Medical versus surgical management of atherosclerotic renal artery stenosis.
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Arteriosclerosis↗
Biomedical subjects
M Pohl
Publications and source records attributed to M Pohl.
Improving the carboligase activity of benzoylformate decarboxylase from Pseudomonas putida by a combination of directed evolution and site-directed mutagenesis.
Benzoylformate decarboxylase (BFD) from Pseudomonas putida was subjected to directed molecular evolution to generate mutants with increased carboligase activity which is a side reaction of the enzyme. After a single round of random mutagenesis mutants were isolated which exhibited a 5-fold increased carboligase activity in aqueous buffer compared to the wild-type enzyme with a high enantiomeric excess of the product (S)-2-hydroxy-1-phenyl-propanone. From the same library, mutants with enhanced carboligase activity in water-miscible organic solvents have been isolated. The selected mutants have been characterized by sequencing, revealing that all mutants carry a mutation at Leu476, which is close to the active site but does not directly interact with the active center. BFD-L476Q has a 5-fold higher carboligase activity than the wild-type enzyme. L476 was subjected to saturation mutagenesis yielding eight different mutants with up to 5-fold increased carboligase activity. Surprisingly, all L476 mutants catalyze the formation of 2-hydroxy-1-phenyl-propanone with significantly higher enantioselectivity than the wild-type enzyme although enantioselectivity was not a selection parameter. Leu476 potentially plays the role of a gatekeeper of the active site of BFD, possibly by controlling the release of the product. The biocatalyst could be significantly improved for its side reaction, the C-C bond formation and for application under conditions that are not optimized in nature.
Amino Acid Substitution↗
Gene therapy of pain: emerging strategies and future directions.
Gene therapy to alleviate pain could appear surprising and perhaps not appropriate when opioids and other active molecules are available. However, the possibility of introducing a therapeutic protein into some targeted structures, where it would be continuously synthesised and exert its biological effect in the near vicinity of, or inside the cells, might avoid some drawbacks of "classical" drugs. Moreover, the gene-transfer techniques might improve present therapies or lead to novel ones. The recent significant and constant advances in vector systems design suggest that these techniques will be available in the near future for safe application in humans. The first experimental protocols attempting the transfer of opioid precursors genes, leading to their overexpression at the spinal level, demonstrated the feasibility and the potential interest of these approaches. Indeed, overproduction of opioid peptides in primary sensory neurones or spinal cord induced antihyperalgesic effects in various animal models of persistent pain. However, numerous other molecules involved in pain processing or associated with chronic pain have been identified and the gene-based techniques might be particularly adapted for the evaluation of the possible therapeutic interest of these new potential targets.
Animals↗
Therapeutic efficacy in experimental polyarthritis of viral-driven enkephalin overproduction in sensory neurons.
Rheumatoid arthritis is characterized by erosive inflammation of the joints, new bone proliferation, and ankylosis, leading to severely reduced locomotion and intense chronic pain. In a model of this disease, adjuvant-induced polyarthritis in the rat, neurons involved in pain transmission and control undergo plastic changes, especially at the spinal level. These changes affect notably neurons that contain opioids, such as enkephalins deriving from preproenkephalin A (PA) precursor protein. Using recombinant herpes simplex virus containing rat PA cDNA, we enhanced enkephalin synthesis in sensory neurons of polyarthritic rats. This treatment markedly improved locomotion and reduced hyperalgesia. Furthermore, the progression of bone destruction slowed down, which is the most difficult target to reach in the treatment of patients suffering from arthritis. These data demonstrate the therapeutic efficacy of enkephalin overproduction in a model of systemic inflammatory and painful chronic disorder.
Animals↗
Continuous production of (R)-phenylacetylcarbinol in an enzyme-membrane reactor using a potent mutant of pyruvate decarboxylase from Zymomonas mobilis.
The optimization of a continuous enzymatic reaction yielding (R)-phenylacetylcarbinol (PAC), an intermediate of the L-ephedrine synthesis, is presented. We compare the suitability of three pyruvate decarboxylases (PDC), PDC from Saccharomyces cerevisiae, PDC from Zymomonas mobilis, and a potent mutant of the latter, PDCW392M, with respect to their application in the biotransformation using acetaldehyde and benzaldehyde as substrates. Among these, the mutant enzyme was the most active and most stable one. The reaction conditions of the carboligation reaction were investigated by determining initial rate velocities with varying substrate concentrations of both aldehydes. From the resulting data a kinetic model was inferred which fits the experimental data with sufficient reliability to deduce the optimal concentrations of both substrates for the enzymatic process. The results demonstrate that the carboligation is most efficiently performed using a continuous reaction system and feeding both aldehydes in equimolar concentration. Initial studies using a continuously operated enzyme-membrane reactor gave (R)-PAC with a space-time yield of 81 g L(-1). d(-1) using a substrate concentration of 50 mM of both aldehydes. The yield was easily increased by cascadation of enzyme-membrane reactors. The new strategy allows the synthesis of (R)-PAC from cheap substrates in an aqueous reaction system. It thereby overcomes the limitation of by-product formation that severely limits the current fermentative process.
