Import of peroxisomal hydroxypyruvate reductase into glyoxysomes

A new procedure was used to purify the peroxisomal matrix enzyme hydroxypyruvate reductase (HPR) from green leaves of pumpkin (Cucurbita pepo L.) and spinach (Spinacia oleracea L.). Monospecific antibodies were prepared against this enzyme in rabbits. Immunoprecipitation of HPR from watermelon (Citrullus vulgaris Schrad.) yielded a single protein with a subunit molecular weight of 45 kDa. Immunohistochemical labeling of HPR was found exclusively in watermelon microbodies. Isolated polyadenylated mRNA from light-grown watermelon cotyledons was injected into Xenopus laevis oocytes. The heterologous in-vivo translation product of HPR exhibited the same molecular weight as the immunoprecipitate from watermelon cotyledons, indicating the lack of a cleavable extra sequence. The watermelon HPR translated in oocytes was imported into isolated glyoxysomes from castor bean (Ricinus communis L.) endosperm and remained resistant to proteolysis after the addition of proteinase K. The HPR did not change its apparent molecular weight during sequestration; however, it may have changed its conformation.Abbreviations HPR hydroxypyruvate reductase - PMSF phenylmethylsulfonyl fluoride - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Combination of DNA-directed immobilization and immuno-PCR: very sensitive antigen detection by means of self-assembled DNA–protein conjugates

An assay for very sensitive antigen detection is described which takes advantage of the self- assembly capabilities of semi-synthetic conjugates of DNA and proteins. The general scheme of this assay is similar to a two-sided (sandwich) enzyme-linked immunoassay (ELISA); however, covalent single-stranded DNA–streptavidin (STV) conjugates, capable of hybridizing to complementary surface-bound DNA oligomers, are utilized for the effective immobilzation of either capture antibodies or antigens, rather than the chemi- or physisorption usually applied in ELISA. Immuno-PCR (IPCR) is employed as a method for signal generation, utilizing oligomeric reagents obtained by self-assembly of STV, biotinylated DNA and antibodies. In three different model systems, detecting human IgG, rabbit IgG or carcinoembryonic antigen, this combination allowed one to increase the sensitivity of the analogous ELISA ~1000-fold. For example, <0.1 amol/µl (15 pg/ml) of rabbit IgG was detectable. The immunoassay can be carried out in a single step by tagging the analyte with both reagents for capture and read-out simultaneously, thereby significantly reducing handling time and costs of analysis. Moreover, as the spatial selectivity of target immobilization is determined by the specificity of DNA base pairing, the assay is particularly suited for miniaturized microfluidics and lab-on-a-chip devices.     

Neuronal Shot Noise and Brownian 1/f2 Behavior in the Local Field Potential

We demonstrate that human electrophysiological recordings of the local field potential (LFP) from intracranial electrodes, acquired from a variety of cerebral regions, show a ubiquitous 1/f² scaling within the power spectrum. We develop a quantitative model that treats the generation of these fields in an analogous way to that of electronic shot noise, and use this model to specifically address the cause of this 1/f² Brownian noise. The model gives way to two analytically tractable solutions, both displaying Brownian noise: 1) uncorrelated cells that display sharp initial activity, whose extracellular fields slowly decay in time and 2) rapidly firing, temporally correlated cells that generate UP-DOWN states.     

Proteomic analysis of the phytopathogenic soilborne fungus Verticillium dahliae reveals differential protein expression in isolates that differ in aggressiveness

Verticillium dahliae is a soilborne fungus that causes a vascular wilt disease of plants and losses in a broad range of economically important crops worldwide. In this study, we compared the proteomes of highly (Vd1396‐9) and weakly (Vs06‐14) aggressive isolates of V. dahliae to identify protein factors that may contribute to pathogenicity. Twenty‐five protein spots were consistently observed as differential in the proteome profiles of the two isolates. The protein sequences in the spots were identified by LC‐ESI‐MS/MS and MASCOT database searches. Some of the identified sequences shared homology with fungal proteins that have roles in stress response, colonization, melanin biosynthesis, microsclerotia formation, antibiotic resistance, and fungal penetration. These are important functions for infection of the host and survival of the pathogen in soil. One protein found only in the highly aggressive isolate was identified as isochorismatase hydrolase, a potential plant‐defense suppressor. This enzyme may inhibit the production of salicylic acid, which is important for plant defense response signaling. Other sequences corresponding to potential pathogenicity factors were identified in the highly aggressive isolate. This work indicates that, in combination with functional genomics, proteomics‐based analyses can provide additional insights into pathogenesis and potential management strategies for this disease.     

Evolution of Integrated Causal Structures in Animats Exposed to Environments of Increasing Complexity

Natural selection favors the evolution of brains that can capture fitness-relevant features of the environment''s causal structure. We investigated the evolution of small, adaptive logic-gate networks (“animats”) in task environments where falling blocks of different sizes have to be caught or avoided in a ‘Tetris-like’ game. Solving these tasks requires the integration of sensor inputs and memory. Evolved networks were evaluated using measures of information integration, including the number of evolved concepts and the total amount of integrated conceptual information. The results show that, over the course of the animats'' adaptation, i) the number of concepts grows; ii) integrated conceptual information increases; iii) this increase depends on the complexity of the environment, especially on the requirement for sequential memory. These results suggest that the need to capture the causal structure of a rich environment, given limited sensors and internal mechanisms, is an important driving force for organisms to develop highly integrated networks (“brains”) with many concepts, leading to an increase in their internal complexity.     

