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H Kuhn

Publications and source records attributed to H Kuhn.

At least 37 records · Page 2Linked to original sources
PubMed2001

Soybean lipoxygenase-1 oxygenates synthetic polyenoic fatty acids with an altered positional specificity: evidence for inverse substrate alignment.

The positional specificity is the decisive enzyme property for classification of lipoxygenases and for the currently used lipoxygenase nomenclature. It has been reported before that soybean lipoxygenase-1, which oxygenates polyenoic fatty acids at alkaline pH to the corresponding n - 6 hydroperoxy derivative, exhibits a different positional specificity when either the reaction conditions or the substrate structure is altered. To investigate the impact of structural substrate modifications on the positional specificity of this enzyme and to force an inverse substrate binding, we synthesized arachidonic acid analogues modified at the omega-terminus. Care was taken that the double bond system remained unchanged so that hydrogen abstraction from all three bisallylic methylenes was theoretically possible. We found that omega-modification of arachidonic acid leads to an impaired substrate affinity and a reduced reaction rate, but we did not detect any 5-lipoxygenation products, suggesting that structural modification of the omega-end may not be sufficient to force an inverse substrate orientation. However, when both ends of the fatty acid chain (omega-terminus and free carboxylate) were modified simultaneously, a considerable share of 5-lipoxygenation products was detected. These results indicate that introduction of polar or bulky groups at the methyl terminus of polyenoic fatty acids was not sufficient to force an inverse substrate orientation. However, simultaneous introduction of an omega-OH group and methylation of the carboxylate led to formation of significant 5-lipoxygenation products, suggesting an inverse head to tail substrate orientation.

Arachidonic Acid↗
PubMed2001

An artificial primosome: design, function, and applications.

Double-stranded (ds) DNA is capable of the sequence-specific accommodation of an additional oligodeoxyribonucleotide strand by the peptide nucleic acid(PNA)-assisted formation of a so-called PD-loop. We demonstrate here that the PD-loop may function as an artificial primosome within linear, nonsupercoiled DNA duplexes. DNA polymerase with its strand displacement activity uses this construct to initiate the primer extension reaction at a designated dsDNA site. The primer is extended by several hundred nucleotides. The efficiency of dsDNA priming by the artificial primosome assembly is comparable to the single-stranded DNA priming used in various assays. The ability of the PD-loop structure to perform like an artificial primosome on linear dsDNA may find applications in biochemistry, molecular biology, and molecular biotechnology, as well as for DNA diagnostics. In particular, multiple labels can be incorporated into a chosen dsDNA site resulting in ultrasensitive direct quantification of specific sequences. Furthermore, nondenaturing dsDNA sequencing proceeds from the PD-loop. This approach opens the way to direct isothermal reading of the DNA sequence against a background of unrelated DNA, thereby eliminating the need for purification of the target DNA.

Base Sequence↗
PubMed2001

Structural basis for lipoxygenase specificity. Conversion of the human leukocyte 5-lipoxygenase to a 15-lipoxygenating enzyme species by site-directed mutagenesis.

Mammalian lipoxygenases constitute a heterogeneous family of lipid-peroxidizing enzymes, and the various isoforms are categorized with respect to their positional specificity of arachidonic acid oxygenation into 5-, 8-, 12-, and 15-lipoxygenases. Structural modeling suggested that the substrate binding pocket of the human 5-lipoxygenase is 20% bigger than that of the reticulocyte-type 15-lipoxygenase; thus, reduction of the active-site volume was suggested to convert a 5-lipoxygenase to a 15-lipoxygenating enzyme species. To test this "space-based" hypothesis of the positional specificity, the volume of the 5-lipoxygenase substrate binding pocket was reduced by introducing space-filling amino acids at critical positions, which have previously been identified as sequence determinants for the positional specificity of other lipoxygenase isoforms. We found that single point mutants of the recombinant human 5-lipoxygenase exhibited a similar specificity as the wild-type enzyme but double, triple, and quadruple mutations led to a gradual alteration of the positional specificity from 5S- via 8S- toward 15S-lipoxygenation. The quadruple mutant F359W/A424I/N425M/A603I exhibited a major 15S-lipoxygenase activity (85-95%), with (8S,5Z,9E,11Z,14Z)-8-hydroperoxyeicosa-5,9 ,11, 14-tetraenoic acid being a minor side product. These data indicate the principle possibility of interconverting 5- and 15-lipoxygenases by site-directed mutagenesis and appear to support the space-based hypothesis of positional specificity.

