Nucleotide variation at the hypervariable esterase 6 isozyme locus of Drosophila simulans

Esterase 6 (Est-6/EST6) is polymorphic in both Drosophila melanogaster and D. simulans for two common allozyme forms, as well as for several other less common variants. Parallel latitudinal clines in the frequencies of the common EST6-F and EST6-S allozymes in these species have previously been interpreted in terms of a shared amino acid polymorphism that distinguishes the two variants and is subject to selection. Here we compare the sequences of four D. simulans Est-6 isolates and show that overall estimates of nucleotide heterozygosity in both coding and 5' flanking regions are more than threefold higher than those obtained previously for this gene in D. melanogaster. Nevertheless, the ratio of replacement to exon silent-site polymorphism in D. simulans is less than the ratio of replacement to silent divergence between D. simulans and D. melanogaster, which could be the result of increased efficiency of selection against replacement polymorphisms in D. simulans or to divergent selection between the two species. We also find that the amino acid polymorphisms separating EST6- F and EST6-S in D. simulans are not the same as those that separate these allozymes in D. melanogaster, implying that the shared clines do not reflect shared molecular targets for selection. All comparisons within and between the two species reveal a remarkable paucity of variation in a stretch of nearly 400 bp immediately 5' of the gene, indicative of strong selective constraint to retain essential aspects of Est-6 promoter function.      

Essential Regulation of Lung Surfactant Homeostasis by the Orphan G Protein-Coupled Receptor GPR116

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Production of endothelial progenitor cells obtained from human Wharton's jelly using different culture conditions

Endothelial progenitor cells (EPC) participate in revascularization and angiogenesis. EPC can be cultured in vitro from mononuclear cells of peripheral blood, umbilical cord blood or bone marrow; they also can be transdifferentiated from mesenchymal stem cells (MSC). We isolated EPCs from Wharton's jelly (WJ) using two methods. The first method was by obtaining MSC from WJ and characterizing them by flow cytometry and their adipogenic and osteogenic differentiation, then applying endothelial growth differentiating media. The second method was by direct culture of cells derived from WJ into endothelial differentiating media. EPCs were characterized by morphology, Dil-LDL uptake/UEA-1 immunostaining and testing the expression of endothelial markers by flow cytometry and RT-PCR. We found that MSC derived from WJ differentiated into endothelial-like cells using simple culture conditions with endothelium induction agents in the medium.     

Human SP-A protein variants derived from one or both genes stimulate TNF-alpha production in the THP-1 cell line

In humans, two functional genes of surfactant protein (SP) A, SP-A1 and SP-A2, and several alleles of each functional gene have been characterized. SP-A is a multimeric molecule consisting of six trimers. Each trimer contains two SP-A1 molecules and one SP-A2 molecule. Until now, it has been unclear whether a single SP-A gene product is functional or whether there are functional differences either among alleles or between single-gene SP-A products and SP-A products derived from both genes. We tested the ability of in vitro expressed SP-A variants to stimulate tumor necrosis factor (TNF)-alpha production by THP-1 cells. We observed that 1) single-gene products and products derived from both genes stimulate TNF-alpha production, 2) there are differences among SP-A1 and SP-A2 alleles in their ability to stimulate TNF-alpha production, and 3) the increases in TNF-alpha production are lower after treatment with the SP-A1 alleles than after treatment with the SP-A2 alleles. Furthermore, coexpressed SP-As from SP-A1 and SP-A2 genes have a higher activity compared with SP-As from individual alleles or mixed SP-As from SP-A1 and SP-A2 genes. These data suggest that the SP-A-induced increases in TNF-alpha levels differ among SP-A variants and appear to be affected by SP-A genotype and whether SP-A is derived from one or both genes.     

Evolution of spore release mechanisms in the saprolegniaceae (Oomycetes): evidence from a phylogenetic analysis of internal transcribed spacer sequences

Classical studies on spore release within the Saprolegniaceae (Oomycetes) led to the proposition that different mechanisms of sporangial emptying represent steps in an evolutionary transition series. We have reevaluated this idea in a phylogenetic framework using internal transcribed spacer sequences of four genera. These data were compared with the response to osmotic stress exhibited by each taxon. Saprolegnia emerges as the most basal genus, sister to Achlya, Thraustotheca, and Dictyuchus. Achlya and Thraustotheca are most closely related, while Dictyuchus appears to have evolved along a separate evolutionary lineage. The resulting phylogenetic framework is consistent with the idea that the mechanism of sporangial emptying exhibited by Saprolegnia represents the plesiomorphic condition from which the other mechanisms were derived independently. These alternative mechanisms of spore release may have resulted from a small number of mutations that inhibited axonemal development and altered the temporal and spatial expression of lytic enzymes that degrade the sporangial wall. Copyright 1998 Academic Press.     

