Studies of intercellular invasion in vitro using rabbit peritoneal neutrophil granulocytes (PMNS). I. Role of contact inhibition of locomotion.

Intercellular invasion is the active migration of cells on one type into the interiors of tissues composed of cells of dissimilar cell types. Contact paralysis of locomotion is the cessation of forward extension of the pseudopods of a cell as a result of its collision with another cell. One hypothesis to account for intercellular invasion proposes that a necessary condition for a cell type to be invasive to a given host tissue is that it lack contact paralysis of locomotion during collision with cells of that host tissue. The hypothesis has been tested using rabbit peritoneal neutrophil granulocytes (PMNs) as the invasive cell type and chick embryo fibroblasts as the host tissue. In organ culture, PMNs rapidly invade aggregates of fibroblasts. The behavior of the pseudopods of PMNs during collision with fibroblasts was analyzed for contact paralysis by a study of time-lapse films of cells in mixed monolayer culture. In monolayer culture, PMNs show little sign of paralysis of the pseudopods upon collision with fibroblasts and thus conform in their behavior to that predicted by the hypothesis

Increased levels of Stress-inducible phosphoprotein-1 accelerates amyloid-β deposition in a mouse model of Alzheimer’s disease

Molecular chaperones and co-chaperones, which are part of the protein quality control machinery, have been shown to regulate distinct aspects of Alzheimer’s Disease (AD) pathology in multiple ways. Notably, the co-chaperone STI1, which presents increased levels in AD, can protect mammalian neurons from amyloid-β toxicity in vitro and reduced STI1 levels worsen Aβ toxicity in C. elegans. However, whether increased STI1 levels can protect neurons in vivo remains unknown. We determined that overexpression of STI1 and/or Hsp90 protected C. elegans expressing Aβ(3–42) against Aβ-mediated paralysis. Mammalian neurons were also protected by elevated levels of endogenous STI1 in vitro, and this effect was mainly due to extracellular STI1. Surprisingly, in the 5xFAD mouse model of AD, by overexpressing STI1, we find increased amyloid burden, which amplifies neurotoxicity and worsens spatial memory deficits in these mutants. Increased levels of STI1 disturbed the expression of Aβ-regulating enzymes (BACE1 and MMP-2), suggesting potential mechanisms by which amyloid burden is increased in mice. Notably, we observed that STI1 accumulates in dense-core AD plaques in both 5xFAD mice and human brain tissue. Our findings suggest that elevated levels of STI1 contribute to Aβ accumulation, and that STI1 is deposited in AD plaques in mice and humans. We conclude that despite the protective effects of STI1 in C. elegans and in mammalian cultured neurons, in vivo, the predominant effect of elevated STI1 is deleterious in AD

Detecting small low emission radiating sources

The article addresses the possibility of robust detection of geometrically small, low emission sources on a significantly stronger background. This problem is important for homeland security. A technique of detecting such sources using Compton type cameras is developed, which is shown on numerical examples to have high sensitivity and specificity and also allows to assign confidence probabilities of the detection. 2D case is considered in detail

Stress-inducible phosphoprotein 1 (HOP/STI1/STIP1) regulates the accumulation and toxicity of α-synuclein in vivo

The predominantly pre-synaptic intrinsically disordered protein α-synuclein is prone to misfolding and aggregation in synucleinopathies, such as Parkinson’s disease (PD) and Dementia with Lewy bodies (DLB). Molecular chaperones play important roles in protein misfolding diseases and members of the chaperone machinery are often deposited in Lewy bodies. Here, we show that the Hsp90 co-chaperone STI1 co-immunoprecipitated α-synuclein, and co-deposited with Hsp90 and Hsp70 in insoluble protein fractions in two mouse models of α-synuclein misfolding. STI1 and Hsp90 also co-localized extensively with filamentous S129 phosphorylated α-synuclein in ubiquitin-positive inclusions. In PD human brains, STI1 transcripts were increased, and in neurologically healthy brains, STI1 and α-synuclein transcripts correlated. Nuclear Magnetic Resonance (NMR) analyses revealed direct interaction of α-synuclein with STI1 and indicated that the STI1 TPR2A, but not TPR1 or TPR2B domains, interacted with the C-terminal domain of α-synuclein. In vitro, the STI1 TPR2A domain facilitated S129 phosphorylation by Polo-like kinase 3. Moreover, mice over-expressing STI1 and Hsp90ß presented elevated α-synuclein S129 phosphorylation accompanied by inclusions when injected with α-synuclein pre-formed fibrils. In contrast, reduced STI1 function decreased protein inclusion formation, S129 α-synuclein phosphorylation, while mitigating motor and cognitive deficits as well as mesoscopic brain atrophy in α-synuclein-over-expressing mice. Our findings reveal a vicious cycle in which STI1 facilitates the generation and accumulation of toxic α-synuclein conformers, while α-synuclein-induced proteostatic stress increased insoluble STI1 and Hsp90

