The evolving biology of small molecules: controlling cell fate and identity

Small molecules have been playing important roles in elucidating basic biology and treatment of a vast number of diseases for nearly a century, making their use in the field of stem cell biology a comparatively recent phenomenon. Nonetheless, the power of biology-oriented chemical design and synthesis, coupled with significant advances in screening technology, has enabled the discovery of a growing number of small molecules that have improved our understanding of stem cell biology and allowed us to manipulate stem cells in unprecedented ways. This review focuses on recent small molecule studies of (i) the key pathways governing stem cell homeostasis, (ii) the pluripotent stem cell niche, (iii) the directed differentiation of stem cells, (iv) the biology of adult stem cells, and (v) somatic cell reprogramming. In a very short period of time, small molecules have defined a perhaps universally attainable naive ground state of pluripotency, and are facilitating the precise, rapid and efficient differentiation of stem cells into somatic cell populations relevant to the clinic. Finally, following the publication of numerous groundbreaking studies at a pace and consistency unusual for a young field, we are closer than ever to completely eliminating the need for genetic modification in reprogramming.     

Quantifying Interactions Within the NADP(H) Enzyme Network in Drosophila melanogaster

In this report, we use synthetic, activity-variant alleles in Drosophila melanogaster to quantify interactions across the enzyme network that reduces nicotinamide adenine dinucleotide phosphate (NADP) to NADPH. We examine the effects of large-scale variation in isocitrate dehydrogenase (IDH) or glucose-6-phosphate dehydrogenase (G6PD) activity in a single genetic background and of smaller-scale variation in IDH, G6PD, and malic enzyme across 10 different genetic backgrounds. We find significant interactions among all three enzymes in adults; changes in the activity of any one source of a reduced cofactor generally result in changes in the other two, although the magnitude and directionality of change differs depending on the gene and the genetic background. Observed interactions are presumably through cellular mechanisms that maintain a homeostatic balance of NADPH/NADP, and the magnitude of change in response to modification of one source of reduced cofactor likely reflects the relative contribution of that enzyme to the cofactor pool. Our results suggest that malic enzyme makes the largest single contribution to the NADPH pool, consistent with the results from earlier experiments in larval D. melanogaster using naturally occurring alleles. The interactions between all three enzymes indicate functional interdependence and underscore the importance of examining enzymes as components of a network.IN traits determined by a network of gene products, the phenotype is a function of the alleles present and of the relative contributions of individual network member genes. Since selection is on phenotype, the total composite genotype, not just individual loci, determines the fitness of an organism. In establishing the connection between genotype and phenotype for such networks, the first challenge is to quantify the relative contribution of each member of the network to the endpoint phenotype. By addressing function on a network-wide basis, interactions and interconnections that may not be apparent in individual gene examinations can be determined (Proulx et al. 2005).In most organisms, reduction of the cofactor nicotinamide adenine dinucleotide phosphate, or NADP, to NADPH is primarily the function of four enzymes: cytosolic malic enzyme (MEN), cytosolic isocitrate dehydrogenase (IDH), and the two oxidative enzymes of the pentose shunt, glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate (6PGD; recently reviewed in Ying 2008). In larval Drosophila melanogaster, MEN produces ∼30% of the available NADPH, IDH ∼20%, and G6PD and 6PGD the remaining ∼40% (Geer et al. 1979a,b). It is believed that these four enzymes interact to maintain the NADP/NADPH balance and supply of reducing power for lipogenesis and antioxidation (Geer et al. 1976, 1978, 1981; Wilton et al. 1982; Bentley et al. 1983; Geer and Laurie-Ahlberg 1984; Merritt et al. 2005; Pollak et al. 2007; Singh et al. 2007; Ying 2008). Dietary induction studies and observations of natural genetic variation have found connections between MEN activity and the activities of the pentose shunt enzymes to be generally straightforward and compensatory; reductions in one lead to increases in the other. The interactions involving IDH activity, however, have been found to be more complicated and at times counterintuitive; reductions in reducing power sometimes lead to decreases in IDH activity.In an earlier study (Merritt et al. 2005), we quantified the impact of genetic variation in Men activity on IDH and G6PD activities and triglyceride (a strong correlate with total lipid; Clark and Keith 1989) concentration. 6PGD was not independently assayed because earlier works suggest that G6PD and 6PGD activities are highly correlated, likely because of their coupled function in the pentose shunt (Wilton et al. 1982). We examined both naturally occurring Men alleles and synthetic alleles created by P-element excision and found significant associations between MEN activity and induction of the activities of both IDH and G6PD. The apparent interactions between MEN and IDH and G6PD across these 10 different third chromosome lines were quantified as mean elasticity coefficients: = −0.76 ± 0.236 and = −0.88 ± 0.208. Because MEN activity was reduced by 20%, both IDH and G6PD activity varied in a compensatory direction, increasing almost 1:1 with the decrease in MEN.The significant change in enzyme activity of two members of the NADPH network in response to our genetic reduction of the activity of a third strongly suggests that a physiological mechanism coregulates the three enzymes. Such functional interdependence would mean that individual members of the network do not act in isolation and should be examined collectively, not as isolated units. In this study, we characterize the effects of the independently varying activity levels of IDH, G6PD, and MEN on the activity of each other and triglyceride concentration in adult flies. We found significant responses to changes in all three enzymes, although the responses to genetic changes in IDH and G6PD were generally small; variation in MEN caused the greatest changes in the other enzymes.     