Inhibition of ozone-induced SP-A oxidation by plant polyphenols

Surfactant protein-A (SP-A) is the best studied and most abundant of the protein components of lung surfactant and plays an important role in host defense of the lung. It has been shown that ozone-induced oxidation of SP-A protein changes its functional and biochemical properties. In the present study, eight plant polyphenols (three flavonoids, three hydroxycinnamic acids, and two hydroxybenzoic acids) known as strong antioxidants, were tested for their ability to inhibit ozone-induced SP-A oxidation as a mechanism for chemoprevention against lung damage. SP-A isolated from alveolar proteinosis patients was exposed to ozone (1 ppm) for 4 h. The flavonoids protected SP-A from oxidation in a dose dependent manner. ( - )-Epicatechin was the most potent flavonoid and exhibited inhibition of ozone-induced formation of carbonyls by 35% at a concentration as low as 5 μM. Hydroxybenzoic acids inhibited SP-A oxidation in a dose-dependent manner although they were less potent than flavonoids. On the other hand, hydroxycinnamic acids exhibited a different inhibitory pattern. Inhibition was observed only at medium concentrations. The results indicate that inhibition of SP-A oxidation by plant polyphenols may be a mechanism accounting for the protective activity of natural antioxidants against the effects of ozone exposure on lungs.     

Effect of coculturing canine notochordal,nucleus pulposus and mesenchymal stromal cells for intervertebral disc regeneration

IntroductionEarly degenerative changes in the nucleus pulposus (NP) are observed after the disappearance of notochordal cells (NCs). Thus, it has been suggested that NCs play an important role in maintaining the NP and may have a regenerative potential on other cells of the NP. As the number of resident NP cells (NPCs) decreases in a degenerating disc, mesenchymal stromal (stem) cells (MSCs) may be used for cell supplementation. In this study, using cells of one species, the regenerative potential of canine NCs was assessed in long-term three-dimensional coculture with canine NPCs or MSCs.MethodsCanine NCs and canine NPCs or MSCs were cocultured in alginate beads for 28 days under hypoxic and high-osmolarity conditions. Cell viability, cell morphology and DNA content, extracellular matrix production and expression of genes related to NC markers (Brachyury, KRT18) and NP matrix production (ACAN, COL2A1, COL1A1) were assessed after 1, 15 and 28 days of culture.ResultsNCs did not completely maintain their phenotype (morphology, matrix production, gene expression) during 28 days of culture. In cocultures of NPCs and NCs, both extracellular matrix content and anabolic gene expression remained unchanged compared with monoculture groups, whereas cocultures of MSCs and NCs showed increased glycosaminoglycan/DNA. However, the deposition of these proteoglycans was observed near the NCs and not the MSCs. Brachyury expression in the MSC and NC coculture group increased in time. The latter two findings indicate a trophic effect of MSCs on NCs rather than vice versa.ConclusionsNo regenerative potential of canine NCs on canine NPCs or MSCs was observed in this study. However, significant changes in NC phenotype in long-term culture may have resulted in a suboptimal regenerative potential of these NCs. In this respect, NC-conditioned medium may be better than coculture for future studies of the regenerative potential of NCs.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0569-6) contains supplementary material, which is available to authorized users.     

<Emphasis Type="Italic">In vivo</Emphasis> rescue of alveolar macrophages from SP-A knockout mice with exogenous SP-A nearly restores a wild type intracellular proteome; actin involvement

Background

Mice lacking surfactant protein-A (SP-A-/-; knockout; KO) exhibit increased vulnerability to infection and injury. Although many bronchoalveolar lavage (BAL) protein differences between KO and wild-type (WT) are rapidly reversed in KO after infection, their clinical course is still compromised. We studied the impact of SP-A on the alveolar macrophage (AM) proteome under basal conditions. Male SP-A KO mice were SP-A-treated (5 micrograms/mouse) and sacrificed in 6 or 18 hr. The AM proteomes of KO, SP-A-treated KO, and WT mice were studied by 2D-DIGE coupled with MALDI-ToF/ToF and AM actin distribution was examined by phalloidon staining.

