Phenylpropanoids, phenylalanine ammonia lyase and peroxidases in elicitor-challenged cassava (Manihot esculenta) suspension cells and leaves

BACKGROUND AND AIMS: Control of diseases in the key tropical staple, cassava, is dependent on resistant genotypes, but the innate mechanisms are unknown. The aim was to study phenylpropanoids and associated enzymes as possible defence components. METHODS: Phenylalanine ammonia-lyase (PAL), phenylpropanoids and peroxidases (POD) were investigated in elicited cassava suspension cells and leaves. Yeast elicitor was the most effective of several microbial and endogenous elicitors. Fungitoxicity was determined against the cassava pathogens Fusarium solani, F. oxysporum and the saprotroph Trichoderma harzianum. KEY RESULTS: A single and rapid (> or =2-3 min) oxidative burst, measured as hydrogen peroxide, occurred in elicited cells. PAL activity was induced maximally at 15 h and was preceded by PAL mRNA accumulation, which peaked at 9 h. Symplasmic POD activity increased four-fold in cells, 48 h post-elicitation. POD isoforms (2-7 isoforms, pI 3.1-8.8) were detected in elicited and unelicited cells, extracellular medium and leaves but two extracellular isoforms were enhanced post-elicitation. Also expression of a cassava peroxidase gene MecPOD1 increased in elicited cells. Only anionic forms oxidized scopoletin, with highest activity by isoform pI 3.6, present in all samples. Unidentified phenolics and possibly scopolin increased post-elicitation, but there was no enhancement of scopoletin, rutin or kaempferol-3-O-rutinoside concentration. Fungal germ tube elongation was inhibited more than germination by esculetin, ferulic acid, quercetin and scopoletin. T. harzianum was generally more sensitive than the pathogens and was inhibited by > or =50 microg mL(-1) of ferulic acid and quercetin and > or =10 microg mL(-1) of scopoletin. CONCLUSIONS: Phenolic levels in cells were not enhanced and were, theoretically, too low to be inhibitory. However, in combination and when oxidized they may contribute to defence, because oxidation of esculetin and scopoletin by peroxidase and of esculetin by tyrosinase enhanced their fungitoxicity up to 20-fold.     

Comparing minimally invasive funnel chest repair versus the conventional technique: an outcome analysis in children

Surgical correction of pectus excavatum in children has gained new momentum since the introduction of the new minimally invasive repair by Nuss. To date, no studies directly evaluate the outcome of the new technique versus that of the conventional technique. From 2000 to 2002, 28 patients underwent pectus excavatum correction in the authors' hospital. Twenty-one were treated by minimally invasive repair of pectus excavatum and seven patients had open correction. Intraoperative and postoperative complications, clinical outcome, and patient satisfaction were evaluated. In the minimally invasive repair of pectus excavatum group, the children were younger (14.4 +/- 2.9 versus 17.8 +/- 3.2 years), had shorter operation times (53 +/- 18 versus 125 +/- 6 minutes), and had less blood loss (minimal versus 380 +/- 175 ml). No intraoperative complications were recorded. In the conventional group, two pleural lacerations occurred. Early postoperative complications in the minimally invasive repair group included two pneumothoraces and one case of pleural effusion. In the conventional group, one pneumothorax and one case of pleural effusion occurred. Late postoperative complications in the Nuss group included one costal erosion, two bar dislocations, one severe wound infection requiring bar removal, one hematothorax, and one case of postpericardiotomy syndrome; in the conventional group, there was one severe wound infection. In both groups, the patients rated their cosmetic results as good to very good. Minimally invasive repair of pectus excavatum is a novel method with clear advantages, such as limited surgical trauma and small scars. The high rate of postoperative complications may decrease with growing experience in the future. In well-selected patients (age, symmetric deformity), the Nuss procedure may become the method of choice. However, there is still a lack of long-term follow-up.     

