Effects of a retaining wall and an artificial embankment on nearshore littoral habitats and biota in a large Alpine lake

Hydrobiologia - The littoral zones of many Central European lakes are severely altered by lake-side retaining walls. These are suspected to impair littoral biota due to the reflection of incoming...     

MS-275, a potent orally available inhibitor of histone deacetylases--the development of an anticancer agent

In the last few years it was found that beside genetic aberrations, epigenetic changes also play an important role in tumorigenesis. Acetylation and deacetylation of histones have been found to contribute to a significant extent to epigenetic regulation of gene expression. Analyses of various tumor models and patient samples revealed that the enzyme class of histone deacetylases is associated with many types of cancer and that, for example, over-expression of these enzymes leads to a disturbed balance between acetylation and deacetylation of histones, resulting in differences in the gene expression patterns between normal and cancer cells. Consequently, this class of enzymes has been considered as a potential target for cancer therapy. Numerous inhibitors have been identified and several are in clinical development. Although, with SAHA, one inhibitor has been approved by the FDA for a tumor indication, many open questions remain regarding the mode of action of these inhibitors. In this review, various aspects of preclinical and clinical research of the HDAC inhibitor MS-275 are described, to provide insight into the development of such a compound.     

Structure of a classical MHC class I molecule that binds "non-classical" ligands

Chicken YF1 genes share a close sequence relationship with classical MHC class I loci but map outside of the core MHC region. To obtain insights into their function, we determined the structure of the YF1*7.1/β₂-microgloblin complex by X-ray crystallography at 1.3 Å resolution. It exhibits the architecture typical of classical MHC class I molecules but possesses a hydrophobic binding groove that contains a non-peptidic ligand. This finding prompted us to reconstitute YF1*7.1 also with various self-lipids. Seven additional YF1*7.1 structures were solved, but only polyethyleneglycol molecules could be modeled into the electron density within the binding groove. However, an assessment of YF1*7.1 by native isoelectric focusing indicated that the molecules were also able to bind nonself-lipids. The ability of YF1*7.1 to interact with hydrophobic ligands is unprecedented among classical MHC class I proteins and might aid the chicken immune system to recognize a diverse ligand repertoire with a minimal number of MHC class I molecules.     

Zipping and Unzipping of Adenylate Kinase: Atomistic Insights into the Ensemble of Open ↔ Closed Transitions

Adenylate kinase (AdK), a phosphotransferase enzyme, plays an important role in cellular energy homeostasis. It undergoes a large conformational change between an open and a closed state, even in the absence of substrate. We investigate the apo-AdK transition at the atomic level both with free-energy calculations and with our new dynamic importance sampling (DIMS) molecular dynamics method. DIMS is shown to sample biologically relevant conformations as verified by comparing an ensemble of hundreds of DIMS transitions to AdK crystal structure intermediates. The simulations reveal in atomic detail how hinge regions partially and intermittently unfold during the transition. Conserved salt bridges are seen to have important structural and dynamic roles; in particular, four ionic bonds that open in a sequential, zipper-like fashion and, thus, dominate the free-energy landscape of the transition are identified. Transitions between the closed and open conformations only have to overcome moderate free-energy barriers. Unexpectedly, the closed state and the open state encompass broad free-energy basins that contain conformations differing in domain hinge motions by up to 40°. The significance of these extended states is discussed in relation to recent experimental Förster resonance energy transfer measurements. Taken together, these results demonstrate how a small number of cooperative key interactions can shape the overall dynamics of an enzyme and suggest an “all-or-nothing” mechanism for the opening and closing of AdK. Our efficient DIMS molecular dynamics computer simulation approach can provide a detailed picture of a functionally important macromolecular transition and thus help to interpret and suggest experiments to probe the conformational landscape of dynamic proteins such as AdK.     

Cutting edge: Chk1 directs senescence and mitotic catastrophe in recovery from G2 checkpoint arrest

Besides the well‐understood DNA damage response via establishment of G₂ checkpoint arrest, novel studies focus on the recovery from arrest by checkpoint override to monitor cell cycle re‐entry. The aim of this study was to investigate the role of Chk1 in the recovery from G₂ checkpoint arrest in HCT116 (human colorectal cancer) wt, p53^–/– and p21^–/– cell lines following H₂O₂ treatment. Firstly, DNA damage caused G₂ checkpoint activation via Chk1. Secondly, overriding G₂ checkpoint led to (i) mitotic slippage, cell cycle re‐entry in G₁ and subsequent G₁ arrest associated with senescence or (ii) premature mitotic entry in the absence of p53/p21^WAF1 causing mitotic catastrophe. We revealed subtle differences in the initial Chk1‐involved G₂ arrest with respect to p53/p21^WAF1: absence of either protein led to late G₂ arrest instead of the classic G₂ arrest during checkpoint initiation, and this impacted the release back into the cell cycle. Thus, G₂ arrest correlated with downstream senescence, but late G₂ arrest led to mitotic catastrophe, although both cell cycle re‐entries were linked to upstream Chk1 signalling. Chk1 knockdown deciphered that Chk1 defines long‐term DNA damage responses causing cell cycle re‐entry. We propose that recovery from oxidative DNA damage‐induced G₂ arrest requires Chk1. It works as cutting edge and navigates cells to senescence or mitotic catastrophe. The decision, however, seems to depend on p53/p21^WAF1. The general relevance of Chk1 as an important determinant of recovery from G₂ checkpoint arrest was verified in HT29 colorectal cancer cells.     

