Kinetic and Spectroscopic Characterization of the H178A Methionyl Aminopeptidase from \u3cem\u3eEscherichia coli\u3c/em\u3e

To gain insight into the role of the strictly conserved histidine residue, H178, in the reaction mechanism of the methionyl aminopeptidase from Escherichia coli (EcMetAP-I), the H178A mutant enzyme was prepared. Metal-reconstituted H178A binds only one equivalent of Co(II) or Fe(II) tightly with affinities that are identical to the WT enzyme based on kinetic and isothermal titration calorimetry (ITC) data. Electronic absorption spectra of Co(II)-loaded H178A EcMetAP-I indicate that the active site divalent metal ion is pentacoordinate, identical to the WT enzyme. These data indicate that the metal binding site has not been affected by altering H178. The effect of altering H178 on activity is, in general, due to a decrease in kcat. The kcat value for Co(II)-loaded H178A decreased 70-fold toward MGMM and 290-fold toward MP-p-NA compared to the WT enzyme, while kcat decreased 50-fold toward MGMM for the Fe(II)-loaded H178A enzyme and 140-fold toward MP-p-NA. The Km values for MGMM remained unaffected, while those for MP-p-NA increased approximately 2-fold for Co(II)- and Fe(II)-loaded H178A. The kcat/Km values for both Co(II)- and Fe(II)-loaded H178A toward both substrates ranged from ∼50- to 580-fold reduction. The pH dependence of log Km, log kcat, and log(kcat/Km) of both WT and H178A EcMetAP-I were also obtained and are identical, within error, for H178A and WT EcMetAP-I. Therefore, H178A is catalytically important but is not required for catalysis. Assignment of one of the observed pKa values at 8.1 for WT EcMetAP-I was obtained from plots of molar absorptivity at λmax(640) vs pH for both WT and H178A EcMetAP-I. Apparent pKa values of 8.1 and 7.6 were obtained for WT and H178A EcMetAP-I, respectively, and were assigned to the deprotonation of a metal-bound water molecule. The data reported herein provide support for the key elements of the previously proposed mechanism and suggest that a similar mechanism can apply to the enzyme with a single metal in the active site

Importance of Haresfoot Clover (\u3ci\u3eTrifolium arvense\u3c/i\u3e) as a Nitrogen Fixer in Semi-Arid Grasslands of New Zealand

The aim of the study was to determine the distribution of annual haresfoot clover (Trifolium arvense) in a depleted indigenous short tussock grassland catchment in the dry subhumid MacKenzie Basin, South Island, New Zealand. Haresfoot clover was present on all landscapes, and widely distributed on lower sunny aspects, suggesting that it is an important component of the nitrogen cycle in these environments even in the absence of fertiliser application. The study was conducted during an atypically moist summer when a bloom of the annual clover occurred. Nitrogen inputs will vary with landscape and spring/summer rainfall and it is likely that significant pulses occur in wetter years

Mapping Nitrogen Fixing Shrubs in Dry Unimproved Tussock Grassland

Global Positioning System (GPS) was used to both map the positions of individual nitrogen-fixing shrub plants and record individual plant height and breadth dimensions, within a small catchment of dry sub-humid unimproved short tussock grassland. The distributions of different species of shrubs varied with different landscape units within the catchment. The information will provide the base data for a first approximation of the role of the shrubs in the nitrogen balance of the ecosystem, which is pivotal to the sustainability of pastoral farming in the tussock grasslands

Environmental Aging of Scotch-Weld(TradeMark) AF-555M Structural Adhesive in Composite to Composite Bonds

Fiber reinforced resin matrix composites have found increased usage in recent years. Due to the lack of service history of these relatively new material systems, their long-term aging performance is not well established. In this study, adhesive bonds were prepared by the secondary bonding of Scotch-Weld(TradeMark) AF-555M between pre-cured adherends comprised of T800H/3900-2 uni-directional laminate. The adherends were co-cured with wet peel-ply for surface preparation. Each bond-line of single-lap-shear (SLS) specimen was measured to determine thickness and inspected visually for voids. A three-year environmental aging plan for the SLS specimens at 82 C and 85% relative humidity was initiated. SLS strengths were measured for both controls and aged specimens at room temperature and 82 C. The aging results of strength retention and failure modes to date are reported

How efficiently can HEPA purifiers remove priority fine and ultrafine particles from indoor air?

