In vitro evolution of horse heart myoglobin to increase peroxidase activity

Random mutagenesis and screening for enzymatic activity has been used to engineer horse heart myoglobin to enhance its intrinsic peroxidase activity. A chemically synthesized gene encoding horse heart myoglobin was subjected to successive cycles of PCR random mutagenesis. The mutated myoglobin gene was expressed in Escherichia coli LE392, and the variants were screened for peroxidase activity with a plate assay. Four cycles of mutagenesis and screening produced a series of single, double, triple, and quadruple variants with enhanced peroxidase activity. Steady-state kinetics analysis demonstrated that the quadruple variant T39I/K45D/F46L/I107F exhibits peroxidase activity significantly greater than that of the wild-type protein with k1 (for H2O2 oxidation of metmyoglobin) of 1.34 × 104 M−1 s−1 (≈25-fold that of wild-type myoglobin) and k3 [for reducing the substrate (2, 2′-azino-di-(3-ethyl)benzthiazoline-6-sulfonic acid] of 1.4 × 106 M−1 s−1 (1.6-fold that of wild-type myoglobin). Thermal stability of these variants as measured with circular dichroism spectroscopy demonstrated that the Tm of the quadruple variant is decreased only slightly compared with wild-type (74.1°C vs. 76.5°C). The rate constants for binding of dioxygen exhibited by the quadruple variant are identical to the those observed for wild-type myoglobin (kon, 22.2 × 10−6 M−1 s−1 vs. 22.3 × 10−6 M−1 s−1; koff, 24.3 s−1 vs. 24.2 s−1; KO2, 0.91 × 10−6 M−1 vs. 0.92 × 10−6 M−1). The affinity of the quadruple variant for CO is increased slightly (kon, 0.90 × 10−6 M−1s−1 vs. 0.51 × 10−6 M−1s−1; koff, 5.08 s−1 vs. 3.51 s−1; KCO, 1.77 × 10−7 M−1 vs. 1.45 × 10−7 M−1). All four substitutions are in the heme pocket and within 5 Å of the heme group.

A link between protein structure and enzyme catalyzed hydrogen tunneling

We present evidence that the size of an active site side chain may modulate the degree of hydrogen tunneling in an enzyme-catalyzed reaction. Primary and secondary kH/kT and kD/kT kinetic isotope effects have been measured for the oxidation of benzyl alcohol catalyzed by horse liver alcohol dehydrogenase at 25°C. As reported in earlier studies, the relationship between secondary kH/kT and kD/kT isotope effects provides a sensitive probe for deviations from classical behavior. In the present work, catalytic efficiency and the extent of hydrogen tunneling have been correlated for the alcohol dehydrogenase-catalyzed hydride transfer among a group of site-directed mutants at position 203. Val-203 interacts with the opposite face of the cofactor NAD+ from the alcohol substrate. The reduction in size of this residue is correlated with diminished tunneling and a two orders of magnitude decrease in catalytic efficiency. Comparison of the x-ray crystal structures of a ternary complex of a high-tunneling (Phe-93 → Trp) and a low-tunneling (Val-203 → Ala) mutant provides a structural basis for the observed effects, demonstrating an increase in the hydrogen transfer distance for the low-tunneling mutant. The Val-203 → Ala ternary complex crystal structure also shows a hyperclosed interdomain geometry relative to the wild-type and the Phe-93 → Trp mutant ternary complex structures. This demonstrates a flexibility in interdomain movement that could potentially narrow the distance between the donor and acceptor carbons in the native enzyme and may enhance the role of tunneling in the hydride transfer reaction.

