Toxicity of Al to Desulfovibrio desulfuricans.

The toxicity of Al to Desulfovibrio desulfuricans G20 was assessed over a period of 8 weeks in a modified lactate C medium buffered at four initial pHs (5.0, 6.5, 7.2, and 8.3) and treated with five levels of added Al (0, 0.01, 0.1, 1.0, and 10 mM). At pH 5, cell population densities decreased significantly and any effect of Al was negligible compared to that of the pH. At pHs 6.5 and 7.2, the cell population densities increased by 30-fold during the first few days and then remained stable for soluble-Al concentrations of <5 x 10(-5) M. In treatments having total-Al concentrations of > or =1 mM, soluble-Al concentrations exceeded 5 x 10(-5) M and limited cell population growth substantially and proportionally. At pH 8.3, soluble-Al concentrations were below the 5 x 10(-5) M toxicity threshold and cell population density increases of 20- to 40-fold were observed. An apparent cell population response to added Al at pH 8.3 was attributed to the presence of large, spirilloidal bacteria (accounting for as much as 80% of the cells at the 10 mM added Al level). Calculations of soluble-Al speciation for the pH 6.5 and 7.2 treatments that showed Al toxicity suggested the possible presence of the Al(13)O(4)(OH)(24)(H(2)O)(12)(7+) "tridecamer" cation and an inverse correlation of the tridecamer concentration and the cell population density. Analysis by (27)Al nuclear magnetic resonance spectroscopy, however, yielded no evidence of this species in freshly prepared samples or those taken 800 days after inoculation. Exclusion of the tridecamer species from the aqueous speciation calculations at pHs 6.5 and 7.2 yielded inverse correlations of the neutral Al(OH)(3) and anionic Al(OH)(4)(-) monomeric species with cell population density, suggesting that one or both of these ions bear primary responsibility for the toxicity observed.

Comparison of transillumination and histologic slide measurements of choroidal melanoma.

OBJECTIVE: To compare transillumination and histologic slide measurements of choroidal melanomas in 479 eyes randomized to enucleation in the Collaborative Ocular Melanoma Study. DESIGN: Transillumination defects were measured during gross examination of enucleated eyes. Tumor basal diameter and height were measured on histologic slides and each tumor was assigned to 1 of 8 distinct shape categories. Comparison of the transillumination and histologic slide measurements revealed 3 categories of difference: underestimation (transillumination measurement more than 4 mm smaller than the histologic slide measurement), overestimation (transillumination measurement more than 4 mm larger than the histologic slide measurement), and agreement within 4 mm. RESULTS: There was good correlation between transillumination and histologic slide estimates of largest basal diameter, particularly when the basal diameter was 16 mm or less. Measurement discrepancies were related to the shape of the tumors but not to the presence of subretinal fluid or fixation. CONCLUSION: Agreement was high between measurements of transillumination defect and histologic sections.

Treatment of spontaneously arising retinoblastoma tumors in transgenic mice with an attenuated herpes simplex virus mutant.

The use of viruses to treat tumors has received renewed interest with the availability of genetically defined attenuated mutants. Herpes simplex virus (HSV) type 1 in particular has been shown to be effective for tumors of neuronal origin. However, the model systems used for these studies rely on the use of explanted tumor cells in immunodeficient animals. We have used a recently developed transgenic mouse model, wherein mice spontaneously develop retinoblastomas, to determine if a mutant HSV has a therapeutic effect against an endogenously arising tumor in an immunocompetent host. The injection of 1 x 10(6) PFU of the neuroattenuated HSV-1/HSV-2 recombinant RE6 into the vitreous of transgenic mice resulted in a significant inhibition of tumor growth compared to injection of medium alone (P = 0.0063). Immunohistochemical analysis of viral antigen showed that viral replication was restricted to focal areas of the tumors and the retinal pigment epithelium. Viral growth was not significantly different in the eyes of transgene-positive and transgene-negative mice, suggesting that enhanced replication in tumor cells may not explain the effects. Tumor cells in the treated eyes were significantly less differentiated than those in the untreated eyes (P = 0.04), suggesting that the virus may replicate better in certain cell types in the tumors. Although the injection of RE6 resulted in a difference in tumor size, the treatment did not result in the elimination of tumors in any of the mice improvements in the efficacy of tumor control are needed if this therapy is to be of use.

