinduced cross-linking reaction of the toxin derivatives with membrane-bound receptor is investigated. The photoactivatable group located at position K25 reacts almost exclusively with the delta subunit of the receptor, whereas the K15 derivative reacts with the alpha and beta subunits. The other derivatives did not react with the receptor to any significant extent. It is shown that, with respect to the receptor subunits, the cross-linking pattern depends on the length and chemical nature of the cross-linking group.

Affinity Labels↗

PubMed1995

Citric acid extracts a specific set of proteins from isolated cell nuclei.

The treatment of isolated cell nuclei with citric acid was described as a method for separating inner and outer nuclear membrane. Using cell nuclei from bovine cerebral cortex, we can show that citric acid does not cause a separation of the two nuclear membranes, but extracts a specific set of proteins from the nuclei. The extraction of proteins is not just an effect of damaging the nuclear membrane or destructing the cytoskeleton, but rather a specific effect of citric acid treatment. One of the extracted proteins, chosen as a marker for the putative outer nuclear membrane fraction, has an apparent molecular weight of 145 kDa and is located in the nucleoplasm as shown by immunofluorescence microscopy. By sequencing tryptic peptides it was identified as RNA helicase A, an abundant nuclear protein assumed to participate in the processing of mRNA.

Amino Acid Sequence↗

PubMed1995

Synthesis of nitrodiazirinyl derivatives of neurotoxin II from Naja naja oxiana and their interaction with the Torpedo californica nicotinic acetylcholine receptor.

Five singly modified nitrodiazirine derivatives of neurotoxin II (NT-II) from Naja naja oxiana were obtained after NT-II reaction with N-hydroxysuccinimide ester of (2-nitro-4-[3-(trifluoromethyl)-3H-diazirin-3yl]phenoxy)acet ic acid followed by chromatographic separation of the products. To localize the label positions, each derivative was first UV-irradiated and then subjected to reduction, carboxymethylation, and trypsinolysis. Tryptic digests were separated by reversed phase-HPLC, the labeled peptides being identified by mass spectrometry. The derivatives containing the photolabel at the position Lys 25, Lys 26, Lys 44, or Lys 46 were [125I]iodinated by the chloramine T procedure. Each iodinated derivative was found to form photoinduced cross-links with the membrane-bound nicotinic acetylcholine receptor (AChR) from Torpedo californica. The pattern of labeling the receptor's alpha, beta, gamma, or delta subunits was dependent on the photolabel position in the NT-II molecule and differed from that obtained earlier with an analogous series of p-azidobenzoyl derivatives of NT-II. The results obtained indicate that (i) different sides of the neurotoxin molecule are involved in the AChR binding, and (ii) fragments of the different AChR subunits are located close together at the neurotoxin-binding sites.

Affinity Labels↗

PubMed1995

The Thrombostat system. A useful method to test antiplatelet drugs and diets.

UNLABELLED: The use of platelet inhibitory drugs, like aspirin, has resulted in a significant reduction of thrombotic complications in primary and secondary prevention of heart attacks. To find more effective substances or better drug combinations, inhibition of primary hemostasis in vitro (Thrombostat system) was investigated, with different drugs and fish diet, using small samples (1 ml) of anticoagulated (Na- citrate 3.8%, 1/9) human blood. RESULTS: 1. In the presence of 1 mM aspirin, which had no effect on bleeding volume, only 0.6 nM iloprost were necessary to show a 50% inhibition, in contrast to 2.5 nM without aspirin. 2. At aspirin concentrations of 1 mM, 50% inhibition of primary hemostasis could be achieved with 20 microM SIN-1, or with 7 microM SIN-1 together with iloprost (500 pM). The same effect was seen only with very high doses of SIN-1 (1000 microM) alone. 3. For 50% inhibition of primary hemostasis in vitro, RGDS concentrations were reduced from 250 microM to 160 microM when blood was pretreated with 1 mM aspirin and to 75 microM when 500 pM iloprost were added additionally. 4. Japanese fishermen (eating 270 g fish/day) demonstrated significantly longer in-vivo bleeding times and in-vitro bleeding volumes (6.49 min/224 microliters), respectively, as compared to Japanese farmers (90 g fish/day, 4.85 min/137 microliters). 5. In Japanese subjects in-vivo bleeding times correlated with in-vitro bleeding volumes (0.69). The Thrombostat system proved to be a sensitive method to detect synergistic effects of various antiplatelet drugs in vitro and of a platelet inhibitory diet ex vivo.

