Differences in PGE2 Production between Primary Human Monocytes and Differentiated Macrophages: Role of IL-1β and TRIF/IRF3

Prostaglandin E2 (PGE₂) is induced in vivo by bacterial products including TLR agonists. To determine whether PGE₂ is induced directly or via IL-1β, human monocytes and macrophages were cultured with LPS or with Pam3CSK4 in presence of caspase-1 inhibitor, ZVAD, or IL-1R antagonist, Kineret. TLR agonists induced PGE₂ in macrophages exclusively via IL-1β-independent mechanisms. In contrast, ZVAD and Kineret reduced PGE₂ production in LPS-treated (but not in Pam3CSK4-treated) monocytes, by 30–60%. Recombinant human IL-1β augmented COX-2 and mPGES-1 mRNA and PGE₂ production in LPS-pretreated monocytes but not in un-primed or Pam3CSK4-primed monocytes. This difference was explained by the finding that LPS but not Pam3CSK4 induced phosphorylation of IRF3 in monocytes suggesting activation of the TRIF signaling pathway. Knocking down TRIF, TRAM, or IRF3 genes by siRNA inhibited IL-1β-induced COX-2 and mPGES-1 mRNA. Blocking of TLR4 endocytosis during LPS priming prevented the increase in PGE₂ production by exogenous IL-1β. Our data showed that TLR2 agonists induce PGE₂ in monocytes independently from IL-1β. In the case of TLR4, IL-1β augments PGE₂ production in LPS-primed monocytes (but not in macrophages) through a mechanism that requires TLR4 internalization and activation of the TRIF/IRF3 pathway. These findings suggest a key role for blood monocytes in the rapid onset of fever in animals and humans exposed to bacterial products and some novel adjuvants.     

Mitochondria structural reorganization during mouse embryonic stem cell derivation

Mouse embryonic stem (ES) cells are widely used in developmental biology and transgenic research. Despite numerous studies, ultrastructural reorganization of inner cell mass (ICM) cells during in vitro culture has not yet been described in detail. Here, we for the first time performed comparative morphological and morphometric analyses of three ES cell lines during their derivation in vitro. We compared morphological characteristics of blastocyst ICM cells at 3.5 and 4.5 days post coitum on feeder cells (day 6, passage 0) with those of ES cells at different passages (day 19, passage 2; day 25, passage 4; and passage 15). At passage 0, there were 23–36% of ES-like cells with various values of the medium cross-sectional area and nucleocytoplasmic parameters, 55% of fibroblast-like (probably trophoblast derivatives), and ~?19% of dying cells. ES-like cells at passage 0 contained autolysosomes and enlarged mitochondria with reduced numerical density per cell. There were three types of mitochondria that differed in matrix density and cristae width. For the first time, we revealed cells that had two and sometimes three morphologically distinct mitochondria types in the cytoplasm. At passage 2, there were mostly ES cells with a high nucleocytoplasmic ratio and a cytoplasm depleted of organelles. At passage 4, ES cell morphology and morphometric parameters were mostly stable with little heterogeneity. According to our data, cellular structures of ICM cells undergo destabilization during derivation of an ES cell line with subsequent reorganization into the structures typical for ES cells. On the basis of ultrastructural analysis of mitochondria, we believe that the functional activity of these organelles changes during early stages of ES cell formation from the ICM.     

Frustule morphogenesis of raphid pennate diatom <Emphasis Type="Italic">Encyonema ventricosum</Emphasis> (Agardh) Grunow

Diatoms stand out among other microalgae due to the high diversity of species-specific silica frustules whose components (valves and girdle bands) are formed within the cell in special organelles called silica deposition vesicles (SDVs). Research on cell structure and morphogenesis of frustule elements in diatoms of different taxonomic groups has been carried out since the 1950s but is still relevant today. Here, cytological features and valve morphogenesis in the freshwater raphid pennate diatom Encyonema ventricosum (Agardh) Grunow have been studied using light and transmission electron microscopy of cleaned frustules and ultrathin sections of cells, and scanning electron and atomic force microscopy of the frustule surface. Data have been obtained on chloroplast structure: the pyrenoid is spherical, penetrated by a lamella (a stack of two thylakoids); the girdle lamella consists of several short lamellae. The basic stages of frustule morphogenesis characteristic of raphid pennate diatoms have been traced, with the presence of cytoskeletal elements near SDVs being observed throughout this process. Degradation of the plasmalemma and silicalemma is shown to take place when the newly formed valve is released into the space between sister cells. The role of vesicular transport and exocytosis in the gliding of pennate diatoms is discussed.     

