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Biomedical subjects

M Hahn

Publications and source records attributed to M Hahn.

At least 145 records · Page 8Linked to original sources
PubMed1996

Influence of healing on the disturbed blood flow regulation in venous ulcers.

Cutaneous blood flow regulation is disturbed in severe chronic venous insufficiency (CVI). The feedback system between the transmural pressure in the postcapillary venules and the precapillary resistance regulating arterioles has gone awry. The upregulated control system causes "luxus"-hyperperfusion. In this study laser Doppler fluxmetry was performed in 6 patients' florid venous ulcers (U) and later in the former ulcer area after healing (H) and in the inner ankle area of 12 healthy volunteers (C). Laser Doppler flux (LDF) changes were measured with the leg elevated and lowered as well as after arterial and during venous occlusion. The results proved a diminished (p < 0.01) flux increase with the leg elevated (U: LDF = -9%, H: LDF = 3.1%, C: LDF = 80%) and after arterial occlusion (U: LDF = 1.3%, H: LDF = 0%; C: 189.6%) in severe CVI both in florid ulcers and after healing. The ability to reduce flux when transmural pressures were increased remained. Leg lowering reduced flux by 46.4% in ulcers and by 36.8% after healing. This postural response to high transmural pressures, however, seemed weakened compared to controls, where LDF decreased by 65.8%. Our findings suggest that the postural feedback system remains disturbed and upregulated even after a venous ulcer has healed.

Adult↗
PubMed1995

Constitutive activation of mitogen-activated protein kinase-activated protein kinase 2 by mutation of phosphorylation sites and an A-helix motif.

A recently described downstream target of mitogen-activated protein kinases (MAPKs) is the MAPK-activated protein (MAPKAP) kinase 2 which has been shown to be responsible for small heat shock protein phosphorylation. We have analyzed the mechanism of MAPKAP kinase 2 activation by MAPK phosphorylation using a recombinant MAPKAP kinase 2-fusion protein, p44MAPK and p38/40MAPK in vitro and using an epitope-tagged MAPKAP kinase 2 in heat-shocked NIH 3T3 cells. It is demonstrated that, in addition to the known phosphorylation of the threonine residue carboxyl-terminal to the catalytic domain, Thr-317, activation of MAPKAP kinase 2 in vitro and in vivo is dependent on phosphorylation of a second threonine residue, Thr-205, which is located within the catalytic domain and which is highly conserved in several protein kinases. Constitutive activation of MAPKAP kinase 2 is obtained by replacement of both of these threonine residues by glutamic acid. A constitutively active form of MAPKAP kinase 2 is also obtained by deletion of a carboxyl-terminal region containing Thr-317 and the A-helix motif or by replacing the conserved residues of the A-helix. These data suggest a dual mechanism of MAPKAP kinase 2 activation by phosphorylation of Thr-205 inside the catalytic domain and by phosphorylation of Thr-317 outside the catalytic domain involving an autoinhibitory A-helix motif.

3T3 Cells↗
PubMed1995

Crystal structure of Bacillus licheniformis 1,3-1,4-beta-D-glucan 4-glucanohydrolase at 1.8 A resolution.

The crystal structure of the 1,3-1,4-beta-D-glucan 4-glucanohydrolase from Bacillus licheniformis is solved at a resolution of 1.8 A and refined to R = 16.5%. The protein has a similar beta-sandwich structure as the homologous enzyme from Bacillus macerans and the hybrid H(A16-M). This demonstrates that the jellyroll fold of these proteins is remarkably rigid and only weakly influenced by crystal contacts. The crystal structure permits to extend mechanistic considerations derived for the B. licheniformis enzyme to the entire class of bacterial 1,3-1,4-beta-D-glucan 4-glucanohydrolases.

Bacillus↗
PubMed1995

Sequence preferences in cleavage of dsDNA and ssDNA by the extracellular Serratia marcescens endonuclease.