The herpes simplex virus type 1 ribonucleotide reductase is required for acute retinal disease.

We have used a herpes simplex virus type 1 (HSV-1) ribonucleotide reductase (RR) null mutant (ICP6 delta) to determine if the HSV-1 RR is required for acute retinal disease. Injection of the ICP6 delta mutant into the vitreous induced mild transient signs of infection (vitreal infiltrate, retinal inflammation, and changes in retinal cytology). In contrast, the parental KOS and a revertant virus (ICP6 delta + 3.1) in which the RR gene had been restored, caused severe retinitis. Injection of media alone also induced mild transient signs of disease. Two months after infection, ICP6 delta injected eyes could not be distinguished from normal eyes. Repeated injection of ICP6 delta (3 times, 2 weeks apart) resulted in vitreal infiltrate near the site of injection but the retina did not appear damaged. The mutant, ICP6 delta, grew to peak titers 1 x 10(3) to 1 x 10(5)-fold lower and cleared faster than KOS or ICP6 delta + 3.1 in the injected eyes suggesting that the reduced virulence was due to reduced ability of the virus to grow. These results show that the viral RR is required for acute retinal disease.

Melanocytes and iris color. Light microscopic findings.

OBJECTIVE: To systematically evaluate morphologic differences in iris stroma that contribute to clinically perceptible differences in iris color, using immunohistochemical identification of stromal melanocytes and fluorescence microscopy. METHODS: Paraffin-embedded sections from 51 human irides were stained with S100a and fluorescein isothiocyanate. Cells were counted and scored as melanocytes or other. Melanocyte number, proportion, and density were determined for light-colored (blue), medium-colored (hazel) and dark-colored (brown) irides and compared. RESULTS: No statistically significant difference was observed for mean total cellularity or mean melanocyte number among the three color groups. Mean total stromal cell count was 1177 +/- 259 (mean +/- SEM), and mean melanocyte number was 778 +/- 196 per 5-micrometer section. In human irides, 65.9% of the iris stroma is composed of melanocytes. Melanocyte density (number of cells per square millimeter) is not related to iris color. CONCLUSION: The number of melanocytes, the proportion of melanocytes, and iris stromal cellularity are not major contributors to iris color.

Use of receiver operating characteristic (ROC) analysis to evaluate sequence matching.

In this paper, we borrow the idea of the receiver operating characteristic (ROC) from clinical medicine and demonstrate its application to sequence comparison. The ROC includes elements of both sensitivity and specificity, and is a quantitative measure of the usefulness of a diagnostic. The ROC is used in this work to investigate the effects of scoring table and gap penalties on database searches. Studies on three families of proteins, 4Fe-4S ferredoxins, lysR bacterial regulatory proteins, and bacterial RNA polymerase sigma-factors lead to the following conclusions: sequence families are quite idiosyncratic, but the best PAM distance for database searches using the Smith-Waterman method is somewhat larger than predicted by theoretical methods, about 200 PAM. The length independent gap penalty (gap initiation penalty) is quite important, but shows a broad peak at values of about 20-24. The length dependent gap penalty (gap extension penalty) is almost irrelevant suggesting that successful database searches rely only to a limited degree on gapped alignments. Taken together, these observations lead to the conclusion that the optimal conditions for alignments and database searches are not, and should not be expected to be, the same.

Neurobehavioral and immunological effects of prenatal cocaine exposure in rat.

