Limiting mutual diffusion coefficients of carbon tetrachloride in methanol and of benzene, toluene, naphthalene, and biphenyl in cyclohexane as well as in ethanol at different temperatures are reported. These new data, together with literature diffusivities for the same probe solutes and for solute mesitylene in various solvents, are utilized to elucidate the effect of solvent on diffusion. Here, the data are consistent with our recent findings [J. Chem. Phys. 2013, 138, 224503] on the effects of free volume fraction, molar density, molecular mass, and fractional viscosity of solvent on diffusion. The results in this study show that the relation developed previously for solvent dependence of diffusion of disc-shaped solutes is also valid for spherical carbon tetrachloride. It is further found in this investigation that diffusivities are weakly dependent on a solvent's dielectric constant. A relation that includes the dielectric effect of solvent is demonstrated to be capable of describing the solvent dependence of diffusion of the nonpolar solutes of different shapes and sizes in this work to within an average deviation of ±2.7%. Comparisons with other diffusion models reveal that the newly developed relation is more accurate for representing the effect of solvent on diffusion. An expression for Zwanzig's "effective hydrodynamic radius" is also presented.
Myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK) has been shown to localize to the lamella of mammalian cells through its interaction with an adaptor protein, leucine repeat adaptor protein 35a (LRAP35a), which links it with myosin 18A (MYO18A) for activation of the lamellar actomyosin network essential for cell migration. Here, we report the identification of another adaptor protein LRAP25 that mediates MRCK association with LIM kinase 1 (LIMK1). The lamellipodium-localized LRAP25-MRCK complex is essential for the regulation of local LIMK1 and its downstream F-actin regulatory factor cofilin. Functionally, inhibition of either MRCK or LRAP25 resulted in a marked suppression of LIMK1 activity and down-regulation of cofilin phosphorylation in response to aluminum fluoride induction in B16-F1 cells, which eventually resulted in deregulation of lamellipodial F-actin and reorganization of cytoskeletal structures causing defects in cell polarization and motility. These biochemical and functional characterizations thus underline the functional relevance of the LRAP25-MRCK complex in LIMK1-cofilin signaling and the importance of LRAP adaptors as key determinants of MRCK cellular localization and downstream specificities.
Progesterone plays an essential role in the maintenance of the endometrium; it prepares the endometrium for pregnancy, promotes decidualization, and inhibits estrogen-dependent proliferation. Progesterone function is often dysregulated in endometrial disease states. In addition, the PI3K/AKT signaling pathway is often overactive in endometrial pathologies and promotes the survival and proliferation of the diseased cells. Understanding how AKT influences progesterone action is critical in improving hormone-based therapies in endometrial pathologies. Here, we summarize recent studies investigating the crosstalk between the AKT pathway and progesterone receptor function in endometriosis and endometrial cancer.
Findings from a year-long exploratory study aimed at describing universal functions of medical social work with interdisciplinary teams in acute care settings are reported here. A universal taxonomy of interdisciplinary social work skills and competencies was empirically identified through a participatory action research framework. Findings support previous conceptual descriptions of medical social work's overarching and historical role to help interdisciplinary teams in acute care to consider patients' home environment, knowledge, beliefs, culture, and resources during assessment, treatment, and discharge planning. The empirically determined taxonomy reported is intended to provide social workers a framework with which to articulate and evaluate their core competencies on interdisciplinary medical teams.
In the last few decades, changing socioeconomic and family structures have increasingly left children alone without adult supervision. Carefully prepared and limited periods of unsupervised time are not harmful for children. However, long unsupervised periods have harmful effects, particularly for those children at high risk for inattention and problem behaviors. In this study, we examined the influence of unsupervised time on behavior problems by studying a sample of elementary school children at high risk for inattention and problem behaviors. The study analyzed data from the Children's Mental Health Promotion Project, which was conducted in collaboration with education, government, and mental health professionals. The child behavior checklist (CBCL) was administered to assess problem behaviors among first- and fourth-grade children. Multivariate logistic regression analysis was used to evaluate the influence of unsupervised time on children's behavior. A total of 3,270 elementary school children (1,340 first-graders and 1,930 fourth-graders) were available for this study; 1,876 of the 3,270 children (57.4%) reportedly spent a significant amount of time unsupervised during the day. Unsupervised time that exceeded more than 2h per day increased the risk of delinquency, aggressive behaviors, and somatic complaints, as well as externalizing and internalizing problems. Carefully planned afterschool programming and care should be provided to children at high risk for inattention and problem behaviors. Also, a more comprehensive approach is needed to identify the possible mechanisms by which unsupervised time aggravates behavior problems in children predisposed for these behaviors.
The genetic architecture of autism spectrum disorder involves the interplay of common and rare variants and their impact on hundreds of genes. Using exome sequencing, here we show that analysis of rare coding variation in 3,871 autism cases and 9,937 ancestry-matched or parental controls implicates 22 autosomal genes at a false discovery rate (FDR) < 0.05, plus a set of 107 autosomal genes strongly enriched for those likely to affect risk (FDR < 0.30). These 107 genes, which show unusual evolutionary constraint against mutations, incur de novo loss-of-function mutations in over 5% of autistic subjects. Many of the genes implicated encode proteins for synaptic formation, transcriptional regulation and chromatin-remodelling pathways. These include voltage-gated ion channels regulating the propagation of action potentials, pacemaking and excitability-transcription coupling, as well as histone-modifying enzymes and chromatin remodellers-most prominently those that mediate post-translational lysine methylation/demethylation modifications of histones.
Mild traumatic brain injury (mTBI) is frequently associated with psychiatric symptoms and cognitive dysfunction, as well as with the receipt of workers' compensation, as many mTBIs occur due to work-related accidents. We hypothesized that depression and insufficient cognitive effort mediate the relationship between sociodemographic variables and cognitive dysfunction in mTBI.
