Feasibility of automating the micronucleus assay

The results of a feasibility study on the automation of the micronucleus assay in whole blood cultures of human lymphocytes are reported. The assay requires determination of the number of lymphocytes with micronuclei among the proliferating population. Using an in-house-assembled image analysis system, a prototype software package was developed that addressed two problems: micronuclei identification and discrimination of nonproliferating cells from proliferating lymphocytes (the only ones that can give rise to micronuclei). The results of manual verification of automated micronucleus scoring showed that 70% of all digitized micronuclei were extracted from the images and 90% of them were correctly classified and paired with a parent nucleus by an "affinity function". The discrimination between proliferating and nonproliferating cells was carried out by linear discriminant analysis of simple nuclear features extracted from Feulgen-stained cells. Among the Feulgen-stained nuclei that were identified by autoradiography as proliferating or not, 85% were correctly classified by a six-feature discriminant function.     

Assembly of F1-ATPase in isolated mitochondria

The assembly of the proton-translocating ATPase complex was studied in isolated mitochondria by incubating yeast mitochondria with radiolabeled precursors of mitochondrial proteins which had been made in a cell-free protein synthesis system. Following such an incubation, the ATPase complex (F1F0) was isolated. Newly assembled F1-ATPase was detected by autoradiography of the isolated enzyme, only peptide subunits which had been made in vitro and imported into the isolated mitochondria could be radioactive. Incorporation of radiolabeled ATPase subunits into the enzyme does not occur in the presence of an uncoupler of oxidative phosphorylation or of a divalent metal chelator, nor does it occur in submitochondrial particles rather than intact mitochondria. Incorporation of labeled ATPase subunits into the enzyme can be completed by unlabeled subunits, provided the unlabeled proteins are added before the mitochondria are incubated with radioactive precursors. These findings suggest that F1-ATPase is assembled from a pool of subunits in mitochondria.     

THE SYNTHESIS OF MICROTUBULE AND OTHER PROTEINS OF THE ORAL APPARATUS IN TETRAHYMENA PYRIFORMIS

Several proteins, including microtubule proteins, have been isolated from the oral apparatus of the ciliate Tetrahymena. The synthesis of these proteins has been studied in relation to formation of this organelle system by the cell. Electron microscopy has shown that the isolated oral apparatus consists primarily of basal bodies, pellicular membranes, and a system of subpellicular microtubules and filaments. Cilia were removed during the isolation; therefore none of the proteins studied was from these structures. Evidence was obtained from the study of total oral apparatus protein which indicates that at least some of the proteins involved in formation of this organelle system may be synthesized and stored in the cytoplasm for use over long periods. This pattern of regulation was found for three individual proteins isolated from the oral apparatus fraction after extraction with a phenol-acetic acid solvent. A different pattern of regulation was found for microtubule proteins isolated from the oral apparatus of Tetrahymena. The data suggest that microtubule proteins, at least in logarithmically growing cells, are not stored in a cytoplasmic pool but are synthesized in the same cell cycle in which they are assembled into oral structures.     

INTRACORPOREAL HEAT DISSIPATION FROM A RADIOISOTOPE-POWERED ARTIFICIAL HEART

The feasibility of radioisotope-fueled circulatory support systems depends on the ability of the body to dissipate the reject heat from the power source driving the blood pump as well as to tolerate chronic intracorporeal radiation. Our studies have focused on the use of the circulating blood as a heat sink. Initial in vivo heat transfer studies utilized straight tube heat exchangers (electrically and radioisotope energized) to replace a segment of the descending aorta. More recent studies have used a left ventricular assist pump as a blood-cooled heat exchanger. This approach minimizes trauma, does not increase the area of prosthetic interface with the blood, and minimizes system volume. Heat rejected from the thermal engine (vapor or gas cycle) is transported from the nuclear power source in the abdomen to the pump in the thoracic cavity via hydraulic lines. Adjacent tissue is protected from the fuel capsule temperature (900 to 1200 degrees F) by vacuum foil insulation and polyurethane foam. The in vivo thermal management problems have been studied using a simulated thermal system (STS) which approximates the heat rejection and thermal transport mechanisms of the nuclear circulatory support systems under development by NHLI. Electric heaters simulate the reject heat from the thermal engines. These studies have been essential in establishing the location, suspension, surgical procedures, and postoperative care for implanting prototype nuclear heart assist systems in calves. The pump has a thermal impedance of 0.12 degrees C/watt. Analysis of the STS data in terms of an electrical analog model implies a heat transfer coefficient of 4.7 x 10(-3) watt/cm(2) degrees C in the abdomen compared to a value of 14.9 x 10(-3) watt/cm(2) degrees C from the heat exchanger plenum into the diaphragm.     

Separation and properties of human brain hexosaminidase C

Hexosaminidase C was separated from human brain supernatant by immunoadsorption of the A and B forms on to a column of immobilized antibody followed by preparative starch-block electrophoresis. There were some differences in the properties of hexosaminidase C preparations after each of these stages, shown by comparison of their heat-inactivation characteristics and filtration through Bio-Gel P-200. The C form prepared by both separation steps had properties which differed markedly from those of the A and B isoenzymes; its molecular weight was much larger, greater than 200000, it had optimum activity between pH6 and 7 and could not be successfully eluted from DEAE-cellulose, even with high salt concentrations, or from Sephadex G-200. These results seem to support the proposal that the C form is under a separate genetic control from the others.     

Biological Activity of 5-Hydroxymethyluracil and Its Deoxynucleoside in Noninfected and Phageinfected Bacillus subtilis

(14)C-hydroxymethyldeoxyuridine (dHMU) is specifically incorporated into the deoxyribonucleic acid (DNA) of bacteriophage SP8. Incorporation experiments demonstrate that the initiation of phage SP8 DNA synthesis occurs between 12.5 to 15 min after infection. Incorporation into host DNA does not occur. (14)C-dHMU can be used as an analytical tool for screening conditionally lethal phage mutants containing hydroxymethyluracil in their DNA to select those that are defective in DNA synthesis under restrictive conditions. The pyrimidine, (14)C-hydroxymethyluracil (HMU), is not incorporated into bacterial or phage DNA. Neither HMU nor dHMU can replace thymine as a growth requirement for Bacillus subtilis 168 Ind(-) Thy(-). HMU does not inhibit the utilization of thymine. Although dHMU inhibits deoxythymidine utilization, the inhibition is not competitive.