Platelet high affinity low density lipoprotein binding and import of lipoprotein derived phospholipids.
The binding of low density lipoprotein (LDL) to the platelet cell membrane could facilitate the transfer of phospholipids from LDL to the platelets. A polyclonal antibody against the platelet glycoproteins IIb/IIIa inhibited the high affinity binding of 125I-LDL by up to 80%. The transfer of pyrene (py)-labeled sphingomyelin (SM), phosphatidylcholine and phosphatidylethanolamine from LDL to the platelets was unaffected by the antibody. The lectin wheat germ agglutinin (WGA) reduced the binding of 125I-LDL to the platelets by approximately 80%. In contrast, the lectin stimulated the transfer of SM from LDL into the platelets by about three-fold. WGA also specifically augmented the transfer of py-SM between lipid vesicles and the platelets, the stimulation being abolished in the presence of N-acetylglucosamine. Dextran sulfate (DS) increased the specific binding of 125I-LDL to the platelets by up to 2.8-fold. On the other hand, the import of LDL-derived py-phospholipids was unaffected by DS. Together, the results indicate that the phospholipid transfer from LDL to the platelets is independent of the high affinity LDL binding to the platelets and is specifically stimulated by WGA. Thus, the interactions of platelets with LDL phospholipids differ markedly from those with the apoprotein components of the lipoproteins. (+info)
Characterization of mannooligosaccharide caps in mycobacterial lipoarabinomannan by capillary electrophoresis/electrospray mass spectrometry.
A new analytical approach based on capillary electrophoresis-electrospray mass spectrometry (CE/ESI-MS) has provided new insight into the characterization of mannooligosaccharide caps from lipoarabinomannans (LAMs), which are key molecules in the immunopathogenesis of tuberculosis. This analytical approach requires oligosaccharide labeling with the fluorophore 1-aminopyrene-3,6,8-trisulfonate (APTS) by reductive amination at the reducing termini. Optimization of the separation and ionization conditions, such as the choice of capillary electrophoresis (CE) electrolyte buffers, is presented and discussed. Anionic separation of the mono and oligosaccharide APTS derivatives was finally achieved with aqueous triethylammonium formate buffer. It was found that in contrast to the triethylammonium phosphate buffer, the triethylammonium formate buffer was appropriate for CE/ESI-MS coupling analysis of APTS-carbohydrate derivatives. In this case, negative ESI-mass spectra of APTS-carbohydrate adducts showed mainly (M-2H)2-pseudomolecular ions and some sequence fragment ions allowing their non-ambiguous structural characterization at the picomolar level. This analytical approach was successfully applied to more complex mixtures of carbohydrates released by mild acid hydrolysis of the lipoarabinomannans from Mycobacterium bovis BCG. The APTS-mannooligosaccharide cap adducts were separated by CE and their structural characterization achieved by CE/ESI-MS analyses. Mannooligosaccharide caps were routinely analyzed by capillary electrophoresis-laser induced fluorescence (CE-LIF) from 50 fmol of lipoarabinomannans with mannosyl capping (ManLAMs) but sensitivity was about 50 times lower using ESI-MS detection. (+info)
HPLC/fluorescence determination of anti-BPDE-DNA adducts in mononuclear white blood cells from PAH-exposed humans.
The aim of this study was to compare (+/-)-r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE)-DNA adduct levels in groups of humans subjected to various levels of polycyclic aromatic hydrocarbon (PAH) (benzo[a]pyrene) exposure. An HPLC/fluorescence method was applied to detect specifically anti-BPDE-DNA adducts in mononuclear white blood cells [lymphocyte plus monocyte fraction (LMF)] from humans exposed to PAHs. A total of 130 subjects comprised the sample population: 26 psoriatic patients (3 days after clinical coal tar treatment of the skin), 15 coke oven workers, 19 chimney sweeps, 36 aluminium anode plant workers and 34 non-occupationally PAH-exposed subjects (controls). PAH exposure was assessed in each group by means of the urinary excretion of 1-pyrenol (mean group levels: 1.2, 0.7, 0.3, 65.0 and 0.1 micromol/mol creatinine in coke oven workers, chimney sweeps, aluminium plant anode workers, psoriatic patients and non-occupationally PAH-exposed subjects, respectively). HPLC/fluorescence analysis of BPDE-DNA adducts showed that the percentage of subjects with adduct levels exceeding the 95 percentile control subject value (8.9 adducts/10(8) nucleotides) was significantly high in coke oven workers (46.7%) and chimney sweeps (21.0%) (chi2 test, P < 0.01 and P < 0.05, respectively) but not in aluminium plant workers (11.1%) and psoriatic patients (0%). The increase in BPDE-DNA adduct levels in LMF (Ln values) was significantly related to chronic inhalatory and high PAH exposure (linear multiple regression analysis, F = 6.37, P < 0.01; t = 4.2, P < 0.001). Skin acute (or short-term) and high PAH exposure, charcoal-grilled meat consumption and smoking habit did not seem to influence BPDE-DNA adduct formation in LMF. (+info)
Comparative metabolism of 1-, 2-, and 4-nitropyrene by human hepatic and pulmonary microsomes.
