This paper evaluates comparative patterns of fertility in new Hispanic destinations and established gateways using pooled cross-sectional data from the 2005–2009 microdata files of the American Community Survey. Changing Hispanic fertility provides a useful indicator of cultural incorporation. Analyses show that high fertility among Hispanics has been driven in part by the Mexican-origin and other new immigrant populations (e.g., noncitizens, those with poor English language skills, etc.). However, high fertility rates among Hispanics – and Mexican-origin Hispanics in particular – cannot be explained entirely by socio-demographic characteristics that place them at higher risk of fertility. For 2005–2009, Hispanic fertility rates were 48 percent higher than fertility among whites; they were roughly 25 percent higher after accounting for differences in key social characteristics, such as age, nativity, county of origin, and education. Contrary to most previous findings of spatial assimilation among in-migrants, fertility rates among Hispanics in new destinations exceeded fertility in established gateways by 18 percent. In the multivariate analyses, Hispanics in new destinations were roughly 10 percent more likely to have had a child in the past year than those living in established gateways. Results are consistent with sub-cultural explanations of Hispanic fertility and raise new questions about the spatial patterning of assimilation and the formation of ethnic enclaves outside traditional settlement areas.
cis -3-Chloroacrylic acid dehalogenase (cis-CaaD) catalyzes the hydrolytic dehalogenation of cis-3-haloacrylates to yield malonate semialdehyde. The enzyme processes other substrates including an allene (2,3-butadienoate) to produce acetoacetate. In the course of a stereochemical analysis of the cis-CaaD-catalyzed reaction using this allene, the enzyme was unexpectedly inactivated in the presence of NaBH4 by the reduction of a covalent enzyme-substrate bond. Covalent modification was surprising because the accumulated evidence for cis-CaaD dehalogenation favored a mechanism involving direct substrate hydration mediated by Pro-1. However, the results of subsequent mechanistic, pre-steady state and full progress kinetic experiments are consistent with a mechanism in which an enamine forms between Pro-1 and the allene. Hydrolysis of the enamine or an imine tautomer produces acetoacetate. Reduction of the imine species is likely responsible for the observed enzyme inactivation. This is the first reported observation of a tautomerase superfamily member functioning by covalent catalysis. The result may suggest that some fraction of the cis-CaaD-catalyzed dehalogenation of cis-3-haloacrylates also proceeds by covalent catalysis.
“Hairpatches” (Hpt) is a naturally occurring, autosomal semi-dominant mouse mutation. Hpt/Hpt homozygotes die in utero, while Hpt/+ heterozygotes exhibit progressive renal failure accompanied by patchy alopecia. This mutation is a model for the rare human disorder “glomerulonephritis with sparse hair and telangiectases" (OMIM 137940). Fine mapping localized the Hpt locus to a 6.7 Mb region of Chromosome 4 containing 62 known genes. Quantitative real time PCR revealed differential expression for only one gene in the interval, T-cell acute lymphocytic leukemia 1 (Tal1), which was highly upregulated in the kidney and skin of Hpt/+ mice. Southern blot analysis of Hpt mutant DNA indicated a new EcoRI site in the Tal1 gene. High throughput sequencing identified an endogenous retroviral class II intracisternal A particle insertion in Tal1 intron 4. Our data suggests that the IAP insertion in Tal1 underlies the histopathological changes in the kidney by three weeks of age, and that glomerulosclerosis is a consequence of an initial developmental defect, progressing in severity over time. The Hairpatches mouse model allows an investigation into the effects of Tal1, a transcription factor characterized by complex regulation patterns, and its effects on renal disease.
