Microparticle enlargement and altered surface proteins after air decompression are associated with inflammatory vascular injuries

Studies in a murine model have shown that decompression stress triggers a progressive elevation in the number of circulating annexin V-coated microparticles derived from leukocytes, erythrocytes, platelets, and endothelial cells. We noted that some particles appeared to be larger than anticipated, and size continued to increase for ≥24 h postdecompression. These observations led to the hypothesis that inert gas bubbles caused the enlargement and particle size could be reduced by hydrostatic pressure. After demonstrating pressure-induced particle size reduction, we hypothesized that annexin V-positive particle changes associated with decompression contributed to their proinflammatory potential. Intravenous injection of naive mice with particles isolated from decompressed mice, but not control mice, caused intravascular neutrophil activation; perivascular neutrophil sequestration and tissue injuries were documented as elevations of vascular permeability and activated caspase-3. These changes were not observed if mice were injected with particles that had been subjected to hydrostatic recompression or particles that had been emulsified by incubation with polyethylene glycol telomere B surfactant. Hydrostatic pressure and surfactant incubation also altered the pattern of proteins expressed on the surface of particles. We conclude that proinflammatory events and vascular damage are due to enlargement of annexin V-coated particles and/or changes in surface marker protein pattern associated with provocative decompression. Injection of annexin V-coated particles from decompressed mice will recapitulate the pathophysiological vascular changes observed following decompression stress.     

Rich Medium Composition Affects Escherichia coli Survival,Glycation, and Mutation Frequency during Long-Term Batch Culture

Bacteria such as Escherichia coli are frequently grown to high density to produce biomolecules for study in the laboratory. To achieve this, cells can be incubated in extremely rich media that increase overall cell yield. In these various media, bacteria may have different metabolic profiles, leading to changes in the amounts of toxic metabolites produced. We have previously shown that stresses experienced during short-term growth can affect the survival of cells during the long-term stationary phase (LTSP). Here, we incubated cells in LB, 2× yeast extract-tryptone (YT), Terrific Broth, or Super Broth medium and monitored survival during the LTSP, as well as other reporters of genetic and physiological change. We observe differential cell yield and survival in all media studied. We propose that differences in long-term survival are the result of changes in the metabolism of components of the media that may lead to increased levels of protein and/or DNA damage. We also show that culture pH and levels of protein glycation, a covalent modification that causes protein damage, affect long-term survival. Further, we measured mutation frequency after overnight incubation and observed a correlation between high mutation frequencies at the end of the log phase and loss of viability after 4 days of LTSP incubation, indicating that mutation frequency is potentially predictive of long-term survival. Since glycation and mutation can be caused by oxidative stress, we measured expression of the oxyR oxidative stress regulator during log-phase growth and found that higher levels of oxyR expression during the log phase are consistent with high mutation frequency and lower cell density during the LTSP. Since these complex rich media are often used when producing large quantities of biomolecules in the laboratory, the observed increase in damage resulting in glycation or mutation may lead to production of a heterogeneous population of plasmids or proteins, which could affect the quality of the end products yielded in some laboratory experiments.     

Isolation and quantitative estimation of diesel exhaust and carbon black particles ingested by lung epithelial cells and alveolar macrophages in vitro

A new procedure for isolating and estimating ingested carbonaceous diesel exhaust particles (DEP) or carbon black (CB) particles by lung epithelial cells and macrophages is described. Cells were incubated with DEP or CB to examine cell-particle interaction and ingestion. After various incubation periods, the cells were separated from free extracellular DEP or CB particles by Ficoll density gradient centrifugation and dissolved in hot sodium dodecyl sulfate detergent. Insoluble DEP or CB residues were isolated by high-speed centrifugation, and the elemental carbon (EC) concentrations in the pellets were estimated by a thermal-optical-transmittance method (i.e., carbon analysis). From the EC concentration, the amount of ingested DEP or CB could be calculated. The described technique allowed the determination of the kinetics and dose dependence of DEP uptake by LA4 lung epithelial cells and MHS alveolar macrophages. Both cell types ingested DEP to a similar degree; however, the MHS macrophages took up significantly more CB than the epithelial cells. Cytochalasin D, an agent that blocks actin polymerization in the cells, inhibited approximately 80% of DEP uptake by both cell types, indicating that the process was actin-dependent in a manner similar to phagocytosis. This technique can be applied to examine the interactions between cells and particles containing EC and to study the modulation of particle uptake in diseased tissue.     