Acetaldehyde↗
Musculo-skeletal loading conditions at the hip during walking and stair climbing.
Musculo-skeletal loading plays an important role in the primary stability of joint replacements and in the biological processes involved in fracture healing. However, current knowledge of musculo-skeletal loading is still limited. In the past, a number of musculo-skeletal models have been developed to estimate loading conditions at the hip. So far, a cycle-to-cycle validation of predicted musculo-skeletal loading by in vivo measurements has not been possible. The aim of this study was to determine the musculo-skeletal loading conditions during walking and climbing stairs for a number of patients and compare these findings to in vivo data. Following total hip arthroplasty, four patients underwent gait analysis during walking and stair climbing. An instrumented femoral prosthesis enabled simultaneous measurement of in vivo hip contact forces. On the basis of CT and X-ray data, individual musculo-skeletal models of the lower extremity were developed for each patient. Muscle and joint contact forces were calculated using an optimization algorithm. The calculated peak hip contact forces both over- and under-estimated the measured forces. They differed by a mean of 12% during walking and 14% during stair climbing. For the first time, a cycle-to-cycle validation of predicted musculo-skeletal loading was possible for walking and climbing stairs in several patients. In all cases, the comparison of in vivo measured and calculated hip contact forces showed good agreement.Thus, the authors consider the presented approach as a useful means to determine valid conditions for the analysis of prosthesis loading, bone modeling or remodeling processes around implants and fracture stability following internal fixation.
Aged↗
Met-enkephalin is preferentially transported into the peripheral processes of primary afferent fibres in both control and HSV1-driven proenkephalin A overexpressing rats.
The demonstration of preproenkephalin A gene expression in rat dorsal root ganglia has raised the question of the physiological role of met-enkephalin-containing primary afferent fibres. Recently, we showed that systemic infection with a recombinant Herpes simplex virus encoding preproenkephalin A (HSVLatEnk1) yielded a marked increase in the density of met-enkephalin-like material synthesising neurons in rat dorsal root ganglia. This study further investigated the synthesis, transport and release of met-enkephalin-like material in the central and/or peripheral processes of primary afferent fibres in HSVLatEnk1-infected and control rats. In controls, dorsal root ganglia neurons containing met-enkephalin-like material were scarce and only a few positively labelled processes were seen at the peripheral output of the dorsal root ganglia. Met-enkephalin-like material accumulated at the proximal side of ligatured sciatic nerve, but not in ligatured L4-L5 dorsal roots. In HSVLatEnk1-infected rats with numerous somas and fibres stained for met-enkephalin-like material in dorsal root ganglia, met-enkephalin immunoreactive material largely accumulated at the proximal side of the ligatured sciatic nerve and few positively stained fibres were also observed in ligatured dorsal roots. Electrical stimulation of L4-L5 dorsal roots attached to a dorsal slice of the lumbar enlargement produced an overflow of met-enkephalin-like material which was approximately 70% higher in HSVLatEnk1-infected rats compared to controls. At the periphery, subcutaneous microdialysis showed higher basal levels of met-enkephalin-like material in the interstitial fluid of hindpaw plantar area in HSVLatEnk1-infected rats, and electrical stimulation of the ipsilateral sciatic nerve resulted in an approximately three-fold-higher overflow of this material than in control rats. These data demonstrated that met-enkephalin synthesised in dorsal root ganglion of both control and preproenkephalin A overexpressing rats is preferentially transported into the peripheral processes of primary afferent fibres where the peptide reaches a releasable compartment, thus providing a neuronal source of peripheral met-enkephalin.
Afferent Pathways↗
[Model fluids of blood for in vitro testing of artificial heart valves].
