Nanostructuring discotic molecules on ITO support.

Patterning of organic compounds on a nanometer length scale is of great interest for solar applications: defined control over the donor-acceptor interface will allow design of an optimized nano-morphology promoting exciton separation and reducing charge recombination. Herein we present an imprinting technique using anodized alumina oxide (AAO) hard templates as stamps. We show an exact pattern transfer of the AAO structures into a solution processable hexa-peri-hexabenzocoronene (HBC), a discotic small molecule with acrylate moieties which is polymerized in situ. Film fabrication is realized for a variety of nanowire dimensions on square centimeter areas. The fabrication directly on conductive glass support and control over the formation of a dense barrier layer render this approach appealing for the fabrication of fully organic nanostructured photovoltaic devices.

Direct imaging of aligned neurofilament networks assembled using in situ dialysis in microchannels.

We report a technique to produce aligned neurofilament networks for direct imaging and diffraction studies using in situ dialysis in a microfluidic device. The alignment is achieved by assembling neurofilaments from protein subunits confined within microchannels. Resulting network structure was probed by polarized optical microscopy and atomic force microscopy, which confirmed a high degree of protein alignment inside the microchannels. This technique can be expanded to facilitate structural studies of a wide range of filamentous proteins and their hierarchical assemblies under varying assembly conditions.

Regulation of aspartate-derived amino acid homeostasis in potato plants (Solanum tuberosum L.) by expression of E. coli homoserine kinase.

The availability of the carbon backbone O-phosphohomoserine (OPHS) is critical to methionine (met) and threonine (thr) synthesis. OPHS derives from homoserine and is formed by homoserine kinase (HSK). To clarify the function of HSK in cellular metabolism, the E. coli HSK ortholog thrB was expressed in potato plants targeting the EcHSK protein to chloroplasts and to the cytosol. Both approaches resulted in up to 11 times increased total HSK enzyme activity. Transgenic plants exhibited reduced homoserine levels while met and thr did not accumulate significantly. However, the precursor cysteine and upstream intermediates of met such as cystathionine and homocysteine did indicating an accelerated carbon flow towards the end products. Coincidently, plants with elevated cytosolic levels of EcHSK exhibited a reduction in transcript levels of the endogenous HSK, as well as of threonine synthase (TS), cystathionine beta-lyase (CbL), and met synthase (MS). In all plants, cystathionine gamma-synthase (CgS) expression remained relatively unchanged from wild type levels, while S-adenosylmethionine synthetase (SAMS) expression increased. Feeding studies with externally supplied homoserine fostered the synthesis of met and thr but the regulation of synthesis of both amino acids retained the wild type regulation pattern. The results indicate that excess of plastidial localised HSK activity does not influence the de novo synthesis of met and thr. However, expression of HSK in the cytosol resulted in the down-regulation of gene expression of pathway genes probably mediated via OPHS. We integrated these data in a novel working model describing the regulatory mechanism of met and thr homeostasis.

Effect of sulfur availability on the integrity of amino acid biosynthesis in plants.

Amino acid levels in plants are regulated by a complex interplay of regulatory circuits at the level of enzyme activities and gene expression. Despite the diversity of precursors involved in amino acid biosynthesis as providing the carbon backbones, the amino groups and, for the amino acids methionine and cysteine, the sulfhydryl group and despite the involvement of amino acids as substrates in various downstream metabolic processes, the plant usually manages to provide relatively constant levels of all amino acids. Here we collate data on how amino acid homeostasis is shifted upon depletion of one of the major biosynthetic constituents, i.e., sulfur. Arabidopsis thaliana seedlings exposed to sulfate starvation respond with a set of adaptation processes to achieve a new balance of amino acid metabolism. First, metabolites containing reduced sulfur (cysteine, glutathione, S-adenosylmethionine) are reduced leading to a number of downstream effects. Second, the relative excess accumulation of N over S triggers processes to dump nitrogen in asparagine, glutamine and further N-rich compounds like ureides. Third, the depletion of glutathione affects the redox and stress response system of the glutathione-ascorbate cycle. Thus, biosynthesis of aromatic compounds is triggered to compensate for this loss, leading to an increased flux and accumulation of aromatic amino acids, especially tryptophan. Despite sulfate starvation, the homeostasis is kept, though shifted to a new state. This adaptation process keeps the plant viable even under an adverse nutritional status.

Engineering of cysteine and methionine biosynthesis in potato.

