Role of calcium in the release of EC 3.4.24.15 and EC 3.4.24.16 from endothelial cells

The two closely related soluble zinc metalloendopeptidases EC 3.4.24.15 (EP24.15) and EC 3.4.24.16 (EP24.16) readily hydrolyze the vasoactive peptide bradykinin in vitro, and therefore may play a role in cardiovascular regulation. Although primarily soluble cytosolic enzymes, both secreted and membrane-associated forms of both peptidases have been reported. However, these enzymes have neither a transmembrane domain nor a signal sequence; thus, the mechanisms of membrane anchoring and secretion are unknown. In the present study, secreted/released EP24.15 and EP24.16 activity from aortic endothelial cells in culture was assessed by the cleavage of a specific quenched fluorescent substrate. An increase in enzyme activity released from endothelial cells, which express both peptidases, was seen following incubation with calcium-free media. In the AtT-20 endocrine cell (mouse pituitary corticotrope), which predominantly expresses EP24.15, the release of activity into media was unaffected by calcium removal. The release of enzyme activity from endothelial cells was inversely proportional to calcium concentrations ranging between 0.01 mM (activity equivalent to calcium-free media) and 0.5 mM (activity equivalent to normal media). Cleavage of the EP24.16-specific substrate AcNT_8-13 indicated that the increase in enzyme activity released upon incubation with calcium-free medium was due at least in part to the release of EP24.16. These results suggest that EP24.15 and EP24.16 are secreted from endothelial cells, and that removal of calcium selectively enhances the release of EP24.16 by an as yet unknown mechanism.     

Commingling in distributions of lipids and related variables.

In a sample of nearly 8,000 Japanese males, the distributions of casual cholesterol and triglyceride, hematocrit, glucose, uric acid, diastolic blood pressure, and weight (covariance adjusted) could not be normalized by a power transform and were significantly better fitted by a mixture of distributions. The evidence for admixture was nonsignificant for systolic blood pressure, significant but unimpressive for height and weight, and strong for the remaining variables. The minor component corresponded to high values, in low frequency except for triglyceride and glucose. These results favor an interpretation of elevated levels in terms of distinct entities, genetic or environmental or both, rather than cumulative small effects only. These entities appear to be megaphenic (i.e., with effects exceeding one phenotypic standard deviation). Consequences of this hypothesis are discussed.     

p34cdc2 kinase is localized to distinct domains within the mitotic apparatus

Antibodies to both the C-terminal and the N-terminal regions of the 34 kd serine-threonine specific protein kinase, p34cdc2, were used to study the distribution of this protein in dividing cells and isolated chromosomes of the Indian muntjac. p34cdc2 was found to be present throughout the cytoplasm of dividing cells. In addition, a portion of cellular p34cdc2 was localized to the centrosome, kinetochore, and intercellular bridge and along kinetochore-to-pole microtubules during cell division. Tubulin-denuded metaphase kinetochores retained their association with p34cdc2. The detection of p34cdc2 within a variety of domains of the mitotic apparatus, in addition to the previous reported association with the centrosome [Bailly et al., EMBO J. 8:3985-3995, 1989; Raibowol et al., Cell 57:393-401, 1989] suggests that p34cdc2 may play a role in events associated with anaphases A and B as well as with the transition between interphase and mitosis.     

Multipoint linkage analysis.

A formula is given for the advantage of n-point sampling, which approaches infinity with n. However, 2-point and 3-point analyses extract nearly all the information in such samples and at the same time communicate this information as lods and chi 2, which can be combined with other data by simple addition without reevaluation of the likelihood. When null interference is assumed, map distances and multiple recombination frequencies are inflated, and there is substantial loss of efficiency and of support for the correct order.     

Influenza A virus infection results in a robust, antigen-responsive, and widely disseminated Foxp3+ regulatory T cell response