The increasing development and implantation of artificial organs subject to perfusion with human blood once implanted (grafts, heart-valve prostheses and assist systems) require extensive testing of hydrodynamic performance in mock circulation models. As human blood ist not always available in the necessary quantities, different fluids (water, saline or glycerine solutions) are employed for measurements of flow characteristics. However, these model fluids do not possess the non-Newtonian rheological properties of blood. In addition, they do not allow estimation of possible blood damage. Aqueous solutions of high molecular weight polyacrylamides (PAA) have rheological properties similar to blood, displaying also molecular degradation due to shear stress in the flow. Therefore, they were used as model fluid for blood. Different model solutions were compared to blood with regard to their influence on characteristic flow parameters of mechanical heart valves. Likewise, the shear damage of erythrocytes could be compared to flow-induced polymer degradation. It was shown that PAA solutions in definite concentrations are suitable models for blood, not only in terms of non-Newtonian rheology, but also in terms of estimation of hemolytic potential of artificial heart valves.
Acrylic Resins↗
Improved biocatalysts by directed evolution and rational protein design.
The efficient application of biocatalysts requires the availability of suitable enzymes with high activity and stability under process conditions, desired substrate selectivity and high enantioselectivity. However, wild-type enzymes often need to be optimized to fulfill these requirements. Two rather contradictory tools can be used on a molecular level to create tailor-made biocatalysts: directed evolution and rational protein design.
Catalysis↗
Different types of aortic stenosis and simulation of their morphological-hydrodynamic interdependence--in vitro study with allografts and stenotic valve models.
Biological valves display a dependence of valve resistance and valve area on flow and a phase shift between systolic flow through the valves and pressure difference across the valves. The pressure-flow relations of stenosed valves raise questions about the "best measure of stenosis". There is a need for quantitative evaluation of the hydrodynamic performance of homografts and allografts. In the present paper, we report on in vitro studies of the hydrodynamic behavior of homografts from human donors, allografts from different animal species as well as three valve models. Valve model I was designed to simulate flow-dependence of valve area, valve model II was designed to simulate restricted valve opening independent of flow, and valve model III was designed to simulate a flow-dependent movement of valve root in flow direction. Among other aspects, the effect of increased viscosity of the test fluid on the pressure difference and the effects of water absorption by valve tissue on valve characteristics were investigated. The results of the present studies clearly indicate that any biological valve may be modelled as a serial connection of a model I type valve and a model II type valve. From the results, the dependence of the characteristic pressure-flow relationship of a valve on valve size and valve distensibility can be clearly seen and the clinical significance of the characteristic coefficients of the pressure-flow relationship of a valve can be elucidated. Further, it was shown that the characteristic phase shift between flow and pressure difference displayed by biological valves is due to their movable valve plane similar to that of valve model III.
Animals↗
A role for Timeless in epithelial morphogenesis during kidney development.
Central to the process of epithelial organogenesis is branching morphogenesis into tubules and ducts. In the kidney, this can be modeled by a very simple system consisting of isolated ureteric bud (UB) cells, which undergo branching morphogenesis in response to soluble factors present in the conditioned medium of a metanephric mesenchyme cell line. By employing a targeted screen to identify transcription factors involved early in the morphogenetic program leading to UB branching, we identified the mammalian ortholog of Timeless (mTim) as a potential immediate early gene (IEG) important in this process. In the embryo, mTim was found to be expressed in patterns very suggestive of a role in epithelial organogenesis with high levels of expression in the developing lung, liver, and kidney, as well as neuroepithelium. In the embryonic kidney, the expression of mTim was maximal in regions of active UB branching, and a shift from the large isoform of mTim to a smaller isoform occurred as the kidney developed. Selective down-regulation of mTim resulted in profound inhibition of embryonic kidney growth and UB morphogenesis in organ culture. A direct effect on the branching UB was supported by the observation that down-regulation of mTim in the isolated UB (cultured in the absence of mesenchyme) resulted in marked inhibition of morphogenesis, suggesting a key role for Tim in the epithelial cell morphogenetic pathway leading to the formation of branching tubules.
Animals↗
Role of hyaluronan and CD44 in in vitro branching morphogenesis of ureteric bud cells.
