Nasal lavage fluid and proteomics as means to identify the effects of the irritating epoxy chemical dimethylbenzylamine.

The aims of this study were to describe the changes in the nasal lavage fluid (NLF) protein pattern after exposure to the irritating epoxy chemical dimethylbenzylamine (DMBA) and to identify the affected proteins using a proteomic approach. The protein patterns of NLF from six healthy subjects and eight epoxy workers with airway irritation were analysed using two-dimensional gel electrophoresis (2-DE) before and after exposure to 100 microg m(-3) DMBA for 2 h in an exposure chamber. NLF proteins were identified by (i) comparison with a 2-DE NLF reference database; (ii) N-terminal amino acid sequencing; and (iii) mass spectrometry. In NLF from healthy subjects, the levels of immunoglobulin A increased and the levels of Clara cell protein 16 (CC16) decreased after chamber exposure, while in NLF from epoxy workers, alpha(2)-macroglobulin and caeruloplasmin increased. Two previously unidentified proteins decreased in NLF from epoxy workers after exposure; these were identified as statherin and calgranulin B. In addition, the subjects who developed high counts of eosinophils in their nasal mucosa after chamber exposure had significantly lower levels of immunoglobulin-binding factor (IgBF) before exposure than subjects with low eosinophil infiltration. These results show that short-term exposure to DMBA causes distinct changes in NLF proteins. Moreover, three proteins that have previously not been associated with upper airway irritation were identified: statherin, calgranulin B and IgBF. Further studies are needed to investigate whether these proteins may be used as biomarkers of airway irritation and to give new insight into the ways in which occupational exposure to irritants causes inflammation of the airways.     

Cigarette smoke-induced DNA-damage: role of hydroquinone and catechol in the formation of the oxidative DNA-adduct, 8-hydroxydeoxyguanosine

This study demonstrates the ability of cigarette smoke condensate to generate hydrogen peroxide and to hydroxylate deoxyguanosine (dG) residues in isolated DNA to 8-hydroxydeoxyguanosine (8-OHdG). Both the formation of hydrogen peroxide and that of 8-OHdG in DNA was significantly decreased when catalase or tyrosinase was added to the smoke condensates, and this also occurred when pure hydroquinone or catechol, two major constitutes in cigarette smoke, was used instead of smoke condensate. Moreover, pure hydroquinone and catechol both caused dose-dependent formation of hydrogen peroxide and 8-OHdG, and there was good correlation between the amounts of hydrogen peroxide and 8-OHdG formed. These findings suggest that (i) hydroquinone and catechol may be responsible for the ability of cigarette smoke to cause 8-OHdG formation in DNA, (ii) this oxidative DNA-damage is due to the action of hydroxyl radicals formed during dissociation of hydrogen peroxide and (iii) the hydrogen peroxide in cigarette smoke is generated via autooxidation of hydroquinone and catechol.     

A lipopolysaccharide mutant of Bradyrhizobium japonicum that uncouples plant from bacterial differentiation.

The Tn5-containing fragment from a non-nodulating mutant of Bradyrhizobium japonicum, strain ML142, was introduced into B. japonicum strain 61A101c by marker exchange to construct strain JS314. Strain JS314 failed to nodulate several soybean varieties tested. However, on a few varieties nodulelike structures were induced to a frequency of 54% of the plants inoculated. The ultrastructure of these nodules was studied in detail by light and electron microscopy. The nodules were devoid of internal bacteria, possessed central vascular tissue (unlike the lateral vascular tissue of a normal nodule), and exhibited localized cell death of epidermal cells. Study of the cell surface polysaccharides of strain JS314 revealed that the exopolysaccharide of this strain was identical to that of the wild type. However, the lipopolysaccharide (LPS) of strain JS314 showed gross differences from that isolated from the wild-type strain. Specifically, the LPS of strain JS314 appeared to lack the high molecular weight LPS I form, strongly suggesting that the LPS lacks the O-chain. Glycosyl-composition analysis showed that the LPS of mutant JS314 lacked 2,3-di-O-methylrhamnose, 3-O-methylrhamnose, fucose, and quinovosamine. These results indicate that LPS I in B. japonicum is essential for bacterial infection of soybean, but is not required to initiate plant cortical cell division, an early plant response to infection.     

