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.     

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.     

ENDOMORPHIC AND ECTOMORPHIC CHARACTERS IN PELECYPHORA AND ENCEPHALOCARPUS

Boke , Norman H. (U. Oklahoma, Norman.) Endomorphic and ectomorphic characters in Pelecyphora and Encephalocarpus. Amer. Jour. Bot. 46(3) : 197-209. Illus. 1959.—Outstanding ectomorphic characters of Pelecyphora valdeziana include its small size; pectinate, hairy spines; broad, truncate, floral buds; dehiscent, berry-like fruits; and black, tuberculate seeds. The leaves are vestigial, and although the areole meristem originates on the adaxial face of the tubercle primordium, it is soon elevated to the summit by intercalary growth. The first primordium of the single, elliptical series of spines is initiated immediately in front of the rudimentary leaf. Others form in acropetal sequence on either side of the areole meristem. The last ones form across the areole, leaving a meristem, which may be floral or vegetative, on the anterior side. Whether areoles of P. valdeziana can be considered dimorphic is doubtful. However, they approach the type of dimorphism found in Epithelantha. Pelecyphora aselliformis has acuminate floral buds; dry, papery fruits; and brown, curved, reticulate seeds. The leaves are reduced almost to extinction. The areole meristem becomes separated into spiniferous and axial portions early in ontogeny, but the 2 parts remain connected by a band of trichomes, which probably represents a vestigial groove. The axial meristem may be reproductive or vegetative. The sequence of spine initiation in P. aselliformis is unusual in that it begins at the anterior side of the spiniferous meristem and proceeds toward the posterior side. Areoles in this species are clearly dimorphic, much as in the mammillarias, but the vestigial groove is reminiscent of Coryphantha and related genera. Although adult specimens of Encephalocarpus strobiliformis bear scale-like tubercles, which are very different from the laterally compressed tubercles of P. aselliformis, their flowers, fruits, and seeds are almost identical. The two species share the same type of areole dimorphism, including the vestigial groove. Tubercles on seedlings and young branches of E. strobiliformis are prismatic rather than scale-like. Since they tend to be laterally compressed at the summit and bear elliptical areoles with many more spines than the adult, they resemble seedling tubercles of P. aselliformis. Tubercles on adult specimens likewise resemble each other in the structure of the epidermis and hypodermis. It does not seem possible that P. valdeziana can be retained in the genus Pelecyphora. If seed structure has any systematic value, the species belongs in or near the genus Thelocactus, to which it was assigned by Bravo. Pelecyphora aselliformis and Encephalocarpus strobiliformis, on the other hand, share so many important characters that they could well be considered cogeneric. Both seed structure and the rudimentary grooves on the tubercles suggest that their affinities may lie with certain coryphanthas or mammillarias rather than with Ariocarpus and Epithelantha.