Homeologous plastid DNA transformation in tobacco is mediated by multiple recombination events.

Efficient plastid transformation has been achieved in Nicotiana tabacum using cloned plastid DNA of Solanum nigrum carrying mutations conferring spectinomycin and streptomycin resistance. The use of the incompletely homologous (homeologous) Solanum plastid DNA as donor resulted in a Nicotiana plastid transformation frequency comparable with that of other experiments where completely homologous plastid DNA was introduced. Physical mapping and nucleotide sequence analysis of the targeted plastid DNA region in the transformants demonstrated efficient site-specific integration of the 7.8-kb Solanum plastid DNA and the exclusion of the vector DNA. The integration of the cloned Solanum plastid DNA into the Nicotiana plastid genome involved multiple recombination events as revealed by the presence of discontinuous tracts of Solanum-specific sequences that were interspersed between Nicotiana-specific markers. Marked position effects resulted in very frequent cointegration of the nonselected peripheral donor markers located adjacent to the vector DNA. Data presented here on the efficiency and features of homeologous plastid DNA recombination are consistent with the existence of an active RecA-mediated, but a diminished mismatch, recombination/repair system in higher-plant plastids.     

Angiopoietin-2 sensitizes endothelial cells to TNF-alpha and has a crucial role in the induction of inflammation

The angiopoietins Ang-1 and Ang-2 have been identified as ligands of the receptor tyrosine kinase Tie-2 (refs. 1,2). Paracrine Ang-1-mediated activation of Tie-2 acts as a regulator of vessel maturation and vascular quiescence. In turn, the antagonistic ligand Ang-2 acts by an autocrine mechanism and is stored in endothelial Weibel-Palade bodies from where it can be rapidly released upon stimulation. The rapid release of Ang-2 implies functions of the angiopoietin-Tie system beyond its established role during vascular morphogenesis as a regulator of rapid vascular responses. Here we show that mice deficient in Ang-2 (encoded by the gene Angpt2) cannot elicit an inflammatory response in thioglycollate-induced or Staphylococcus aureus-induced peritonitis, or in the dorsal skinfold chamber model. Recombinant Ang-2 restores the inflammation defect in Angpt2(-/-) mice. Intravital microscopy showed normal TNF-alpha-induced leukocyte rolling in the vasculature of Angpt2(-/-)mice, but rolling cells did not firmly adhere to activated endothelium. Cellular experiments showed that Ang-2 promotes adhesion by sensitizing endothelial cells toward TNF-alpha and modulating TNF-alpha-induced expression of endothelial cell adhesion molecules. Together, these findings identify Ang-2 as an autocrine regulator of endothelial cell inflammatory responses. Ang-2 thereby acts as a switch of vascular responsiveness exerting a permissive role for the activities of proinflammatory cytokines.     

Anther cap retention prevents self-pollination by elaterid beetles in the South African orchid Eulophia foliosa

BACKGROUND AND AIMS: Pollination by insects that spend long periods visiting many flowers on a plant may impose a higher risk of facilitated self-pollination. Orchids and asclepiads are particularly at risk as their pollen is packaged as pollinia and so can be deposited on self-stigmas en masse. Many orchids and asclepiads have adaptations to limit self-deposition of pollinia, including gradual reconfiguration of pollinaria following removal. Here an unusual mechanism--anther cap retention--that appears to prevent self-pollination in the South African orchid Eulophia foliosa is examined. METHODS: Visits to inflorescences in the field were observed and pollinators collected. Visitation rates to transplanted inflorescences were compared between a site where putative pollinators were abundant and a site where they were rare. Anther cap retention times were determined for removed pollinaria and atmospheric vapour pressure deficit was recorded concurrently. Anther cap anatomy was examined using light microscopy. KEY RESULTS: Eulophia foliosa is pollinated almost exclusively by Cardiophorus obliquemaculatus (Elateridae) beetles, which remain on the deceptive inflorescences for on average 301 s (n = 18). The anther cap that covers the pollinarium is retained for an average of 512 s (n = 24) after pollinarium removal by beetles. In all populations measured, anther cap dimensions are greater than those of the stigmatic cavity, thus precluding the deposition of self-pollinia until after the anther cap has dropped. An anatomical investigation of this mechanism suggests that differential water loss from regions of the anther cap results in opening of the anther cap flaps. This is supported by observations that as atmospheric vapour pressure deficits increased, the duration of anther cap retention was reduced. CONCLUSIONS: Flowers of E. foliosa are specialized for pollination by elaterid beetles. Retention of anther caps for a period exceeding average visit times by beetles to inflorescences appears to prevent facilitated self-pollination in E. foliosa effectively.     

Phenotype microarray profiling of Staphylococcus aureus menD and hemB mutants with the small-colony-variant phenotype

Standard biochemical tests have revealed that hemin and menadione auxotrophic Staphylococcus aureus small-colony variants (SCVs) exhibit multiple phenotypic changes. To provide a more complete analysis of the SCV phenotype, two genetically defined mutants with a stable SCV phenotype were comprehensively tested. These mutants, generated via mutations in menD or hemB that yielded menadione and hemin auxotrophs, were subjected to phenotype microarray (PM) analysis of over 1,500 phenotypes (including utilization of different carbon, nitrogen, phosphate, and sulfur sources; growth stimulation or inhibition by amino acids and other nutrients, osmolytes, and metabolic inhibitors; and susceptibility to antibiotics). Compared to parent strain COL, the hemB mutant was defective in utilization of a variety of carbon sources, including Krebs cycle intermediates and compounds that ultimately generate ATP via electron transport. The phenotype of the menD mutant was similar to that of the hemB mutant, but the defects in carbon metabolism were more pronounced than those seen with the hemB mutant. In both mutant strains, hexose phosphates and other carbohydrates that provide ATP in the absence of electron transport stimulated growth. Other phenotypes of SCV mutants, such as hypersensitivity to sodium selenite, sodium tellurite, and sodium nitrite, were also uncovered by the PM analysis. Key results of the PM analysis were confirmed in independent growth studies and by using Etest strips for susceptibility testing. PM technology is a new and efficient technology for assessing cellular phenotypes in S. aureus.     