Methionine and cysteine, two amino acids containing reduced sulfur, are not only an important substrate of protein biosynthesis but are also precursors of various other metabolites such as glutathione, phytochelatines, S-adenosylmethionine, ethylene, polyamines, biotin, and are involved as methyl group donor in numerous cellular processes. While methionine is an essential amino acid due to an inability of monogastric animals and human beings to synthesise this metabolite, animals are still able to convert methionine consumed with their diet into cysteine. Thus, a balanced diet containing both amino acids is necessary to provide a nutritionally favourable food or feed source. Because the concentrations of methionine and cysteine are often low in edible plant sources, e.g. potato, considerable efforts in plant breeding and research have been and are still performed to understand the physiological, biochemical, and molecular mechanisms that contribute to their synthesis, transport, and accumulation in plants. During the last decade molecular tools have enabled the isolation of most of the genes involved in cysteine and methionine biosynthesis, and the efficient plant transformation technology has allowed the creation of transgenic plants that are altered in the activity of individual genes. The physiological analysis of these transgenic plants has contributed considerably to our current understanding of how amino acids are synthesised. We focused our analysis on potato (Solanum tuberosum cv. Désirée) as this plant provides a clear separation of source and sink tissues and, for applied purposes, already constitutes a crop plant. From the data presented here and in previous work we conclude that threonine synthase and not cystathionine gamma-synthase as expected from studies of Arabidopsis constitutes the main regulatory control point of methionine synthesis in potato. This article aims to cover the current knowledge in the area of molecular genetics of sulfur-containing amino acid biosynthesis and will provide new data for methionine biosynthesis in solanaceous plants such as potato.

Antisense inhibition of threonine synthase leads to high methionine content in transgenic potato plants.

Methionine (Met) and threonine (Thr) are members of the aspartate family of amino acids. In plants, their biosynthetic pathways diverge at the level of O-phosphohomo-serine (Ser). The enzymes cystathionine gamma-synthase and Thr synthase (TS) compete for the common substrate O-phosphohomo-Ser with the notable feature that plant TS is activated through S-adenosyl-Met, a metabolite derived from Met. To investigate the regulation of this branch point, we engineered TS antisense potato (Solanum tuberosum cv Désirée) plants using the constitutive cauliflower mosaic virus 35S promoter. In leaf tissues, these transgenics exhibit a reduction of TS activity down to 6% of wild-type levels. Thr levels are reduced to 45% wild-type controls, whereas Met levels increase up to 239-fold depending on the transgenic line and environmental conditions. Increased levels of homo-Ser and homo-cysteine indicate increased carbon allocation into the aspartate pathway. In contrast to findings in Arabidopsis, increased Met content has no detectable effect on mRNA or protein levels or on the enzymatic activity of cystathionine gamma-synthase in potato. Tubers of TS antisense potato plants contain a Met level increased by a factor of 30 and no reduction in Thr. These plants offer a major biotechnological advance toward the development of crop plants with improved nutritional quality.

Approaches towards understanding methionine biosynthesis in higher plants.

Plants are able to synthesise all amino acids essential for human and animal nutrition. Because the concentrations of some of these dietary constituents, especially methionine, lysine, and threonine, are often low in edible plant sources, research is being performed to understand the physiological, biochemical, and molecular mechanisms that contribute to their transport, synthesis and accumulation in plants. This knowledge can be used to develop strategies allowing a manipulation of crop plants, eventually improving their nutritional quality. This article is intended to serve two purposes. The first is to provide a brief review on the physiology of methionine synthesis in higher plants. The second is to highlight some recent findings linked to the metabolism of methionine in plants due to its regulatory influence on the aspartate pathway and its implication in plant growth. This information can be used to develop strategies to improve methionine content of plants and to provide crops with a higher nutritional value.

Manipulation of thiol contents in plants.