Amino Acid Sequence↗
PubMed2001

Peptide nucleic acid-assisted topological labeling of duplex dna.

Peptide nucleic acids (PNAs) are a family of synthetic polyamide mimics of nucleic acids that offer a variety of applications. Pyrimidine bis-PNAs can be used for rational design of novel interlocked DNA nanostructures, earring labels, representing locked pseudorotaxanes or locked catenanes. These structures are created through DNA ligase-mediated catenation of duplex DNA with a circularized oligonucleotide tag at a designated DNA site. The assembly is performed via formation of the PD-loop consisting of a pair of bis-PNA openers and the probe oligonucleotide. The openers locally expose one of the two strands of duplex DNA for hybridizing the probe, whose termini are complementary to the displaced DNA strand. After hybridization, they are in juxtaposition and can subsequently be linked by DNA ligase. As a result, a true topological link forms at a precise position on the DNA double helix yielding locked, earring-like label. DNA topological labeling can be done both in solution and, for longer templates, within the agarose gel plug. Accordingly, highly localized DNA detection with rolling circle amplification of hybridization signal and effective micromanipulations with DNA duplexes become possible through precise spatial positioning of various ligands on the DNA scaffold.

DNA↗
PubMed2001

Gene transfer into solid tumours--is a special application device beneficial?

The replacement of inactivated tumour suppressor genes is a promising approach in cancer therapy. The aim of this study was to evaluate the influence of technical determinants on the efficiency of adenoviral-mediated gene transfer into solid tumours. Therefore, we compared the efficacy of two different injection needle types, a conventional needle and a modified needle characterised by perforations at the side of the shaft in vivo. The total amount of adenoviral vector DNA and the activity of the transferred reporter gene were quantitatively analysed by polymerase chain reaction (PCR) specific for the E4 region of the Ad vector genome and the beta-galactosidase assay, respectively. The levels of adenoviral DNA, as well as the total beta-galactosidase activity, varied widely, but were not significantly different for the two groups. These results suggest, that the efficiency of intratumoral gene transfer cannot be improved by the design of the application device.

Adenoviridae↗
PubMed2001

Alterations of lipoxygenase specificity by targeted substrate modification and site-directed mutagenesis.

BACKGROUND: Mammalian lipoxygenases (LOXs) are categorised with respect to their positional specificity of arachidonic acid oxygenation. However, the mechanistic basis for this classification is not well understood. To gain a deeper insight into the structural basis of LOX specificity we determined the reaction characteristics of wild-type and mutant mammalian LOX isoforms with native and synthetic fatty acids substrates. RESULTS: The rabbit 15-LOX is capable of catalysing major 12-lipoxygenation when the volume of the substrate-binding pocket is enlarged. These alterations in the positional specificity can be reversed when bulky residues are introduced at the omega end of the substrate. Simultaneous derivatisation of both ends of fatty acids forces a 15-LOX-catalysed 5-lipoxygenation and this reaction involves an inverse head-to-tail substrate orientation. In contrast, for arachidonic acid 5-lipoxygenation by the human 5-LOX the substrate fatty acid may not be inversely aligned. The positional specificity of this isoenzyme may be related to its voluminous substrate-binding pocket. Site-directed mutagenesis, which leads to a reduction of active site volume, converts the 5-LOX to a 15-lipoxygenating enzyme species. CONCLUSIONS: The positional specificity of LOXs is not an invariant enzyme property but depends on the substrate structure and the volume of the substrate-binding pocket. 15-LOX-catalysed 5-lipoxygenation involves an inverse substrate alignment but this may not be the case for 5-LOXs. Thus, both theories for the mechanistic basis of 5-lipoxygenation (straight and inverse substrate orientation) appear to be correct for different LOX isoforms.

Animals↗
PubMed2001

Factors influencing breath condensate volume.