Antiangiogenic and antivascular effects of a recombinant tumstatin-derived peptide in a corneal neovascularization model

Tumstatin, a cleavage fragment of collagen IV, is a potent endogenous inhibitor of angiogenesis. Tumstatin-derived peptide T8 possesses all angiostatic properties of full-length tumstatin and indirectly suppresses tumor growth. The potential of T8 to block pathological angiogenesis in the eye has not been explored yet. Here we assess antiangiogenic effects of a recombinant T8 peptide in rabbit corneal neovascularization models. The fusion protein consisting of T8 and thioredoxin was synthesized in a highly efficient Escherichia coli expression system, isolated using ion-exchange chromatography and cleaved with TEV (tobacco etch virus) protease. The target peptide was purified on an anion-exchange resin and by reversed phase high-performance liquid chromatography. The recombinant peptide suppressed the proliferation of basic fibroblast growth factor-induced SVEC-4-10 endothelial cells (simian virus 40-immortalized murine endothelial cells) and inhibited tube formation in these cells in a dose-dependent manner. In rabbit corneal neovascularization models T8 demonstrated the ability to prevent pathological angiogenesis (when injected simultaneously with the induction of neovascularization) and, moreover, to promote the regression of newly-formed blood vessels (when injected on day 8 after angiogenesis stimulation). Our results suggest that T8 may have a therapeutic potential in the treatment of ocular neovascular diseases.     

The mechanism by which the mitochondrial ATP-sensitive K+ channel opening and H2O2 inhibit the mitochondrial permeability transition

Myocardial infarction is a manifestation of necrotic cell death as a result of opening of the mitochondrial permeability transition (MPT). Receptor-mediated cardioprotection is triggered by an intracellular signaling pathway that includes phosphatidylinositol 3-kinase, endothelial nitric-oxide synthase, guanylyl cyclase, protein kinase G (PKG), and the mitochondrial K(ATP) channel (mitoK(ATP)). In this study, we explored the pathway that links mitoK(ATP) with the MPT. We confirmed previous findings that diazoxide and activators of PKG or protein kinase C (PKC) inhibited MPT opening. We extended these results and showed that other K(+) channel openers as well as the K(+) ionophore valinomycin also inhibited MPT opening and that this inhibition required reactive oxygen species. By using isoform-specific peptides, we found that the effects of K(ATP) channel openers, PKG, or valinomycin were mediated by a PKCepsilon. Activation of PKCepsilon by phorbol 12-myristate 13-acetate or H(2)O(2) resulted in mitoK(ATP)-independent inhibition of MPT opening, whereas activation of PKCepsilon by PKG or the specific PKCepsilon agonist psiepsilon receptor for activated C kinase caused mitoK(ATP)-dependent inhibition of MPT opening. Exogenous H(2)O(2) inhibited MPT, because of its activation of PKCepsilon, with an IC(50) of 0.4 (+/-0.1) microm. On the basis of these results, we propose that two different PKCepsilon pools regulate this signaling pathway, one in association with mitoK(ATP) and the other in association with MPT.     

Mate‐finding failure as an important cause of Allee effects along the leading edge of an invading insect population

The movement of humans and goods has facilitated the arrival of non‐native insects, some of which successfully establish and cause negative consequences to the composition, services, and functioning of ecosystems. The gypsy moth, Lymantria dispar (L.) (Lepidoptera: Lymantriidae), is currently invading North American forests at variable rates, spreading by local and long‐distance movement in a process known as stratified dispersal. Newly arriving colonizers often occur considerably ahead of the population front, and a key question is the degree to which they successfully establish. Prior research has highlighted mate‐finding failures in sparse populations as a cause of an Allee effect (positive density dependence). We explored this mechanism by measuring the relationship between female mating success and background male moth densities along the gypsy moth western front in Northern Wisconsin (USA) over 2 years. The mating results were then compared with analogous previous studies in southern Wisconsin, and the southern front in West Virginia and Virginia (USA). Mate‐finding failures in low‐density populations were consistently observed to be density‐dependent across all years and locations. Mate‐finding failures in low‐density populations have important ramifications to invasive species management, particularly in predicting species invasiveness, preventing successful establishment by small founder populations, and concentrating eradication efforts where they are most likely to succeed.     

A keystone-like grain in hornet comb cells: its nature and physical properties

In the roof of cells built by the Oriental hornet, Vespa orientalis (Vespinae, Hymenoptera), there is one or more grains differing in their composition and shape from the surrounding structures. These grains have a diameter of 100-200 microm and protrude slightly from the roof inward. They contain primarily a large percentage of Ti but also Fe and occasionally Si. The mineral makeup in the grains is still uncertain, but it seems that the minerals belong to the group called perovskite. These grains in the hornet's comb may originate from the soil around the nest, that is, comprised of ceramic matter, but it is also possible that they are secreted from a gland within the frons plate of the hornet because the fungus-like secretion in this gland contains elements similar to those in the roof of the comb cells. We conjecture that the grain in question serves as a reference point for the hornets that build the cell walls in the direction of gravity. The cement whereby the building hornets conjoin the various building materials, whether organic (cellulose and the like) or inorganic (sand grains or tiny stones), is a saliva that is secreted from their oral gland, which upon release hardens into a polymeric glue. The ceramic grain embedded within this polymeric matrix is endowed with different physical properties and can therefore respond differently to the tension or pressure of the surrounding walls.