A simplified hydroethidine method for fast and accurate detection of superoxide production in isolated mitochondria

Because superoxide is involved in various physiological processes, many efforts have been made to improve its accurate quantification. We optimized and validated a superoxide-specific and -sensitive detection method. The protocol is based on fluorescence detection of the superoxide-specific hydroethidine (HE) oxidation product, 2-hydroxyethidium. We established a method for the quantification of superoxide production in isolated mitochondria without the need for acetone extraction and purification chromatography as described in previous studies.     

Induced tyramine overproduction in transgenic rice plants expressing a rice tyrosine decarboxylase under the control of methanol-inducible rice tryptophan decarboxylase promoter

Tyramine, one of the various biogenic amines found in plants, is derived from the aromatic L-amino acid tyrosine through the catalytic reaction of tyrosine decarboxylase (TYDC). Tyramine overproduction by constitutive expression of TYDC in rice plants leads to stunted growth, but an increased number of tillers. To regulate tyramine production in rice plants, we expressed TYDC under the control of a methanol-inducible plant tryptophan decarboxylase (TDC) promoter and generated transgenic T(2) homozygous rice plants. The transgenic rice plants showed normal growth phenotypes with slightly increased levels of tyramine in seeds relative to wild type. Upon treatment with 1% methanol, the transgenic rice leaves produced large amounts of tyramine, whereas no increase in tyramine production was observed in wild-type plants. The methanol-induced accumulation of tyramine in the transgenic rice leaves was inversely correlated with the tyrosine level. These data indicate that tyramine production in rice plants can be artificially controlled using the methanol-inducible TDC promoter, suggesting that this promoter could be used to selectively induce the expression of other proteins or metabolites in rice plants.     

Coupled oxygen transport analysis in the avascular wall of a coronary artery stenosis during angioplasty

The coupled oxygen transport in the avascular wall of a coronary artery stenosis is studied numerically by solving the convection-diffusion equations. Two geometries replicating stenosis before and after percutaneous transluminal coronary angioplasty (PTCA) are used for the analysis. The results are compared to evaluate the effect of the degree of stenosis on oxygen transport. Important physiological aspects, such as oxygen consumption in the wall, oxygen carried by the hemoglobin, non-Newtonian viscosity of the blood, and supply of oxygen from the vasa vasorum are included. The results show that the PO2 in the medial region of the arterial wall is approximately 10mmHg. The oxygen flux to the wall increases in the flow acceleration region, whereas it decreases at the flow reattachment zone. Near the location of flow separation, there is a small rise followed by a sharp fall in the oxygen flux. The drop in the oxygen flux to the wall at the point of flow reattachment for pre-PTCA stenosis is four times that for post-PTCA stenosis. The minimum PO2 in the avascular wall, PO2,min, at this location decreases to approximately 6.0 and 4.2mmHg for post- and pre-PTCA stenosis, respectively. The drop in PO2,w and PO2,min at the point of flow reattachment for pre-PTCA is approximately 2 times that for post-PTCA stenosis. Thus, the present study quantifies the oxygen transport to the arterial wall before and after cardiovascular intervention.     

Functional analysis of the amine substrate specificity domain of pepper tyramine and serotonin N-hydroxycinnamoyltransferases

Pepper (Capsicum annuum) serotonin N-hydroxycinnamoyltransferase (SHT) catalyzes the synthesis of N-hydroxycinnamic acid amides of serotonin, including feruloylserotonin and p-coumaroylserotonin. To elucidate the domain or the key amino acid that determines the amine substrate specificity, we isolated a tyramine N-hydroxycinnamoyltransferase (THT) gene from pepper. Purified recombinant THT protein catalyzed the synthesis of N-hydroxycinnamic acid amides of tyramine, including feruloyltyramine and p-coumaroyltyramine, but did not accept serotonin as a substrate. Both the SHT and THT mRNAs were found to be expressed constitutively in all pepper organs. Pepper SHT and THT, which have primary sequences that are 78% identical, were used as models to investigate the structural determinants responsible for their distinct substrate specificities and other enzymatic properties. A series of chimeric genes was constructed by reciprocal exchange of DNA segments between the SHT and THT cDNAs. Functional characterization of the recombinant chimeric proteins revealed that the amino acid residues 129 to 165 of SHT and the corresponding residues 125 to 160 in THT are critical structural determinants for amine substrate specificity. Several amino acids are strongly implicated in the determination of amine substrate specificity, in which glycine-158 is involved in catalysis and amine substrate binding and tyrosine-149 plays a pivotal role in controlling amine substrate specificity between serotonin and tyramine in SHT. Furthermore, the indisputable role of tyrosine is corroborated by the THT-F145Y mutant that uses serotonin as the acyl acceptor. The results from the chimeras and the kinetic measurements will direct the creation of additional novel N-hydroxycinnamoyltransferases from the various N-hydroxycinnamoyltransferases found in nature.     