The neuropeptides oxytocin and vasopressin are evolutionarily conserved regulators of social perception and behavior. Evidence is building that they are critically involved in the development of social recognition skills within rodent species, primates, and humans. We investigated whether common polymorphisms in the genes encoding the oxytocin and vasopressin 1a receptors influence social memory for faces. Our sample comprised 198 families, from the United Kingdom and Finland, in whom a single child had been diagnosed with high-functioning autism. Previous research has shown that impaired social perception, characteristic of autism, extends to the first-degree relatives of autistic individuals, implying heritable risk. Assessments of face recognition memory, discrimination of facial emotions, and direction of gaze detection were standardized for age (7-60 y) and sex. A common SNP in the oxytocin receptor (rs237887) was strongly associated with recognition memory in combined probands, parents, and siblings after correction for multiple comparisons. Homozygotes for the ancestral A allele had impairments in the range -0.6 to -1.15 SD scores, irrespective of their diagnostic status. Our findings imply that a critical role for the oxytocin system in social recognition has been conserved across perceptual boundaries through evolution, from olfaction in rodents to visual memory in humans.
Nucleoside analogs are an important class of anticancer agent that historically show better efficacy against hematological cancers versus solid tumors. This report describes the development and characterization of a new class of nucleoside analog that displays anticancer effects against both hematological and adherent cancer cell lines. These new analogs lack canonical hydrogen-bonding groups yet are effective nucleotide substrates for several high-fidelity DNA polymerases. Permutations in the position of the non-hydrogen-bonding functional group greatly influence the kinetic behavior of these nucleosides. One particular analog designated 4-nitroindolyl-2-deoxynucleoside triphosphate (4-NITP) is unique as it is incorporated opposite C and T with high catalytic efficiencies. In addition, this analog functions as a nonobligate chain terminator of DNA synthesis, since it is poorly elongated. Consistent with this mechanism, the corresponding nucleoside, 4-nitroindolyl-2-deoxynucleoside (4-NIdR), produces antiproliferative effects against leukemia cells. 4-NIdR also produces cytostatic and cytotoxic effects against several adherent cancer cell lines, especially those that are deficient in mismatch repair and p53. Cell death in this case appears to occur via mitotic catastrophe, a specialized form of apoptosis. Mass spectroscopy experiments performed on nucleic acid isolated from cells treated with 4-NIdR validate that the non-natural nucleoside is stably incorporated into DNA. Xenograft mouse studies demonstrate that administration of 4-NIdR delays tumor growth without producing adverse side effects such as anemia and thrombocytopenia. Collectively, the results of in vitro, cell-based, and animal studies provide evidence for the development of a novel nucleoside analog that shows enhanced effectiveness against solid tumors.
Processive protein degradation is a common feature found in ATP-dependent proteases. This study utilized a physiological substrate of Escherichia coli Lon protease known as the lambda N protein (?N) to initiate the first kinetic analysis of the proteolytic mechanism of this enzyme. To this end, experiments were designed to determine the timing of three selected scissile sites in ?N approaching the proteolytic site of ELon and their subsequent cleavages to gain insight into the mechanism by which ATP-dependent proteases attain processivity in protein degradation. The kinetic profile of peptide bond cleavage at different regions of ?N was first detected by the iTRAQ/mass spectrometry technique. Fluorogenic ?N constructs were then generated as reporter substrates for transient kinetic characterization of the ATP- versus AMPPNP-dependent peptide bond cleavage and the delivery of the scissile sites near the amino- versus carboxyl-terminal of the ?N protein to the proteolytic site of ELon. Collectively, our results support a mechanism by which the cleavage of multiple peptide bonds awaits the "almost complete" delivery of all the scissile sites in ?N to the proteolytic site in an ATP-dependent manner. Comparing the time courses of delivery to the active site of the selected scissile sites further implicates the existence of a preferred directionality in the final stage of substrate delivery, which begins at the carboxyl-terminal. The subsequent cleavage of the scissile sites in ?N, however, appears to lack a specific directionality and occurs at a much faster rate than the substrate delivery step.
Pregnant women infected with influenza virus are more likely to experience severe complications when compared with their non-pregnant peers. Yet influenza vaccine uptake is low among pregnant women. The purpose of this study was to assess the prevalence of seasonal influenza vaccine uptake among pregnant women in Hong Kong and to identify predictors of vaccine uptake.
We evaluated the effect of osmotic-release oral system (OROS) methylphenidate on learning skills in adolescents with attention-deficit/hyperactivity disorder (ADHD). In an open-label study, 121 adolescents with ADHD were administered flexible doses of OROS methylphenidate for 12 weeks. The efficacy of methylphenidate on ADHD symptoms was evaluated by ADHD Rating Scale (ARS) and Clinical Global Impression Scale (CGI). Learning Skills Test (LST) was used to measure the learning skills of the participants at the baseline and the endpoint. Continuous performance test, visuospatial and verbal working memory, verbal fluency, and inhibition were evaluated before and after the 12 weeks of treatment. The mean total and subscores of LST were significantly increased after the 12-week treatment with OROS methylphenidate. Executive functions were also improved during the trial, with the exception of inhibition measured by the Stroop Test. To the best of our knowledge, this is the first study to examine the influence of OROS methylphenidate on learning skill. As a result, OROS methylphenidate was effective in enhancing learning skills in adolescents with ADHD.
This report describes the use of several isosteric non-natural nucleotides as probes to evaluate the roles of nucleobase shape, size, solvation energies, and ?-electron interactions as forces influencing key kinetic steps of the DNA polymerization cycle. Results are provided using representative high- and low-fidelity DNA polymerases. Results generated with the E. coli Klenow fragment reveal that this high-fidelity polymerase utilizes hydrophobic nucleotide analogues with higher catalytic efficiencies compared to hydrophilic analogues. These data support a major role for nucleobase desolvation during nucleotide selection and insertion. In contrast, the low-fidelity HIV-1 reverse transcriptase discriminates against hydrophobic analogues and only tolerates non-natural nucleotides that are capable of hydrogen-bonding or ?-stacking interactions. Surprisingly, hydrophobic analogues that function as efficient substrates for the E. coli Klenow fragment behave as noncompetitive or uncompetitive inhibitors against HIV-1 reverse transcriptase. In these cases, the mode of inhibition depends upon the absence or presence of a templating nucleobase. Molecular modeling studies suggest that these analogues bind to the active site of reverse transcriptase as well as to a nearby hydrophobic binding pocket. Collectively, the studies using these non-natural nucleotides reveal important mechanistic differences between representative high- and low-fidelity DNA polymerases during nucleotide selection and incorporation.