Time-pregnant Sprague-Dawley rats were injected subcutaneously with 20 mg/kg of cocaine HCl or 0.9% saline daily from gestation days 15 through 21. Maternal plasma levels of approximately 720 ng/ml of cocaine did not alter maternal weight gain during treatment, duration of pregnancy, any of the litter variables or several indices of maternal behavior. Offsprings' body weight from birth to 30 days of age and physical maturation were not generally affected by prenatal cocaine exposure. While the development of surface righting, cliff avoidance, and the startle response was accelerated in cocaine-exposed offspring, acquisition of a preference for a social odor was unaltered. Prenatal cocaine also attenuated the locomotor response of the offspring to d-amphetamine and cocaine at PND 15; at PND 30 both of these catecholaminergic agonists increased activity in prenatal saline and prenatal cocaine offspring. However, the difference in plasma levels of cocaine at PND 30 suggests a possible down-regulation of adrenergic receptors following prenatal cocaine exposure. Decreased thymus/body weight ratios and splenomegaly were observed in prenatal cocaine animals at 55 days of age. Although complete neutralization of herpes simplex virus-type 1 was not observed, sera from prenatal cocaine offspring showed an increased rate of appearance of cytopathic effect, while sera from animals given cocaine postnatally showed a reduction in the rate at which viral infectivity was expressed in culture. These results indicate that prenatal cocaine exposure can alter neurobehavioral ontogeny and humoral immune responsitivity in the offspring.

In vitro toxicity of gentamicin to corneal epithelial cells.

We report morphologic and viability changes in the rabbit corneal epithelium exposed in vitro to gentamicin sulfate. Rabbit corneal epithelial cells (3 x 10(5)) were seeded in replicate 24-well plates at their first in vitro passage, and gentamicin in concentrations of 0, 50, 250, 500, 1,000, or 5,000 micrograms/ml was added to the tissue culture medium beginning 7 days after subculture. By phase contrast microscopy, changes in cell morphologic appearance, particularly increased cytoplasmic granularity, were observed in the 5,000-micrograms/ml groups as early as 24 h after introduction of the drug. At 48 h, similar findings were observed in the 250-micrograms/ml group and at all higher concentrations. The cytoplasmic granularity was not noted in the 0- or 50-micrograms/ml groups. By electron microscopy, these observations correlated with ultrastructural findings of increased accumulations of intralysosomal bodies beginning in the 250-micrograms/ml group after 48 h of exposure to gentamicin. Furthermore, a statistically significant difference (p = 0.001) was demonstrated between the total number of viable cells in the low-dose group (50-micrograms/ml) and the high-dose groups (greater than or equal to 250-micrograms/ml) for exposure periods of 48 h or more. These findings demonstrate aminoglycoside toxicity to corneal epithelial cells in vitro similar to that seen in the human kidney and conjunctiva.

Immunocytochemical Localization of ADPglucose Pyrophosphorylase in Developing Potato Tuber Cells.

The subcellular localization of ADPglucose pyrophosphorylase, a key regulatory enzyme in starch biosynthesis, was determined in developing potato tuber cells by immunocytochemical localization techniques at the light microscopy level. Specific labeling of ADPglucose pyrophosphorylase by either immunofluorescence or immunogold followed by silver enhancement was detected only in the amyloplasts and indicates that this enzyme is located exclusively in the amyloplasts in developing potato tuber cells. Labeling occurred on the starch grains and, in some instances, specific labeling patterns were evident which may be related to sites active in starch deposition.

Sink metabolism in tomato fruit : I. Developmental changes in carbohydrate metabolizing enzymes.

In developing tomato (Lycopersicon esculentum Mill.) fruit, starch levels reach a peak early in development with soluble sugars (hexoses) gradually increasing in concert with starch degradation. To determine the enzymic basis of this transient partitioning of carbon to starch, the activities of key carbohydrate-metabolizing enzymes were investigated in extracts from developing fruits of three varieties (cv VF145-7879, cv LA1563, and cv UC82B), differing in final soluble sugar accumulation. Of the enzymes analyzed, ADPglucose pyrophosphorylase and sucrose synthase levels were temporally correlated with the transient accumulation of starch, having highest activities in cv LA1563, the high soluble sugar accumulator. Of the starch-degrading enzymes, phosphorylase levels were fivefold higher than those of amylase, and these activities did not increase during the period of starch degradation. Fiften percent of the amylase activity and 45 to 60% of the phosphorylase activity was localized in the chloroplast in cv VF145-7879. These results suggest that starch degradation in tomato fruit is predominantly phosphorolytic. The results suggest that starch biosynthetic capacity, as determined by levels of ADPglucose pyrophosphorylase rather than starch degradative capacity, regulate the transient accumulation of starch that occurs early in tomato fruit development. The results also suggest that ADPglucose pyrophosphorylase and sucrose synthase levels correlated positively with soluble sugar accumulation in the three varieties examined.

