Western red cedar dust exposure and lung function: a dose-response relationship.

The relationship between levels of cumulative red cedar dust exposure and decline in lung function was explored in an 11-yr follow-up study of 243 sawmill workers who participated in at least two occasions. We also studied 140 office workers in a similar manner as control subjects. Workers with asthma were excluded from the analysis. During the period of the study, 916 personal and 216 area samples of dust were collected from the sawmill. Cumulative wood dust exposure was calculated for each sawmill worker according to the duration and exposure in each job, based on the geometric mean of all dust measurements for that job. Average daily dust exposure was calculated by dividing the total cumulative exposure by the number of days of work. Workers were divided into low-, medium-, and high-exposure groups with mean daily level of exposure of < 0.2, 0.2 to 0.4, and > 0.4 mg/m3, respectively. Sawmill workers had significantly greater declines in FEV1 and FVC compared with office workers adjusted for age, smoking, and initial lung function. A dose-response relationship was observed between the level of exposure and the annual decline in FVC. We conclude that exposure to Western red cedar dust is associated with a greater decline in lung function which may lead to development of chronic airflow limitation.

Comparison of dust related respiratory effects in Dutch and Canadian grain handling industries: a pooled analysis.

OBJECTIVES: Four previously conducted epidemiological studies in more than 1200 grain workers were used to compare exposure-response relations between exposure to grain dust and respiratory health. METHODS: The studies included Dutch workers from an animal feed mill and a transfer grain elevator and Canadian workers from a terminal grain elevator and the docks. Relations between forced expiratory volume in one second (FEV1) and exposure were analysed with multiple regression analysis corrected for smoking, age, and height. Exposure variables examined included cumulative and current dust exposure and the numbers of years a subject was employed in the industry. Sampling efficiencies of the Dutch and Canadian measurement techniques were compared in a pilot study. Results of this study were used to correct slopes of exposure-response relations for differences in dust fractions sampled by Dutch and Canadian personal dust samplers. RESULTS: Negative exposure-response relations were shown for regressions of FEV1 on cumulative and current exposure and years employed. Slopes of the exposure-response relations differed by a factor of three to five between industries, apart from results for cumulative exposure. Here the variation in slopes differed by a factor of 100, from -1 to -0.009 ml/mg.y/m3. The variation in slopes between industries reduced to between twofold to fivefold when the Dutch transfer elevator workers were not considered. There was evidence that the small exposure-response slope found for this group is caused by misclassification of exposure and a strong healthy worker effect. Alternative, but less likely explanations for the variation in slopes were differences in exposure concentrations, composition of grain dust, exposure characteristics, and measurement techniques. CONCLUSION: In conclusion, this study showed moderately similar negative exposure-response relations for four different populations from different countries, despite differences in methods of exposure assessment and exposure estimation.

Possible role of a short extra loop of the long-chain flavodoxin from Azotobacter chroococcum in electron transfer to nitrogenase: complete 1H, 15N and 13C backbone assignments and secondary solution structure of the flavodoxin.

The 1H, 15N and 13C backbone and 1H and 13C beta resonance assignments of the long-chain flavodoxin from Azotobacter chroococcum (the 20-kDa nifF product, flavodoxin-2) in its oxidized form were made at pH 6.5 and 30 degrees C using heteronuclear multidimensional NMR spectroscopy. Analysis of the NOE connectivities, together with amide exchange rates, 3JHNH alpha coupling constants and secondary chemical shifts, provided extensive solution secondary structure information. The secondary structure consists of a five-stranded parallel beta-sheet and five alpha-helices. One of the outer regions of the beta-sheet shows no regular extended conformation, whereas the outer strand beta 4/6 is interrupted by a loop, which is typically observed in long-chain flavodoxins. Two of the five alpha-helices are nonregular at the N-terminus of the helix. Loop regions close to the FMN are identified. Negatively charged amino acid residues are found to be mainly clustered around the FMN, whereas a cluster of positively charged residues is located in one of the alpha-helices. Titration of the flavodoxin with the Fe protein of the A. chroococcum nitrogenase enzyme complex revealed that residues Asn11, Ser68 and Asn72 are involved in complex formation between the flavodoxin and Fe protein. The interaction between the flavodoxin and the Fe protein is influenced by MgADP and is of electrostatic nature.

