Embryonic stem cells induce pluripotency in somatic cell fusion through biphasic reprogramming

It is a long-held paradigm that cell fusion reprograms gene expression but the extent of reprogramming and whether it is affected by the cell types employed remain unknown. We recently showed that the silencing of somatic genes is attributable to either trans-acting cellular environment or cis-acting chromatin context. Here, we examine how trans- versus cis-silenced genes in a somatic cell type behave in fusions to another somatic cell type or to embryonic stem cells (ESCs). We demonstrate that while reprogramming of trans-silenced somatic genes occurs in both cases, reprogramming of cis-silenced somatic genes occurs only in somatic-ESC fusions. Importantly, ESCs reprogram the somatic genome in two distinct phases: trans-reprogramming occurs rapidly, independent of DNA replication, whereas cis-reprogramming occurs with slow kinetics requiring DNA replication. We also show that pluripotency genes Oct4 and Nanog are cis-silenced in somatic cells. We conclude that cis-reprogramming capacity is a fundamental feature distinguishing ESCs from somatic cells.     

3D models of human ERα and ERβ complexed with 5-androsten-3β,17β-diol

Recently, binding of 5-androsten-3β,17β-diol (Δ(5)-androstenediol) to human estrogen receptor-beta (ERβ) was found to repress microglia-mediated inflammation, which is associated with various neurodegenerative diseases, such as multiple sclerosis. In contrast, binding of estradiol to ERβ resulted in little or no repression of microglia-mediated inflammation. Binding of Δ(5)-androstenediol to ERβ, as well as to ERα, is unexpected because unlike estradiol, Δ(5)-androstenediol has a saturated A ring and a C19 methyl group. To begin to elucidate the interaction of Δ(5)-androstenediol with both ERs, we constructed 3D models of Δ(5)-androstenediol with human ERα and ERβ for comparison with the crystal structures of estradiol in ERα and ERβ. Conformational flexibility in human ERα and ERβ accommodates the C19 methyl on Δ(5)-androstenediol. This conformational flexibility may be relevant for binding of other Δ(5)-steroids with C19 methyl substituents, such as 25-hydroxycholesterol and 27-hydroxycholesterol, to ERs.     

Progesterone and placentation increase secreted phosphoprotein one (SPP1 or osteopontin) in uterine glands and stroma for histotrophic and hematotrophic support of ovine pregnancy

Secreted phosphoprotein one (SPP1, osteopontin) may regulate conceptus implantation and placentation. We investigated effects of progesterone (P(4)) and the conceptus on expression and localization of SPP1 in the ovine uterus. Steady-state levels of SPP1 mRNA in the endometrium of unilaterally pregnant ewes did not differ significantly between nongravid and gravid horns within their respective days of pregnancy; however, levels did increase as pregnancy progressed. SPP1 mRNA was detectable in the glandular epithelium (GE) of both nongravid and gravid horns via in situ hybridization. SPP1 protein was localized to the apical surface of the luminal epithelium of both nongravid and gravid uterine horns. Gravid horns exhibited extensive stromal SPP1 on Days 40 through 120, whereas SPP1 was markedly lower in the stroma of nongravid uterine horns through Day 80 of pregnancy. By Day 120, stromal expression of SPP1 between nongravid and gravid horns was similar. Long-term P(4) treatment of ovariectomized ewes induced SPP1 in the uterine stroma and GE. A bioactive 45-kDa SPP1 fragment was purified from uterine secretions and promoted ovine trophectoderm cell attachment in vitro. Interestingly, increased stromal cell expression of SPP1 was positively associated with vascularization as assessed by von Willebrand factor staining. Finally, ovine uterine artery endothelial cells produced SPP1 during outgrowth into three-dimensional collagen matrices in an in vitro model system that recapitulates angiogenesis. Collectively, P(4) induces and the conceptus further stimulates SPP1 in uterine GE and stroma, where SPP1 likely influences histotrophic and hematotrophic support of conceptus development.     

