The Association of Defensin HNP-2 with Negatively Charged Membranes: A Combined Fluorescence and Linear Dichroism Study

The association of defensin HNP-2 with negatively charged membranes has been studied using a new approach that combines fluorescence and linear dichroism (LD) spectroscopies with simulated LD spectra in order to characterise the binding kinetics and bound configurations of the peptide. Binding to membranes composed of mixtures of diacylglycerophosphocholines (PC) with either diacylglycerophosphoglycerol (PG) or diacylglycerophosphoserine (PS) was conducted at lipid:peptide ratios that yielded binding, but not membrane fusion. HNP-2 association with membranes under these conditions was a 2 stage-process, with both stages exhibiting first order kinetics. The fast initial step, with a half-life of 3 min. Conversion between the states was estimated to have an enthalpy of activation of approximately 10 kJ mol− 1 and an entropy of activation of − 0.2 kJ K mol− 1. LD spectra corresponding to each of the membrane bound states were generated by non-linear regression using a standard kinetic model. These spectra are interpreted in comparison with spectra calculated using the program Dichrocalc and reveal that the peptide associates with membranes in a small number of stable configurations. All of these configurations have a significant proportion of β-sheet structure residing in the plane of the membrane. Two configurations support structures previously proposed for defensins in membranes

ESCRT-III-associated proteins and spastin inhibit protrudin-dependent polarised membrane traffic

Funder: National Institute for Health Research; doi: http://dx.doi.org/10.13039/501100000272Funder: Gates Cambridge Trust; doi: http://dx.doi.org/10.13039/501100005370Abstract: Mutations in the gene encoding the microtubule severing ATPase spastin are the most frequent cause of hereditary spastic paraplegia, a genetic condition characterised by length-dependent axonal degeneration. Here, we show that HeLa cells lacking spastin and embryonic fibroblasts from a spastin knock-in mouse model become highly polarised and develop cellular protrusions. In HeLa cells, this phenotype was rescued by wild-type spastin, but not by forms unable to sever microtubules or interact with endosomal ESCRT-III proteins. Cells lacking the spastin-interacting ESCRT-III-associated proteins IST1 or CHMP1B also developed protrusions. The protrusion phenotype required protrudin, a RAB-interacting protein that interacts with spastin and localises to ER–endosome contact sites, where it promotes KIF5-dependent endosomal motility to protrusions. Consistent with this, the protrusion phenotype in cells lacking spastin also required KIF5. Lack or mutation of spastin resulted in functional consequences for receptor traffic of a pathway implicated in HSP, as Bone Morphogenetic Protein receptor distribution became polarised. Our results, therefore, identify a novel role for ESCRT-III proteins and spastin in regulating polarised membrane traffic

The Synergistic Action of Melittin and Phospholipase A2 with Lipid Membranes: Development of Linear Dichroism for Membrane-Insertion Kinetics

Here we present data on the kinetics of insertion of melittin, a peptide from bee venom, into lipid membranes of different composition. Another component of bee venom is the enzyme phospholipase A2 (PLA₂). We have examined the interaction of melittin and PLA₂ with liposomes both separately and combined and demonstrate that they work synergistically to disrupt the membranes. A dramatic difference in the action of melittin and PLA₂ is observed when the composition of the membrane is altered. Temperature also has a large effect on the kinetics of insertion and membrane disruption. We use a combination of techniques to measure liposome size (dynamic light scattering), peptide secondary structure (circular dichroism spectroscopy), peptide orientation relative to the membrane (linear dichroism spectroscopy) and enzymatic digestion of the lipids (mass spectrometry)

Factors Associated with Survival of Veterans with Gastrointestinal Neuroendocrine Tumors

