Cytochemical localization of mercury in Saccharomyces cerevisiae treated with mercuric chloride.

We describe a cytochemical method for localizing mercury at the electron microscopic level in the yeast Saccharomyces cerevisiae. After addition of a lethal concentration of mercuric chloride to growing yeast cells, mercury was associated with the cell wall and cytoplasmic membrane. Little or no mercury was present in the cytoplasm. Electron probe X-ray microanalysis (EPMA) confirmed that the cytochemical reaction, visualized as mercury-silver complexes, was localized in dense bodies consisting of a core of mercury sulfide polymers surrounded by a shell of silver atoms.

Learning patterns of eye motion for foveal pursuit.

Two human subjects fixated a small light oscillating sinusoidally. After the light disappeared, sinusoidal post-pursuit eye motion (PPEM) continued to follow the unexpected trajectory of targets oscillating at 0.8 and 1.0 Hz. Saccades corrected differences between eye and expected target position during PPEM in complete darkness. Predictive tracking, the ability of primates to accurately fixate even rapidly moving targets, may thus involve learning specific eye movement patterns that mimic target motion.

Eccentric fixation with macular scotoma.

People with macular scotoma tend to read and visually scan more slowly than others with equivalently reduced visual acuity but intact central fields. We measured fixation eye movements and considered the contribution of fixation variability and centripetal eye drift to poor visual performance. These factors might confound efforts to consistently use an optimum retinal locus outside of the macula. We measured monocular horizontal and vertical eye movements using a search coil eyetracker while subjects with naturally occurring central scotomata or control subjects with simulated scotomata eccentrically fixated a single character that was sized to their visual acuity. Motivated subjects with long-standing stable maculopathies were chosen to estimate attainable performance limits. During attempts to eccentrically fixate, an ubiquitous foveal pursuit or centripetal drift tendency was not found; rather a pattern of drift was idiosyncratic from subject to subject. This finding was confirmed by an analysis of eye drift of 32 eyes with long-standing bilateral macular scotomata. Moreover, the eye drift speeds (15-200 minarc/sec) were too low to be of functional significance. Drift speeds during eccentric fixation with a visible target were not significantly different than those after the target was extinguished; however, drift speeds were greater than during foveal fixation. This suggests that the fovea has a specialized control of slow eye movements. Fixation variability increased with scotoma size for both simulated and real scotomata, with an abrupt rise when scotomata diameters exceeded 20 degrees C. A significant minority of subjects (39%) adopted two or more distinct preferred retinal loci (PRL) during fixation. Multiple PRL were also more likely if scotoma size exceeded 20 degrees C. Reasonably steady fixation is thus attainable when central scotoma sizes are smaller than approximately 20 degrees C.

Foveating saccades.

A review of the literature revealed that foveating saccades were found to be faster than other fast eye movements (FEMs) except optokinetic nystagmus (OKN) quick phase. In the present experiment, foveating saccades were compared to OKN quick phase in humans and were found to have higher maximum speeds and shorter durations. Unlike previous experiments, foveating saccades were made to targets at unpredictable distances, and active pursuit during OKN was discouraged. Previously reported differences between the speeds of foveating saccades and saccades to remembered target positions were replicated. Foveating saccades, therefore, can be distinguished from other FEMs on the basis of speed. This behavioral difference suggests that a distinctive mechanism exists for foveating targets.

Saccade control without a fovea.

Prior research has suggested that two types of fast eye movements (FEMs) can be distinguished behaviorally. Foveating saccades respond to salient peripheral targets by directing the target image to the fovea. Non-foveating saccades include other FEMs such as nystagmus quick phase, saccades without visual stimuli and visually-directed saccades that direct target images to eccentric retina. Foveating saccades have a shorter initiation latency and are faster than non-foveating saccades. Following adaptation to central scotoma, patients tend to use preferred retinal loci for fixation (PRL). If PRL acquire the foveal characteristic of a retino-motor center then visually guided saccades would acquire the properties of foveating saccades. Using an objectively-calibrated 2-dimensional search coil, we measured saccades in response to salient, unpredictable targets. The saccades of normal observers were compared to the saccades of patients with long-standing macular scotomas. Although the saccades of patients consistently directed images to PRL, the saccades still had the latency and dynamic characteristics of non-foveating saccades. Moreover, the non-foveating saccades of patients were found to be less accurate than foveating saccades, showing a range effect (larger saccades undershoot with greater error than do smaller saccades). Apparently, patients with macular scotoma suppress rather than adapt a foveating saccade mechanism.

Detection of induced mitotic chromosome loss in Saccharomyces cerevisiae--an interlaboratory assessment of 12 chemicals.