Conversion of phenol derivatives to hydroxylated products by phenol hydroxylase from Trichosporon cutaneum. A comparison of regioselectivity and rate of conversion with calculated molecular orbital substrate characteristics.

This study describes the regioselective hydroxylation and the rates of conversion of a series of fluorinated phenol derivatives by phenol hydroxylase from the yeast Trichosporon cutaneum. The natural logarithm of the kcat value for the conversion of the phenolic substrates correlates with the calculated energy of the reactive electrons in the highest occupied molecular orbital of the substrate (r = 0.85). This observation supports the hypothesis that at physiological pH (7.6) and 25 degrees C, in the absence of monovalent anions, the nucleophilic attack of the electrons in the highest occupied molecular orbital of the substrate on the C(4a)-hydroperoxyflavin enzyme intermediate is of major importance in determining the overall rate of catalysis. Results from 19F-NMR analysis of the incubation mixtures demonstrate for phenols with two identical ortho substituents, that the ortho position which becomes preferentially hydroxylated is the one with the highest density of the reactive electrons in the highest occupied molecular orbital. A halogen substituent at a meta position decreases the chances for hydroxylation at the adjacent ortho position further than expected on the basis of the calculated reactivity. This result indicates a contribution of a protein/substrate dipolar interaction, influencing the time-averaged orientation of the substrate with respect to the reactive C(4a)-hydroperoxyflavin intermediate.

Two-dimensional NMR studies of the flavin binding site of Desulfovibrio vulgaris flavodoxin in its three redox states.

The riboflavin 5'-monophosphate (FMN) binding site of Desulfovibrio vulgaris flavodoxin in the diamagnetic oxidized and two-electron reduced form was investigated using two-dimensional proton NMR. The NMR results are compared to existing X-ray crystallographic data. In the paramagnetic one-electron reduced redox state resonances of protons which are close to the FMN ring are strongly broadened due to the paramagnetic properties of the flavin ring. From comparison of the NMR spectra of the three redox states it could be concluded that outside the FMN binding site no structural changes occur upon reduction. Strong hydrogen bonds are observed between the N(1) and C(2) carbonyl of the isoalloxazine ring and the amide protons of D95 and C102, respectively. The amide resonances of D95 and C102 are strongly downfield shifted upon two-electron reduction, caused by the negative charge in the N(1)-C(2) carbonyl region in the two-electron reduced FMN. It is suggested that the ring current of the central pyrazine ring of the FMN molecule in the two-electron reduced flavodoxin is decreased compared to the oxidized flavodoxin. The decrease in ring current is apparently caused by the loss of aromaticity of this pyrazine moiety due to protonation of N(5). Strong hydrogen bonds between the flavin phosphate group and amide and hydroxyl protons of the apoprotein are observed. Resonances of protons involved in this hydrogen bonding network are downfield shifted up to 3.5 ppm. It is suggested that the negative charges of the dianionic FMN phosphate group are stabilized by local peptide dipoles. On reduction of the protein from the oxidized to the one-electron reduced form, a conformational change occurs in the FMN binding region. No conformational change can be observed between the one-electron and the two-electron reduced state.

Molecular orbital-based quantitative structure-activity relationship for the cytochrome P450-catalyzed 4-hydroxylation of halogenated anilines.

The cytochrome P450 (P450) catalyzed 4-hydroxylation of halogenated anilines was investigated with special emphasis on possible relationships between kinetic parameters and physicochemical and electronic characteristics of the substrates. The most important observation of the present study was a correlation (r = 0.96) between the natural logarithm of the apparent maximum reaction rate kcats for 4-hydroxylation of the aniline substrates in a iodosobenzene-supported microsomal cytochrome P450-catalyzed reaction and the energy of the highest molecular orbital [E(HOMO)] of the anilines. This result is in accordance with a mechanism that proceeds by an initial electrophilic attack of the P450 (FeO)3+ intermediate on the frontier pi electrons of the aniline substrates. In the iodosobenzene-supported aniline 4-hydroxylation this electrophilic attack is the rate-limiting step. In the NADPH/oxygen-supported cytochrome P450-catalyzed 4-hydroxylation of the anilines a correlation of the natural logarithm of kcats with E(HOMO) was not observed and the kcats values were lower than observed in the iodosobenzene-supported reaction. From this result it is concluded that, although the NADPH/oxygen-supported microsomal 4-hydroxylation of the halogenated anilines proceeds by the same cytochrome P450 (FeO)3+ intermediate and, thus, by a similar electrophilic attack of the (FeO)3+ on the pi electrons of the substrate, this attack is no longer the rate-limiting step of the reaction. Additional results of the present study demonstrate that the apparent Michaelis constant Kms of the NADPH/oxygen-supported 4-hydroxylation of the anilines decreases with increasing hydrophobicity of the aniline derivatives.(ABSTRACT TRUNCATED AT 250 WORDS)

