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Biomedical subjects

H Kuhn

Publications and source records attributed to H Kuhn.

At least 55 records · Page 3Linked to original sources
PubMed1999

The diversity of the lipoxygenase family. Many sequence data but little information on biological significance.

Lipoxygenases form a family of lipid peroxidising enzymes, which oxygenate free and esterified polyenoic fatty acids to the corresponding hydroperoxy derivatives. They are widely distributed in both the plant and animal kingdoms. During the last couple of years more and more lipoxygenase isoforms have been discovered but for most of them the biological significance remains unclear. This review attempts to classify the currently known mammalian lipoxygenase isoforms and critically reviews the concepts for their biological importance.

Animals↗
PubMed1999

An experimental study of mechanism and specificity of peptide nucleic acid (PNA) binding to duplex DNA.

We investigated the mechanism and kinetic specificity of binding of peptide nucleic acid clamps (bis-PNAs) to double-stranded DNA (dsDNA). Kinetic specificity is defined as a ratio of initial rates of PNA binding to matched and mismatched targets on dsDNA. Bis-PNAs consist of two homopyrimidine PNA oligomers connected by a flexible linker. While complexing with dsDNA, they are known to form P-loops, which consist of a [PNA]2-DNA triplex and the displaced DNA strand. We report here a very strong pH-dependence, within the neutral pH range, of binding rates and kinetic specificity for a bis-PNA consisting of only C and T bases. The specificity of binding reaches a very sharp and high maximum at pH 6.9. In contrast, if all the cytosine bases in one of the two PNA oligomers within the bis-PNA are replaced by pseudoisocytosine bases (J bases), which do not require protonation to form triplexes, a weak dependence on pH of the rates and specificity of the P-loop formation is observed. A theoretical analysis of the data suggests that for (C+T)-containing bis-PNA the first, intermediate step of PNA binding to dsDNA occurs via Hoogsteen pairing between the duplex target and one oligomer of bis-PNA. After that, the strand invasion occurs via Watson-Crick pairing between the second bis-PNA oligomer and the homopurine strand of the target DNA, thus resulting in the ultimate formation of the P-loop. The data for the (C/J+T)-containing bis-PNA show that its high affinity to dsDNA at neutral pH does not seriously compromise the kinetic specificity of binding. These findings support the earlier expectation that (C/J+T)-containing PNA constructions may be advantageous for use in vivo.

Base Pair Mismatch↗
PubMed1999

The earliest invasion of macrophages into the developing brain and eye of the tree shrew Tupaia belangeri.

The earliest occurrence of macrophages was investigated in the brain and optic anlagen of the tree shrew Tupaia belangeri. Nineteen serially sectioned embryos, belonging to five phases of programmed neuroepithelial cell death previously found during optic cup formation, were used. Macrophages were identified by structural criteria and by labelling with the lectin Griffonia simplicifolia I-B(4). Macrophages, most probably derived from the yolk sac, are present in the perineural vessels of the phase 1 embryo (V-shaped optic evagination). Within this compartment, their number increases up to phase 4 (advanced invagination) and drops during phase 5. This first wave of macrophages is followed by a second one occurring within the perineural mesenchyme and within the neuroepithelium of the brain and eyes from phase 3 onwards. In the phase 4 embryos, a considerable rise in the number of intraventricular macrophages is noted. During phase 5 (far advanced invagination), marked vascularization of the brain starts, and a peak of macrophages is noted in the neuroepithelium and in the ventricular lumen of the brain. This spatiotemporal pattern suggests that, in Tupaia, the earliest macrophages are simultaneously shifted from perineural vessels into the neuroepithelial walls of the developing brain and, at earlier stages than previously described in other vertebrate species, of the eye anlagen.

Animals↗
PubMed1999

Changes in the left ventricular outflow tract after transcoronary ablation of septal hypertrophy (TASH) for hypertrophic obstructive cardiomyopathy as assessed by transoesophageal echocardiography and by measuring myocardial glucose utilization and perfusion.

