Biochemistry

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Biochemistry

United States
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Wang S.-S.,University of Texas Southwestern Medical Center | Wang S.-S.,University of Texas at Dallas | Gu Y.-F.,University of Texas Southwestern Medical Center | Gu Y.-F.,University of Texas at Dallas | And 17 more authors.
Proceedings of the National Academy of Sciences of the United States of America | Year: 2014

Why different species are predisposed to different tumor spectra is not well understood. In particular, whether the physical location of tumor suppressor genes relative to one another influences tumor predisposition is unknown. Renal cancer presents a unique opportunity to explore this question. Renal cell carcinoma (RCC) of clearcell type (ccRCC), the most common type, begins with an intragenic mutation in the von Hippel-Lindau (VHL) gene and loss of 3p (where VHL is located). Chromosome 3p harbors several additional tumor suppressor genes, including BRCA1-associated protein-1 (BAP1). In the mouse, Vhl is on a different chromosome than Bap1. Thus, whereas loss of 3p in humans simultaneously deletes one copy of BAP1, loss of heterozygosity in the corresponding Vhl region in the mouse would not affect Bap1. To test the role of BAP1 in ccRCC development, we generated mice deficient for either Vhl or Vhl together with one allele of Bap1 in nephron progenitor cells. Six2- Cre;VhlF/F;Bap1F/+ mice developed ccRCC, but Six2-Cre;VhlF/F mice did not. Kidneys from Six2-Cre;VhlF/F;Bap1F/+ mice resembled kidneys from humans with VHL syndrome, containing multiple lesions spanning from benign cysts to cystic and solid RCC. Although the tumors were small, they showed nuclear atypia and exhibited features of human ccRCC. These results provide an explanation for why VHL heterozygous humans, but not mice, develop ccRCC. They also explain why a mouse model of ccRCC has been lacking. More broadly, our data suggest that differences in tumor predisposition across species may be explained, at least in part, by differences in the location of two-hit tumor suppressor genes across the genome.


Ma L.,Internal Medicine Nephrology | Shelness G.S.,Pathology Lipid science | Snipes J.A.,Internal Medicine Nephrology | Murea M.,Internal Medicine Nephrology | And 14 more authors.
Journal of the American Society of Nephrology | Year: 2015

Although APOL1 gene variants are associated with nephropathy in African Americans, little is known about APOL1 protein synthesis, uptake, and localization in kidney cells. To address these questions, we examined APOL1 protein and mRNA localization in human kidney and human kidney-derived cell lines. Indirect immunofluorescence microscopy performed on nondiseased nephrectomy cryosections from persons with normal kidney function revealed that APOL1 protein was markedly enriched in podocytes (colocalized with synaptopodin and Wilms' tumor suppressor) and present in lower abundance in renal tubule cells. Fluorescence in situ hybridization detected APOL1 mRNA in glomeruli (podocytes and endothelial cells) and tubules, consistent with endogenous synthesis in these cell types. When these analyses were extended to renal-derived cell lines, quantitative RT-PCR did not detect APOL1 mRNA in human mesangial cells; however, abundant levels of APOL1 mRNA were observed in proximal tubule cells and glomerular endothelial cells, with lower expression in podocytes. Western blot analysis revealed corresponding levels of APOL1 protein in these cell lines. To explain the apparent discrepancy between the marked abundance of APOL1 protein in kidney podocytesobserved in cryosections versus the lesser abundance in podocyte cell lines, we explored APOL1 cellular uptake. APOL1 protein was taken up readily by human podocytes in vitro but was not taken up efficiently by mesangial cells, glomerular endothelial cells, or proximal tubule cells. We hypothesize that the higher levels of APOL1 protein in human cryosectioned podocytes may reflect both endogenous protein synthesis and APOL1 uptake from the circulation or glomerular filtrate. Copyright © 2015 by the American Society of Nephrology doi:.


Shirley M.D.,Biochemistry | Shirley M.D.,Kennedy Krieger Institute | Tang H.,Duke University | Gallione C.J.,Duke University | And 8 more authors.
New England Journal of Medicine | Year: 2013

BACKGROUND: The Sturge-Weber syndrome is a sporadic congenital neurocutaneous disorder characterized by a port-wine stain affecting the skin in the distribution of the ophthalmic branch of the trigeminal nerve, abnormal capillary venous vessels in the leptomeninges of the brain and choroid, glaucoma, seizures, stroke, and intellectual disability. It has been hypothesized that somatic mosaic mutations disrupting vascular development cause both the Sturge-Weber syndrome and port-wine stains, and the severity and extent of presentation are determined by the developmental time point at which the mutations occurred. To date, no such mutation has been identified. METHODS: We performed whole-genome sequencing of DNA from paired samples of visibly affected and normal tissue from 3 persons with the Sturge-Weber syndrome. We tested for the presence of a somatic mosaic mutation in 97 samples from 50 persons with the Sturge-Weber syndrome, a port-wine stain, or neither (controls), using amplicon sequencing and SNaPshot assays, and investigated the effects of the mutation on downstream signaling, using phosphorylation-specific antibodies for relevant effectors and a luciferase reporter assay. RESULTS: We identified a nonsynonymous single-nucleotide variant (c.548G→A, p.Arg183Gln) in GNAQ in samples of affected tissue from 88% of the participants (23 of 26) with the Sturge-Weber syndrome and from 92% of the participants (12 of 13) with apparently nonsyndromic port-wine stains, but not in any of the samples of affected tissue from 4 participants with an unrelated cerebrovascular malformation or in any of the samples from the 6 controls. The prevalence of the mutant allele in affected tissues ranged from 1.0 to 18.1%. Extracellular signal-regulated kinase activity was modestly increased during transgenic expression of mutant Gαq. CONCLUSIONS: The Sturge-Weber syndrome and port-wine stains are caused by a somatic activating mutation in GNAQ. This finding confirms a long-standing hypothesis. Copyright © 2013 Massachusetts Medical Society.