Bleeding Time↗

PubMed1995

Subclinical thought disorder in first-degree relatives of schizophrenic patients. Results from a matched-pairs study with the Thought Disorder Index.

To assess the frequency and quality of formal thought disorder in schizophrenic patients and their first-degree relatives, a consecutive series of 36 unmedicated patients, 20 siblings of these patients and 37 normal control subjects were examined with Holzman's Thought Disorder Index (TDI). As a proof of the internal validity of this tool, the patients demonstrated significantly more thought disorder than the controls as measured by the TDI total score and various subscores, which proved the internal validity of this tool in a German-speaking sample. In addition, in a pairwise comparison with controls who were individually matched by age and sex the patients' siblings had a significantly higher total TDI score and nonsignificant elevations on several subscores (combinatory and flippant). As to the level of severity of the thought disorder, the siblings mostly took an intermediate position between patients and controls. In conjunction with previous reports from other authors, our findings lend further support to the notion of subclinical thought disorder as an indicator of familial vulnerability to schizophrenia.

Adult↗

PubMed1995

Personality patterns in subjects at risk for affective disorders.

The main conclusions of this study on the familial links between personality patterns and affective disorders are: (1) The personality features with the greatest degree of symptomatic overlap with unipolar depression were more common among the first-degree relatives of probands with this diagnosis: thus dysthymic temperament and neuroticism are enhanced in this group of relatives compared to controls. Likewise personality features with a high degree of symptomatic overlap with bipolar affective disorder were more common among the first-degree relatives of probands with this diagnosis. Thus levels of dysthymic and cyclothymic temperament were elevated in this group of relatives compared to controls, whereas a familial link between neuroticism and bipolar disorder was not observed. (2) In addition, personality traits with only limited similarity with the syndromes of depression and mania were also found to be linked with affective disorders (obsessive-compulsive PD to bipolar disorders, rigidity to both subtypes of affective disorders). These associations have different implications regarding the association between personality/temperament conditions and disorders depending on the degree of overlap between symptoms and the personality trait. Those with substantial overlap may indicate the presence of a unitary disease process in which the enhancement of subthreshold affective traits may result in aggravation of behavior characteristics fulfilling the criteria of fullblown episodes of the disease. This relationship was particularly stressed by Kraepelin, Kretschmer, Clayton et al. and Akiskal. In this view personality and temperament patterns represent minor variants of the associate acute disorders. On the other hand, the relationship between dissimilar conditions may indicate the presence of a risk of underlying vulnerability factors which led to affective disorder only in the presence of additional risk factors. An example of the latter relationship in the present study is the aggregation of obsessive-compulsive and anancastic traits in families with affective disorders. Tellenbach focussed on this particular constellation. Epidemiological and family studies including these personality traits are too rare to fully appreciate the relevance of this particular relationship. Future prospective studies and family genetic studies which investigate the relationship between temperament, personality traits and disorders and affective syndromes are clearly indicated by the results presented herein.

Humans↗

PubMed1994

All potential glycosylation sites of the nicotinic acetylcholine receptor delta subunit from Torpedo californica are utilized.

All possible N-glycosylation sites of the delta subunit of the nicotinic acetylcholine receptor from Torpedo californica electric tissue are utilized. By a combination of microsequencing and mass spectrometry, it was shown that a high-mannose-type oligosaccharide is bound at Asn143 of the delta subunit. The oligosaccharides at positions Asn70 and Asn208 of the delta subunit are probably of the complex type. The utilized glycosylation sites pose restrictions on possible transmembrane folding models of the subunit.

