bluuebunny/crossref_metadata_2025_split huggingface dataset:
Endothelial Nitric Oxide Synthase Haplotypes Are Associated with Features of Metabolic Syndrome
Authors: González-Sánchez José L , Martínez-Larrad María T , Sáez María E , Zabena Carina , Martínez-Calatrava María J , Serrano-Ríos Manuel 
Published: November 2006

Abstract
Background: The metabolic syndrome, a cluster of several metabolic disorders, is increasingly being recognized as a risk factor for cardiovascular disease. Endothelium-derived nitric oxide facilitates skeletal muscle glucose uptake, and data from animal models indicate that endothelial nitric oxide synthase (eNOS) gene–null mice present with a phenotype of insulin resistance, hypertension, and hypertriglyceridemia, much like that observed in humans with metabolic syndrome. We used haplotype tagging single nucleotide polymorphisms (htSNPs) to investigate the role of genetic variation in the eNOS gene (NOS3) in metabolic syndrome in humans.
Methods: We recruited 738 unrelated persons from a cross-sectional population-based epidemiological survey in the province of Segovia in Central Spain (Castille). Metabolic syndrome was defined according to the recently modified National Cholesterol Education Program Adult Treatment Panel III guidelines.
Results: Haplotype analysis showed a statistically significant association between some NOS3 gene variants and features of metabolic syndrome. Relative to the most common haplotype, 121, the haplotype 212 was associated with an increased odds ratio (OR) for metabolic syndrome [OR = 1.81, 95% confidence interval (CI) 1.15–2.84], and for decreased HDL-cholesterol concentrations (OR 1.52, 95% CI 1.01–2.29), and with increased mean values for the homeostasis model assessment of insulin resistance (P = 0.043), and triglycerides (P = 0.026).
Conclusions: Our results suggest that genetic variation at the eNOS locus is associated with features of metabolic syndrome, and might represent a new genetic susceptibility component for insulin resistance, hypertriglyceridemia, and low HDL-cholesterol concentrations.
Molecular Beacon–Based Temperature Control and Automated Analyses for Improved Resolution of Melting Temperature Analysis Using SYBR I Green Chemistry
Authors: Nellåker Christoffer , Wållgren Ulf , Karlsson Håkan 
Published: November 2006

AbstractBackground: Melting temperature analysis of products amplified with SYBR I Green chemistry is a cheap and effective method for identification of sequence differences. When used in conventional quantitative real-time PCR instruments (qPCR), this method is limited by temperature variations over the heating block and low numbers of fluorescence measurements during the dissociation step, which hamper the ability of most instruments to report accurate and precise melting temperatures.Methods: We designed a molecular beacon–based temperature indicator probe (Tm-probe) to control for variations in temperatures over the heating block of the instrument. In addition, we wrote an automated curve-fit analysis algorithm of dissociation data to use multiple data points with a gaussian curve fit to extrapolate precise melting temperatures.Results: Use of the Tm-probe in conjunction with the analysis algorithm and multiple dissociations improved SDs of melting temperatures over a 96-well plate from 0.19 to 0.06 °CConclusions: Melting temperature analyses with SYBR I Green chemistry on conventional qPCR instruments can be improved by the use of a Tm-probe in conjunction with curve-fit analysis of data. Resolution improvement up to 3-fold is possible and allows additional melting temperatures to be identified.
Factitious Diarrhea Induced by Stimulant Laxatives: Accuracy of Diagnosis by a Clinical Reference Laboratory Using Thin Layer Chromatography
Authors: Shelton Joseph H , Santa Ana Carol A , Thompson Donald R , Emmett Michael , Fordtran John S 
Published: November 2006

