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BackgroundNitric oxide (NO) produced by nitric oxide synthase (NOS) in human red blood cells (RBCs) was shown to depend on shear stress and to exhibit important biological functions, such as inhibition of platelet activation. In the present study we hypothesized that exercise-induced shear stress stimulates RBC-NOS activation pathways, NO signaling, and deformability of human RBCs.Methods/FindingsFifteen male subjects conducted an exercise test with venous blood sampling before and after running on a treadmill for 1 hour. Immunohistochemical staining as well as western blot analysis were used to determine phosphorylation and thus activation of Akt kinase and RBC-NOS as well as accumulation of cyclic guanylyl monophosphate (cGMP) induced by the intervention. The data revealed that activation of NO upstream located enzyme Akt kinase was significantly increased after the test. Phosphorylation of RBC-NOSSer1177 was also significantly increased after exercise, indicating activation of RBC-NOS through Akt kinase. Total detectable RBC-NOS content and phosphorylation of RBC-NOSThr495 were not affected by the intervention. NO production by RBCs, determined by DAF fluorometry, and RBC deformability, measured via laser-assisted-optical-rotational red cell analyzer, were also significantly increased after the exercise test. The content of the NO downstream signaling molecule cGMP increased after the test. Pharmacological inhibition of phosphatidylinositol 3 (PI3)-kinase/Akt kinase pathway led to a decrease in RBC-NOS activation, NO production and RBC deformability.Conclusion/SignificanceThis human in vivo study first-time provides strong evidence that exercise-induced shear stress stimuli activate RBC-NOS via the PI3-kinase/Akt kinase pathway. Actively RBC-NOS-produced NO in human RBCs is critical to maintain RBC deformability. Our data gain insights into human RBC-NOS regulation by exercise and, therefore, will stimulate new therapeutic exercise-based approaches for patients with microvascular disorders.

SummaryCullin proteins, which belong to multigenic families in all eukaryotes, associate with other proteins to form ubiquitin protein ligases (E3s) that target substrates for proteolysis by the 26S proteasome. Here, we present the molecular and genetic characterization of a plant Cullin3. In contrast to fungi and animals, the genome of the model plant Arabidopsis thaliana contains two related CUL3 genes, called CUL3A and CUL3B. We found that CUL3A is ubiquitously expressed in plants and is able to interact with the ring-finger protein RBX1. A genomic search revealed the existence of at least 76 BTB-domain proteins in Arabidopsis belonging to 11 major families. Yeast two-hybrid experiments indicate that representative members of certain families are able to physically interact with both CUL3A and CUL3B, suggesting that Arabidopsis CUL3 forms E3 protein complexes with certain BTB domain proteins. In order to determine the function of CUL3A, we used a reverse genetic approach. The cul3a null mutant flowers slightly later than the control plants. Furthermore, this mutant exhibits a reduced sensitivity of the inhibition of hypocotyl growth in far-red light and miss-expresses COP1. The viability of the mutant plants suggests functional redundancy between the two CUL3 genes in Arabidopsis.

Matrix protein (36,500 daltons), one of the major polypeptides of the Escherichia coli cell envelope, is arranged in a periodic monolayer which covers the outer surface of the peptidoglycan. Although its association with the peptidoglycan layer is probably tight, the periodic structure is maintained in the absence of peptidoglycan, and is therefore based on strong protein-protein interactions. A detailed analysis of the ultrastructure of the matrix protein array by electron microscopy and image processing of specimens prepared by negative staining or by freezedrying and shadowing shows that the molecules are arranged according to threefold symmetry on a hexagonal lattice whose repeat is 7.7 nm. The most pronounced feature of the unit cell, which probably contains three molecules of matrix protein, is a triplet of indentations, each approx. 2 nm in diameter, with a center-to-center spacing of 3 nm. They are readily penetrated by stain and may represent channels which span the protein monolayer.The envelopes of Gram-negative bacteria have a multilaminar organization consisting of a cytoplasmic membrane, a rigid peptidoglycan layer, and an outer membrane. The latter is composed of phospholipids, lipopolysaccharides, and proteins. These include the matrix protein 1 (16) which, in Escherichia coli, has a mol wt of 36,500. The cellular complement of approx. 105 molecules forms a periodic array in close association with the underlying peptidoglycan layer. Numerically, the most abundant outer membrane protein is a lipoprotein (7,000 daltons) which has been extensively characterized (4). Each cell has approx. 6 • 105 copies, of which a third (11) are covalently linked to the peptide cross-bridges of the peptidoglycan layer (4). In addition, the outer membrane contains several other major (9) as well as a number of minor protein species, which have not yet 1 This protein has been designated by various names (cf. reference 7). been so well characterized and will not be considered further here.The structural organization of the outer membrane and the interactions between its components are not yet well understood. Likewise, the functions of its major polypeptides are still unclear. Motivated by the idea that information on the architecture of the periodic arrangement of matrix protein which effectively covers the peptidoglycan cell wall might afford insight into these questions, we have undertaken a study of it by electron microscopy combined with image processing. Our findings are reported in this communication.

Freeze-etch electron microscopy was used to show that colchicine interacts with membranes of the ciliate protozoan Tetrahymena pyriformis. Colchicine impairs the temperature-induced translational and vertical mobility of the membrane-intercalating particles of the freeze-fractured alveolar membranes lying just below the plasma membranes.