Epithelial-mesenchymal transition (EMT), proliferation and migration of RPE cells characterize the development of proliferative vitreoretinopathy (PVR) and other fibro-proliferative eye diseases leading to blindness. A common event in these pathologies is the alteration of the BRB which allows the interaction of RPE cells with thrombin, a pro-inflammatory protease contained in serum. Thrombin promotion of cytoskeletal reorganization, proliferation, and migration has been reported in different cell types, although the molecular mechanisms involved in these processes remain poorly understood. Our previous work demonstrated that thrombin promotes RPE cell proliferation, cytoskeletal remodeling and migration, hallmark processes in the development of PVR. Thrombin induction of RPE cell proliferation requires PI3K, PDK1, and Akt/PKB (Akt) signaling leading to cyclin D1 gene expression. Since Akt functions as an upstream activator of mechanistic target of rapamycin complex 1 (mTORC1) and is also a downstream target for mTORC2, the aim of this work was to determine whether mTOR is involved in thrombin-induced RPE cell proliferation by regulating cyclin D1 expression in immortalized rat RPE-J cell line. Results demonstrate that thrombin-induced cyclin D1 expression and cell proliferation require Akt-independent phosphorylation/activation of mTOR at Ser 2448 mediated by PI3K/PKC-?/ERK1/2 signaling, concomitant to Akt-dependent activation of p70S6K carried by mTORC1.
The misreplication of damaged DNA is an important biological process that produces numerous adverse effects on human health. This report describes the synthesis and characterization of a non-natural nucleotide, designated 3-ethynyl-5-nitroindolyl-2-deoxyriboside triphosphate (3-Eth-5-NITP), as a novel chemical reagent that can probe and quantify the misreplication of damaged DNA. We demonstrate that this non-natural nucleotide is efficiently inserted opposite an abasic site, a commonly formed and potentially mutagenic non-instructional DNA lesion. The strategic placement of the ethynyl moiety allows the incorporated nucleoside triphosphate to be selectively tagged with an azide-containing fluorophore using click chemistry. This reaction provides a facile way to quantify the extent of nucleotide incorporation opposite non-instructional DNA lesions. In addition, the incorporation of 3-Eth-5-NITP is highly selective for an abasic site, and occurs even in the presence of a 50-fold molar excess of natural nucleotides. The biological applications of using 3-Eth-5-NITP as a chemical probe to monitor and quantify the misreplication of non-instructional DNA lesions are discussed.
A bienzymatic electrode incorporating trehalase (Tre) and glucose oxidase (GOx) covalently bound to the surface of Pt through a functionalized thiol linker (Tre|GOx|Pt) has been designed and assembled and its catalytic properties examined by chemical and electrochemical methods in aqueous phosphate buffer solutions (PBS, pH 7.4). Exposure of Tre|GOx|Pt to PBS containing trehalose (Tr) and subsequent polarization at 0.6 V versus Ag/AgCl yielded after a few minutes well-defined steady-state currents ascribed to the oxidation of hydrogen peroxide generated by the GOx-mediated oxidation of glucose (Gl) produced by the Tre-mediated dissociation of Tr. Plots of the steady-state currents versus [Tr] over the range examined, i.e., 5-25 mM, were found to be linear. Implications of these results toward the development of an implantable biofuel cell as an autonomous energy conversion device for insects are discussed.
The AAA(+) Lon protease is a soluble single-ringed homo-oligomer, which represents the most streamlined operational unit mediating ATP-dependent proteolysis. Despite its simplicity, the architecture of Lon proteases exhibits a species-specific diversity. Homology modeling provides insights into the structural features that distinguish bacterial and human Lon proteases as hexameric complexes from yeast Lon, which is uniquely heptameric. The best-understood functions of mitochondrial Lon are linked to maintaining proteostasis under normal metabolic conditions, and preventing proteotoxicity during environmental and cellular stress. An intriguing property of human Lon is its specific binding to G-quadruplex DNA, and its association with the mitochondrial genome in cultured cells. A fraction of Lon preferentially binds to the control region of mitochondrial DNA where transcription and replication are initiated. Here, we present an overview of the diverse functions of mitochondrial Lon, as well as speculative perspectives on its role in protein and mtDNA quality control.
A general method has been developed to determine the ionization constants of polymer thin films based on the stimuli-responsiveness of the polymer. Robust polymer films were fabricated on silicon wafers and gold slides using perfluorophenyl azide (PFPA) as the coupling agent. The ionization constants were measured by a number of techniques including ellipsometry, dynamic contact angle goniometry, and surface plasmon resonance imaging (SPRi). Using poly(4-vinylpyridine) (P4VP) as the model system, P4VP thin films were fabricated and the ionization constants of the films were measured taking advantage of the pH responsive property of the polymer. The pK(a) determined by ellipsometry, ~4.0, reflects the swelling of the polymer film in response to pH. The pK(a) value calculated from the dynamic contact angle measurements, ~5.0, relies on the change in hydrophilicity/hydrophobicity of the films as the polymer undergoes protonation/deprotonation. The pK(a) value measured by SPRi, ~4.9, monitors in situ the change of refractive index of the polymer thin film as it swells upon protonation. This was the first example where SPRi was used to measure the ionization constants of polymers.