Protective effects of <Emphasis Type="SmallCaps">l</Emphasis>-selenomethionine on space radiation induced changes in gene expression

Ionizing radiation can produce adverse biological effects in astronauts during space travel. Of particular concern are the types of radiation from highly energetic, heavy, charged particles known as HZE particles. The aims of our studies are to characterize HZE particle radiation induced biological effects and evaluate the effects of l-selenomethionine (SeM) on these adverse biological effects. In this study, microarray technology was used to measure HZE radiation induced changes in gene expression, as well as to evaluate modulation of these changes by SeM. Human thyroid epithelial cells (HTori-3) were irradiated (1 GeV/n iron ions) in the presence or in the absence of 5 μM SeM. At 6 h post-irradiation, all cells were harvested for RNA isolation. Gene Chip U133Av2 from Affymetrix was used for the analysis of gene expression, and ANOVA and EASE were used for a determination of the genes and biological processes whose differential expression is statistically significant. Results of this microarray study indicate that exposure to small doses of radiation from HZE particles, 10 and 20 cGy from iron ions, induces statistically significant differential expression of 196 and 610 genes, respectively. In the presence of SeM, differential expression of 77 out of 196 genes (exposure to 10 cGy) and 336 out of 610 genes (exposure to 20 cGy) is abolished. In the presence or in the absence of SeM, radiation from HZE particles induces differential expression of genes whose products have roles in the induction of G1/S arrest during the mitotic cell cycle, as well as heat shock proteins. Some of the genes, whose expressions were affected by radiation from HZE particles and were unchanged in irradiated cells treated with SeM, have been shown to have altered expression levels in cancer cells. The conclusions of this report are that radiation from HZE particles can induce differential expression of many genes, some of which are known to play roles in the same processes that have been shown to be activated in cells exposed to radiation from photons (like cell cycle arrest in G1/S), and that supplementation with SeM abolishes HZE particle-induced differential expression of many genes. Understanding the roles that these genes play in the radiation-induced transformation of cells may help to decipher the origins of radiation-induced cancer.     

EFFECT OF INCUBATION TEMPERATURE ON THE MACROMOLECULAR AND LIPID CONTENT OF THE PHYTOFLAGELLATE OCHROMONAS DANICA1

Ochromonas danica grown in a chemically defined medium under controlled conditions at different incubation temperatures showed marked changes in chemical composition and secretory pattern. As the incubation temperature rose from 15 to 30 C, the cell number per unit volume of medium increased; the cell mass reached a maximum and then declined slightly, and the weight per single cell decreased. The chemical composition of the cells changed with increasing temperature: carbohydrates decreased dramatically; proteins quadrupled at 20 C and then declined 25%; lipids increased; and RNA increased to 25 C and then decreased to about 40% of maximum at 30 C. The extracellular secretion by the cells of carbohydrates seemed to increase and then decline; RNA decreased; proteins increased; and lipids remained approximately constant, as the temperature increased. The implications of these findings for thermal pollution are discussed.     

Elevated miR-155 promotes inflammation in cystic fibrosis by driving hyperexpression of interleukin-8