Assessment of decorin-binding protein A to the infectivity of <Emphasis Type="Italic">Borrelia burgdorferi</Emphasis> in the murine models of needle and tick infection

Background  

Decorin-binding proteins (Dbps) A and B of Borrelia burgdorferi, the agent of Lyme disease, are surface-exposed lipoproteins that presumably bind to the extracellular matrix proteoglycan, decorin. B. burgdorferi infects various tissues including the bladder, heart, joints, skin and the central nervous system, and the ability of B. burgdorferi to bind decorin has been hypothesized to be important for this disseminatory pathogenic strategy.     

Evolution to environmental contamination ablates the circadian clock of an aquatic sentinel species

Environmental contamination is a common cause of rapid evolution. Recent work has shown that Daphnia pulex, an important freshwater species, can rapidly evolve increased tolerance to a common contaminant, sodium chloride (NaCl) road salt. While such rapid evolution can benefit organisms, allowing them to adapt to new environmental conditions, it can also be associated with unforeseen tradeoffs. Given that exposure to environmental contaminants can cause circadian disruption, we investigated whether the circadian clock was affected by evolving a tolerance to high levels of road salt. By tracking the oscillations of a putative clock gene, period, we demonstrated that D. pulex express per mRNA with approximately 20‐hr oscillations under control conditions. This putative circadian rhythm was ablated in response to high levels of salinity; populations adapted to high NaCl concentrations exhibited an ablation of period oscillation. Moreover, we showed that while gene expression is increased in several other genes, including clock, actin, and Na⁺/K⁺‐ATPase, upon the adaptation to high levels of salinity, per expression is unique among the genes we tracked in that it is the only gene repressed in response to salt adaptation. These results suggest that rapid evolution of salt tolerance occurs with the tradeoff of suppressed circadian function. The resultant circadian disruption may have profound consequences to individuals, populations, and aquatic food webs by affecting species interactions. In addition, our research suggests that circadian clocks may also be disrupted by the adaptation to other environmental contaminants.     

A comparison of protein extraction methods optimizing high protein yields from marine algae and cyanobacteria

Algae are a diverse group of aquatic autotrophs that represent important models for studying morphology, physiology, and molecular biology. Many, because of their adaptation to the marine environment, cell wall composition, and secondary metabolites, present difficulties when trying to extract significant amounts of protein for analyses. In this study, we compared protein extraction methods for 39 different species of marine algae and cyanobacteria. Since successful protein detection requires the liberation of protein from a cell, the goal of the current investigation was to establish a high yield protein extraction method that would allow for protein extraction from different organisms ranging from cyanobacteria, unicellular, multicellular, and coenocytic algae. Six protein extraction assays were tested (three from literature citations and three from commercially available kits). A modification of the technique originally described by Barbarino and Lourenço (J Appl Phycol 17:447–460, 2005) displayed significantly higher yields (p > 0.001, average of 6.7–30.87% dry weight) in all algal and cyanobacterial species examined. Protein yields using this modified procedure were especially successful in the Trebouxiophyceae, Bangiophyceae, Phaeophyceae, and Cyanobacteria.     

V490M, a common mutation in 3-phosphoglycerate dehydrogenase deficiency, causes enzyme deficiency by decreasing the yield of mature enzyme.

A deficiency of 3-phosphoglycerate dehydrogenase (PHGDH) is a disorder of serine biosynthesis identified in children with congenital microcephaly, seizures, and severe psychomotor retardation. We report here the identification of the 1468G-->A (V490M) mutation of this gene in two siblings of an Ashkenazi Jewish family, providing further evidence that the V490M mutation is a common, panethnic cause of this deficiency. Using a novel, DNA-based diagnostic test, the mutation was not detected in 400 non-Jewish controls; one heterozygote was found among 400 persons of Ashkenazi Jewish ethnicity. Extensive biochemical studies were undertaken to characterize the effect of this mutation on enzyme activity, turnover, and stability. The V490M PHGDH yielded less than 35% of the activity observed for the wild-type enzyme when overexpressed by transient transfection or when comparing the endogenous activity in fibroblast cells from the patients with controls. Immunoblotting studies showed a comparable reduction in the level of immunoreactive PHGDH in cells expressing the mutant enzyme. Pulse-chase experiments with metabolically labeled PHGDH indicated that this resulted from an increased rate of degradation of the mutant enzyme following its synthesis. Thermolability analyses of mutant and wild-type enzyme activity revealed no significant differences. While others have proposed that the V490M mutation decreases the V(max) of the enzyme, we conclude that this mutation impairs the folding and/or assembly of PHGDH but has minimal effects on the activity or stability of that portion of the V490M mutant that reaches a mature conformation.     

