Gene Coexpression Networks Drive and Predict Reproductive Effects in Daphnia in Response to Environmental Disturbances.

Increasing effects of anthropogenic stressors and those of natural origin on aquatic ecosystems have intensified the need for predictive and functional models of their effects. Here, we use gene expression patterns in combination with weighted gene coexpression networks and generalized additive models to predict effects on reproduction in the aquatic microcrustacean Daphnia. We developed models to predict effects on reproduction upon exposure to different cyanobacteria, different insecticides and binary mixtures of cyanobacteria and insecticides. Models developed specifically for groups of stressors (e.g., either cyanobacteria or insecticides) performed better than general models developed on all data. Furthermore, models developed using in silico generated mixture gene expression profiles from single stressor data were able to better predict effects on reproduction compared to models derived from the mixture exposures themselves. Our results highlight the potential of gene expression data to quantify effects of complex exposures at higher level organismal effects without prior mechanistic knowledge or complex exposure data.

Single toxin dose-response models revisited.

The goal of this paper is to offer a rigorous analysis of the sigmoid shape single toxin dose-response relationship. The toxin efficacy function is introduced and four special points, including maximum toxin efficacy and inflection points, on the dose-response curve are defined. The special points define three phases of the toxin effect on mortality: (1) toxin concentrations smaller than the first inflection point or (2) larger then the second inflection point imply low mortality rate, and (3) concentrations between the first and the second inflection points imply high mortality rate. Probabilistic interpretation and mathematical analysis for each of the four models, Hill, logit, probit, and Weibull is provided. Two general model extensions are introduced: (1) the multi-target hit model that accounts for the existence of several vital receptors affected by the toxin, and (2) model with a nonzero mortality at zero concentration to account for natural mortality. Special attention is given to statistical estimation in the framework of the generalized linear model with the binomial dependent variable as the mortality count in each experiment, contrary to the widespread nonlinear regression treating the mortality rate as continuous variable. The models are illustrated using standard EPA Daphnia acute (48h) toxicity tests with mortality as a function of NiCl or CuSO4 toxin.

Functional characterization of four metallothionein genes in Daphnia pulex exposed to environmental stressors.

We characterized the metallothionein genes (Mt1, Mt2, Mt3, and Mt4) in Daphnia pulex on both molecular and ecotoxicological level. We therefore conducted a bioinformatical analysis of the gene location and predicted protein sequence, and screened the upstream flanking region for regulatory elements. The number of these elements and their positions relative to the start codon varied strongly among the four genes and even among two gene duplicates (Mt1A and Mt1B), suggesting different roles of the four proteins in the organisms' response to stress. We subsequently conducted a chronic 16-day exposure of D. pulex to different environmental stressors (at sublethal levels causing approximately 50% reduction in reproduction). Based on prior knowledge, we exposed them to the metals Cd, Cu, and Ni, the moulting hormone hydroxyecdysone (20E), and the oxidative stressors cyanobacteria (Microcystis aeruginosa), and paraquat (Pq). We then compared mRNA expression levels of the four Mt genes under these stress conditions with control conditions in "The Chosen One" clone (TCO), for which the full genome was sequenced and annotated. All together, the mRNA expression results under the different stress regimes indicate that different Mt genes may play different and various roles in the response of D. pulex to stress and that some (but not all) of the differences among the four genes could be related to the pattern of regulatory elements in their upstream flanking region.

Maize genes encoding the small subunit of ADP-glucose pyrophosphorylase.

Plant ADP-glucose pyrophosphorylase (AGP) is a heterotetrameric enzyme composed of two large and two small subunits. Here, we report the structures of the maize (Zea mays) genes encoding AGP small subunits of leaf and endosperm. Excluding exon 1, protein-encoding sequences of the two genes are nearly identical. Exon 1 coding sequences, however, possess no similarity. Introns are placed in identical positions and exhibit obvious sequence similarity. Size differences are primarily due to insertions and duplications, hallmarks of transposable element visitation. Comparison of the maize genes with other plant AGP small subunit genes leads to a number of noteworthy inferences concerning the evolution of these genes. The small subunit gene can be divided into two modules. One module, encompassing all coding information except that derived from exon 1, displays striking similarity among all genes. It is surprising that members from eudicots form one group, whereas those from cereals form a second group. This implies that the duplications giving rise to family members occurred at least twice and after the separation of eudicots and monocot cereals. One intron within this module may have had a transposon origin. A different evolutionary history is suggested for exon 1. These sequences define three distinct groups, two of which come from cereal seeds. This distinction likely has functional significance because cereal endosperm AGPs are cytosolic, whereas all other forms appear to be plastid localized. Finally, whereas barley (Hordeum vulgare) reportedly employs only one gene to encode the small subunit of the seed and leaf, maize utilizes the two genes described here.