Sink Metabolism in Tomato Fruit : III. Analysis of Carbohydrate Assimilation in a Wild Species.

Carbohydrate composition and key enzymes involved in carbohydrate metabolism were assayed throughout development of Lycopersicon esculentum and L. chmielewskii fruit. Translocation and assimilation of asymmetric sucrose and total soluble solids content was also determined in both species. The data showed that L. chmielewskii accumulated less starch than L. esculentum, and this was related to a lower level of ADPglucose pyrophosphorylase and a higher level of phosphorylase in L. chmielewskii. L. chmielewskii accumulated sucrose throughout fruit development rather than glucose and fructose which were accumulated by L. esculentum. A low level of invertase and nondetectable levels of sucrose synthase were associated with the high level of sucrose in L. chmielewskii. Translocation and assimilation of asymmetrically labeled sucrose indicated that sucrose accumulated in L. chmielewskii fruit was imported and stored directly in the fruit without intervening metabolism along the translocation path. In contrast, the relatively low level of radioactive sucrose found in L. esculentum fruit appeared to arise from hydrolysis and resynthesis of sucrose. The possible relationship between the level of soluble solids and differences in carbohydrate metabolism in sink tissue of the two species is discussed.

Localization of Carbohydrate Metabolizing Enzymes in Guard Cells of Commelina communis.

The localization of enzymes involved in the flow of carbon into and out of starch was determined in guard cells of Commelina communis. The guard cell chloroplasts were separated from the rest of the cellular components by a modification of published microfuge methods. The enzymes of interest were then assayed in the supernatant and chloroplast fractions. The chloroplast yield averaged 75% with 10% cytoplasmic contamination. The enzymes involved in starch biosynthesis, ADPglucose pyrophosphorylase, starch synthase, and branching enzyme, are located exclusively in the chloroplast fraction. The enzymes involved in starch degradation show a more complex distribution. Phosphorylase is located in both the supernatant and chloroplast fraction, 50% in each fraction. Most of the amylase and debranching enzyme activity is present in the supernatant (70%) fraction. The majority of the rest of the enzymes involved in the degradation of starch to malate and synthesis of starch from a hexose precursor were also investigated. All of the enzymes were present in the chloroplast except for hexokinase and phosphofructokinase. The inability to assay these enzymes could possibly have been due to the lack of or low activity of the enzymes or to nonoptimal assay conditions.

Glial cell component in retinoblastoma.

The right eye of a 4-month-old girl with a large, unilateral, sporadic retinoblastoma was enucleated. The tumor was unusual because it contained Flexner-Wintersteiner rosettes with extremely large lumina. Smaller rosettes and undifferentiated tumor cells were observed within the lumina. Also of importance were cells resembling glial cells which were intermixed with more typical cuboidal retinoblastoma cells. These cells had electron microscopic features typical of glial cells and stained positively for glial fibrillary acidic protein in immunohistochemical studies. Rosettes and glial cells continued to be observed in the tumor carried in tissue culture through two passages over a 7-month period. This tumor is presented because of its unusual rosette structures and because it confirms recent reports describing a glial cell component in retinoblastoma.

Establishment of cell lines of uveal melanoma. Methodology and characteristics.