A 55-kDa protein isolated from human cells shows DNA glycosylase activity toward 3,N4-ethenocytosine and the G/T mismatch

Etheno adducts in DNA arise from multiple endogenous and exogenous sources. Of these adducts we have reported that, 1,N6-ethenoadenine (ɛA) and 3,N4-ethenocytosine (ɛC) are removed from DNA by two separate DNA glycosylases. We later confirmed these results by using a gene knockout mouse lacking alkylpurine-DNA-N-glycosylase, which excises ɛA. The present work is directed toward identifying and purifying the human glycosylase activity releasing ɛC. HeLa cells were subjected to multiple steps of column chromatography, including two ɛC-DNA affinity columns, which resulted in >1,000-fold purification. Isolation and renaturation of the protein from SDS/polyacrylamide gel showed that the ɛC activity resides in a 55-kDa polypeptide. This apparent molecular mass is approximately the same as reported for the human G/T mismatch thymine-DNA glycosylase. This latter activity copurified to the final column step and was present in the isolated protein band having ɛC-DNA glycosylase activity. In addition, oligonucleotides containing ɛC⋅G or G/T(U), could compete for ɛC protein binding, further indicating that the ɛC-DNA glycosylase is specific for both types of substrates in recognition. The same substrate specificity for ɛC also was observed in a recombinant G/T mismatch DNA glycosylase from the thermophilic bacterium, Methanobacterium thermoautotrophicum THF.

The Yck2 Yeast Casein Kinase 1 Isoform Shows Cell Cycle-specific Localization to Sites of Polarized Growth and Is Required for Proper Septin Organization

Casein kinase 1 protein kinases are ubiquitous and abundant Ser/Thr-specific protein kinases with activity on acidic substrates. In yeast, the products of the redundant YCK1 and YCK2 genes are together essential for cell viability. Mutants deficient for these proteins display defects in cellular morphogenesis, cytokinesis, and endocytosis. Yck1p and Yck2p are peripheral plasma membrane proteins, and we report here that the localization of Yck2p within the membrane is dynamic through the cell cycle. Using a functional green fluorescent protein (GFP) fusion, we have observed that Yck2p is concentrated at sites of polarized growth during bud morphogenesis. At cytokinesis, GFP–Yck2p becomes associated with a ring at the bud neck and then appears as a patch of fluorescence, apparently coincident with the dividing membranes. The bud neck association of Yck2p at cytokinesis does not require an intact septin ring, and septin assembly is altered in a Yck-deficient mutant. The sites of GFP–Yck2p concentration and the defects observed for Yck-deficient cells together suggest that Yck plays distinct roles in morphogenesis and cytokinesis that are effected by differential localization.

Expression of homeobox genes shows chelicerate arthropods retain their deutocerebral segment

Expression patterns of six homeobox containing genes in a model chelicerate, the oribatid mite Archegozetes longisetosus, were examined to establish homology of chelicerate and insect head segments and to investigate claims that the chelicerate deutocerebral segment has been reduced or lost. engrailed (en) expression, which has been used to demonstrate the presence of segments in insects, fails to demonstrate a reduced deutocerebral segment. Expression patterns of the chelicerate homologs of the Drosophila genes Antennapedia (Antp), Sex combs reduced (Scr), Deformed (Dfd), proboscipedia (pb), and orthodenticle (otd) confirm direct correspondence of head segments. The chelicerate deutocerebral segment has not been reduced or lost. We make further inferences concerning the evolution of heads and Hox genes in arthropods.

An un-mounted horse care group for children and youth with an Autism Spectrum Disorder: Recommendations for program development and treatment intervention

Equine Assisted Activities and Therapies (EAAT) including Therapeutic Horseback Riding (THR) and un-mounted equine assisted activities are interventions aimed at improving the daily functioning and success of individuals with disabilities, including those with an autism spectrum disorder (ASD). While THR is frequently utilized as a treatment intervention for children with ASD, there are many limitations (individual's weight, horse health, weather, physical limitations, health conditions, etc.) that prevent this population from participating in mounted programs. Un-mounted equine assisted activities are often utilized as an alternative, but they are not informed by empirical research or a standardized treatment model. This paper provides a comprehensive review of the literature for EAAT including un-mounted programs, examination of organizational guidelines as they apply to un-mounted programs, and consultation with program directors regarding current practices in the field, and finally it establishes recommendations for the development of a standard curriculum that would strengthen un-mounted horse care group programs serving children with ASD.