Genetic variation in nine Shorea species (Dipterocarpaceae) in Indonesia revealed by AFLPs

Shorea is the largest and most important genus of the Dipterocarpaceae. The genetic diversity and structure of nine Shorea species from two different locations, namely Nanjak Makmur in Sumatra and Sumalindo in Borneo, were evaluated using amplified fragment length polymorphism (AFLP) markers. A total of 274 trees were investigated at 85 polymorphic AFLP loci. Levels of genetic diversity of these species ranged from  = 0.100 for S. acuminata to  = 0.165 for S. blumutensis. The population of rare species S. blumutensis possessed the highest genetic diversity suggesting that geographically restricted species can have levels of genetic variation comparable to closely related widespread common congeners. Analyses of molecular variance revealed that the genetic variation was mainly found among species in both locations (57.7% in Sumatra; 56.3% in Borneo). The unweighted pairgroup method using arithmetic averages dendrogram of all samples revealed an almost complete separation of species. Thus, AFLP markers proved appropriate for phylogenetic studies of Shorea species. Specific markers have been detected showing high-frequency differences among species and between regions within species. Sequence information of these markers can be used to develop specific polymerase chain reaction markers for wood identification. The possibility of interspecific hybridization was discussed.     

Depolarization-induced ERK phosphorylation depends on the cytosolic Ca2+ level rather than on the Ca2+ channel subtype of chromaffin cells

The contribution of Ca2+ entry through different voltage-activated Ca2+ channel (VACC) subtypes to the phosphorylation of extracellular signal regulated kinase (ERK) was examined in bovine adrenal-medullary chromaffin cells. High K+ depolarization (40 mM, 3 min) induced ERK phosphorylation, an effect that was inhibited by specific mitogen-activated protein kinase kinase inhibitors. By using selective inhibitors, we observed that depolarization-induced ERK phosphorylation completely depended on protein kinase C-alpha (PKC-alpha), but not on Ca2+/calmodulin-dependent protein kinase nor cyclic AMP-dependent protein kinase. Blockade of L-type Ca2+ channels by 3 microm furnidipine, or blockade of N channels by 1 micromomega-conotoxin GVIA reduced ERK phosphorylation by 70%, while the inhibition of P/Q channels by 1 micromomega-agatoxin IVA only caused a 40% reduction. The simultaneous blockade of L and N, or P/Q and N channels completely abolished this response, yet 23% ERK phosphorylation remained when L and P/Q channels were simultaneously blocked. Confocal imaging of cytosolic Ca2+ elevations elicited by 40 mm K+, showed that Ca2+ levels increased throughout the entire cytosol, both in the presence and the absence of Ca2+ channel blockers. Fifty-eight percent of the fluorescence rise depended on Ca2+ entering through N channels. Thus, ERK phosphorylation seems to depend on a critical level of Ca2+ in the cytosol rather than on activation of a given Ca2+ channel subtype.     

Neutrophil-derived heparin-binding protein (HBP/CAP37) deposited on endothelium enhances monocyte arrest under flow conditions

In acute inflammation, infiltration of neutrophils often precedes a second phase of monocyte invasion, and data in the literature suggest that neutrophils may directly stimulate mobilization of monocytes via neutrophil granule proteins. In this study, we present a role for neutrophil-derived heparin-binding protein (HBP) in monocyte arrest on endothelium. Adhesion of neutrophils to bovine aorta endothelial cells (ECs) or HUVEC-triggered secretion of HBP and binding of the protein to the EC surface. Blockade of neutrophil adhesion by treatment with a mAb to CD18 greatly reduced accumulation of HBP. In a flow chamber model, immobilized recombinant HBP induced arrest of human monocytes or monocytic Mono Mac 6 (MM6) cells to activated EC or plates coated with recombinant adhesion molecules (E-selectin, P-selectin, VCAM-1). However, immobilized recombinant HBP did not influence arrest of neutrophils or lymphocytes. Treatment of MM6 cells with recombinant HBP evoked a rapid and clear-cut increase in cytosolic free Ca(2+) that was found to be critical for the HBP-induced monocyte arrest inasmuch as pretreatment with the intracellular calcium chelating agent BAPTA-AM abolished the evoked increase in adhesion. Thus, secretion of a neutrophil granule protein, accumulating on the EC surface and promoting arrest of monocytes, could contribute to the recruitment of monocytes at inflammatory loci.     

Transduction of human embryonic stem cells by ecotropic retroviral vectors

The steadily increasing availability of human embryonic stem (hES) cell lines has created strong interest in applying available tools for gene transfer in murine cells to human systems. Here we present a method for the transduction of hES cells with ecotropic retroviral vectors. hES cells were transiently transfected with a construct carrying the murine retrovirus receptor mCAT1. Subsequently, the cells were exposed to replication-deficient Moloney murine leukemia virus (MoMuLV) derivatives or pseudotyped lentiviral vectors. With oncoretroviral vectors, this procedure yields overall transduction efficiencies of up to 20% and permits selection of permanently transduced clones with high frequency. Selected clones maintained expression of pluripotency-associated markers and exhibited multi-germ layer differentiation both in vitro and in vivo. HES cell-derived somatic cells including neural progeny maintained high levels of transgene expression. Lentiviral vectors pseudotyped with the MoMuLV envelope could be introduced in the same manner with efficiencies of up to 33%. Transgene expression of lentivirally transduced hES cells remained permanent after differentiation even without selection pressure. Bypassing the regulatory issues associated with the use of amphotropic retroviral systems and exploiting the large pool of existing murine vectors, this method provides a safe and versatile tool for gene transfer and lineage analysis in hES cells and their progeny.