PACAP enhances the expression of CD11b,CD66b and CD63 in human neutrophils

Pituitary adenylate cyclase-activating peptide (PACAP) is a neuropeptide with strong bronchodilator capacity, present in the human airways. There is recent evidence that PACAP decreases the release of proinflammatory cytokines. We have previously shown that PACAP inhibits neutrophil chemotaxis, but altogether little is known about the effects of PACAP on granulocytes. The present study was designed to investigate if PACAP and the closely related peptide vasoactive intestinal peptide (VIP) could affect the cell surface expression of CD11b, CD63 and CD66b in human neutrophils. Neutrophils isolated from 12 healthy blood donors were incubated with either PACAP or VIP, and the expression of neutrophil cell surface markers was assessed using flowcytometry. Neutrophils incubated with PACAP38 exhibited a marked, concentration-dependent increase in their expression of CD11b, CD63 and CD66b. In contrast, neutrophils incubated with VIP showed no increase of the investigated surface markers. This indicates a role for PACAP in granulocyte activation, mediated via a pathway not shared with VIP. Together with the previously presented data on leukocyte migration it suggests that PACAP acts as a regulator of neutrophil inflammation.     

Differential responsiveness of CRF receptor subtypes to N-terminal truncation of peptidic ligands

The role of the N-terminal domains of corticotropin-releasing factor (CRF) and CRF-like peptides in receptor subtype selectivity, ligand affinity and biological potency was investigated. Therefore, human CRF(12-41), human URP(12-38) and antisauvagine-30 (aSvg) were N-terminally prolonged by consecutive addition of one or two amino acids. The peptides obtained were tested for their binding affinities to rat CRF1 and murine CRF(2beta) receptor, and their capability to stimulate cAMP-release by HEK cells producing either receptor.It was observed that human CRF N-terminally truncated by eight residues was bound with high affinity to CRF2 receptor (Ki=5.4nM), whereas affinity for CRF1 receptor was decreased (Ki=250 nM). A similar shift of affinity was found with sauvagine (Svg) analogs. Truncation of human URP analogs did not affect their preference for CRF(2beta) receptor, but reduced their affinity. Changes in affinity were positively correlated with changes in potency. These results indicated that CRF1 receptor was more stringent in its structural requirements for ligands to exhibit high affinity binding than CRF(2beta) receptor.     

Mathematical modelling in the post-genome era: understanding genome expression and regulation--a system theoretic approach

This paper introduces a mathematical framework for modelling genome expression and regulation. Starting with a philosophical foundation, causation is identified as the principle of explanation of change in the realm of matter. Causation is, therefore, a relationship, not between components, but between changes of states of a system. We subsequently view genome expression (formerly known as 'gene expression') as a dynamic process and model aspects of it as dynamic systems using methodologies developed within the areas of systems and control theory. We begin with the possibly most abstract but general formulation in the setting of category theory. The class of models realised are state-space models, input--output models, autoregressive models or automata. We find that a number of proposed 'gene network' models are, therefore, included in the framework presented here. The conceptual framework that integrates all of these models defines a dynamic system as a family of expression profiles. It becomes apparent that the concept of a 'gene' is less appropriate when considering mathematical models of genome expression and regulation. The main claim of this paper is that we should treat (model) the organisation and regulation of genetic pathways as what they are: dynamic systems. Microarray technology allows us to generate large sets of time series data and is, therefore, discussed with regard to its use in mathematical modelling of gene expression and regulation.     

Importance of phosphate contacts for sequence recognition by EcoRI restriction enzyme

We have studied the importance of charge and hydrogen-bonding potential of the phosphodiester backbone for binding and cleavage by EcoRI restriction endonuclease. We used 12-mer oligodeoxynucleotide substrates with single substitutions of phosphates by chiral methylphosphonates at each position of the recognition sequence -pGpApApTpTpCp-. Binding was moderately reduced between 4- and 400-fold more or less equally for the R(P) and S(P)-analogues mainly caused by missing charge interaction. The range of cleavage effects was much wider. Four substrates were not cleaved at all. At both flanking positions and in the purine half of the sequence up to the central position, cleavage was more impaired than binding and differences between R(P) and S(P) diastereomeres were more pronounced. These effects are easily interpreted by direct phosphate contacts seen in the crystal structure. For the effects of substitutions in the pyrimidine half of the recognition sequence, more indirect effects have to be discussed.     