Inbred strain variants of the Cdh23 gene have been shown to influence the onset and progression of age-related hearing loss (AHL) in mice. In linkage backcrosses, the recessive Cdh23 allele (ahl) of the C57BL/6J strain, when homozygous, confers increased susceptibility to AHL, while the dominant allele (Ahl+) of the CBA/CaJ strain confers resistance. To determine the isolated effects of these alleles on different strain backgrounds, we produced the reciprocal congenic strains B6.CBACa-Cdh23Ahl+and CBACa.B6-Cdh23ahl and tested 15-30 mice from each for hearing loss progression. ABR thresholds for 8 kHz, 16 kHz, and 32 kHz pure-tone stimuli were measured at 3, 6, 9, 12, 15 and 18 months of age and compared with age-matched mice of the C57BL/6J and CBA/CaJ parental strains. Mice of the C57BL/6N strain, which is the source of embryonic stem cells for the large International Knockout Mouse Consortium, were also tested for comparisons with C57BL/6J mice. Mice of the C57BL/6J and C57BL/6N strains exhibited identical hearing loss profiles: their 32 kHz ABR thresholds were significantly higher than those of CBA/CaJ and congenic strain mice by 6 months of age, and their 16 kHz thresholds were significantly higher by 12 months. Thresholds of the CBA/CaJ, the B6.CBACa-Cdh23Ahl+, and the CBACa.B6-Cdh23ahl strain mice differed little from one another and only slightly increased throughout the 18-month test period. Hearing loss, which corresponded well with cochlear hair cell loss, was most profound in the C57BL/6J and C57BL/6NJ strains. These results indicate that the CBA/CaJ-derived Cdh23Ahl+ allele dramatically lessens hearing loss and hair cell death in an otherwise C57BL/6J genetic background, but that the C57BL/6J-derived Cdh23ahl allele has little effect on hearing loss in an otherwise CBA/CaJ background. We conclude that although Cdh23ahl homozygosity is necessary, it is not by itself sufficient to account for the accelerated hearing loss of C57BL/6J mice.
age-related hearing loss; Cdh23; ahl; C57BL/6J; C57BL/6N; CBA/CaJ; inbred mouse strains; congenic mouse strains; cochleograms; ABR thresholds; hair cells
Asbestos is classified as a human carcinogen, and studies have consistently demonstrated that workplace exposure to it increases the risk of developing lung cancer. Few studies have evaluated risks in population-based settings where there is a greater variety in the types of occupations, and exposures.
This was a population based case–control study with 1,681 incident cases of lung cancer, and 2,053 controls recruited from 8 Canadian provinces between 1994 and 1997. Self-reported questionnaires were used to elicit a lifetime occupational history, including general tasks, and information for other risk factors. Occupational hygienists, who were blinded to case–control status, assigned asbestos exposures to each job on the basis of (i) concentration (low, medium, high), (ii) frequency (<5%, 5-30%, and >30% of the time in a normal work week), and (iii) reliability (possible, probable, definite). Logistic regression was used to estimate odds ratios (ORs) and their corresponding 95% confidence intervals (CI).
Those occupationally exposed to (i) low, and (ii) medium or high concentrations of asbestos had ORs for lung cancer of 1.17 (95% CI=0.92 – 1.50) and 2.16 (95% CI=1.21-3.88), respectively, relative to those who were unexposed. Medium or high exposure to asbestos roughly doubled the risk for lung cancer across all three smoking pack-year categories. The joint relationship between smoking and asbestos was consistent with a multiplicative risk model.
Our findings provide further evidence that exposure to asbestos has contributed to an increased risk of lung cancer in Canadian workplaces, and suggests that nearly 3% of lung cancers among Canadian men are caused by occupational exposure to asbestos.