Amino Acid Sequence↗

PubMed1994

Detection of 4'-phosphopantetheine at the thioester binding site for L-valine of gramicidinS synthetase 2.

Biosynthesis of gramicidinS in Bacillus brevis is catalysed by a multienzyme system consisting of two multifunctional proteins, gramicidinS synthetase 1 and 2 codified by the grsA and grsB genes, respectively. GramicidinS synthetase 2 shows a modular architecture of four amino acid-activating domains each containing a thioester binding motif LGG H/D S L/I highly conserved in its C-terminal region, as demonstrated by sequence analysis of the grsB gene [W. Schlumbohm et al. (1991) J. Biol. Chem. 266, 23135-23141]. This multienzyme was specifically labeled at the thioester binding site of L-valine with [3H]N-ethylmaleimide using a substrate protection technique. After enzymatic digestion a labeled active site peptide was isolated in pure form by multistep methodology. This fragment was identified by gas-phase sequencing as the active site peptide of the thiotemplate site for L-Val by comparison with the grsB gene sequence. By mass spectrometry in combination with amino acid analysis it was demonstrated that a 4'-phosphopantetheine carrier was attached to the active serine in this motif. Our results give evidence that multiple peripheral 4'-phosphopantetheine carriers are involved in the formation of gramicidinS in contrast to a central carrier arm as assumed in the original version of the thiotemplate mechanism. A 'Multiple Carrier Model' of nonribosomal peptide biosynthesis is proposed.

Amino Acid Isomerases↗

PubMed1994

Degeneration model of the porcine lumbar motion segment: effects of various intradiscal procedures.

In an experimental study with 18 minipigs, we have tried to establish a model for the standardized evaluation of mechanical, histological and radiological phenomena of degenerative and reparative processes within the lumbar motion segment. Comparing different operative techniques revealed that the intradiscal application of hyaluronic acid into the nuclear defect is likely to enhance the regeneration process. Using the measurement of disc compliance, a semiautomatic picture analyzer and a new semiquantitative disc score could make future studies more comparable. From this basis, the intradiscal application of hyaluronic acid deserves further evaluation.

Animals↗

PubMed1994

Attentional abilities and measures of schizotypy: their variation and covariation in schizophrenic patients, their siblings, and normal control subjects.

Thirty-five schizophrenic patients in the early stages of illness, 26 of their healthy siblings, and 35 normal control subjects performed the Continuous Performance Test, Identical Pairs version (CPT-IP). Both schizophrenic patients and their siblings were significantly impaired in their attentional performance compared with normal subjects. These results support impaired attention as a vulnerability marker of schizophrenia and indicate that at-risk siblings of schizophrenic patients display attentional deficits comparable to those found for the offspring of schizophrenic parents. By contrast, a decline in performance with the onset of a distraction condition (auditory and visual stimuli) was seen only in schizophrenic patients; siblings and normal control subjects did not differ from one another in response to experimental distraction. Therefore, it was concluded that differential distractibility is likely to be a state marker of schizophrenia. In clinical assessments, healthy siblings rated themselves as experiencing significantly more physical anhedonia than did normal control subjects, but the siblings did not differ from normal control subjects in self-rated perceptual aberrations. Contrary to expectation, performance on the CPT-IP did not correlate significantly with either anhedonia or perceptual aberration in high-risk siblings. These results suggest that psychometrically measured "psychosis proneness" and neuropsychologically detected deficits may tap two nonoverlapping sources of vulnerability to schizophrenia.

Attention↗

PubMed1994

Reaction time paradigms in subjects at risk for schizophrenia.

