Spontaneous immortalization of mouse embryo cells: strain differences and changes in gene expression with particular reference to retroviral gag-pol genes.

We have studied the kinetics with which cultures of primary mouse embryo cells pass through the crisis period, escape their terminal differentiation (cellular senescence), and give rise to an immortal cell line. The process is strain-dependent, with cells from the outbred Swiss CD-1 mouse being considerably more adept at forming an immortal 3T3 line than cells from the inbred SWR line; Balb/c cells appeared intermediate in their behavior. The continued presence of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate or the poly(ADPribose)polymerase inhibitor 3-aminobenzamide affected the kinetics but did not seem to alter the outcome. Changes in expression of various genes, including those encoding mitogen-regulated protein (proliferin), endogenous gag-pol retrovirus sequences, insulin-like growth factor II, and a variety of protooncogenes, were monitored during the process of immortalization, and although certain changes were reproducibly characteristic of cells from a given mouse strain passed according to a specific regimen, none of the observed changes were reproducibly characteristic under all conditions of immortalization. In particular, our data indicate the absence of a strict correlation between cellular immortalization and the activation of endogenous gag-pol expression. We conclude from our observations that the establishment of permanent lines from primary mouse embryo cells in serum-containing medium reflects the selection of a variant subpopulation of cells that did not preexist but rather arose in response to the specific culture conditions by a process resembling differentiation. Multiple and complex changes in gene expression occur that are affected by the culture conditions and the strain (genotype) of the mouse.     

Genetic Connectivity of the Moth Pollinated Tree Glionnetia sericea in a Highly Fragmented Habitat

Long-distance gene flow is thought to be one prerequisite for the persistence of plant species in fragmented environments. Human influences have led to severe fragmentation of native habitats in the Seychelles islands, with many species surviving only in small and isolated populations. The endangered Seychelles endemic tree Glionnetia sericea is restricted to altitudes between 450 m and 900 m where the native forest vegetation has been largely lost and replaced with exotic invasives over the last 200 years. This study explores the genetic and ecological consequences of population fragmentation in this species by analysing patterns of genetic diversity in a sample of adults, juveniles and seeds, and by using controlled pollination experiments. Our results show no decrease in genetic diversity and no increase in genetic structuring from adult to juvenile cohorts. Despite significant inbreeding in some populations, there is no evidence of higher inbreeding in juvenile cohorts relative to adults. A Bayesian structure analysis and a tentative paternity analysis indicate extensive historical and contemporary gene flow among remnant populations. Pollination experiments and a paternity analysis show that Glionnetia sericea is self-compatible. Nevertheless, outcrossing is present with 7% of mating events resulting from pollen transfer between populations. Artificial pollination provided no evidence for pollen limitation in isolated populations. The highly mobile and specialized hawkmoth pollinators (Agrius convolvuli and Cenophodes tamsi; Sphingidae) appear to promote extensive gene flow, thus mitigating the potential negative ecological and genetic effects of habitat fragmentation in this species. We conclude that contemporary gene flow is sufficient to maintain genetic connectivity in this rare and restricted Seychelles endemic, in contrast to other island endemic tree species with limited contemporary gene flow.     

A Synechococcus leopoliensis SAUG 1402-1 operon harboring the 1-deoxyxylulose 5-phosphate synthase gene and two additional open reading frames is functionally involved in the dimethylallyl diphosphate synthesis

Experiments have been performed to prove the existence and the functionality of the novel mevalonate independent 1-deoxyxylulose 5-phosphate isoprenoid biosynthesis pathway in cyanobacteria. For this purpose, a segment of the 1-deoxyxylulose 5-phosphate synthase gene (dxs) was amplified from Synechococcus leopoliensis SAUG 1402-1 DNA via PCR using oligonucleotides for conserved regions of dxs. Subsequent hybridization screening of a genomic cosmid library of S. leopoliensis with this segment has led to the identification of an 18.7 kbp segment of the S. leopoliensis genome on which a dxs homologous gene and two adjacent open reading frames organized in one operon could be localized by DNA sequencing. The three genes of the operon were separately expressed in Escherichia coli, proving that the identified cyanobacterial dxs is functionally involved in the formation of dimethylallyl diphosphate, one basic intermediate of isoprenoid biosynthesis.     

Effects of Chronic D-Serine Elevation on Animal Models of Depression and Anxiety-Related Behavior

NMDA receptors are activated after binding of the agonist glutamate to the NR2 subunit along with a co-agonist, either L-glycine or D-serine, to the NR1 subunit. There is substantial evidence to suggest that D-serine is the most relevant co-agonist in forebrain regions and that alterations in D-serine levels contribute to psychiatric disorders. D-serine is produced through isomerization of L-serine by serine racemase (Srr), either in neurons or in astrocytes. It is released by astrocytes by an activity-dependent mechanism involving secretory vesicles. In the present study we generated transgenic mice (SrrTg) expressing serine racemase under a human GFAP promoter. These mice were biochemically and behaviorally analyzed using paradigms of anxiety, depression and cognition. Furthermore, we investigated the behavioral effects of long-term administration of D-serine added to the drinking water. Elevated brain D-serine levels in SrrTg mice resulted in specific behavioral phenotypes in the forced swim, novelty suppression of feeding and olfactory bulbectomy paradigms that are indicative of a reduced proneness towards depression-related behavior. Chronic dietary D-serine supplement mimics the depression-related behavioral phenotype observed in SrrTg mice. Our results suggest that D-serine supplementation may improve mood disorders.     