AbstractBackground: Surreptitious ingestion of laxatives can lead to serious factitious diseases that are difficult to diagnose. Most cases involve ingestion of bisacodyl or senna. Thin layer chromatography (TLC) of urine or stool is the only commercially available test for these laxatives. Such testing is considered highly reliable, but its accuracy in clinical practice is unknown. Our aim was to evaluate the reliability of TLC laxative testing by a clinical reference laboratory in the United States.Methods: Diarrhea was induced in healthy volunteers by ingestion of bisacodyl, senna, or a control laxative (n = 11 for each laxative group). Samples of urine and diarrheal stool were sent in blinded fashion to the clinical reference laboratory for bisacodyl and senna analysis.Results: TLC testing for bisacodyl-induced diarrhea revealed a sensitivity of 73% and specificity of 91% when urine was tested and sensitivity and specificity of 91% and 96%, respectively, when stool was analyzed. When diarrhea was induced by senna, the TLC assay for senna failed to identify even a single urine or stool specimen as positive (zero% sensitivity).Conclusions: Considering the expected prevalence of surreptitious laxative abuse in patients with chronic idiopathic diarrhea (2.4%–25%, depending on the clinical setting), TLC of urine or stool for bisacodyl by this reference laboratory would often produce misleading results, and testing for senna would have no clinical value. The major problems are false-positive tests for bisacodyl and false-negative tests for senna.
Rates of Recombination in the Ribosomal DNA of Apomictically Propagated Daphnia obtusa Lines
Authors: McTaggart Seanna J , Dudycha Jeffry L , Omilian Angela , Crease Teresa J 
Published: November 2006

Abstract
Ribosomal (r)DNA undergoes concerted evolution, the mechanisms of which are unequal crossing over and gene conversion. Despite the fundamental importance of these mechanisms to the evolution of rDNA, their rates have been estimated only in a few model species. We estimated recombination rate in rDNA by quantifying the relative frequency of intraindividual length variants in an expansion segment of the 18S rRNA gene of the cladoceran crustacean, Daphnia obtusa, in four apomictically propagated lines. We also used quantitative PCR to estimate rDNA copy number. The apomictic lines were sampled every 5 generations for 90 generations, and we considered each significant change in the frequency distribution of length variants between time intervals to be the result of a recombination event. Using this method, we calculated the recombination rate for this region to be 0.02–0.06 events/generation on the basis of three different estimates of rDNA copy number. In addition, we observed substantial changes in rDNA copy number within and between lines. Estimates of haploid copy number varied from 53 to 233, with a mean of 150. We also measured the relative frequency of length variants in 30 lines at generations 5, 50, and 90. Although length variant frequencies changed significantly within and between lines, the overall average frequency of each length variant did not change significantly between the three generations sampled, suggesting that there is little or no bias in the direction of change due to recombination.
Thermodynamics of Neutral Protein Evolution
Authors: Bloom Jesse D , Raval Alpan , Wilke Claus O 
Published: November 2006

Abstract
Naturally evolving proteins gradually accumulate mutations while continuing to fold to stable structures. This process of neutral evolution is an important mode of genetic change and forms the basis for the molecular clock. We present a mathematical theory that predicts the number of accumulated mutations, the index of dispersion, and the distribution of stabilities in an evolving protein population from knowledge of the stability effects (ΔΔG values) for single mutations. Our theory quantitatively describes how neutral evolution leads to marginally stable proteins and provides formulas for calculating how fluctuations in stability can overdisperse the molecular clock. It also shows that the structural influences on the rate of sequence evolution observed in earlier simulations can be calculated using just the single-mutation ΔΔG values. We consider both the case when the product of the population size and mutation rate is small and the case when this product is large, and show that in the latter case the proteins evolve excess mutational robustness that is manifested by extra stability and an increase in the rate of sequence evolution. All our theoretical predictions are confirmed by simulations with lattice proteins. Our work provides a mathematical foundation for understanding how protein biophysics shapes the process of evolution.
A Genetic Screen in Drosophila for Genes Interacting With senseless During Neuronal Development Identifies the Importin moleskin
Authors: Pepple Kathryn L , Anderson Aimée E , Frankfort Benjamin J , Mardon Graeme 
Published: November 2006

Abstract
Senseless (Sens) is a conserved transcription factor required for normal development of the Drosophila peripheral nervous system. In the Drosophila retina, sens is necessary and sufficient for differentiation of R8 photoreceptors and interommatidial bristles (IOBs). When Sens is expressed in undifferentiated cells posterior to the morphogenetic furrow, ectopic IOBs are formed. This phenotype was used to identify new members of the sens pathway in a dominant modifier screen. Seven suppressor and three enhancer complementation groups were isolated. Three groups from the screen are the known genes Delta, lilliputian, and moleskin/DIM-7 (msk), while the remaining seven groups represent novel genes with previously undefined functions in neural development. The nuclear import gene msk was identified as a potent suppressor of the ectopic interommatidial bristle phenotype. In addition, msk mutant adult eyes are extremely disrupted with defects in multiple cell types. Reminiscent of the sens mutant phenotype, msk eyes demonstrate reductions in the number of R8 photoreceptors due to an R8 to R2,5 fate switch, providing genetic evidence that Msk is a component of the sens pathway. Interestingly, in msk tissue, the loss of R8 fate occurs earlier than with sens and suggests a previously unidentified stage of R8 development between atonal and sens.
A Proteomic Strategy for Quantifying Polyubiquitin Chain Topologies
Authors: Xu Ping , Cheng Dongmei , Duong Duc M. , Rush John , Roelofs Jeroen , Finley Daniel , Peng Junmin 
Published: November 2006

