Performance of 133 compounds in the lambda prophage induction endpoint of the Microscreen assay and a comparison with S. typhimurium mutagenicity and rodent carcinogenicity assays.

The Microscreen assay was developed as a means of testing very small samples, as in complex mixture fractionation. It is a multi-endpoint assay which utilizes E. coli WP2s(lambda). Exposure takes place to serial dilutions of the test compound in microtitre wells (250 microliters) followed by sampling from wells in which growth has occurred ('non-toxic wells'). Although a number of different endpoints can be measured, only the prophage induction endpoint (the first one developed) has been extensively tested. Results with 133 compounds are presented. These include 111 compounds which have been tested in the S. typhimurium assay and 66 compounds for which both rodent bioassay and S. typhimurium assay data exists. The concordance for the Microscreen assay and the S. typhimurium assay was 71%. For this group of compounds, the sensitivity of the Microscreen assay in detecting carcinogens was 76% compared with 58% for the S. typhimurium assay. However, the S. typhimurium assay was somewhat more specific (69%) compared with the Microscreen (56%). The overall association between carcinogenicity and Microscreen results was statistically significant (p = 0.029), whereas for the S. typhimurium assay the association with carcinogenicity was non-significant (p = 0.086). The Microscreen assay was able to detect halogenated compounds better than the S. typhimurium assay. The Microscreen assay should prove useful in complex mixture fractionation, or in other situations where sample size is limiting.     

Automated microanalysis of adenosine phosphates, phosphocreatine, creatine, and lactate in muscle

An automated enzymatic procedure suitable for determination of ATP, ADP, AMP, phosphocreatine, creatine, and lactate in needle biopsies of human skeletal muscle (ca. 30 mg dry wt) using a fast centrifugal analyzer (Multistat III, Instrumentation Laboratory Inc.) is presented. Coefficients of variation ranged from 0.7 to 4.2% for multiple determinations of ATP, ADP, phosphocreatine, and creatine; from 6 to 24% for lactate; and from 9 to 20% for AMP. The procedure should be usable, with appropriate modification, with other tissues and with other fast centrifugal analyzers. Muscle samples are collected into liquid freon, lyophilized, and extracted with 600 microliter of 0.65 M perchloric acid. Neutralized supernatants can be stored for up to 3 years at -80 degrees C with no significant deterioration. The procedure takes much less time than similar manual procedures and gives better reproducibility, particularly for ADP and AMP.     

Characterization of compost biofiltration system degrading dichloromethane

The effects of acclimatization of microbial populations, compound concentration, and media pH on the biodegradation of low concentration dichloromethane emissions in biofiltration systems was evaluated. Greater than 98% removal efficiency was achieved for dichloromethane at superficial velocities from 1 to 1.5 m(3)/m(3). min (reactor residence times of 1 and 0.7 min, respectively) and inlet concentrations of 3 and 50 ppm Although acclimatization of microbial populations to toluene occurred within 2 weeks of operation start-up, initial dichloromethane acclimatization took place over a period of 10 weeks. This period was shortened to 10 days when a laboratory grown consortium of dichloromethane degrading organism, isolated from a previously acclimatized column, was introduced into fresh biofilter media. The mixed culture consisted to 12 members, which together were able to degrade dichloromethane at concentrations up to 500 mg/L. Only one member of the consortium was able to degrade dichloromethane were sustained for more than 4 months in a biofilter column receiving an inlet gas stream with 3 ppm(v) of dichloromethane acidification of the column and resulting decline in performance occurred when a 50-ppm(v) inlet concentration was used. A biofilm model incorporating first order biodegradation kinetics provided a good fit to observed concentration profiles, and may prove to be a useful tool for designing biofiltration systems for low concentration VOC emissions. (c) 1994 John Wiley & Sons, Inc.     

Cloning of a higher-plant plastid omega-6 fatty acid desaturase cDNA and its expression in a cyanobacterium.

Oligomers based on amino acids conserved between known plant omega-3 and cyanobacterium omega-6 fatty acid desaturases were used to screen an Arabidopsis cDNA library for related sequences. An identified clone encoding a novel desaturase-like polypeptide was used to isolate its homologs from Glycine max and Brassica napus. The plant deduced amino acid sequences showed less than 27% similarity to known plant omega-6 and omega-3 desaturases but more than 48% similarity to cyanobacterial omega-6 desaturase, and they contained putative plastid transit sequences. Thus, we deduce that the plant cDNAs encode the plastid omega-6 desaturase. The identity was supported by expression of the B. napus cDNA in cyanobacterium. Synechococcus transformed with a chimeric gene that contains a prokaryotic promoter fused to the rapeseed cDNA encoding all but the first 73 amino acids partially converted its oleic acid fatty acid to linoleic acid, and the 16:1(9c) fatty acid was converted primarily to 16:2(9c, 12) in vivo. Thus, the plant omega-6 desaturase, which utilizes 16:1(7c) in plants, can utilize 16:1(9c) in the cyanobacterium. The plastid and cytosolic homologs of plant omega-6 desaturases are much more distantly related than those of omega-3 desaturases.     

