Mutants for photosystem I subunit D of Arabidopsis thaliana: effects on photosynthesis, photosystem I stability and expression of nuclear genes for chloroplast functions

In Arabidopsis thaliana, the D-subunit of photosystem I (PSI-D) is encoded by two functional genes, PsaD1 and PsaD2, which are highly homologous. Knock-out alleles for each of the loci have been identified by a combination of forward and reverse genetics. The double mutant psad1-1 psad2-1 is seedling-lethal, high-chlorophyll-fluorescent and deficient for all tested PSI subunits, indicating that PSI-D is essential for photosynthesis. In addition, psad1-1 psad2-1 plants show a defect in the accumulation of thylakoid multiprotein complexes other than PSI. Of the single-gene mutations, psad2 plants behave like wild-type (WT) plants, whereas psad1-1 markedly affects the accumulation of PsaD mRNA and protein, and photosynthetic electron flow. Additional effects of the psad1-1 mutation include a decrease in growth rate under greenhouse conditions and downregulation of the mRNA expression of most genes involved in the light phase of photosynthesis. In the same mutant, a marked decrease in the levels of PSI and PSII polypeptides is evident, as well as a light-green leaf coloration and increased photosensitivity. Increased dosage of PsaD2 in the psad1-1 background restores the WT phenotype, indicating that PSI-D1 and PSI-D2 have redundant functions.     

Modelling of the provisional side-branch stenting approach for the treatment of atherosclerotic coronary bifurcations: effects of stent positioning

The most common approach to treat atherosclerosis in coronary bifurcations is the provisional side-branch (PSB) stenting, which consists sequentially of the insertion of a stent in the main branch (MB) of the bifurcation and a dilatation of the side branch (SB) passing through the struts of the stent at the bifurcation. This approach can be followed by a redilatation of the MB only or by a Final Kissing Balloon (FKB) inflation, both strategies leading to a minor stent distortion in the MB. The positioning of the stent struts in the bifurcation and the stresses generated in the stent and vessel wall are worthy of investigation for a better understanding of the mechanobiology of the system. For this purpose, a computer model of an atherosclerotic coronary bifurcation based on the finite element method was developed; the effects of performing the final redilatation with the two strategies utilising one or two balloons and those created by a different stent strut positioning around the SB were investigated. Results correlate well with previous experimental tests regarding the deformation following balloon expansion. Furthermore, results confirm firstly that the re-establishment of an optimal spatial configuration of the stent after the PSB approach is achieved with both strategies; secondly, results show that case of stent positioning with one cell placed centrally (with regard to the SB) should be preferred, avoiding the presence of struts inside the vessel lumen, which may reduce hemodynamic disturbances. The central positioning also resulted in a better solution in terms of lower stresses in the stent struts and, more importantly, in the vascular tissues.     

Biotechnical Characteristics of Root Systems of Typical Mediterranean Species

Quantifying patterns of fine root dynamics is crucial to the understanding of ecosystem structure and function, and in predicting how ecosystems respond to disturbance. Part of this understanding involves consideration of the carbon lost through root turnover. In the context of the rainfall pattern in the tropics, it was hypothesised that rainfall would strongly influence fine root biomass and longevity. A field study was conducted to determine root biomass, elemental composition and the influence of rainfall on longevity of fine roots in a tropical lowland evergreen rainforest at Danum Valley, Sabah, Malaysia. A combination of root coring, elemental analysis and rhizotron observation methods were used. Fine (less than 2 mm diameter) root biomass was relatively low (1700 kg ha −1) compared with previously described rainforest data. Standing root biomass was positively correlated with preceding rainfall, and the low fine root biomass in the dry season contained higher concentrations of N and lower concentrations of P and K than at other times. Observations on rhizotrons demonstrated that the decrease in fine root biomass in the dry season was a product of both a decrease in fine root length appearance and an increase in fine root length disappearance. Fitting an overall model to root survival time showed significant effects of rainfall preceding root disappearance, with the hazard of root disappearance decreasing by 8 for each 1 mm increase in the average daily (30 day) rainfall preceding root disappearance. While it is acknowledged that other factors have a part to play, this work demonstrates the importance of rainfall and soil moisture in influencing root biomass and root disappearance in this tropical rainforest.     

The Caenorhabditis elegans Elongator Complex Regulates Neuronal α-tubulin Acetylation

Although acetylated α-tubulin is known to be a marker of stable microtubules in neurons, precise factors that regulate α-tubulin acetylation are, to date, largely unknown. Therefore, a genetic screen was employed in the nematode Caenorhabditis elegans that identified the Elongator complex as a possible regulator of α-tubulin acetylation. Detailed characterization of mutant animals revealed that the acetyltransferase activity of the Elongator is indeed required for correct acetylation of microtubules and for neuronal development. Moreover, the velocity of vesicles on microtubules was affected by mutations in Elongator. Elongator mutants also displayed defects in neurotransmitter levels. Furthermore, acetylation of α-tubulin was shown to act as a novel signal for the fine-tuning of microtubules dynamics by modulating α-tubulin turnover, which in turn affected neuronal shape. Given that mutations in the acetyltransferase subunit of the Elongator (Elp3) and in a scaffold subunit (Elp1) have previously been linked to human neurodegenerative diseases, namely Amyotrophic Lateral Sclerosis and Familial Dysautonomia respectively highlights the importance of this work and offers new insights to understand their etiology.     