Induced mitotic chromosome loss was assayed using diploid yeast strain S. cerevisiae D61.M. The test relies upon the uncovering and expression of multiple recessive markers reflecting the presumptive loss of the chromosome VII homologue carrying the corresponding wild-type alleles. An interlaboratory study was performed in which 12 chemicals were tested under code in 2 laboratories. The results generated by the Berkeley and the Darmstadt laboratories were in close agreement. The solvents benzonitrile and methyl ethyl ketone induced significantly elevated chromosome loss levels. However, a treatment regime that included overnight storage at 0 degree C was required to optimize chromosome loss induction. Hence, these agents are postulated to induce chromosome loss via perturbation of microtubular assembly. Fumaronitrile yielded inconsistent results: induction of chromosome loss and respiratory deficiency was observed in both laboratories, but the response was much more pronounced in the Darmstadt trial than that observed in Berkeley. The mammalian carcinogens, benzene, acrylonitrile, trichloroethylene, 1,1,1-trichloroethane and 1,1,1,2-tetrachloroethane failed to induce chromosome loss but elicited high levels of respiratory deficiency, reflecting anti-mitochondrial activity. Trifluralin, cyclophosphamide monohydrate, diazepam and diethylstilbestrol dipropionate failed to induce any detectable genetic effects. These data suggest that the D61.M system is a reproducible method for detecting induced chromosome loss in yeast.

Quantitative approaches for assessing chromosome loss in Saccharomyces cerevisiae: general methods for analyzing downturns in dose response.

Statistical methods are considered for analysis of data arising from a mitotic chromosome loss assay in Saccharomyces cerevisiae strain D61.M. The methods make use of reproducibility trial data from the assay (presented herein) and previous data, which suggest a unimodal, 'umbrella-patterned' dose response. Computer simulations are employed to illustrate the operating characteristics of the umbrella response methods. These methods are generally applicable to any toxicity assay that exhibits a downturn in dose response. Experimental design considerations are also discussed. These include applications of 2-stage sampling rules to first gauge the dose window of peak response, then test if the response deviates significantly from untreated levels.

Detection of induced mitotic chromosome loss in Saccharomyces cerevisiae--an interlaboratory study.

The diploid yeast strain D61.M was used to study induction of mitotic chromosome loss. The test relies upon the uncovering and expression of multiple recessive markers reflecting the presumptive loss of the chromosome VII homologue carrying the corresponding wild-type alleles. The underlying 'loss event' is probably complex since the predicted centromere-linked lethal tetrad segregations for chromosome VII are not recovered. Instead, the homologue bearing the multiple recessive markers is patently homozygous. An interlaboratory study was performed in which 16 chemicals were tested under code in 2 laboratories. The results generated by the Berkeley and Darmstadt laboratories were in close agreement. Acetonitrile, ethyl acetate, 4-acetylpyridine, propionitrile and nocodazole were identified as potent inducers of mitotic chromosome loss. Acetone, dimethyl sulfoxide and 2-methoxyethyl acetate either elicited weak responses or yielded ambiguous results. Water, carbon tetrachloride, 4-fluoro-D,L-phenylalanine, amphotericin B, griseofulvin, cadmium chloride, ethyl methanesulfonate and methylmercury(II) chloride failed to induce chromosome loss. These data suggest that the system described herein represents a reliable assay for chemically induced chromosome loss in yeast.

The detection of mitotic and meiotic chromosome gain in the yeast Saccharomyces cerevisiae: effects of methyl benzimidazol-2-yl carbamate, methyl methanesulfonate, ethyl methanesulfonate, dimethyl sulfoxide, propionitrile and cyclophosphamide monohydrate.

The diploid yeast strain BR1669 was used to study induction of mitotic and meiotic chromosome gain by selected chemical agents. The test relies on a gene dosage selection system in which hyperploidy is detected by the simultaneous increase in copy number of two alleles residing on the right arm of chromosome VIII: arg4-8 and cup1S (Rockmill and Fogel. 1988; Whittaker et al., 1988). Methyl methanesulfonate (MMS) induced mitotic, but not meiotic, chromosome gain. Methyl benzimidazol-2-yl carbamate (MBC) and ethyl methanesulfonate (EMS) induced both mitotic and meiotic chromosome gain. Propionitrile, a polar aprotic solvent, induced only mitotic chromosome gain; a reliable response was only achieved by overnight incubation of treated cultures at 0 degrees C. MBC is postulated to act by binding directly to tubulin. The requirement for low-temperature incubation suggests that propionitrile also induces aneuploidy by perturbation of microtubular dynamics. The alkylating agents MMS and EMS probably induce recombination which might in turn perturb chromosome segregation. Cyclophosphamide monohydrate and dimethyl sulfoxide (DMSO) failed to induce mitotic or meiotic chromosome gain.

Effects of benzimidazole analogs on cultures of differentiating rodent embryonic cells.