Cloning of higher plant omega-3 fatty acid desaturases.

Arabidopsis thaliana T-DNA transformants were screened for mutations affecting seed fatty acid composition. A mutant line was found with reduced levels of linolenic acid (18:3) due to a T-DNA insertion. Genomic DNA flanking the T-DNA insertion was used to obtain an Arabidopsis cDNA that encodes a polypeptide identified as a microsomal omega-3 fatty acid desaturase by its complementation of the mutation. Analysis of lipid content in transgenic tissues demonstrated that this enzyme is limiting for 18:3 production in Arabidopsis seeds and carrot hairy roots. This cDNA was used to isolate a related Arabidopsis cDNA, whose mRNA is accumulated to a much higher level in leaf tissue relative to root tissue. This related cDNA encodes a protein that is a homolog of the microsomal desaturase but has an N-terminal extension deduced to be a transit peptide, and its gene maps to a position consistent with that of the Arabidopsis fad D locus, which controls plastid omega-3 desaturation. These Arabidopsis cDNAs were used as hybridization probes to isolate cDNAs encoding homologous proteins from developing seeds of soybean and rapeseed. The high degree of sequence similarity between these sequences suggests that the omega-3 desaturases use a common enzyme mechanism.     

Pyridine nucleotide cycle of Salmonella typhimurium: in vitro demonstration of nicotinamide mononucleotide deamidase and characterization of pnuA mutants defective in nicotinamide mononucleotide transport.

The enzyme nicotinamide mononucleotide deamidase, an integral component of the proposed four-membered pyridine nucleotide cycle (PNC IV), has been demonstrated in extracts of Salmonella typhimurium LT2. The enzyme has an optimum pH of 8.7 and deamidates nicotinamide mononucleotide, forming nicotinic acid mononucleotide. Sigmoidal kinetic data suggest that this enzyme may be allosteric and therefore an important regulatory component of pyridine nucleotide cycle metabolism. Mutants previously designated pncC in anticipation of their lacking nicotinamide mononucleotide deamidase were examined and found to have normal levels of this enzyme. [14C]nicotinamide mononucleotide uptake studies, however, revealed a defect in the transport of this compound. Accordingly, the genetic designation for this locus was changed to pnuA to reflect its involvement in pyridine nucleotide uptake. Evidence is presented for the existence of two separate nicotinamide mononucleotide transport systems.     

Molecular evolution of the maize sex-determining gene TASSELSEED2 in Bouteloua (Poaceae)

The majority of angiosperms produce hermaphrodite flowers, while a lesser number (20–30%) produce unisexual flowers. Little is known about the molecular biology of sex-determination in angiosperms, however, a few sex-determining genes have been cloned from the model system Zea mays. One of these genes is Tasselseed2 (Ts2) which has been shown to be involved in the arrest of developing pistils in male flowers. In this study, we sequenced a putative homologue of Ts2 in species of Bouteloua, a genus in the grass subfamily Chloridoideae. We found significant genetic variation at Ts2 in Bouteloua relative to other developmental genes characterized in maize and other grass species. We also found that in Bouteluoua, Ts2 is evolving non-neutrally in the hermaphrodite-flowered Bouteloua hirsuta while no difference from neutral expectation was detected at Ts2 in the monoecious/dioecious Bouteloua dimorpha. The putatively neutral gene Alcohol Dehydrogenase1 (Adh1) was also examined for the same species of Bouteloua, and no departure from neutral expectation was detected. Our results suggest that purifying selection may be acting on Ts2 in the hermaphrodite-flowered B. hirsuta while no evidence of selection was detected at Ts2 in the monoecious/dioecious B. dimorpha.     

Calcium as a branching signal in Neurospora crassa

The divalent cation ionophore A23187 was found to induce apical branching in Neurospora crassa. Optimal effects were obtained by treatment with 0.1 mM ionophore for 30 min. Branching first became manifest during or shortly after treatment; successive rounds of branching could be observed at later times. Calcium starvation of the mycelium markedly reduced its subsequent response to the ionophore, whereas starvation for other divalent cations had no detectable effect. The branching response was markedly reduced in the presence of 10 to 30 mM cyclic AMP or derivatives thereof.