Population genetic structure of turbot (Scophthalmus maximus L., 1758) in the Black Sea

Turbot, Scophthalmus maximus, is a commercially important demersal flatfish species distributed throughout the Black Sea. Several studies performed locally with a limited number of specimens using both mitochondrial DNA (mtDNA) and microsatellite markers evidenced notable genetic variation among populations. However, comprehensive population genetic studies are required to help management of the species in the Black Sea. In the present study eight microsatellite loci were used to resolve the population structure of 414 turbot samples collected from 12 sites across the Black Sea. Moreover, two mtDNA genes, COI and Cyt-b, were used for taxonomic identification. Microsatellite markers of Smax-04 and B12-I GT14 were excluded from analysis due to scoring issues. Data analysis was performed with the remaining six loci. Loci were highly polymorphic (average of 17.8 alleles per locus), indicating high genetic variability. Locus 3/20CA17, with high null allele frequency (>30%), significantly deviated from HW equilibrium. Pairwise comparison of the F_ST index showed significant differences between most of the surveyed sampling sites (P < 0.01). Cluster analysis evidenced the presence of three genetic groups among sampling sites. Significant genetic differentiation between Northern (Sea of Azov and Crimea) and Southern (Turkish Black Sea Coast) Black Sea sampling sites were detected. The Mantel test supported an isolation by distance model of population structure. These findings are vital for long-term sustainable management of the species and development of conservation programs. Moreover, generated mtDNA sequences would be useful for the establishment of a database for S. maximus.     

Phosphatidic Acid Mediates the Nem1-Spo7/Pah1 Phosphatase Cascade in Yeast Lipid Synthesis

In the yeast Saccharomyces cerevisiae, the PAH1-encoded Mg²⁺-dependent phosphatidate (PA) phosphatase Pah1 regulates the bifurcation of PA to diacylglycerol (DAG) for triacylglycerol (TAG) synthesis and to CDP-DAG for phospholipid synthesis. Pah1 function is mainly regulated via control of its cellular location by phosphorylation and dephosphorylation. Pah1 phosphorylated by multiple protein kinases is sequestered in the cytosol apart from its substrate PA in the membrane. The phosphorylated Pah1 is then recruited and dephosphorylated by the protein phosphatase complex Nem1 (catalytic subunit)-Spo7 (regulatory subunit) in the endoplasmic reticulum. The dephosphorylated Pah1 hops onto and scoots along the membrane to recognize PA for its dephosphorylation to DAG. Here, we developed a proteoliposome model system that mimics the Nem1-Spo7/Pah1 phosphatase cascade to provide a tool for studying Pah1 regulation. Purified Nem1-Spo7 was reconstituted into phospholipid vesicles prepared in accordance with the phospholipid composition of the nuclear/endoplasmic reticulum membrane. The Nem1-Spo7 phosphatase reconstituted in the proteoliposomes, which were measured 60 nm in an average diameter, was catalytically active on Pah1 phosphorylated by Pho85-Pho80, and its active site was located at the external side of the phospholipid bilayer. Moreover, we determined that PA stimulated the Nem1-Spo7 activity, and the regulatory effect was governed by the nature of the phosphate headgroup but not by the fatty acyl moiety of PA. The reconstitution system for the Nem1-Spo7/Pah1 phosphatase cascade, which starts with the phosphorylation of Pah1 by Pho85-Pho80 and ends with the production of DAG, is a significant advance to understand a regulatory cascade in yeast lipid synthesis.Supplementary key words: phosphatidate, diacylglycerol, triacylglycerol, phosphatidate phosphatase, Pho85-Pho80, Nem1-Spo7 protein phosphatase, endoplasmic reticulum, phospholipid bilayer, reconstitution, proteoliposome     

