Poly(ADP-ribose) biosynthesis and suicidal NAD+ depletion following carcinogen exposure of mammalian cells

Hepatocytes were found to be remarkably resistant to suicidal NAD+ depletion due to consumption for chromatin-associated poly(ADP-ribose) biosynthesis, which normally follows infliction of DNA damage in mammalian cells. N-methyl-N'-nitro-N-nitrosoguanidine treatment, which depleted NAD+ levels of confluent fibroblasts to about 40% of controls, did not reduce hepatocellular NAD+ pools, although poly(ADP-ribose) concentrations were concomitantly elevated by 21-fold. This differential behavior, demonstrable also with other carcinogens, can be attributed to the different NAD+ biosynthetic capacities of these cells.     

Bulk separation and long-term culture of oligodendrocytes from adult pig brain. I. Morphological studies

Abstract: A method is described by which oligodendrocytes from adult pig brains can be isolated. It results in a cellular preparation suitable for long-term culture. The entire procedure can be accomplished within 2–3 h. The purity of oligodendrocytes ranges between 80 and 95% depending on the Percoll gradient used and on the time in vitro . Yields between 2.5 and 4 × 10⁷ cells per brain and plating efficiencies on the order of 60% make the system very useful for biochemical investigations. It was shown by immunocytochemical studies that oligodendrocytes produce extensive networks of processes, some of them having elaborate membranous expansions. Anti-galactocerebroside (GC) antibodies as well as anti-myelin basic protein (MBP), anti-Wolfgram protein (WP), antiglial fibrillary acidic protein (GFAP), and monoclonal antibodies O1 and O4 are used to identify the cell types and to characterize the cellular composition of the cultures. Anti-GC and O1 are suitable markers for these oligodendrocytes. Both antibodies label similar cells, and the staining intensities are equally strong. In the case of O4, variable staining intensities are observed, and a few additional cells are labeled that are anti-GC⁻. After 3¹/2 weeks in culture, about 60% of the cells can be labeled by anti-MBP. Here too differences in staining intensities are observed. The anti-WP stain is too weak to be defined as positive. The percentage of GFAP⁺ cells lies in the range 15–20% at maximum. Cells were also mixed into collagen gels. This method appears to be more useful for outgrowth and branching of fibers than are monolayer systems. Drawbacks, however, include limited access for the antibodies and poor recovery of undamaged cells with their fibers.     

Bulk Separation and Long-Term Culture of Oligodendrocytes from Adult Pig Brain.

Oligodendroglial proteins labeled with radioactive amino acids were subjected to one- and two-dimensional polyacrylamide electrophoresis. Bands comigrating with myelin proteins, the basic protein (MBP), the proteolipid protein (PLP), and the Wolfgram protein (WP) doublet, were detected by Coomassie Blue staining and by autoradiography. The identity of the MBP and WP in the cellular material is evidenced by immunoblotting with specific antibodies. A comparative study of myelin samples from rat and pig CNS reveals that WP can be detected immunochemically in both species. Different protein patterns, however, are observed. Three protein bands are found with antibodies against the myelin-associated glycoprotein (MAG). The high-molecular-weight component prevails in pig myelin, whereas the medium-molecular-weight component is predominant in rat myelin. Moreover, two protein bands, of molecular weights 35,000 and 33,000 (Ol 1 and Ol 2), are present in high amounts in oligodendroglial particulate material but are not detectable in myelin. These oligodendroglial characteristic proteins are not species-specific, since they are found in preparations of cat oligodendrocytes as well. Activities of cerebroside sulfotransferase (EC 2.8.2.11) are low in freshly isolated cells and increase during the first week of culture. A reverse course of enzyme activities is observed with 2',3'-cyclic nucleotide 3'-phosphohydrolase (EC 3.1.4.37). Values reach a minimum about day 5 in culture and recover their initial values. At day 10 they remain stable until the end of the third week of the culture period.     