Background. Gastrointestinal (GI) neuroendocrine tumor (NET) incidence has been increasing; however, GI NET within the national Veterans Affairs (VA) health system has not been described. Methods. We used the VA Central Cancer Registry to identify the cohort of patients diagnosed with GI NET in 1995–2009. Cox regression models were constructed to explore factors associated with survival. Results. We included 1793 patients with NET of the stomach (9%), duodenum (10%), small intestine (24%), colon (19%) or rectum (38%). Twenty percent were diagnosed in 1995–1999, 35% in 2000–2004, and 45% in 2005–2009. Unadjusted 5-year survival rates were: stomach 56%, duodenum 66%, small intestine 52%, colon 67%, and rectum 84%. Factors associated with shorter survival were increasing age, hazard ratio (HR) 1.05 (95% CI 1.04–1.06), NET location [compared to rectum: stomach HR 2.26 (95% CI 1.68–3.05), duodenum HR 1.70 (95% CI 1.26–2.28), small intestine HR 1.85 (95% CI 1.42–2.42), and colon 1.83 (95% CI 1.41–2.39)], stage [compared to in situ/local: regional HR 1.15 (95% CI 0.90–1.47), distant HR 2.38 (95% CI 1.87–3.05)], and earlier period of diagnosis [compared to 1995–1999: 2000–2004 HR 0.70 (95% CI 0.59–0.85), 2005–2009 HR 0.43 (95% CI 0.34–0.54)]. Conclusions. The incidence of GI NET has also increased over time in the VA system with similar survival rates to those observed in non-VA settings. Worsened survival was associated with older age, tumor site, advanced stage, and earlier year of diagnosis

Diesel exhaust particulate induces pulmonary and systemic inflammation in rats without impairing endothelial function ex vivo or in vivo

<p>Abstract</p> <p>Background</p> <p>Inhalation of diesel exhaust impairs vascular function in man, by a mechanism that has yet to be fully established. We hypothesised that pulmonary exposure to diesel exhaust particles (DEP) would cause endothelial dysfunction in rats as a consequence of pulmonary and systemic inflammation.</p> <p>Methods</p> <p>Wistar rats were exposed to DEP (0.5 mg) or saline vehicle by intratracheal instillation and hind-limb blood flow, blood pressure and heart rate were monitored <it>in situ </it>6 or 24 h after exposure. Vascular function was tested by administration of the endothelium-dependent vasodilator acetylcholine (ACh) and the endothelium-independent vasodilator sodium nitroprusside (SNP) <it>in vivo </it>and <it>ex vivo </it>in isolated rings of thoracic aorta, femoral and mesenteric artery from DEP exposed rats. Bronchoalveolar lavage fluid (BALF) and blood plasma were collected to assess pulmonary (cell differentials, protein levels & interleukin-6 (IL-6)) and systemic (IL-6), tumour necrosis factor alpha (TNFα) and C-reactive protein (CRP)) inflammation, respectively.</p> <p>Results</p> <p>DEP instillation increased cell counts, total protein and IL-6 in BALF 6 h after exposure, while levels of IL-6 and TNFα were only raised in blood 24 h after DEP exposure. DEP had no effect on the increased hind-limb blood flow induced by ACh <it>in vivo </it>at 6 or 24 h. However, responses to SNP were impaired at both time points. In contrast, <it>ex vivo </it>responses to ACh and SNP were unaltered in arteries isolated from rats exposed to DEP.</p> <p>Conclusions</p> <p>Exposure of rats to DEP induces both pulmonary and systemic inflammation, but does not modify endothelium-dependent vasodilatation. Other mechanisms <it>in vivo </it>limit dilator responses to SNP and these require further investigation.</p

Laboring to Mother in the Context of Past Trauma: The Transition to Motherhood

The occurrence of interpersonal trauma is a reality for many women, with effects that often persist long after the traumatic events end. The purpose of this feminist grounded theory study was to examine how past trauma shaped the lives of women as they became new mothers. We recruited a purposive sample of 32 women from two Canadian communities and conducted semistructured, dialogic interviews during the second trimester of pregnancy. We analyzed data using thematic content analytic methods, including open coding whereby we read transcripts line by line and applied codes to portions of text that illustrated concepts or themes. The substantive grounded theory, “laboring to mother in the context of past trauma,” describes the exceedingly difficult emotional and cognitive work undertaken by pregnant women with histories of trauma as they anticipate becoming mothers. In this article, we present key components of the theory and offer recommendations for health and social service providers

Understanding young people's transitions in university halls through space and time

This article contributes to the theoretical discussion about young people's transitions through space and time. Space and time are complex overarching concepts that have creative potential in deepening understanding of transition. The focus of this research is young people's experiences of communal living in university halls. It is argued that particular space-time concepts draw attention to different facets of experience and in combination deepen the understanding of young people's individual and collective transitions. The focus of the article is the uses of the space-time concepts 'routine', 'representation', 'rhythm' and 'ritual' to research young people's experiences. The article draws on research findings from two studies in the North of England. © 2010 SAGE Publications