Validation of 1,3-butadiene exposure estimates for workers at a synthetic rubber plant

PURPOSE: This investigation assessed the validity of estimates of exposure to 1,3-butadiene (BD) developed for a plant included in a study of mortality among synthetic rubber industry workers. The estimates were developed without using historical measurement data and have not been validated previously. METHODS: Personal BD measurements came from an exposure-monitoring program initiated in 1977. For each job, we computed the year-specific difference between the BD estimate and the mean of BD measurements. We also computed rank correlation coefficients and calculated the mean, across all measurements, of the difference between the estimate and the measurement. RESULTS: The mean BD concentration was 5.2 ppm for 4978 measurements and 4.7 ppm for the corresponding estimates. The mean difference between estimates and measurements was -0.50 ppm (standard deviation, 26.5 ppm) overall and ranged from -227.9 to +27.0 ppm among all 306 job/year combinations. Estimates were correlated with measurements for all 306 combinations (rank correlation coefficient, r=0.45, p<0.0001), for 82 combinations pertaining to jobs that were well-defined by a specific set of tasks and typically found in styrene-BD rubber (SBR) plants (r=0.81, p<0.0001), for 70 combinations pertaining to jobs that were well-defined but not typical (r=0.29, p=0.01) and for 92 combinations pertaining to poorly-defined jobs typically found in SBR plants (r=0.56, <0.0001). Estimates were not correlated with measurements for poorly defined jobs not typically found in SBR plants (r=0.01, p=0.93). For well-defined typical SBR jobs with measurement means that were over 7.0 ppm, estimates were consistently lower than measurements. CONCLUSIONS: Possible reasons for differences between estimates and measurements included faulty assumptions used in developing BD estimates, unstable or nonrepresentive measurements and errors in linking measurement data to the job-exposure matrix. Exposure misclassification may have been more severe for subjects from the validation study plant than for subjects from other plants in the mortality study. BD estimates for typical SBR jobs, which comprise most operations at all but one of the plants in the mortality study, appeared to be useful for ranking workers by cumulative exposure. Uncertainty analyses would enhance the utility of the BD exposure estimates for quantitative risk assessment.     

Batch and continuous culture-based selection strategies for acetic acid tolerance in xylose-fermenting Saccharomyces cerevisiae

Acetic acid tolerance of Saccharomyces cerevisiae is crucial for the production of bioethanol and other bulk chemicals from lignocellulosic plant-biomass hydrolysates, especially at a low pH. This study explores two evolutionary engineering strategies for the improvement of acetic acid tolerance of the xylose-fermenting S. cerevisiae RWB218, whose anaerobic growth on xylose at pH 4 is inhibited at acetic acid concentrations >1 g L(-1) : (1) sequential anaerobic, batch cultivation (pH 4) at increasing acetic acid concentrations and (2) prolonged anaerobic continuous cultivation without pH control, in which acidification by ammonium assimilation generates selective pressure for acetic acid tolerance. After c. 400 generations, the sequential-batch and continuous selection cultures grew on xylose at pH≤4 with 6 and 5 g L(-1) acetic acid, respectively. In the continuous cultures, the specific xylose-consumption rate had increased by 75% to 1.7 g xylose g(-1) biomass h(-1) . After storage of samples from both selection experiments at -80 °C and cultivation without acetic acid, they failed to grow on xylose at pH 4 in the presence of 5 g L(-1) acetic acid. Characterization in chemostat cultures with linear acetic acid gradients demonstrated an acetate-inducible acetic acid tolerance in samples from the continuous selection protocol.     

BK channel beta1-subunit regulation of calcium handling and constriction in tracheal smooth muscle

