Identification and biochemical characterization of the fructokinase gene family in Arabidopsis thaliana.

BACKGROUND: Fructose is an abundant sugar in plants as it is a breakdown product of both major sucrose-cleaving enzymes. To enter metabolism, fructose is phosphorylated by a fructokinase (FRK). Known FRKs are members of a diverse family of carbohydrate/purine kinases known as the phosphofructokinase B (pfkB) family. The complete complement of active fructokinases has not been reported for any plant species. RESULTS: Protein sequence analysis of the 22 Arabidopsis thaliana pfkB members identified eight highly related predicted proteins, including one with previously demonstrated FRK activity. For one, At1g50390, the predicted open reading frame is half the size of active FRKs, and only incompletely spliced RNAs were identified, which led to a premature stop codon, both indicating that this gene does not produce active FRK. The remaining seven proteins were expressed in E. coli and phosphorylated fructose specifically in vitro leading us to propose a unifying nomenclature (FRK1-7). Substrate inhibition was observed for fructose in all FRKs except FRK1. Fructose binding was on the same order of magnitude for FRK1-6, between 260 and 480 µM. FRK7 was an outlier with a fructose Km of 12 µM. ATP binding was similar for all FRKs and ranged between 52 and 280 µM. YFP-tagged AtFRKs were cytosolic, except plastidic FRK3. T-DNA alleles with non-detectable wild-type RNAs in five of the seven active FRK genes produced no overt phenotype. We extended our sequence comparisons to include putative FRKs encoded in other plant sequenced genomes. We observed that different subgroups expanded subsequent to speciation. CONCLUSIONS: Arabidopsis thaliana as well as all other plant species analyzed contain multiple copies of genes encoding FRK activity. Sequence comparisons among multiple species identified a minimal set of three distinct FRKs present on all species investigated including a plastid-localized form. The selective expansion of specific isozymes results in differences in FRK gene number among species. AtFRKs exhibit substrate inhibition, typical of their mammalian counterparts with the single AtFRK1 lacking this property, suggesting it may have a distinct in vivo role. Results presented here provide a starting point for the engineering of specific FRKs to affect biomass production.

Arabidopsis fructokinase-like protein associations are regulated by ATP.

The Arabidopsis thaliana fructokinase-like proteins FLN1 and FLN2 are required for the differentiation of plastids into photosynthetically competent chloroplasts. However, their specific roles are unknown. FLN1 and FLN2 localize in a multisubunit prokaryotic-type polymerase (plastid-encoded RNA polymerase) complex that transcribes genes encoding components of photosynthesis-related assemblies. Despite sequence identity with fructokinases, which are members of the pfkB (phosphofructokinase B) family of enzymes, kinase activity of FLN1 and FLN2 has not been demonstrated. Homology modeling using pfkB X-ray structures, sequence comparisons, and mutational analyses suggests that FLN proteins may bind their substrates differently from other pfkB proteins. We provide evidence that purified recombinant FLN1 undergoes an ATP-mediated change in binding affinity with both itself and recombinant FLN2. The ATP-mediated change in the affinity of FLN1 for FLN2 is not affected by mutations in conserved active-site residues known to affect catalysis in active pfkB enzymes. In contrast, recombinant FLN2 hetero-oligomerizes independently of ATP concentration. At ATP concentrations that promote FLN1 homomeric interactions, the FLN1-FLN2 hetero-oligomer is the dominant form in vitro We further present evidence that FLN1 associates with a large protein complex in chloroplasts independently of ATP. Given that ATP levels fluctuate between light-dark cycles in the 1-5 mM range, we propose that changes in FLN1 and FLN2 interactions are biologically meaningful.

Identification of the Plant Ribokinase and Discovery of a Role for Arabidopsis Ribokinase in Nucleoside Metabolism.

Ribose can be used for energy or as a component of several important biomolecules, but for it to be used in either capacity it must first be phosphorylated by ribokinase (RBSK). RBSK proteins are part of the phosphofructokinase-B (pfkB) family of carbohydrate kinases. Sequence comparisons of pfkB proteins from the model plant Arabidopsis thaliana with the human and Escherichia coli RBSK identified a single candidate RBSK, At1g17160 (AtRBSK). AtRBSK is more similar to predicted RBSKs from other plant species and known mammalian and prokaryotic RBSK than to all other PfkB proteins in Arabidopsis AtRBSK contains a predicted chloroplast transit peptide, and we confirmed plastid localization using AtRBSK fused to YFP. Structure prediction software verified that the AtRBSK sequence mapped onto a known RBSK structure. Kinetic parameters of purified recombinant AtRBSK were determined to be Kmribose = 150 µm ± 17 µm, KmATP = 45 µm ± 5.6 µm, and kcat = 2.0 s-1 Substrate inhibition was observed for AtRBSK (KiATP = 2.44 mm ± 0.36 mm), as has been demonstrated for other RBSK proteins. Ribose accumulated in Arabidopsis plants lacking AtRBSK. Such plants grew normally unless media was supplemented with ribose, which led to chlorosis and growth inhibition. Both chlorosis and ribose accumulation were abolished upon the introduction of a transgene expressing AtRBSK-MYC, demonstrating that the loss of protein is responsible for ribose hypersensitivity. Ribose accumulation in plants lacking AtRBSK was reduced in plants also deficient in the nucleoside ribohydrolase NSH1, linking AtRBSK activity to nucleoside metabolism.