AIMS AND METHODS: Transcoronary ablation of septal hypertrophy (TASH) leads to marked clinical and haemodynamic improvement in patients with hypertrophic obstructive cardiomyopathy. In order to obtain more detailed information about changes in the outflow tract after TASH, transoesophageal echocardiography and a repeat invasive investigation were conducted before as well as 2 weeks and 6 months after TASH (n=62). In a subset of patients (n=11), metabolism and perfusion of the myocardium ((18)F-FDG-PET and(99m)Tc-MIBI-SPET) were investigated. RESULTS: After TASH there was a typical regional subaortic contraction disorder. It was quantified by a significant decrease in the fractional shortening of the left ventricular end-diastolic diameter, which declined from an average of 40.6% to 18.0%. The end-diastolic diameter increased from an average of 39.1 to 40.6 mm. There was also a significant reduction in septal thickness, which continued for up to 6 months after TASH, from an average of 20.0 mm to 11.1 mm in the region of ablation and from 23. 2 to 21.7 mm outside this region. The decrease in the gradient post TASH corresponded with a concomitant significant increase in the outflow tract area from a mean value of 1.04 cm(2)before the process to a value of 3.0 cm(2)after. In contrast to coronary heart disease, these changes were accompanied by non-diffuse, well demarcated subaortic-septal necrosis verified by(18)F-FDG-PET and(99m)Tc-MIBI-SPET. On average the TASH induced necrotic area comprised 6.6% of the left ventricle and correlated significantly with echocardiographic changes in the outflow tract. CONCLUSIONS: Alterations post TASH indicated that this catheter interventional treatment for hypertrophic obstructive cardiomyopathy affects the specific region of obstruction. The changes reflect a 'therapeutic remodelling' of the outflow tract of the left ventricle. They were demonstrable over the entire 6 months investigation period and obviously constituted the basis of post TASH clinical and haemodynamic improvement. Progressive alterations post TASH (post TASH reduction of subaortic septal thickness and an increase in the end-diastolic diameter) need special consideration during long-term follow up.

Cardiac Catheterization↗
PubMed1999

PNA openers as a tool for direct quantification of specific targets in duplex DNA.

We report a new approach for target quantification directly within DNA duplex. Our assay is based on the formation of a new biomolecular structure, the PD-loop. The approach takes advantage of a selective hybridization of a probe to double-stranded DNA (dsDNA), which is locally opened by a pair of bis-PNA oligomers. To optimize the technique, several experimental formats are tested with the use of PNA and oligonucleotide probes. The highest sensitivity is achieved when the hybridized probe is extended and multiply labeled with 125I-dCTP by DNA polymerase via strand displacement in the presence of single-strand binding (SSB) protein. In this case, the PNA-assisted probe hybridization combined with the method of multiphoton detection (MPD) allows to monitor sub-attomolar amounts of the HIV-1 target on the background of unrelated DNA at sub-nCi level of radioactivity. The developed robust methodology is highly discriminative to single mutations, thus being of practical use for DNA analysis.

Base Sequence↗
PubMed1999

Inverse regulation of lipid-peroxidizing and hydroperoxyl lipid-reducing enzymes by interleukins 4 and 13.

12/15-lipoxygenases and phospholipid hydroperoxide glutathione peroxidases are opposite enzymes balancing the intracellular concentration of hydroperoxy lipids. We studied the regulation of both enzymes by interleukins 4 and 13 and found an inverse response. When human lung carcinoma cells A549 were cultured in vitro in the presence of these cytokines, an up-regulation of the 12/15-lipoxygenase and a down-regulation of the phospholipid hydroperoxide glutathione peroxidase were observed. A similar inverse regulation was found in human peripheral monocytes. Interleukin 4-treated A549 cells exhibited an impaired capability of reducing exogenous hydroperoxyl lipids and their levels of endogenous lipid hydroperoxides were markedly increased. To find out whether these regulatory processes also occur in vivo, arachidonic acid oxygenase and phospholipid hydroperoxide glutathione peroxidase activity was assayed in various tissues of transgenic mice that systemically overexpress interleukin 4. In lung, spleen, kidney, and heart, an increased arachidonic acid oxygenase activity was detected when transgenic mice were compared with inbred controls. The phospholipid hydroperoxide glutathione peroxidase activity was impaired in lung, liver, and spleen of the transgenic animals. These data indicate that lipid-peroxidizing and lipid peroxide-reducing enzymes are inversely regulated in various mammalian cells. Up-regulation of the 12/15-lipoxygenase and simultaneous down-regulation of the phospholipid hydroperoxide glutathione peroxidase may lead to an increased oxidizing potential, which is reflected by an augmented intracellular peroxide tone.

