Probing the Reaction Mechanism of the D-ala-D-ala Dipeptidase, VanX, by Using Stopped-Flow Kinetic and Rapid-Freeze Quench EPR Studies on the Co(II)-Substituted Enzyme

In an effort to probe the reaction mechanism of VanX, the D-ala-D-ala dipeptidase required for high-level vancomycin resistance in bacteria, stopped-flow kinetic and rapid-freeze quench EPR studies were conducted on the Co(II)-substituted enzyme when reacted with d-ala-d-ala. The intensity of the Co(II) ligand field band at 550 nm decreased (ε550 = 140 to 18 M-1 cm-1) when VanX was reacted with substrate, suggesting that the coordination number of the metal increases from 5 to 6 upon substrate binding. The stopped-flow trace was fitted to a kinetic mechanism that suggests the presence of an intermediate whose breakdown is rate-limiting. Rapid-freeze quench EPR studies verified the presence of a reaction intermediate that exhibits an unusually low hyperfine constant (33 G), which suggests a bidentate coordination of the intermediate to the metal center. The EPR studies also identified a distinct enzyme product complex. The results were used to offer a detailed reaction mechanism for VanX that can be used to guide future inhibitor design efforts

College students’ perceptions regarding sensory aspects of conventionally produced and unconventionally produced foods: implications for marketing to the Millennial generation

Consumers vote every day on which products line the shelves of grocery stores, co-ops, and niche markets. Public unrest with regard to the environmental, animal welfare, food purity, and human health impacts of agricultural production practices have led to the rise of unconventionally produced (UP) food products. While the sales of UP foods is increasing, studies regarding the qualities of such products that impact consumer purchases have yielded inconsistent results. This study examined students’ perceptions of sensory aspects of conventionally produced (CP) and UP foods to better understand how sensory aspects impact decisions to purchase. Students reported consistent perceptions regarding the favorability of each sensory aspect of chicken and apples; the UP versions of the products yielded higher mean scores on every sensory aspect. However, students’ perceptions of the sensory qualities of chocolate, milk, and beef were not consistent; for example, they reported more favorable perceptions of the appearance and smell of CP milk, but perceived a more favorable texture and flavor from the UP milk. The results of this study imply that when making purchasing decisions, consumers may value specific sensory attributes over others. One approach to marketing UP products is to focus on valued extrinsic aspects designed to attract consumers to purchase products even though they may have less favorable perceptions of certain sensory qualities

Structural Studies on a Mitochondrial Glyoxalase II

Glyoxalase 2 is a β-lactamase fold-containing enzyme that appears to be involved with cellular chemical detoxification. Although the cytoplasmic isozyme has been characterized from several organisms, essentially nothing is known about the mitochondrial proteins. As a first step in understanding the structure and function of mitochondrial glyoxalase 2 enzymes, a mitochondrial isozyme (GLX2-5) from Arabidopsis thaliana was cloned, overexpressed, purified, and characterized using metal analyses, EPR and 1H NMR spectroscopies, and x-ray crystallography. The recombinant enzyme was shown to bind 1.04 ± 0.15 eq of iron and 1.31 ± 0.05 eq of Zn(II) and to exhibit kcat and Km values of 129 ± 10 s-1 and 391 ± 48 μm, respectively, when using S-d-lactoylglutathione as the substrate. EPR spectra revealed that recombinant GLX2-5 contains multiple metal centers, including a predominant Fe(III)Z-n(II) center and an anti-ferromagnetically coupled Fe(III)Fe(II) center. Unlike cytosolic glyoxalase 2 from A. thaliana, GLX2-5 does not appear to specifically bind manganese. 1H NMR spectra revealed the presence of at least eight paramagnetically shifted resonances that arise from protons in close proximity to a Fe(III)Fe(II) center. Five of these resonances arose from solvent-exchangeable protons, and four of these have been assigned to NH protons on metal-bound histidines. A 1.74-Å resolution crystal structure of the enzyme revealed that although GLX2-5 shares a number of structural features with human GLX2, several important differences exist. These data demonstrate that mitochondrial glyoxalase 2 can accommodate a number of different metal centers and that the predominant metal center is Fe(III)Zn(II)