Lung cancer; Asbestos; Cigarette smoking; Case–control; Occupational epidemiology
Nearly 100 years ago Michaelis and Menten published their now classic paper (Michaelis, L., and Menten, M. L. (1913) Die Kinetik der Invertinwirkung, Biochemische Zeitschrift 49, 333–369), in which they show that the rate of an enzyme-catalyzed reaction is proportional to the concentration of enzyme-substrate complex predicted by the Michaelis-Menten equation. Because the original text was written in German, yet is often quoted by English speaking authors, we undertook a complete translation of the 1913 publication, which we provide as an online supplement (http://pubs.acs.org). Here we introduce the translation, describe the historical context of the work, and show a new analysis of the original data. In doing so, we uncovered several surprises that reveal an interesting glimpse into the early history of enzymology. In particular, our re-analysis of Michaelis and Menten’s data using modern computational methods revealed an unanticipated rigor and precision in the original publication and uncovered a sophisticated, comprehensive analysis that has been overlooked in the century since their work was published. Michaelis and Menten not only analyzed initial velocity measurements, but they also fit their full time course data to the integrated form of the rate equations, including product inhibition, and derived a single global constant to represent all of their data. That constant was not the Michaelis constant, but rather, Vmax/Km, the specificity constant times the enzyme concentration (kcat/Km*E0).
The human mitochondrial DNA polymerase (pol γ) is responsible for the replication of the mitochondrial genome. Mutation Y955C in the active site of pol γ results in early onset progressive external ophthalmoplegia, premature ovarian failure, and Parkinson’s disease. In single turnover kinetic studies, we show that the Y955C mutation resulted in a decrease in the maximum rate of polymerization and an increase in the Km for correct incorporation. The mutation decreased the specificity constant for correct incorporation of dGTP, TTP, and ATP to values of 1.5, 0.35, and 0.044 μM−1s−1, respectively, representing reductions of 30-, 110- and 1300-fold relative to wild-type enzyme. The fidelity of incorporation was reduced 6- to 130-fold; largely due the significant decrease in the specificity constant for correct dATP:T incorporation. For example, kcat/Km for forming a TTP:T mismatch was decreased tenfold from 0.0002 to 0.00002 μM−1s−1 by the Y955C mutant, but the 1300-fold slower incorporation of the correct dATP:T relative to wild-type led to a 130-fold lower fidelity. While correct incorporation of 8-oxo-dGTP was largely unchanged, incorporation of 8-oxo-dG with dA in the template strand was reduced 500-fold. These results support a role of Y955 in stabilizing A:T base pairs at the active site of pol γ and suggest that the severe clinical symptoms of patients with this mutation may be due, in part, to the reduced efficiency of dATP incorporation opposite T, and that the autosomal dominant phenotype may arise from the resulting higher mutation frequency.
The anomalous dispersion signal of the bromine-containing kinase inhibitor H-89 was used to characterize discrete binding modes of the compound when complexed with the catalytic subunit of protein kinase A.
With its ability to show the interactions between drug-target proteins and small-molecule ligands, X-ray crystallography is an essential tool in drug-discovery programmes. However, its usefulness can be limited by crystallization artifacts or by the data resolution, and in particular when assumptions of unimodal binding (and isotropic motion) do not apply. Discrepancies between the modelled crystal structure and the physiological range of structures generally prevent quantitative estimation of binding energies. Improved crystal structure resolution will often not aid energy estimation because the conditions which provide the highest rigidity and resolution are not likely to reflect physiological conditions. Instead, strategies must be employed to measure and model flexibility and multiple binding modes to supplement crystallographic information. One useful tool is the use of anomalous dispersion for small molecules that contain suitable atoms. Here, an analysis of the binding of the kinase inhibitor H-89 to protein kinase A (PKA) is presented. H-89 contains a bromobenzene moiety that apparently binds with multiple conformations in the kinase ATP pocket. Using anomalous dispersion methods, it was possible to resolve these conformations into two distinct binding geometries.