Oxidative stress responses during cassava post-harvest physiological deterioration

A major constraint to the development of cassava (Manihot esculenta Crantz) as a crop to both farmers and processors is its starchy storage roots’ rapid post-harvest deterioration, which can render it unpalatable and unmarketable within 24–72 h. An oxidative burst occurs within 15 min of the root being injured, that is followed by the altered regulation of genes, notably for catalase and peroxidase, related to the modulation of reactive oxygen species, and the accumulation of secondary metabolites, some of which show antioxidant properties. The interactions between these enzymes and compounds, in particular peroxidase and the coumarin, scopoletin, are largely confined to the vascular tissues where the visible symptoms of deterioration are observed. These, together with other data, are used to develop a tentative model of some of the principal events involved in the deterioration process.     

Proteomic approach: Identification of <Emphasis Type="Italic">Medicago truncatula</Emphasis> proteins induced in roots after infection with the pathogenic oomycete <Emphasis Type="Italic">Aphanomyces euteiches</Emphasis>

The legume root rot disease caused by the oomycete pathogen Aphanomyces euteiches is one major yield reducing factor in legume crop production. A comparative proteomic approach was carried out in order to identify proteins of the model legume Medicago truncatula which are regulated after an infection with A. euteiches. Several proteins were identified by two dimensional gel electrophoresis to be differentially expressed after pathogen challenge. Densitometric evaluation of expression values showed different regulation during the time-course analysed. Proteins regulated during the infection were identified by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Among the differentially expressed proteins, two encoded putative cell wall proteins and two were designated as small heat shock proteins. Furthermore, an isoform of the chalcone-O-methyltransferase was found to be increased in infected roots. The majority of induced proteins belonged to the family of class 10 of pathogenesis related proteins (PR10). Previously, various PR10-like proteins have been shown to be regulated by general stress or abscisic acid (ABA). Therefore, these proteins were further investigated concerning their regulation in response to drought stress and exogenous ABA-application. Complex regulation patterns were identified: three of the A. euteiches-induced PR10-like proteins were also induced by exogenous ABA- but none of them is induced after drought stress. In contrast, three of these proteins are down-regulated by drought stress. Hence, the strong expression of different PR10-family members and their regulation profiles indicates that this set of proteins plays a major role during root adaptations to various stress conditions.     

Bone marrow angiogenesis: methods of quantification and changes evolving in chronic myeloproliferative disorders

Until now little information is available about bone marrow (BM) angiogenesis in chronic myeloproliferative disorders (CMPDs). Amongst the various immunohistochemical markers for endothelial cells CD34 and CD105 have proven to be most reliable since they exhibit no relevant co-staining. Determination of vascularity has to include pathophysiological aspects of perfusion. Therefore, quantification of the microvascular density (MVD) by the so-called hot spot method has to be improved by parameters that characterize blood flow more properly like microvessel area (luminal distension), shape (form factor), tortuosity, and branching (maximal vessel length). In comparison to the normal BM chronic myeloid leukemia (CML) revealed a significant increase in MVD which was functionally associated with elevated levels of angiogenic cytokines. Structure of vessels was significantly altered by showing an enhanced irregularity of shape and tortuosity and increase in fibers was conspicuously accompanied by a higher degree of MVD. Contrasting the group of patients with Imatinib (STI571) therapy interferon failed to reduce the number of vessels. Following bone marrow transplantation a significant enhancement of the MVD was found in the early post-transplant period, but after about 6 months normalization occurred. Anomalies of microvascular architecture were easily demonstrable by three-dimensional reconstruction and consisted of a complex branching network of irregular shaped sinuses. Chronic idiopathic myelofibrosis displayed a significant increase in the MVD only in the advanced fibrosclerotic stages. This feature was accompanied by an enhanced luminal distension and tortuosity, thus contrasting the prefibrotic and early fibrotic phases of this disorder. Similar to CML a relationship between evolving myelofibrosis and change in vascular architecture was encountered. This feature may present a possible target for future anti-angiogenic therapy. In essential thrombocythemia there is only a mild increase in MVD detectable while in polycythemia vera besides an enlarged number, a luminal dilation due to the densely packed erythrocytes is recognizable. In conclusion, contrasting the usually applied quantification technique more elaborate morphometrical methods are warranted to obtain a better insight into the vascular architecture of the BM. In CMPDs angiogenesis is significantly associated with the evolution of myelofibrosis and may be altered by therapeutic regimens probably due to changes in cytokine release.     