As sulfur constitutes one of the macronutrients necessary for the plant life cycle, sulfur uptake and assimilation in higher plants is one of the crucial factors determining plant growth and vigour, crop yield and even resistance to pests and stresses. Inorganic sulfate is mostly taken up as sulfate from the soil through the root system or to a lesser extent as volatile sulfur compounds from the air. In a cascade of enzymatic steps inorganic sulfur is converted to the nutritionally important sulfur-containing amino acids cysteine and methionine (Hell, 1997; Hell and Rennenberg, 1998; Saito, 1999). Sulfate uptake and allocation between plant organs or within the cell is mediated by specific transporters localised in plant membranes. Several functionally different sulfate transporters have to be postulated and have been already cloned from a number of plant species (Clarkson et al., 1993; Hawkesford and Smith, 1997; Takahashi et al., 1997; Yamaguchi, 1997). Following import into the plant and transport to the final site of reduction, the plastid, the chemically relatively inert sulfate molecule is activated through binding to ATP forming adenosine-5'-phosphosulfate (APS). This enzymatic step is controlled through the enzyme ATP-sulfurylase (ATP-S). APS can be further phosphorylated to form 3'-phosphoadenosine-5'-phosphosulfate (PAPS) which serves as sulfate donor for the formation of sulfate esters such as the biosynthesis of sulfolipids (Schmidt and Jäger, 1992). However, most of the APS is reduced to sulfide through the enzymes APS-reductase (APR) and sulfite reductase (SIR). The carbon backbone of cysteine is provided through serine, thus directly coupling photosynthetic processes and nitrogen metabolism to sulfur assimilation. L-serine is activated by serine acetyltransferase (SAT) through the transfer to an acetyl-group from acetyl coenzyme A to form O-acetyl-L-serine (OAS) which is then sulhydrylated using sulfide through the enzyme O-acetyl-L-serine thiol lyase (OAS-TL) forming cysteine. Cysteine is the central precursor of all organic molecules containing reduced sulfur ranging from the amino acid methionine to peptides as glutathione or phytochelatines, proteines, vitamines, cofactors as SAM and hormones. Cysteine and derived metabolites display essential roles within plant metabolism such as protein stabilisation through disulfide bridges, stress tolerance to active oxygen species and metals, cofactors for enzymatic reactions as e.g. SAM as major methylgroup donor and plant development and signalling through the volatile hormone ethylene. Cysteine and other metabolites carrying free sulfhydryl groups are commonly termed thioles (confer Fig. 1). The physiological control of the sulfate reduction pathway in higher plants is still not completely understood in all details. The objective of this paper is to summarise the available data on the molecular analysis and control of cysteine biosynthesis in plants, and to discuss potentials for manipulating the pathway using transgenic approaches.

Indirect RT-PCR in-situ hybridization: a novel non-radioactive method for detecting glucose-dependent insulinotropic peptide.

To establish indirect in-situ PCR for the detection of intestinal peptide hormones, rat intestine and a murine intestinal tumor cell line, STC 1, were used. The results exhibited intensive staining of GIP-producing K-cells. Paraformaldehyde-fixed cryostat sections yielded the best results in signal to background ratio with RT-PCR in-situ hybridization. Moreover, it was possible to elevate the positive staining signal and to reduce background staining. Digoxigenin-labeled in-situ hybridization served as a control for specificity and sensitivity of GIP (glucose-dependent insulinotropic peptide) mRNA expression on cryostat as well as paraffin sections. In conclusion, this RT-PCR in-situ hybridization protocol proves to be a specific, sensitive and reliable non-radioactive technique for the detection of intestinal peptide hormone mRNA, especially in tissues or tumor cells where the application of ISH is limited.

Enhanced cystathionine beta-lyase activity in transgenic potato plants does not force metabolite flow towards methionine.

Cystathionine beta-lyase (CbL) catalyses the second step in higher-plant methionine biosynthesis. To further characterise the role of CbL in methionine biosynthesis, transgenic potato (Solanum tuberosum L.) plants were generated that express a potato cystathionine beta-lyase (StCbL; EC 4.4.1.8) under the control of the cauliflower mosaic virus 35 S promoter. Transgenic potato lines showed no visible phenotype but revealed an accumulation of both CbL transcript and protein. The enzymatic activity of CbL in these lines was up to 2.5-fold higher than that of wild-type plants. GC-MS measurements of aspartate-derived metabolites, however, showed no significant changes in content of amino acids and pathway intermediates when transgenic and wild-type plants were compared. CbL over-expression did not change the expression patterns and gene products of other pathway-relevant genes as evident from RNA and protein blot analyses. Despite the essential role of CbL in plant growth and development, the data presented indicate that the homologous over-expression of CbL is not in itself able to enhance metabolic flux towards methionine biosynthesis.

Transgenic potato plants reveal the indispensable role of cystathionine beta-lyase in plant growth and development.