The fashionable horse : Ute saddlebags and saddle blankets 1870-1925

This research paper examines the creation, design, and functions of ten Ute double saddlebags and four beaded Ute saddle blankets fabricated between 1870 and 1925. Based on this sample and comparison with additional Basin and Plains tribal dressings, it appears that Ute saddlebags and blankets possess a combination of characteristics that reflect Ute territory, lifestyle, and aesthetics. These attributes, including fabrication from hide; similar rectangular dimensions; simple geometric beadwork patterns that emphasize triangles; preference for blue, white, and yellow beads; largely solid colored backgrounds; and back entries into the bags, work together to create a style that is specifically Ute.

Preliminary data on horse mackerel (Trachurus spp) landings from Mauritanian waters

European fishery off Mauritania (one of the most productive and most intensively exploited regions in the world) targeted to small pelagic fish accounts for 30% of the total catches in the area. The Instituto Español de Oceanografía (IEO), through its Centro Oceanográfico de Canarias, follows up the activity of these vessels that land at Spanish port (Las Palmas de Gran Canaria, Spain), under the European Project Data Collection (Regulation (EC) 1543/2000). One of the target groups in this fishery is Trachurus spp, commercially named “JAX”. During 2008 and 2009, several samples of this group from these landings were analysed, and Caranx rhonchus was the second species of importance. The mixture of these species in the landings of this UE fleet should be determined to improve the data provided to the international Working Groups responsible for assessment of those resources.

Gulf of Mexico and Caribbean Sea Region, ca. 1715 (Raster Image)

This layer is a georeferenced raster image of the historic paper map entitled: A map of the West-Indies or the islands of America in the North Sea : with ye adjacent countries, explaning [sic] what belongs to Spain, England, France, Holland &c. also ye trade winds, and ye several tracts made by ye galeons and flota from place to place : according to ye newest and most exact observations, by Herman Moll, geographer. It was printed for Tho. Bowles in St. Pauls Church Yard and John Bowles at the Black Horse in Cornhill ca. 1715. Scale [ca. 1:4,300,000]. Covers the Gulf of Mexico and Caribbean Sea Region including parts of southern United States, Mexico, Central America, West Indies, and northern South America.The image inside the map neatline is georeferenced to the surface of the earth and fit to the North American Lambert Conformal Conic coordinate system. All map collar and inset information is also available as part of the raster image, including any inset maps, profiles, statistical tables, directories, text, illustrations, index maps, legends, or other information associated with the principal map. This map shows features such as drainage, cities and other human settlements, territorial boundaries, shoreline features, and more. Relief shown pictorially. Includes also historical notes and insets.This layer is part of a selection of digitally scanned and georeferenced historic maps from the Harvard Map Collection. These maps typically portray both natural and manmade features. The selection represents a range of originators, ground condition dates, scales, and map purposes.

Burma, Bangladesh, & South Asia, 1825 (Raster Image)

This layer is a georeferenced raster image of the historic paper map entitled: Burmese empire, drawn by L. Hebert. It was published by Quarr. Mastr. Genls.' Office, Horse Guards in 1825. Scale [ca. 1:2,050,000]. Covers Burma, Bangladesh, and portions of Bhutan, India, China, Laos, Thailand, and Vietnam.The image inside the map neatline is georeferenced to the surface of the earth and fit to the Asia North Lambert Conformal Conic coordinate system. All map collar and inset information is also available as part of the raster image, including any inset maps, profiles, statistical tables, directories, text, illustrations, index maps, legends, or other information associated with the principal map. This map shows features such as drainage, cities and other human settlements, roads, territorial and administrative boundaries, shoreline features, and more. Relief shown by hachures. Includes notes. This layer is part of a selection of digitally scanned and georeferenced historic maps from the Harvard Map Collection. These maps typically portray both natural and manmade features. The selection represents a range of originators, ground condition dates, scales, and map purposes.

