Anne E. Cowan

Annexin I is an endogenous ligand that mediates apoptotic cell engulfment

Engulfment of apoptotic cells requires presentation of new cell surface ligands by the dying cells. Using a differential proteomics technology, we identify that annexin I is a caspase-dependent engulfment ligand; it is recruited from the cytosol and exported to the outer plasma membrane leaflet, colocalizes with phosphatidylserine, and is required for efficient clearance of apoptotic cells. Furthermore, phosphatidylserine receptor (PSR) clustering around apoptotic cells indicates a requirement for annexin I. In the nematode Caenorhabditis elegans, downregulation of the annexin homolog prevents efficient engulfment of pharyngeal cell corpses. These results provide novel mechanistic insights into how apoptotic cells are removed and may explain a pathogenic mechanism of chronic inflammatory diseases where annexin I autoantibodies have been described.

Essential Role for Prolyl Hydroxylase Domain Protein 2 in Oxygen Homeostasis of the Adult Vascular System

Background— Prolyl hydroxylase domain (PHD) proteins, including PHD1, PHD2, and PHD3, mediate oxygen-dependent degradation of hypoxia-inducible factor (HIF)-α subunits. Although angiogenic roles of hypoxia-inducible factors are well known, the roles of PHDs in the vascular system remain to be established. Methods and Results— We evaluated angiogenic phenotypes in mice carrying targeted disruptions in genes encoding different PHD isoforms. Although Phd1−/− and Phd3−/− mice did not display apparent angiogenic defects, broad-spectrum conditional knockout of Phd2 led to hyperactive angiogenesis and angiectasia. Blood vessels in PHD2-deficient mice were highly perfusable. Furthermore, examination of medium-sized vessels in subendocardial layer in the heart demonstrated successful recruitment of vascular smooth muscle cells. Surprisingly, increased vascular growth was independent of local efficiency of Phd2 disruption. Mice carrying significant Phd2 disruption in multiple organs, including the liver, heart, kidney, and lung, displayed excessive vascular growth not only in these organs but also in the brain, where Phd2 disruption was very inefficient. More surprisingly, increased accumulation of hypoxia-inducible factor-1α and angiectasia in the liver were not accompanied by corresponding increases in hepatic expression of Vegfa or angiopoietin-1. However, the serum vascular endothelial growth factor-A level was significantly increased in PHD2-deficient mice. Conclusions— PHD2, but not PHD1 and PHD3, is a major negative regulator for vascular growth in adult mice. Increased angiogenesis in PHD2-deficient mice may be mediated by a novel systemic mechanism.

Luteinizing hormone causes MAP kinase-dependent phosphorylation and closure of connexin 43 gap junctions in mouse ovarian follicles: one of two paths to meiotic resumption

Luteinizing hormone (LH) acts on ovarian follicles to reinitiate meiosis in prophase-arrested mammalian oocytes, and this has been proposed to occur by interruption of a meioisis-inhibitory signal that is transmitted through gap junctions into the oocyte from the somatic cells that surround it. To investigate this idea, we microinjected fluorescent tracers into live antral follicle-enclosed mouse oocytes, and we demonstrate for the first time that LH causes a decrease in the gap junction permeability between the somatic cells, prior to nuclear envelope breakdown (NEBD). The decreased permeability results from the MAP kinase-dependent phosphorylation of connexin 43 on serines 255, 262 and 279/282. We then tested whether the inhibition of gap junction communication was sufficient and necessary for the reinitiation of meiosis. Inhibitors that reduced gap junction permeability caused NEBD, but an inhibitor of MAP kinase activation that blocked gap junction closure in response to LH did not prevent NEBD. Thus, both MAP kinase-dependent gap junction closure and another redundant pathway function in parallel to ensure that meiosis resumes in response to LH.

Mechanisms of killing spores of Bacillus subtilis by acid, alkali and ethanol

Aims: To determine the mechanisms of killing of Bacillus subtilis spores by ethanol or strong acid or alkali.

How moist heat kills spores of Bacillus subtilis.

Populations of Bacillus subtilis spores in which 90 to 99.9% of the spores had been killed by moist heat gave only two fractions on equilibrium density gradient centrifugation: a fraction comprised of less dense spores that had lost their dipicolinic acid (DPA), undergone significant protein denaturation, and were all dead and a fraction with the same higher density as that of unheated spores. The latter fraction from heat-killed spore populations retained all of its DPA, but >/=98% of the spores could be dead. The dead spores that retained DPA germinated relatively normally with nutrient and nonnutrient germinants, but the outgrowth of these germinated spores was significantly compromised, perhaps because they had suffered damage to some proteins such that metabolic activity during outgrowth was greatly decreased. These results indicate that DPA release takes place well after spore killing by moist heat and that DPA release during moist-heat treatment is an all-or-nothing phenomenon; these findings also suggest that damage to one or more key spore proteins causes spore killing by moist heat.