AbstractWe present a method to probe the structure of polyubiquitin (polyUb) chains by mass spectrometry. As polyUb chains can be linked by any of seven lysine residues in the sequence of ubiquitin, the structural diversity of the chains could be enormous (e.g., as many as 70 configurations for tri‐Ub). To quantify the frequency of every lysine linkage, polyUb chains were digested by trypsin and analyzed by tandem mass spectrometry using synthetic isotope‐labeled peptides as internal standards. Employing a purified ubiquitin dimer to optimize conditions for trypsinization, we found that all tryptic sites except K33 could be reliably cleaved under insolution and in‐gel conditions. Because K33 miscleavage led to large underestimation of K29 linkage, its digestion efficiency was calculated to correct the measurement. The reverse‐phase chromatography was also adjusted to capture the small hydrophilic peptide of K29 linkage. In addition, the analysis of the K6‐linkage peptide was complicated by methionine oxidation and therefore the sample was treated with hydrogen peroxide to fully oxidize methionine to sulfoxide. While the method was sensitive enough to detect K48 and K63 linkages directly from total yeast extract, all linkages were quantified from pre‐enriched Ub‐conjugates, revealing that the concentration ratio of K6:K11:K27:K29:K33:K48:K63 was ˜40:120:20:15:5:100:40 in yeast during the log growth phase, and the sum of non‐K48 linkages was more than twice as abundant as the canonical K48 linkage. Moreover, increasing the concentration of ubiquitin in yeast raised the level of almost all linkages. Inhibiting proteasome activity augmented only K48‐ and K29‐linked chains, recapitulating their roles in proteolysis and suggesting non‐proteolytic functions of other linkages in vivo.
Intracellular Protein Degradation: From a Vague Idea, through the Lysosome and the Ubiquitin—Proteasome System, and onto Human Diseases and Drug Targeting Nobel Lecture
Authors: Ciechanover Aaron 
Published: November 2006

AbstractBetween the 1950s and 1980s, scientists were focusing mostly on how the genetic code is transcribed to RNA and translated to proteins, but how proteins are degraded has remained a neglected research area. With the discovery of the lyso‐some by Christian de Duve it was assumed that cellular proteins are degraded within this organelle. Yet, several independent lines of experimental evidence strongly suggested that intracellular proteolysis is largely non‐lysosomal, but the mechanisms involved had remained obscure. The discovery of the ubiquitin‐proteasome system resolved this enigma. We now recognize that ubiquitin‐ and proteasome‐mediated degradation of intracellular proteins is involved in regulation of a broad array of cellular processes, such as cell cycle and division, regulation of transcription factors, and assurance of the cellular quality control. Not surprisingly, aberrations in the system have been implicated in the pathogenesis of many human diseases, malignancies and neurodegenerative disorders among them, which led subsequently to an increasing effort to develop mechanism‐based drugs; one is already in use.
Synthetic Genetic Interaction Between the Ubiquitin‐Conjugating Enzyme Cdc34 and a tRNA Mutant
Authors: Holtzman Tzvi , Meimoun Ariella , Kornitzer Daniel 
Published: November 2006