Determination of nitric oxide metabolites, nitrate and nitrite, in Anopheles culicifacies mosquito midgut and haemolymph by anion exchange high-performance liquid chromatography: plausible mechanism of refractoriness

<p>Abstract</p> <p>Background</p> <p>The diverse physiological and pathological role of nitric oxide in innate immune defenses against many intra and extracellular pathogens, have led to the development of various methods for determining nitric oxide (NO) synthesis. NO metabolites, nitrite (NO₂^-) and nitrate (NO₃^-) are produced by the action of an inducible <it>Anopheles culicifacies </it>NO synthase (AcNOS) in mosquito mid-guts and may be central to anti-parasitic arsenal of these mosquitoes.</p> <p>Method</p> <p>While exploring a plausible mechanism of refractoriness based on nitric oxide synthase physiology among the sibling species of <it>An. culicifacies</it>, a sensitive, specific and cost effective high performance liquid chromatography (HPLC) method was developed, which is not influenced by the presence of biogenic amines, for the determination of NO₂^-and NO₃^-from mosquito mid-guts and haemolymph.</p> <p>Results</p> <p>This method is based on extraction, efficiency, assay reproducibility and contaminant minimization. It entails de-proteinization by centrifugal ultra filtration through ultracel 3 K filter and analysis by high performance anion exchange liquid chromatography (Sphereclone, 5 μ SAX column) with UV detection at 214 nm. The lower detection limit of the assay procedure is 50 pmoles in all midgut and haemolymph samples. Retention times for NO₂^-and NO₃^-in standards and in mid-gut samples were 3.42 and 4.53 min. respectively. Assay linearity for standards ranged between 50 n<it>M </it>and 1 m<it>M</it>. Recoveries of NO₂^-and NO₃^-from spiked samples (1–100 μ<it>M</it>) and from the extracted standards (1–100 μ<it>M</it>) were calculated to be 100%. Intra-assay and inter assay variations and relative standard deviations (RSDs) for NO₂^-and NO₃^-in spiked and un-spiked midgut samples were 5.7% or less. Increased levels NO₂^-and NO₃^-in midguts and haemolymph of <it>An. culicifacies </it>sibling species B in comparison to species A reflect towards a mechanism of refractoriness based on AcNOS physiology.</p> <p>Conclusion</p> <p>HPLC is a sensitive and accurate technique for identification and quantifying pmole levels of NO metabolites in mosquito midguts and haemolymph samples that can be useful for clinical investigations of NO biochemistry, physiology and pharmacology in various biological samples.</p

Aggravation of allergic airway inflammation by cigarette smoke in mice is CD44-dependent

Background : Although epidemiological studies reveal that cigarette smoke (CS) facilitates the development and exacerbation of allergic asthma, these studies offer limited information on the mechanisms involved. The transmembrane glycoprotein CD44 is involved in cell adhesion and acts as a receptor for hyaluronic acid and osteopontin. We aimed to investigate the role of CD44 in a murine model of CS-facilitated allergic airway inflammation. Methods : Wild type (WT) and CD44 knock-out (KO) mice were exposed simultaneously to house dust mite (HDM) extract and CS. Inflammatory cells, hyaluronic acid (HA) and osteopontin (OPN) levels were measured in bronchoalveolar lavage fluid (BALF). Proinflammatory mediators, goblet cell metaplasia and peribronchial eosinophilia were assessed in lung tissue. T-helper (Th) 1, Th2 and Th17 cytokine production was evaluated in mediastinal lymph node cultures. Results : In WT mice, combined HDM/CS exposure increased the number of inflammatory cells and the levels of HA and OPN in BALF and Th2 cytokine production in mediastinal lymph nodes compared to control groups exposed to phosphate buffered saline (PBS)/CS, HDM/Air or PBS/Air. Furthermore, HDM/CS exposure significantly increased goblet cell metaplasia, peribronchial eosinophilia and inflammatory mediators in the lung. CD44 KO mice exposed to HDM/CS had significantly fewer inflammatory cells in BALF, an attenuated Th2 cytokine production, as well as decreased goblet cells and peribronchial eosinophils compared to WT mice. In contrast, the levels of inflammatory mediators were similar or higher than in WT mice. Conclusion : We demonstrate for the first time that the aggravation of pulmonary inflammation upon combined exposure to allergen and an environmental pollutant is CD44-dependent. Data from this murine model of concomitant exposure to CS and HDM might be of importance for smoking allergic asthmatics