The large-conductance, Ca2+-activated K+ (BK) channels are regulators of voltage-dependent Ca2+ entry in many cell types. The BK channel accessory beta1-subunit promotes channel activation in smooth muscle and is required for proper tone in the vasculature and bladder. However, although BK channels have also been implicated in airway smooth muscle function, their regulation by the beta1-subunit has not been investigated. Utilizing the gene-targeted mice for the beta1-subunit gene, we have investigated the role of the beta1-subunit in tracheal smooth muscle. In mice with the beta1-subunit-knockout allele, BK channel activity was significantly reduced in excised tracheal smooth muscle patches and spontaneous BK currents were reduced in whole tracheal smooth muscle cells. Knockout of the beta1-subunit resulted in an increase in resting Ca2+ levels and an increase in the sustained component of Ca2+ influx after cholinergic signaling. Tracheal constriction studies demonstrate that the level of constriction is the same with knockout of the beta1-subunit and BK channel block with paxillin, indicating that BK channels contribute little to airway relaxation in the absence of the beta1-subunit. Utilizing nifedipine, we found that the increased constriction caused by knockout of the beta1-subunit could be accounted for by an increased recruitment of L-type voltage-dependent Ca2+ channels. These results indicate that the beta1-subunit is required in airway smooth muscle for control of voltage-dependent Ca2+ influx during rest and after cholinergic signaling in BK channels.     

Tolerance of switchgrass to extreme soil moisture stress: Ecological implications

Switchgrass (Panicum virgatum L.), a native of eastern and central North America, is a leading candidate as a dedicated biofuel feedstock in the US due to its broad adaptability, rapid growth rate, and ability to grow in low production soils. To begin to characterize the important agronomic and ecological traits related to environmental tolerance of switchgrass, we evaluated fitness under stressful growing conditions. We assessed the germination, establishment, performance, and reproductive potential of four common accessions, both upland and lowland ecotypes, at various levels of soil moisture availability (moisture deficit to flooded) in the greenhouse. Seeds emerged and established (55–90% survival) under all soil moisture conditions (−0.3 MPa to flooded). Transplants of lowland ecotypes performed as well in flooded conditions as in field capacity controls, though flooding reduced performance of upland ecotypes. Drought treatments (−4.0 and −11.0 MPa) reduced tiller length and number, leaf area, and biomass production by up to 80%. However, once established, all plants survived at −4.0 MPa and had the same proportion of tillers in flower as at field capacity. The ability of switchgrass to germinate, establish, and flower in low moisture and flooded conditions, particularly lowland ecotypes, may increase the range of environments suitable for biofuel cultivation, and can serve as a baseline for further ecological studies and genetic improvement.     

Novel lipoic acid analogues that inhibit nitric oxide synthase

The synthesis and biological activity of novel lipoic acid analogues are reported. Lipoic acid and structural homologues coupled to arylthiophene amidine via carboxamide linkers are metabolic antioxidants capable of protecting neuronal cells against glutamate cytotoxicity, preventing loss of intracellular glutathione, and inhibit nitric oxide synthase.     

ADP-glucose pyrophosphorylase from potato tuber: site-directed mutagenesis of homologous aspartic acid residues in the small and large subunits

Asp142 in the homotetrameric ADP-glucose pyrophosphorylase (ADP-Glc PPase) enzyme from Escherichia coli was demonstrated to be involved in catalysis of this enzyme [Frueauf, J.B., Ballicora, M.A. and Preiss J. (2001) J. Biol. Chem., 276, 46319-46325]. The residue is highly conserved throughout the family of ADP-Glc PPases, as well as throughout the super-family of sugar-nucleotide pyrophosphorylases. In the heterotetrameric ADP-Glc PPase from potato (Solanum tuberosum L.) tuber, the homologous residue is present in both the small (Asp145) and the large (Asp160) subunits. It has been proposed that the small subunit of plant ADP-Glc PPases is catalytic, while the large subunit is modulatory; however, no catalytic residues have been identified. To investigate the function of these conserved Asp residues in the ADP-Glc PPase from potato tuber, we used site-directed mutagenesis to introduce either an Asn or a Glu. Kinetic analysis in the direction of synthesis or pyrophosphorolysis of ADP-Glc showed a significant decrease (more than four orders of magnitude) in the specific activity of the SD145NLwt, SD145NLD160N, and SD145NLD160E mutants, while the effect was smaller (approximately two orders of magnitude) with the SD145ELwt, SD145ELD160N, and SD145ELD160E mutants. By contrast, mutation of the large subunit alone did not affect the specific activity but did alter the apparent affinity for the activator 3-phosphoglycerate, showing two types of apparent roles for this residue in the different subunits. These results show that mutation of Asp160 of the large subunit does not affect catalysis, thus the large subunit is not catalytic, and that the negative charge of Asp145 in the small subunit is necessary for enzyme catalysis.