Selective suppression of the catalytic activity of cDNA-expressed cytochrome P4502B1 toward polycyclic hydrocarbons in the microsomal membrane: modification of this effect by specific amino acid substitutions.

Human hepatoma HEPG2 cells were infected with recombinant vaccinia virus vectors containing cDNAs encoding both known and variant rat cytochromes P450 (CYP). CYP2B1 and CYP2B2 cytochromes were equally well expressed (110-140 pmol/mg of microsomal protein) and catalyzed metabolism of 7,12-dimethylbenz[a]anthracene (DMBA). Their regioselectivity for DMBA metabolism paralleled that of the respective purified rat liver enzymes and reproduced previously reported regioselective differences between CYP2B1 and CYP2B2 [Wilson et al. (1984) Carcinogenesis 5, 1475-1483]. CYP2A1 and CYP2A2 expressed in HEPG2 microsomes exhibited nearly equal DMBA-metabolizing activities that closely matched that of purified CYP2A1. Although purified rat liver CYP2B1 was 3 times more active than purified rat liver CYP2B2, the expressed recombinant microsomal CYP2B1 (rCYP2B1) was 20 times less active than rCYP2B2, where activity matched that of the purified cytochrome. Microsomal suppression of rCYP2B1 catalytic activity was also observed for benzo[a]pyrene. Specific amino acid substitutions at equivalent positions of the completely homologous NH2-terminal halves of rCYP2B1 and rCYP2B2 changed this suppression effect. Thus, a L58----F, I114----F double mutant exhibited 3 times the normal activity for rCYP2B1 while remaining inhibitory for rCYP2B2. The single substitutions produced very different effects. The L58----F substitution prevented expression of rCYP2B1, while the I114----F substitution was inhibitory for both rCYP2B1 and rCYP2B2 (40 and 70%). A single E282----V mutation produced a stimulation of rCYP2B1 activity comparable to that of the L58----F, I114----F double substitution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Site-directed mutagenesis of cytochrome P450s CYP2A1 and CYP2A2: influence of the distal helix on the kinetics of testosterone hydroxylation.

Cytochrome P450s CYP2A1 and CYP2A2 exhibit 88% sequence similarity, yet CYP2A1 metabolizes testosterone almost exclusively (90%) at the 7 alpha-position, whereas CYP2A2 forms several metabolites, with 15 alpha-hydroxytestosterone as a major metabolite. One of the regions with relatively low sequence homology corresponds by sequence alignment to the I and J helices of P450cam. Since this region is known to be part of the active site for P450cam, 26 single point and two double point mutants were prepared where the amino acid for one form was substituted with that of the other. Mutant and wild-type enzymes were expressed in Hep G2 cells using the vaccinia virus vector. Analysis of testosterone regioselectivity revealed that 25 of the mutants show the same regioselectivity as the parent wild-type enzymes and three are inactive, suggesting that no single amino acid in this region is totally responsible for the different selectivities of CYP2A1 and CYP2A2. Kinetic analysis of the CYP2A1 mutants showed that four of the mutants with changes near the conserved oxygen-binding region had Km values with much higher and Vmax values much lower than those of the wild-type enzyme and one mutant had a Vmax value twice as high as that of the wild-type enzyme. Deuterium isotope effects on 7 alpha-hydroxxylation were used to determine changes in the rate of reduction and estimate the relative amount of excess water formation. Changes in reduction rates and the amount of water produced are not sufficient to account for the differences in Vmax values, suggesting that the amount of hydrogen peroxide released is a primary determinant for changes in Vmax.     

Compensation of respiratory alkalosis induced after acclimation to simulated altitude

Conscious intact rats previously acclimated for 3 wk to barometric pressure of 370-380 Torr (3WHx) were made alkalotic for 3 h by a decrease in inspired O2 fraction from 0.10 to 0.075 at ambient barometric pressure (730-740 Torr). Controls were normoxic littermates (Nx) in which inspired O2 fraction was lowered from approximately 0.21 to 0.10 for 3 h. Arterial PCO2 decreased progressively and similarly in both groups (65-70% of control at 15 min). Initially, arterial pH increased less in 3WHx (0.09 +/- 0.004 vs. 0.15 +/- 0.008). As hypocapnia continued, delta[HCO3-]/delta pH (mmol.l-1.pH) became more negative in Nx, from -15.2 +/- 2.5 at 15 min to -37.0 +/- 2.9 at 3 h, indicating nonrespiratory compensation of alkalosis. In 3WHx, delta[HCO3-]/delta pH did not change during alkalosis. Cumulative renal excretion of base (mueq/100 g) during alkalosis increased by 73.2 +/- 11.1 in Nx and 25.4 +/- 7.3 in 3WHx. This difference was mainly due to a larger increase in HCO3- excretion in Nx. The data suggest that the smaller compensation of hypocapnic alkalosis in 3WHx is partly due to the smaller increase in renal base excretion. Because base availability limits renal base excretion, the smaller renal response of 3WHx may be secondary to the low plasma HCO3- concentration that accompanies altitude acclimation.     

Clean technology in aquaculture — a production without waste products?

A method has been developed for the determination of H₂S and FeS in sediments. FeS is converted into H₂S which is flushed from the samples directly into an excess of chlorine bleach, NaC1O or KClO with some Zn²⁺ added. Either the excess can be titrated back potentiometrically with As₂O₃, or the sulphate formed can be measured colorimetrically. The precision is primarily controlled by the homogeneity of the sediment suspensions and can be better than 99%.     