The preferred cleavage sites in dsDNA and ssDNA for the extracellular Serratia marcescens endonuclease (commercially available as BENZONASE) were identified by limited digestion of PCR-generated substrates. Two different dsDNA substrates were synthesized by using either radioactively or fluorescent dye labeled primers. ssDNA of identical sequence to one of the fluorescent dye labeled duplex strands was prepared by affinity chromatography. Cleavage experiments carried out under single hit conditions demonstrate that the enzyme shows preferences for GC-rich regions in dsDNA, in particular d(G).d(C)-tracts, and avoids cleavage of d(A).d(T)-tracts. There is a correlation between cleavage at a given position in one strand with cleavage at the same position in the other strand of the duplex. ssDNA cleavage occurs at somewhat different preferred sites than observed in dsDNA. On dsDNA, the Serratia nuclease produces a very different cleavage pattern compared to bovine pancreatic DNase I, with the notable exception that both enzymes avoid d(A).d(T)-tracts. In general, the Serratia nuclease compared to DNase I is a slightly more nonspecific endonuclease that attacks a particular substrate more evenly under standard reaction conditions. At high ionic strength or in the presence of DMSO, it becomes more nonspecific. Addition of urea, however, makes the enzyme more selective than observed under standard conditions. From these results which were confirmed by the results of cleavage experiments with synthetic oligodeoxynucleotides, we conclude that the Serratia nuclease like DNase I is sensitive to global features of the DNA, for example, the width of the minor groove. In addition, localized sequence-dependent interactions between substrate and nuclease determine whether a site is cleaved preferentially. Some of these interactions seem to be the same for ds- and ssDNA.

Base Sequence↗
PubMed1995

Crystal and molecular structure at 0.16-nm resolution of the hybrid Bacillus endo-1,3-1,4-beta-D-glucan 4-glucanohydrolase H(A16-M).

H(A16-M) is a hybrid endo-1,3-1,4-beta-D-glucan 4-glucanohydrolase from Bacillus. Its crystal structure was refined using synchrotron X-ray diffraction data up to a maximal resolution of 0.16 nm. The R value of the resulting model is 14.3% against 21,032 reflections > 2 sigma. 93% of the amino acid residues are in the most favorable regions of the Ramachandran diagram, and geometrical parameters are in accordance with other proteins solved at high resolution. As shown earlier [Keitel, T., Simon, O., Borriss, R. & Heinemann, U. (1993) Proc. Natl Acad. Sci. USA 90, 5287-5291], the protein folds into a compact jellyroll-type beta-sheet structure. A systematic analysis of the secondary structure reveals the presence of two major antiparallel beta-sheets and a three-stranded minor mixed sheet. Amino acid residues involved in catalysis and substrate binding are located inside a deep channel spanning the surface of the protein. To investigate the stereochemical cause of the observed specificity of endo-1,3-1,4-beta-D-glucan 4-glucanohydrolases towards beta-1,4 glycosyl bonds adjacent to beta-1,3 bonds, the high-resolution crystal structure has been used to model an enzyme-substrate complex. It is proposed that productive substrate binding to the subsites p1, p2 and p3 of H(A16-M) requires a beta-1,3 linkage between glucose units bound to p1 and p2.

Bacillus↗
PubMed1995

The race-specific elicitor, NIP1, from the barley pathogen, Rhynchosporium secalis, determines avirulence on host plants of the Rrs1 resistance genotype.

NIP1, a small phytotoxic protein secreted by the barley pathogen Rhynchosporium secalis, is a race-specific elicitor of defense responses in barley cultivars carrying the resistance gene, Rrs1. Co-inoculation employing spores from a virulent fungal race together with the NIP1 protein converted the phenotype of the interaction from compatible to incompatible only on Rrs1-containing plants. In addition, transformation of a virulent fungal race with the nip1 gene yielded avirulent transformants. This demonstrated that the protein is the product of a fungal avirulence gene. The fungal genome was found to contain a single copy of the nip1 gene. Sequence analysis of nip1 cDNA and genomic clones revealed that the gene consists of two exons and one intron. The derived amino acid sequence comprised a secretory signal peptide of 22 amino acids and a cysteine-rich mature protein of 60 amino acids. All fungal races that were avirulent on barley cultivars of the Rrs1 resistance genotype carry and express the nip1 gene and secrete an elicitor-active NIP1 polypeptide. In contrast, races lacking this gene were virulent. In addition, single nucleotide exchanges were detected in the coding region of the nip1 alleles in one virulent fungal race and in a race whose interaction with barley is not controlled by the Rrs1 gene. The resulting exchanges of single amino acids render the gene products elicitor-inactive. Thus, the R.secalis-barley interaction provides the first example of a pathosystem conforming to the gene-for-gene hypothesis in which a plant with a particular resistance gene recognizes a pathogen by a virulence factor, i.e. one of its offensive weapons. On the fungal side, in turn, recognition by the host plant is eluded by either deletion of the encoding gene or alteration of the primary structure of the gene product.