Analysis of breath condensate (BC) has received interest recently due to the need for easy and repetitive monitoring or airway and pulmonary disease. While many authors have used custom built systems, commercial systems are now available and will probably be used more widely. Early studies of markers and mediators in BC have reported concentrations following varying periods of sampling time. However, factors that influence the generation of BC have not been analysed and it is unclear whether breathing rate, tidal volume, lung function, body weight, height or age influence the amount of BC collected. We therefore studied the influence of these factors on breath condensate volume and breath condensate urea and protein concentrations in 22 healthy volunteers and 23 COPD patients. A strong correlation of total respired volume and breath condensate volume was observed for both groups (volunteers: r=0.952, p < 0.0001, COPD: r=0.883, p < 0.001) while no significant correlation existed for breath condensate volume and TLC, RV, Vc, FEV1, R(tot), height or body weight, As long as ventilation remained fairly constant, breath condensate volume increased linearly with time. The fraction of breath condensate extracted from total vapour contained in the exhalate was estimated by measuring relative atmospheric humidity before and after the collecting tube. The amount calculated by the change in temperature and saturation corresponded closely to the amount actually collected. We conclude from these results that breath condensate volume is primarily dependent on V(E), and does not seem to depend on lung function parameters. For standardisation it is suggested to report breath condensate measurements per volume respired. Both, urea and protein are present in measurable quantities in breath condensate and protein as well as BCV may be helpful denominators for comparison with e.g. cytokines in lung disease.

Asthma↗
PubMed2001

p53 in surgically treated and pathologically staged cervical cancer: correlation with local tumor progression, but not with lymphatic spread.

There is only limited information about the prognostic value of p53 immunostaining in cervical cancer. The purpose of this study was to assess the clinical significance of p53 and prognosis in operatively treated cervical carcinoma. A hundred and fourteen primary surgically treated cervical carcinomas (CX) were obtained from the so called Wertheim Archive in the Department of Obstetrics and Gynecology at the University of Leipzig. These included 105 squamous cell cancer (SCC) and nine adenocarcinomas (AC). No cases received neoadjuvant therapy. For immunohistochemical analysis, the cases were tested with the monoclonal antibody DO-7 (DAKO Diagnostics, Denmark). Two hundred tumor cell nuclei were counted for positive nuclear immunostaining, regardless of staining intensity. Cases were stated as positive when a minimum of 10% nuclei showed positive staining. Fresh frozen tissue was available from 21 CX for p53-mutation analysis (exons 4-9) using PCR-based amplification and SSCP-analysis. Of the squamous cell cancers (SCC), 63.8% showed positive nuclear p53-immunostaining; adenocarcinomas (AC) were completely negative (P = 0.0000, Chi2-test). Stage-by-stage analysis revealed no differences in p53-expression. However, combining pT1b- and pT2-cases, the difference in positive immunostaining reached statistical significance (44.4% vs. 71.7%; P = 0.007). There were no differences in p53-reactivity regarding the presence of pelvic lymph node metastases, tumor grading, relapse-free survival and tumor recurrence. In addition, only 5% of CX with positive p53-immunostaining showed genomic alterations in mutational analysis. p53-immunoreactivity showed significant correlation with local tumor progression but not with lymphatic spread, lacking any prognostic impact in surgically treated cervical cancer. There is no correlation of p53-immunostaining with the occurrence of p53-gene mutations in cervical cancer.

Adenocarcinoma↗
PubMed2001

High-purity preparation of a large DNA dumbbell.

We report on the efficient biochemical synthesis of a large DNA dumbbell starting from a pair of short DNA hairpins with long single-stranded tails of arbitrary sequence. The DNA dumbbell is obtained by enzymatic ligation yielding a 94-bp duplex stem closed at both termini by single-stranded loops of 5 nt. Following ligation, all unligated precursors and monoligated by-products were multiply biotinylated via nick-translation or primer-extension or both. Thus, they could readily be removed from the DNA dumbbell preparation by a mild biomagnetic separation procedure. The closed conformation of the purified DNA dumbbell was verified by its altered gel mobility as compared with unligated or monoligated samples and by an exonuclease assay. Considering the promising therapeutic potential of DNA dumbbells, the developed biosynthetic approach could be used for high-purity preparation of longer, covalently closed DNA decoys.