Studies of the Inheritance of Human Ribosomal DNA Variants Detected in Two-Dimensional Separations of Genomic Restriction Fragments

We have investigated the variation in human ribosomal DNA repeat units as revealed in two-dimensional electrophoretic separations of genomic restriction fragments that were end-labeled at NotI cleavage sites. The transcribed portion of the ribosomal DNA results in ~20 labeled fragments visible on each gel as multicopy spots. We have mapped these spots to the sequences responsible for their appearance on the gels, based on their migration positions and direct sequencing of spots, and describe several previously unreported sources of variation. By studying mother/father/child families we gained information on how much of the between-repeats variation is due to differences between and within repeat arrays on homologous chromosomes. Two instances in which a child exhibited more copies of a particular fragment than were present in the parents are described and hypothesized to be due to events such as multiple unequal sister-chromatid exchanges or gene conversions.     

Daphnids respond to algae-associated odours

Using a Y-tube olfactometer, it was found that Daphnia galeatax hyalina moves to the arm with odour from either of two ediblealgal species (Scenedesmus acuminatus and Oscillatoria limnetica)rather than to the alternative arm with clean water. However,no differential response was observed when odours of the toxiccyanobacterium (Oscillatoria agardhu) were tested. We suggestthat odours associated with edible algae attract Daphnia whereasnon-edible algae do not elicit attraction of Daphnia.     

Grain yield, symbiotic N2 fixation and interspecific competition for inorganic N in pea-barley intercrops

The effect of mixed intercropping of field pea (Pisum sativum L.) and spring barley (Hordeum vulgare L.), compared to monocrop cultivation, on the yield and crop-N dynamics was studied in a 4-yr field experiment using ¹⁵N-isotope dilution technique. Crops were grown with or without the supply of 5 g ¹⁵N-labeled N m^-2. The effect of intercropping on the dry matter and N yields, competition for inorganic N among the intercrop components, symbiotic fixation in pea and N transfer from pea to barley were determined. As an average of four years the grain yields were similar in monocropped pea, monocropped and fertilized barley and the intercrop without N fertilizer supply. Nitrogen fertilization did not influence the intercrop yield, but decreased the proportion of pea in the yield. Relative yield totals (RYT) showed that the environmental sources for plant growth were used from 12 to 31% more efficiently by the intercrop than by the monocrops, and N fertilization decreased RYT-values. Intercrop yields were less stable than monocrop barley yields, but more stable than the yield of monocropped pea. Barley competed strongly for soil and fertilizer N in the intercrop, and was up to 30 times more competitive than pea for inorganic N. Consequently, barley obtained a more than proportionate share of the inorganic N in the intercrop. At maturity the total recovery of fertilizer N was not significantly different between crops, averaging 65% of the supplied N. The fertilizer N recovered in pea constituted only 9% of total fertilizer-N recovery in the intercrop. The amount of symbiotic N₂ fixation in the intercrop was less than expected from its composition and the fixation in monocrop. This indicates that the competition from barley had a negative effect on the fixation, perhaps via shading. At maturity, the average amount of N₂ fixation was 17.7 g N m^-2 in the monocrop and 5.1 g N m^-2 in the intercropped pea. A higher proportion of total N in pea was derived from N₂ fixation in the intercrop than in the monocrop, on average 82% and 62%, respectively. The ¹⁵N enrichment of intercropped barley tended to be slightly lower than of monocropped barley, although not significantly. Consequently, there was no evidence for pea N being transferred to barley. The intercropping advantage in the pea-barley intercrop is mainly due to the complimentary use of soil inorganic and atmospheric N sources by the intercrop components, resulting in reduced competition for inorganic N, rather than a facilitative effect, in which symbiotically fixed N₂ is made available to barley.Abbreviations MC monocrop - IC intercrop - PMC pea monocrop - BMC barley monocrop - PIC pea in intercrop - BIC barley in intercrop     

Inheritance and genetic mapping of resistance to Alternaria alternata f. sp. lycopersici in Lycopersicon pennellii

The fungal pathogen Alternaria alternata f. sp. lycopersici produces AAL-toxins that function as chemical determinants of the Alternaria stem canker disease in the tomato (Lycopersicon esculentum). In resistant cultivars, the disease is controlled by the Asc locus on chromosome 3. Our aim was to characterize novel sources of resistance to the fungus and of insensitivity to the host-selective AAL-toxins. To that end, the degree of sensitivity of wild tomato species to AAL-toxins was analyzed. Of all members of the genus Lycopersicon, only L. cheesmanii was revealed to be sensitive to AAL-toxins and susceptible to fungal infection. Besides moderately insensitive responses from some species, L. pennellii and L. peruvianum were shown to be highly insensitive to AAL-toxins as well as resistant to the pathogen. Genetic analyses showed that high insensitivity to AAL-toxins from L. pennellii is inherited in tomato as a single complete dominant locus. This is in contrast to the incomplete dominance of insensitivity to AAL-toxins of L. esculentum. Subsequent classical genetics, RFLP mapping and allelic testing indicated that high insensitivity to AAL-toxins from L. pennellii is conferred by a new allele of the Asc locus.