Negative impact of DEP exposure on human airway epithelial cell adhesion,stiffness, and repair

Epidemiological and experimental studies suggest that diesel exhaust particles (DEPs) may be associated with increased respiratory mortality and morbidity. Several recent studies have also shown that DEPs increase the production of inflammatory cytokines by human bronchial epithelium (HBE) cells in vitro. The present study investigates the effects of DEPs on the interaction of l-HBE cells (16HBE14o-) with the cell and matrix microenvironment based on evaluation of integrin-type cell/matrix ligand expression, cytoskeleton (CSK) stiffness, and matrix remodeling via matrix metalloproteinase (MMP)-1, MMP-2, and MMP-9 expression. The results showed that DEP exposure induced: 1) a net dose-dependent decrease in CSK stiffness through actin fibers, 2) a concomitant specific reduction of both alpha(3)- and beta(1)-integrin subunits extensively expressed on the HBE cell surface, 3) a decrease in the level of CD44, which is a major HBE cell-cell and HBE cell-matrix adhesion molecule; and 4) an isolated decrease in MMP-1 expression without any change in tissue inhibitor of matrix metalloproteinase (TIMP)-1 or TIMP-2 tissue inhibitors. Restrictive modulation of cell-matrix interaction, cell-cell connection, CSK stiffness, and fibrillary collagen remodeling results in a decreased wound closure capacity and an increased deadhesion capacity. In conclusion, on the basis of these results, we can propose that, in addition to their ability to increase the production of inflammatory cytokines, DEPs could also alter the links between actin CSK and the extracellular matrix, suggesting that they might facilitate HBE cell detachment in vivo.     

Metapopulation structure of the specialized herbivore Macrosiphoniella tanacetaria (Homoptera,Aphididae)

We investigated population dynamics, genetic diversity and spatial structure in the aphid species Macrosiphoniella tanacetaria, a specialist herbivore feeding on tansy, Tanacetum vulgare. Tansy plants (genets) consist of many shoots (ramets), and genets are grouped in sites. Thus, aphids feeding on tansy can cluster at the level of ramets, genets and sites. We studied aphid population dynamics in 1997 and 2001 and found that within sites: (i). at any time, aphids used only a fraction of the available ramets and genets; (ii). at the level of ramets, most aphid colonies survived only one week; (iii). at the level of genets, mean survival time was less than 4 weeks; and (iv). colonization and extinction events occurred throughout the season. We sampled aphids in seven sites in the Alsace region, France (4-45 km apart) and two sites in Germany in 1999 to study genetic structure within and between populations. Genetic analyses using nine microsatellite loci showed that: (i). genotypic variability was high, (ii). none of the populations was in Hardy-Weinberg equilibrium, (iii). heterozygote deficits and linkage disequilibria were frequent, and (iv). all populations were genetically differentiated, even at a small geographical scale. Renewed sampling of the Alsace sites in 2001 showed that three populations had become extinct and significant genetic changes had occurred in the remaining four populations. The frequencies of extinction and colonization events at several spatial scales suggest a hierarchical metapopulation structure for M. tanacetaria. Frequent population turnover and drift are likely causes for the genetic differentiation of M. tanacetaria populations.     

Pattern of recovery of species-specific cuticular hydrocarbon mixtures by Reticulitermes santonensis and Reticulitermes lucifugus grassei after being removed from a mixed group. Is the acquisition of allospecific hydrocarbons reversible?

Each of the termite species Reticulitermes santonensis and Reticulitermes lucifugus grassei has its own particular cuticular chemical profile. When members of the two species are placed together to form artificially mixed species groups, their chemical profiles undergo changes: Each species acquires all the hydrocarbons which initially characterized the other species. When the members of a mixed group which had been kept together for 24 h were split into two homospecific groups, the cuticular profiles of the members of both groups immediately showed a sharp drop in both the homospecific and allospecific components. In R. santonensis, the homospecific hydrocarbons subsequently increased in quantity, reaching values which were higher on the 33rd day after the separation than those initially recorded in this species; whereas in R. lucifugus grassei, the homospecific hydrocarbon proportions were still lower on the 33rd day than the initial values. In both species, the allospecific hydrocarbon levels began to increase sharply on the 5th day after separation, and the homospecific products still showed no tendency to return to the initial proportions 33 days after separation. In the light of these results, some hypotheses are put forward as to what mechanisms might posibly regulate the hydrocarbon profiles of these two species. Arch. Insect Biochem. Physiol. 35:237-259, 1997.© 1997 Wiley-Liss, Inc.     

