PKM2, cancer metabolism,and the road ahead

A major metabolic aberration associated with cancer is a change in glucose metabolism. Isoform selection of the glycolytic enzyme pyruvate kinase has been implicated in the metabolic phenotype of cancer cells, and specific pyruvate kinase isoforms have been suggested to support divergent energetic and biosynthetic requirements of cells in tumors and normal tissues. PKM2 isoform expression has been closely linked to embryogenesis, tissue repair, and cancer. In contrast, forced expression of the PKM1 isoform has been associated with reduced tumor cell proliferation. Here, we discuss the role that PKM2 plays in cells and provide a historical perspective for how the study of PKM2 has contributed to understanding cancer metabolism. We also review recent studies that raise important questions with regard to the role of PKM2 in both normal and cancer cell metabolism.     

Efficient disruption and replacement of an effector gene in the oomycete Phytophthora sojae using CRISPR/Cas9

Phytophthora sojae is an oomycete pathogen of soybean. As a result of its economic importance, P. sojae has become a model for the study of oomycete genetics, physiology and pathology. The lack of efficient techniques for targeted mutagenesis and gene replacement have long hampered genetic studies of pathogenicity in Phytophthora species. Here, we describe a CRISPR/Cas9 system enabling rapid and efficient genome editing in P. sojae. Using the RXLR effector gene Avr4/6 as a target, we observed that, in the absence of a homologous template, the repair of Cas9‐induced DNA double‐strand breaks (DSBs) in P. sojae was mediated by non‐homologous end‐joining (NHEJ), primarily resulting in short indels. Most mutants were homozygous, presumably as a result of gene conversion triggered by Cas9‐mediated cleavage of non‐mutant alleles. When donor DNA was present, homology‐directed repair (HDR) was observed, which resulted in the replacement of Avr4/6 with the NPT II gene. By testing the specific virulence of several NHEJ mutants and HDR‐mediated gene replacements in soybean, we have validated the contribution of Avr4/6 to recognition by soybean R gene loci, Rps4 and Rps6, but also uncovered additional contributions to resistance by these two loci. Our results establish a powerful tool for the study of functional genomics in Phytophthora, which provides new avenues for better control of this pathogen.     

What Is the Tree of Life?

A universal Tree of Life (TOL) has long been a goal of molecular phylogeneticists, but reticulation at the level of genes and possibly at the levels of cells and species renders any simple interpretation of such a TOL, especially as applied to prokaryotes, problematic.One of the several ways in which microbiology puts the neo-Darwinian synthesis in jeopardy is by the threatening to “uproot the Tree of Life (TOL)” [1]. Lateral gene transfer (LGT) is much more frequent than most biologists would have imagined up until about 20 years ago, so phylogenetic trees based on sequences of different prokaryotic genes are often different. How to tease out from such conflicting data something that might correspond to a single, universal Tree of Life becomes problematic. Moreover, since many important evolutionary transitions involve lineage fusions at one level or another, the aptness of a tree (a pattern of successive bifurcations) as a summary of life’s history is uncertain [2–4].     

Genetic structure of a disjunct peripheral population of mountain sucker <Emphasis Type="Italic">Pantosteus jordani</Emphasis> in the Black Hills,South Dakota,USA

A peripheral population of mountain sucker, Pantosteus jordani, located in the Black Hills of South Dakota, USA, represents the eastern-most range of the species and is completely isolated from other populations. Over the last 50 years, mountain sucker populations have declined in the Black Hills, and now only occur in 40 % of the historic local range, with densities decreasing by more than 84 %.We used microsatellite DNA markers to estimate genetic diversity and to assess population structure across five streams where mountain suckers persist. We evaluated results in the context of recent ecological surveys to inform decisions about mountain sucker conservation. Significant allele frequency differences existed among sample streams (Global F_ST = 0.041) but there was no evidence of isolation by distance. Regionally, genetic effective size, N_e, was estimated to be at least 338 breeding individuals, but N_e within streams was expected to be less. Despite almost complete demographic isolation and reduced population size, there appears to be little evidence of inbreeding, but genetic drift and local isolation due to fragmentation probably best explains genetic structure in this peripheral mountain sucker population. Recommended strategies for population enhancement include restoration of stream connectivity and habitat improvement. Moreover, repatriation and assisted movement (i.e., gene flow) of fishes should maximize genetic diversity in stream fragments in the Black Hills region.     