Metabolism of cationized lipoproteins by human fibroblasts: biochemical and morphologic correlations

Human plasma low density lipoprotein (LDL) that had been rendered polycationic by coupling with N, N-dimethyl-1, 3-propanediamine (DMPA) was shown by electron microscopy to bind in clusters to the surface of human fibroblasts. The clusters resembled those formed by polycationic ferritin (DMPA-feritin), a visual probe that binds to anionic site on the plasma membrane. Biochemical studies with (125)I-labeled DMPA-LDL showed that the membrane-bound lipoprotein was internalized and hydrolyzed in lysosomes. The turnover time for cell bound (125)I-DMPA-LDL, i.e., the time in which the amount of (125)I-DMPA-LDL degraded was equal to the steady-state cellular content of the lipoprotein, was about 50 h. Because the DMPA-LDL gained access to fibroblasts by binding nonspecifically to anionic sites on the cell surface rather than by binding to the physiologic LDL receptor, its uptake failed to be regulated under conditions in which the uptake of native LDL was reduced by feedback suppression of the LDL receptor. As a result, unlike the case with native LDL, the DMPA-LDL accumulated progressively within the cell, and this led to a massive increase in the cellular content of both free and esterified cholesterol. Studies with (14)C-oleate showed that at least 20 percent of the accumulated cholesteryl esters represented cholesterol that had been esterified within the cell. After 4 days of incubation with 10 μg/ml of DMPA-LDL, fibroblasts had accumulated so much cholesteryl ester that neutral lipid droplets were visible at the light microscope level with Oil Red O staining. By electron microscopy, these intracellular lipid droplets were observed to lack a tripartite limiting membrane. The ability to cause the overaccumulation of cholesteryl esters within cells by using DMPA-LDL provides a model system for study of the pathologic consequences at the cellular level of massive deposition of cholesteryl ester.     

Physicochemical consequences of opsonization: perturbation of liposomal membranes by Salmonella typhimurium 395 MS opsonized with IgG antibodies.

When phagocytosis-resistant Salmonella typhimurium 395 MS bacteria sensitized with anti-MS IgG antibodies were incubated with liposomes composed of phosphatidylcholine, cholesterol, and dicetylphosphate, a previously sequestered liposomal marker (4-methylumbelliferylphosphate) was released. Unsensitized or F(ab')2-sensitized bacteria had no such effect. This perturbation was neither dependent upon negatively charged dicetylphosphate nor upon cholesterol. It was further evident that palmitoyl-poly(ethyleneglycol) caused release of the trapped marker in a similar way as sensitized bacteria. These findings demonstrate a similarity between sensitized bacteria and a hydrophobic probe and lend support to the hypothesis that the perturbation was brought about by hydrophobic interaction. The observations indicate that liposomes, like phagocytes, possess "receptor sites" for the activated part of IgG and raise the possibility that phagocytic effectors can operate in a relatively nonspecific manner.     

Molecular evolution of a multigene family in group A streptococci

The emm genes are members of a gene family in group A streptococci (GAS) that encode for antiphagocytic cell-surface proteins and/or immunoglobulin-binding proteins. Previously sequenced genes in this family have been named "emm," "fcrA," "enn," "arp," "protH," and "mrp"; herein they will be referred to as the "emm gene family." The genes in the emm family are located in a cluster occupying 3-6 kb between the genes mry and scpA on the chromosome of Streptococcus pyogenes. Most GAS strains contain one to three tandemly arranged copies of emm-family genes in the cluster, but the alleles within the cluster vary among different strains. Phylogenetic analysis of the conserved sequences at the 3' end of these genes differentiates all known members of this family into four evolutionarily distinct emm subfamilies. As a starting point to analyze how the different subfamilies are related evolutionarily, the structure of the emm chromosomal region was mapped in a number of diverse GAS strains by using subfamily-specific primers in the polymerase chain reaction. Nine distinct chromosomal patterns of the genes in the emm gene cluster were found. These nine chromosomal patterns support a model for the evolution of the emm gene family in which gene duplication followed by sequence divergence resulted in the generation of four major-gene subfamilies in this locus.      

Relationships between lake ecology and morphological characters in Icelandic Arctic charr,Salvelinus alpinus

The common occurrence of parallel phenotypic patterns suggests that a strong relationship exists between ecological dynamics and micro‐evolution. Comparative studies from a large number of populations under varying sets of ecological drivers could contribute to a better understanding of this relationship. We used data on morphology of arctic charr (Salvelinus alpinus) and ecological factors from 35 Icelandic lakes to test the hypothesis that morphological patterns among monomorphic charr populations from different lakes are related to interlake variation in ecological characteristics. There is extensive phenotypic diversity among populations of Icelandic charr, and populations are easily distinguished based on overall body morphology. The results obtained in the present study showed that the morphological diversity of charr was related to large‐scale diversity in lake ecology. Variation in charr morphology was related to water origin (e.g. spring fed versus run‐off), bedrock age, and fish community structure. The present study shows how various ecological factors can shape the biological diversity that we observe. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 103 , 761–771.