Acute and chronic physiological effects of silver exposure in three marine teleosts.

This study evaluated the physiological effects of waterborne silver (added as AgNO(3)) on seawater fish, using acute (48-72 h) high level exposures (250-650 microg/l Ag) on tidepool sculpins (Oligocottus maculosus), and chronic (up to 21 day) low level exposures (1.5-50 microg/l Ag) on tidepool sculpins, plainfin midshipmen (Porichthys notatus), and rainbow trout (Oncorhynchus mykiss). Sculpins were tested at different salinities. Acclimation to lower salinity (18 vs 30 ppt) led to altered physiology, with higher ammonia excretion (J(Amm)), lower oxygen consumption, and lower branchial and intestinal Na(+)/K(+)-ATPase activities, but no difference in drinking rate. Short-term exposure to high silver levels tended to stimulate M(O(2)), J(Amm), and drinking rate. However, long-term exposure to low levels of silver depressed both J(Amm) and M(O(2)), and also led to decreased drinking rates. Both inhibition and stimulation of Na(+)/K(+)-ATPase activity occurred, dependent upon length and concentration of exposure, salinity (18 vs 30 ppt), tissue (gill vs intestine), and fish species (sculpin vs midshipmen vs rainbow trout). While the effects were variable, due to differing balances between inhibitory and compensatory responses, chronic silver exposure significantly altered Na(+)/K(+)-ATPase activity levels in almost all tests. In total, these findings reinforce the view that intestinal osmoregulatory function (drinking, Na(+)/K(+)-ATPase activity) is an important site of toxic impact for waterborne silver, that gill Na(+)/K(+)-ATPase activity is also a site of impact, and that chronic exposures at silver concentrations (1.5, 14.5 microg/l Ag) close to current or proposed water quality guidelines (albeit much higher than normal environmental levels), exert a variety of sublethal effects on marine teleosts.

Physiology of acute silver toxicity in the starry flounder (Platichthys stellatus) in seawater.

Physiological effects of exposure to silver (AgClnn-1; 250 micrograms Ag l-1 or 1000 micrograms Ag l-1) in seawater fish were investigated using adult starry flounders. While all fish survived up to 10 days in 250 micrograms Ag l-1, flounders started to die after day 4 in 1000 micrograms l-1. Dose-dependent increases in plasma and hepatic silver concentrations showed that silver was available for uptake. There were minimal negative effects on hematological parameters, acid-base status, and blood gases. Plasma ammonia showed a pronounced (three- to four-fold), but transient increase in flounders exposed to either 250 micrograms Ag l-1 or 1000 micrograms Ag l-1. Whole body ammonia and acid equivalent efflux measurements indicated that ammonia retention was due to a combination of stimulated production and inhibited excretion. In the 1000-microgram Ag l-1 group there was a similar transient increase in plasma [magnesium], which was restored by day 4. In contrast, plasma chloride and sodium levels increased gradually towards the point when fish began to die. At 250 micrograms Ag l-1, the Na+/K(+)-ATPase activity of the intestine was unaffected but there was a two-fold increase in branchial Na+/K(+)-ATPase activity. The latter effect was interpreted as compensation for an elevated chloride and sodium load. The increases in plasma chloride and sodium concentrations were accompanied by a marked suppression of drinking, thereby indicating that acute silver toxicity was likely caused by a combination of elevated electrolyte concentrations and dehydration.

A splice site mutant of maize activates cryptic splice sites, elicits intron inclusion and exon exclusion, and permits branch point elucidation.