Animals↗
PubMed1998

Probing the substrate alignment at the active site of 15-lipoxygenases by targeted substrate modification and site-directed mutagenesis. Evidence for an inverse substrate orientation.

For oxygenation of polyenoic fatty acids by 12- and 15-lipoxygenases the methyl terminus of the substrate constitutes the signal for the initial hydrogen abstraction. In contrast, for 5-lipoxygenases an inverse head to tail substrate orientation has been proposed. However, recent structure-based sequence alignments suggested a conserved uniform substrate orientation for 5S- and 15S-lipoxygenation. Oxygenation of 15S-HETE derivatives by various wild-type and mutant lipoxygenases was investigated, and the evidence proved an inverse substrate orientation: (i) Substrate affinity and Vmax of 15S-HETE oxygenation by arachidonic acid 15-lipoxygenases are >1 order of magnitude lower than the corresponding data for polyenoic fatty acids. 5S,15S- and 14R, 15S-DiH(P)ETE were identified as major reaction products. (ii) Methylation of the carboxylate group of 15S-HETE augmented the reaction rate and shifted the reaction specificity strongly toward 5S-lipoxygenation. In contrast, methyl arachidonate was less effectively oxygenated than the free acid. Methylation of 15S-HETrE(8,11,14), which lacks the C5-C6 double bond, was without major impact on the oxygenation rate and on the product specificity. (iii) Introduction of a bulky glycerol moiety at the carboxylic group of 15S-HETE reversed the kinetic effects of methylation and led to a 14R-oxygenation of the substrate. (iv) When the product pattern of 15S-HETE oxygenation by the recombinant wild-type rabbit 15-lipoxygenase was compared with that formed by the Arg403Leu mutant, 5S- and 8S-lipoxygenations were augmented and 14R, 15S-DiH(P)ETE formation was impaired. (v) Phe353Leu or Ile418Ala mutation of the same enzyme, which favored 12S-HETE formation from arachidonic acid, strongly augmented 8S-lipoxygenation of 15S-HETE methyl ester. These kinetic data and the alterations in the product specificity are consistent with the concept of an inverse head to tail substrate orientation during the oxygenation of 15S-HETE methyl ester and/or of free 15S-HETE by 15-LOXs. For 5S- and 8S-lipoxygenation, 15-HETE may slide into the substrate binding pocket with its carboxy terminus approaching the doubly allylic methylenes C-7 or C-10 to the non-heme iron.

Animals↗
PubMed1998

Kinetic sequence discrimination of cationic bis-PNAs upon targeting of double-stranded DNA.

Strand displacement binding kinetics of cationic pseudoisocytosine-containing linked homopyrimidine peptide nucleic acids (bis-PNAs) to fully matched and singly mismatched decapurine targets in double-stranded DNA (dsDNA) are reported. PNA-dsDNA complex formation was monitored by gel mobility shift assay and pseudo-first order kinetics of binding was obeyed in all cases studied. The kinetic specificity of PNA binding to dsDNA, defined as the ratio of the initial rates of binding to matched and mismatched targets, increases with increasing ionic strength, whereas the apparent rate constant for bis-PNA-dsDNA complex formation decreases exponentially. Surprisingly, at very low ionic strength two equally charged bis-PNAs which have the same sequence of nucleobases but different linkers and consequently different locations of three positive charges differ in their specificity of binding by one order of magnitude. Under appropriate experimental conditions the kinetic specificity for bis-PNA targeting of dsDNA is as high as 300. Thus multiply charged cationic bis-PNAs containing pseudoisocytosines (J bases) in the Hoogsteen strand combined with enhanced binding affinity also exhibit very high sequence specificity, thereby making such reagents extremely efficient for sequence-specific targeting of duplex DNA.