19F-NMR study on the pH-dependent regioselectivity and rate of the ortho-hydroxylation of 3-fluorophenol by phenol hydroxylase from Trichosporon cutaneum. Implications for the reaction mechanism.

The regioselectivity and rate of the ortho-hydroxylation of 3-fluorophenol by phenol hydroxylase from Trichosporon cutaneum (EC 1.14.13.7) was studied using 19F-NMR. The regioselective hydroxylation as well as the rate of ortho-hydroxylation are pH dependent with a pKa of 6.5. At pH values below 6.5, 3-fluorophenol preferentially becomes hydroxylated at the C6 ortho position, resulting in a maximum C6/C2 hydroxylation ratio of 6.7. Upon increasing the pH, the total rate of conversion increases. Also, the C2 ortho-hydroxylation increases relatively to the C6 ortho-hydroxylation and yields a minimum C6/C2 hydroxylation ratio of 2.2 at pH values above 7.5. Based on data from 19F-NMR binding studies and molecular orbital calculations, a hypothesis is put forward which explains the pH-dependent effects observed. A mechanism is proposed involving an active-site amino acid residue acting as a base in the reduced form of the protein. Deprotonation of this residue results in hydrogen bond formation with the hydroxyl moiety of the phenolic substrate, leading to (partial) deprotonation of the substrate. Molecular orbital calculations demonstrate that such a (partial) deprotonation increases (a) the overall reactivity of 3-fluorophenol for an electrophilic attack and (b) the reactivity of C2 relative to the C6 position. The hypothesis may explain the decrease in the C6/C2 hydroxylation ratio. Furthermore the increased amount of ortho-hydroxylated products formed with increasing pH can also be explained by this hypothesis.

Stoichiometry, selectivity, and exchange dynamics of lipid-protein interaction with bacteriophage M13 coat protein studied by spin label electron spin resonance. Effects of protein secondary structure.

Bacteriophage M13 major coat protein has been isolated with cholate and reconstituted in dimyristoyl- and dioleoylphosphatidylcholine (DMPC and DOPC, respectively) bilayers by dialysis. Fourier transform infrared spectra of DMPC/coat protein recombinants confirmed that, whereas the protein isolated by phenol extraction was predominantly in a beta-sheet conformation, the cholate-isolated coat protein contained a higher proportion of the alpha-helical conformation [cf. Spruijt, R. B., Wolfs, C. J. A. M., & Hemminga, M. A. (1989) Biochemistry 28, 9158-9165]. The cholate-isolated coat protein/lipid recombinants gave different electron spin resonance (ESR) spectral line shapes of incorporated lipid spin labels, as compared with those from recombinants with the phenol-extracted protein that were studied previously [Wolfs, C. J. A. M., Horváth, L. I., Marsh, D., Watts, A., & Hemminga, M. A. (1989) Biochemistry 28, 9995-10001]. Plots of the ratio of the fluid/motionally restricted components in the ESR spectra of spin-labeled phosphatidylglycerol were linear with respect to the lipid/protein ratio in the recombinants up to 20 mol/mol. The corresponding values of the relative association constants, Kr, and number of association sites, N1, on the protein were Kr approximately 1 and N1 approximately 4 for DMPC recombinants and Kr approximately 1 and N1 approximately 5 for DOPC recombinants. Simulation of the two-component lipid spin label ESR spectra with the exchange-coupled Bloch equations gave values for the off-rate of the lipids leaving the protein surface of 2.0 x 10(7) s-1 at 27 degrees C in DMPC recombinants and 3.0 x 10(7) s-1 at 24 degrees C in DOPC recombinants.(ABSTRACT TRUNCATED AT 250 WORDS)