Progesterone is a growth inhibitory hormone in the endometrium. While progestins can be used for the treatment of well-differentiated endometrial cancers, resistance to progestin therapy occurs for reasons that remain unclear. We have previously demonstrated that progesterone receptors (PR) A and B differentially regulate apoptosis in response to overexpression of the forkhead transcription factor, FOXO1. In this study, we further examined the PR-isoform-dependent cellular response to the AKT pathway. Treatment of PRA and PRB-expressing Ishikawa cells (PRA14, PRB23), with an AKT inhibitor API-59CJ-OMe (API-59) promoted apoptosis in the presence and absence of the ligand, R5020 preferentially in PRA14 cells. Upon PR knockdown using small interfering RNA, an increase in apoptosis was observed in PRB23 cells treated with API-59 with or without R5020 while there was no influence in PRA14 cells. Using an apoptosis-focused real-time PCR array, genes regulated by API-59 and R5020 were identified both common and unique to PRA14 and PRB23 cells. BIRC3 was identified as the only gene regulated by R5020 which occurred only in PRB cells. Knockdown of BIRC3 in PRB23 cells promoted a decrease in cell viability in response to API-59 + R5020. Furthermore, the important role of inhibitors of apoptosis (IAPs) in the PRB23 cells to promote cell survival was demonstrated using an antagonist to IAPs, a second mitochondria-derived activator of caspase (Smac also known as DIABLO) mimetic. Treatment of PRB23 cells with Smac mimetic increased apoptosis in response to API-59 + R5020. In summary, our findings indicate a mechanism by which PRB can promote cell survival in the setting of high AKT activity in endometrial cancer cells.
The World Health Organization (WHO) developed the Baby-Friendly Hospital Initiative to improve hospital maternity care practices that support breastfeeding. In Hong Kong, although no hospitals have yet received the Baby-Friendly status, efforts have been made to improve breastfeeding support. The aim of this study was to examine the impact of Baby-Friendly hospital practices on breastfeeding duration.
Lon and ClpXP are the only soluble ATP-dependent proteases within the mammalian mitochondria matrix, which function in protein quality control by selectively degrading misfolded, misassembled, or damaged proteins. Chemical tools to study these proteases in biological samples have not been identified, thereby hindering a clear understanding of their respective functions in normal and disease states. In this study, we applied a proteolytic site-directed approach to identify a peptide reporter substrate and a peptide inhibitor that are selective for Lon but not ClpXP. These chemical tools permit quantitative measurements that distinguish Lon-mediated proteolysis from that of ClpXP in biochemical assays with purified proteases, as well as in intact mitochondria and mitochondrial lysates. This chemical biology approach provides needed tools to further our understanding of mitochondrial ATP-dependent proteolysis and contributes to the future development of diagnostic and pharmacological agents for treating diseases associated with defects in mitochondrial protein quality.
The Autism Genome Project (AGP) Consortium recently reported genome-wide significant association between autism and an intronic single nucleotide polymorphism marker, rs4141463, within the MACROD2 gene. In the present study we attempted to replicate this finding using an independent case-control design of 1,170 cases with autism spectrum disorder (ASD) (874 of which fulfilled narrow criteria for Autism (A)) from five centers within Europe (UK, Germany, the Netherlands, Italy, and Iceland), and 35,307 controls. The combined sample size gave us a non-centrality parameter (NCP) of 11.9, with 93% power to detect allelic association of rs4141463 at an alpha of 0.05 with odds ratio of 0.84 (the best odds ratio estimate of the AGP Consortium data), and for the narrow diagnosis of autism, an NCP of 8.9 and power of 85%. Our case-control data were analyzed for association, stratified by each center, and the summary statistics were combined using the meta-analysis program, GWAMA. This resulted in an odds ratio (OR) of 1.03 (95% CI 0.944-1.133), with a P-value of 0.5 for ASD and OR of 0.99 (95% CI 0.88-1.11) with P-value = 0.85 for the Autism (A) sub-group. Therefore, this study does not provide support for the reported association between rs4141463 and autism.
The FMR1 gene, mapping to an area of the X chromosome closely associated with autoimmunity also affects ovarian reserve, with specific genotypes associated with distinct ovarian aging patterns. They, therefore, could also be associated with differences of in vitro fertilization (IVF) outcomes, reported between races/ethnicities. We analyzed 339 consecutive IVF patients, 232 Caucasian, 59 African and 48 Asian, for FMR1 genotypes, and tested by multiple logistic regressions for associations between race/ethnicity, FMR1 genotype, autoimmunity and pregnancy chances with IVF. FMR1 genotypes were predictive of pregnancy (P?=?0.046), het-norm/low most significantly and with decreasing chance in comparison to norm genotypes (OR 0.44; 95% CI 0.23-0.85; P?=?0.014). Race/ethnicity was, overall, independently associated (P?=?0.03), African demonstrating decreased odds in comparison to Caucasian (OR 0.33. 95%CI 0.13-0.79; P?=?0.014). Autoimmunity did not differ but interaction of autoimmunity with FMR1 genotype almost reached significance (P?=?0.07). Logistic regression with race/ethnicity and interaction between FMR1 genotype and autoimmunity in the model, demonstrated 2.5-times the odds of being associated with autoimmune positivity (OR 2.5, 1.34-4.55; P?=?0.004). FMR1 genotypes offer a possible explanation for differences in IVF outcomes between races/ethnicities.
This multicenter, randomized, open-label, parallel trial aimed to provide a detailed dose-response profile for atomoxetine in Korean pediatric outpatients with attention-deficit/hyperactivity disorder (ADHD).
The purpose of the present study was to develop reliable and valid parent and teacher scales for measurement of functional impairment in children and adolescents in order to assist the diagnosis of attention-deficit/hyperactivity disorder (ADHD).