Concerted Spatio-Temporal Dynamics of Imported DNA and ComE DNA Uptake Protein during Gonococcal Transformation

Competence for transformation is widespread among bacterial species. In the case of Gram-negative systems, a key step to transformation is the import of DNA across the outer membrane. Although multiple factors are known to affect DNA transport, little is known about the dynamics of DNA import. Here, we characterized the spatio-temporal dynamics of DNA import into the periplasm of Neisseria gonorrhoeae. DNA was imported into the periplasm at random locations around the cell contour. Subsequently, it was recruited at the septum of diplococci at a time scale that increased with DNA length. We found using fluorescent DNA that the periplasm was saturable within minutes with ∼40 kbp DNA. The DNA-binding protein ComE quantitatively governed the carrying capacity of the periplasm in a gene-dosage-dependent fashion. As seen using a fluorescent-tagged derivative protein, ComE was homogeneously distributed in the periplasm in the absence of external DNA. Upon addition of external DNA, ComE was relocalized to form discrete foci colocalized with imported DNA. We conclude that the periplasm can act as a considerable reservoir for imported DNA with ComE governing the amount of DNA stored potentially for transport through the inner membrane.     

Ischemic post-conditioning reduces infarct size of the in vivo rat heart: role of PI3-K, mTOR, GSK-3β, and apoptosis

Post-conditioning by repetitive cycles of reperfusion/ischemia after prolonged ischemia protects the heart from infarction. The objectives of this study were: Are kinases (PI3-kinase, mTOR, and GSK-3β) involved in the signaling pathway of post-conditioning? Does post-conditioning result in a diminished necrosis or apoptosis? In open chest rats the infarct size was determined after 30 min of regional ischemia and 30 min of reperfusion using propidium iodide and microspheres. Post-conditioning was performed by three cycles of 30 s reperfusion and reocclusion each, immediately upon reperfusion. PI3-kinase and mTOR were blocked using wortmannin (0.6 mg/kg) or rapamycin (0.25 mg/kg), respectively. The phosphorylation of GSK-3β and p70S6K was determined with phospho-specific antibodies. TUNEL staining and detection of apoptosis-inducing factor (AIF) were used for the determination of apoptosis. Control hearts had an infarct size of 49 ± 3%, while post-conditioning significantly reduced it to 29 ± 3% (P < 0.01). Wortmannin as well as rapamycin completely blocked the infarct size reduction of post-conditioning (51 ± 2% and 54 ± 5%, respectively). Western blot analysis revealed that post-conditioning increased the phosphorylation of GSK-3β by 2.3 times (P < 0.01), and this increase could be blocked by wortmannin, a PI3-kinase inhibitor. Although rapamycin blocked the infarct size reduction, phosphorylation of p70S6K was not increased in post-conditioned hearts. After 2 h of reperfusion, the post-conditioned hearts had significantly fewer TUNEL-positive nuclei (35 %) compared to control hearts (53%; P < 0.001). AIF was equally reduced in post-conditioned rat hearts (P < 0.05 vs. control). Infarct size reduction by ischemic post-conditioning of the in vivo rat heart is PI3-kinase dependent and involves mTOR. Furthermore, GSK-3β, which is thought to be a regulator of the mPTP, is part of the signaling pathway of post-conditioning. Finally, apoptosis was inhibited by post-conditioning, which was shown by two independent methods. The role of apoptosis and/or autophagy in post-conditioning has to be further elucidated to find therapeutic targets to protect the heart from the consequences of acute myocardial infarction.     

Development of a microtargeting device for particle bombardment of plant meristems

A gene transfer system for meristem cells was developed on the basis of a ballistic approach. In order to meet some important prerequisites for an efficient transfer system, such as for example aiming at small tissues and control of penetration of the microprojectiles, we developed an acceleration system fundamentally different from the usual macroprojectile driven approach. Instead of a macroprojectile, microtargeting uses the law of Bernoulli for accleration of highly uniform-sized gold particles. The system is able to deliver 80% of the particles to an area as small as 150 micron in diameter, which corresponds to the size of a meristem. Microtargeting yields gene delivery (measured as number of transiently GUS expressing cells to up to 3% of the cells exposed in the target area or up to 35 × 10³ cells per cm². Stable transformation of tobacco microcolonies with the microtargeting device was shown to have an efficiency up to one stable transformant per 1000 cells exposed to the shot, or up to one transformant per shot. We perform 4 or 5 shots per min. After 30 to 40 shots, reloading can take up to 2 min. Microtargeting is very flexible and allows for the adjustment of the important parameters to fit the requirements of the respective tissue.     

Integrin-mediated invasion of Staphylococcus aureus into human cells requires Src family protein-tyrosine kinases

Staphylococcus aureus, a common cause of nosocomial infections, is able to invade eukaryotic cells by indirectly engaging beta1 integrin-containing host receptors, whereas non-pathogenic Staphylococcus carnosus is not invasive. Here, we identify intracellular signals involved in integrin-initiated internalization of S. aureus. In particular, the host cell actin cytoskeleton and Src family protein-tyrosine kinases (PTKs) are essential to mediate S. aureus invasion. Src PTKs are activated in response to pathogenic S. aureus, but not S. carnosus. In addition, pharmacological and genetic interference with Src PTK function reduces bacterial internalization. Importantly, Src PTK-deficient cells are resistant to S. aureus invasion, demonstrating the essentiality of host Src PTKs in integrin-mediated uptake of this pathogen.