DNA sequence analysis of the bt2-7503 mutant allele of the maize brittle-2 gene revealed a point mutation in the 5' terminal sequence of intron 3 changing GT to AT. This lesion completely abolishes use of this splice site, activates two cryptic splice sites, and alters the splicing pattern from extant splice sites. One activated donor site, located nine nt 5' to the normal splice donor site, begins with the dinucleotide GC. While non-consensus, this sequence still permits both trans-esterification reactions of pre-mRNA splicing. A second cryptic site located 23 nt 5' to the normal splice site and beginning with GA, undergoes the first trans-esterification reaction leading to lariat formation, but lacks the ability to participate in the second reaction. Accumulation of this splicing intermediate and use of an innovative reverse transcriptase-polymerase chain reaction technique (J. Vogel, R.H. Wolfgang, T. Borner [1997] Nucleic Acids Res 25: 2030-2031) led to the identification of 3' intron sequences needed for lariat formation. In most splicing reactions, neither cryptic site is recognized. Most mature transcripts include intron 3, while the second most frequent class lacks exon 3. Traditionally, the former class of transcripts is taken as evidence for the intron definition of splicing, while the latter class has given credence to the exon definition of splicing.

Release of cytochrome c, Bax migration, Bid cleavage, and activation of caspases 2, 3, 6, 7, 8, and 9 during endothelial cell apoptosis.

Although the executioner phase of apoptosis has been well defined in many cell types, the subcellular events leading to apoptosis in endothelial cells remain undefined. In the current study, apoptosis was induced in primary human umbilical venous endothelial cells by the photosensitizer verteporfin and light. Release of mitochondrial cytochrome c into the cytosol was detectable immediately and accumulated over 2 hours after treatment while cytosolic levels of the proapoptotic Bcl-2 family member, Bax, decreased reciprocally over the same time period. Cleavage of another proapoptotic Bcl-2 family member, Bid, was observed by 2 hours after treatment. Although Bid cleavage has been shown to occur as an upstream event responsible for inducing cytochrome c release, we demonstrate that Bid cleavage can also occur after cytochrome c release. Activation of caspases 2, 3, 6, 7, 8, and 9 occurred following the release of cytochrome c, and cleavage of downstream substrates was observed. In summary, endothelial cell death involves the cellular redistribution of Bax and cytochrome c, followed by the activation of multiple caspases which manifest the apoptotic phenotype.

Effect of endodontic instrument handle diameter on operator performance.

A phantom head simulator was used by 30 dental students to determine the effect of instrument handle diameter on the time taken to complete a manipulative task. No significant differences were found for handles between 2.5 and 5 mm diameter. Almost one-third of the participants thought that gloves were a hindrance, and it was considered that "glove wrap," where layers of glove roll over one another, might be a problem with small handles. Despite these subjective reports, the students performed no better without gloves when using the 2.5 mm handle.

Structural features of the maize sus1 gene and protein.

Genomic clones, cDNA clones, and protein of the maize (Zea mays L.) Suc synthase1 (sus1) gene were isolated and sequenced. Termini (5' and 3') of the transcribed unit were identified. The SUS1 protein was purified from tissue culture cells as a phosphorylated protein. The overall structure of sus1 is virtually identical with that of the paralogous gene, shrunken1 (sh1); however, the last intron of sh1 is missing in sus1. This intron bears much sequence similarity with the adjacent exon, suggesting that the intron arose from an internal duplication. Although the placement of the other 14 introns is identical in both genes, the introns exhibit markedly greater differences in size and sequence relative to that shown by the exons. An explanation for the differential rate of divergence of exons and introns is selection pressure for gene function. Additionally, comparisons of coding regions of plant sucrose synthases show that sh1-like and sus1-like genes can be found in all monocots so far analyzed. These latter observations point to an important role played by both genes in this group of plants.

Tactile sensitivity of three endodontic instrument handles.

Three currently available endodontic instrument handles, introduced over the past four decades, were investigated for tactility. Larger handles have been promoted to overcome sensitivity loss from wearing gloves. Smaller handles were preferred in this experiment. While the largest diameter handle appeared to give enhanced tactility, significance was not reached in any of the tests.

Comparative study of the knowledge, attitudes, and behaviors of large animal veterinarians, dairy farmers, and dairy processors in Michigan on bovine somatotropin.