Determining the capability of humans to metabolize the mononitropyrene (mono-NP) isomers 1-, 2-, and 4-NP and understanding which human cytochrome P450 (P450) enzymes are involved in their activation and/or detoxification is important in the assessment of individual susceptibility to these environmental carcinogens. We compared the ability of 15 human hepatic and 8 pulmonary microsomal samples to metabolize each of the three isomers. Human hepatic microsomes were competent in metabolizing all three isomers. Qualitatively similar metabolic patterns were observed, although at much lower levels, upon incubating mono-NP with pulmonary microsomes. Ring-oxidized metabolites (phenols and trans-dihydrodiols) were produced from all three isomers. However, the nitroreductive metabolism leading to the formation of aminopyrene was evident only with 4-NP. The role of specific P450 enzymes in the human hepatic microsomal metabolism of mono-NP was investigated by correlating the P450-dependent catalytic activities in each microsomal sample with the levels of individual metabolites formed by the same microsomes and by examining the effects of agents that can either inhibit or stimulate specific P450 enzymes in mono-NP metabolism. On the basis of these studies, we attribute most of the hepatic microsomal metabolism of 1- and 4-NP to P450 3A4, although a minor role for P450 1A2 cannot be ruled out. Specifically, P450 3A4 was responsible for the formation of 3-hydroxy-1nitropyrene from 1-NP and the formation of trans-9,10-dihydro-9,10dihydroxy-4-nitropyrene, 9(10)-hydroxy-4-nitropyrene, and 4-aminopyrene from 4-NP. None of the P450 enzymes examined (P450s 3A4, 1A2, 2E1, 2A6, 2D6, and 2C9) appeared to be involved in catalyzing the formation of trans-4,5-dihydro-4,5-dihydroxy-2-nitropyrene and 6-hydroxy-2-nitropyrene from 2-NP in human hepatic microsomes. These results, the first report on the comparative metabolism of mono-NP in humans, clearly demonstrate that the role of specific human P450 enzymes in catalyzing oxidative and reductive pathways of mono-NP is dependent upon the position of the nitro group. (+info)
Genotoxic exposures of potroom workers.
OBJECTIVES: Potroom workers in aluminum reduction plants have increased risks for bladder and lung cancer due to exposure from polycyclic aromatic hydrocarbons (PAH). In this study correlations between measures of the external, internal, and biological effective dose have been studied for PAH. METHODS: Venous blood samples were obtained from 98 male potroom workers and 55 unexposed male blue-collar workers, for the analysis of aromatic adducts to DNA (deoxyribonucleic acid) in lymphocytes, using the 32P-postlabeling technique. 1-Hydroxypyrene in urine was analyzed with high-pressure liquid chromatography. Personal sampling of both particulate and gas phase PAH was performed during a full workday for the potroom workers and for 5 referents. Individual PAH congeners were determined with liquid chromatographic-mass spectrometric and gas chromatographic-mass spectrometric techniques. RESULTS: The respiratory-zone airborne level of the sum of 22 particulate (median 13.2 micro/m3) and the 7 gas phase PAH-congeners (median 16.3 microg/m3) among the potroom workers was a hundred times higher than among the referents. The urinary concentration of 1-hydroxypyrene before work was 30 times higher for the potroom workers (median 3.43 micromol/mol creatinine) than for the referents. Most airborne PAH congeners correlated with the excretion of 1-hydroxypyrene in urine. The frequency of aromatic DNA adducts did not, however, differ between the potroom workers and the referents, and no correlation was found for 1-hydroxypyrene in urine. CONCLUSIONS: Despite an obvious occupational exposure to PAH, no increase in aromatic DNA adducts in lymphocytes was found among the potroom workers. (+info)
Molecular mechanisms of peptide loading by the tumor rejection antigen/heat shock chaperone gp96 (GRP94).