Methionine (Met) is an essential amino acid that is often unavailable at sufficient dietary levels. In order to better understand Met pathway regulation, a cDNA encoding cystathionine beta-lyase (CbL; EC 4.4.1.8) has been cloned from Solanum tuberosum. An antisense construct of this gene was used to generate transgenic potato plants with reduced CbL levels. Transgenic plants exhibiting leaf CbL activity levels of up to 50% below wild-type levels were obtained. Metabolite analysis revealed a reduction in Met levels in these CbL antisense plants, as well as remarkable increases in the pathway intermediates cystathionine, homoserine and cysteine. Unexpectedly, an increase in homocysteine was also observed. Levels of aspartate amino acid pathway intermediates (including aspartate, lysine and threonine) remained essentially unaffected. Neither transcript levels nor protein products of other pathway-relevant genes were altered significantly in these plants. CbL antisense plants exhibited an altered phenotype characterized by a bushy growth habit, small light-green leaves and small tubers. This phenotype could be alleviated upon Met supplementation, suggesting that low Met levels, rather than pathway intermediate accumulation, is responsible for the phenotypic effects of CbL transgene expression. These data unequivocally demonstrate the central role of CbL in Met biosynthesis, and, subsequently, in plant growth and development.

Expression of threonine synthase from Solanum tuberosum L. is not metabolically regulated by photosynthesis-related signals or by nitrogenous compounds.

Although the control of carbon fixation and nitrogen assimilation has been studied in detail, little is known about the regulation of carbon and nitrogen flow into amino acids. In this paper the isolation of a cDNA encoding threonine synthase is reported (TS; EC 4.2.99.2) from a leaf lambda ZAP II-library of Solanum tuberosum L. and the transcriptional regulation of the respective gene expression in response to metabolic changes. The pattern of expression of TS by feeding experiments of detached petioles revealed that TS expression is regulated neither by photosynthesis-related metabolites nor by nitrogenous compounds. The present study suggests that the regulation of the conversion of aspartate to threonine is not controlled at the transcript level of TS. The nucleotide and deduced amino acid sequences of potato TS show homology to other known sequences from Arabidopsis thaliana and microorganisms. TS is present as a low copy gene in the genome of potato as demonstrated in Southern blot analysis. When cloned into a bacterial expression vector, the cDNA did functionally complement the Escherichia coli mutant strain Gif41. TS transcript was found in all tissues of potato and was most abundant in flowers and source leaves.

Expression of a bacterial serine acetyltransferase in transgenic potato plants leads to increased levels of cysteine and glutathione.

The coding sequence of the wild-type, cys-sensitive, cysE gene from Escherichia coli, which encodes an enzyme of the cysteine biosynthetic pathway, namely serine acetyltransferase (SAT, EC 2.3.1. 30), was introduced into the genome of potato plants under the control of the cauliflower mosaic virus 35S promoter. In order to target the protein into the chloroplast, cysE was translationally fused to the 5'-signal sequence of rbcS from Arabidopsis thaliana. Transgenic plants showed a high accumulation of the cysE mRNA. The chloroplastic localisation of the E. coli SAT protein was demonstrated by determination of enzymatic activities in enriched organelle fractions. Crude leaf extracts of these plants exhibited up to 20-fold higher SAT activity than those prepared from wild-type plants. The transgenic potato plants expressing the E. coli gene showed not only increased levels of enzyme activity but also exhibited elevated levels of cysteine and glutathione in leaves. Both were up to twofold higher than in control plants. However, the thiol content in tubers of transgenic lines was unaffected. The alterations observed in leaf tissue had no effect on the expression of O-acetylserine(thiol)-lyase, the enzyme which converts O-acetylserine, the product of SAT, to cysteine. Only a minor effect on its enzymatic activity was observed. In conclusion, the results presented here demonstrate the importance of SAT in plant cysteine biosynthesis and show that production of cysteine and related sulfur-containing compounds can be enhanced by metabolic engineering.

Molecular cloning and expression analyses of mitochondrial and plastidic isoforms of cysteine synthase (O-acetylserine(thiol)lyase) from Arabidopsis thaliana.