Lam S.M.,CAS Institute of Genetics and Developmental Biology | Tong L.,Singapore Eye Research Institute | Tong L.,Singapore National Eye Center | Tong L.,National University of Singapore | And 4 more authors.
Journal of Lipid Research | Year: 2014

The tear film covers the anterior eye and the precise balance of its various constituting components is critical for maintaining ocular health. The composition of the tear film amphiphilic lipid sublayer, in particular, has largely remained a matter of contention due to the limiting concentrations of these lipid amphiphiles in tears that render their detection and accurate quantitation tedious. Using systematic and sensitive lipidomic approaches, we validated dif ferent tear collection techniques and report the most comprehensive human tear lipidome to date; comprising more than 600 lipid species from 17 major lipid classes. Our study confers novel insights to the compositional details of the existent tear film model, in particular the disputable amphiphilic lipid sublayer constituents, by demonstrating the presence of cholesteryl sulfate, O-Acyl- α -hydroxy fatty acids, and various sphingolipids and phospholipids in tears. The discovery and quantitation of the relative abundance of various tear lipid amphiphiles reported herein are expected to have a profound impact on the current understanding of the existent human tear film model. -Lam, S. M., L. Tong, X. Duan, A. Petznick, M. R. Wenk, and G. Shui. Extensive characterization of human tear fl uid collected using different techniques unravels the presence of novel lipid amphiphiles. Copyright © 2014 by the American Society for Biochemistry and Molecular Biology, Inc..


Andersen C.J.,University of Connecticut | Blesso C.N.,University of Connecticut | Lee J.,University of Connecticut | Barona J.,University of Connecticut | And 4 more authors.
Lipids | Year: 2013

We recently demonstrated that daily whole egg consumption during moderate carbohydrate restriction leads to greater increases in plasma HDL-cholesterol (HDL-C) and improvements in HDL profiles in metabolic syndrome (MetS) when compared to intake of a yolk-free egg substitute. We further investigated the effects of this intervention on HDL composition and function, hypothesizing that the phospholipid species present in egg yolk modulate HDL lipid composition to increase the cholesterol-accepting capacity of subject serum. Men and women classified with MetS were randomly assigned to consume either three whole eggs (EGG, n = 20) per day or the equivalent amount of egg substitute (SUB, n = 17) throughout a 12-week moderate carbohydrate-restricted (25-30 % of energy) diet. Relative to other HDL lipids, HDL-cholesteryl ester content increased in all subjects, with greater increases in the SUB group. Further, HDL-triacylglycerol content was reduced in EGG group subjects with normal baseline plasma HDL-C, resulting in increases in HDL-CE/TAG ratios in both groups. Phospholipid analysis by mass spectrometry revealed that HDL became enriched in phosphatidylethanolamine in the EGG group, and that EGG group HDL better reflected sphingomyelin species present in the whole egg product at week 12 compared to baseline. Further, macrophage cholesterol efflux to EGG subject serum increased from baseline to week 12, whereas no changes were observed in the SUB group. Together, these findings suggest that daily egg consumption promotes favorable shifts in HDL lipid composition and function beyond increasing plasma HDL-C in MetS. © 2013 AOCS.


Heupel S.,Karlsruhe Institute of Technology | Roser B.,Karlsruhe Institute of Technology | Kuhn H.,Karlsruhe Institute of Technology | Lebrun M.-H.,French National Center for Scientific Research | And 2 more authors.
Molecular Plant-Microbe Interactions | Year: 2010

Comparative analyses of genome sequences from several plant-infecting fungi have shown conservation and expansion of protein families with plant disease-related functions. Here, we show that this hypothesis can be extended to mutualistic symbiotic fungi. We have identified a gene encoding an Era (Escherichia coli Ras)-like GTPase in the rice blast fungus Magnaporthe oryzae and found that it is orthologous to the mature amino terminal part of the Gin1 protein from the arbuscular mycorrhizal (AM) fungus Glomus intraradices. M. oryzae Erl1 is required for full root virulence. Appressoria formation was not severely affected in Δerllstraias, but invasive hyphae grew slower than in the wild type. Root browning defect of δerll strains could be complemented by the AM gene under the control of the ERL1 promoter. Erl1 and Gin-N localized to the nucleus when carboxy-terminally labeled with green fluorescent protein (GFP). However, amino-terminal GFP-tagged versions of the proteins expressed in Aspergillus nidulans were shown to localize in the cytoplasm and to cause polarity defects. These data suggest that Erl1 and Gin-N are orthologs and might be involved in the control of hyphal growth in planta. This is the first characterization of an Era-like GTPase in filamentous fungi. © 2010 The American Phytopathological Society.