protein kinase A; kinases; ligands; inhibitors; flexibility; anomalous dispersion; SAD; bromine
DNA recombinases (RecA in bacteria, Rad51 in eukarya and RadA in archaea) catalyse strand-exchange between homologous DNA molecules, the central reaction of homologous recombination, and are among the most conserved DNA repair proteins known. In bacteria, RecA is the sole protein responsible for this reaction, whereas, in eukaryotes, there are several RAD51 paralogs that cooperate to catalyse strand exchange. All archaea have at least one (and as many as four) RadA paralogs, but their function remains unclear. Here we show the three RadA paralogs encoded by the Sulfolobus solfataricus genome are expressed under normal growth conditions, and are not UV-inducible. We demonstrate that one of these proteins, Sso2452, which is representative of the large aRadC sub-family of archaeal RadA paralogs, functions as an ATPase that binds tightly to ssDNA. However, Sso2452 is not an active recombinase in vitro, and inhibits D-loop formation by RadA. We present the high-resolution crystal structure of Sso2452, which reveals key structural differences from the canonical RecA family recombinases that may explain its functional properties. The possible roles of the archaeal RadA paralogs in vivo are discussed.
Archaea; Recombinase; RadA; Homologous Recombination; Strand Exchange
Single turnover kinetic studies were conducted using fluorescently labeled HIV Reverse Transcriptase (RT) to evaluate the role of nucleotide-induced changes in enzyme structure in the selectivity against AZT in order to explore why AZT-resistant forms of the enzyme fail to significantly discriminate against AZT. Fluorescent labeling of HIV RT provided a signal to monitor the isomerization from “open” to “closed” states following nucleotide binding. We measured the rate constants governing nucleotide binding and enzyme isomerization for TTP and AZT-triphosphate by the wild-type and AZT-resistant forms of the enzyme containing the thymidine analog mutations (TAMs). We show that the TAMs alter the kinetics of AZT incorporation by weakening ground state nucleotide binding and decreasing the rate of chemistry relative to the wild-type enzyme. However, the slower rate of incorporation of AZT by the TAMs HIV RT is counterbalanced a lower Km resulting from the equilibration of the conformational change step. In contrast, the Km for the wild-type enzyme reflects the balance between rates of binding and incorporation so the conformational change step does not come to equilibrium. These data once again demonstrate that the rate of substrate release, limited by the reverse of the substrate-induced conformational change, is the key determinant of the role of induced-fit in enzyme specificity. Mutations leading to slower rates of incorporation have the unfortunate consequence of lowering the Km value by allowing the conformational change step to come to equilibrium.
In order to measure the kinetics of pyrophosphate release, we have coupled pyrophosphatase to the fluorescently labeled phosphate binding protein developed by Webb. We show that the assay is capable of measuring rates of pyrophosphate release up to 100 s−1.
A long-lived and sequence specific ligand-DNA complex would make possible the modulation of biological processes for extended periods. We have been investigating the threading polyintercalation approach to DNA recognition in which chains of aromatic units thread back and forth repeatedly through the double helix. Here we report the preliminary sequence specificity and detailed kinetic analysis of a structurally characterized threading tetraintercalator. Specific binding on a relatively long DNA strand was observed, strongly favoring a predicted 14-base pair sequence. Kinetic studies revealed a multi-step association process and specificity was found to derive primarily from large differences in dissociation rates. Importantly, the rate-limiting dissociation rate constant of the tetraintercalator complex dissociating from its preferred binding site was extremely slow, corresponding to a 16 day half-life, making it one of the longer non-covalent complex half-lives yet measured, and, to the best of our knowledge, the longest for a DNA binding molecule.