Endogenous mammalian histone H3.3 exhibits chromatin-related functions during development.

BACKGROUND: The histone variant H3.3 plays key roles in regulating chromatin states and transcription. However, the role of endogenous H3.3 in mammalian cells and during development has been less thoroughly investigated. To address this gap, we report the production and phenotypic analysis of mice and cells with targeted disruption of the H3.3-encoding gene, H3f3b. RESULTS: H3f3b knockout (KO) mice exhibit a semilethal phenotype traceable at least in part to defective cell division and chromosome segregation. H3f3b KO cells have widespread ectopic CENP-A protein localization suggesting one possible mechanism for defective chromosome segregation. KO cells have abnormal karyotypes and cell cycle profiles as well. The transcriptome and euchromatin-related epigenome were moderately affected by loss of H3f3b in mouse embryonic fibroblasts (MEFs) with ontology most notably pointing to changes in chromatin regulatory and histone coding genes. Reduced numbers of H3f3b KO mice survive to maturity and almost all survivors from both sexes are infertile. CONCLUSIONS: Taken together, our studies suggest that endogenous mammalian histone H3.3 has important roles in regulating chromatin and chromosome functions that in turn are important for cell division, genome integrity, and development.

Cost-effectiveness analysis of rubella screening strategies using electronic medical records.

OBJECTIVE: The redundancy of routine laboratory tests in medicine has become increasingly more apparent in the age of electronic medical records (EMRs). The purpose of this study was to determine whether targeted screening strategies are more cost-effective than the current standard of universal screening of pregnant women for immunity to rubella. STUDY DESIGN: A decision analysis model was used to evaluate three strategies: universal screening, screening if a previous titer was not available, and use of an "alert" in the EMR to prompt screening. Cost, probability, and utility values were derived from the literature and institutional data from Lyndon B. Johnson General Hospital. One-way sensitivity analyses were performed on all cost and probability values. RESULTS: The strategy of an EMR alert was most cost-effective, with a cost of $0.27 per quality-adjusted life years (QALY). The model was robust to all costs and probability values over their respective ranges. CONCLUSIONS: Although all strategies were cost-effective compared with traditional industry benchmarks of $50,000/QALY, the EMR alert strategy is most cost-effective. Implementing an EMR alert may lead to a more cost-effective approach to prenatal evaluation of rubella immunity.

Induced pluripotency and oncogenic transformation are related processes.

Induced pluripotent stem cells (iPSCs) have the potential for creating patient-specific regenerative medicine therapies, but the links between pluripotency and tumorigenicity raise important safety concerns. More specifically, the methods employed for the production of iPSCs and oncogenic foci (OF), a form of in vitro produced tumor cells, are surprisingly similar, raising potential concerns about iPSCs. To test the hypotheses that iPSCs and OF are related cell types and, more broadly, that the induction of pluripotency and tumorigenicity are related processes, we produced iPSCs and OF in parallel from common parental fibroblasts. When we compared the transcriptomes of these iPSCs and OF to their parental fibroblasts, similar transcriptional changes were observed in both iPSCs and OF. A significant number of genes repressed during the iPSC formation were also repressed in OF, including a large cohort of differentiation-associated genes. iPSCs and OF shared a limited number of genes that were upregulated relative to parental fibroblasts, but gene ontology analysis pointed toward monosaccharide metabolism as upregulated in both iPSCs and OF. iPSCs and OF were distinct in that only iPSCs activated a host of pluripotency-related genes, while OF activated cellular damage and specific metabolic pathways. We reprogrammed oncogenic foci (ROF) to produce iPSC-like cells, a process dependent on Nanog. However, the ROF had reduced differentiation potential compared to iPSC, suggesting that oncogenic transformation leads to cellular changes that impair complete reprogramming. Taken together, these findings support a model in which OF and iPSCs are related, yet distinct cell types, and in which induced pluripotency and induced tumorigenesis are similar processes.

Induced pluripotent stem cells show metabolomic differences to embryonic stem cells in polyunsaturated phosphatidylcholines and primary metabolism.