Entropy as a factor in the binding of γ-aminobutyric acid and nipecotic acid to the γ-aminobutyric acid transport system

Nipecotic acid is one of the most potent competitive inhibitors and alternative substrates for the high-affinity -aminobutyric acid transport system in neurons, but the structural basis of this potency is unclear. Because -aminobutyrate is a highly flexible molecule in solution, it would be expected to lose rotational entropy upon binding to the transport system, a change which does not favor binding. Nipecotic acid, in contrast, is a much less flexible molecule, and one would expect the loss of conformational entropy upon binding to be smaller thus favoring the binding of nipecotic acid over -aminobutyric acid. To investigate this possibility, the thermodynamic parameters, G°, H°, and S°, were determined for the binding of -aminobutyrate and nipecotic acid to the high affinity GABA transport system in synaptosomes. In keeping with expectations, the apparent entropy change for nipecotic acid binding (112±13 J·K^–1) was more favorable than the apparent entropy change for -aminobutyric acid binding (61.3±6.6 J·K^–1). The results suggest that restricted conformation per se is an important contributory factor to the affinity of nipecotic acid for the high-affinity transport system for -aminobutyric acid.This work was conducted when both authors were at the Department of Chemistry, University of Maryland, College Park.Special issue dedicated to Dr. Elling Kvamme.     

Vertically stratified frugivore community composition and interaction frequency in a liana fruiting across forest strata

Vertical stratification is a key feature of tropical forests and structures plant–frugivore interactions. However, it is unclear whether vertical differences in plant-frugivore interactions are due to differences among strata in plant community composition or inherent preferences of frugivores for specific strata. To test this, we observed fruit removal of a diverse frugivore community on the liana Marcgravia longifolia in a Peruvian rain forest. Unlike most other plants, Marcgravia longifolia produces fruits across forest strata. This enabled us to study effects of vertical stratification on fruit removal without confounding effects of plant species and stratum. We found a high number of visits of a few frugivore species in the understorey and a low number of visits of many different frugivores in the canopy and midstorey. Whereas partial and opportunistic frugivores foraged across strata with differing frequencies, obligate frugivores were only found eating fruits in the higher strata. Avian frugivores foraging in the canopy were mainly large species with pointed wings, whereas under- and midstorey avian foragers were smaller with rounded wings. Our findings suggest a continuous shift in the frugivore community composition along the vertical gradient, from a few generalized frugivores in the understorey to a diverse set of specialized frugivores in the canopy. This shift in the frugivore community leads to correlated, reciprocal changes from specialized to generalized plant-frugivore interactions. Thus, we conclude that vertical niche differentiation between species in tropical forests persists even when food resources are available across strata. This highlights its role for promoting biodiversity and ecosystem functioning.     

Assessment of interleukin 1 and interleukin 2 effects on cycling and noncycling murine thymocytes

When mouse thymocytes are stimulated with PHA, the proliferative response is very low, unless the culture medium is enriched with interleukin 1 (IL-1)- or interleukin 2 (IL-2)-containing supernatants. Cytofluorometric analyses show, however, that PHA stimulation generates a significant number of cells with increased RNA content (transition from the G₀ to G₁ phase of the cell cycle). If IL-2 is added to such cultures, the activated cells complete their process of RNA synthesis and then enter the S phase. The use of IL-2-containing culture medium thus permits one to obtain a high correlation between the number of g₁ cells and [³H]thymidine incorporation (r = 0.97). Enrichment with IL-1-containing supernatants also results in a statistically significant correlation (r = 0.68), but the regression lines are markedly different for the two interleukins (s = 20.3 for IL-2 and s = 9.2 for IL-1), when analyzed after 48 hr of incubation. These observations suggest that the G₁ phase must be divided into two subcompartments, G_1a and G_1b, the G_1a-G_1b transition being an IL-2-dependent event. If the number of G_1b cells is used to establish correlations with [³H]thymidine incorporation, all values fall on the same regression line, regardless of culture conditions and of the addition of interleukins. It is concluded that IL-2 regulates lymphocyte proliferation at the level of RNA synthesis (G_1a-G_1b transition) rather than that of DNA synthesis (G₁-S transition).     

Differences in relative air humidity affect responses to soil salinity in freshwater and salt marsh populations of the dominant grass species Phragmites australis

Hydrobiologia - Soil salinity diminishes the dominance of species and affects their distribution. Phragmites australis is a dominant ecosystem engineer with broad distribution, high intraspecific...