A new method for the determination of thrombocyte function in patients and in preserved blood]

In a circulating thrombocytic plasma the pressure in front of a defined mesh filter depends on the combined capacity of adhesion and aggregation of thrombocytes. A new clinical in vitro test is based on this principle which comes nearest to the conditions to be found in hemostasis dependent on thrombocytes. Judging from the preliminary results of investigations performed on stored blood, blood donors, patients with thrombocytopenia, disturbances of blood flow and patients under anticoagulant therapy, the time until the pressure rise after administering ADP and the maximal pressure amplitude proved to be the most significant parameters of the new procedure.     

The molecular organization of the beta-globin complex of the deer mouse, Peromyscus maniculatus

Recombinant DNA clones have been isolated that contain 80 kb of the beta-globin complex from the deer mouse, Peromyscus maniculatus. Comparisons of this complex with that from the laboratory mouse, Mus domesticus (with an order 5'-Hbby, Hbb-bhO, Hbb-bhl, Hbb-bh2, Hbb-bh3, Hbb-bl, Hbb-b2 3') highlight organizational trends in the beta-globin complex since the two species diverged. Unlike other mammals studied thus far, the deer mouse possesses three adult genes. Partial sequence analysis indicates that each of the three adult genes is intact and hence may be functional. Hybridization of one of the two Mus pseudogenes, Hbb-bh3, to genomic blots from Peromyscus reveals that it has a homologous counterpart in Peromyscus. Homologous genes to the two gamma-like Mus genes, Hbb-bhO and Hbb-bhl, are also found in Peromyscus. The strong hybridization between the Hbb-bhl genes and significant nucleotide similarity between the Hbb-bhO genes suggest that both pairs are important for the ontogeny of these mice although no known product has been identified for the Hbb-bhO genes. The presence of Hbb-bhO and Hbb-bhl in Peromyscus suggests that the duplication that created this related gene set occurred before the two lineages diverged. A single gene for Hbb-y has been isolated from Peromyscus. The adult region in Peromyscus has undergone significant divergence from the same region in Mus, having three rather than two adult genes, the acquisition of at least 15 kb of extra DNA relative to Mus, and possibly the loss of the Hbb-bh2 pseudogene. The nonadult region of the complex, in contrast, contains the same set of genes apparently distributed over the same amount of DNA as in the Mus beta- globin complex. This observation suggests that the embryonic region of the complex is more evolutionarily stable than the adult region.      

Cell-free production and characterisation of human uncoupling protein 1–3

The uncoupling proteins (UCPs) leak protons across the inner mitochondrial membrane, thus uncoupling the proton gradient from ATP synthesis. The main known physiological role for this is heat generation by UCP1 in brown adipose tissue. However, UCPs are also believed to be important for protection against reactive oxygen species, fine-tuning of metabolism and have been suggested to be involved in disease states such as obesity, diabetes and cancer.Structural studies of UCPs have long been hampered by difficulties in sample preparation with neither expression in yeast nor refolding from inclusion bodies in E. coli yielding sufficient amounts of pure and stable protein. In this study, we have developed a protocol for cell-free expression of human UCP1, 2 and 3, resulting in 1 mg pure protein per 20 mL of expression media. Lauric acid, a natural UCP ligand, significantly improved protein thermal stability and was therefore added during purification. Secondary structure characterisation using circular dichroism spectroscopy revealed the proteins to consist of mostly α-helices, as expected. All three UCPs were able to bind GDP, a well-known physiological inhibitor, as shown by the Fluorescence Resonance Energy Transfer (FRET) technique, suggesting that the proteins are in a natively folded state.     

Arabidopsis MSL10 Has a Regulated Cell Death Signaling Activity That Is Separable from Its Mechanosensitive Ion Channel Activity

Members of the MscS superfamily of mechanosensitive ion channels function as osmotic safety valves, releasing osmolytes under increased membrane tension. MscS homologs exhibit diverse topology and domain structure, and it has been proposed that the more complex members of the family might have novel regulatory mechanisms or molecular functions. Here, we present a study of MscS-Like (MSL)10 from Arabidopsis thaliana that supports these ideas. High-level expression of MSL10-GFP in Arabidopsis induced small stature, hydrogen peroxide accumulation, ectopic cell death, and reactive oxygen species- and cell death-associated gene expression. Phosphomimetic mutations in the MSL10 N-terminal domain prevented these phenotypes. The phosphorylation state of MSL10 also regulated its ability to induce cell death when transiently expressed in Nicotiana benthamiana leaves but did not affect subcellular localization, assembly, or channel behavior. Finally, the N-terminal domain of MSL10 was sufficient to induce cell death in tobacco, independent of phosphorylation state. We conclude that the plant-specific N-terminal domain of MSL10 is capable of inducing cell death, this activity is regulated by phosphorylation, and MSL10 has two separable activities—one as an ion channel and one as an inducer of cell death. These findings further our understanding of the evolution and significance of mechanosensitive ion channels.