The CholecystokininB Receptor Is Coupled to Two Effector Pathways Through Pertussis Toxin-Sensitive and -Insensitive G Proteins

Previous binding studies have suggested the existence of two affinity states for type B cholecystokinin receptors (CCK(B)R), which could correspond to different coupling states of the receptor to G proteins. To test this hypothesis, we have further investigated signal transduction pathways coupled to rat CCK(B)R stably transfected in Chinese hamster ovary cells. We show that CCK(B)R are coupled to two distinct transduction pathways involving two different G proteins, a pertussis toxin-insensitive/phospholipase C pathway leading to the production of inositol phosphate and arachidonic acid, and a pertussis toxin-sensitive/phospholipase A2 pathway leading to the release of arachidonic acid. We further demonstrate that the relative degree of activation of each effector pathway by different specific CCK(B)R agonists is the same, and that a specific CCK(B)R antagonist, RB213, can differentially antagonize the two signal transduction pathways elicited by these agonists. Taken all together, these data could be explained by the recently proposed theory assuming that the receptor can exist in a three-state model in which two active conformations corresponding to the complex formed by the receptor with two different G proteins coexist. According to this model, agonists or antagonists could recognize preferentially either conformation of the activated receptor, leading to variable behavior in a system containing a single receptor type.     

Annonaceous acetogenins: Precursors from the seeds of Annona squamosa

From the less polar fractions of an Annona squamosa (Annonaceae) seeds extract, four new annonaceous acetogenins, dieposabadelin (1), squamocenin (2), lepirenin (3) and dotistenin (4), were isolated, along with sixteen known acetogenins: corepoxylone, diepomuricanins A and B, dieporeticenine, tripoxyrollin, bullatencin, glabrencin B, reticulatains-1, -2, uvariamicins I, II, III, erythrosolamin, annotemoyin-1 and -2, and solamin. Fourteen are reported for the first time in this species.     

Proteome analysis of separated male and female gametocytes reveals novel sex-specific Plasmodium biology

Gametocytes, the precursor cells of malaria-parasite gametes, circulate in the blood and are responsible for transmission from host to mosquito vector. The individual proteomes of male and female gametocytes were analyzed using mass spectrometry, following separation by flow sorting of transgenic parasites expressing green fluorescent protein, in a sex-specific manner. Promoter tagging in transgenic parasites confirmed the designation of stage and sex specificity of the proteins. The male proteome contained 36% (236 of 650) male-specific and the female proteome 19% (101 of 541) female-specific proteins, but they share only 69 proteins, emphasizing the diverged features of the sexes. Of all the malaria life-cycle stages analyzed, the male gametocyte has the most distinct proteome, containing many proteins involved in flagellar-based motility and rapid genome replication. By identification of gender-specific protein kinases and phosphatases and using targeted gene disruption of two kinases, new sex-specific regulatory pathways were defined.     

NG2 proteoglycan mediates beta1 integrin-independent cell adhesion and spreading on collagen VI

Collagens V and VI have been previously identified as specific extracellular matrix (ECM) ligands for the NG2 proteoglycan. In order to study the functional consequences of NG2/collagen interactions, we have utilized the GD25 cell line, which does not express the major collagen-binding beta(1) integrin heterodimers. Use of these cells has allowed us to study beta(1) integrin-independent phenomena that are mediated by binding of NG2 to collagens V and VI. Heterologous expression of NG2 in the GD25 line endows these cells with the capability of attaching to surfaces coated with collagens V and VI. The specificity of this effect is emphasized by the failure of NG2-positive GD25 cells to attach to other collagens or to laminin-1. More importantly, NG2-positive GD25 cells spread extensively on collagen VI. beta(1) integrin-independent extension of ruffling lamellipodia demonstrates that engagement of NG2 by the collagen VI substratum triggers signaling events that lead to rearrangement of the actin cytoskeleton. In contrast, even though collagens V and VI each bind to the central segment of the NG2 ectodomain, collagen V engagement of NG2 does not trigger cell spreading. The distinct morphological consequences of NG2/collagen VI and NG2/collagen V interaction indicate that closely-related ECM ligands for NG2 differ in their ability to initiate transmembrane signaling via engagement of the proteoglycan.     