Copper amine oxidases (CAOs) are a family of redox active enzymes containing a 2,4,5-trihydroxyphenylalanine quinone (TPQ) cofactor generated from post translational modification of an active site tyrosine residue. The Arthrobacter globiformis amine oxidase (AGAO) has been widely used as a model to guide the design and development of selective inhibitors of CAOs. In this study, two aryl 2,3-butadienamine analogs, racemic 5-phenoxy-2,3-pentadienylamine (POPDA) and racemic 6-phenyl-2,3-hexadienylamine (PHDA), were synthesized and evaluated as mechanism-based inactivators of AGAO. Crystal structures show that both compounds form a covalent adduct with the amino group of the substrate-reduced TPQ, and that the chemical structures of the rac-PHDA and rac-POPDA modified TPQ differ by the allenic carbon that is attached to the cofactor. A chemical mechanism accounting for the formation of the respective TPQ derivative is proposed. Under steady-state conditions, no recovery of enzyme activity is detected when AGAO pre-treated with rac-PHDA or rac-POPDA is diluted with excess amount of the benzylamine substrate (100-fold K(m)). Comparing the IC(50) values further reveals that the phenoxy substituent in POPDA offers an approximately 4-fold increase in inhibition potency, which can be attributed to a favourable binding interaction between the oxygen atom in the phenoxy group and the active site of AGAO as revealed by crystallographic studies. This hypothesis is corroborated by the observed >3-fold higher partition ratio of PHDA compared to POPDA. Taken together, the results presented in this study reveal the mechanism by which aryl 2,3-butadienamines act as mechanism-based inhibitors of AGAO, and the potency of enzyme inactivation could be fine-tuned by optimizing binding interaction between the aryl substituent and the enzyme active site.
This report examines the molecular mechanism by which high-fidelity DNA polymerases select nucleotides during the replication of an abasic site, a non-instructional DNA lesion. This was accomplished by synthesizing several unique 5-substituted indolyl 2-deoxyribose triphosphates and defining their kinetic parameters for incorporation opposite an abasic site to interrogate the contributions of ?-electron density and solvation energies. In general, the K(d, app) values for hydrophobic non-natural nucleotides are ?10-fold lower than those measured for isosteric hydrophilic analogs. In addition, k(pol) values for nucleotides that contain less ?-electron densities are slower than isosteric analogs possessing higher degrees of ?-electron density. The differences in kinetic parameters were used to quantify the energetic contributions of desolvation and ?-electron density on nucleotide binding and polymerization rate constant. We demonstrate that analogs lacking hydrogen-bonding capabilities act as chain terminators of translesion DNA replication while analogs with hydrogen bonding functional groups are extended when paired opposite an abasic site. Collectively, the data indicate that the efficiency of nucleotide incorporation opposite an abasic site is controlled by energies associated with nucleobase desolvation and ?-electron stacking interactions whereas elongation beyond the lesion is achieved through a combination of base-stacking and hydrogen-bonding interactions.
Here we describe the design, synthesis, and evaluation of physicochemical and pharmacological properties of D(4) dopamine receptor ligands related to N-[2-[4-(4-chlorophenyl)piperazin-1-yl]ethyl]-3-methoxybenzamide (2). Structural features were incorporated to increase affinity for the target receptor, to improve selectivity over D(2) and ?(1) receptors, to enable labeling with carbon-11 or fluorine-18, and to adjust lipophilicity within the range considered optimal for brain penetration and low nonspecific binding. Compounds 7 and 13 showed the overall best characteristics: nanomolar affinity for the D(4) receptor, >100-fold selectivity over D(2) and D(3) dopamine receptors, 5-HT(1A), 5-HT(2A), and 5-HT(2C) serotonin receptors and ?(1) receptors, and log?P = 2.37-2.55. Following intraperitoneal administration in mice, both compounds rapidly entered the central nervous system. The methoxy of N-[2-[4-(3-cyanopyridin-2-yl)piperazin-1-yl]ethyl]-3-methoxybenzamide (7) was radiolabeled with carbon-11 and subjected to PET analysis in non-human primate. [(11)C]7 time-dependently accumulated to saturation in the posterior eye in the region of the retina, a tissue containing a high density of D(4) receptors.
The FMR1 gene partially appears to control ovarian reserve, with a specific ovarian sub-genotype statistically associated with a polycystic ovary (PCO)- like phenotype. Some forms of PCO have been associated with autoimmunity. We, therefore, investigated in multiple regression analyses associations of ovary-specific FMR1 genotypes with autoimmunity and pregnancy chances (with in vitro fertilization, IVF) in 339 consecutive infertile women (455 IVF cycles), 75 with PCO-like phenotype, adjusted for age, race/ethnicity, medication dosage and number of oocytes retrieved. Patients included 183 (54.0%) with normal (norm) and 156 (46%) with heterozygous (het) FMR1 genotypes; 133 (39.2%) demonstrated laboratory evidence of autoimmunity: 51.1% of het-norm/low, 38.3% of norm and 24.2% het-norm/high genotype and sub-genotypes demonstrated autoimmunity (p=0.003). Prevalence of autoimmunity increased further in PCO-like phenotype patients with het-norm/low genotype (83.3%), remained unchanged with norm (34.0%) and decreased in het-norm/high women (10.0%; P<0.0001). Pregnancy rates were significantly higher with norm (38.6%) than het-norm/low (22.2%, p=0.001). FMR1 sub-genotype het-norm/low is strongly associated with autoimmunity and decreased pregnancy chances in IVF, reaffirming the importance of the distal long arm of the X chromosome (FMR1 maps at Xq27.3) for autoimmunity, ovarian function and, likely, pregnancy chance with IVF.
The synthesis and development of N-terminal peptidic boronic acids as protease inhibitors is reported. N-Terminal peptidic boronic acids interrogate the S sites of the target protein for selectivity and provide a new strategy that complements the currently known peptidic alpha-amino boronic acids (C-terminal boronic acids). After screening a series of N-terminal peptidic boronic acids, the first selective inhibitor of human ClpXP, an ATP-dependent serine protease present in the mitochondrial matrix, was discovered. This should facilitate the understanding of the physiological function of this protease.
Breastfeeding provides optimal and complete nutrition for newborn babies. Although new mothers in Hong Kong are increasingly choosing to breastfeed their babies, rates of exclusive breastfeeding are low and duration remains short. The purpose of this study was to describe the breastfeeding and weaning practices of Hong Kong mothers over the infants first year of life to determine the factors associated with early cessation.