Specific Detection of Leuconostoc mesenteroides subsp. mesenteroides with DNA Primers Identified by Randomly Amplified Polymorphic DNA Analysis

Randomly amplified polymorphic DNA analysis using primer 239 (5′ CTGAAGCGGA 3′) was performed to characterize Leuconostoc sp. strains. All the strains of Leuconostoc mesenteroides subsp. mesenteroides (with the exception of two strains), two strains formerly identified as L. gelidum, and one strain of Leuconostoc showed a common band at about 1.1 kb. This DNA fragment was cloned and sequenced in order to verify its suitability for identifying L. mesenteroides subsp. mesenteroides strains.     

Evaluation of genotoxic effects induced by exposure to antineoplastic drugs in lymphocytes and exfoliated buccal cells of oncology nurses and pharmacy employees

The continuous introduction of new antineoplastic drugs and their use as complex mixture emphasize the need to carry out correct health risk assessment. The aim of this study was to evaluate genotoxic effects of antineoplastic drugs in nurses (n=25) and pharmacy technicians (n=5) employed in an oncology hospital. The nurses administered antineoplastic drugs in the day-care hospital (n=12) and in the wards (n=13), and pharmacy technicians prepared the drugs in the central pharmacy. We performed the micronucleus (MN) test with lymphocytes and exfoliated buccal cells and conducted traditional analysis of chromosomal aberrations (CA). Thirty healthy subjects were selected as controls. Monitoring of surface contamination with cyclophosphamide, 5-fluorouracil, ifosfamide, cytarabine, and gemcitabine showed the presence of detectable levels only for cyclophosphamide, 5-fluorouracil and ifosfamide. In addition, we measured the 5-fluorouracil metabolite alpha-F-betaalanine in the urine of all subjects and found significant concentrations only in 3 out of 25 nurses. The micronucleus assay with lymphocytes did not show significant differences between exposed and control groups, while the same test with exfoliated buccal cells found higher values in nurses administering antineoplastic drugs than in pharmacy employees. In the CA analysis, we detected in exposed groups a significant increase (about 2.5-fold) of structural CA, particularly breaks (up to 5.0-fold). Our results confirm the genotoxic effect of antineoplastic drugs in circulating blood lymphocytes. Moreover, in exfoliated buccal cells the data show more consistent genetic damage induced during administration of the antineoplastic drugs than during their preparation. The data also stress the use of this non-invasive sampling, to assess occupational exposure to mixture of chemicals at low doses.     

Identification of a new chemical class of potent angiogenesis inhibitors based on conformational considerations and database searching

The vascular endothelial growth factor (VEGF) tyrosine kinase receptors KDR and Flt-1 are targets of current interest in anticancer drug research. PTK787/ZK222584 is a potent inhibitor of these enzymes in clinical evaluation as an antiangiogenic agent. The development of a hypothesis concerning the bioactive conformation of this compound has led to the discovery of a new class of potent inhibitors of KDR and Flt-1, the anthranilamides. This could be achieved with a limited experimental effort, which only involved the testing of one archive compound and the synthesis and testing of one appropriate analogue.     

Glutamine Supplementation Alleviates Vasculopathy and Corrects Metabolic Profile in an In Vivo Model of Endothelial Cell Dysfunction

Endothelial Cell Dysfunction (ECD) is a recognized harbinger of a host of chronic cardiovascular diseases. Using a mouse model of ECD triggered by treatment with L-Nω-methylarginine (L-NMMA), we previously demonstrated that renal microvasculature displays a perturbed protein profile, including diminished expression of two key enzymes of the Krebs cycle associated with a Warburg-type suppression of mitochondrial metabolism. We hypothesized that supplementation with L-glutamine (GLN), that can enter the Krebs cycle downstream this enzymatic bottleneck, would normalize vascular function and alleviate mitochondrial dysfunction. To test this hypothesis, mice with chronic L-NMMA-induced ECD were co-treated with GLN at different concentrations for 2 months. Results confirmed that L-NMMA led to a defect in acetylcholine-induced relaxation of aortic rings that was dose-dependently prevented by GLN. In caveolin-1 transgenic mice characterized by eNOS inactivation, L-NMMA further impaired vasorelaxation which was partially rescued by GLN co-treatment. Pro-inflammatory profile induced by L-NMMA was blunted in mice co-treated with GLN. Using an LC/MS platform for metabolite profiling, we sought to identify metabolic perturbations associated with ECD and offset by GLN supplementation. 3453 plasma molecules could be detected with 100% frequency in mice from at least one treatment group. Among these, 37 were found to be differentially expressed in a 4-way comparison of control vs. LNMMA both with and without GLN. One of such molecules, hippuric acid, an “uremic toxin” was found to be elevated in our non-uremic mice receiving L-NMMA, but normalized by treatment with GLN. Ex vivo analysis of hippuric acid effects on vasomotion demonstrated that it significantly reduced acetylcholine-induced vasorelaxation of vascular rings. In conclusion, functional and metabolic profiling of animals with early ECD revealed macrovasculopathy and that supplementation GLN is capable of improving vascular function. Metabolomic analyses reveal elevation of hippuric acid, which may further exacerbate vasculopathy even before the development of uremia.