Infectivity of Proviral DNA from Avian Sarcoma Virus-Transformed Mammalian Cells

The number of Rous viral genomes in the cellular DNA from two subclones (RS2/3, RS2/6) derived from the same clone of hamster BHK-21 cells transformed by Rous sarcoma virus was determined by hybridization with viral complementary DNA made in vitro, and the capacity of the cellular DNA to infect (transfect) chicken embryo fibroblasts was compared before and after shearing this DNA to about the size of the provirus (6 x 10(6) to 7 x 10(6) daltons). The two subclones differed widely both in their capacity to give rise to virus (inducibility) after fusion with chicken embryo fibroblasts and in level of expression of viral proteins. It was shown that cells of both subclones contain a single copy of Rous DNA and yield infectious DNA. However, whereas transfection of chicken embryo fibroblasts was successful with both unsheared (>/=18 x 10(6) daltons) and sheared DNA from the most inducible subclone (RS2/3 subclone), which also expresses viral proteins to an appreciable amount, transfection with DNA from the least inducible subclone (RS2/6 subclone), in which viral proteins are not expressed, succeeded only with sheared DNA. It was then about as successful as with sheared or unsheared RS2/3 DNA. The lack of infectivity of unsheared RS2/6 DNA may be explained by the hypothesis proposed by Cooper and Temin (G. M. Cooper and H. T. Temin, J. Virol. 17:422-430, 1976) to explain the lack of infectivity of DNA from certain chicken cells producing spontaneously low amounts of RAV-0 and resistant to exogenous RAV-0 infection, that is, that the viral genome (proviral DNA) is linked to a cis-acting control element which blocks its expression. This linkage might originate, in RS2/6 cells, from translocation of cellular DNA containing the single proviral copy.     

Effects of four agents that modify microtubules and microfilaments (vinblastine,colchicine, lidocaine,and cytochalasin B) on macrophage-mediated cytotoxicity to tumor cells

Summary The effects of vinblastine, colchicine, lidocaine, and cytochalasin B on tumor cell killing by BCG-activated macrophages were examined. These four drugs were selected for their action on membrane-associated cytoskeletal components, microtubules, and microfilaments. Colchicine and vinblastine, which block microtubular synthesis, inhibit macrophage-mediated tumor-cell cytotoxicity at a concentration of 10^–6 M. Cytochalasin B, which disrupts microfilaments, enhances tumor cell lysis and stasis due to activated macrophages at a concentration of 10^–7 M. Lidocaine, which may induce the disappearance of both microtubules and microfilaments, has the same inhibiting effect as vinblastine at a concentration of 5×10^–7 M. Whereas vinblastine and lidocaine seem to act on the macrophage itself, cytochalasin B exerts its effect predominantly on the tumor cell. These results suggest that microtubules and microfilaments play a role in the destruction of tumor cells by activated macrophages.     

Bactericidal activity of tuftsin

Summary The biological activities of the phagocytosis stimulating tetrapeptide, Thr-Lys-Pro-Arg are discussed. A brief account on the stimulation by tuftsin of phagocytosis of various particles, including bacteria was reported. Stimulation of bactericidal activity by this tetrapeptide was investigated in vitro as well as in vivo. The potency of tuftsin to enhance blood clearing of Staphylococcus aureus, Listeria monocytogenes, Escherichia coli and Serratia marcescens by mouse peritoneal macrophages was demonstrated.Bactericidal activity and effects of tuftsin on this phenomenon were studied in liver and spleen of mice. Tuftsin stimulates these activities. Same experiments were performed in infected leukemic mice by Serratia marcescens or Escherichia coli. Results on blood clearing and bactericidal activities in liver and spleen were reported and compared to those of healthy and leukemic untreated animals. Tuftsin was found to present interesting stimulatory effects on the bactericidal activity of phagocytes.     

The ultrastructure of the sinus gland of the crayfish Astacus leptodactylus (Nordmann)

Summary An ultrastructural study of the sinus gland of the crayfish Astacus leptodactylus demonstrates that this gland is mainly composed of glial cells, axons and axon terminals. On the basis of the size, shape and electron density of the neurosecretory granules, we could distinguish five different types of axon terminals.     

