Molecular structure of DNA by scanning tunneling microscopy.

Uncoated DNA molecules marked with an activated tris(l-aziridinyl) phosphine oxide (TAPO) solution were deposited on gold substrates and imaged in air with the use of a high-resolution scanning tunneling microscope (STM). Constant-current and gap-modulated STM images show clear evidence of the helicity of the DNA structure: pitch periodicity ranges from 25 to 35 angstroms, whereas the average diameter is 20 angstroms. Molecular structure within a single helix turn was also observed.

Reduced maturation of Friend virus in adhesive mutants of Friend leukemia cells.

The replication of Friend Leukemia virus (FLV) has been investigated in adhesive clones (FF) of Friend Leukemia cells which were selected via cultivation on top of human fibroblast monolayers. In these adhesive clones a shut-down of FLV production is observed under conditions of culture confluency; this finding is not due either to a reduced number of cell divisions nor to a defective expression of FLV genome as assessed by Northern blot and immunofluorescence studies. Ultrastructural studies showed that virus budding and release into the medium is not detectable under these conditions. Conversely, in confluent FF cell monolayers abundant imperfect type-A enveloped particles were visible, possibly originating from stacks of granular endoplasmic reticulum with thickened membranes. It is postulated that the reduced virus production in adhesive FF monolayers is due to as yet undetermined events taking place during virus maturation at a time coincident with that of cell-cell adhesion under conditions of culture confluency.

Induction and maintenance of flattened morphology in highly adhesive Friend leukemia clones depends on the time- and space-specific assembly of microtubular networks.

Indirect immunofluorescent staining with anti-tubulin antibodies, SEM and TEM were applied to study microtubule (MT) assembly in clones isolated from Friend leukemia cells (FLC, 745 A strain) on the basis of their sensitivity to exogenous fibronectin (FN). Kinetics of cell spreading and elongation were studied using computerized image analysis and SEM. In contrast to 745 A cells, FN-sensitive clones (referred to as FF clones) showed elaborate MT networks when observed by immunofluorescent staining as well as by TEM. A good correlation was found between the degree of spreading and elongation of FF cells and the degree and cellular distribution of their MT. The highest concentration of MT networks oriented parallel to the main cellular axis was observed in very elongated FF cells. The majority of MT in interphase FF cells radiated from the centrosomes; some MT apparently originated from the nuclear membranes. TEM showed the existence of morphological differences between centrosomes of 745 A and FF cells. The characteristic ultrastructure of the centrosomes of FF cells was maintained in trypsinized cells, even if such FF cells lost MT's and acquired a spherical morphology. FF cells, treated with a wide spectrum of MT-disrupting agents, promptly acquired a rounded morphology with rapid dissolution of polymerized tubulin. Removal of MT-disrupting agents from the culture medium rapidly restored a flattened morphology with concurrent regeneration of MT's. During recovery from MT-disrupting agents, FF cells showed increased numbers of centrosomes per cell. We conclude that MT networks cooperate in the attachment, spreading and elongation of FF cells isolated from FLC. Moreover, we hypothesize the existence in FF cells of a variant form of centrosomes as compared with those of 745 A cells.

Acquisition of sensitivity to exogenous fibronectin by Friend leukemia cells correlates with reduction of their tumorigenic potential.

Strongly adhesive, highly flattened clones (FF clones) can be selected from Friend leukemia cells (FLC) cultivated on top of monolayers of human embryonic fibroblasts (HEF). The flattened phenotype of FF clones is stable during cell replication either in soft agar or in vivo. With the number of passages of FLC on HEF the fibronectin (FN) sensitivity of FF clones increases with a proportional reduction of their tumorigenicity. The FN-sensitivity was defined as the ability of a certain dose of FN to flatten 50% of cells of a given FF clone. Tumorigenicity was defined as the number of FF cells able to give palpable tumors, in 50% of inoculated mice. Exogenous FN does not modify the duplication time of FF clones but strongly influences their growth behavior. In the presence of FN, the growth rate of FF cells is controlled by the size of the growth are available. Highly FN-sensitive FF cells grown on FN-coated substrata die at confluency while FF cells not adherent to substrata escape cell death and grow in suspension. We conclude that the high intrinsic FN-sensitivity of FF cells and FN availability at the site of tumor inoculation could be responsible for the reduced tumorigenicity of highly flattened FF clones.

Morphometric studies in triacetyloleandomycin-induced ultrastructural modifications of rat hepatocyte mitochondria.

Morphometric parameters of mitochondria such as volumetric density, surface density of envelopes and cristae and numerical density were calculated for control untreated and triacetyloleandomycin (TAO)-treated rat hepatocytes using a point-counting technique. Moreover, morphometric parameters calculated experimentally and those computed on the basis of the least square interpolation and the agreement with the chi2 test, were compared. The equations modelling each main morphometric parameter of experimental motochondria were also computed. Morphometric study revealed that TAO produces a highly reproducible pattern of morphological alterations in the mitochondrial population of rat hepatocytes, and a good agreement between experimental and theoretical data was found. The response of hepatocyte mitochondria to TAO may be modelled by parabolic functions described by equations of the second degree. During the whole experimental period, the number of mitochondria decreases, but the specific volume of mitochondria increases. The area of the cristae surface per mitochondrion does not change substantially during the whole experimental period but since the quantity of internal mitochondrial membranes per hepatocyte is less in the later experimental period than in control material, it can be assumed that the oxidizing capacity per hepatocyte has diminished. The morphometric model based on TAO-treated hepatocyte mitochondria is different from those so far described in the literature for rat liver.

Study on the reinitiation of transcription in 37 RC cells after actinomycin D inhibition. Spectrum of major RNA species resynthesized after maximal suppression by the drug.

The reinitiation of the synthesis of major RNA species has been studied in 37 RC cells after maximal inhibition of RNA synthesis by actinomycin D (AMD). During the period of recovery from AMD, resynthesized RNA (rec-RNA) is initially composed of almost exclusively light (4-14S) heterogeneous RNA species. All normal species of RNA can be detected in the rec-RNA spectrum as early as 3 h after AMD removal. The synthesis of low molecular weight methylated RNA species increases slightly during the early period after AMD removal, while the increase of low molecular weight unmethylated species is more significant during the same period. Much of the radioactivity in the polyribosomal fraction is EDTA and puromycin sensitive. Since polysomal, puromycin-sensitive RNA is polyadenylated (as evidenced by the binding of poly-U filters), and is heterogenous in size, it belongs to the m-RNA class. The synthesis of m-RNA increases immediately after AMD removal, whereas the reinitiation of the r-RNA synthesis occurs after a lag period of about 2 h. The kinetics of recovery of the synthesis of major RNA species from AMD inhibition show a size dependency comparable to the size-related sensitivity to AMD inhibition in other cellular systems. This dependency is most clearly seen in HnRNA, the AMD sensitivity of which is measured by the length of the lag period between AMD removal and the appearance of HnRNA fractions in a sucrose density gradient. Low molecular weight HnRNA reappears first, whereas heavier fractions of HnRNA appear in the spectrum after a lag period, the length of which is in direct relation to the position of the HnRNA fraction in the gradient.