Amino Acid Sequence↗
PubMed1995

Quantitative polymerase chain reaction with enzyme-linked immunosorbent assay detection of selectively digested amplified sample and control DNA.

A quantitative polymerase chain reaction (PCR) method for the exact quantitation of DNA is described that does not require radioactive labeling or electrophoretic separation of product species, thereby avoiding hazardous and time-consuming procedures which have so far impeded routine use of PCR, in particular in the clinical laboratory. Sample and internal control DNA are competitively amplified in a one-tube nested PCR. The control DNA differs from the sample DNA by only two base pairs which change a single restriction site for a new one. Thereby, it is possible to discriminate the two PCR product species by selective restriction enzyme digestion (RED). Because inner PCR primers were labeled with biotin and digoxigenin, respectively, nested PCR products can be immobilized on avidin-coated microtiter plates and quantitated separately by enzyme-linked immunosorbent assay (ELISA) techniques. We demonstrate here that with the combination of selective restriction enzyme digestion and ELISA (RED-ELISA) sample DNA ranging from nanomolar to attomolar concentrations can be quantitated within +/- 10%. This procedure can be easily adapted for quantitation of other PCR products. It is suitable for rapid and automatic screening of many samples in parallel, e.g., for detection and quantitation of pathogens, for quantitation of gene copy numbers or for gene expression after reverse transcription.

Base Sequence↗
PubMed1995

Receptor surface models. 1. Definition and construction.

A receptor site model is a hypothetical model that characterizes the putative active site of a receptor. This paper describes a type of receptor site model called a receptor surface model, which is based on the construction of surfaces to represent spatial and electrostatic properties of the receptor active site. A receptor surface model is visually intuitive and is modifiable as the hypothesis is refined. It allows computations comparable to those that can be performed with traditional atomistic models. Structures can be energy minimized within the receptor surface model to arrive at conformations that are consistent with the model, and interaction energies can be estimated. Such calculations facilitate the evaluation of new candidate structures and provide a means to assess the predictive ability of a model.

Binding Sites↗
PubMed1995

Receptor surface models. 2. Application to quantitative structure-activity relationships studies.

A new technique for using receptor surface models in quantitative structure-activity relationship (QSAR) analysis is described. Receptor surface models provide compact, quantitative descriptors which capture three-dimensional information about putative receptor/ligand interactions. Receptor surface models can be constructed quickly, which allows the construction of multiple plausible models; a variable selection technique such as genetic function approximation (GFA) can then be used to suggest which receptor surface models provide the most valuable descriptors for QSAR. Advantages of this approach are shown by applying it against two previously-published and well-studied QSAR data sets. Our results indicate that the approach can model data as effectively as established 3D-QSAR techniques.

Adrenal Cortex Hormones↗
PubMed1995

Cholest-3,5-dien-7-one formation in peroxidized human plasma as an indicator of lipoprotein cholesterol peroxidation potential.

Lipoprotein peroxidation susceptibility is routinely evaluated using products of unsaturated fatty acids as markers (e.g., malonaldehyde). The significance and factors influencing peroxidation of cholesterol moiety of lipoproteins are relatively unknown due to lack of a reliable marker product which can be measured easily. Under the influence of Cu2+ ions, the major product of lipoprotein cholesterol peroxidation (isolated after saponification) was cholest-3-5-dien-7-one (CSD). Apart from gas-liquid chromatography, this compound lends itself for measurement by alternative methods. Due to lack of the 3 beta-hydroxyl group, CSD was separated from the rest of the oxysterols and cholesterol by passing through digitonin-coated silica-gel G and its concentration was determined by absorption at 283 nm. The recovery of CSD by this method exceeded by 87%. The formation of CSD was also sensitive to vitamin E and therefore could be used as an index of lipoprotein cholesterol susceptibility to peroxidation.

