Automated protein crystallization and a new crystal form of a subtilisin:eglin complex

A procedure is described for automating labour-intensive steps of the 'hanging drop' protein crystallization method. An automatic sample changer is employed to fill the wells in a multi-well plate so that concentration gradients in various components are obtained. The sample changer is also used for preparing droplets on a second multi-well plate. Subsequently, this second plate is manually turned around and placed on top of the first multi-well plate such that a large number of chambers with different conditions is obtained simultaneously. During initial trials a new crystal form of a subtilisin:eglin complex was obtained. The crystals have space group P2(1), contain two enzyme inhibitor complexes per asymmetric unit and diffract beyond 2.2 A.     

Spatial Structure Facilitates Cooperation in a Social Dilemma: Empirical Evidence from a Bacterial Community

Cooperative organisms are ubiquitous in nature, despite their vulnerability to exploitation by cheaters. Although numerous theoretical studies suggest that spatial structure is critical for cooperation to persist, the spatial ecology of microbial cooperation remains largely unexplored experimentally. By tracking the community dynamics of cooperating (rpoS wild-type) and cheating (rpoS mutant) Escherichia coli in well-mixed flasks and microfabricated habitats, we demonstrate that spatial structure stabilizes coexistence between wild-type and mutant and thus facilitates cooperator maintenance. We develop a method to interpret our experimental results in the context of game theory, and show that the game wild-type and mutant bacteria play in an unstructured environment changes markedly over time, and eventually obeys a prisoner’s dilemma leading to cheater dominance. In contrast, when wild-type and mutant E. coli co-inhabit a spatially-structured habitat, cooperators and cheaters coexist at intermediate frequencies. Our findings show that even in microhabitats lacking patchiness or spatial heterogeneities in resource availability, surface growth allows cells to form multi-cellular aggregates, yielding a self-structured community in which cooperators persist.     

Hexamers of the Type II Secretion ATPase GspE from Vibrio cholerae with Increased ATPase Activity

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Exogenously applied 20-hydroxyecdysone increases the net photosynthetic rate but does not affect the photosynthetic electron transport or the content of photosynthetic pigments in Tetragonia tetragonioides L.

Phytoecdysteroids are steroid compounds present in many plant species (sometimes in rather large amounts), but their biological role is still far from being clear. We have found that the exogenous application of 20-hydroxyecdysone (20E) to leaves of Tetragonia tetragonioides L. causes stimulation of its net photosynthetic rate (P _N) but does not positively affect the photosynthetic electron transport or the content of photosynthetic pigments. The increase in P _N was observed shortly after 20E treatment and was statistically significant during the 4th and 6th hours after treatment but not later, which could be perhaps caused by a strictly short-term window of opportunity for ecdysteroids to significantly affect photosynthetic processes. To our knowledge, these results are the first to suggest a new potential biological function of phytoecdysteroids—regulation of photosynthesis.     

Detection of cell type and marker specificity of nuclear binding sites for anionic carbohydrate ligands

The emerging functionality of glycosaminoglycan chains engenders interest in localizing specific binding sites using cytochemical tools. We investigated nuclear binding of labeled heparin, heparan sulfate, a sulfated fucan, chondroitin sulfate, and hyaluronic acid in epidermal keratinocytes, bone marrow stromal cells, 3T3 fibroblasts and glioma cells using chemically prepared biotinylated probes. Binding of the markers was cell-type specific and influenced by extraction of histones, but was not markedly affected by degree of proliferation, differentiation or malignancy. Cell uptake of labeled heparin and other selected probes and their transport into the nucleus also was monitored. Differences between keratinocytes and bone marrow stromal cells were found. Preincubation of permeabilized bone marrow stromal cells with label-free heparin reduced the binding of carrier-immobilized hydrocortisone to its nuclear receptors. Thus, these tools enabled binding sites for glycosaminoglycans to be monitored in routine assays.     

A radiological study of the central circulation in the lungfish, Protopterus aethiopicus

The dipnoan heart is only in part structurally developed to support a separated circulation in pulmonary and systemic circuits. In the present investigation biplane angiocardiography has been used to describe the extent of such a double circulation and the factors which may modify it in the African lungfish, Protopterus aethiopicus. Contrast injections in the pulmonary vein revealed a clear tendency for aerated blood returing from the lungs to be selectively dispatched to the anterior branchial arteries giving rise to the major systemic circulation. Contrast injections in the vena cava delineated the sinus venosus as a large receiving chamber for systemic venous blood. Contraction of the sinus venosus discharged blood into the right, posterior part of the partially divided atrial space. Contrast injection in the pulmonary vein showed that vessel to pass obliquely from right to left such that blood was emptied distinctly into the left side of the atrium. During contraction the atrial space tended to retain a residual volume in its anterior undivided part which minized mixing. Ventricular filling occurred through separate right and left atrio-ventricular connections. Right-left separation in most of the ventricle was maintained by the partial ventricular septum, the trabeculated, spongelike myocardium and the mode of inflow from the atria. Mixing in the anterior undivided portion of the ventricle during the ejection phase was slight due to a streamlined ejection pattern. The outflow through the bulbus cordis occurred in discrete streams which in part were structurally separated by well developed spiral folds. In the anterior bulbus segment the spiral folds are fused and make completely separate dorsal and ventral outflow tracts. The ventral bulbus channel provides blood to the three anterior branchial arteries. The second and third branchial arteries are large and represent direct shunts to the dorsal aorta. The fourth and fifth branchial arteries are gill bearing and receive blood form the dorsal bulbus channel. The most posterior epibranchial vessels give rise to the pulmonary arteries.     

Loss of CDK5RAP2 affects neural but not non-neural mESC differentiation into cardiomyocytes

Biallelic mutations in the gene encoding centrosomal CDK5RAP2 lead to autosomal recessive primary microcephaly (MCPH), a disorder characterized by pronounced reduction in volume of otherwise architectonical normal brains and intellectual deficit. The current model for the microcephaly phenotype in MCPH invokes a premature shift from symmetric to asymmetric neural progenitor-cell divisions with a subsequent depletion of the progenitor pool. The isolated neural phenotype, despite the ubiquitous expression of CDK5RAP2, and reports of progressive microcephaly in individual MCPH cases prompted us to investigate neural and non-neural differentiation of Cdk5rap2-depleted and control murine embryonic stem cells (mESC). We demonstrate an accumulating proliferation defect of neurally differentiating Cdk5rap2-depleted mESC and cell death of proliferative and early postmitotic cells. A similar effect does not occur in non-neural differentiation into beating cardiomyocytes, which is in line with the lack of non-central nervous system features in MCPH patients. Our data suggest that MCPH is not only caused by premature differentiation of progenitors, but also by reduced propagation and survival of neural progenitors.