Arctic and subarctic environmental analyses utilizing ERTS-1 imagery

The author has identified the following significant results. ERTS-1 imagery provides a means of distinguishing and monitoring estuarine surface water circulation patterns and changes in the relative sediment load of discharging rivers on a regional basis. Physical boundaries mapped from ERTS-1 imagery in combination with ground truth obtained from existing small scale maps and other sources resulted in improved and more detailed maps of permafrost terrain and vegetation for the same area. Snowpack cover within a research watershed has been analyzed and compared to ground data. Large river icings along the proposed Alaska pipeline route from Prudhoe Bay to the Brooks Range have been monitored. Sea ice deformation and drift northeast of Point Barrow, Alaska have been measured during a four day period in March and shore-fast ice accumulation and ablation along the west coast of Alaska have been mapped for the spring and early summer seasons

A Five-coordinate Metal Center in Co(II)-substituted VanX

In an effort to structurally probe the metal binding site in VanX, electronic absorption, EPR, and extended x-ray absorption fine structure (EXAFS) spectroscopic studies were conducted on Co(II)-substituted VanX. Electronic spectroscopy revealed the presence of Co(II) ligand field transitions that had molar absorptivities of ∼100 m–1 cm–1, which suggests that Co(II) is five-coordinate in Co(II)-substituted VanX. Low temperature EPR spectra of Co(II)-substituted VanX were simulated using spin Hamiltonian parameters of M = |±½〉, E/D = 0.14, greal(x,y) = 2.37, and grealS(z) = 2.03. These parameters lead to the prediction that Co(II) in the enzyme is five-coordinate and that there may be at least one solvent-derived ligand. Single scattering fits of EXAFS data indicate that the metal ions in both native Zn(II)-containing and Co(II)-substituted VanX have the same coordination number and that the metal ions are coordinated by 5 nitrogen/oxygen ligands at ∼ 2.0 Å. These data demonstrate that Co(II) (and Zn(II) from EXAFS studies) is five-coordinate in VanX in contrast to previous crystallographic studies (Bussiere, D. E., Pratt, S. D., Katz, L., Severin, J. M., Holzman, T., and Park, C. H. (1998) Mol. Cell 2, 75–84). These spectroscopic studies also demonstrate that the metal ion in Co(II)-substituted VanX when complexed with a phosphinate analog of substrate d-Ala-d-Ala is also five-coordinate

College Students\u27 Perceptions regarding Sensory Aspects of Conventionally Produced and Unconventionally Produced Foods: Implications for Marketing to the Millennial Generation

Every day consumers vote which products line the shelves of grocery stores, co-ops, and niche markets by use of their dollars. Public unrest with regard to the environment, animal welfare, food purity, and human health impacts of agricultural production practices have led to the rise of alternatively produced food products. While the sales of alternatively produced foods are increasing, studies regarding the qualities of such products impact consumer purchases have yielded inconsistent results. This study examined students’ perceptions of sensory aspects of conventionally produced and alternatively produced foods to better understand how sensory aspects impact decisions to purchase. Students reported consistent perceptions regarding the favorability of each sensory aspect of chicken and apples; the alternatively produced versions of the products yielded higher mean scores on every sensory aspect. However, students’ perceptions of the sensory qualities of chocolate, milk, and beef were not consistent; for example, they reported more favorable perceptions of the appearance and smell of conventionally produced milk, but perceived a more favorable texture and flavor from the alternatively produced milk. The results of this study imply when making purchasing decisions, consumers may value specific sensory attributes over others. An alternative approach to marketing alternatively produced products is to focus on valued extrinsic aspects designed to attract consumers to purchase products in spite of their perhaps less valued perceptions of sensory aspects