Developing pulsatile flow in a deployed coronary stent

A major consequence of stent implantation is restenosis that occurs due to neointimal formation. This patho-physiologic process of tissue growth may not be completely eliminated. Recent evidence suggests that there are several factors such as geometry and size of vessel, and stent design that alter hemodynamic parameters, including local wall shear stress distributions, all of which influence the restenosis process. The present three-dimensional analysis of developing pulsatile flow in a deployed coronary stent quantifies hemodynamic parameters and illustrates the changes in local wall shear stress distributions and their impact on restenosis. The present model evaluates the effect of entrance flow, where the stent is placed at the entrance region of a branched coronary artery. Stent geometry showed a complex three-dimensional variation of wall shear stress distributions within the stented region. Higher order of magnitude of wall shear stress of 530 dyn/cm2 is observed on the surface of cross-link intersections at the entrance of the stent. A low positive wall shear stress of 10 dyn/cm2 and a negative wall shear stress of -10 dyn/cm2 are seen at the immediate upstream and downstream regions of strut intersections, respectively. Modified oscillatory shear index is calculated which showed persistent recirculation at the downstream region of each strut intersection. The portions of the vessel where there is low and negative wall shear stress may represent locations of thrombus formation and platelet accumulation. The present results indicate that the immediate downstream regions of strut intersections are areas highly susceptible to restenosis, whereas a high shear stress at the strut intersection may cause platelet activation and free emboli formation.     

Hemodynamic diagnostics of epicardial coronary stenoses: <Emphasis Type="Italic">in-vitro</Emphasis> experimental and computational study

Background  

The severity of epicardial coronary stenosis can be assessed by invasive measurements of trans-stenotic pressure drop and flow. A pressure or flow sensor-tipped guidewire inserted across the coronary stenosis causes an overestimation in true trans-stenotic pressure drop and reduction in coronary flow. This may mask the true severity of coronary stenosis. In order to unmask the true severity of epicardial stenosis, we evaluate a diagnostic parameter, which is obtained from fundamental fluid dynamics principles. This experimental and numerical study focuses on the characterization of the diagnostic parameter, pressure drop coefficient, and also evaluates the pressure recovery downstream of stenoses.     

Long‐term effects of drainage and initial effects of hydrological restoration on rich fen vegetation

Questions: What vegetational changes does a boreal rich fen (alkaline fen) undergo during a time period of 24 years after drainage? How is plant species richness affected, and what are the changes in composition of ecological groups of species? Is it possible to recover parts of the original flora by rewetting the rich fen? Which are the initial vegetation changes in the flora after rewetting? What are the major challenges for restoration of rich fen flora after rewetting? Location: Eastern central Sweden, southern boreal vegetational zone. Previously rich fen site, drained for forestry purposes during 1978–1979. The site was hydrologically restored (rewetted) in 2002. Method: Annual vegetation survey in permanent plots during a period of 28 years. Results: There were three successional stages in the vegetational changes. In the first stage there was a rapid (< 5 years) loss of rich fen bryophytes. The second step was an increase of sedges and early successional bryophytes, which was followed by an increase of a few emerging dominants, such as Molinia caerulea, Betula pubescens and Sphagnum spp. After rewetting, there are indications of vegetation recovery, albeit at slow rates. Depending on, for instance, initial species composition different routes of vegetation change were observed in the flora after drainage, although after 24 years, species composition became more homogenous and dominated by a few species with high cover. Conclusion: Major changes have occurred after changes in the hydrology (drainage and rewetting) with a severe impact on the biodiversity among vascular plants and bryophytes. Several rich fen bryophytes respond quickly to the changes in water level (in contrast to vascular plants). The recovery after rewetting towards the original rich fen vegetation is slow, as delayed by substrate degradation, dispersal limitation and presence of dominant species.