Cysteine synthase, the key enzyme for fixation of inorganic sulfide, catalyses the formation of cysteine from O-acetylserine and inorganic sulfide. Here we report the cloning of cDNAs encoding cysteine synthase isoforms from Arabidopsis thaliana. The isolated cDNA clones encode for a mitochondrial and a plastidic isoform of cysteine synthase (O-acetylserine (thiol)-lyase, EC 4.2.99.8), designated cysteine synthase C (AtCS-C, CSase C) and B (AtCS-B; CSase B), respectively. AtCS-C and AtCS-B, having lengths of 1569-bp and 1421-bp, respectively, encode polypeptides of 430 amino acids (approximately 45.8 kD) and of 392 amino acids (approximately 41.8 kD), respectively. The deduced amino acid sequences of the mitochondrial and plastidic isoforms exhibit high homology even with respect to the presequences. The predicted presequence of AtCS-C has a N-terminal extension of 33 amino acids when compared to the plastidic isoform. Northern blot analysis showed that AtCS-C is higher expressed in roots than in leaves whereas the expression of AtCS-B is stronger in leaves. Furthermore, gene expression of both genes was enhanced by sulfur limitation which in turn led to an increase in enzyme activity in crude extracts of plants. Expression of the AtCS-B gene is regulated by light. The mitochondrial, plastidic and cytosolic (Hesse and Altmann, 1995) isoforms of cysteine synthase of Arabidopsis are able to complement a cysteine synthase-deficient mutant of Escherichia coli unable to grow on minimal medium without cysteine, indicating synthesis of functional plant proteins in the bacterium. Two lines of evidence proved that AtCS-C encodes a mitochondrial form of cysteine synthase; first, import of in vitro translation products derived from AtCS-C in isolated intact mitochondria and second, Western blot analysis of mitochondria isolated from transgenic tobacco plants expressing AtCS-C cDNA/c-myc DNA fusion protein.

Elevation of the pacing threshold: a side effect in a patient with pacemaker undergoing therapy with doxorubicin and vincristine.

A 56-year-old female patient with an IgG plasmocytoma first diagnosed 5 years before underwent 3 cycles of chemotherapy according to the VAD scheme. A VVI pacemaker had been implanted 3 years earlier. After each cycle, the output of the pacemaker had to be increased as the pacing threshold had increased, resulting in a slow pulse. This case demonstrates that patients with a pacemaker that undergo chemotherapy with cardiotoxic agents must be followed carefully.

Interactions between pacemakers and security systems.

Electromagnetic fields arising from a variety of different sources have been shown to interfere with normal pacemaker function. This study evaluated the possible interactions between two modern security systems and different pacemaker types. Fifty-three patients (27 single chamber pacemakers, 25 dual chamber pacemakers) have been tested routinely for their pacemaker function. Thirty-eight patients presented with unipolar sensing and 15 with bipolar sensing. The patients were asked to walk through an installed security system, an antitheft device, and electromagnetic access device with different field strengths while a six-channel ECG monitored the patients. The pacemaker systems were first measured in their basic programmed modes, then the intervention frequency was changed to 100/min and, thereafter, the maximum sensitivity without T wave oversensing was added. In the security system with the highest field strength (2,700 mA/m), a pacemaker malfunction could be observed in 13% of the monitored patients. In one case, a pacemaker (VVIR) switched to ventricular safety pacing (VOO mode). In the security system with the lower field strength (1,600 mA/m) we found a pacemaker malfunction in 4% of the tested patients. In the antitheft device (50 mA/m), in the electromagnetic access device (300 mA/m), and in pacemaker systems with bipolar sensing, none of these dysfunctions were observed. Phantom programming as described previously did not occur in any of the systems. Persons who are often in the vicinity of security systems should be equipped with a bipolar pacemaker system. Our findings indicate that patients with pacemakers should avoid contact with security systems.

Central somatosensory conduction time in severely growth-stunted children.

To examine the effects of chronic malnutrition on central nervous system function, we used the somatosensory evoked potential to measure the central conduction time of 20 children aged 7-8 y with heights below the third percentile for their age and 20 control children in Honduras. The two groups differed significantly in socioeconomic status, achievement in Bender's neurointegrative test, and hematocrit, but not in birth weight. After median nerve stimulation, the mean central conduction time (interpeak latency between N13 and N20) for the growth-stunted group (6.19 +/- 0.52 ms) did not differ significantly from that of the control subjects (6.30 +/- 0.58 ms), suggesting appropriate myelination and fiber diameter. Somatosensory tracts may escape damage resulting from postnatal dietary deficiencies because myelination in these tracts is almost complete at birth.

Papillary muscle injury after blunt chest trauma.

In many cases blunt chest trauma involves cardiac lesions, such as pericardial effusion, aneurysma dissecans, or valvular rupture. Early diagnosis with routine transthoracic and/or transesophageal echocardiography is essential to prevent a fatal outcome. In the case reported, a previously healthy 68-year-old woman fell 7 meters from the roof of a barn and sustained blunt injury to the chest as well as fractures of the face. Physical examination revealed a systolic murmur at the cardiac apex, and chest x-ray film showed a severe pulmonary edema. Transesophageal echocardiography demonstrated a ruptured anterolateral papillary muscle with fourth degree mitral insufficiency. An immediate mitral valve replacement was necessary.