Cellulose acetate electrophoresis of protein kinases: Detection of the active forms using various substrates

Electrophoresis on cellulose acetate strips was used to analyze protein kinases from normal rat liver. In addition to already well-characterized cAMP-dependent protein kinases type I and II and cAMP-independent casein kinases I and II, this method enabled the detection of several supplementary bands corresponding to kinases which were investigated according to their substrate specificity, activation by cAMP, and inhibition by the specific inhibitor of the catalytic subunit of cAMP-dependent protein kinases or by heparin. Using this rapid, sensitive, and resolutive electrophoretic method, different isozyme patterns could be obtained starting from minute amounts of different types of biological material.     

Spontaneous insertion of plant plasma membrane (H+)ATPase into a preformed bilayer

Summary The purified (H⁺ATPase from corn roots plasma membrane inserted spontaneously into preformed bilayer from soybean lipids. The yield of the protein insertion, as measured from its H⁺-pumping activity, increased as a function of lipids and protein concentrations. In optimum conditions, all the (H⁺)ATPase molecules were closely associated with liposomes, exhibiting a high H⁺-pumping activity (150,000% quenching· min^–1·mg^–1 protein of the probe 9-amino-6-chloro-2-methoxyacridine). The insertion was achieved within a few seconds. No latency of the (H⁺)ATPase hydrolytic activity was revealed when lysophosphatidylcholine was added to permeabilize the vesicles. This indicated that the (H⁺)ATPase molecules inserted unidirectionally, the catalytic sites being exposed outside the vesicles (inside-out orientation), and thus freely accessible to Mg-ATP. The nondelipidated (H⁺)ATPase could also functionally insert into bilayer from PCPEPG or PCPEPI, due to the presence of both hydrophobic defects promoted by PE, and negative phospholipids specifically required by the (H⁺)ATPase from corn roots. The detergent octylglucoside facilitated the delipidated (H⁺)ATPase reinsertion probably by promoting both a proper protein conformation and hydrophobic defects in the bilayer. Lysophosphatidylcholine facilitated the delipidated protein insertion only when hydrophobic defects were already present, and thus seemed only capable to ensure a proper protein conformation     

Mitochondrial DNA evolution in lagomorphs: Origin of systematic heteroplasmy and organization of diversity in European rabbits

Summary A characterization was conducted on mitochondrial DNA (mtDNA) molecules extracted separately from 107 European rabbits (Oryctolagus cuniculus) both wild and domestic, 13 European hares (Lepus capensis), and 1 eastern cottontail (Sylvilagus floridanus). Experimentally this study took into account restriction site polymorphism, overall length variation of the noncoding region, and numbers of repeated sequences. Nucleotide divergences indicate that the mtDNAs from the three species derived from a common ancestor some 6–8 million years (Myr) ago. Every animal appeared heteroplasmic for a set of molecules with various lengths of the noncoding region and variable numbers of repeated sequences that contribute to them. This systematic heteroplasmy, most probably generated by a rate of localized mtDNA rearrangements high enough to counterbalance the cellular segregation of rearranged molecules, is a shared derived character of leporids.The geographic distribution of mtDNA polymorphism among wild rabbit populations over the western European basin shows that two molecular lineages are represented, one in southern Spain, the second over northern Spain, France, and Tunisia. These two lineages derived from a common ancestor some 2 Myr ago. Their present geographical distribution may be correlated to the separation of rabbits into two stocks at the time of Mindel glaciation.Finally the distribution of mtDNA diversity exhibits a mosaic pattern both at inter- and intrapopulation levels.     

Amphibian oocytes and sphere organelles: are the U snRNA genes amplified?

The sphere organelles (spheres) ofXenopus and other amphibian oocytes are known to contain small nuclear ribonucleoprotein particles (snRNPs) and have been suggested to play a role in snRNP complex assembly. Coupled with the similarities that exist between spheres and nucleoli and the quantitative and kinetic aspects of snRNA synthesis in theXenopus oocyte, we have investigated whether or not the U snRNA encoding genes are amplified inXenopus oogenesis, the spheres being possible sites for the location of such extrachromosomal gene copies. By applying a number of quantitative nucleic acid hybridization procedures to both total and fractionated oocyte and somatic DNA, employing both homologous and heterologous U snRNA gene probes and suitable amplification and non-amplification control probes, we show that the U snRNA genes do not undergo any major amplification inXenopus oogenesis. Therefore, the analogy between the sphere organelles and nucleoli appears to be limited. The role of the spheres and their relationship to other snRNP containing structures, specifically B snurposomes, and the sphere organizer loci remains obscure.by A. Spradling