Base Sequence↗
PubMed2001

PNA beacons for duplex DNA.

We report here on the hybridization of peptide nucleic acid (PNA)-based molecular beacons (MB) directly to duplex DNA sites locally exposed by PNA openers. Two stemless PNA beacons were tested, both featuring the same recognition sequence and fluorophore-quencher pair (Fluorescein and DABCYL, respectively) but differing in arrangement of these groups and net electrostatic charge. It was found that one PNA beacon rapidly hybridized, with the aid of openers, to its complementary target within duplex DNA at ambient conditions via formation of a PD-like loop. In contrast, the other PNA beacon bound more slowly to preopened duplex DNA target and only at elevated temperatures, although it readily hybridized to single-stranded (ss) DNA target. Besides a higher selectivity of hybridization provided by site-specific PNA openers, we expect this approach to be very useful in those MB applications when denaturation of the duplex DNA analytes is unfavorable or undesirable. Furthermore, we show that PNA beacons are advantageous over DNA beacons for analyzing unpurified/nondeproteinized DNA samples. This feature of PNA beacons and our innovative hybridization strategy may find applications in emerging fluorescent DNA diagnostics.

Base Pairing↗
PubMed2000

Alterations in leukotriene synthase activity of the human 5-lipoxygenase by site-directed mutagenesis affecting its positional specificity.

The positional specificity of arachidonic acid oxygenation is currently the decisive parameter for classification of lipoxygenases. Although the mechanistic basis of lipoxygenase specificity is not completely understood, sequence determinants for the positional specificity have been identified for various isoenzymes. In this study we altered the positional specificity of the human 5-lipoxygenase by multiple site-directed mutagenesis and assayed the leukotriene A(4) synthase activity of the mutant enzyme species with (5S,6E,8Z,11Z,14Z)-5-hydroperoxy-6,8,11,14-eicos atetraenoic acid (5S-HpETE) as substrate. The wild-type 5-lipoxygenase converts 5S-HpETE almost exclusively to leukotriene A(4) as indicated by the dominant formation of leukotriene A(4) hydrolysis products. Since leukotriene synthesis involves a hydrogen abstraction from C(10), it was anticipated that the 15-lipoxygenating quadruple mutant F359W + A424I + N425M + A603I might not exhibit a major leukotriene A(4) synthase activity. Surprisingly, we found that this quadruple mutant exhibited a similar leukotriene synthase activity as the wild-type enzyme in addition to its double oxygenation activity. The leukotriene synthase activity of the 8-lipoxygenating double mutant F359W + A424I was almost twice as high, and similar amounts of leukotriene A(4) hydrolysis products and double oxygenation derivatives were detected with this enzyme species. These data indicate that site-directed mutagenesis of the human 5-lipoxygenase that leads to alterations in the positional specificity favoring arachidonic acid 15-lipoxygenation does not suppress the leukotriene synthase activity of the enzyme. The residual 8-lipoxygease activity of the mutant enzyme and its augmented rate of 5-HpETE conversion may be discussed as major reasons for this unexpected result.

Animals↗
PubMed2000

Bacterial and nonbacterial expression of wild-type and mutant human phospholipid hydroperoxide glutathione peroxidase and purification of the mutant enzyme in the milligram scale.