Six continuous cell lines have been established from choroidal and ciliary body melanomas. These lines have been maintained in culture for at least 100 in vitro population doublings for periods over 1 year. They were established initially using a human diploid fibroblast strain MRC-5 as a feeder layer. Cells were grown in Ham's F-12 medium containing fetal bovine and horse sera and supplemented with glucose, cholera toxin, and epidermal growth factor. Culture doubling times ranged from 72-96 hr; cloning efficiencies ranged from 1-5% in the absence of a feeder layer. Six cell lines were studied in detail by electron microscopy, and all were found to have evidence of melanosomes and/or premelanosomes. The morphology of the cells was characteristic of melanomas as defined by the Callender classification, with cell types ranging from spindle A to epithelioid. Karyotypic studies revealed the presence of only human chromosomes with modal numbers ranging from 48-54 in the different lines.

Intraocular paragonimiasis.

A case of intraocular paragonimiasis is reported in a 13-year-old Chinese boy. The disease manifested as repeated attacks of acute intraocular pain associated with panuveitis. A combination of inflammatory reaction and ocular findings mimicking both perforating and contusion injuries caused by the migration of the fluke within the eye characterises the infection. The living fluke was successfully extracted from the anterior chamber and identified as Paragonimus westermani.

Retinoblastoma and angiogenesis activity.

Aqueous humor from 30 enucleated retinoblastoma-bearing eyes and 9 cell cultures of retinoblastoma were tested for angiogenesis activity using capillary endothelial cell migration assays. Aqueous from 90% of retinoblastoma eyes stimulated endothelial cell migration, compared with 25% of control eyes. Five of nine retinoblastoma cultures stimulated endothelial cell migration and five showed chorioallantoic membrane (CAM) neovascularization when compared with positive controls for angiogenesis activity. These results suggest an inverse relation between the length of time in culture and the ability of these cells to stimulate angiogenesis using these assays. Cells in culture for short periods of time gave positive test results, whereas continuous cell lines of retinoblastoma were consistently negative. While aqueous humor from retinoblastoma-bearing eyes demonstrated significant angiogenesis activity, cultured retinoblastoma cells show this only in early passages. Several possible explanations for this are considered.

Regulation of ADPGlucose Synthesis in Guard Cells of Commelina communis.

The activator specificity of the ADPglucose pyrophosphorylase from Commelina communis guard cells is the same as observed for the mesophyll cell enzyme. 3-Phosphoglycerate was found to be the most effective activator. Fifty per cent of maximal stimulation was observed at about 100 micromolar. Inorganic phosphate was found to be a potent inhibitor giving 50% inhibition at 0.3 millimolar. These results are discussed with respect to regulation of starch synthesis in guard cells.

Capillary endothelial cell migration: stimulating activity of aqueous humor from patients with ocular cancers.

In double-masked studies, various concentrations of aqueous humor (AH) from 157 patients (208 samples) with ocular cancers, nonmalignant ocular lesions, and normal eyes were added to bovine capillary endothelial (BCE) cells plated onto gold-coated cover slips. The phagokinetic tracks made by 100 cells at each concentration were traced, and the mean area of migration plus or minus the standard error of the mean was determined. Data are expressed as the percentages of increase in mean track area made by 100 cells incubated in medium that contained AH samples beyond the mean area of 100 cells incubated in medium alone. The percentage increases in migration-stimulating activity were as follows: a) malignant ocular disease--retinoblastoma (30 samples), 34 +/- 2; malignant melanoma (55 samples), 37 +/- 3; b) nonmalignant ocular disease--cataracts, glaucoma, pseudoglioma, and diabetic retinopathy (36 samples), 14 +/- 2; c) control AH--no ocular disease (51 samples), 9 +/- 1; normal eyes and systemic cancer (36 samples), 38 +/- 6. The percentage increase in endothelial cell migration was as great in cases of systemic cancer as it was in cases of ocular cancer. The endothelial cell migration-stimulating activity in AH from patients with intraocular cancers was significantly higher than the levels in the other groups of patients having no systemic cancer (P much less than .001). In addition, when the results were compared in the control group and the group with benign ocular disease, no significant differences were detected (P greater than .01).