Neisseria meningitidis expressing lgtB lipopolysaccharide targets DC-SIGN and modulates dendritic cell function

Neisseria meningitidis lipopolysaccharide (LPS) has been identified as a major determinant of dendritic cell (DC) function. Here we report that one of a series of meningococcal mutants with defined truncations in the lacto-N-neotetraose outer core of the LPS exhibited unique strong adhesion and internalization properties towards DC. These properties were mediated by interaction of the GlcNAc(beta1-3)-Gal(beta1-4)-Glc-R oligosaccharide outer core of lgtB LPS with the dendritic-cell-specific ICAM-3 grabbing non-integrin (DC-SIGN) lectin receptor. Activation of DC-SIGN with this novel oligosaccharide ligand skewed T-cell responses driven by DC towards T helper type 1 activity. Thus, the use of lgtB LPS may provide a powerful instrument to selectively induce the desired arm of the immune response and potentially increase vaccine efficacy.     

HotSwap for bioinformatics: A STRAP tutorial

Background

Bioinformatics applications are now routinely used to analyze large amounts of data. Application development often requires many cycles of optimization, compiling, and testing. Repeatedly loading large datasets can significantly slow down the development process. We have incorporated HotSwap functionality into the protein workbench STRAP, allowing developers to create plugins using the Java HotSwap technique.

Results

Users can load multiple protein sequences or structures into the main STRAP user interface, and simultaneously develop plugins using an editor of their choice such as Emacs. Saving changes to the Java file causes STRAP to recompile the plugin and automatically update its user interface without requiring recompilation of STRAP or reloading of protein data. This article presents a tutorial on how to develop HotSwap plugins. STRAP is available at http://strapjava.de and http://www.charite.de/bioinf/strap.

Conclusion

HotSwap is a useful and time-saving technique for bioinformatics developers. HotSwap can be used to efficiently develop bioinformatics applications that require loading large amounts of data into memory.     

Neuron firing in driven nonlinear integrate-and-fire models

Statistical properties of neuron firing are studied in the framework of a nonlinear leaky integrate-and-fire model that is driven by a slow periodic subthreshold signal. The firing events are characterized by first passage time densities. The experimentally better accessible interspike interval density generally depends on the sojourn times in a refractory state of the neuron. This aspect is not part of the integrate-and-fire model and must be modelled additionally. For a large class of refractory dynamics, a general expression for the interspike interval density is given and further evaluated for the two cases with an instantaneous resetting (i.e. no refractory state) and a refractory state possessing a deterministic lifetime. First passage time densities and interspike interval densities following from the proposed theory compare favorably with precise numerical simulations.     

Sustained phosphorylation of tyrosine hydroxylase at serine 40: a novel mechanism for maintenance of catecholamine synthesis

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine synthesis. Its activity is known to be controlled acutely (minutes) by phosphorylation and chronically (days) by protein synthesis. Using bovine adrenal chromaffin cells we found that nicotine, acting via nicotinic receptors, sustained the phosphorylation of TH at Ser40 for up to 48 h. Nicotine also induced sustained activation of TH, which for the first 24 h was completely independent of TH protein synthesis, and the phosphorylation of TH at Ser31. Imipramine did not inhibit the acute phosphorylation of TH at Ser40 or TH activation induced by nicotine, but did inhibit the sustained responses to nicotine seen at 24 h. The protein kinase(s) responsible for TH phosphorylation at Ser40 switched from being protein kinase C (PKC) independent in the acute phase to PKC dependent in the sustained phase. Sustained phosphorylation and activation of TH were also observed with histamine and angiotensin II. Sustained phosphorylation of TH at Ser40 provides a novel mechanism for increasing TH activity and this leads to increased catecholamine synthesis. Sustained phosphorylation of TH may be a selective target for drugs or pathology in neurons that contain TH and synthesize dopamine, noradrenaline or adrenaline.     

Classical anticytokinins do not interact with cytokinin receptors but inhibit cyclin-dependent kinases

Cytokinins are a class of plant hormones that regulate the cell cycle and diverse developmental and physiological processes. Several compounds have been identified that antagonize the effects of cytokinins. Based on structural similarities and competitive inhibition, it has been assumed that these anticytokinins act through a common cellular target, namely the cytokinin receptor. Here, we examined directly the possibility that various representative classical anticytokinins inhibit the Arabidopsis cytokinin receptors CRE1/AHK4 (cytokinin response 1/Arabidopsis histidine kinase 4) and AHK3 (Arabidopsis histidine kinase 3). We show that pyrrolo[2,3-d]pyrimidine and pyrazolo[4,3-d]pyrimidine anticytokinins do not act as competitors of cytokinins at the receptor level. Flow cytometry and microscopic analyses revealed that anticytokinins inhibit the cell cycle and cause disorganization of the microtubular cytoskeleton and apoptosis. This is consistent with the hypothesis that they inhibit regulatory cyclin-dependent kinase (CDK) enzymes. Biochemical studies demonstrated inhibition by selected anti-cytokinins of both Arabidopsis and human CDKs. X-ray determination of the crystal structure of a human CDK2-anticytokinin complex demonstrated that the antagonist occupies the ATP-binding site of CDK2. Finally, treatment of human cancer cell lines with anticytokinins demonstrated their ability to kill human cells with similar effectiveness as known CDK inhibitors.