Animals↗
PubMed1998

Disk formation in retinal cones of Tupaia belangeri (Scandentia).

Existing hypotheses on the mode of disk formation in the photoreceptor cells of mammals appear to be incompatible: (1) plasma membranes of adjacent evaginations form a disk which, subsequently, is internalized by a disk rim; (2) pinocytotic vesicles are pinched off from the plasma membrane and fuse into a larger vesicle, which flattens and forms a disk. We have studied the development of the cone outer segment and the disk formation in Tupaia belangeri by transmission electron microscopy. During the first two postnatal weeks, the distal part of the single cilium, which is inserted apically on the inner segment, becomes balloon-shaped. Apical to the axoneme, it contains tubular and vesicular material, which, most probably, has been detached from the axonemal microtubules. These tubules and vesicles do not contribute to disks. The balloon-shaped expansion, later retained as the ciliary backbone, establishes the contact with the pigment epithelium. Formation of disks, from the 12-day-old Tupaia onwards, occurs between adjacent evaginations at the outer segment base. The initial disk rims are "hooked" to the ciliary axonemal microtubules. The axonemal microtubules are involved in the initiation and in the alignment of the disks. Disk rim formation and, thus, internalization of disks proceeds from the base to the apex of the outer segment, that is, from the younger to the older disks. In the adult Tupaia, an uneven progression of disk rim formation on both sides of the axoneme is found among consecutive disks. The seemingly incompatible hypotheses on the mode of disk formation reflect a heterochrony of the internalization of membranes and of the disk formation among different mammals and, possibly, between cones and rods.

Animals↗
PubMed1997

Primary angioplasty versus thrombolysis in the treatment of acute myocardial infarction. ALKK Study Group.

This study investigates the hypothesis if primary angioplasty is superior to intravenous thrombolysis in the treatment of acute myocardial infarction (AMI). Small prospective randomized studies did not demonstrate a significant benefit regarding total mortality. A total of 14,980 patients with AMI were registered by "The 60-Minutes Myocardial Infarction Project," a prospective multicenter observational study: 210 of these patients were treated with primary angioplasty. A matched pair analysis comparing 1 primary angioplasty patient with 3 intravenous thrombolysis patients could be performed in 156 primary angioplasty patients. Criteria for matching were age, sex, location of AMI, systolic blood pressure, previous AMI, and prehospital delay. Patients with a bundle branch block or requiring resuscitation were excluded from analysis. Because of matching, both groups showed similar baseline characteristics. Patients with primary angioplasty had more relative contraindications for thrombolysis (ulcers: 10.3% vs 2.3%, recent intramuscular injections: 6.4% vs 1.6%, recent surgical interventions: 5.1% vs 1.1%, central punctures: 9% vs 3.9%). There was a tendency toward less combined adverse events in the primary angioplasty group (3.2% vs 5.7%, odds ratio [OR] = 0.55, 95% confidence interval [CI] = 0.21 to 1.44). In-hospital mortality rates in the primary angioplasty group and thrombolysis group were 4.3% and 10.3%, respectively (OR = 0.39, 95% CI = 0.17 to 0.92). The difference in mortality could already be demonstrated within the first 48 hours with 1.9% versus 5.3% deaths (OR = 0.35, 95% CI = 0.11 to 1.14). Thus this study indicates a superiority of primary angioplasty in comparison to intravenous thrombolysis in AMI even in a clinical routine setting, with a reduction of hospital mortality of about 60%.

Aged↗
PubMed1997

Modulation of hu/mu severe combined immunodeficient (SCID) mouse arthritis by local application of human recombinant IL-1beta, IL-2 and IL-6.