AbstractThe ubiquitin‐conjugating enzyme Cdc34, in conjunction with the SCF ubiquitin ligase complexes, catalyzes the ubiquitination of protein substrates involved in various cellular pathways, including the cell cycle and amino acid synthesis. A synthetic lethality screen was carried out in S. cerevisiae with the hypomorphic allelele cdc34‐2 to uncover new genetic interactions with this component of the ubiquitin system. Three temperature‐sensitive mutants that are synthetically lethal with cdc34‐2 at its permissive temperature were characterized. They represent new alleles of the F‐box protein gene CDC4, of the ubiquitin‐activating enzyme Uba1, and an allele of SUP70/CDC65, encoding the unique tRNAGln(CUG). The uba1‐118 mutant displayed extremely enlarged cells and defects in cell cycle regulation. The N‐end rule substrate Arg‐lacZ and the UFD pathway substrate Pro‐LacZ were stabilized in the uba1‐118 strain. However, no defects were detected in either in vitro activity of Uba1‐118, or in degradation of several natural substrates of the ubiquitin system at the non‐permissive temperature, suggesting that the uba1‐118 allele differentially affects degradation of specific substrates. The sup70‐65 allele, which is synthetically lethal with cdc34‐2, consists of a basepair‐disrupting mutation at the top of the anticodon stem of tRNAGln(CUG). This allele was suggested to disrupt a regulatory role of tRNAGln(CUG) in nitrogen metabolism and morphogenesis at permissive temperature. At restrictive temperature, sup70‐65 cells arrest predominantly in G1, possibly reflecting the requirement for protein synthesis in cell‐cycle START control. The synthetic interaction of cdc34‐2 and sup70‐65 suggests that common targets are affected by both Cdc34 activity and tRNAGln(CUG).
Phosphorylation by COP9 Signalosome‐Associated CK2 Promotes Degradation of p27 during the G1 Cell Cycle Phase
Authors: Huang Xiaohua , Wagner Erika , Dumdey Renate , Peth Andreas , Berse Matthias , Dubiel Wolfgang , Berndt Christoph 
Published: November 2006

AbstractThe cell cycle regulator p27Kip1 (p27) is controlled by 26S proteasome‐mediated proteolysis by two different pathways. From the S till the G2 phase of the cell cycle, degradation of p27 takes place in the nucleus and is initiated by CDK2‐dependent phosphorylation of threonine 187 with subsequent ubiquitination by the SCFSkp2 ubiquitin ligase. During the G1 cell cycle phase (G1), p27 breakdown is cytosolic and is initiated by nuclear export with subsequent ubiquitination by a RING finger ligase called kip1 ubiquitination complex. Here we show that the COP9 signalosome (CSN) is a regulator of p27 proteolysis during G1. The CSN interacts with p27 and the CSN‐associated kinase CK2 phosphorylates p27 at two regions. One is central to the protein (amino acids 101–113), and the other was mapped near to the C‐terminus (amino acids 170–189). Elimination of the putative C‐terminal phosphorylation sites stabilizes ectopic p27 towards proteasomal degradation and abolishes CSN–p27 binding. Inhibition of CSN‐associated kinase activity by curcumin attenuates loss of p27 upon cell cycle re‐entry. Similar but not additive effects of the phosphoinositol‐3‐kinase blocker LY 290042 may point to a common pathway of CSN‐associated CK2 and protein kinase B/Akt (Akt) in regulating p27 abundance. Akt is found in Flag pulldowns of lysates obtained from cells permanently expressing Flag‐tagged CSN2, indicating that Akt is a novel kinase associated with the CSN. Thus, the CSN seems to regulate p27 proteolysis at G1 downstream of Ras‐mediated signal pathways.
Protein Quality Control by the Ubiquitin Proteolytic Pathway: Roles in Resistance to Oxidative Stress and Disease
Authors: Shang Fu , Dudek Edward , Liu Qing , Boulton Michael E. , Taylor Allen 
Published: November 2006