Cystic Fibrosis (CF) is characterized by a massive proinflammatory phenotype in the lung arising from profound expression of inflammatory genes, including interleukin-8 (IL-8). We have previously reported that IL-8 mRNA is stabilized in CF lung epithelial cells, resulting in concomitant hyperexpression of IL-8 protein. However, the mechanistic link between mutations in CFTR and acquisition of the proinflammatory phenotype in the CF airway has remained elusive. We hypothesized that specific microRNAs (miRNAs) might mediate this linkage. To identify the potential link, we screened an miRNA library for differential expression in ΔF508-CFTR and wild type CFTR lung epithelial cell lines. Of 22 differentially and significantly expressed miRNAs, we found that expression of miR-155 was more than 5-fold elevated in CF IB3-1 lung epithelial cells in culture, compared with control IB3-1/S9 cells. Clinically, miR-155 was also highly expressed in CF lung epithelial cells and circulating CF neutrophils biopsied from CF patients. We report here that high levels of miR-155 specifically reduced levels of SHIP1, thereby promoting PI3K/Akt activation. However, overexpressing SHIP1 or inhibition of PI3K in CF cells suppressed IL-8 expression. Finally, we found that phospho-Akt levels were elevated in CF lung epithelial cells and were specifically lowered by either antagomir-155 or elevated expression of SHIP1. We therefore suggest that elevated miR-155 contributes to the proinflammatory expression of IL-8 in CF lung epithelial cells by lowering SHIP1 expression and thereby activating the PI3K/Akt signaling pathway. These data suggest that miR-155 may play an important role in the activation of IL-8-dependent inflammation in CF.     

Divalent metal transporter-1 decreases metal-related injury in the lung

Exposure to airborne particulates makes the detoxification of metals a continuous challenge for the lungs. Based on the fate of iron in airway epithelial cells, we postulated that divalent metal transporter-1 (DMT1) participates in detoxification of metal associated with air pollution particles. Homozygous Belgrade rats, which are functionally deficient in DMT1, exhibited diminished metal transport from the lower respiratory tract and greater lung injury than control littermates when exposed to oil fly ash. Preexposure of normal rats to iron in vivo increased expression of the isoform of DMT1 protein that lacked an iron-response element (-IRE), accelerated metal transport out of the lung, and decreased injury after particle exposure. In contrast, normal rats preexposed to vanadium showed less expression of the -IRE isoform of DMT1, decreased metal transport, and greater pulmonary injury after particle instillation. Respiratory epithelial cells in culture gave similar results. Also, DMT1 mRNA and protein expression for the -IRE isoform increased or decreased in these cells when exposed to iron or vanadium, respectively. These results thus demonstrate for the first time a primary role for DMT1 in lung metal transport and detoxification.     

Contributions of freshwater mussels (Unionidae) to nutrient cycling in an urban river: filtration,recycling, storage,and removal

Physical separation of soil into different soil organic matter (SOM) fractions is widely used to identify organic carbon pools that are differently stabilized and have distinct chemical composition. However, the mechanisms underlying these differences in stability and chemical composition are only partly understood. To provide new insights into the stabilization of different chemical compound classes in physically-separated SOM fractions, we assessed shifts in the biomolecular composition of bulk soils and individual particle size fractions that were incubated in the laboratory for 345 days. After the incubation, also the incubated bulk soil was fractionated. The chemical composition of organic matter in bulk soils and fractions was characterized by ¹³C-CPMAS nuclear magnetic resonance spectroscopy and sequential chemical extraction followed by GC/MS measurements. Plant-derived lipids and lignin were abundant in particulate organic matter (POM) fractions of sand-, silt-, and clay-size and the mineral-bound, clay-sized organic matter. These results indicate that recent conceptualizations of SOM stabilization probably understate the contribution of plant-derived organic matter to stable SOM pools. Although our data indicate that inherent recalcitrance could be important in soils with limited aggregation, organo-mineral interactions and aggregation were responsible for long-term SOM stabilization. In particular, we observed consistently higher concentrations of plant-derived lipids in POM fractions that were incubated individually, where aggregates were disrupted, as compared to those incubated as bulk soil, where aggregates stayed intact. This finding emphasizes the importance of aggregation for the stabilization of less ‘recalcitrant’ biomolecules in the POM fractions. Because also the abundance of lipids and lignin in clay-sized, mineral-associated SOM was substantially influenced by aggregation, the bioavailability of mineral-associated SOM likely increases after the destruction of intact soil structures.     