Trade-off between thermal sensitivity,hypoxia tolerance and growth in fish

One outcome of contemporary climate trends is that the involvement of hypoxia and heat tolerance in determining individual fitness will increase in many fish populations. Large fish are believed to be more tolerant to hypoxia than small fish (Nilsson and Östlund-Nilsson, 2008) whereas thermal sensitivity is thought to decrease with body size (Clark et al., 2008). To better understand the bases of inter-individual variation in environmental adaptation performance, the current study examined hypoxia and heat tolerance in a fast growing (FGS; 288.3 ±14.4 g, 26.04±0.49 cm) and a slow growing (SGS; 119.95±6.41 g; 20.98±0.41 cm) strain of 1-year old rainbow trout (Oncorhynchus mykiss). This examination was conducted using two standardized challenge tests aimed at assessing individual incipient lethal oxygen saturation and incipient upper lethal temperature. Results to these tests were then cross-correlated with swim tests during which individual basal and active metabolic rate values were also measured. Measurements of permeabilized ventricular myofibers oxygen consumption were also conducted, as well as various organ-to-body-mass ratios. Experimental data showed that FGS was more hypoxia tolerant than SGS (13.4 to 16.7% air sat versus 14.7 to 18.9% air sat respectively). On the other hand, FGS was found less tolerant to heat than SGS (24.7–27.6 °C versus 28.5 to 29.7 °C respectively). Adding to the body size effect, another source of inter-individual variation in environmental tolerance was found. Residual analysis highlighted that whereas none of the individual morphometric and energetic traits correlated with hypoxia tolerance, permeabilized ventricular myofibers maximal oxygen consumption correlated well with individual tolerance to heat.     

Mitochondrial injury by disulfiram: two different mechanisms of the mitochondrial permeability transition.

Disulfiram (Ds), a clinically employed alcohol deterrent of the thiuram disulfide (TD) class of compounds, is known to cause hepatitis and neuropathies. Although this drug has been shown to inhibit different thiol-containing enzymes, the actual mechanism of Ds toxicity is not clear. We have previously demonstrated that Ds impairs the permeability of inner mitochondrial membrane (IMM) [Arch. Biochem. Biophys. 356 (1998) 46]. In this report, the effect of Ds and its structural analogue thiram (Th) on mitochondrial functions was studied in detail. We found that mitochondria metabolize TDs in a NAD(P)H- and GSH-dependent manner. At the concentration above characteristic threshold, TDs induced irreversible oxidation of NAD(P)H and glutathione (GSH) pools, collapse of transmembrane potential, and inhibition of oxidative phosphorylation. The presence of Ca(2+) and exhaustion of mitochondrial glutathione (GSH+GSSG) decreased the threshold concentration of TDs. Swelling of the mitochondria and leakage of non-transported fluorescent dye BCECF from the matrix indicated that TDs induced the mitochondrial permeability transition (MPT). Mitochondrial permeabilization by TDs involves two, apparently distinct mechanisms. In the presence of Ca(2+), TDs produced cylosporin A-sensitive swelling of mitochondria, which was inhibited by ADP and accelerated by carboxyatractyloside (CATR) and phosphate. In contrast, the swelling produced by TDs in the absence of Ca(2+) was not sensitive to cyclosporin A (CsA), ADP and CATR but was inhibited by phosphate. Titration with N-ethylmaleimide revealed that these two mechanisms involve different SH-groups and probably different transport proteins on the IMM. Our findings indicate that at pharmacologically relevant concentrations TDs may cause an irreversible mitochondrial injury as a result of induction of the MPT.     

The Salmonella mutagenicity test: evaluation of 29 drug candidates

The Salmonella mutagenicity test (Ames assay) is part of the routine screening battery applied to all new drugs at The Upjohn Company. The purpose of this paper is to report results for 29 compounds. These compounds are very diverse in chemical structure and represent classes of compounds selected because of known biological activity and other reasons. None of the compounds reported here produced an increase in revertant colonies in the Salmonella strains employed (TA98, TA100, TA1535, TA1537 and TA1538) and therefore the Salmonella mutagenicity results with these materials do not suggest potential for mutagenesis or carcinogenesis.     

Ant diversity decreases during the dry season: A meta-analysis of the effects of seasonality on ant richness and abundance

Tropical studies traditionally describe insect diversity variation throughout the year. The temporally structured responses of insect assemblages to climate seasonality vary across ecosystems due to gradients of resource availability and limiting ecological factors. These idiosyncratic responses might be particularly true across the vast geographical range of the Brazilian territory, including various environments that harbor one of the most diverse ant faunas worldwide. This study addressed the relationship between ant diversity and climatic seasonality, performing a quantitative review of the published data on ant diversity collected in Brazil. We investigated the seasonality effect on ant abundance and richness described in the literature in 47 papers published between 2000 and 2018. These studies were developed mainly in the Atlantic Forest biome and collected ants with pitfall traps on the soil/litter stratum. We initially carried out a vote-counting procedure by comparing the number of significant results describing seasonal differences in the ant assemblage. We found that most papers described a similar pattern of ant abundance, richness, and species composition between seasons. However, when we performed a meta-analysis, we observed a clear pattern of higher ant abundance and richness in the wet/summer season compared with the dry/winter season. Our meta-analysis reveals that the ant diversity decreases in the dry season, strongly in the Cerrado biome. Additionally, we point out differences in the sampling effort across biomes, indicating the need for further investments in studies focused on temporal diversity patterns, including seasonal effects, on the insect assemblage in biomes less investigated so far. Abstract in Portuguese is available with online material.