Stereocilia, the modified microvilli projecting from the apical surfaces of the sensory hair cells of the inner ear, are essential to the mechanoelectrical transduction process underlying hearing and balance. The actin-filled stereocilia on each hair cell are tethered together by fibrous links to form a highly patterned hair bundle. Although many structural components of hair bundles have been identified, little is known about the signaling mechanisms that regulate their development, morphology, and maintenance. Here, we describe two naturally occurring, allelic mutations that result in hearing and balance deficits in mice, named roundabout (rda) and roundabout-2J (rda2J). Positional cloning identified both as mutations of the mouse ELMO domain containing 1 gene (Elmod1), a poorly characterized gene with no previously reported mutant phenotypes. The rda mutation is a 138 kb deletion that includes exons 1–5 of Elmod1, and rda2J is an intragenic duplication of exons 3–8 of Elmod1. The deafness associated with these mutations is caused by cochlear hair cell dysfunction, as indicated by conspicuous elongations and fusions of inner hair cell stereocilia and progressive degeneration of outer hair cell stereocilia. Mammalian ELMO-family proteins are known to be involved in complexes that activate small GTPases to regulate the actin cytoskeleton during phagocytosis and cell migration. ELMOD1 and ELMOD2 recently were shown to function as GTPase-activating proteins (GAPs) for the Arf family of small G proteins. Our finding connecting ELMOD1 deficiencies with stereocilia dysmorphologies thus establishes a link between the Ras superfamily of small regulatory GTPases and the actin cytoskeleton dynamics of hair cell stereocilia.
Cathepsin L mutants with the ability to condense silica from solution have been generated and a 1.5 Å crystal structure of one of these chimeras allows us to rationalize the catalytic mechanism of silicic acid condensation.
Mammalian RNF4 is a dimeric RING ubiquitin E3 ligase that ubiquitylates poly-SUMOylated proteins. We found that RNF4 bound ubiquitin-charged UbcH5a tightly but free UbcH5a weakly. To provide insight into the mechanism of RING-mediated ubiquitylation we docked the UbcH5~ubiquitin thioester onto the RNF4 RING structure. This revealed that with E2 bound to one monomer of RNF4, the thioester-linked ubiquitin could reach across the dimer to engage the other monomer. In this model the “Ile44 hydrophobic patch” of ubiquitin is predicted to engage a conserved tyrosine located at the dimer interface of the RING and mutation of these residues blocked ubiquitylation activity. Thus, dimeric RING ligases are not simply inert scaffolds that bring substrate and E2-loaded ubiquitin into close proximity. Instead, they facilitate ubiquitin transfer by preferentially binding the E2~ubiquitin thioester across the dimer and activating the thioester bond for catalysis.
The XPD helicase (Rad3 in Saccharomyces cerevisiae) is a component of transcription factor IIH (TFIIH), which functions in transcription initiation and Nucleotide Excision Repair in eukaryotes, catalysing DNA duplex opening localised to the transcription start site or site of DNA damage, respectively. XPD has a 5′ to 3′ polarity and the helicase activity is dependent on an iron-sulfur cluster binding domain, a feature that is conserved in related helicases such as FancJ. The xpd gene is the target of mutation in patients with xeroderma pigentosum, trichothiodystrophy and Cockayne’s syndrome, characterised by a wide spectrum of symptoms ranging from cancer susceptibility to neurological and developmental defects. The 2.25 Å crystal structure of XPD from the crenarchaeon Sulfolobus tokodaii, presented here together with detailed biochemical analyses, allows a molecular understanding of the structural basis for helicase activity and explains the phenotypes of xpd mutations in humans.