Proteomic analysis of serum opsonins impacting biodistribution and cellular association of porous silicon microparticles

Mass transport of drug delivery vehicles is guided by particle properties, such as size, shape, composition, and surface chemistry, as well as biomolecules and serum proteins that adsorb to the particle surface. In an attempt to identify serum proteins influencing cellular associations and biodistribution of intravascularly injected particles, we used two-dimensional gel electrophoresis and mass spectrometry to identify proteins eluted from the surface of cationic and anionic silicon microparticles. Cationic microparticles displayed a 25-fold greater abundance of Ig light variable chain, fibrinogen, and complement component 1 compared to their anionic counterparts. Anionic microparticles were found to accumulate in equal abundance in murine liver and spleen, whereas cationic microparticles showed preferential accumulation in the spleen. Immunohistochemistry supported macrophage uptake of both anionic and cationic microparticles in the liver, as well as evidence of association of cationic microparticles with hepatic endothelial cells. Furthermore, scanning electron micrographs supported cellular competition for cationic microparticles by endothelial cells and macrophages. Despite high macrophage content in the lungs and tumor, microparticle uptake by these cells was minimal, supporting differences in the repertoire of surface receptors expressed by tissue-specific macrophages. In summary, particle surface chemistry drives selective binding of serum components impacting cellular interactions and biodistribution.     

Increases in the particle size of high-density lipoproteins induced by purified lecithin: cholesterol acyltransferase: effect of low-density lipoproteins

Homogeneous subpopulations of human high-density lipoproteins subfraction-3 (HDL3) have been incubated at 37 degrees C with purified lecithin: cholesterol acyltransferase, human serum albumin and varying concentrations of human low-density lipoproteins (LDL). Changes in HDL particle size and composition during these incubations were monitored. Incubation of HDL3a (particle radius 4.3 nm) in the absence of LDL resulted in an esterification of more than 70% of the HDL free cholesterol after 24 h of incubation. This, however, was sufficient to increase the HDL cholesteryl ester by less than 10% and was not accompanied by any change in particle size. When this mixture was incubated in the presence of progressively increasing concentrations of LDL, which donated free cholesterol to the HDL, the molar rate of production of cholesteryl ester was much greater; at the highest LDL concentration HDL cholesteryl ester content was almost doubled after 24 h and there was an increase in the HDL particle size up to the HDL2 range. In the case of HDL3b (radius 3.9 nm), there were again only minimal changes in particle size in incubations not containing LDL. In the presence of the highest concentration of LDL tested, however, the particles were again enlarged into the HDL2 size range after 24 h incubation. These HDL2-like particles were markedly enriched with cholesteryl ester but depleted of phospholipid and free cholesterol when compared with native HDL2. Furthermore, the ratio of apolipoprotein A-I to apolipoprotein A-II resembled that in the parent-HDL3 and was very much lower than that in native HDL2. It has been concluded that purified lecithin: cholesterol acyltransferase is capable of increasing the size of HDL3 towards that of HDL2 but that other factors must operate in vivo to modulate the chemical composition of the enlarged particles.     

High-Performing Dry Powder Inhalers of Paclitaxel DPPC/DPPG Lung Surfactant-Mimic Multifunctional Particles in Lung Cancer: Physicochemical Characterization,In Vitro Aerosol Dispersion,and Cellular Studies

Inhalable lung surfactant-based carriers composed of synthetic phospholipids, dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG), along with paclitaxel (PTX), were designed and optimized as respirable dry powders using organic solution co-spray-drying particle engineering design. These materials can be used to deliver and treat a wide variety of pulmonary diseases with this current work focusing on lung cancer. In particular, this is the first time dry powder lung surfactant-based particles have been developed and characterized for this purpose. Comprehensive physicochemical characterization was carried out to analyze the particle morphology, surface structure, solid-state transitions, amorphous character, residual water content, and phospholipid bilayer structure. The particle chemical composition was confirmed using attenuated total reflectance-Fourier-transform infrared (ATR-FTIR) spectroscopy. PTX loading was high, as quantified using UV-VIS spectroscopy, and sustained PTX release was measured over weeks. In vitro cellular characterization on lung cancer cells demonstrated the enhanced chemotherapeutic cytotoxic activity of paclitaxel from co-spray-dried DPPC/DPPG (co-SD DPPC/DPPG) lung surfactant-based carrier particles and the cytotoxicity of the particles via pulmonary cell viability analysis, fluorescent microscopy imaging, and transepithelial electrical resistance (TEER) testing at air-interface conditions. In vitro aerosol performance using a Next Generation Impactor™ (NGI™) showed measurable powder deposition on all stages of the NGI and was relatively high on the lower stages (nanometer aerodynamic size). Aerosol dispersion analysis of these high-performing DPIs showed mass median diameters (MMADs) that ranged from 1.9 to 2.3 μm with excellent aerosol dispersion performance as exemplified by high values of emitted dose, fine particle fractions, and respirable fractions.

