Best practice procedures for the establishment of a C(4) cycle in transgenic C(3) plants

C(4) plants established a mechanism for the concentration of CO(2) in the vicinity of ribulose-1,5-bisphosphate carboxylase/oxygenase in order to saturate the enzyme with substrate and substantially to reduce the alternative fixation of O(2) that results in energy losses. Transfer of the C(4) mechanism to C(3) plants has been repeatedly tested, but none of the approaches so far resulted in transgenic plants with enhanced photosynthesis or growth. Instead, often deleterious effects were observed. A true C(4) cycle requires the co-ordinated activity of multiple enzymes in different cell types and in response to diverse environmental and metabolic stimuli. This review summarizes our current knowledge about the most appropriate regulatory elements and coding sequences for the establishment of C(4) protein activities in C(3) plants. In addition, technological breakthroughs for the efficient transfer of the numerous genes probably required to transform a C(3) plant into a C(4) plant will be discussed.     

Patchy bed disturbance and fish predation independently influence the distribution of stream invertebrates and algae

1. The identification of factors determining the patchy distribution of organisms in space and time is a central concern of ecology. Predation and abiotic disturbance are both well-known drivers of this patchiness, but their interplay is still poorly understood, especially for communities dominated by mobile organisms in frequently disturbed ecosystems. 2. We investigated the separate and interactive influences of bed disturbance by floods and predation by fish on the benthic community in a flood-prone stream. Electric fields excluded fish predators from half of 48 stream bed patches (area 0·49 m(2) ) with contrasting disturbance treatments. Three types of bed disturbance were created by either scouring or filling patches to a depth of 15-20 cm or by leaving the patches undisturbed, thus mimicking the mosaic of scour and fill caused by a moderate flood. Benthic invertebrates and algae were sampled repeatedly until 57 days after the disturbance. 3. Disturbance influenced all ten investigated biological response variables, whereas predation affected four variables. Averaged across time, invertebrate taxon richness and total abundance were highest in stable patches. Algal biomass and densities of five of the seven most common invertebrate taxa (most of which were highly mobile) were higher in fill than in scour patches, whereas two taxa were more abundant in scour and stable than in fill patches. Furthermore, two common invertebrate grazers were more abundant and algal biomass tended to be reduced in fish exclusion patches, suggesting a patch-scale trophic cascade from fish to algae. 4. Our results highlight the importance of patchy physical disturbance for the microdistribution of mobile stream organisms and indicate a notable, but less prevalent, influence of fish predation at the patch scale in this frequently disturbed environment. Disturbance and predation treatments interacted only once, suggesting that the observed predation effects were largely independent of local bed disturbance patterns.     

Metalloprotease meprin beta generates nontoxic N-terminal amyloid precursor protein fragments in vivo

Identification of physiologically relevant substrates is still the most challenging part in protease research for understanding the biological activity of these enzymes. The zinc-dependent metalloprotease meprin β is known to be expressed in many tissues with functions in health and disease. Here, we demonstrate unique interactions between meprin β and the amyloid precursor protein (APP). Although APP is intensively studied as a ubiquitously expressed cell surface protein, which is involved in Alzheimer disease, its precise physiological role and relevance remain elusive. Based on a novel proteomics technique termed terminal amine isotopic labeling of substrates (TAILS), APP was identified as a substrate for meprin β. Processing of APP by meprin β was subsequently validated using in vitro and in vivo approaches. N-terminal APP fragments of about 11 and 20 kDa were found in human and mouse brain lysates but not in meprin β(-/-) mouse brain lysates. Although these APP fragments were in the range of those responsible for caspase-induced neurodegeneration, we did not detect cytotoxicity to primary neurons treated by these fragments. Our data demonstrate that meprin β is a physiologically relevant enzyme in APP processing.     

Aberrant epigenetic and genetic marks are seen in myelodysplastic leukocytes and reveal Dock4 as a candidate pathogenic gene on chromosome 7q

Myelodysplastic syndromes (MDS) are characterized by abnormal and dysplastic maturation of all blood lineages. Even though epigenetic alterations have been seen in MDS marrow progenitors, very little is known about the molecular alterations in dysplastic peripheral blood cells. We analyzed the methylome of MDS leukocytes by the HELP assay and determined that it was globally distinct from age-matched controls and was characterized by numerous novel, aberrant hypermethylated marks that were located mainly outside of CpG islands and preferentially affected GTPase regulators and other cancer-related pathways. Additionally, array comparative genomic hybridization revealed that novel as well as previously characterized deletions and amplifications could also be visualized in peripheral blood leukocytes, thus potentially reducing the need for bone marrow samples for future studies. Using integrative analysis, potentially pathogenic genes silenced by genetic deletions and aberrant hypermethylation in different patients were identified. DOCK4, a GTPase regulator located in the commonly deleted 7q31 region, was identified by this unbiased approach. Significant hypermethylation and reduced expression of DOCK4 in MDS bone marrow stem cells was observed in two large independent datasets, providing further validation of our findings. Finally, DOCK4 knockdown in primary marrow CD34(+) stem cells led to decreased erythroid colony formation and increased apoptosis, thus recapitulating the bone marrow failure seen in MDS. These findings reveal widespread novel epigenetic alterations in myelodysplastic leukocytes and implicate DOCK4 as a pathogenic gene located on the 7q chromosomal region.     