15-Lipoxygenases and phospholipid hydroperoxide glutathione peroxidases are counterparts in the metabolism of hydroperoxy lipids and a balanced regulation of both enzymes is essential for normal cell function. Glutathione peroxidases contain selenocysteine as catalytically active amino acid and this selenocysteine is encoded by a TGA stop codon. Detailed protein chemical investigations on phospholipid hydroperoxide glutathione peroxidases and crystal trials have been hampered in the past by limited protein supply. There is no efficient natural source for large-scale enzyme preparation and overexpression of the functional protein in recombinant systems has not been reported so far. To avoid problems with recognition of the selenocysteine stop codon we mutated the selenocysteine to a cysteine and expressed the Sec46Cys mutant in milligram amounts in the baculovirus/insect cell system and as His-tag fusion protein in Escherichia coli. The recombinant enzyme species were purified by conventional fast protein liquid chromatography (nonfusion protein) or by affinity chromatography on a nickel matrix (His-tag protein). Surprisingly, we found that both protein variants were functional although their specific activities were reduced when compared with the wild-type enzyme. Basic protein chemical and enzymatic properties of the purified enzyme species were determined and monoclonal antibodies which recognize the native phospholipid hydroperoxide glutathione peroxidases were raised using our enzyme preparations as antigen. The described strategies for overexpression of mutant phospholipid hydroperoxide glutathione peroxidase species and their purification from recombinant sources provide sufficient amounts of enzyme for future protein chemical investigations and detailed crystal trials.

Animals↗
PubMed2000

Transcoronary ablation of septal hypertrophy (TASH): a new treatment option for hypertrophic obstructive cardiomyopathy.

In 1991, our group started to develop a catheter interventional therapy for hypertrophic obstructive cardiomyopathy (HOCM). The new concept was proposed in 1994. It is based on the conventional PTCA technique with the aim of inducing an artificial myocardial infarction by instillation of 96% ethanol into the most proximally situated septal branch of the left anterior descending coronary artery. This leads to a subaortic contraction disorder with subsequent decrease of the intraventricular pressure gradient, shrinkage of the hypertrophied septal bulge and widening of the outflow tract ("therapeutic remodeling"). The subaortic defect is small and well demarcated as assessed by left ventricular angiography, transesophageal echocardiography and 18 F-glucose positron emission tomography. The term transcoronary ablation of septum hypertrophy (TASH) was suggested. Our patient cohort that now comprises 215 therapeutic procedures in 187 patients underwent a large variety of prospective studies (maximum follow-up 4.5 years) including invasive controls at regular intervals, investigation of hemodynamics at rest and at exercise, transesophageal and transthoracic echocardiography, Doppler echocardiography during bicycle exercise, electrophysiologic testing, Holter monitoring and measurement of myocardial metabolism and perfusion, assessment of microembolic events by transcranial Doppler sonography and histological examinations. This article gives an overview and reports our increasing experience in applying TASH. The following post-TASH findings were obtained: significant hemodynamic and clinical improvement at rest and at exercise, decrease of septum thickness, increase of outflow tract area and decrease of induced ventricular tachycardia. There were well-demarcated, histologically atypical subaortic myocardial defects, no microembolic events, abnormal early peak of infarct related enzymes, and no change of baroreflex sensitivity. Pre-/post-TASH evaluations of the patients should be based in particular on clinical symptoms correlated to the intraventricular gradient measured by bicycle exercise Doppler echocardiography and to outflow tract area as assessed by transesophageal echocardiography. Since 1994, as a roughly estimate, worldwide 1000 patients in 20 countries have been treated. According to published articles, abstract presentations and workshops, TASH consistently leads to a pronounced clinical and hemodynamic benefit for patients with HOCM. TASH has become an established technique. At least in centers with a high level of expertise, it is no longer experimental but a routinely performed alternative to surgical treatment for HOCM, i.e., the previous gold standard of therapy. Of course, patient outcome needs further careful clinical and prognostic evaluation. With respect to complications, TASH appears to be superior to surgery (transaortic septal myectomy) for HOCM. Like surgical treatment, TASH is currently indicated in critically ill patients with typical HOCM (subaortic form), who exhibit with drug refractory symptoms, including patients, who preferred DDD pacemaker therapy as a first therapeutic step but in whom this produced no subsequent clinical benefit.

Adult↗
PubMed2000

[Transcoronary ablation of septal hypertrophy (TASH): a 5-year experience].