Foxp3(+) CD4(+) regulatory T cells (Tregs) represent a highly suppressive T cell subset with well-characterized immunosuppressive effects during immune homeostasis and chronic infections, although the role of these cells in acute viral infections is poorly understood. The present study sought to examine the induction of Foxp3(+) CD4(+) Tregs in a nonlethal murine model of pulmonary viral infection by the use of the prototypical respiratory virus influenza A. We establish that influenza A virus infection results in a robust Foxp3(+) CD4(+) T cell response and that regulatory T cell induction at the site of inflammation precedes the effector T cell response. Induced Foxp3(+) CD4(+) T cells are highly suppressive ex vivo, demonstrating that influenza virus-induced Foxp3(+) CD4(+) T cells are phenotypically regulatory. Influenza A virus-induced regulatory T cells proliferate vigorously in response to influenza virus antigen, are disseminated throughout the site of infection and primary and secondary lymphoid organs, and retain Foxp3 expression in vitro, suggesting that acute viral infection is capable of inducing a foreign-antigen-specific Treg response. The ability of influenza virus-induced regulatory T cells to suppress antigen-specific CD4(+) and CD8(+) T cell proliferation and cytokine production correlates closely to their ability to respond to influenza virus antigens, suggesting that virus-induced Tregs are capable of attenuating effector responses in an antigen-dependent manner. Collectively, these data demonstrate that primary acute viral infection is capable of inducing a robust, antigen-responsive, and suppressive regulatory T cell response.     

Standing genetic variance for female resistance to harm from males and its relationship to intralocus sexual conflict

Interlocus sexual conflict theory predicts that some male adaptations are harmful to their mates. Females are therefore expected to evolve resistance to this harm. Using cytogenetic cloning techniques, we tested for heritable genetic variation among females for resistance to harm from males and determined whether propensity to remate, female body size, and intralocus conflict contributes to this variation. We found low but significant heritability for female resistance, but this variation accounted for more than half of the standing genetic variation for net fitness among females. We found no association between female resistance and female body size or level of intralocus sexual conflict. Reluctance to remate was found to be an important factor contributing to the female resistance phenotype, and we found a positive selection gradient on this trait. However, we observed only a nonsignificant positive correlation between a female's resistance and her net fitness. One factor contributing to the observed nominal level of selection on female resistance was that males cause the greatest amount of harm to females with the highest intrinsic fecundity.     

TMAO promotes fibrillization and microtubule assembly activity in the C-terminal repeat region of tau

Alzheimer's disease most closely correlates with the appearance of the neurofibrillary tangles (NFTs), intracellular fibrous aggregates of the microtubule-associated protein, tau. Under native conditions, tau is an unstructured protein, and its physical characterization has revealed no clues about the three-dimensional structural determinants essential for aggregation or microtubule binding. We have found that the natural osmolyte trimethylamine N-oxide (TMAO) induces secondary structure in a C-terminal fragment of tau (tau(187)) and greatly promotes both self-aggregation and microtubule (MT) assembly activity. These processes could be distinguished, however, by a single-amino acid substitution (Tyr(310) --> Ala), which severely inhibited aggregation but had no effect on MT assembly activity. The inability of this mutant to aggregate could be completely reversed by TMAO. We propose a model in which TMAO induces partial order in tau(187), resulting in conformers that may correspond to on-pathway intermediates of either aggregation or tau-dependent MT assembly or both. These studies set the stage for future high-resolution structural characterization of these intermediates and the basis by which Tyr(310) may direct pathologic versus normal tau function.     

2-Aminobenzimidazoles as potent Aurora kinase inhibitors

This Letter describes the discovery and key structure–activity relationship (SAR) of a series of 2-aminobenzimidazoles as potent Aurora kinase inhibitors. 2-Aminobenzimidazole serves as a bioisostere of the biaryl urea residue of SNS-314 (1c), which is a potent Aurora kinase inhibitor and entered clinical testing in patients with solid tumors. Compared to SNS-314, this series of compounds offers better aqueous solubility while retaining comparable in vitro potency in biochemical and cell-based assays; in particular, 6m has also demonstrated a comparable mouse iv PK profile to SNS-314.     

Anaerobic degradation pathway and kinetics of domestic wastewater at low temperatures

The effect of temperatures below 20 °C (20, 15 and 10 °C) on the anaerobic degradation pathway and kinetics of domestic wastewater fractionated at different sizes was studied in a fluidized-bed batch reactor. The overall degradation pathway was characterized by a soluble fraction degrading according to zero-order kinetics and a colloidal fraction (between 0.45 and 4.5 μm) that first disintegrates into a particulate fraction smaller than 0.45 μm before finally degrading. The colloidal degradation processes follow a first-order kinetic. In contrast, suspended solids (bigger than 4.5 μm) degrade to soluble and colloidal fractions according to first-order kinetics. The colloidal fraction originating from suspended solids further degrades into soluble fraction. These soluble fractions have the same degradation kinetics as the original soluble fraction. The suspended solids degradation was highly affected by temperature, whereas the soluble fraction slightly affected and the colloidal fraction was not affected at all. On the other hand, the colloidal non-degradable fraction increased significantly with the decrease in temperature while the suspended solids slowly increased. The soluble non-degradable fraction was little affected by temperatures changes.