Induced pluripotent stem cells are different from embryonic stem cells as shown by epigenetic and genomics analyses. Depending on cell types and culture conditions, such genetic alterations can lead to different metabolic phenotypes which may impact replication rates, membrane properties and cell differentiation. We here applied a comprehensive metabolomics strategy incorporating nanoelectrospray ion trap mass spectrometry (MS), gas chromatography-time of flight MS, and hydrophilic interaction- and reversed phase-liquid chromatography-quadrupole time-of-flight MS to examine the metabolome of induced pluripotent stem cells (iPSCs) compared to parental fibroblasts as well as to reference embryonic stem cells (ESCs). With over 250 identified metabolites and a range of structurally unknown compounds, quantitative and statistical metabolome data were mapped onto a metabolite networks describing the metabolic state of iPSCs relative to other cell types. Overall iPSCs exhibited a striking shift metabolically away from parental fibroblasts and toward ESCs, suggestive of near complete metabolic reprogramming. Differences between pluripotent cell types were not observed in carbohydrate or hydroxyl acid metabolism, pentose phosphate pathway metabolites, or free fatty acids. However, significant differences between iPSCs and ESCs were evident in phosphatidylcholine and phosphatidylethanolamine lipid structures, essential and non-essential amino acids, and metabolites involved in polyamine biosynthesis. Together our findings demonstrate that during cellular reprogramming, the metabolome of fibroblasts is also reprogrammed to take on an ESC-like profile, but there are select unique differences apparent in iPSCs. The identified metabolomics signatures of iPSCs and ESCs may have important implications for functional regulation of maintenance and induction of pluripotency.

Biodistribution and pharmacokinetics of a telodendrimer micellar paclitaxel nanoformulation in a mouse xenograft model of ovarian cancer.

BACKGROUND: A multifunctional telodendrimer-based micelle system was characterized for delivery of imaging and chemotherapy agents to mouse tumor xenografts. Previous optical imaging studies demonstrated qualitatively that these classes of nanoparticles, called nanomicelles, preferentially accumulate at tumor sites in mice. The research reported herein describes the detailed quantitative imaging and biodistribution profiling of nanomicelles loaded with a cargo of paclitaxel. METHODS: The telodendrimer was covalently labeled with ¹²5I and the nanomicelles were loaded with ¹4C-paclitaxel, which allowed measurement of pharmacokinetics and biodistribution in the mice using microSPECT/CT imaging and liquid scintillation counting, respectively. RESULTS: The radio imaging data showed preferential accumulation of nanomicelles at the tumor site along with a slower clearance rate than paclitaxel formulated in Cremophor EL (Taxol®). Liquid scintillation counting confirmed that ¹4C-labeled paclitaxel sequestered in nanomicelles had increased uptake by tumor tissue and slower pharmacokinetics than Taxol. CONCLUSION: Overall, the results indicate that nanomicelle-formulated paclitaxel is a potentially superior formulation compared with Taxol in terms of water solubility, pharmacokinetics, and tumor accumulation, and may be clinically useful for both tumor imaging and improved chemotherapy applications.

Comparing homeless and domiciled patients' utilization of the Harris County, Texas public hospital system.

Homeless individuals have mortality rates three to six times higher than their housed counterparts and have elevated rates of mental illness, substance abuse, and co-morbidities that increase their need for health services. Data on the utilization of Harris County, Texas' public hospital system by 331 homeless individuals and a random sample of 17,824 domiciled patients were obtained from June 2008 to July 2009. Homeless individuals had increased readmission rates, especially within 30 days of discharge, resulting in significantly higher total annual length of stay. Homeless patients also more frequently utilize public hospitals for mental illness and HIV. Lack of community health services contributes to an increased dependence and preventable over-utilization of public hospital systems. Case management interventions integrating primary and behavioral care into health homes, medical respite programs, and training for health care professionals who provide indigent care will improve health outcomes of this population and reduce costs.

Outcomes of term vaginal breech delivery.

In December 2001, the American College of Obstetricians and Gynecologists revised their recommendations for breech delivery. These recommendations acknowledge that although a planned vaginal delivery may no longer be appropriate, there are instances in which vaginal breech delivery is inevitable. Moreover, there continues to be patients who for any number of reasons will choose vaginal over cesarean delivery when faced with a fetus in the breech presentation. We sought to review maternal and fetal outcomes in such circumstances when vaginal breech delivery occurs, and compare these outcomes to elective cesarean deliveries for breech presentation. We performed a retrospective review of all singleton breech deliveries at our county hospital from January 2002 through June 2003. We reviewed maternal age, ethnicity, gestational age, gravity, parity, birthweight, mode of delivery, Apgar scores, umbilical arterial blood gases, and maternal and infant complications of both cesarean deliveries and vaginal breech deliveries. Univariate and logistic regression statistical analyses were performed with NCSS software. We had a total of 150 term breech deliveries with gestational ages between 37 and 42 weeks. Of these, 41 were vaginal breech and 109 were cesarean deliveries. Greater than 95% of patients are of Hispanic origin. There were no statistically significant differences in maternal age, ethnicity, gravity, or gestational age. Mean birthweight was significantly lower and parity was significantly higher in the vaginal delivery group. There was also a higher proportion of patients who underwent labor induction/augmentation in the vaginal group. We found no differences in the outcomes of 5-minute Apgar scores, umbilical arterial blood gas values, neonatal intensive care unit admissions, deaths or maternal/fetal complications reported between the two groups. Mean umbilical arterial blood gas values were greater than 7.18 in both groups. Vaginal breech delivery cannot always be avoided. Moreover, at our county hospital several patients continue to choose vaginal breech delivery. Our data would suggest that vaginal breech delivery remains a viable option in selected patients.