Cholestenones↗
PubMed1995

Crystal structure and site-directed mutagenesis of Bacillus macerans endo-1,3-1,4-beta-glucanase.

In beta-glucans those beta-1,4 glycosidic bonds which are adjacent to beta-1,3 bonds are cleaved by endo-1,3-1,4-beta-glucanases (beta-glucanases). Here, the relationship between structure and activity of the beta-glucanase of Bacillus macerans is studied by x-ray crystallography and site-directed mutagenesis of active site residues. Crystal structure analysis at 2.3-A resolution reveals a jelly-roll protein structure with a deep active site channel harboring the amino acid residues Trp101, Glu103, Asp105, and Glu107 as in the hybrid Bacillus beta-glucanase H(A16-M) (Keitel, T., Simon, O., Borriss, R., and Heinemann, U. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 5287-5291). Different mutant proteins with substitutions in these residues are generated by site-directed mutagenesis, isolated, and characterized. Compared with the wild-type enzyme their activity is reduced to less than 1%. Several mutants with isosteric substitutions in Glu103 and Glu107 are completely inactive, suggesting a direct role of these residues in glycosyl bond hydrolysis. The kinetic properties of mutant beta-glucanases and the crystal structure of the wild-type enzyme are consistent with a mechanism where Glu103 and Glu107 are the catalytic amino acid residues responsible for cleavage of the beta-1,4 glycosidic bond within the substrate molecule.

Amino Acid Sequence↗
PubMed1995

Microcallus formations of the cancellous bone: a quantitative analysis of the human spine.

Microcallus formations are demonstrable in nearly all spongy bone by means of suitable preparation techniques. Histologically, these structures are immature fibrous bone. Their genesis, frequency, and importance are largely unknown. To address these issues, 26 normal human spines, 11 osteoporotic spines, and different parts of the skeleton (femur head, iliac crest) were investigated for microcallus using a new preparation technique--allowing a combined 2- and 3-dimensional analysis. According to our analysis, microcallus formation occurs frequently in persons older than 45 years of age. These formations are mainly localized in the lower thoracic and lumbar spine and are obviously more frequent in females than in males. In individuals with a trabecular bone volume (BV/TV) in the spine below 11%, microcallus formations occur regularly. But the number of microcallus formations depends more on the microarchitecture of the cancellous bone (trabecular bone pattern factor, TBPf), than on individual trabecular parameters (trabecular number, TbN; trabecular bone volume, BV/TV; and trabecular thickness, TbTh). In about 33% of cases microfractures are demonstrable in the center of the microcallus formation. It is unclear whether microcallus may be the result of a nontraumatic process. In therapy studies the bone mass could be misrepresented due to the amount of microcallus. Although it indicates instability of the bone structure, microcallus formation is not only a negative mechanism, but stabilizes and regenerates the bone tissue. Furthermore, complete new trabeculae can be formed due to bridges of microcallus between the remnant trabeculae. Osteoporosis is not the result of an inability to form microcallus formations.

Adolescent↗
PubMed1995

An improved method for the species-specific assessment of mycobacteria in routinely formalin-fixed and paraffin-embedded tissues.

A polymerase chain reaction (PCR) assay for the rapid and species-specific diagnosis of mycobacterial infections in paraffin-embedded clinical specimens was developed using oligonucleotide primers to amplify a fragment of the DNA coding for the ribosomal 16S RNA of mycobacteria. The oligonucleotide primers amplified DNA from all 14 species of mycobacteria tested. By means of a reamplification protocol, as few as one to two mycobacteria could be detected in the presence of human DNA. The method of DNA isolation and amplification was applied on sections of routinely formalin-fixed and paraffin-embedded tissues. PCR for the beta-actin gene served as a control for successful DNA isolation. Mycobacterial DNA could be detected in cases of mycobacterial infections. The mycobacterial species was determined by additional sequencing of the PCR fragment. This PCR method may be a powerful tool for the diagnosis of mycobacterial infections from histopathological material and for the assessment of those mycobacteria that cannot readily be cultured, such as Mycobacterium leprae.

Base Sequence↗
PubMed1995

Trabecular bone structure in patients with primary hyperparathyroidism.