The contribution of interleukins produced by most inflammatory cells to chronic arthritis is not well understood. Therefore, we investigated the influence of several human recombinant interleukins (IL-1beta, IL-2 and IL-6) on joint swelling, on the inflammatory process, and on serological parameters in a novel animal model of arthritis, the human/murine SCID arthritis. In this model an arthritis is induced by implanting human synovial tissue from patients with rheumatoid arthritis (RA) into the knee joint of mice with SCID. These mice tolerate the xenogeneic implant and develop a mixed human/murine pannus tissue. The interleukins were injected daily for 7 or 14 days after implantation. IL-1beta led to a significant increase in joint swelling. It intensified the inflammatory process accompanied by enhanced migration of murine inflammatory cells into the knee joint. The production of human IL-6 in the transplanted tissue was stimulated through the application of IL-1beta, and the serum level of human IL-6 was thus significantly higher than in controls. We could not observe a significant influence of IL-1beta on the production of human IgG or IgM by the implant. The application of human IL-2 had a weak effect similar to that of IL-1beta, but without statistical significance. Although IL-6 is a good marker for inflammation in RA, the application of recombined human IL-6 had no influence on the inflammatory process in this model.

Administration, Topical↗
PubMed1997

Induction of subaortic septal ischaemia to reduce obstruction in hypertrophic obstructive cardiomyopathy. Studies to develop a new catheter-based concept of treatment.

AIM: To develop a new catheter-based method of treatment in patients with hypertrophic obstructive cardiomyopathy. METHOD: Does abolition of the blood supply to the subaortic part of the septum lead to regional myocardial ischaemia and a decrease in the left ventricular outflow tract gradient? To find this out, in 10 consecutive patients the first larger septal branch of the left anterior descending coronary artery was temporarily occluded with conventional percutaneous transluminal coronary angioplasty. The intracoronary electrocardiogram was registered for objective verification of the intended ischaemia. The intraventricular pressure was measured at rest and at the post extrasystolic beat under programmed electrostimulation of the right ventricle. RESULTS: During occlusion, regional ischaemia was observed in all patients. Simultaneously, there was a significant reduction of the intraventricular gradient from 56.2 mmHg to 32.2 mmHg (P < 0.05) followed by an increase from 32.2 mmHg to 61.1 mmHg (P < 0.01) after release of occlusion of the septal branch. During ischaemia there was no increase in left ventricular end-diastolic pressure. CONCLUSION: We conclude that the results form the basis for a new catheter interventional therapy in hypertrophic obstructive cardiomyopathy.

Adult↗
PubMed1997

Myocardial perfusion and metabolism after transcoronary ablation of septum hypertrophy (TASH) in hypertrophic obstructive cardiomyopathy.

Outflow obstruction constitutes a major problem in hypertrophic obstructive cardiomyopathy (HOCM) and may be treated by transcoronary injection of ethanol into septal arteries (transcoronary ablation of septum hypertrophy, TASH). We report on myocardial perfusion and glucose metabolism in a 50 year old man in whom TASH led to a reduction of septal thickness (9 vs. 26 mm), a sustained elimination of the outflow tract obstruction (resting gradient 0 vs. 60 mmHg; post-ES-gradient 10 vs. 145 mmHg) and a substantial clinical improvement (NYHA stage II vs. II-III) without impairment of global ventricular function (left ventricular ejection fraction 0.62 vs. 0.64). After TASH, perfusion and glucose metabolism were assessed by positron emission tomography (PET) using F-18-fluorodeoxyglucose (F-18-FDG) and Tc-99m-MIBI single photon emission tomography (SPECT). TASH results in matched reduction of perfusion and glucose consumption in a circumscribed area fed by the septal branch used for ethanol injection. No remote effects were observed. TASH leads to a sharply delineated septal reduction of perfusion and metabolism with consecutive reduction of septal thickness, a sustained elimination of the outflow tract obstruction, and a substantial clinical improvement without impairment of global ventricular function.

Cardiomyopathy, Hypertrophic↗