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.     

Differential retrotranslocation of mitochondrial Bax and Bak

The Bcl-2 proteins Bax and Bak can permeabilize the outer mitochondrial membrane and commit cells to apoptosis. Pro-survival Bcl-2 proteins control Bax by constant retrotranslocation into the cytosol of healthy cells. The stabilization of cytosolic Bax raises the question whether the functionally redundant but largely mitochondrial Bak shares this level of regulation. Here we report that Bak is retrotranslocated from the mitochondria by pro-survival Bcl-2 proteins. Bak is present in the cytosol of human cells and tissues, but low shuttling rates cause predominant mitochondrial Bak localization. Interchanging the membrane anchors of Bax and Bak reverses their subcellular localization compared to the wild-type proteins. Strikingly, the reduction of Bax shuttling to the level of Bak retrotranslocation results in full Bax toxicity even in absence of apoptosis induction. Thus, fast Bax retrotranslocation is required to protect cells from commitment to programmed death.     

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.     

Specific Human Astrocyte Subtype Revealed by Affinity Purified GFAP+1 Antibody; Unpurified Serum Cross-Reacts with Neurofilament-L in Alzheimer

The human GFAP splice variants GFAPΔ164 and GFAPΔexon6 both result in a GFAP protein isoform with a unique out-of-frame carboxy-terminus that can be detected by the GFAP⁺¹ antibody. We previously reported that GFAP⁺¹ was expressed in astrocytes and in degenerating neurons in Alzheimer''s disease brains. In this study we aimed at further investigating the neuronal GFAP⁺¹ expression and we started by affinity purifying the GFAP⁺¹ antibody. The purified antibody resulted in a loss of neuronal GFAP⁺¹ signal, although other antibodies directed against the amino- and carboxy-terminus of GFAPα still revealed GFAP-immunopositive neurons, as described before. With an in-depth analysis of a western blot, followed by mass spectrometry we discovered that the previously detected neuronal GFAP⁺¹ expression was due to cross-reactivity of the antibody with neurofilament-L (NF-L). This was confirmed by double-label fluorescent immunohistochemistry and western blotting with the unpurified GFAP⁺¹ antibody and an antibody against NF-L. Our data imply that NF-L can accumulate in some tangle-like structures in Alzheimer brains. More importantly, the purified GFAP⁺¹ antibody clearly revealed a specific subtype of astrocytes in the adult human brain. These large astrocytes are present throughout the brain, e.g., along the subventricular zone, in the hippocampus, in the striatum and in the spinal cord of controls, Alzheimer, and Parkinson patients. The presence of a specific GFAP-isoform suggests a specialized function of these astrocytes.     

Heterologous expression of L. major proteins in S. cerevisiae: a test of solubility, purity, and gene recoding

High level expression of many eukaryotic proteins for structural analysis is likely to require a eukaryotic host since many proteins are either insoluble or lack essential post-translational modifications when expressed in E. coli. The well-studied eukaryote Saccharomyces cerevisiae possesses several attributes of a good expression host: it is simple and inexpensive to culture, has proven genetic tractability, and has excellent recombinant DNA tools. We demonstrate here that this yeast exhibits three additional characteristics that are desirable in a eukaryotic expression host. First, expression in yeast significantly improves the solubility of proteins that are expressed but insoluble in E. coli. The expression and solubility of 83 Leishmania major ORFs were compared in S. cerevisiae and in E. coli, with the result that 42 of the 64 ORFs with good expression and poor solubility in E. coli are highly soluble in S. cerevisiae. Second, the yield and purity of heterologous proteins expressed in yeast is sufficient for structural analysis, as demonstrated with both small scale purifications of 21 highly expressed proteins and large scale purifications of 2 proteins, which yield highly homogeneous preparations. Third, protein expression can be improved by altering codon usage, based on the observation that a codon-optimized construct of one ORF yields three-fold more protein. Thus, these results provide direct verification that high level expression and purification of heterologous proteins in S. cerevisiae is feasible and likely to improve expression of proteins whose solubility in E. coli is poor.     

Role of ultrasonography in diagnosing early rheumatoid arthritis and remission of rheumatoid arthritis - a systematic review of the literature

Introduction

Ultrasonography (US) might have an added value to clinical examination in diagnosing early rheumatoid arthritis (RA) and assessing remission of RA. We aimed to clarify the added value of US in RA in these situations performing a systematic review.

Methods

A systematic literature search was performed for RA, US, diagnosis and remission. Methodological quality was assessed; the wide variability in the design of studies prohibited pooling of results.

Results

Six papers on the added value of US diagnosing early RA were found, in which at least bilateral metacarpophalangeal (MCP), wrists and metatarsophalangeal (MTP) joints were scanned. Compared to clinical examination, US was superior with regard to detecting synovitis and predicting progression to persistent arthritis or RA. Eleven papers on assessing remission were identified, in which at least the wrist and the MCP joints of the dominant hand were scanned. Often US detected inflammation in patients clinically in remission, irrespective of the remission criteria used. Power Doppler signs of synovitis predicted X-ray progression and future flare in patients clinically in remission.

Conclusions

US appears to have added value to clinical examination for diagnosing of RA when scanning at least MCP, wrist and MTP joints, and, when evaluating remission of RA, scanning at least wrist and MCP joints of the dominant hand. For both purposes primarily power Doppler US might be used since its results are less equivocal than those of greyscale US.