John N. Telford

Ca++-induced fusion of proteoliposomes: Dependence on transmembrane osmotic gradient

SummaryThe fusion of cytochrome oxidase liposomes with liposomes reconstituted with mitochondrial hydrophobic protein is dependent on the presence of an acidic phospholipid in the liposomes and on the addition of Ca++ ions. Liposomes which have grown, by fusion, to diameters in excess of 1000 Å lose the ability to fuse further, unless an osmotic gradient across the liposome membrane is established, with the internal osmotic pressure higher than the external. At a given Ca++ concentration, the extent to which this second fusion step takes place is determined by the ratio of internal to external osmolarity. Single-walled liposomes with diameters exceeding 1 μm have been produced by this technique. The data suggest that the thermodynamic driving force for the Ca++-induced fusion is an excess surface free energy which can be supplied by membrane curvature or transmembrane osmotic gradients.

Dimensions of active cytochrome c oxidase in reconstituted liposomes using a gold ball shadow width standard: a freeze-etch electron microscopy study.

The preparation and characterization of a distribution of gold balls on a thin, flat carbon film is described. The relation of the platinum carbon shadow width distribution means to a gold ball size is reported.

Toxicologic studies with male sheep grazing on municipal sludge‐amended soil

Growing sheep were grazed for 152 d on grass-legume forage growing on soil that had been amended with municipal sewage sludge from Syracuse, N.Y., at 224 metric tons per hectare. Cadmium was higher, but not significantly (p greater than 0.05), in tissues of sheep fed the sludge-grown forage as compared to controls. No significant differences between the sludge or control treatments were found in weight of the complete or cauda epididymis or in percent progressive motility of cauda epididymal sperm. The sludge-treatment group had significantly larger testes (p less than 0.025) when expressed as a percentage of body weight, and higher blood uric acid values (p less than 0.05). There were no observable changes in tissue ultrastructure of liver, kidney, muscle, or testes as examined by electron microscopy in either of the treatment groups. There were no significant differences for rate of animal weight gain, carcass weight, dressing percentage, or quality or yield grade of the carcases between the treatment groups.

Toxicologic studies in growing sheep fed silage corn cultured on municipal sludge‐amended acid subsoil

Field corn was grown on subsoil, pH 5.5, that had been amended with 100 dry tons per acre (224 metric tons per hectare) of municipal sewage sludge from Syracuse, New York. The corn plants containing 3.88 ppm dry weight of cadmium were field-chopped and ensiled, and the silage was fed to growing sheep for 225 d. The sheep fed the sludge-grown corn silage showed a significantly (10 higher feed efficiency, (2) higher hepatic microsomal p-nitroanisole O-demethylase activity, and (3) higher concentrations of cadmium in liver and kidney and nickel in kidney as compared to the control animals. No significant treatment effects were observed in mutagenic responses for animal feed or feces samples. No consistent treatment effects were noted during histopathologic examination of sheep tissues.

Safety Evaluation of Vegetables Cultured on Municipal Sewage Sludge-amended Soil

Cabbage, beets, green beans, and butternut squash were grown in control and municipal sewage sludge-treated experimental plots. Freezedried edible portions of each vegetable fed at 25% of the diet to male Fischer rats for 12 weeks, indicated no differences in dietary intake or weight gain, nor were there any differences in blood levels of alpha-fetoprotein, a marker for hepatic preneoplastic transformation. Sludge-grown vegetables did not cause any differences in relative liver weight or in activity of the hepatic mono-oxygenases, aminopyrene-N-demethylase, orp-nitroanisole-O-demethylase, when compared to the same vegetable grown on control soil. There were no effects attributable to the sewage sludge treatment on liver cell ultrastructure. The level of cadmium in the kidneys of rats fed sludge-grown beets was twice that of the controls. The results of mutagenesis testing withS. typhimurium and microsomes (S-9) activation indicated a positive dose response with extracts of sludge-grown beans and in the urine of rats consuming sludge-grown beets. Although no overt toxicologic responses were observed in rats fed sludge-grown vegetables, these positive mutagenic changes create uncertainties in evaluation of potential risks involved in using municipal sludge on food-producing land.

Response of Japanese quail fed seed meal from sunflowers grown on a municipal sludge-amended soil: elevation of cadmium in tissues.

