A comparative evaluation of five hazard screening tools.

An increasing number of hazard assessment tools and approaches are being used in the marketplace as a means to differentiate products and ingredients with lower versus higher hazards or to certify what some call greener chemical ingredients in consumer products. Some leading retailers have established policies for product manufacturers and their suppliers to disclose chemical ingredients and their related hazard characteristics often specifying what tools to use. To date, no data exists that show a tool's reliability to provide consistent, credible screening-level hazard scores that can inform greener product selection. We conducted a small pilot study to understand and compare the hazard scoring of several hazard screening tools to determine if hazard and toxicity profiles for chemicals differ. Seven chemicals were selected that represent both natural and man-made chemistries as well as a range of toxicological activity. We conducted the assessments according to each tool provider's guidelines, which included factors such as endpoints, weighting preferences, sources of information, and treatment of data gaps. The results indicate the tools varied in the level of discrimination seen in the scores for these 7 chemicals and that tool classifications of the same chemical varied widely between the tools, ranging from little or no hazard or toxicity to very high hazard or toxicity. The results also highlight the need for transparency in describing the basis for the tool's hazard scores and suggest possible enhancements. Based on this pilot study, tools should not be generalized to fit all situations because their evaluations are context-specific. Before choosing a tool or approach, it is critical that the assessment rationale be clearly defined and matches the selected tool or approach. Integr Environ Assess Manag 2017;13:139-154. © 2016 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals, Inc. on behalf of SETAC.

A conserved family of calcineurin regulators.

The protein phosphatase calcineurin mediates many cellular responses to calcium signals. Using a genetic screen in yeast, we identified a new family of proteins conserved in fungi and animals that inhibit calcineurin function when overexpressed. Overexpression of the yeast protein Rcn1p or the human homologs DSCR1 or ZAKI-4 inhibited two independent functions of calcineurin in yeast: The activation of the transcription factor Tcn1p and the inhibition of the H(+)/Ca(2+) exchanger Vcx1p. Purified recombinant Rcn1p and DSCR1 bound calcineurin in vitro and inhibited its protein phosphatase activity. Signaling via calmodulin, calcineurin, and Tcn1p induced Rcn1p expression, suggesting that Rcn1p operates as an endogenous feedback inhibitor of calcineurin. Surprisingly, rcn1 null mutants exhibited phenotypes similar to those of Rcn1p-overexpressing cells. This effect may be due to lower expression of calcineurin in rcn1 mutants during signaling conditions. Thus, Rcn1p levels may fine-tune calcineurin signaling in yeast. The structural and functional conservation between Rcn1p and DSCR1 suggests that the mammalian Rcn1p-related proteins, termed calcipressins, will modulate calcineurin signaling in humans and potentially contribute to disorders such as Down Syndrome.

DSCR1, overexpressed in Down syndrome, is an inhibitor of calcineurin-mediated signaling pathways.

Down syndrome is one of the major causes of mental retardation and congenital heart malformations. Other common clinical features of Down syndrome include gastrointestinal anomalies, immune system defects and Alzheimer's disease pathological and neurochemical changes. The most likely consequence of the presence of three copies of chromosome 21 is the overexpression of its resident genes, a fact which must underlie the pathogenesis of the abnormalities that occur in Down syndrome. Here we show that DSCR1, the product of a chromosome 21 gene highly expressed in brain, heart and skeletal muscle, is overexpressed in the brain of Down syndrome fetuses, and interacts physically and functionally with calcineurin A, the catalytic subunit of the Ca(2+)/calmodulin-dependent protein phosphatase PP2B. The DSCR1 binding region in calcineurin A is located in the linker region between the calcineurin A catalytic domain and the calcineurin B binding domain, outside of other functional domains previously defined in calcineurin A. DSCR1 belongs to a family of evolutionarily conserved proteins with three members in humans: DSCR1, ZAKI-4 and DSCR1L2. We further demonstrate that overexpression of DSCR1 and ZAKI-4 inhibits calcineurin-dependent gene transcription through the inhibition of NF-AT translocation to the nucleus. Together, these results suggest that members of this newly described family of human proteins are endogenous regulators of calcineurin-mediated signaling pathways and as such, they may be involved in many physiological processes.

Saccharomyces cerevisiae genes required in the absence of the CIN8-encoded spindle motor act in functionally diverse mitotic pathways.