Mutual interaction between the metanephric mesenchyme (MM) and the ureteric bud (UB) in the developing kidney leads to branching morphogenesis and the formation of the ureteric tree. A UB-derived cell line, stimulated by conditioned medium derived from an embryonic MM cell line (or, similarly, by 10% fetal calf serum), forms branching tubules under three-dimensional culture conditions (H. Sakurai et al., 1997, Proc. Natl. Acad. Sci. USA 94, 6279-6284). The formation of branching tubules in this simple in vitro system for early nephrogenesis is highly sensitive to the matrix environment, a key component of which is the glycosaminoglycan hyaluronan (HA). Consistent with this, we found that HA in the extracellular environment markedly stimulated the formation of cellular processes and multicellular cords (early steps in branching morphogenesis) and also acted as a cell survival factor. Inhibition of HA binding to the cells by addition of blocking antibodies to CD44, the principal cell surface receptor for HA, or degradation of HA by the addition of Streptomyces hyaluronidase resulted in decreased cell survival and diminished morphogenesis, indicating that the HA-CD44 axis plays a central role in in vitro branching morphogenesis. Analysis of the expression of a large number of genes displayed on a cDNA array revealed that significant changes in gene expression in cells undergoing morphogenesis in the presence of HA were limited to a small subset of genes regulating apoptosis, proliferation, and morphogenesis. This included upregulation by HA of its receptor, CD44, which was found to largely localize to the tips of branching cellular processes. In the embryonic kidney, HA was found near the developing ureteric tree and CD44 was expressed basolaterally in UB-derived structures. In addition, both UB and MM appear to express HA synthase, suggesting their ability to secrete HA. We propose that HA promotes branching morphogenesis by creating a positive feedback loop that results in (1) enhanced interaction of HA-CD44 at branching tips (possibly leading to localization of HA binding morphoregulatory factors at the tips) and (2) an activated transcriptional program favoring cell survival/proliferation and migration/morphogenesis of cells through matrix by the expression of key morphoregulatory molecules. Furthermore, since HA, hyaluronidase, and CD44 have been functionally implicated in branching morphogenesis in this model, and since HA, CD44, and HA synthase are all expressed in an appropriate spatiotemporal fashion in the developing kidney, we propose that these molecules may, together, constitute a morphoregulatory pathway that plays a key role in sequential cycles of branching morphogenesis in the UB.
Animals↗
Benzoylformate decarboxylase from Pseudomonas putida as stable catalyst for the synthesis of chiral 2-hydroxy ketones.
The thiamin diphosphate- and Mg2+-dependent enzyme benzoylformate decarboxylase (BFD) from Pseudomonas putida was characterized with respect to its suitability to catalyze the formation of chiral 2-hydroxy ketones in a benzoin-condensation type reaction. Carboligation constitutes a side reaction of BFD, whereas the predominant physiological task of the enzyme is the non-oxidative decarboxylation of benzoylformate. For this purpose the enzyme was obtained in sufficient purity from Pseudomonas putida cells in a one-step purification using anion-exchange chromatography. To facilitate the access to pure BFD for kinetical studies, stability investigations, and synthetical applications, the coding gene was cloned into a vector allowing the expression of a hexahistidine fusion protein. The recombinant enzyme shows distinct activity maxima for the decarboxylation and the carboligation beside a pronounced stability in a broad pH and temperature range. The enzyme accepts a wide range of donor aldehyde substrates which are ligated to acetaldehyde as an acceptor in mostly high optical purities. The enantioselectivity of the carboligation was found to be a function of the reaction temperature, the substitution pattern of the donor aldehyde and, most significantly, of the concentration of the donor aldehyde substrate. Our data are consistent with a mechanistical model based on the X-ray crystallographic data of BFD. Furthermore we present a simple way to increase the enantiomeric excess of (S)-2-hydroxy-1-phenyl-propanone from 90% to 95% by skillful choice of the reaction parameters. Enzymatic synthesis with BFD are performed best in a continuously operated enzyme membrane reactor. Thus, we have established a new enzyme tool comprising a vast applicability for stereoselective synthesis.
Amino Acid Sequence↗
Insertion pain in needle electromyography can be reduced by simultaneous finger slapping.
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Electromyography↗
Delta-opioid receptor-mediated increase in cortical extracellular levels of cholecystokinin-like material by subchronic morphine in rats.
Numerous pharmacological data indirectly support the idea that interactions between cholecystokinin (CCK) and opioids participate in the development of tolerance to morphine. Biochemical investigations were performed with the aim of directly assessing the status of such interactions in morphine treated rats. Tolerance to the alkaloid after s.c. implantation of morphine pellets for three days was not associated with any change in the levels of both CCK like-material (CCKLM) and proCCK mRNA in the frontal cortex. However, microdialysis in the freely moving rat showed that this morphine treatment produced a significant increase (+40%) of the cortical spontaneous CCKLM outflow, which could be completely prevented by intracortical infusion of naloxone (10 microM). The opioid receptors responsible for morphine-induced cortical CCKLM overflow appeared to be of the delta type because intracortical infusion of selective delta-opioid receptor antagonists such as naltriben (10 microM) and 7-benzylidenenaltrexone (10 microM) also prevented the effect of morphine, whereas CTOP (10 microM), a selective mu-opioid receptor antagonist, and nor-binaltorphimine (10 microM), a selective K-opioid receptor antagonist, were inactive. These data indicate that morphine tolerance is associated with delta-opioid receptor mediated activation of cortical CCKergic systems in rats.