The Schizosaccharomyces pombe JmjC-Protein,Msc1, Prevents H2A.Z Localization in Centromeric and Subtelomeric Chromatin Domains

Eukaryotic genomes are repetitively packaged into chromatin by nucleosomes, however they are regulated by the differences between nucleosomes, which establish various chromatin states. Local chromatin cues direct the inheritance and propagation of chromatin status via self-reinforcing epigenetic mechanisms. Replication-independent histone exchange could potentially perturb chromatin status if histone exchange chaperones, such as Swr1C, loaded histone variants into wrong sites. Here we show that in Schizosaccharomyces pombe, like Saccharomyces cerevisiae, Swr1C is required for loading H2A.Z into specific sites, including the promoters of lowly expressed genes. However S. pombe Swr1C has an extra subunit, Msc1, which is a JumonjiC-domain protein of the Lid/Jarid1 family. Deletion of Msc1 did not disrupt the S. pombe Swr1C or its ability to bind and load H2A.Z into euchromatin, however H2A.Z was ectopically found in the inner centromere and in subtelomeric chromatin. Normally this subtelomeric region not only lacks H2A.Z but also shows uniformly lower levels of H3K4me2, H4K5, and K12 acetylation than euchromatin and disproportionately contains the most lowly expressed genes during vegetative growth, including many meiotic-specific genes. Genes within and adjacent to subtelomeric chromatin become overexpressed in the absence of either Msc1, Swr1, or paradoxically H2A.Z itself. We also show that H2A.Z is N-terminally acetylated before, and lysine acetylated after, loading into chromatin and that it physically associates with the Nap1 histone chaperone. However, we find a negative correlation between the genomic distributions of H2A.Z and Nap1/Hrp1/Hrp3, suggesting that the Nap1 chaperones remove H2A.Z from chromatin. These data describe H2A.Z action in S. pombe and identify a new mode of chromatin surveillance and maintenance based on negative regulation of histone variant misincorporation.     

Triglyceride pools, flight and activity variation at the Gpdh locus in Drosophila melanogaster

We have created a set of P-element excision-derived Gpdh alleles that generate a range of GPDH activity phenotypes ranging from zero to full activity. By placing these synthetic alleles in isogenic backgrounds, we characterize the effects of minor and major activity variation on two different aspects of Gpdh function: the standing triglyceride pool and glycerol-3-phosphate shuttle-assisted flight. We observe small but statistically significant reductions in triglyceride content for adult Gpdh genotypes possessing 33-80% reductions from normal activity. These small differences scale to a notable proportion of the observed genetic variation in triglyceride content in natural populations. Using a tethered fly assay to assess flight metabolism, we observed that genotypes with 100 and 66% activity exhibited no significant difference in wing-beat frequency (WBF), while activity reductions from 60 to 10% showed statistically significant reductions of approximately 7% in WBF. These studies show that the molecular polymorphism associated with GPDH activity could be maintained in natural populations by selection in the triglyceride pool.     

Modulation of hydrogen sulfide gasotransmitter limits the proven benefits of garlic

Phytochemistry Reviews - Garlic has been used as a medicinal food for centuries. The biological activity of garlic relies on the presence of highly reactive sulfur compounds. Widespread in vitro...     

The limited role of NH2-terminal c-Jun phosphorylation in neuronal apoptosis: identification of the nuclear pore complex as a potential target of the JNK pathway

c-Jun is induced in many neuronal death paradigms. A critical step in c-Jun regulation involves phosphorylation of Ser63/Ser73 located in the NH2-terminal transactivation domain. To determine the importance of this phosphorylation for neuronal apoptosis, we analyzed the sympathetic neurons of mice carrying a mutant c-Jun gene that lacks Ser63/Ser73 phosphorylation sites (jun aa). Trophic factor-deprivation or DNA damage-induced death was significantly delayed in jun aa/aa neurons. Neuronal c-Jun induction was only partially inhibited, demonstrating that phosphorylation of Ser63/73 is not required for c-Jun activation. The inductions of proapoptotic BH3-only proteins, Bim and PUMA/Bbc3, were delayed during neuronal apoptosis in mutant neurons. These results demonstrate that NH2-terminal c-Jun phosphorylation is important, but not necessary, for the induction of proapoptotic genes and neuronal apoptosis. Thus, additional JNK substrates may be critical for neuronal death. As potential mediators, we identified additional nuclear MLK/JNK substrates, including Nup214 subunit of the nuclear pore complex.