Rafii S.,New York Medical College | Kloss C.C.,Biochemistry | Kloss C.C.,Sloan Kettering Cancer Center | Butler J.M.,New York Medical College | And 13 more authors.
Blood | Year: 2013

Several studies have demonstrated that hematopoietic cells originate from endothelium in early development; however, the phenotypic progression of progenitor cells during human embryonic hemogenesis is not well described. Here, we define the developmental hierarchy among intermediate populations of hematopoietic progenitor cells (HPCs) derived from human embryonic stem cells (hESCs). We genetically modified hESCs to specifically demarcate acquisition of vascular (VE-cadherin) and hematopoietic (CD41a) cell fate and used this dual-reporting transgenic hESC line to observe endothelial to hematopoietic transition by real-time confocal microscopy. Live imaging and clonal analyses revealed a temporal bias in commitment of HPCs that recapitulates discrete waves of lineage differentiation noted during mammalian hemogenesis. Specifically, HPCs isolated at later time points showed reduced capacity to form erythroid/megakaryocytic cells and exhibited a tendency toward myeloid fate that was enabled by expression of the Notch ligand Dll4 on hESC-derived vascular feeder cells. These data provide a framework for defining HPC lineage potential, elucidate a molecular contribution from the vascular niche in promoting hematopoietic lineage progression, and distinguish unique subpopulations of hemogenic endothelium during hESC differentiation. © 2013 by The American Society of Hematology.


Grasshopper communities were sampled in three associated habitats of the Central Basin of Tennesee: cedar glades, xeric limestone prairies, and cedar hardwood forest. Twenty-five grasshopper species were collected across all three habitats. Eleven species were found in the cedar glades, 12 species were collected in the xeric limestone prairies, and six species were collected from the cedar-hardwood forests. A Principal Component Analysis of the resulting species lists indicated that grasshopper community composition differed significantly between each habitat. Four new state records for Tennessee were documented during this survey. An annotated species list is presented.


Obesity and gestational diabetes mellitus continue to increase worldwide and span the spectrum of age, race, ethnicity, and socioeconomic status. Alarmingly, 1 in 10 infants and toddlers is obese, and 1 in 5 youths is both obese and at risk formetabolic syndrome prior to puberty. The mechanisms underlying how poor maternal health imparts risk for future metabolic disease in the offspring are beginning to emerge in deeply phenotyped human and nonhuman primate models. Maternal diet and obesity impact fuels, hormones, and inflammation with powerful effects on fetal metabolic systems. These are accompanied by persistent changes in the infant microbiome and epigenome and in offspring behavior. These results suggest that gestational and lactational dietary exposures are driving health risks in the next generation. Whether maternal diet can prevent changes in the womb to alter infant life-course disease risk is still unknown. Controlled, mechanistic studies to identify interventions are sorely needed for a healthier next generation. ©2015 by the American Diabetes Association.


Jacob D.A.,Lipscomb University | Mercer S.L.,Lipscomb University | Osheroff N.,Biochemistry | Osheroff N.,Vanderbilt University | Deweese J.E.,Lipscomb University
Biochemistry | Year: 2011

Etoposide is a topoisomerase II poison that is used to treat a variety of human cancers. Unfortunately, 2-3% of patients treated with etoposide develop treatment-related leukemias characterized by 11q23 chromosomal rearrangements. The molecular basis for etoposide-induced leukemogenesis is not understood but is associated with enzyme-mediated DNA cleavage. Etoposide is metabolized by CYP3A4 to etoposide catechol, which can be further oxidized to etoposide quinone. A CYP3A4 variant is associated with a lower risk of etoposide-related leukemias, suggesting that etoposide metabolites may be involved in leukemogenesis. Although etoposide acts at the enzyme-DNA interface, several quinones poison topoisomerase II via redox-dependent protein adduction. The effects of etoposide quinone on topoisomerase IIα-mediated DNA cleavage have been examined previously. Although findings suggest that the activity of the quinone is slightly greater than that of etoposide, these studies were carried out in the presence of significant levels of reducing agents (which should reduce etoposide quinone to the catechol). Therefore, we examined the ability of etoposide quinone to poison human topoisomerase IIα in the absence of reducing agents. Under these conditions, etoposide quinone was ∼5-fold more active than etoposide at inducing enzyme-mediated DNA cleavage. Consistent with other redox-dependent poisons, etoposide quinone inactivated topoisomerase IIα when incubated with the protein prior to DNA and lost activity in the presence of dithiothreitol. Unlike etoposide, the quinone metabolite did not require ATP for maximal activity and induced a high ratio of double-stranded DNA breaks. Our results support the hypothesis that etoposide quinone contributes to etoposide-related leukemogenesis. © 2011 American Chemical Society.

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