Crystallization studies of Pictet-Spenglerases

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2008.Vita.Includes bibliographical references.Natural products are a rich source of medicinally important molecules. Monoterpene indole alkaloids from plants are an especially important source of therapeutic molecules. Due to the complexity of these molecules, biosynthesis of derivatives is an attractive way of obtaining molecules with potentially new or improved functionality. The rational design of mutants with altered/expanded substrate scope is an important step in engineering organisms to produce such compounds. In monoterpene indole alkaloid biosynthesis, the enzyme strictosidine synthase catalyzes the first committed reaction. This reaction is a Pictet-Spengler coupling between tryptamine and secologanin and produces the biosynthetic intermediate strictosidine, common to all monoterpene indole alkaloids. To better understand the structural features that impart binding selectivity, crystallization studies of this enzyme were performed. The native enzyme and several interesting mutants were studied; co-crystallization experiments with inhibitors and substrates were also performed. Diffraction quality crystals of the native enzyme were obtained following optimization by grid screening, additive screens, and macroseeding. Data on the optimized crystals was collected at the Argonne National Labs synchrotron radiation source. In addition to monoterpene indole alkaloids, the benzylisoquinoline alkaloids are another class of medicinally important plant derived natural products. In a reaction analogous to that catalyzed by strictosidine synthase, the first committed step of benzylisoquinoline biosynthesis is a Pictet-Spengler reaction between 4-hydroxyphenylactetaldehyde and dopamine, catalyzed by the enzyme norcoclaurine synthase. Two different forms of this enzyme have been identified, neither of which shows any homology to strictosidine synthase.(cont.) Structural information for these enzymes could provide general structural features required for enzymatic Pictet-Spengler reactions. Before crystallization, the enzymes were expressed and tested for activity. Once active preparations of protein were available, crystallization studies were performed and crystals were obtained.by William C. Hillmann.S.M

Core Services in the Architecture of the National Digital Library for Science Education (NSDL)

We describe the core components of the architecture for the (NSDL) National Science, Mathematics, Engineering, and Technology Education Digital Library. Over time the NSDL will include heterogeneous users, content, and services. To accommodate this, a design for a technical and organization infrastructure has been formulated based on the notion of a spectrum of interoperability. This paper describes the first phase of the interoperability infrastructure including the metadata repository, search and discovery services, rights management services, and user interface portal facilities

Plasma 25-Hydroxyvitamin D and Progression to Diabetes in Patients at Risk for Diabetes

OBJECTIVE To investigate the association between vitamin D status, assessed by plasma 25-hydroxyvitamin D, and risk of incident diabetes. RESEARCH DESIGN AND METHODS Prospective observational study with a mean follow-up of 2.7 years in the Diabetes Prevention Program (DPP), a multicenter trial comparing different strategies for prevention of diabetes in patients with prediabetes. We assessed the association between plasma 25-hydroxyvitamin D, measured repeatedly during follow-up, and incident diabetes in the combined placebo (n = 1,022) and intensive lifestyle (n = 1,017) randomized arms of the DPP. Variables measured at multiple study time points (25-hydroxyvitamin D, BMI, and physical activity) entered the analyses as time-varying “lagged” covariates, as the mean of the previous and current visits at which diabetes status was assessed. RESULTS After multivariate adjustment, including for the DPP intervention, participants in the highest tertile of 25-hydroxyvitamin D (median concentration, 30.1 ng/mL) had a hazard ratio of 0.72 (95% CI 0.56–0.90) for developing diabetes compared with participants in the lowest tertile (median concentration, 12.8 ng/mL). The association was in the same direction in placebo (0.70; 0.52–0.94) versus lifestyle arm (0.80; 0.54–1.17). CONCLUSIONS Higher plasma 25-hydroxyvitamin D, assessed repeatedly, was associated with lower risk of incident diabetes in high-risk patients, after adjusting for lifestyle interventions (dietary changes, increased physical activity, and weight loss) known to decrease diabetes risk. Because of the observational nature of the study, the potential association between vitamin D and diabetes needs to be confirmed in intervention studies

Movement Behaviour of Traditionally Managed Cattle in the Eastern Province of Zambia Captured Using Two-Dimensional Motion Sensors

Two-dimensional motion sensors use electronic accelerometers to record the lying, standing and walking activity of cattle. Movement behaviour data collected automatically using these sensors over prolonged periods of time could be of use to stakeholders making management and disease control decisions in rural sub-Saharan Africa leading to potential improvements in animal health and production. Motion sensors were used in this study with the aim of monitoring and quantifying the movement behaviour of traditionally managed Angoni cattle in Petauke District in the Eastern Province of Zambia. This study was designed to assess whether motion sensors were suitable for use on traditionally managed cattle in two veterinary camps in Petauke District in the Eastern Province of Zambia. In each veterinary camp, twenty cattle were selected for study. Each animal had a motion sensor placed on its hind leg to continuously measure and record its movement behaviour over a two week period. Analysing the sensor data using principal components analysis (PCA) revealed that the majority of variability in behaviour among studied cattle could be attributed to their behaviour at night and in the morning. The behaviour at night was markedly different between veterinary camps; while differences in the morning appeared to reflect varying behaviour across all animals. The study results validate the use of such motion sensors in the chosen setting and highlight the importance of appropriate data summarisation techniques to adequately describe and compare animal movement behaviours if association to other factors, such as location, breed or health status are to be assessed