Guanidinoacetate Is More Effective than Creatine at Enhancing Tissue Creatine Stores while Consequently Limiting Methionine Availability in Yucatan Miniature Pigs

Creatine (Cr) is an important high-energy phosphate buffer in tissues with a high energy demand such as muscle and brain and is consequently a highly consumed nutritional supplement. Creatine is synthesized via the S-adenosylmethionine (SAM) dependent methylation of guanidinoacetate (GAA) which is not regulated by a feedback mechanism. The first objective of this study was to determine the effectiveness of GAA at increasing tissue Cr stores. Because SAM is required for other methylation reactions, we also wanted to determine whether an increased creatine synthesis would lead to a lower availability of methyl groups for other methylated products. Three month-old pigs (n = 18) were fed control, GAA- or Cr-supplemented diets twice daily. On day 18 or 19, anesthesia was induced 1–3 hours post feeding and a bolus of [methyl-³H]methionine was intravenously infused. After 30 minutes, the liver was analyzed for methyl-³H incorporation into protein, Cr, phosphatidylcholine (PC) and DNA. Although both Cr and GAA led to higher hepatic Cr concentration, only supplementation with GAA led to higher levels of muscle Cr (P < 0.05). Only GAA supplementation resulted in lower methyl-³H incorporation into PC and protein as well as lower hepatic SAM concentration compared to the controls, suggesting that Cr synthesis resulted in a limited methyl supply for PC and protein synthesis (P < 0.05). Although GAA is more effective than Cr at supporting muscle Cr accretion, further research should be conducted into the long term consequences of a limited methyl supply and its effects on protein and PC homeostasis.     

Microtubule depolymerization rapidly collapses capillary tube networks in vitro and angiogenic vessels in vivo through the small GTPase Rho

Maintenance of endothelial cell tube integrity is dependent on an intact cytoskeleton. We present data indicating that rapid collapse of endothelial tubular networks in vitro occurs in a dose-dependent manner after administration of microtubule-depolymerizing reagents but not after actin depolymerization. Pretreatment of endothelial cell networks with C3 exoenzyme or recombinant adenoviruses expressing dominant negative RhoA resulted in complete blockade of tube collapse, indicating a role for RhoA in these events. Microtubule depolymerization also resulted in activation of RhoA, whereas increased expression of constitutively active RhoA induced cell rounding and apoptosis of endothelial cells. Furthermore, following treatment with the chemotherapeutic agent vinblastine, rapid capillary tube network collapse occurred followed by endothelial cell apoptosis. Vinblastine, but not control agents, induced cleavage of procaspase-3, procaspase-9, and procaspase-8, along with the known caspase targets p21-activated kinase-2 and gelsolin, indicating that tube collapse caused a defined apoptotic response. Using a model of vascular endothelial growth factor-stimulated angiogenesis in vivo, vinblastine treatment also resulted in collapse and apoptosis of angiogenic blood vessels. Apoptotic endothelial cells stained strongly for cleaved caspase-3, and terminal dUTP nick-end labeling staining revealed fragmented nuclei in vinblastine-treated but not control angiogenic areas. Together, these findings indicate that microtubule-depolymerizing agents directly induce endothelial network collapse in vitro and in vivo leading to endothelial cell apoptosis in a manner dependent on the small GTPase, RhoA. In addition, these findings reveal a novel function for microtubule disrupting chemotherapeutic agents, namely their ability to rapidly collapse newly formed angiogenic vessels, which may contribute to their effectiveness in limiting angiogenesis and tumor growth.