Graphical Abstract

Open in a separate windowᅟKEY WORDS: lung surfactant, NBD-PC fluorescent microscopy imaging, Next Generation Impactor (NGI), particle engineering design, pulmonary cell lines     

The effect of a lipid-rich diet on the properties and composition of lipoprotein particles from the Golgi apparatus of guinea-pig liver

1. A cell fraction rich in Golgi apparatus was isolated from the livers of guinea pigs fed on a lipid-rich diet (1.6% cholesterol, 15% corn oil). 2. The Golgi cisternae and secretory vesicles contained electron-dense particles which were tentatively identified as VLD (very-low-density) and LD (low-density) lipoproteins. Particles of moderate electron density, 150–500nm in diameter, were seen associated with membranous elements of the Golgi-apparatus cell fraction. Disruption of this cell fraction permitted the release of these three species of particles, which were separated into particulate lipid, and VLD and LD lipoproteins. 3. The large particles of moderate electron density, isolated as particulate lipid, were distinct from both species of Golgi particles in their chemical composition and in possessing an immunochemically unreactive apolipoprotein(s). Morphological observations suggest that the particulate lipid arose from cytoplasmic lipid droplets which were present as contaminants of the Golgi-rich fraction. 4. The chemical and immunochemical results are consistent with the suggestion that the Golgi LD particles are precursors of the VLD particles, into which they may be transformed by the addition of both triglyceride and cholesteryl ester. The present results provide further support for the proposal that the Golgi VLD particles are precursors of the serum VLD lipoproteins in the guinea pig. 5. Hepatic Golgi VLD particles isolated from guinea pigs fed on the lipid-rich diet contained significantly higher molar amounts (relative to protein) of both cholesteryl ester and triglyceride than similar particles from animals fed on a normal diet. These results suggest that the type of Golgi VLD particle produced from the LD particle is a direct consequence of the amount and composition of the dietary lipid. 6. Hepatic Golgi LD particles isolated from guinea pigs fed on different diets were similar in chemical composition and contained approx. 50% by weight of phospholipid. We conclude that the Golgi LD particle is normally present in the Golgi-apparatus cell fraction from guinea-pig liver, and may represent the end product of lipoprotein biosynthesis in the smooth endoplasmic reticulum. 7. The serum LD lipoproteins and Golgi LD particles were quite distinct in chemical composition. However, these two lipoprotein species were immunochemically identical and exhibited a similar range of flotation rate. It appears unlikely that the Golgi LD particles are secreted as the precursors of the serum LD lipoproteins.     

The development of membrane specializations in the receptor-bipolar-horizontal cell synapse of the chick embryo retina

Retinae of chick embryos and chicks one to six weeks after hatching were examined in ultrathin sections and in freeze-etch specimens. The development of the synaptic contacts between receptor cells and bipolar cells starts at the end of the second week of incubation with the enclosure of the dendritic prolongations, invaginating receptor terminals accompanied by the appearance of electron dense material at the synaptic contact sites. Subsequently receptor terminals become filled with synaptic vesicles which surround the synaptic lamellae that appear on the 16th day of incubation. The application of the freeze-fracture technique demonstrates that the differentiation of the synaptic membranes continues into the first week post hatching. E-fracture faces of the presynaptic membranes are characterized by crater-like structures, called synaptopores. Their number is rather small during incubation and increases after hatching. In the P-fracture faces of the dendrites, which are enclosed by the receptor terminals, small particle aggregations appear on the 16th day of incubation. These small particle clusters increase by the apposition of further particles which become arranged in lines and bring out a lattice-like aspect. This arrangement of particles in the inner part of the cell membrane is the morphological expression of the maturation process. The significance of these aggregations as a postsynaptic receptor for neurotransmitters in excitatory cells is discussed.     