Pyrrolnitrin is a commonly used and clinically effective treatment for fungal infections and provides the structural basis for the more widely used fludioxinil. The pyrrolnitrin biosynthetic pathway consists of four chemical steps, the second of which is the rearrangement of 7-chloro-tryptophan by the enzyme PrnB, a reaction that is so far unprecedented in biochemistry. When expressed in Pseudomonas fluorescens, PrnB is red in color due to the fact that it contains 1 mole of heme b per mole of protein. The crystal structure unexpectedly establishes PrnB as a member of the heme-dependent dioxygenase superfamily with significant structural but not sequence homology to the two-domain indoleamine 2,3-dioxygenase enzyme (IDO). The heme-binding domain is also structurally similar to that of tryptophan 2,3-dioxygenase (TDO). Here we report the binary complex structures of PrnB with D- and L-tryptophan and D- and L-chloro-tryptophan. The structures identify a common hydrophobic pocket for the indole ring but exhibit unusual heme ligation and substrate binding when compared with that observed in the TDO crystal structures. Our solution studies support the heme ligation observed in the crystal structures. Purification of the hexahistidine-tagged PrnB yields homogeneous protein that only displays in vitro activity with 7-chloro-L-tryptophan after reactivation with crude extract from the host strain, suggesting that an as yet unknown cofactor is required for activity. Mutation of the proximal heme ligand results, not surprisingly, in inactive enzyme. Redox titrations show that PrnB displays a significantly different reduction potential to that of IDO or TDO, indicating possible differences in the PrnB catalytic cycle. This is confirmed by the absence of tryptophan dioxygenase activity in PrnB, although a stable oxyferrous adduct (which is the first intermediate in the TDO/IDO catalytic cycle) can be generated. We propose that PrnB shares a key catalytic step with TDO and IDO, generation of a tryptophan hydroperoxide intermediate, although this species suffers a different fate in PrnB, leading to the eventual formation of the product, monodechloroaminopyrrolnitrin.
Few epidemiological studies of air pollution have used residential histories to develop long-term retrospective exposure estimates for multiple ambient air pollutants and vehicle and industrial emissions. We present such an exposure assessment for a Canadian population-based lung cancer case-control study of 8353 individuals using self-reported residential histories from 1975 to 1994. We also examine the implications of disregarding and/or improperly accounting for residential mobility in long-term exposure assessments.
National spatial surfaces of ambient air pollution were compiled from recent satellite-based estimates (for PM2.5 and NO2) and a chemical transport model (for O3). The surfaces were adjusted with historical annual air pollution monitoring data, using either spatiotemporal interpolation or linear regression. Model evaluation was conducted using an independent ten percent subset of monitoring data per year. Proximity to major roads, incorporating a temporal weighting factor based on Canadian mobile-source emission estimates, was used to estimate exposure to vehicle emissions. A comprehensive inventory of geocoded industries was used to estimate proximity to major and minor industrial emissions.
Calibration of the national PM2.5 surface using annual spatiotemporal interpolation predicted historical PM2.5 measurement data best (R2 = 0.51), while linear regression incorporating the national surfaces, a time-trend and population density best predicted historical concentrations of NO2 (R2 = 0.38) and O3 (R2 = 0.56). Applying the models to study participants residential histories between 1975 and 1994 resulted in mean PM2.5, NO2 and O3 exposures of 11.3 μg/m3 (SD = 2.6), 17.7 ppb (4.1), and 26.4 ppb (3.4) respectively. On average, individuals lived within 300 m of a highway for 2.9 years (15% of exposure-years) and within 3 km of a major industrial emitter for 6.4 years (32% of exposure-years). Approximately 50% of individuals were classified into a different PM2.5, NO2 and O3 exposure quintile when using study entry postal codes and spatial pollution surfaces, in comparison to exposures derived from residential histories and spatiotemporal air pollution models. Recall bias was also present for self-reported residential histories prior to 1975, with cases recalling older residences more often than controls.
We demonstrate a flexible exposure assessment approach for estimating historical air pollution concentrations over large geographical areas and time-periods. In addition, we highlight the importance of including residential histories in long-term exposure assessments.
For submission to: Environmental Health
Air pollution; Canada; Exposure assessment; Lung cancer; Residential mobility; Spatiotemporal
Ranasmurfin is an unusual blue protein isolated from the nests of a Malaysian tree frog, Polypedates leucomystax, showing the rich chemical diversity displayed by biomolecular foams. Many species of tropical frogs use foams to protect delicate eggs and developing embryos against environmental challenges. These nests act as miniature ecosystems containing a spectrum of novel proteins and other macromolecules with functions related to foam stabilization and adhesion, resistance to microbial degradation, predation, or dehydration, providing a biocompatible environment for embryonic development.Thisworkformspartofourwiderstudyofthe intriguing physical and chemical properties of biofoams as unusual examples of biological soft matter.