Kinesin-related Cin8p is the most important spindle-pole-separating motor in Saccharomyces cerevisiae but is not essential for cell viability. We identified 20 genes whose products are specifically required by cell deficient for Cin8p. All are associated with mitotic roles and represent at least four different functional pathways. These include genes whose products act in two spindle motor pathways that overlap in function with Cin8p, the kinesin-related Kip1p pathway and the cytoplasmic dynein pathway. In addition, genes required for mitotic spindle checkpoint function and for normal microtubule stability were recovered. Mutant alleles of eight genes caused phenotypes similar to dyn1 (encodes the dynein heavy chain), including a spindle-positioning defect. We provide evidence that the products of these genes function in concept with dynein. Among the dynein pathway gene products, we found homologues of the cytoplasmic dynein intermediate chain, the p150Glued subunit of the dynactin complex, and human LIS-1, required for normal brain development. These findings illustrate the complex cellular interactions exhibited by Cin8p, a member of a conserved spindle motor family.

Tcn1p/Crz1p, a calcineurin-dependent transcription factor that differentially regulates gene expression in Saccharomyces cerevisiae.

Ca2+ signals regulate gene expression in animal and yeast cells through mechanisms involving calcineurin, a protein phosphatase activated by binding Ca2+ and calmodulin. Tcn1p, also named Crz1p, was identified as a transcription factor in yeast required for the calcineurin-dependent induction of PMC1, PMR1, PMR2A, and FKS2 which confer tolerance to high Ca2+, Mn2+, Na+, and cell wall damage, respectively. Tcn1p was not required for other calcineurin-dependent processes, such as inhibition of a vacuolar H+/Ca2+ exchanger and inhibition of a pheromone-stimulated Ca2+ uptake system, suggesting that Tcn1p functions downstream of calcineurin on a branch of the calcium signaling pathway leading to gene expression. Tcn1p contains three zinc finger motifs at its carboxyl terminus resembling the DNA-binding domains of Zif268, Swi5p, and other transcription factors. When fused to the transcription activation domain of Gal4p, the carboxy terminal domain of Tcn1p directed strong calcineurin-independent expression of PMC1-lacZ and other target genes. The amino-terminal domain of Tcn1p was found to function as a calcineurin-dependent transcription activation domain when fused to the DNA-binding domain of Gal4p. This amino-terminal domain also formed Ca2+-dependent and FK506-sensitive interactions with calcineurin in the yeast two-hybrid assay. These findings suggest that Tcn1p functions as a calcineurin-dependent transcription factor. Interestingly, induction of Tcn1p-dependent genes was found to be differentially controlled in response to physiological Ca2+ signals generated by treatment with mating pheromone and high salt. We propose that different promoters are sensitive to variations in the strength of Ca2+ signals generated by these stimuli and to effects of other signaling pathways.

Characterization of the interaction of the glp repressor of Escherichia coli K-12 with single and tandem glp operator variants.

The glp operons of Escherichia coli are negatively controlled by the glp repressor. Comparison of the repressor-binding affinities for consensus and altered consensus operators in vivo showed that all base substitutions at positions 3, 4, 5, and 8 from the center of the palindromic operator caused a striking decrease in repressor binding. Substitutions at other positions had a severe to no effect on repressor binding, depending on the base substitution. The results obtained indicate that the repressor binds with highest affinity to operators with the half-site WATKYTCGWW, where W is A or T, K is G or T, and Y is C or T. Strong cooperative binding of the repressor to tandem operators was demonstrated in vivo. Cooperativity was maximal when two 20-bp operators were directly repeated or when 2 bp separated the two operators. Cooperativity decreased with the deletion of 2 bp or the addition of 4 bp between the individual operators. Cooperativity was eliminated with a 6-bp insertion between the operators.

Inhalation regional cerebral blood flow: the use of tidal CO2 data to find radionuclide activity associated with exhaled alveolar gas.

When calculating cerebral blood flow by the inhalation regional cerebral blood flow technique, radionuclide activity associated with exhaled alveolar gas is used to represent the arterial input function for each brain region. In this study, tidal CO2 data are used to identify respiratory gas samples that contain alveolar gas. Traditional methods identify alveolar gas samples by searching for maxima and minima in the raw air curve. The raw air curve is determined by sequentially counting radionuclide activity in respiratory gases sampled at the mouth. Traditional methods sometimes erroneously identify and use maxima or minima that do not represent alveolar gas. The use of CO2 data is advantageous since the range of CO2 during exhalation can identify those exhalations that approach the functional reserve capacity and hence represent alveolar gas. The arterial input function is represented by counting intervals from the raw air curve which coincide with exhalation of alveolar gas as identified by CO2 data. This approach for representing the arterial input function is fully automatic, accurate, and reproducible.

The rCBF response to Diamox in normal subjects and cerebrovascular disease patients.