AbstractThere is now consensus that the accumulation of oxidatively modified proteins is cytotoxic and causally related to several age‐related diseases including the amyloid diseases and age‐related cataracts. There is also general agreement that the ubiquitin proteolytic pathway (UPP) provides a quality control mechanism to limit accumulation of modified proteins. We asked if and how oxidative stress is related to the function of the ubiquitin proteolytic pathway, and vice versa, with the objective of obtaining information that can lead to the development of strategies to delay age‐related “amyloid” or “protein precipitation” diseases such as cataracts and age‐related macular degeneration. Elevated levels of ubiquitin conjugates were observed when human, rabbit, bovine, and rat lens, retina, liver cells or tissues were exposed to mild oxidative stress, which was created by exposure to paraquat, diamide, peroxide, light together with lipofuscin, and radiomimetic drugs. The increase in ubiquitin conjugates derived from an increase in substrates as well as by hyperactivation of E1, rather than inactivation of the proteasome. Using a novel glutathiolated substrate, γC‐crystallin, we demonstrated that the UPP shows a previously unrecognized selectivity for such specifically oxidatively modified proteins. Selectivity of the pathway for other oxidatively modified proteins, specifically for protein carbonyls, was indicated in assays that employed the ubiquitin conjugation‐competent, but degradation‐resistant ubiquitin variant K6W‐ubiquitin. These experiments showed that failure to execute ubiquitin‐dependent proteolysis renders cells more susceptible to oxidative‐stress‐related cytotoxicity. Activity of the pathway is regulated in part by cellular redox status, specifically as affected by GSSG. Ubiquitination is enhanced when GSSG/GSH ratios are 0.02–0.15. Since there is potentiation of ubiquitination even when GSSG/GSH ratios are indistinguishable from basal levels, it appears that ubiquitination provides one of the most sensitive indicators of oxidative stress. Ubiquitination is attenuated when GSSG/GSH rises >0.2 and does not occur when GSSG/GSH ⩾ 2.9. The data indicate that inhibition of the pathway, which occurs upon aging, is associated with accumulation rather than the timely degradation of ubiquitin conjugates. They further suggest that if the system fails to keep up with production of substrates, high mass ubiquitin conjugates may accumulate and precipitate in cytotoxic aggregates such as are seen in many age‐related syndromes, including lens cataracts or in lipofuscin and drusen in the aging retina.
Proteasome Channel Opening as a Rate‐Limiting Step in the Ubiquitin‐Proteasome System
Authors: Rabinovich Efrat , Bajorek Monika , Glickman Michael , Bar‐Nun Shoshana 
Published: November 2006

AbstractThe 26S proteasome eliminates multiubiquitinated proteins in cytosol and nucleus, and from the secretory pathway by a mechanism known as ER‐associated degradation (ERAD). Access to the proteasomal 20S catalytic core particle is hindered by conserved N‐terminal tails of α‐subunits that form a gated pore into the central channel. Hence, the isolated 20S core particle possesses slower peptide hydrolysis rates and cannot degrade multiubiquitinated proteins. Purified catalytic particles from an α3α7ΔN open channel double mutant, in which the N‐terminal tails of α‐subunits from opposite sites of the α ring are deleted, showed significantly enhanced peptidase activity and proteolytic properties. Here we show that also in vivo the access of substrates to the proteasomal catalytic chamber partially limits the overall rate of protein elimination. This regulation applies to unstable cytosolic proteins of the N‐end rule and ubiquitin fusion degradation (UFD) pathways, as well as to ERAD substrates that must dislocate from the ER back to the cytosol in order to become ubiquitinated and degraded by the proteasome. Hence, even for a complicated multistep process such as ERAD, traffic through the proteasome itself is partially rate limiting for the entire proteolytic process. However, proteasome gating can be added to a growing list of phenomena that distinguish membrane ERAD substrates from lumenal ones because while gating hinders access of lumenal substrates, it is less effective in controlling the entry of membrane substrates. The open channel mutant is a new class of proteasome mutant, which is unrelated to the catalytic protease active sites or to the “classical” regulatory particle mutants. Its improved performance at high temperatures is in stark contrast to the behavior of the “classical” mutants, suggesting that the α3α7ΔN mutant adapts better to mild stress conditions.
Juvenile Coho Salmon Growth and Survival across Stream Network Seasonal Habitats
Authors: Ebersole Joseph L. , Wigington Parker J. , Baker Joan P. , Cairns Michael A. , Church M. Robbins , Hansen Bruce P. , Miller Bruce A. , LaVigne Henry R. , Compton Jana E. , Leibowitz Scott G. 
Published: November 2006

AbstractUnderstanding watershed‐scale variation in juvenile salmonid survival and growth can provide insights into factors influencing demographics and can help target restoration and mitigation efforts for imperiled fish populations. We assessed growth, movement, and apparent overwinter survival of individually tagged juvenile coho salmon Oncorhynchus kisutch in a coastal Oregon watershed from June 2002 to June 2003 and related growth and survival parameters to stream characteristics. Fall body size of juvenile coho salmon was a good predictor of smolt size and survival, but smolt size was also influenced by overwintering location. This was due to strong spatial patterns in winter growth rates associated with residency and movement into a small intermittent tributary. Though nearly dry in midsummer, this stream supported high densities of spawning coho salmon in the fall, and juveniles rearing there exhibited relatively high growth rates and emigrated as larger smolts. Improved winter growth and survival of juvenile coho salmon utilizing tributary habitats underscore the importance of maintaining connectivity between seasonal habitats and providing a diversity of sheltering and foraging opportunities, particularly where main‐stem habitats have been simplified by human land uses.