Scanning irradiation device for mice in vivo with pulsed and continuous proton beams

A technical set-up for irradiation of subcutaneous tumours in mice with nanosecond-pulsed proton beams or continuous proton beams is described and was successfully used in a first experiment to explore future potential of laser-driven particle beams, which are pulsed due to the acceleration process, for radiation therapy. The chosen concept uses a microbeam approach. By focusing the beam to approximately 100 × 100 μm(2), the necessary fluence of 10(9) protons per cm(2) to deliver a dose of 20 Gy with one-nanosecond shot in the Bragg peak of 23 MeV protons is achieved. Electrical and mechanical beam scanning combines rapid dose delivery with large scan ranges. Aluminium sheets one millimetre in front of the target are used as beam energy degrader, necessary for adjusting the depth-dose profile. The required procedures for treatment planning and dose verification are presented. In a first experiment, 24 tumours in mice were successfully irradiated with 23 MeV protons and a single dose of 20 Gy in pulsed or continuous mode with dose differences between both modes of 10%. So far, no significant difference in tumour growth delay was observed.     

Combining metabolic pathway analysis with Evolutionary Game Theory: explaining the occurrence of low-yield pathways by an analytic optimization approach

Elementary-mode analysis is a powerful method for detecting all potential pathways in a metabolic network and computing the associated molar yields. Metabolic pathways can be interpreted as different strategies of organisms. Thus, methods from Evolutionary Game Theory can be employed. In Flux Balance Analysis (FBA), it is usually assumed that molar yields of relevant products (such as biomass or ATP) have been maximized during evolution. This has been questioned on game-theoretical grounds. In particular, in situations that can be characterized as a Prisoner's Dilemma, maximization of flux is not in line with maximization of yield. Under other conditions (that is, for other parameter values of maximal velocities), a Harmony game can result, where the above two maximization criteria give the same result. Here, we analyse the optimal situations under varying conditions. In particular, we consider the case where the cell can allocate a certain amount of protein on several enzymes in a varying distribution and model this by a linear programming problem in which not only the rates but also the maximal velocities are variable. It turns out that in the case of low or moderate synthesis costs for the enzymes of the high-yield pathway, maximizing pathway flux is in line with maximizing molar yield while in the case of high costs, it is not. This may explain the observation that many cells such as striated muscle cells, tumour cells, activated lymphocytes and several yeasts do not reallocate protein away from glycolytic enzymes towards TCA cycle and respiratory chain enzymes, in spite of the higher efficiency of respiration. This provides a straightforward explanation of the Warburg effect in tumour cells.     

Catalytic properties and classification of cellobiose dehydrogenases from ascomycetes

Putative cellobiose dehydrogenase (CDH) genes are frequently discovered in various fungi by genome sequencing projects. The expression of CDH, an extracellular flavocytochrome, is well studied in white rot basidiomycetes and is attributed to extracellular lignocellulose degradation. CDH has also been reported for plant-pathogenic or saprotrophic ascomycetes, but the molecular and catalytic properties of these enzymes are currently less investigated. This study links various ascomycetous cdh genes with the molecular and catalytic characteristics of the mature proteins and suggests a differentiation of ascomycete class II CDHs into two subclasses, namely, class IIA and class IIB, in addition to the recently introduced class III of hypothetical ascomycete CDHs. This new classification is based on sequence and biochemical data obtained from sequenced fungal genomes and a screening of 40 ascomycetes. Thirteen strains showed CDH activity when they were grown on cellulose-based media, and Chaetomium atrobrunneum, Corynascus thermophilus, Dichomera saubinetii, Hypoxylon haematostroma, Neurospora crassa, and Stachybotrys bisbyi were selected for detailed studies. In these strains, one or two cdh-encoding genes were found that stem either from class IIA and contain a C-terminal carbohydrate-binding module or from class IIB without such a module. In several strains, both genes were found. Regarding substrate specificity, class IIB CDHs show a less pronounced substrate specificity for cellobiose than class IIA enzymes. A pH-dependent pattern of the intramolecular electron transfer was also observed, and the CDHs were classified into three groups featuring acidic, intermediate, or alkaline pH optima. The pH optimum, however, does not correlate with the CDH subclasses and is most likely a species-dependent adaptation to different habitats.     

Sports anthropological investigations on somatotypology of elite karateka

The goal of this work was to find differences between the somatotypes of elite karateka. For this purpose the somatotypes of Conrad, Heath & Carter were used. This sports anthropological study was carried out on the assumption that constant karate training causes changes in body composition. It should clarify whether different body types, including sexual dimorphism, are to be found in people practicing the karate disciplines kata and kumite. 80 male and female elite karateka were tested. As a comparison group, 62 leisure sports persons and 66 hobby karateka were used as control groups. The measurements were taken under standardised conditions and the results were examined statistically (ANOVA). The body types of Conrad, which were confirmed by the somatocharts of Heath & Carter, showed differences between the elite karateka in comparison to the control group. According to this, the typical elite karateka is more athletic and smaller. He weighs less than the sports persons of the comparison fitness group. Moreover, within the karate disciplines, the kata practisers are more endomorph than their colleagues. The kumite athletes take more ektomorph positions in the somatocharts (Heath & Carter 1990). On the basis of these results the assumption of a differentiated somatotype can be confirmed in the two groups and also within karate between the disciplines kata and kumite. This study concludes that there is both a kata and a kumite somatotype among karate practisers. Further research is required regarding longitudinal measurements of the change in body composition of young karateka in the course of their competitive careers. The career-affecting factors of training frequency, training age, and frequency of injury, related to the level of achievement, leave room for further investigation. This also applies to possible ethnic body-build differences and the athletes' level of achievement.