Most models accounting for the efficiency and fidelity of DNA polymerization invoke the use of either hydrogen bonding contacts or complementarity of shape and size between the formed base pair. This report evaluates these mechanisms by quantifying the ability of a high-fidelity DNA polymerase to replicate 5-nitroindole, a purine mimetic devoid of classic hydrogen bonding capabilities. 5-NITP acts as a universal nucleotide since it is incorporated opposite any of the four natural nucleobases with nearly equal efficiencies. Surprising, the polymerization reaction is not reciprocal as natural nucleotides are poorly incorporated opposite 5-nitroindole in the template strand. Incorporation opposite 5-nitroindole is more efficient using natural nucleotides containing various modifications that increase their base stacking potential. However, 5-substituted indolyl nucleotides that contain pi-electron and/or hydrophobic groups are incorporated opposite the non-natural nucleobase with the highest catalytic efficiencies. The collective data set indicate that replication of a non-natural nucleobase is driven by a combination of the hydrophobic nature and pi-electron surface area of the incoming nucleotide. In this mechanism, the overall hydrophobicity of the incoming nucleobase overcomes the lack of hydrogen bonding groups that are generally required for optimal DNA polymerization. However, the lack of hydrogen bonds between base pairs prevents primer extension. This final aspect is manifest by the appearance of unusually high pyrophosphorolysis activity by the T4 DNA polymerase that is only observed with the non-natural nucleobase in the template. These results highlight the importance of hydrogen bonding interactions during primer extension and pyrophosphorolysis.
Lon protease, also known as protease La, is an ATP-dependent serine protease. Despite the presence of a proteolytic Ser-Lys dyad, the enzyme only catalyzes protein degradation in the presence of ATP. Lon possesses an intrinsic ATPase activity that is stimulated by protein and certain peptide substrates. Through sequence alignment and analysis, it is concluded that Lon belongs to the AAA+ protein family. Previous kinetic characterization of the ATPase domain of Escherichia coli Lon protease implicates a half-site reactivity model in which only 50% of the ATP bound to Lon are hydrolyzed to yield ADP; the remaining ATPase sites remain bound with ATP and are considered non-catalytic. In this model, it is implied that ATP hydrolysis is irreversible. To further evaluate the proposed half-site reactivity model, the reversibility of the ATPase activity of E. coli Lon was evaluated by positional isotope exchange experiments. The ATPase reactions were conducted in the 18O-enriched buffer such that the extent of 18O incorporation into inorganic phosphate generated from ATP hydrolysis could be used to evaluate the extent of reversibility in ATP hydrolysis. Collectively, our experimental data reveal that the ATPase reaction catalyzed by E. coli Lon in the presence and absence of peptide substrate that stimulated the enzymes ATPase activity is irreversible. Therefore, the half-site ATPase reactivity of E. coli Lon is validated, and can be used to account for the kinetic mechanism of the ATP-dependent peptidase activity of the enzyme.
Oxidant stress contributes to the pathogenesis of multiple conditions and can be assessed by measuring plasma F(2)-isoprostane concentrations. We hypothesized that oxidant stress is associated with plasma homocysteine concentration and risk factors for atherosclerosis in HIV-infected women.
A robotic virtual-reality (VR) simulator has been developed to improve robot-assisted training for laparoscopic surgery and to enhance surgical performance in laparoscopic skills. The simulated VR training environment provides an effective approach to evaluate and improve surgical performance. This study presents our findings of the VR training environment for robotic laparoscopy.
DNA is a remarkable macromolecule that functions primarily as the carrier of the genetic information of organisms ranging from viruses to bacteria to eukaryotes. The ability of DNA polymerases to efficiently and accurately replicate genetic material represents one of the most fundamental yet complex biological processes found in nature. The central dogma of DNA polymerization is that the efficiency and fidelity of this biological process is dependent upon proper hydrogen-bonding interactions between an incoming nucleotide and its templating partner. However, the foundation of this dogma has been recently challenged by the demonstration that DNA polymerases can effectively and, in some cases, selectively incorporate non-natural nucleotides lacking classic hydrogen-bonding capabilities into DNA. In this review, we describe the results of several laboratories that have employed a variety of non-natural nucleotide analogs to decipher the molecular mechanism of DNA polymerization. The use of various non-natural nucleotides has lead to the development of several different models that can explain how efficient DNA synthesis can occur in the absence of hydrogen-bonding interactions. These models include the influence of steric fit and shape complementarity, hydrophobicity and solvation energies, base-stacking capabilities, and negative selection as alternatives to rules invoking simple recognition of hydrogen-bonding patterns. Discussions are also provided regarding how the kinetics of primer extension and exonuclease proofreading activities associated with high-fidelity DNA polymerases are influenced by the absence of hydrogen-bonding functional groups exhibited by non-natural nucleotides.
Lon, also known as protease La, is an ATP-dependent protease functioning to degrade many unstructured proteins. Currently, very little is known about the substrate determinants of Lon at the proteolytic site. Using synthetic peptides constituting different regions of the endogenous protein substrate lambdaN, we demonstrated that the proteolytic site of Escherichia coli Lon exhibits a certain level of localized sequence specificity. Using an alanine positional scanning approach, we discovered a set of discontinuous substrate determinants surrounding the scissile Lon cleavage site in a model peptide substrate, which function to influence the k(cat) of the peptidase activity of Lon. We further investigated the mode of peptide interaction with the proteolytically inactive Lon mutant S679A in the absence and presence of ADP or AMPPNP by 2-dimensional nuclear magnetic resonance spectroscopy, and discovered that the binding interaction between protein and peptide varies with the nucleotide bound to the enzyme. This observation is suggestive of a substrate translocation step, which likely limits the turnover of the proteolytic reaction. The contribution of the identified substrate determinants towards the kinetics of ATP-dependent degradation of lambdaN and truncated lambdaN mutants by Lon was also examined. Our results indicated that Lon likely recognizes numerous discontinuous substrate determinants throughout lambdaN to achieve substrate promiscuity.