Deviant response patterns in experimental reaction time paradigms in schizophrenic probands are well documented. Although simple reaction times are strongly influenced by the current psychopathological status of the proband (e.g. florid psychotic patients versus remitted patients) these influences are less clear for measures obtained from more complex reaction time paradigms. These include the crossover paradigm (reaction time to stimuli presented after constant preparatory intervals in comparison to reaction time to stimuli presented after irregular preparatory intervals) and the modality shift paradigm (reaction time to a stimulus (light or tone) when the modality of the stimulus on the preceding trial was the same compared to when it was different). It is not clear if these peculiarities of response patterns occur as a consequence of the disease or if they represent vulnerability markers for schizophrenia. Both crossover reaction time and modality shift reaction time paradigms were applied to 56 drug free schizophrenics, 45 healthy siblings of these patients and 68 healthy controls. The results indicate that retarded reaction times and the occurrence of the crossover effect as well as of the modality shift effect distinguish schizophrenics and controls. Healthy siblings of schizophrenics differed from healthy controls with regard to the crossover effect but not with regard to the modality shift effect. Therefore only the crossover effect represents a vulnerability marker for schizophrenia. Correlations between the modality shift and the crossover effect revealed strong correlations in the schizophrenic group only.

Adolescent↗

PubMed1994

Personality variations in healthy relatives of schizophrenics.

A familial relationship between schizophrenia and schizotypal personality disorder is widely acknowledged; the familial relationship between schizophrenia and the broad continuum of schizoid personality variation is less clear. In a comprehensive family study healthy relatives of schizophrenics were compared by self rated personality features with relatives of unipolar depressed patients and with relatives of controls. The dimension of schizoidia was not able to distinguish the groups of relatives. However, relatives of schizophrenics (in particular male relatives) scored higher on 'normalized' personality dimensions such as 'rigidity' and 'neuroticism'. Healthy relatives of probands with unipolar depression revealed a similar deviant pattern.

Adult↗

PubMed1994

[Structure of peptide fragments of the complex (Lys(Abz)26) neurotoxin II from Naja naja oxiana cross-linked with the nicotinic acetylcholine receptor from Torpedo californica].

After irradiating the acetylcholine receptor complex with the title neurotoxin derivative, the labeled delta-subunit was separated by preparative SDS-PAGE, reduced-carboxymethylated and cleaved with LysC endoproteinase. One of the radioactive peptides isolated by HPLC was further purified by electrophoresis in a tricin gel. Edman degradation of the radioactive fractions yielded the sequence of the delta-subunit fragment starting from Phe148. The AspN-cleavage of the radioactive peptide from the LysC digest gave on HPLC a radioactive peak which eluted similarly to peptide 33-44 generated by LysC/AspN-cleavage of 125I-neurotoxin II. In model experiments, irradiation of the photoactivable derivative was found to produce a heterogeneous mixture of reaction products. Unusually low initial and repetitive yields were observed for neurotoxin II and its fragments containing the photolabeled and radiolabeled residues. These results might explain why the neurotoxin sequence was not detected on Edman degradation of the cross-linked products available at a low picomole level.

Amino Acid Sequence↗

PubMed1994

[Pharmacokinetics and biotransformation of 3-cyan-2-morpholino-5-(pyrid-4-yl)pyridine (AWD 122-14) in the rat].

A sensitive isocratic HPLC method for the analysis of AWD 122-14 (1) in plasma and urine was developed. The extraction was processed on-line on a short column. The pharmacokinetics of 1 was studied in rats. The plasma concentration-time course was described by an 1-compartment-model. The pharmacokinetic parameters were estimated. The absorption and elimination of 1 are relatively fast. A single oral dose (D = 1 x 10(-5) mol/kg) was rapidly absorbed (absorption rate constant: kI = 6.85 h-1). The elimination rate constant is kE = 1.59 h-1. The absolute bioavailability of a single oral dose equals 11%. In rats 1 is mainly metabolized. Less than 5% of the dose is excreted in the urine as unchanged drug. The chemical structures of 10 of the 12 isolated metabolites were determined using high-resolution mass spectrometry.

Administration, Oral↗