Micromass cell culture systems for rat embryo midbrain (CNS) and limb bud (LB) cells were employed to assess the in vitro developmental toxicity of the benzimidazole analogs, mebendazole (MBZ), thiabendazole (TBZ), and nocodazole (NCZ), in addition to the classic microtubule inhibitor, colchicine. Comparison was made to albendazole (ABZ), a previously studied benzimidazole anthelmintic. Two parameters for assessing developmental toxicity were measured: differentiation and cytotoxicity. The relative potencies of the benzimidazole analogs in the micromass system (NCZ greater than MBZ approximately ABZ much greater than TBZ) mirrored their effectiveness in an assay for in vitro inhibition of mammalian tubulin polymerization. Colchicine also exhibits a high affinity for mammalian tubulin and was a potent inhibitor of cell proliferation, chondrogenesis, and neuronal differentiation. Immunofluorescent staining of Day 1 LB cultures with a monoclonal antibody to beta-tubulin revealed that these agents elicited mitotic arrest. Many anti-tubulin agents are teratogenic in rats and their in vivo developmental toxicity may reflect perturbation of microtubular structure or function. With the exception of TBZ, these agents should be considered potential developmental toxicants since they inhibit cell growth and differentiation of micromass cultures at nanomolar concentrations.

Origin of wavelets in the visual evoked potential.

Low amplitude high frequency wavelets have been demonstrated to be ubiquitous in the visual system of animals and are observed in the ERG of man. Wavelets have also been observed superimposed upon large slow waves obtained from electrodes on occipital scalp. Presently, the rather stereotypic wavelet repetition rate permitted the use of active analog filters tuned to 100 Hz, with a 60-200 Hz bandpass which produced no measurable distortion at 100 Hz. With this method we recorded a series of wavelets in 15 of 16 subjects that usually began from 35 to 40 msec following the onset of a 10(4) troland, 25 degrees visual angle 100 msec flash. We then sought to determine the origin of these wavelets. Wavelets recorded between occiput (Oz) and vertex (Cz) had photopic spectral sensitivity that differed from that of ERG wavelets simultaneously recorded. Moreover, in a topographical study, wavelets recorded between Cz and various lateral positions reached a maximum at Oz; and when adjacent bipolar electrode pairs were used, wavelet polarities inverted as pairs were moved across the occipital region. Thus wavelets recorded between 35 and 70 msec were likely generated from occipital cortex rather than retinal, sub cortical, or diffuse cortical sites. In addition, the topographical distribution of slow wave ('P100') and wavelets differed. Wavelet latencies had a different relation to retinal illuminance than P100 latencies, suggesting that P100 and wavelets have different neurogeneses. Wavelets recorded between Oz and Cz thus reflect the earliest cortical visual processing recorded in man.

Effects of albendazole and albendazole sulfoxide on cultures of differentiating rodent embryonic cells.

Micromass cell culture systems for rat embryo midbrain (CNS) and limb bud (LB) cells were employed to assess the in vitro developmental toxicity of the human and veterinary anthelmintic albendazole (ABZ) and its sulfoxide metabolite (SOABZ). ABZ is reported to be teratogenic in rats, and is extensively metabolized to the sulfoxide derivative. It has been postulated that SOABZ is the reactive metabolite responsible for albendazole's developmental toxicity and anthelmintic activity in vivo. Three parameters for assessing developmental toxicity were measured: cell growth, differentiation, and cytotoxicity. CNS and LB cultures were equivalent in their sensitivities to both ABZ and SOABZ. ABZ was approximately 50-fold more potent than SOABZ. Immunohistochemical determinations of tubulin organization revealed that both ABZ and its sulfoxide metabolite elicit an accumulation of cells in the mitotic phase of the cell cycle. Since ABZ is one of the most potent agents tested in the micromass system to date, this anthelmintic should be considered a potential developmental toxicant.

Visual requirements for reading.

We have applied research on the visual psychophysics of reading to low vision assessment. Research on different aspects of the reading process found that reading rate rather than reading comprehension is more sensitive to variations in a subject's visual functioning or the stimulus properties of print. The research identified four different visual factors that significantly affect reading rate: (1) acuity reserve [print size relative to acuity threshold], (2) contrast reserve [print contrast relative to contrast threshold], (3) field of view [number of letters visible], and (4) in cases of maculopathy, central scotoma size. Our research indicates that fluent reading rates can be attained with a restricted field of view, as little as four characters. However, attainment of fluent reading levels requires that print size and contrast should be several times threshold and the diameter of a central scotoma should be less than 22 degrees. Although important clinical studies are lacking, we derived specific visual requirements for different reading rates from published experimental research to provide a starting point and to illustrate how visual requirements could be derived, even with poor correlations. Research has made significant progress toward the development of a comprehensive low vision assessment that will allow the practitioner to identify visual impediments to reading, other than reduced visual acuity. Having more fully characterized a visual impairment, the practitioner may tailor devices or interventions to the individual's needs and capabilities.