A cross-sectional epidemiologic study was conducted to evaluate two objectives. The first was to determine the knowledge, attitudes, and behaviors of large animal (dairy) veterinarians, dairy farmers, and dairy processors in Michigan toward the use of bovine somatotropin (BST). The second was to compare and contrast the data from the large animal veterinarians, dairy farmers, and dairy processors according to the knowledge, attitude, and behavior variables. A written questionnaire was used as an instrument to collect data relating to the objectives. Three basic types of questions were asked of each group: (1) how much do you know about BST? (knowledge-based question), (2) how do you feel about BST? (attitude-based question), and (3) how would you use BST or use dairy products produced through BST biotechnology (behavior-based question)?

The cloning, genetic mapping, and expression of the constitutive sucrose synthase locus of maize.

Two differentially expressed genes encode isoenzymes of sucrose synthase in Zea mays. A clone of the shrunken 1 (Sh1) locus, the structural gene for the major endosperm form of sucrose synthase, was used to isolate a genomic clone of constitutive sucrose synthase (Css), the structural gene for the isoenzyme expressed in embryo and other tissues. The Css clone was positively identified by RNA blot analysis of RNA from wild type and a sh1 deletion stock and by analysis of the in vitro translation product of hybrid-selected mRNA. Southern blot analysis of DNA from monosomic plants derived from an r-x1 stock, coupled with restriction fragment length polymorphism mapping, placed the Css gene 32 map units from Sh1 on chromosome 9. In seedling tissues, Css mRNA is present at higher levels than Sh1 mRNA. Expression of both Sh1 and Css in root tissue is enhanced by anaerobic conditions, although Css is induced to a lesser extent than is Sh1. Thus, Css appears to be expressed constitutively, whereas Sh1 is expressed at high levels only in response to specific developmental and environmental stimuli.

An alteration in tubulin expression detected in SV40 transformed 3T3 cells.

Tubulin expression was analysed in normal and simian virus-40 (SV40) transformed 3T3 cells by two-dimensional polyacrylamide gel electrophoresis and immunoblotting studies using monoclonal antibodies raised to alpha- and beta-tubulin subunits. The ratio of alpha- to beta-tubulin recognised was calculated for both cell lines and found to shift from 2.50 in normal cells to 0.52 in virally transformed cells. beta-Tubulin was thereby shown to be the predominant subunit in SV40-transformed 3T3 cells in contrast to normal 3T3 cells.

Studies of sugars and sorbitol in developing corn kernels.

Sugars and sorbitol were determined on corn (Zea mays L.) kernels harvested at various developmental stages, using sugary (su), sugary-sugary enhancer (su se), and starchy (Su) cultivars. In all cultivars tested, the sorbitol content increased from trace amounts in unpollinated ovules to a maximum at about the time that rapid starch synthesis was proceeding. Thereafter, sorbitol and sugars decreased continuously to the mature dry stage. Sorbitol in the su se kernels was higher than that of other cultivars from 28 days postpollination onwards; sucrose and maltose were higher from 21 days onwards. [(14)C]Sorbitol was recovered from kernel base, pedicel, and endosperm of IL677a (su se) kernels after allowing a flag leaf to fix (14)CO(2) photosynthetically. No [(14)C]sorbitol was detected in the shank of the ear, and none was detected by the gas chromatograph. [(14)C]Sucrose was the predominant labeled substance recovered from the kernel base, pedicel, and endosperm tissues during the 10-h chase period, as well as from the shank of the ear, and nonradioactive sucrose was the predominant ethanol-soluble compound detected by the gas chromatograph. Hence, sorbitol appears not to be translocated from corn leaves as it is in certain woody plants of the rose family. The altered sugar profile of su se kernels may be related to reduced starch synthesis, but the biochemical mechanism is not yet known.

The treatment of experimental schistosomiasis with a combination of oxamniquine and praziquantel.

The object of the study was to identify any potential therapeutic advantage in using the two antischistosomal drugs, oxamniquine and praziquantel, in combination rather than separately, in the treatment of schistosomiasis. The efficacy of the drugs was studied in albino mice with mature Schistosoma mansoni infections. It was found that the efficacy of combinations of the two drugs was markedly superior to that expected on the basis of a simple additive effect. The significance of this synergistic action is discussed in the context of schistosomiasis therapy in man.