Mano a Mano Online Information System: A Technical Report

From Morse Code, to the rotary telephone, to the internet, communication among individuals has been and still is the champion for human growth and development. Moreover, human communication is key for educational, professional, and personal development. Mano a Mano (Spanish for "Hand to Hand"), an online system designed for UNC-CH students, staff, faculty and organizations to meet, greet, and achieve serves a vital role in the educational, professional, and personal development of such campus affiliates. In addition, it strives to weaken the campus population barrier to communication by providing a means in which campus affiliates can make contacts among a diverse body of individuals. Designed in accordance with the conceptual framework of the iterative method proposed by Mullen and Nigay (1996), Mano a Mano and its underlying objectives intend to set UNC-CH apart from other UNC system colleges and universities by introducing a novel, technological platform of campus communication

Ionic partitioning of KCl in AOT reverse micelles from molecular dynamics simulations

Reverse micelles are an important class of nanoreactors providing an array of applications. The structure and dynamics of aqueous reverse micelles have been intensely investigated, yet there are many subtleties involved in characterizing the arrangement of water and ionic species within these aggregates. The interfacial arrangement of water and dopants added to bis(2-ethylhexyl) sulfosuccinate sodium salt (AOT) reverse micelles were investigated by fully atomistic molecular dynamics simulations. Reverse micelles of increasing water to surfactant ratio were doped with concentrations of KCl from 0.0 to 1.0 M to analyze the partitioning of ions and water. Detailed analysis of pair density functions reveals that Cl− ions are embedded deep in the aqueous interior, and the K+ ions display a remarkable affinity for the interface. Our results confirm the existence of multiple layers of water, and an ordered multi-layer ionic arrangement that is driven by the negatively charged surfactant headgroup

Are We Missing Important Areas in Pelagic Marine Conservation? Redefining Conservation Hotspots in the Ocean

The protection of biodiversity is one of the most important goals in terrestrial and marine conservation. Marine conservation approaches have traditionally followed the example of terrestrial initiatives. However, patterns, processes, habitats, and threats differ greatly between the 2 systems - and even within the marine environment. As a result, there is still a lack of congruence as to how to best identify and prioritize conservation approaches moving from the static terrestrial and nearshore realm into a more fluid, 3-dimensional pelagic realm. To address this problem, we investigate how the conservation science literature has been used to inform and guide management strategies in the marine system from coastal to pelagic environments. As cumulative impacts on the health of the oceans continue to increase, conservation priorities have shifted to include highly dynamic areas of the pelagic marine system. By evaluating whether priorities match science with current place-based management approaches (i.e. marine protected areas, MPAs), we identify important gaps that must be considered in current conservation schemes. Effective pelagic MPA design requires monitoring and evaluation across multiple physical, biological, and human dimensions. Because many threatened and exploited marine species move through an ephemeral and ever-changing environment, our results highlight the need to move beyond traditional, 2-dimensional approaches to marine conservation, and into dynamic management approaches that incorporate metrics of biodiversity as well as oceanographic features known to promote multilevel, trophic productivity

Glyoxalase II from A. thaliana requires Zn(II) for catalytic activity

AbstractCytosolic glyoxalase II from Arabidopsis thaliana, GLX2-2, was overexpressed and purified to homogeneity using Q-sepharose chromatography. MALDI-TOF mass spectrometry studies indicated a molecular weight of 28 767 Da. Using steady-state kinetics studies, the purified enzyme exhibited a Km of 660±100 μM and a kcat of 484±92 s−1 at 37°C. Metal analyses demonstrated that the enzyme binds 2.1±0.5 moles of Zn(II) per monomer; the binding of Zn(II) is essential for enzyme viability and activity. Sequence comparison of glyoxalase II enzymes from human, A. thaliana, and yeast and the metallo-β-lactamases reveal that all metal binding ligands of the metallo-β-lactamases are conserved in glyoxalase II enzymes, suggesting that all glyoxalase II enzymes are Zn(II) metalloenzymes. These results and their implications are discussed in light of previous studies on glyoxalase II, and an active site for the glyoxalase II enzymes is proposed