Germination of spores of Bacillus subtilis with dodecylamine

Aims: To determine the properties of Bacillus subtilis spores germinated with the alkylamine dodecylamine, and the mechanism of dodecylamine‐induced spore germination.

A soluble protein is immobile in dormant spores of Bacillus subtilis but is mobile in germinated spores: Implications for spore dormancy

Fluorescence redistribution after photobleaching has been used to show that a cytoplasmic GFP fusion is immobile in dormant spores of Bacillus subtilis but becomes freely mobile in germinated spores in which cytoplasmic water content has increased ≈2-fold. The GFP immobility in dormant spores is not due to the high levels of dipicolinic acid in the spore cytoplasm, because GFP was also immobile in germinated cwlD spores that had excreted their dipicolinic acid but where cytoplasmic water content had only increased to a level similar to that in dormant spores of several other Bacillus species. The immobility of a normally mobile protein in dormant wild-type spores and germinated cwlD spores is consistent with the lack of metabolism and enzymatic activity in these spores and suggests that protein immobility, presumably due to low water content, is a major reason for the metabolic dormancy of spores of Bacillus species.

Studies on the mechanism of killing of Bacillus subtilis spores by hydrogen peroxide

Aims: To determine the mechanism of killing of Bacillus subtilis spores by hydrogen peroxide.

Fluorescence correlation spectroscopy analysis of serotonin, adrenergic, muscarinic, and dopamine receptor dimerization: the oligomer number puzzle.

The issue of G protein–coupled receptor (GPCR) oligomer status has not been resolved. Although many studies have provided evidence in favor of receptor-receptor interactions, there is no consensus as to the exact oligomer size of class A GPCRs. Previous studies have reported monomers, dimers, tetramers, and higher-order oligomers. In the present study, this issue was examined using fluorescence correlation spectroscopy (FCS) with photon counting histogram (PCH) analysis, a sensitive method for monitoring diffusion and oligomer size of plasma membrane proteins. Six different class A GPCRs were selected from the serotonin (5-HT2A), adrenergic (α1b-AR and β2-AR), muscarinic (M1 and M2), and dopamine (D1) receptor families. Each GPCR was C-terminally labeled with green fluorescent protein (GFP) or yellow fluorescent protein (YFP) and expressed in human embryonic kidney 293 cells. FCS provided plasma membrane diffusion coefficients on the order of 7.5 × 10−9 cm2/s. PCH molecular brightness analysis was used to determine the GPCR oligomer size. Known monomeric (CD-86) and dimeric (CD-28) receptors with GFP and YFP tags were used as controls to determine the molecular brightness of monomers and dimers. PCH analysis of fluorescence-tagged GPCRs revealed molecular brightness values that were twice the monomeric controls and similar to the dimeric controls. Reduced χ2 analyses of the PCH data best fit a model for a homogeneous population of homodimers, without tetramers or higher-order oligomers. The homodimer configuration was unaltered by agonist treatment and was stable over a 10-fold range of receptor expression level. The results of this study demonstrate that biogenic amine receptors freely diffusing within the plasma membrane are predominantly homodimers.

Primary care physician perspectives on reimbursement for childhood immunizations

OBJECTIVES. The purpose of this research was to explore physicians’ attitudes and behaviors related to vaccine financing issues within their practice. Amid the increasing number of vaccine doses recommended for children and adolescents, anecdotal reports suggest that physicians are facing increasing financial pressures from vaccine purchase and administration and may stop providing vaccines altogether to privately insured children. Whether these sentiments are widely held among immunization providers is unknown. METHODS. We conducted a cross-sectional mail survey from July to September 2007 of a random sample of 1280 US pediatricians and family physicians engaged in direct patient care. Main outcome measures included delay in the purchase of specific vaccines for financial reasons; reported decrease in profit margin from immunizations; and practice consideration of whether to stop providing all vaccines to privately insured children. RESULTS. The response rate was 70% for pediatricians and 60% for family physicians. Approximately half of the respondents reported that their practice had delayed the purchase of specific vaccines for financial reasons (49%) and experienced decreased profit margin from immunizations (53%) in the previous 3 years. Twenty-one percent of respondents strongly disagreed that “reimbursement for vaccine purchase is adequate,” and 17% strongly disagreed that “reimbursement for vaccine administration is adequate.” Eleven percent of respondents said their practice had seriously considered whether to stop providing all vaccines to privately insured children in the previous year. CONCLUSIONS. Physicians who provide vaccines to children and adolescents report dissatisfaction with reimbursement levels and increasing financial strain from immunizations. Although large-scale withdrawal of immunization providers does not seem to be imminent, efforts to address root causes of financial pressures should be undertaken.