To evaluate the effects of increased parathyroid hormone (PTH)-secretion on trabecular bone structure in patients with primary hyperparathyroidism (PHPT) we have analysed iliac crest biopsies from 84 patients (38 male, 46 female, age 20-85 years) with surgically proven PHPT by using quantitative histomorphometry. As there might be an influence of age or sex, subjects were divided into 4 subgroups according to age (younger than 50 years, older than 50 years) and sex. Eighty four age and sex matched autopsy cases of accidental death served as controls. When compared with age matched controls, trabecular bone volume (BV/TV) and trabecular diameter in PHPT were increased. Trabecular density in PHPT was elevated significantly in the older age-groups. These results suggest a preservation of intertrabecular connectivity, which we were able to confirm by measurement of the number of intertrabecular nodes and trabecular bone pattern factor (TBPf). No differences were demonstrated between male and female subjects. Furthermore, no variations according to the histological type (with or without endosteal fibrosis) could be demonstrated in our material. We conclude that increased trabecular bone volume in PHPT is the result not only of thicker trabecula but also of a reduction of the age-dependent loss of complete trabecular plates due to perforations. Thus PHPT leads to a substantial preservation of intertrabecular connectivity.

Adult↗
PubMed1995

Blockade of receptor-operated calcium channels by mibefradil (Ro 40-5967): effects on intracellular calcium and platelet aggregation.

The goal of the present study was to evaluate if mibefradil, a novel nondihydropyridine Ca2+ antagonist, could block receptor-operated calcium channels (ROCC) present in human platelets and to determine the functional consequences of this blockade. Therefore, the effect of mibefradil on increases in intracellular Ca2+ concentrations and aggregation of human platelets induced by platelet activating factor (PAF) was examined. In order to differentiate effects on Ca2+ mobilization from intracellular stores from those on Ca2+ influx through ROCC, intracellular Ca2+ concentrations were measured either in fura-2-loaded platelets or in cells loaded with both BAPTA and fura-2. Mibefradil totally and dose dependently inhibited PAF-induced Ca2+ influx with a maximal effective concentration of 10 microM, but at this concentration only reduced Ca2+ mobilization from intracellular stores. A similar effect was observed when platelets were stimulated with ADP, suggesting that mibefradil was indeed interfering with ROCC and not specifically with PAF receptors. In the same range of concentrations, mibefradil inhibited Ca(2+)-dependent platelet aggregation induced by PAF. This effect was most likely due to the inhibition of ROCC, as Ca(2+)-independent aggregation induced by phorbol-myristyl-acetate (PMA) was insensitive to mibefradil. We conclude that mibefradil, which has previously been described to be an antagonist for L- and T-Type Ca2+ channels, also blocks receptor-operated Ca2+ channels. This blockade seems to be functionally relevant for platelet aggregation.

Benzimidazoles↗
PubMed1995

[Laser Doppler flowmetry in treatment follow-up of circumscribed scleroderma].

A 38-year-old male patient presented with circumscribed scleroderma (10 x 12 cm) in the left pectoral area. Hyperaemia of the skin in the area of the lilac ring (morphoea) was quantified by means of Laser-Doppler flux (LDF). At the lilac border of the morphoea LDF was increased to 420% of the reference value recorded for the unaffected skin of the contralateral side. Immediately after 13 days of antibiotic therapy (3 x 10 Mega penicillin G per day, i.v.) LDF normalized, though clinical changes were hardly apparent at this stage. By 6 months later, LDF was still in the normal range, and clinical signs, such as induration and hyperaemic redness, had also significantly improved. LDF measurements allow assessment of the therapeutic response to antibiotic therapy at a very early stage while the clinical situation is still unchanged.

Adult↗
PubMed1995

[New possibilities for structural analysis of bone biopsies using microcomputer tomography (muCT)].

The combined histological and microcomputed analysis of human iliac crest biopsies leads to major advances in our understanding of three-dimensional bone architecture. Microcomputed tomography avoids the time-consuming reconstruction and artifacts of serial sections. Furthermore, its high resolution allows the recording of structural differences as low as 10 microns. Thus, three-dimensional analysis in combination with histological evaluation of cellular dynamics facilitates earlier and easier recording of changes of cancellous bone.

Biopsy↗