Changes in synapse structure occur in frontal neocortex with HIV encephalitis (HIVE) and may contribute to HIV-associated neurocognitive disorders (HAND). A postmortem survey was conducted to determine if mRNAs involved in synaptic transmission are perturbed in dorsolateral prefrontal cortex (DLPFC) in subjects with HIVE or HAND. Expression of the opioid neurotransmitter preproenkephalin mRNA (PENK) was significantly decreased in a sampling of 446 brain specimens from HIV-1 infected people compared to 67 HIV negative subjects. Decreased DLPFC PENK was most evident in subjects with HIVE and/or increased expression of interferon regulatory factor 1 mRNA (IRF1). Type 2 dopamine receptor mRNA (DRD2L) was decreased significantly, but not in the same set of subjects with PENK dysregulation. DRD2L downregulation occurred primarily in the subjects without HIVE or neurocognitive impairment. Subjects with neurocognitive impairment often failed to significantly downregulate DRD2L and had abnormally high IRF1 expression. Conclusion: Dysregulation of synaptic preproenkephalin and DRD2L in frontal neocortex can occur with and without neurocognitive impairment in HIV-infected people. Downregulation of DRD2L in the prefrontal cortex was associated with more favorable neuropsychological and neuropathological outcomes; the failure to downregulate DRD2L was significantly less favorable. PENK downregulation was related neuropathologically to HIVE, but was not related to neuropsychological outcome independently. Emulating endogenous synaptic plasticity pharmacodynamically could enhance synaptic accommodation and improve neuropsychological and neuropathological outcomes in HIV/AIDS.
Dopamine receptor; Enkephalin; HAND; HIVE; HIV encephalitis; HIV-associated neurocognitive disorders; Interferon regulatory factor; Opiate; Opioid; Synaptic plasticity
Background: Previous studies have failed to reconstitute an active replication complex with hepatitis C virus (HCV) RNA-dependent RNA polymerase.
Results: The replication complex from HCV was assembled, purified, and characterized.
Conclusion: A highly active replication complex can be formed with HCV polymerase that catalyzes fast and processive RNA replication.
Significance: A purified and active replication complex is essential for mechanistic studies and drug discovery.
NS5B is the RNA-dependent RNA polymerase responsible for replicating hepatitis C virus (HCV) genomic RNA. Despite more than a decade of work, the formation of a highly active NS5B polymerase·RNA complex suitable for mechanistic and structural studies has remained elusive. Here, we report that through a novel way of optimizing initiation conditions, we were able to generate a productive NS5B·primer·template elongation complex stalled after formation of a 9-nucleotide primer. In contrast to previous reports of very low proportions of active NS5B, we observed that under optimized conditions up to 65% of NS5B could be converted into active elongation complexes. The elongation complex was extremely stable, allowing purification away from excess nucleotide and abortive initiation products so that the purified complex was suitable for pre-steady-state kinetic analyses of polymerase activity. Single turnover kinetic studies showed that CTP is incorporated with apparent Kd and kpol values of 39 ± 3 μm and 16 ± 1 s−1, respectively, giving a specificity constant of kpol/Kd of 0.41 μm−1 s−1. The kinetics of multiple nucleotide incorporation during processive elongation also were determined. This work establishes a novel way to generate a highly active elongation complex of the medically important NS5B polymerase for structural and functional studies.