Analgesics, Opioid↗
Hemodynamic effects of immunoadsorption and subsequent immunoglobulin substitution in dilated cardiomyopathy: three-month results from a randomized study.
OBJECTIVES: The objective of our study was to assess the hemodynamic effects of immunoadsorption (IA) and subsequent immunoglobulin G (IgG) substitution in comparison with the effects of conventional medical treatment in patients with dilated cardiomyopathy (DCM). BACKGROUND: Various circulating cardiac autoantibodies have been detected among patients suffering from DCM. These antibodies are extractable by IA. METHODS: Patients with DCM (n = 18, New York Heart Association III-IV, left ventricular ejection fraction <30%) and who were on stable medication participated in the study. Hemodynamic measurements were performed using a Swan-Ganz thermodilution catheter. The patients were randomly assigned either to the treatment group with IA and subsequent IgG substitution (IA/IgG group, n = 9) or to the control group without IA/IgG (n = 9). In the IA/IgG group, the patients were initially treated in one IA session daily on three consecutive days. After the final IA session, 0.5 g/kg of polyclonal IgG was substituted. At one-month intervals, IA was then repeated for three further courses with one IA session daily on two consecutive days, until the third month. RESULTS: After the first IA course and IgG substitution, cardiac index (CI) increased from 2.1 (+/-0.1) to 2.8 (+/-0.1) L/min/m2 (p < 0.01) and stroke volume index (SVI) increased from 27.8 (+/-2.3) to 36.2 (+/-2.5) ml/m2 (p < 0.01). Systemic vascular resistance (SVR) decreased from 1,428 (+/-74) to 997 (+/-55) dyne x s x cm(-5) (p < 0.01). The improvement in CI, SVI and SVR persisted after three months. In contrast, hemodynamics did not change throughout the three months in the control group. CONCLUSIONS: Immunoadsorption and subsequent IgG substitution improves cardiovascular function in DCM.
Adult↗
Monitoring gene flow from transgenic sugar beet using cytoplasmic male-sterile bait plants.
One of the most discussed environmental effects associated with the use of transgenic plants is the flow of genes to plants in the environment. The flow of genes may occur through pollen since it is the reproductive system that is designed for gene movement. Pollen-mediated gene escape is hard to control in mating plants. Pollen from a wind pollinator can move over distances of more than 1000 m. To investigate the efficiency of transgenic pollen movement under realistic environmental conditions, the use of bait plants might be an effective tool. In this study, cytoplasmic male-sterile (CMS) sugar beets were tested with regard to their potential for monitoring transgene flow. As the pollen source, transgenic sugar beets were used that express recombinant DNA encoding viral (beet necrotic yellow vein virus) resistance, and antibiotic (kanamycin) and herbicide (glufosinate) tolerance genes. In a field trial, the effectiveness of a hemp (Cannabis sativa) stripe containment strategy was tested by measuring the frequency of pollinated CMS bait plants placed at different distances and directions from a transgenic pollen source. The results demonstrated the ineffectiveness of the containment strategy. Physiological and molecular tests confirmed the escape and production of transgenic offspring more than 200 m behind the hemp containment. Since absolute containment is unlikely to be effective, the CMS-bait plant detection system is a useful tool for other monitoring purposes.
Aminobutyrates↗
Stabilization of NAD-dependent formate dehydrogenase from Candida boidinii by site-directed mutagenesis of cysteine residues.
The gene of the NAD-dependent formate dehydrogenase (FDH) from the yeast Candida boidinii was cloned by PCR using genomic DNA as a template. Expression of the gene in Escherichia coli yielded functional FDH with about 20% of the soluble cell protein. To confirm the hypothesis of a thiol-coupled inactivation process, both cysteine residues in the primary structure of the enzyme have been exchanged by site-directed mutagenesis using a homology model based on the 3D structure of FDH from Pseudomonas sp. 101 and from related dehydrogenases. Compared to the wt enzyme, most of the mutants were significantly more stable towards oxidative stress in the presence of Cu(II) ions, whereas the temperature optima and kinetic constants of the enzymatic reaction are not significantly altered by the mutations. Determination of the Tm values revealed that the stability at temperatures above 50 degrees C is optimal for the native and the recombinant wt enzyme (Tm 57 degrees C), whereas the Tm values of the mutant enzymes vary in the range 44-52 degrees C. Best results in initial tests concerning the application of the enzyme for regeneration of NADH in biotransformation of trimethyl pyruvate to Ltert leucine were obtained with two mutants, FDHC23S and FDHC23S/C262A, which are significantly more stable than the wt enzyme.
Amino Acid Sequence↗