Factors involved in the generation and replication of rhabdovirus defective T particles.

Previous indications that cloned B virions might be genetically predisposed to generate a particular defective T particle are shown to be inaccurate. T particle generation was found to be a much more random process than was previously believed. We show that the previously observed generation of particular sizes of T particles by B virion pools is due to the random generation of T particles during preparation of first-passage pools of cloned B virions, and these breed true during the additional passages needed to produce visible quantities of T particles. It is also shown that different host cell lines selectively amplify different T particles, suggesting a strong role of host cell factors in T particle replication. Surprisingly, our line of HeLa cells did not generate or replicate detectable T particles of vesicular stomatitis virus (VSV) Indiana after either serial undiluted passage or direct addition of T particles, even though the added T particles strongly interfered with B virion replication. In contrast to VSV, rabies virus generates large amounts of T particles during the first passage of cloned B virions, and every rabies-infected baby hamster kidney-21 cell culture evolves into a persistent carrier state. We find that T particle RNA is biologically inactive although T particle nucleocapsid ribonucleoprotein replicates and interferes in cells coinfected with B virions. Efforts to study the mechanism of T particle generation by in vitro attempts to generate T particles or modify their size (using sheared ribonucleoprotein or chemical or UV mutagenesis) were unsuccessful. The kinetics of UV and nitrous acid inactivation of T particles indicate a smaller target size relative to B virions, even after correcting for lengths of RNA molecules. The intercalating dye proflavine does not photosensitize VSV B virions or T particles when present during replication, indicating that there is little or no RNA base pairing in the helical nucleocapsids of either.     

Biological responses to diesel exhaust particles (DEPs) depend on the physicochemical properties of the DEPs

Diesel exhaust particles (DEPs) are the main components of ambient particulate materials, including polyaromatic hydrocarbons (PAHs), n-PAHs, heavy metals, and gaseous materials. Many epidemiological, clinical, and toxicological studies have shown that ambient particles, including DEPs, are associated with respiratory disorders, such as asthma, allergic rhinitis, and lung cancer. However, the relationship between the biological response to DEPs and their chemical composition remains unclear. In this study, we investigated the physicochemical properties of DEPs before toxicological studies, and then administered a single intratracheal instillation of DEPs to mice. The mice were then killed 1, 7, 14 and 28 days after DEP exposure to observe the biological responses induced by DEPs over time. Our findings suggest that DEPs engulfed into cells induced a Th2-type inflammatory response followed by DNA damage, whereas DEPs not engulfed into cells induced a Th1-type inflammatory response. Further, the physicochemical properties, including surface charge, particle size, and chemical composition, of DEPs play a crucial role in determining the biological responses to DEPs. Consequently, we suggest that the biological response to DEPs depend on cell-particle interaction and the physicochemical properties of the particles.     

MOLECULAR,ENZYMATIC AND ULRASTRUCURE CHARACTERIZATION OF THE PYRENOID OF THE SCALY GREEN MONAD MICROMONAS SQUAMATA1,2

Pyrenoid material of micromonas squamata Manton & Parke was obtained free of cell and subcellular particle conamination by differential centrifugation of brei from osmoically lysed cells. The isolated pyrenoid particles were characterized by transmission and scanning electron microscopy. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of pyrenoid extracts revealed a compled polypeptide composition with major components of 12, 54 and 66 kilodalton mol wt. Whole pyrenoids possessed the enzymatic properties of ribulose diphosphate carboxylase and fixed carbon dioxide with specific activity 10 times greaer than that of a pyrenoid-free high speed supenaant fracion of cell brei. Energy dispersive X-ray microanalysis revealed he presence of copper in masses of cryo-impacted pyrenoid material. Ultrastrucural cytochemistry was employed o determine he chemical nature of the reserve carbohydrate shell. Also, the pyrenoid of the intact cell was characterized by transmission electron microscopy.