More than 1 million premature deaths in Asia annually are estimated to be associated with indoor air quality. HEPA (high-efficiency particulate air) filter air purifiers (APs) are widely used in urban Chinese residences by the growing middle class, as public awareness of air pollution increases. Currently, understanding of how particle size affects particle removal is inconsistent, and the rate at which different particle types are removed remains largely unknown. Therefore, this investigation aimed to determine the relationship between particle size and the removal efficiency of particles, and how efficiently ambient air is filtered compared to standard particle types which are typically used for such tests (tobacco smoke, dust and pollen). Three of the most popular AP models in China were tested in China’s largest indoor controlled chamber laboratory and the removal efficiencies of particles in the 18-514nm range were identified. Each AP had a distinct profile of removal efficiency against particle size, but the three APs shared similarities in performance, with removal efficiency consistently lowest at 200-250nm. This size fraction is important in an exposure context as these particles are abundant in ambient air in mega-cities, can penetrate through building shells effectively, remain airborne for long periods of time and can penetrate the deepest areas of the lungs. Ambient air particles were removed at a similar rate to test particles; this confirms that the Association of Home Appliance Manufacturers’ (AHAM) standards are a suitable proxy for “real world” performance

The Effects of Temperature, Humidity and Aircraft Fluid Exposure on T800H/3900-2 Composites Bonded with AF-555M Adhesive

Fiber reinforced resin matrix composites and structural adhesives have found increased usage on commercial and military aircraft in recent years. Due to the lack of service history of these relatively new material systems, their long-term aging performance has not been well established. In this study, single lap shear specimens (SLS) were fabricated by secondary bonding of Scotch-Weld(TradeMark) AF-555M between pre-cured adherends comprised of T800H/3900-2 uni-directional laminates. The adherends were co-cured with wet peel-ply for surface preparation. Each bond-line of the SLS specimen was measured to determine thickness and inspected visually using an optical microscope for voids. A three-year environmental aging plan for the SLS specimens at 82 C (180 F) and 85% relative humidity was initiated. SLS strengths were measured for both controls and aged specimens at room temperature and 82 C. The effect of this exposure on lap shear strength and failure modes to date is reported. In addition, the effects of water, saline water, deicing fluid, JP-5 jet fuel and hydraulic fluid on both the composite material and the adhesive bonds were investigated. The up to date results on the effects of these exposures will be discussed

Proteomics and phylogenetic analysis of the cathepsin L protease family of the helminth pathogen Fasciola hepatica: Expansion of a repertoire of virulence-associated factors

Cathepsin L proteases secreted by the helminth pathogen Fasciola hepatica have functions in parasite virulence including tissue invasion and suppression of host immune responses. Using proteomics methods alongside phylogenetic studies we characterized the profile of cathepsin L proteases secreted by adult F. hepatica and hence identified those involved in host-pathogen interaction. Phylogenetic analyses showed that the Fasciola cathepsin L gene family expanded by a series of gene duplications followed by divergence that gave rise to three clades associated with mature adult worms (Clades 1, 2, and 5) and two clades specific to infective juvenile stages (Clades 3 and 4). Consistent with these observations our proteomics studies identified representatives from Clades 1, 2, and 5 but not from Clades 3 and 4 in adult F. hepatica secretory products. Clades 1 and 2 account for 67.39 and 27.63% of total secreted cathepsin Ls, respectively, suggesting that their expansion was positively driven and that these proteases are most critical for parasite survival and adaptation. Sequence comparison studies revealed that the expansion of cathepsin Ls by gene duplication was followed by residue changes in the S2 pocket of the active site. Our biochemical studies showed that these changes result in alterations in substrate binding and suggested that the divergence of the cathepsin L family produced a repertoire of enzymes with overlapping and complementary substrate specificities that could cleave host macromolecules more efficiently. Although the cathepsin Ls are produced as zymogens containing a prosegment and mature domain, all secreted enzymes identified by MS were processed to mature active enzymes. The prosegment region was highly conserved between the clades except at the boundary of prosegment and mature enzyme. Despite the lack of conservation at this section, sites for exogenous cleavage by asparaginyl endopeptidases and a Leu-Ser ↓ His motif for autocatalytic cleavage by cathepsin Ls were preserved. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc

Structural and Biochemical Studies of Human 4-hydroxy-2-oxoglutarate Aldolase: Implications for Hydroxyproline Metabolism in Primary Hyperoxaluria

4-hydroxy-2-oxoglutarate (HOG) aldolase is a unique enzyme in the hydroxyproline degradation pathway catalyzing the cleavage of HOG to pyruvate and glyoxylate. Mutations in this enzyme are believed to be associated with the excessive production of oxalate in primary hyperoxaluria type 3 (PH3), although no experimental data is available to support this hypothesis. Moreover, the identity, oligomeric state, enzymatic activity, and crystal structure of human HOGA have not been experimentally determined.In this study human HOGA (hHOGA) was identified by mass spectrometry of the mitochondrial enzyme purified from bovine kidney. hHOGA performs a retro-aldol cleavage reaction reminiscent of the trimeric 2-keto-3-deoxy-6-phosphogluconate aldolases. Sequence comparisons, however, show that HOGA is related to the tetrameric, bacterial dihydrodipicolinate synthases, but the reaction direction is reversed. The 1.97 Å resolution crystal structure of hHOGA bound to pyruvate was determined and enabled the modeling of the HOG-Schiff base intermediate and the identification of active site residues. Kinetic analyses of site-directed mutants support the importance of Lys196 as the nucleophile, Tyr168 and Ser77 as components of a proton relay, and Asn78 and Ser198 as unique residues that facilitate substrate binding.The biochemical and structural data presented support that hHOGA utilizes a type I aldolase reaction mechanism, but employs novel residue interactions for substrate binding. A mapping of the PH3 mutations identifies potential rearrangements in either the active site or the tetrameric assembly that would likely cause a loss in activity. Altogether, these data establish a foundation to assess mutant forms of hHOGA and how their activity could be pharmacologically restored

High Kinetic Energy Penetrator Shielding and High Wear Resistance Materials Fabricated with Boron Nitride Nanotubes (BNNTS) and BNNT Polymer Composites

Boron nitride nanotubes (BNNTs), boron nitride nanoparticles (BNNPs), carbon nontubes (CNTs), graphites, or their combinations, are incorporated into matrices of polymer, ceramic or metals. Fibers, yarns, and woven or nonwoven mates of BNNTs are uses as toughening layers in penetration resistant materials to maximize energy absorption and/or high hardness layers to rebound or deform penetrators. They can be also uses as reinforcing inclusions combining with other polymer matrices to create composite layer like typical reinforcing fibers such as Kevlar (Registered Trademark), Spectra (Registered Trademark) ceramics and metals. Enhanced wear resistance and prolonged usage time, even under harsh conditions, are achieved by adding boron nitride nanomaterials because both hardness and toughness are increased. Such materials can be used in high temperature environments since the oxidation temperature of BNNTs exceeds 800 C in air. Boron nitride based composite materials are useful as strong structural materials for anti-micrometeorite layers for spacecraft and space suits, ultra strong tethers, protective gear for the human body as well as for vehicles, helmets, shields and safety suits/helmets for industry

High Kinetic Energy Penetrator Shielding and High Wear Resistance Materials Fabricated with Boron Nitride Nanotubes (BNNTS) and BNNT Polymer Composites

Boron nitride nanotubes (BNNTs), boron nitride nanoparticles (BNNPs), carbon nanotubes (CNTs), graphites, or combinations, are incorporated into matrices of polymer, ceramic or metals. Fibers, yarns, and woven or nonwoven mats of BNNTs are used as toughening layers in penetration resistant materials to maximize energy absorption and/or high hardness layers to rebound or deform penetrators. They can be also used as reinforcing inclusions combining with other polymer matrices to create composite layers like typical reinforcing fibers such as Kevlar.RTM., Spectra.RTM., ceramics and metals. Enhanced wear resistance and usage time are achieved by adding boron nitride nanomaterials, increasing hardness and toughness. Such materials can be used in high temperature environments since the oxidation temperature of BNNTs exceeds 800.degree. C. in air. Boron nitride based composites are useful as strong structural materials for anti-micrometeorite layers for spacecraft and space suits, ultra strong tethers, protective gear, vehicles, helmets, shields and safety suits/helmets for industry