Methylene blue as an antimalarial agent

Methylene blue has intrinsic antimalarial activity and it can act as a chloroquine sensitizer. In addition, methylene blue must be considered for preventing methemoglobinemia, a serious complication of malarial anemia. As an antiparasitic agent, methylene blue is pleiotropic: it interferes with hemoglobin and heme metabolism in digestive organelles, and it is a selective inhibitor of Plasmodium falciparum glutathione reductase. The latter effect results in glutathione depletion which sensitizes the parasite for chloroquine action. At the Centre de Recherche en Santé de Nouna in Burkina Faso, we study the combination of chloroquine with methylene blue (BlueCQ) as a possible medication for malaria in endemic regions. A pilot study with glucose-6-phosphate dehydrogenase-sufficient adult patients has been conducted recently.     

Whole genome shotgun sequencing guided by bioinformatics pipelines--an optimized approach for an established technique

While the sequencing of bacterial genomes has become a routine procedure at major sequencing centers, there are still a number of genome projects at small- or medium-size facilities. For these facilities a maximum of control over sequencing, assembling and finishing is essential. At the same time, facilities have to be able to co-operate at minimum costs for the overall project. We have established a pipeline for the distributed sequencing of Alcanivorax borkumensis SK2, Azoarcus sp. BH72, Clavibacter michiganensis subsp. michiganensis NCPPB382, Sorangium cellulosum So ce56 and Xanthomonas campestris pv. vesicatoria 85-10. Our pipeline relies on standard tools (e.g. PHRED/PHRAP, CAP3 and Consed/Autofinish) wherever possible, supplementing them with new tools (BioMake and BACCardI) to achieve the aims described above.     

The apparent uptake of fluorescently labeled siRNAs by electroporated cells depends on the fluorochrome

Transfection of mammalian cells with preformed small interfering RNAs (siRNAs) permits a transient and often specific reduction of gene expression. It is possible to rapidly examine the uptake of siRNAs by transfection with fluorescently labeled siRNAs. We examined the apparent uptake of such siRNAs by several leukemic cell lines after electroporation. We show that Cy3 and Cy5-labeled siRNAs cause a significant amount of cell fluorescence, as judged by flow cytometry. In contrast, several fluorescein-labeled siRNAs could not be detected. Nevertheless, such fluoresceinated siRNAs efficiently suppressed a leukemic target gene, demonstrating that siRNA uptake must have taken place. Therefore, for cell electroporation, fluorescein-labeled siRNAs may lead to false negative results and should not be used to examine electroporation-mediated siRNA uptake.     

FtsH is involved in the early stages of repair of photosystem II in Synechocystis sp PCC 6803

When plants, algae, and cyanobacteria are exposed to excessive light, especially in combination with other environmental stress conditions such as extreme temperatures, their photosynthetic performance declines. A major cause of this photoinhibition is the light-induced irreversible photodamage to the photosystem II (PSII) complex responsible for photosynthetic oxygen evolution. A repair cycle operates to selectively replace a damaged D1 subunit within PSII with a newly synthesized copy followed by the light-driven reactivation of the complex. Net loss of PSII activity occurs (photoinhibition) when the rate of damage exceeds the rate of repair. The identities of the chaperones and proteases involved in the replacement of D1 in vivo remain uncertain. Here, we show that one of the four members of the FtsH family of proteases (cyanobase designation slr0228) found in the cyanobacterium Synechocystis sp PCC 6803 is important for the repair of PSII and is vital for preventing chronic photoinhibition. Therefore, the ftsH gene family is not functionally redundant with respect to the repair of PSII in this organism. Our data also indicate that FtsH binds directly to PSII, is involved in the early steps of D1 degradation, and is not restricted to the removal of D1 fragments. These results, together with the recent analysis of ftsH mutants of Arabidopsis, highlight the critical role played by FtsH proteases in the removal of damaged D1 from the membrane and the maintenance of PSII activity in vivo.     

Novel versus unsupported clades: assessing the qualitative support for clades in MRP supertrees

Matrix representation with parsimony (MRP) supertree construction has been criticized because the supertree may specify clades that are contradicted by every source tree contributing to it. Such unsupported clades may also occur using other supertree methods; however, their incidence is largely unknown. In this study, I investigated the frequency of unsupported clades in both simulated and empirical MRP supertrees. Here, I propose a new index, QS, to quantify the qualitative support for a supertree and its clades among the set of source trees. Results show that unsupported clades are very rare in MRP supertrees, occurring most often when there are few source trees that all possess the same set of taxa. However, even under these conditions the frequency of unsupported clades was <0.2%. Unsupported clades were absent from both the Carnivora and Lagomorpha supertrees, reflecting the use of large numbers of source trees for both. The proposed QS indices are correlated broadly with another measure of quantitative clade support (bootstrap frequencies, as derived from resampling of the MRP matrix) but appear to be more sensitive. More importantly, they sample at the level of the source trees and thus, unlike the bootstrap, are suitable for summarizing the support of MRP supertree clades.