Changes in extracellular dopamine in the rat globus pallidus induced by typical and atypical antipsychotic drugs

Typical antipsychotic drugs with a high extrapyramidal motor side-effects liability markedly increase extracellular dopamine in the caudate-putamen, while atypical antipsychotic drugs with a low incidence of extrapyramidal motor side-effects have less pronounced stimulating actions on striatal dopamine. Therefore, it has been suggested that the extrapyramidal motor side-effects liability of antipsychotic drugs (APD) is correlated with their ability to increase extracellular dopamine in the caudate-putamen. The globus pallidus (GP) is another basal ganglia structure probably mediating extrapyramidal motor side-effects of typical antipsychotic drugs. Therefore, the present study sought to determine whether extracellular dopamine in the globus pallidus might be a further indicator to differentiate neurochemical actions of typical and atypical antipsychotic drugs. Using in vivo microdialysis we compared effects on pallidal dopamine induced by typical and atypical antipsychotic drugs in rats. Experiment I demonstrated that systemic administration of haloperidol (1 mg/kg; i.p.) and clozapine (20 mg/kg; i.p.) induced a significant pallidal dopamine release to about 160 and 180% of baseline, respectively. Experiment II revealed that reverse microdialysis of raclopride and clozapine using a cumulative dosing regimen did not stimulate extracellular dopamine in the globus pallidus if low (1microM) or intermediate (10 and 100 microM) concentrations were used. Only at a high concentration (1,000 microM), raclopride and clozapine induced a significant pallidal dopamine release to about 130 and 300% of baseline values, respectively. Thus, effects of typical and atypical antipsychotic drugs on pallidal dopamine were similar and thus, may not be related to their differential extrapyramidal motor side-effects liability. Furthermore, the finding that reverse microdialysis of raclopride over a wide range of concentrations did not stimulate pallidal dopamine concentrations tentatively suggests that pallidal dopamine release under basal conditions is not regulated by D2 autoreceptors.     

Mechanistic changes during phytoremediation of perchlorate under different root-zone conditions

Two types of hydroponic bioreactors were used to investigate the mechanisnistic changes during phytoremediation of perchlorate under different root-zone conditions. The bioreactors included: (1) an aerobic ebb-and-flow system planted with six willow trees, and (2) individual willow trees grown in sealed root-zone bioreactors. Rhizosphere probes were used to monitor for the first time during phytoremediation of perchlorate, diurnal swings in oxidation-reduction potential (E(H)), dissolved oxygen (DO), and pH. Radiolabeled (36Cl-labeled) perchlorate was used as a tracer in a subset of the sealed bioreactor experiments to quantify the contribution of phytodegradation and rhizodegradation mechanisms. Rhizodegradation accounted for the removal of 96.1 +/- 4.5% (+/-95% CI) of the initial perchlorate dose in experiments conducted in sealed hydroponic bioreactors with low DO and little or no nitrate N. Meanwhile, the contribution of rhizodegradation decreased to 76 +/- 14% (+/-95% CI) when nitrate (a competing terminal electron acceptor) was provided as the nitrogen source. Slower rates of phytoremediation by uptake and phytodegradation were observed under high nitrate concentrations and aerobic conditions, which allowed perchlorate to persist in solution and resulted in a higher fraction uptake by the plant. Specifically, the rate of removal of perchlorate from bulk solution ranged from 5.4 +/- 0.54 to 37.1 +/- 2.25 mg/L/d (+/-SE) in the absence of nitrate to 1.78 +/- 0.27 to 0.46 +/- 0.02 mg/L/d (+/-SE) at high nitrate concentration. The results of this study indicate that the root-zone environment of plants can be manipulated to optimize rhizodegradation and to minimize undesirable processes such as uptake, temporal phytoaccumulation, and slow phytodegradation during phytoremediation of perchlorate. Rhizodegradation is desired because contaminants resident in plant tissue may remain an ecological risk until completely phytodegraded.