Sunflowers were grown on soil amended with 224 metric tons/ha of municipal sewage sludge from Syracuse, N.Y. The yield of sunflower seeds was reduced by 47.2% by the sludge addition. The harvested seeds contained 1.71 ppm dry weight of cadmium. Deoiled seed meal was incorporated as 25 and 50% of semipurified diet and fed to male and female Japanese quail. The concentrations of cadmium were higher in kidney, liver, muscle, and eggs of birds fed the sludge-grown seed meal as compared to control quail. Tissue concentrations of cadmium increased with increasing dietary levels of sludge-grown seed meal. No significant differences (p greater than 0.05) were observed between dietary treatments in the activity of hepatic microsomal p-nitroanisole O-demethylase or aminopyrine N-demethylase in the male birds. Additionally, no mutagenic activity, either direct or with metabolic activation, was found in quail eggs. No observable changes in tissue ultrastructure were observed under electron microscopy in any of the treatment groups. There were no significant (p greater than 0.05) differences among the dietary treatment groups in feed intake, growth rate, egg production, or egg hatchability.

Toxicologic studies with japanese quail fed winter wheat grown on municipal sludge-amended soil

Winter wheat was grown on soil amended with 100 dry tons per acre (224 metric tons/ha) of municipal sewage sludge from Syracuse, New York. The grain contained 1.43 and 0.98 ppm (mg/kg) dry weight, respectively, of cadmium and nickel. This wheat was incorporated as 60% of a semi-synthetic diet and fed to male and female Japanese quail for two generations. Male quail from the F1 generation fed sludge-grown wheat showed induction of hepatic microsomal enzymes,i.e., aminopyrene-N-demethylase and aniline hydroxylase, that indicated foreign compounds present in the wheat. Cadmium was significantly higher (p < 0.05) than controls in kidney, liver, and testes and nickel in the liver of the male quail (F0) fed the sludge-grown grain. Cadmium in kidney and liver (but not in eggs) and nickel in liver was significantly higher (p < 0.05) than controls in the females fed the sludge-grown wheat. Birds from the F1 generation showed no significantly different (p > 0.05) concentrations of cadmium in kidney, liver, or eggs between the two dietary treatments. There were no observable changes in the tissue ultrastructure of liver and kidney as examined by electron microscopy in any of the treatment groups.

Toxicologic studies with pregnant goats fed grass-legume silage grown on municipal sludge-amended subsoil

Pregnant goats were fed grass-legume silage grown on soil amended with 112 dry metric tons per hectare of municipal sewage sludge from Syracuse, New York for 135 days. Whereas PCBs were not detectable in control or sludge-grown silage, cadmium was much higher (3.81 ppm) in the sludge-grown silage as compared to the control silage (0.14 ppm). There were no consistent differences in residues of cadmium in the goats milk or body tissues of the kids when compared to controls. The concentration of cadmium in the livers of the adult animals was significantly higher (p < 0.05) in the sludge treatment than controls. No significant treatment effects were observed in mutagenic responses for the goats milk. Interestingly, the pregnant goats fed the sludge-grown silage produced far too little milk for their kids while the control animals produced sufficient milk. There were no observable changes in the tissue ultrastructure as examined by electron microscopy for both the adults and the kids from either ration treatment.

The platinum‐carbon replication of a homogenous population of gold particles makes log‐normal distributions of shadow widths and lengths

Gold particles were prepared, dried on grids and shadowed at 45° with a 1.2 nm platinum‐carbon (Pt‐C) film using the shadowing conditions previously described for the freeze‐fracture of gastric parietal cell membranes. The particle diameters and the particle shadow widths and lengths were measured using an image analysis system. Statistical analysis of 2000 diameters, shadow widths and shadow lengths indicated that a homogenous population of particles had a normal frequency distribution of diameters (mean diameter 14.5 ± 1.5 nm) and that the Pt‐C shadowing transformed that normal curve into a log‐normal frequency distribution of shadow widths. The frequency distribution of shadow lengths was log‐normal too. We conclude that a statistical partition of experimental frequency distributions of particle shadow widths and lengths of natural membranes to determine the number and parameters of individual components should involve log‐normal subdistributions rather than normal ones.