Subpopulations of patients with adjustment disorder are at increased risk for suicide. The current study investigated whether personality traits, including alexithymia, temperament, and character, are associated with an increased risk of suicide in individuals with adjustment disorder. Age- and sex-matched patients meeting the diagnostic and statistical manual of mental disorders (DSM-IV) criteria for adjustment disorder with (n=92) and without (n=92) a history of suicide attempts were recruited for the present study. Ninety-two healthy individuals who did not meet diagnostic criteria for Axis I or II diagnoses were used as controls. The Toronto alexithymia scale-20 (TAS-20) and the temperament and character inventory (TCI) were used to assess personality traits. Significantly higher total and subscale scores on the TAS-20, including on the difficulty-identifying-feelings (DIF) and difficulty-describing-feelings (DDF) subscales, and lower scores on the TCI cooperativeness subscale were noted in adjustment-disorder patients with previous suicide attempts. In the multivariate regression analysis, high DDF and DIF and low cooperativeness increased the risk of suicide attempts in adjustment-disorder patients. A subsequent path analysis revealed that high DDF had a direct effect on suicide attempts, whereas high DIF had an indirect effect on suicide attempts via low cooperativeness.
Personality is an important clinical factor for successful adjustment in stressful situations. The aim of this study was to examine possible differences in temperament and character dimensions between patients with adjustment disorder with depressed mood and healthy controls. Among the young male conscripts, 86 subjects with adjustment disorder with depressed mood and 86 healthy controls were included. The mean scores in the 7 dimensions and 25 subscales of the Temperament and Character Inventory were compared between the patients with adjustment disorder with depressed mood and the control group by an independent t-test. The patients with adjustment disorder with depressed mood had significantly higher scores on harm-avoidance and lower scores on self-directedness, cooperativeness, and self-transcendence than did the controls. There were no differences in novelty seeking, reward dependence, and persistence in temperament between the two groups. The results of this study suggest that the personality traits of the subjects with adjustment disorder with depressed mood would make them vulnerable to stressful situations and less skilled in coping with conscription.
High fidelity DNA polymerases maintain genomic fidelity through a series of kinetic steps that include nucleotide binding, conformational changes, phosphoryl transfer, polymerase translocation, and nucleotide excision. Developing a comprehensive understanding of how these steps are coordinated during correct and pro-mutagenic DNA synthesis has been hindered due to lack of spectroscopic nucleotides that function as efficient polymerase substrates. This report describes the application of a non-natural nucleotide designated 5-naphthyl-indole-2-deoxyribose triphosphate which behaves as a fluorogenic substrate to monitor nucleotide incorporation and excision during the replication of normal DNA versus two distinct DNA lesions (cyclobutane thymine dimer and an abasic site). Transient fluorescence and rapid-chemical quench experiments demonstrate that the rate constants for nucleotide incorporation vary as a function of DNA lesion. These differences indicate that the non-natural nucleotide can function as a spectroscopic probe to distinguish between normal versus translesion DNA synthesis. Studies using wild-type DNA polymerase reveal the presence of a fluorescence recovery phase that corresponds to the formation of a pre-excision complex that precedes hydrolytic excision of the non-natural nucleotide. Rate constants for the formation of this pre-excision complex are dependent upon the DNA lesion, and this suggests that the mechanism of exonuclease proofreading is regulated by the nature of the formed mispair. Finally, spectroscopic evidence confirms that exonuclease proofreading competes with polymerase translocation. Collectively, this work provides the first demonstration for a non-natural nucleotide that functions as a spectroscopic probe to study the coordinated efforts of polymerization and exonuclease proofreading during correct and translesion DNA synthesis.
Progestin resistance is a major obstacle to treating early stage, well-differentiated endometrial cancer as well as recurrent endometrial cancer. The mechanism behind the suboptimal response to progestin is not well understood. The PTEN tumor suppressor gene is frequently mutated in type I endometrial cancers and this mutation results in hyperactivation of the PI3K/AKT pathway. We hypothesized that increased activation of AKT promotes an inadequate response to progestins in endometrial cancer cells. Ishikawa cells stably transfected with progesterone receptor B (PRB23 cells) were treated with the AKT inhibitor, MK-2206, which effectively decreased levels of p(Ser473)-AKT in a dose-dependent (10 nM to 1 uM) and time-dependent manner (0.5 h to 24 h). MK-2206 inhibited levels of p(Thr308)-AKT and a downstream target, p(Thr246)-PRAS40, but did not change levels of p(Thr202/Tyr204)ERK or p(Thr13/Tyr185)SAPK/JNK, demonstrating specificity of MK-2206 for AKT. Additionally, MK-2206 treatment of PRB23 cells resulted in a significant increase in levels of progesterone receptor B (PRB) protein. Microarray analysis of PRB23 cells identified PDK4 as the most highly upregulated gene among 70 upregulated genes in response to R5020. Inhibition of AKT further upregulated progestin-mediated expression of PDK4 but did not affect another progestin-responsive gene, SGK1. Treatment of PRB23 cells with R5020 and MK-2206 independently decreased viability of cells while the combination of R5020 and MK-2206 caused the greatest decrease in cell viability. Furthermore, mice with xenografted tumors treated with MK-2206 alone or with progesterone alone exhibited modest reductions in their tumor volume. The largest decrease in tumor size was observed in the mice treated with both MK-2206 and progesterone; these tumors exhibited the least proliferation (Ki67) and the most apoptosis (cleaved caspase-3) of all the treatment groups. In summary, inhibition of AKT stabilizes the Progesterone Receptor B and augments progesterone response in endometrial cancer cells that have hyperactivated AKT.