Scanning characters and reading with a central scotoma.

Among the patients we tested, most of those who had lost normal macular function developed extrafoveal retinal loci which resulted in strongly preferred viewing angles for steady fixation. These patients used these extrafoveal retinal loci to scan lines of characters in patterns similar to patients with normal macular function. Although the scanning speed of these patients was adversely affected by their large size scotomas, their reading accuracy was not necessarily reduced. The assessment of scanning and reading performance in this population requires careful measurement of the visual skills involved in reading.

Characterization of cytoskeletal and neuronal markers in micromass cultures of rat embryonic midbrain cells.

Micromass cultures of rat embryonic midbrain cells were characterized with regard to the immunolocalization of neuronal and cytoskeletal markers. Cells taken from gestational day-12 embryos and cultured for 5 days in vitro comprise at least two morphologically distinct cells types: fibroblast-like cells and neurons. Antibodies to the following markers yielded preferential staining of neuronal cells: A2B5 (GQ ganglioside), gamma-aminobutyric acid (GABA), microtubule-associated protein 2 (MAP2), MAP5, neuron-specific enolase (NSE), neural cell adhesion molecule (N-CAM), and tau. Antibodies to beta-tubulin, c-neu, MAP1, and neurofilament (NF-H) stained both neuronal and fibroblast-like cells. Antibodies to glial fibrillary acidic protein (GFAP) and vimentin failed to immunoreact with any cells in day-5 CNS cultures. SDS-PAGE and Western analysis were employed to determine the specificity of the antibodies and determine the electrophoretic profiles of the markers. We conclude that the pattern of neuronal differentiation in CNS micromass cultures exhibits certain similarities to that observed in vivo. In addition, certain markers identified in this study may be of potential utility as (1) biomarkers of chemically-induced developmental neurotoxicity, and (2) indicators of differential toxicity toward the diverse cell types that comprise the mammalian central nervous system.

The detection of mitotic and meiotic aneuploidy in yeast using a gene dosage selection system.

A system is described in which spontaneous and chemically-induced mitotic and meiotic hyperploidy can be assayed in the same diploid culture of Saccharomyces cerevisiae. Monitoring gene dosage changes at two loci on chromosome VIII, the test utilizes a leaky temperature-sensitive allele arg4-8 and low level copper resistance conferred by the single copy allele cup1s. An extra chromosome VIII provides simultaneous increased dosage for both genes, resulting in colonies that are both prototrophic for arginine at 30 degrees C and copper resistant. During mitotic cell divisions in diploids, spontaneous chromosome VIII hyperploids (trisomes and tetrasomes) occur at a frequency of 6.4 x 10(-6) per viable cell. Among ascospores, the spontaneous chromosome VIII disome frequency is 5.5 x 10(-6) per viable spore. The tubulin-binding reagent methyl benzimidazol-2-yl carbamate (MBC) elicits enhanced levels of mitotic and meiotic aneuploidy relative to control levels. The system represents a novel model for examining chromosome behavior during mitosis and meiosis and provides a sensitive and quantifiable procedure for examining chemically induced aneuploidy.

Effects of methyl mercury on the cell cycle of primary rat CNS cells in vitro.

Methyl mercury (MeHg) may interfere with cell cycle progression in a number of ways, most notably through an inhibition of protein synthesis or through effects on mitotic spindle performance; both mechanisms have experimental support. Results from investigations into the effects of MeHg exposure on cell cycle progression in a primary fetal rat CNS culture are presented here. Colchicine was also investigated because it is a well-characterized mitotic inhibitor. Flow cytometric DNA content analysis was utilized to determine the cell cycle distribution histograms of control and treated cultures. In addition, a flow cytometric technique which involves the incorporation of 5-bromo-2'-deoxyuridine into newly synthesized DNA was used to discriminate between successive cell cycles. Exposure of the CNS cell cultures to MeHg (2 and 4 microM) over a period of 0-48 hr led to a G2/M-phase inhibition as determined by flow cytometric DNA content analysis. Whereas exposure to 2 microM MeHg resulted in G2/M-phase inhibition, an analysis of cell cycle progression demonstrated an inhibition of cell cycling through any phase following exposure to 4 microM MeHg; these effects occurred in the first round of cell division following plating. Exposure to colchicine (25 nM) resulted in a G2/M-phase arrest similar to that observed with MeHg (2 microM). However, a comparison of the cytotoxicity patterns between MeHg-treated and colchicine-treated cultures suggests that MeHg-induced cytotoxicity cannot be solely ascribed to G2/M-phase arrest, since at equivalent levels of G2/M-phase arrest, MeHg was more cytotoxic than colchicine. These results are consistent with the hypothesis that microtubules, and the mitotic spindle, are especially sensitive to MeHg exposure.