Manhattan, New York, N.Y., ca. 1883 (Raster Image)

This layer is a georeferenced raster image of the historic paper map entitled: Map of the city of New York : with street directory showing house-numbers, hotels, churches, banks, theatres, ferries, house-car, steam and elevated R.R'ds, &c. It was published by M. Dripps ca. 1883. Scale [ca. 1:12,670]. Covers Manhattan below 61st St. and portions of Brooklyn and Queens. The image inside the map neatline is georeferenced to the surface of the earth and fit to the Universal Transverse Mercator (UTM) Zone 18N NAD83 projection. All map collar and inset information is also available as part of the raster image, including any inset maps, profiles, statistical tables, directories, text, illustrations, index maps, legends, or other information associated with the principal map. This map shows features such as roads, railroads, drainage, selected public buildings, parks, wharves, ferry lines, city wards, and more. Depths shown by soundings. Includes directories of streets and places of interest, table of dimensions of the East River Bridge, and horse car routes. This layer is part of a selection of digitally scanned and georeferenced historic maps from The Harvard Map Collection as part of the Imaging the Urban Environment project. Maps selected for this project represent major urban areas and cities of the world, at various time periods. These maps typically portray both natural and manmade features at a large scale. The selection represents a range of regions, originators, ground condition dates, scales, and purposes.

Chicago and vicinity, Illinois, 1893 (Raster Image)

This layer is a georeferenced raster image of the historic paper map entitled: Rand McNally and Co.'s standard map of Chicago, Rand, McNally & Co., engravers. It was published by Rand McNally & Co. in 1893. Scale [ca. 1:20,000]. The image inside the map neatline is georeferenced to the surface of the earth and fit to the Illinois East State Plane Coordinate System NAD83 (in Feet) (Fipszone 1201). All map collar and inset information is also available as part of the raster image, including any inset maps, profiles, statistical tables, directories, text, illustrations, index maps, legends, or other information associated with the principal map. This map shows features such as roads, railroads (steam and elevated), depots, cable and horse car lines, drainage, boulevards and parks, ward boundaries, selected public buildings, and more. Includes index to streets, avenues, and parks and list of railroads and their depots. This layer is part of a selection of digitally scanned and georeferenced historic maps from The Harvard Map Collection as part of the Imaging the Urban Environment project. Maps selected for this project represent major urban areas and cities of the world, at various time periods. These maps typically portray both natural and manmade features at a large scale. The selection represents a range of regions, originators, ground condition dates, scales, and purposes.

Kansas City, Missouri, 1898 (Raster Image)

This layer is a georeferenced raster image of the historic paper map entitled: Rand, McNally & Co.'s Kansas City. It was published by Rand, McNally & Co. in 1898. Scale [ca. 1:10,000]. Covers Kansas City, Missouri, and Kansas City, Kansas. The image inside the map neatline is georeferenced to the surface of the earth and fit to the Missouri West State Plane Coordinate System NAD83 (in Feet) (Fipszone 2403). All map collar and inset information is also available as part of the raster image, including any inset maps, profiles, statistical tables, directories, text, illustrations, index maps, legends, or other information associated with the principal map. This map shows features such as roads, railroads, electric railroads, cable car lines, horse car lines, elevated, electric, and surface cable lines, drainage, selected public buildings, parks, and more. Includes inset: Kansas City, MO., Northeast Portion. This layer is part of a selection of digitally scanned and georeferenced historic maps from The Harvard Map Collection as part of the Imaging the Urban Environment project. Maps selected for this project represent major urban areas and cities of the world, at various time periods. These maps typically portray both natural and manmade features at a large scale. The selection represents a range of regions, originators, ground condition dates, scales, and purposes.