Age-related norms for the regional cerebral blood flow (rCBF) response to Diamox (acetazolamide) were based on studies of 55 normal subjects at rest and on studies of 33 of these 55 normal subjects following an intravenous injection of Diamox (22 mg/kg). After the Diamox injection, rCBF increased at all locations measured in all subjects. On average, rCBF increased 1.7 times. The following were found for rCBF in both resting and Diamox-treated subjects: 1) rCBF decreased significantly with increasing age; 2) slope and intercept for the regression of rCBF on age were largest for frontal detectors, intermediate for parietal detectors, and smallest for occipital detectors; 3) rCBF hyperfrontality was most noticeable in younger subjects; 4) in subjects of any age, 95% confidence intervals for rCBF were relatively large (expected value +/- 30%) and lower 95% confidence intervals for Diamox rCBF tended to overlap the upper 95% confidence intervals for resting rCBF; and 5) side-to-side percentage difference in rCBF did not have a significant regression on age and tended to be less than 10% to 20%. Diamox did not have an important effect on blood pressure, pulse rate, or respiratory rate. The normative data for the rCBF response to Diamox was used in evaluating 20 patients with cerebrovascular disease. Forty percent of these patients, all of whom exhibited angiographic evidence of potentially hemodynamically significant lesions, had normal rCBF at rest and after Diamox injection. Twenty percent had normal resting flows with abnormal Diamox-activated flows. Asymmetry in rCBF was the most sensitive indicator of a potential abnormality in cerebral perfusion. Thirty percent of the abnormal studies showed only significant asymmetry. It is suggested that rCBF studies at rest and after Diamox treatment, with age-related norms, may be useful in the management of patients with cerebrovascular disease.

Analysis of inhalation rCBF data.

Relative to other approaches that have been recommended, fitting all head data, solving for a time shift, and including an air passage artifact term in the model significantly improved the estimate of gray matter blood flow by the inhalation technique. A robust algorithm, which incorporates these features, has been developed. Formulas which facilitate implementation of this algorithm are reported. An artifact from large scalp arteries was not significant and does not need to be included in the model.

Improvements in the technique of spinal subarachnoid recirculatory perfusion.

With 14C-labeled dextran as the tracer, studies of the original configuration of spinal recirculatory perfusion and the original model for data analysis demonstrated that this approach does not yield acceptable accuracy in determining cerebrospinal fluid (CSF) formation (Fcsf) and absorption (Acsf) rates. A significant component of this error was due to the fact that the method of data analysis used originally is not based on a realistic mathematical model of the system. A more realistic mathematical model resulted in two simultaneous differential equations that did not have simple analytical solutions and, therefore, could not be used easily for data analysis. By computer simulation, a comparison of the more realistic model with the original model demonstrated that, under ideal conditions, there was a 10% error inherent in the original data analysis method. In the experimental setting, the magnitude of this inherent error is probably 20%. There were three other major problems with the original system: (a) one could not tell when enough data had been collected to ensure convergence of the data analysis algorithm; (b) calibration of the syringe pump in the external circuit was not accurate for short infusion periods; and (c) the presence of the syringe in the external circuit unnecessarily lengthened the period of nonhomogeneous mixing. A new system configuration and new data analysis methods have been developed. In the new system, the syringe is removed from the external circuit and intracranial pressure is controlled by infusion from a separate reservoir where the pressure head is maintained at any desired level by feedback control. Spectrophotometry is used to measure tracer concentration in the external circuit. Data collection and analysis are fully automated under computer control so that, during an experimental run, the investigators are updated at 1- to 2-second intervals as to the convergence of the data analysis routine. Data analysis methods for the new system are superior to previous methods because the models are realistic and no extrapolation is required. In addition, all of the data during the initial period of nonhomogeneous mixing are used to calculate Fcsf and Acsf. With the new system and a simple phantom of the CSF system, the mean error in finding Acsf was 1.7 +/- 1.2% for 27 determinations covering a wide range of absorption rates. Fcsf could be determined to within 0.001 ml/minute. In up to six sequential pressure plateaus, the magnitude of error did not increase with each subsequent run.(ABSTRACT TRUNCATED AT 400 WORDS)

Photochemotherapy of psoriasis using methoxsalen and sunlight. A controlled study.

Fifty-one patients with psoriasis were treated with oral methoxsalen and sunlight exposure. Twelve of these patients received either methoxsalen or placebo prior to whole-body exposure. The remainder were treated with methoxsalen and sunlight to one side of the body and sunlight alone to the other. The conventional dose of methoxsalen (0.6 mg/kg) was compared with a low dose (0.3 mg/kg). Oral methoxsalen when used in the higher dose followed by sun exposure is an effective treatment for psoriasis. Accurate ultraviolet dosimetry is essential to avoid phototoxic burns. The advantages and disadvantages of solar photochemotherapy are discussed.