The anatomy of the larynx of the bowhead whale, Balaena mysticetus, and its sound-producing functions

This study describes the morphology of the laryngeal apparatus in bowhead whales (Balaena mysticetus) with respect to respiration, deglutition, and vocalization. We also examined the intrinsic cricoarytenothyroid muscle (Musculus (M.) diverticuli laryngei) which forms the laryngeal diverticulum, to ascertain its interactions with the laryngeal cartilages during respiration and sound production. Five fetal larynges and four from adult whales were studied using noninvasive imaging, as well as macroscopic and microscopic techniques. The larynx extends from the skull base into the thoracic inlet. The dorsally curved laryngeal stalk, supported by epiglottis and the corniculate processes of arytenoid cartilages, is situated within the nasopharynx. The epiglottic cartilage exhibits a prominent medial ridge. The arytenoid cartilages are rod-shaped, and extend through the laryngeal cavity. The thyroid cartilage possesses a prominent caudal horn with a fibrous articulation to the ventrally incomplete cricoid cartilage. The M. thyroepiglotticus forms the connection between epiglottic and thyroid cartilages. The M. cricothyroideus lateralis connects the caudal horn of the thyroid cartilage with the cricoid cartilage and the M. cricothyroideus medialis connects the cricoid and thyroid cartilage. An extensive laryngeal diverticulum (Diverticulum laryngis), formed by the laryngeal mucosa and M. diverticuli laryngei, is positioned caudo-ventral to the laryngeal vestibule. The mucosa thickens into a fold medial to the vocal processes of the arytenoid cartilages. Experiments with airflow combined with histological and anatomical evidence strongly suggest a sound producing function for these (vocal) folds. This analysis provides the first account of sound producing structures and function in bowhead whales

Decline of the Maurepas Swamp, Pontchartrain Basin, Louisiana, and Approaches to Restoration

The Maurepas swamp is the second largest contiguous coastal forest in Louisiana but it is highly degraded due to subsidence, near permanent flooding, nutrient starvation, nutria herbivory, and saltwater intrusion. Observed tree mortality rates at study sites in the Maurepas swamp are very high (up to 100% tree mortality in 11 years) and basal area decreased with average salinities of <1 ppt. Habitat classification, vegetation productivity and mortality, and surface elevation changes show a clear trajectory from stagnant, nearly permanently flooded forests with broken canopy to degraded forests with sparse baldcypress and dominated by herbaceous species and open water to open water habitat for most of the Maurepas swamp without introduction of fresh water to combat saltwater intrusion and stimulate productivity and accretion. Healthy forests in the Maurepas are receiving fresh water containing nutrients and sediments from urban areas, high quality river water, or secondarily treated municipal effluent. Currently, two proposed diversions into the swamp are via Hope Canal (57 m3·s−1) and Blind River (142 m3·s−1). These diversions would greatly benefit their immediate area but they are too small to influence the entire Maurepas sub-basin, especially in terms of accretion. A large diversion (>1422 m3·s−1) is needed to deliver the adequate sediments to achieve high accretion rates and stimulate organic soil formation

Restoration and Management of a Degraded Baldcypress Swamp and Freshwater Marsh in Coastal Louisiana

The Central Wetlands Unit (CWU), covering 12,000 hectares in St. Bernard and Orleans Parishes, Louisiana, was once a healthy baldcypress–water tupelo swamp and fresh and low salinity marsh before construction of levees isolated the region from Mississippi River floodwaters. Construction of the Mississippi River Gulf Outlet (MRGO), which funneled saltwater inland from the Gulf of Mexico, resulted in a drastic ecosystem change and caused mortality of almost all trees and low salinity marsh, but closure of the MRGO has led to decreases in soil and surface water salinity. Currently, the area is open water, brackish marsh, and remnant baldcypress stands. We measured hydrology, soils, water and sediment chemistry, vegetation composition and productivity, accretion, and soil strength to determine relative health of the wetlands. Vegetation species richness is low and above- and belowground biomass is up to 50% lower than a healthy marsh. Soil strength and bulk density are low over much of the area. A baldcypress wetland remains near a stormwater pumping station that also has received treated municipal effluent for about four decades. Based on the current health of the CWU, three restoration approaches are recommended, including: (1) mineral sediment input to increase elevation and soil strength; (2) nutrient-rich fresh water to increase productivity and buffer salinity; and (3) planting of freshwater forests, along with fresh and low salinity herbaceous vegetation