Complexes of gp96/GRP94 and peptides have been shown to elicit immunogenicity. We used fluorescence to understand peptide association with gp96. A pyrene-peptide conjugate was complexed with gp96 under a variety of conditions. At room temperature in low salt (20 mM NaCl), the peptide binds gp96 with a strong affinity (approximately 100-150 nM) and forms pyrene excimers, suggesting that the peptides were assembled as dimers. In high salt (2.2 M NaCl), although peptide binding was stronger (Ka approximately 55 nM) than in low salt, pyrene excimers were absent, implying that peptides were farther apart in the complex. Heat shock-activated peptide binding exhibited characteristics of both low salt and high salt modes of binding. Anisotropy and average lifetime of the bound pyrene suggested that peptides were probably located in a solvent-accessible hydrophobic binding pocket in low salt, whereas in high salt, the peptide may be buried in a less hydrophobic (more hydrophilic) environment. These results suggested that peptide-gp96 complexes were assembled in several different ways, depending on the assembly conditions. Resonance energy transfer between the intrinsic tryptophan(s) in gp96 and pyrene suggested that one or more tryptophan residues were within the critical Forster distance of 27-30 A from the pyrene in the bound peptide. It is proposed that peptides are assembled within higher order gp96 complexes (dimers, etc.) in a hydrophobic pocket and that there may be a conformational change in gp96 leading to an open configuration for peptide loading. (+info)
Pathologic changes induced in respiratory tract mucosa by polycyclic hydrocarbons of differing carcinogenic activity.
Seven aromatic polycyclic hydrocarbons (PCHs) were investigated for their toxic effects on respiratory mucosa: benzo(e)pyrene (BeP), pyrene, anthracene, benz(a)anthracene(BaA), dibenz(a,c)anthracene(DBacA), benzo (a)pyrene (BaP), and dimethylbenz(a)anthracene (DMBA). The compounds were chosen because they comprise a spectrum of PCHs ranging from noncarcinogens, to initiators, to weak and strong carcinogens. All of them except DMBA are environmentally relevant chemicals. The chemicals were tested over an 8-week period. Heterotopic tracheal transplants were continously exposed and the histopathologic effects induced by the various PCHs were periodically assessed semiquantitatively. All PCHs exhibited varying degrees of toxicity for respiratory epithelium and submucosa. BeP clearly showed the least toxicity followed by pyrene and anthracene. BaA and DBacA caused marked epithelial and submucosal changes. In addition to epithelial hyperplasia, undifferentiated epithelium and squamous metaplasia developed. Marked mononuclear infiltration occurred in the subepithelial connective tissue. With BaP the epithelial and submucosal changes were similar but were much stronger. DMBA was the most toxic substance, causing epithelial necrosis followed by generalized keratinizing squamous metaplasia; the subepithelial changes consisted of an early acellular exudate and, later (at 8 weeks), marked condensation and hyalinization of the lamina propria. The toxic response pattern of the tracheal mucosa to carcinogenic agents was characterized by the chronicity of epithelial and connective tissue damage, as opposed to the short-lived hyperplastic and inflammatory response elicited by the noncarcinogens and weak initiators. (+info)
2'-Pyrene modified oligonucleotide provides a highly sensitive fluorescent probe of RNA.
Oligonucleotide 9mers containing 2'-O-(1-pyrenylmethyl)uridine [U(pyr)] at the center position were synthesized by using a protected U(pyr) phosphoramidite. The UV melting behaviors indicate that the pyrene-modified oligonucleotides can bind to both their complementary DNA and RNA in aqueous solution. When compared with the unmodified oligonucleotides, the pyrene-modified oligonucleotides showed higher affinity for DNA while exhibiting lower affinity for RNA. The pyrene-modified oligonucleotides in diluted solution exhibited fluorescence typical of pyrene monomer emission [lambdamax 378 (band I) and 391 nm (band III)]. When these oligomers bound to DNA, the fluorescence intensity ratio of band III/band I was increased. With this fluorescence change, a new broad emission (lambdamax 450 nm) due to exciplex between the pyrene and an adjacent nucleobase appeared. In contrast, addition of RNA to the pyrene oligonucleotides resulted in enhancement of the pyrene monomer emission with decrease in the fluorescence band ratio. The extent of the emission enhancement was found to be highly dependent on the nucleobase adjacent to the U(pyr) in the pyrene oligomers. The pyrene oligonucleotide containing dC at the 3'-site of the modification showed remarkable increase (approximately 250 times) in fluorescence (375 nm) upon binding to complementary RNA. The present findings would open the way to the design of a highly sensitive fluorescent probe of RNA. (+info)