Sil1, a nucleotide exchange factor for BiP,is not required for antibody assembly or secretion

Sil1 is a nucleotide exchange factor for the endoplasmic reticulum chaperone BiP, and mutations in this gene lead to Marinesco–Sjögren syndrome (MSS), a debilitating autosomal recessive disease characterized by multisystem defects. A mouse model for MSS was previously produced by disrupting Sil1 using gene-trap methodology. The resulting Sil1^Gt mouse phenocopies several pathologies associated with MSS, although its ability to assemble and secrete antibodies, the best-characterized substrate of BiP, has not been investigated. In vivo antigen-specific immunizations and ex vivo LPS stimulation of splenic B cells revealed that the Sil1^Gt mouse was indistinguishable from wild-type age-matched controls in terms of both the kinetics and magnitude of antigen-specific antibody responses. There was no significant accumulation of BiP-associated Ig assembly intermediates or evidence that another molecular chaperone system was used for antibody production in the LPS-stimulated splenic B cells from Sil1^Gt mice. ER chaperones were expressed at the same level in Sil1^WT and Sil1^Gt mice, indicating that there was no evident compensation for the disruption of Sil1. Finally, these results were confirmed and extended in three human EBV-transformed lymphoblastoid cell lines from individuals with MSS, leading us to conclude that the BiP cofactor Sil1 is dispensable for antibody production.     

Model of Fission Yeast Cell Shape Driven by Membrane-Bound Growth Factors and the Cytoskeleton

Fission yeast serves as a model for how cellular polarization machinery consisting of signaling molecules and the actin and microtubule cytoskeleton regulates cell shape. In this work, we develop mathematical models to investigate how these cells maintain a tubular shape of approximately constant diameter. Many studies identify active Cdc42, found in a cap at the inner membrane of growing cell tips, as an important regulator of local cell wall remodeling, likely through control of exocyst tethering and the targeting of other polarity-enhancing structures. First, we show that a computational model with Cdc42-dependent local cell wall remodeling under turgor pressure predicts a relationship between spatial extent of growth signal and cell diameter that is in agreement with prior experiments. Second, we model the consequences of feedback between cell shape and distribution of Cdc42 growth signal at cell tips. We show that stability of cell diameter over successive cell divisions places restrictions on their mutual dependence. We argue that simple models where the spatial extent of the tip growth signal relies solely on geometrical alignment of confined microtubules might lead to unstable width regulation. Third, we study a computational model that combines a growth signal distributed over a characteristic length scale (as, for example, by a reaction-diffusion mechanism) with an axis-sensing microtubules system that places landmarks at positions where microtubule tips touch the cortex. A two-dimensional implementation of this model leads to stable cell diameter for a wide range of parameters. Changes to the parameters of this model reproduce straight, bent, and bulged cell shapes, and we discuss how this model is consistent with other observed cell shapes in mutants. Our work provides an initial quantitative framework for understanding the regulation of cell shape in fission yeast, and a scaffold for understanding this process on a more molecular level in the future.     

Purine-less death in Plasmodium falciparum induced by immucillin-H, a transition state analogue of purine nucleoside phosphorylase.

Plasmodium falciparum is responsible for the majority of life-threatening cases of malaria. Plasmodia species cannot synthesize purines de novo, whereas mammalian cells obtain purines from de novo synthesis or by purine salvage. Hypoxanthine is proposed to be the major source of purines for P. falciparum growth. It is produced from inosine phosphorolysis by purine nucleoside phosphorylase (PNP). Immucillins are powerful transition state analogue inhibitors of mammalian PNP and also inhibit P. falciparum PNP as illustrated in the accompanying article (Kicska, G. A., Tyler, P. C., Evans, G. B., Furneaux, R. H., Kim, K., and Schramm, V. L. (2002) J. Biol. Chem. 277, 3219-3225). This work tests the hypothesis that erythrocyte and P. falciparum PNP are essential elements for growth and survival of the parasite in culture. Immucillin-H reduces the incorporation of inosine but not hypoxanthine into nucleic acids of P. falciparum and kills P. falciparum cultured in human erythrocytes with an IC(50) of 35 nm. Growth inhibition by Imm-H is reversed by the addition of hypoxanthine but not inosine, demonstrating the metabolic block at PNP. The concentration of Imm-H required for inhibition of parasite growth varies as a function of culture hematocrit, reflecting stoichiometric titration of human erythrocyte PNP by the inhibitor. Human and P. falciparum PNPs demonstrate different specificity for inhibition by immucillins, with the 2'-deoxy analogues showing marked preference for the human enzyme. The IC(50) values for immucillin analogue toxicity to P. falciparum cultures indicate that inhibition of PNP in both the erythrocytes and the parasite is necessary to induce a purine-less death.