Enzyme Kinetics; Hepatitis C Virus; Pre-steady-state Kinetics; RNA Polymerase; Viral Polymerase; Viral Replication
Quantitative trait locus (QTL) analysis of genetic crosses has proven to be a useful tool for identifying loci associated with specific phenotypes and for dissecting genetic components of complex traits. Inclusion of a mutation that interacts epistatically with QTLs in genetic crosses is a unique and potentially powerful method of revealing the function of novel genes and pathways. Although we know that a mutation within the novel tub gene leads to obesity and cochlear and retinal degeneration, the biological function of the gene and the mechanism by which it induces its phenotypes are not known. In the current study, a QTL analysis for auditory brainstem response (ABR) thresholds, which indicates hearing ability, was performed in tubby mice from F2 intercrosses between C57BL/6J-tub/tub and AKR/J-+/+ F1, hybrids (AKR intercross) and between C57BL/6J-tub/tub and CAST/Ei.B6-tub/tub F1 hybrids (CAST intercross). A major QTL, designated as modifier of tubby hearing1 (moth1), was identified on chromosome 2 with a LOD score of 33.4 (P <; 10−33) in the AKR intercross (181 mice) and of 6.0 (P <; 10−6) in the CAST intercross (46 mice). This QTL is responsible for 57 and 43% of ABR threshold variance, respectively, in each strain combination. In addition, a C57BL/6J congenic line carrying a 129/Ola segment encompassing the described QTL region when made homozygous for tubby also exhibits normal hearing ability. We hypothesize that C57BL/6J carries a recessive mutation of the moth1 gene which interacts with the tub mutation to cause hearing loss in tub/tub mice. A moth1 allele from either AKR/J, CAST/Ei or 129/Ola is sufficient to protect C57BL/6J-tub/tub mice from hearing loss.
The effect of Ocean Acidification (OA) on marine biota is quasi-predictable at best. While perturbation studies, in the form of incubations under elevated pCO2, reveal sensitivities and responses of individual species, one missing link in the OA story results from a chronic lack of pH data specific to a given species' natural habitat. Here, we present a compilation of continuous, high-resolution time series of upper ocean pH, collected using autonomous sensors, over a variety of ecosystems ranging from polar to tropical, open-ocean to coastal, kelp forest to coral reef. These observations reveal a continuum of month-long pH variability with standard deviations from 0.004 to 0.277 and ranges spanning 0.024 to 1.430 pH units. The nature of the observed variability was also highly site-dependent, with characteristic diel, semi-diurnal, and stochastic patterns of varying amplitudes. These biome-specific pH signatures disclose current levels of exposure to both high and low dissolved CO2, often demonstrating that resident organisms are already experiencing pH regimes that are not predicted until 2100. Our data provide a first step toward crystallizing the biophysical link between environmental history of pH exposure and physiological resilience of marine organisms to fluctuations in seawater CO2. Knowledge of this spatial and temporal variation in seawater chemistry allows us to improve the design of OA experiments: we can test organisms with a priori expectations of their tolerance guardrails, based on their natural range of exposure. Such hypothesis-testing will provide a deeper understanding of the effects of OA. Both intuitively simple to understand and powerfully informative, these and similar comparative time series can help guide management efforts to identify areas of marine habitat that can serve as refugia to acidification as well as areas that are particularly vulnerable to future ocean change.
The biological and palaeontological communities have approached the problem of informatics separately, creating a divide between communities that is both technological and sociological in nature. In this paper we describe one new advance towards solving this problem - expanding the Scratchpads platform to deal with geological time. In creating this system we have attempted to make our work open to existing communities by providing a webservice of geological time data via the GBIF Vocabularies site. We have also ensured that our system can adapt to changes in the definition of geological time intervals and is capable of querying datasets independently of the format of geological age data used.
Palaeontology; Biodiversity Informatics; Scratchpads; web services; GBIF
The on and off rates corresponding to the binding of two test anions (acetate, AcO− and dihydrogen phosphate, H2PO4− studied as their tetrabutylammonium salts) to diprotonated cyclopyrrole have been determined in CH3CN using stopped-flow analyses carried out at various temperatures. For dihydrogen phosphate, this afforded the activation enthalpies and entropies associated with both off and on processes. The different dynamic behavior seen for these test anions underscores the utility of kinetic analyses as a possible new tool for the advanced characterization of anion receptors.
Anion Binding; Kinetics; Stopped-Flow