Quantitative gas chromatographic-mass spectrometric assay of 4-hydroxynonenal bound to thiol proteins in ischemic/reperfused rat hearts

Increasing evidence indicates that protein-aldehyde adducts involving mostly 4-hydroxynonenal could be causally involved in both pathophysiological and adaptive events following an oxidative stress insult such as ischemia/reperfusion. The goal of this study was to assess if isotope dilution chromatography-mass spectrometry can be used to quantitate changes in the cardiac levels of 4-hydroxynonenal and 1,4-dihydroxynonene, one of its major metabolites, bound to thiol proteins during ischemia/reperfusion. For this purpose, we modified a previously published method to include treatment with Raney Nickel, which specifically cleaves thioether linkages. Our study model was the isolated Langendorff-perfused rat heart subjected to various ischemia/reperfusion protocols. Hearts perfused under normoxia contained small amounts of protein-bound 4-hydroxynonenal and 1,4-dihydroxynonene (1.38 +/- 0.29 and 2.69 +/- 0.17 nmol/g wet weight, respectively). The accumulation of these adducts after global ischemia depended on the severity of the ischemic insult up to a plateau and was not exacerbated by reperfusion. In conclusion, our method allows the quantification of time-dependent changes in 4-hydroxynonenal and 1,4-dihydroxynonene bound to proteins via thioether linkage in ischemic/reperfused heart tissues. The presence of protein-bound 1,4-dihydroxynonene in heart tissues suggests that this organ can detoxify protein-bound 4-hydroxynonenal.     

Cross‐species amplification of microsatellite loci in aphids: assessment and application

Despite the relative ease of isolating microsatellites, their development still requires substantial inputs of time, money and expertise. For this reason there is considerable interest in using existing microsatellites on species from which markers were not cloned. We tested cross‐species amplification of 48 existing aphid loci in species of the following genera: Aphidinae: Aphidini: Aphis and Rhopalosiphum; Aphidinae: Macrosiphini: Acyrthosiphum, Brevicoryne, Diuraphis, Illinoia, Macrosiphoniella, Macrosiphum, Metopeurum, Metapolophium, Myzus, Phorodon, Sitobion and Uroleucon and Neuquenaphidinae: Neuquenaphis. Our results show cross‐species application of known microsatellite loci is a highly promising source of codominant markers for population genetic and evolutionary studies in aphids.     

Remodeling of Aorta Extracellular Matrix as a Result of Transient High Oxygen Exposure in Newborn Rats: Implication for Arterial Rigidity and Hypertension Risk

Neonatal high-oxygen exposure leads to elevated blood pressure, microvascular rarefaction, vascular dysfunction and arterial (aorta) rigidity in adult rats. Whether structural changes are present in the matrix of aorta wall is unknown. Considering that elastin synthesis peaks in late fetal life in humans, and early postnatal life in rodents, we postulated that transient neonatal high-oxygen exposure can trigger premature vascular remodelling. Sprague Dawley rat pups were exposed from days 3 to 10 after birth to 80% oxygen (vs. room air control) and were studied at 4 weeks. Blood pressure and vasomotor response of the aorta to angiotensin II and to the acetylcholine analogue carbachol were not different between groups. Vascular superoxide anion production was similar between groups. There was no difference between groups in aortic cross sectional area, smooth muscle cell number or media/lumen ratio. In oxygen-exposed rats, aorta elastin/collagen content ratio was significantly decreased, the expression of elastinolytic cathepsin S was increased whereas collagenolytic cathepsin K was decreased. By immunofluorescence we observed an increase in MMP-2 and TIMP-1 staining in aortas of oxygen-exposed rats whereas TIMP-2 staining was reduced, indicating a shift in the balance towards degradation of the extra-cellular matrix and increased deposition of collagen. There was no significant difference in MMP-2 activity between groups as determined by gelatin zymography. Overall, these findings indicate that transient neonatal high oxygen exposure leads to vascular wall alterations (decreased elastin/collagen ratio and a shift in the balance towards increased deposition of collagen) which are associated with increased rigidity. Importantly, these changes are present prior to the elevation of blood pressure and vascular dysfunction in this model, and may therefore be contributory.