Results

We observed: a) significant differences from KO in WT or exogenous SP-A-treated in 45 of 76 identified proteins (both increases and decreases). These included actin-related/cytoskeletal proteins (involved in motility, phagocytosis, endocytosis), proteins of intracellular signaling, cell differentiation/regulation, regulation of inflammation, protease/chaperone function, and proteins related to Nrf2-mediated oxidative stress response pathway; b) SP-A-induced changes causing the AM proteome of the KO to resemble that of WT; and c) that SP-A treatment altered cell size and F-actin distribution.

Conclusions

These differences are likely to enhance AM function. The observations show for the first time that acute in vivo SP-A treatment of KO mice, under basal or unstimulated conditions, affects the expression of multiple AM proteins, alters F-actin distribution, and can restore much of the WT phenotype. We postulate that the SP-A-mediated expression profile of the AM places it in a state of "readiness" to successfully conduct its innate immune functions and ensure lung health.

     

Proteomic profiling of human plasma by iTRAQ reveals down-regulation of ITI-HC3 and VDBP by cigarette smoking

Biomarkers in noninvasive fluids indicative of cigarette smoke's effects are urgently needed. In this pilot study, we utilized the proteomic approach, isobaric Tags for Relative and Absolute Quantitation (iTRAQ), to identify differentially expressed plasma proteins in healthy cigarette smokers compared to healthy nonsmokers; select proteins were further confirmed by immunoblot analysis. Significant, differentially expressed proteins identified in the plasma separated subjects based on their condition as smokers or nonsmokers. Several of the proteins identified in this study are associated with immunity and inflammatory responses and have been shown to be associated with tobacco-related diseases, including chronic obstructive pulmonary disease (COPD) and lung cancer. Proteins up-regulated in smokers included complement component 8 polypeptide chains α, β, and γ, and mannose-binding protein C, and proteins down-regulated included inter-α-trypsin inhibitor heavy chain H3 (ITI-HC3) and vitamin D-binding protein (VDBP). In addition, gelsolin and vitronectin, known tissue leakage proteins, were up- and down-regulated, respectively. Our results demonstrate for the first time that chronic cigarette smoking can influence the expression profile of the human plasma proteome. Proteins identified in this pilot study may serve as candidate biomarkers of diseases resulting from exposure to cigarette smoke in future molecular epidemiological studies.     

Modulatory effects of ozone on THP-1 cells in response to SP-A stimulation

Ozone (O(3)), a major component of air pollution and a strong oxidizing agent, can lead to lung injury associated with edema, inflammation, and epithelial cell damage. The effects of O(3) on pulmonary immune cells have been studied in various in vivo and in vitro systems. We have shown previously that O(3) exposure of surfactant protein (SP)-A decreases its ability to modulate proinflammatory cytokine production by cells of monocyte/macrophage lineage (THP-1 cells). In this report, we exposed THP-1 cells and/or native SP-A obtained from bronchoalveolar lavage of patients with alveolar proteinosis to O(3) and studied cytokine production and NF-kappaB signaling. The results showed 1) exposure of THP-1 cells to O(3) significantly decreased their ability to express TNF-alpha in response to SP-A; TNF-alpha production, under these conditions, was still significantly higher than basal (unstimulated) levels in filtered air-exposed THP-1 cells; 2) exposure of both THP-1 cells and SP-A to O(3) did not result in any significant differences in TNF-alpha expression compared with basal levels; 3) O(3) exposure of SP-A resulted in a decreased ability of SP-A to activate the NF-kappaB pathway, as assessed by the lack of significant increase and decrease of the nuclear p65 subunit of NF-kappaB and cytoplasmic IkappaBalpha, respectively; and 4) O(3) exposure of THP-1 cells resulted in a decrease in SP-A-mediated THP-1 cell responsiveness, which did not seem to be mediated via the classic NF-kappaB pathway. These findings indicate that O(3) exposure may mediate its effect on macrophage function both directly and indirectly (via SP-A oxidation) and by involving different mechanisms.