From 1991 to 1993, our group carried out extensive studies in the development of a new catheter-interventional concept of treatment for HOCM and suggested in 1994 the injection of absolute ethanol into the first major septal branch using the common PTCA technique. This approach was first used therapeutically by Sigwart in London and by our group in Bielefeld. Based on rhythmologic studies of other authors we suggested the designation Transcoronary Ablation of Septal Hypertrophy (TASH). Since 1995, an estimated 1000 patients (about 600 from Germany) in some 20 countries have been treated by this method. In 1997 a national multicenter TASH-registry was created by the German Cardiac Society. During the last five years extensive studies have been performed by different groups. Significant clinical and hemodynamic improvements were consistently found. This was true for both young and old patients, for patients with resting gradient and with provocable gradient. Clinical improvement (rate of improved patients about 90%) was seen under conditions of rest and stress, as well as during long-term follow-up. Specifically, there was a significant decrease of functional class, a reduced rate of syncope, and a reduced degree of mitral insufficiency. Exercise capacity, oxygen uptake and the cardiac index increased, intraventricular gradient, the end diastolic pressure of the left ventricle and the left atrial size decreased. Also there was a decrease in the septal thickness with increase in the cross sectional area of the outflow tract and with local contraction disorder. PET examination showed an ablation induced local myocardial defect. There was also evidence for improved diastolic function. Electrophysiological and Holter monitor studies indicated no increased arrhythmogenicity. The prognosis seems to be improved. Histologically, an atypical infarct pattern was found, i.e., the common round cell infiltration and the fibrotic process were surrounding but not penetrating the alcohol induced myocardial center of necrosis. The in-hospital mortality rate amounts to about 1.8%. The rate of permanent total av-block after TASH is less than 10%. In 15% a second TASH is necessary. In summary, during the last five years TASH has become a promising new catheter interventional therapy for HOCM, which compares favorably with surgical treatment. In experienced centers it has become a routinely performed procedure. The indication should be limited to severely symptomatic patients with typical (subaortic) HOCM. Further careful clinical examinations after TASH are required. All patients should be enrolled into a registry. TASH also constitutes a new area for comprehensive clinical research.

Angioplasty, Balloon, Coronary↗
PubMed2000

Structural basis for the positional specificity of lipoxygenases.

The positional specificity of arachidonic acid oxygenation is currently the decisive parameter for classification of mammalian lipoxygenases but, unfortunately, the structural reasons for lipoxygenase specificity are not well understood. Although there are no direct structural data on lipoxygenase/substrate interaction, experiments with modified fatty acid substrates and mutagenesis studies suggest that for 12- and 15-lipoxygenases, arachidonic acid slides into the substrate-binding pocket with its methyl end ahead. For arachidonate 5- and/or 8-lipoxygenation two alternative models for the enzyme/substrate interaction have been developed: 1) The orientation-determined model and 2) the space-determined model. This review explores the experimental data available on the mechanistic reasons for lipoxygenase specificity and concludes that each of the above-mentioned hypotheses may be valid for arachidonate 5-lipoxygenation under certain circumstances.

Animals↗
PubMed2000

Determinants of umbilical cord arterial 8-iso-prostaglandin F2alpha concentrations.

OBJECTIVE: To determine the concentration of 8-isoPGF(2alpha) in cord blood as a measure of oxidative stress during labour, and to compare them with other established parameters of in vivo lipid peroxidation and with the acid-base status of the newborn. METHOD: Umbilical cord arterial and venous blood samples were collected from 81 singleton term deliveries for determination of 8-isoPGF(2alpha), malondialdehyde and organic hydroperoxides. In addition, metabolites derived from the oxidative metabolism of purines during hypoxia-reoxygenation and routine cord blood of oxygen saturation, pH, pO2, pCO2, HCO3 and base excess were measured. RESULTS: Arterial concentrations of 8-isoPGF(2alpha) were significantly higher in cases with fetal distress, tight nuchal cord (P < 0.001), the umbilical coiling index, and male sex (P < 0.05) (R2 = 0.48). No correlation was found with any parameter of acid-base status. In arterial and venous blood the concentrations of organic hydroperoxides and hypoxanthine significantly correlated with the fetal nuchal cord (P < 0.001) (R2 = 0.26 and 0.16, respectively). CONCLUSION: Our findings indicate that 8-isoPGF2(alpha) in cord arterial blood is a suitable parameter to quantify a possible oxidative stress in the fetus during labour. Measurements of the F2-isoprostane concentrations in cord blood at labour provide a clinically useful method to assess the perinatal outcome.

Acid-Base Imbalance↗