Acute lymphoblastic leukemia (ALL) is the most common type of childhood cancer, presenting with approximately 5,000 new cases each year in the United States. An interesting enzyme implicated in this disease is terminal deoxynucleotidyl transferase (TdT), a specialized DNA polymerase involved in V(D)J recombination. TdT is an excellent biomarker for ALL as it is overexpressed in ~90% of ALL patients, and these higher levels correlate with a poor prognosis. These collective features make TdT an attractive target to design new selective anti-cancer agents against ALL. In this report, we evaluate the anti-leukemia activities of two non-natural nucleotides designated 5-nitroindolyl-2-deoxynucleoside triphosphate (5-NITP) and 3-ethynyl-5-nitroindolyl-2-deoxynucleoside triphosphate (3-Eth-5-NITP). Using purified TdT, we demonstrate that both non-natural nucleotides are efficiently utilized as TdT substrates. However, 3-Eth-5-NITP is poorly elongated, and this observation validates its activity as a chain-terminator for blunt-end DNA synthesis. Cell-based experiments validate that the corresponding non-natural nucleoside produces robust cytostatic and cytotoxic effects against leukemia cells that overexpress TdT. The strategic placement of the ethynyl moiety allows the incorporated nucleoside triphosphate to be selectively tagged with an azide-containing fluorophore via "click" chemistry. This reaction allows the extent of nucleotide incorporation to be quantified such that the anti-cancer effects of the corresponding non-natural nucleoside can be self-assessed. The applications of this novel nucleoside are discussed, focusing on its use as a "theranostic" agent that can improve the accuracy of dosing regimens and accelerate clinical decisions regarding therapeutic intervention.
Lon, also known as protease La, is an AAA+ protease machine that contains the ATPase and proteolytic domain within each enzyme subunit. Three truncated Escherichia coli Lon (ELon) mutants were generated based on a previous limited tryptic digestion result and hydrogen-deuterium exchange mass spectrometry analyses performed in this study. Using methods developed for characterizing wild-type (WT) Lon, we compared the ATPase, ATP-dependent protein degradation and ATP-dependent peptidase activities. With the exception of not degrading a putative structured substrate known as CcrM (cell-cycle-regulated DNA methyltransferase), the mutant lacking the first 239 residues behaved like WT ELon. Comparing the activity data of WT and ELon mutants reveals that the first 239 residues are not needed for minimal enzyme catalysis. The mutants lacking the first 252 residues or residues 232-252 displayed compromised ATPase, protein degradation and ATP-dependent peptide translocation abilities but retained WT-like steady-state peptidase activity. The binding affinities of WT and Lon mutants were evaluated by determining the concentration of ? N (K(?N)) needed to achieve 50% maximal ATPase stimulation. Comparing the K(?N) values reveals that the region encompassing 232-252 of ELon could contribute to ? N binding, but the effect is modest. Taken together, results generated from this study reveal that the region constituting residues 240-252 of ELon is important for ATPase activity, substrate translocation and protein degradation.
To report a study of the relations of prenatal psychosocial adaptation, social support, demographic and obstetric characteristics, uncertainty, information-seeking behaviour, motherhood normalization, self-efficacy, and commitment to pregnancy.
The rod-derived cone viability factors, RdCVF and RdCVF2, have potential therapeutical interests for the treatment of inherited photoreceptor degenerations. In the mouse lacking Nxnl2, the gene encoding RdCVF2, the progressive decline of the visual performance of the cones in parallel with their degeneration, arises due to the loss of trophic support from RdCVF2. In contrary, the progressive loss of rod visual function of the Nxnl2-/- mouse results from a decrease in outer segment length, mediated by a cell autonomous mechanism involving the putative thioredoxin protein RdCVF2L, the second spliced product of the Nxnl2 gene. This novel signaling mechanism extends to olfaction as shown by the progressive impairment of olfaction in aged Nxnl2-/- mice and the protection of olfactory neurons by RdCVF2. This study shows that Nxnl2 is a bi-functional gene involved in the maintenance of both the function and the viability of sensory neurons.
The photoinitiated radical reactions between thiols and alkenes/alkynes (thiol-ene and thiol-yne chemistry) have been applied to a functionalization methodology to produce carbohydrate-presenting surfaces for analyses of biomolecular interactions. Polymer-coated quartz surfaces were functionalized with alkenes or alkynes in a straightforward photochemical procedure utilizing perfluorophenylazide (PFPA) chemistry. The alkene/alkyne surfaces were subsequently allowed to react with carbohydrate thiols in water under UV-irradiation. The reaction can be carried out in a drop of water directly on the surface without photoinitiator, and any disulfide side products were easily washed away after the functionalization process. The resulting carbohydrate-presenting surfaces were evaluated in real-time studies of protein-carbohydrate interactions using a quartz crystal microbalance (QCM) flow-through system with recurring injections of selected lectins, with intermediate regeneration steps using low pH buffer. The resulting methodology proved fast, efficient and scalable to high-throughput analysis formats, and the produced surfaces showed significant protein binding with expected selectivities of the lectins used in the study.
This report describes the design and application of several distinct gold-containing indoles as anticancer agents. When used individually, all gold-bearing compounds display cytostatic effects against leukemia and adherent cancer cell lines. However, two gold-bearing indoles show unique behavior by increasing the cytotoxic effects of clinically relevant levels of ionizing radiation. Quantifying the amount of DNA damage demonstrates that each gold-indole enhances apoptosis by inhibiting DNA repair. Both Au(I)-indoles were tested for inhibitory effects against various cellular targets including thioredoxin reductase, a known target of several gold compounds, and various ATP-dependent kinases. While neither compound significantly inhibits the activity of thioreoxin reductase, both showed inhibitory effects against several kinases associated with cancer initiation and progression. The inhibition of these kinases provides a possible mechanism for the ability of these Au(I)-indoles to potentiate the cytotoxic effects of ionizing radiation. Clinical applications of combining Au(I)-indoles with ionizing radiation are discussed as a new strategy to achieve chemosensitization of cancer cells.
A biofuel cell incorporating a bienzymatic trehalase|glucose oxidase trehalose anode and a bilirubin oxidase dioxygen cathode using Os complexes grafted to a polymeric backbone as electron relays was designed and constructed. The specific power densities of the biofuel cell implanted in a female Blaberus discoidalis through incisions into its abdomen yielded maximum values of ca. 55 ?W/cm(2) at 0.2 V that decreased by only ca. 5% after ca. 2.5 h of operation.
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