Background: Glutathione S-transferases (GSTs) comprise a multifunctional group of enzymes that play a critical role in the cellular detoxification process. These enzymes reduce the reactivity of ...toxic compounds by catalyzing their conjugation with glutathione. As a result of their role in detoxification, GSTs have been implicated in the development of cellular resistance to antibiotics, herbicides and clinical drugs and their study is therefore of much interest. In mammals, the cytosolic GSTs can be divided into five distinct classes termed alpha, mu, pi, sigma and theta. The human theta class GST, hGST T2-2, possesses several distinctive features compared to GSTs of other classes, including a long C-terminal extension and a specific sulfatase activity. It was hoped that the determination of the structure of hGST T2-2 may help us to understand more about this unusual class of enzymes.
Results: Here we present the crystal structures of hGST T2-2 in the apo form and in complex with the substrates glutathione and 1-menaphthyl sulfate. The enzyme adopts the canonical GST fold with a 40-residue C-terminal extension comprising two helices connected by a long loop. The extension completely buries the substrate-binding pocket and occludes most of the glutathione-binding site. The enzyme has a purpose-built novel sulfate-binding site. The crystals were shown to be catalytically active: soaks with 1-menaphthyl sulfate result in the production of the glutathione conjugate and cleavage of the sulfate group.
Conclusions: hGST T2-2 shares less than 15% sequence identity with other GST classes, yet adopts a similar three-dimensional fold. The C-terminal extension that blocks the active site is not disordered in either the apo or complexed forms of the enzyme, but nevertheless catalysis occurs in the crystalline state. A narrow tunnel leading from the active site to the surface may provide a pathway for the entry of substrates and the release of products. The results suggest a molecular basis for the unique sulfatase activity of this GST.
Sheep infected with the triclabendazole-susceptible, Cullompton isolate of Fasciola hepatica were dosed with 15 mg/kg of compound alpha at 12 weeks post-infection. Adult flukes were recovered from ...the bile ducts at 24, 48 and 72 h post-treatment (p.t.). Ultrastructural changes to the flukes were assessed using transmission electron microscopy (TEM), with a view to gathering information on the mechanism(s) of action for compound alpha and on the possible route of its entry into F. hepatica. The tegumental syncytium was more severely affected than the gut at all time-points p.t. with compound alpha, suggesting a predominantly trans-tegumental route of uptake. Disruption to the tegumental system became increasingly severe over time. A stress response was observed at 24 h p.t. and took the form of blebbing and increases in the production and transport of secretory bodies. By 72 h p.t., extensive tegumental loss and degeneration of the tegumental cell bodies had occurred. Degeneration of subtegumental tissues and internal flooding were also observed. Changes in the gastrodermal cells were slow to develop: reduced secretory activity was evident at 72 h p.t.. There was progressive disruption to the somatic muscle layers, with disorganization of the muscle blocks and loss of muscle fibres.
WEPP-predicting water erosion using a process-based model Laflen, J.M. (National Soil Tilth Laboratory, USDA, ARS, Ames, IA.); Elliot, W.J; Flanagan, D.C ...
Journal of soil and water conservation,
03/1997, Volume:
52, Issue:
2
Journal Article
Ecosystem models are important tools for diagnosing the carbon cycle and projecting its behavior across space and time. Despite the fact that ecosystems respond to drivers at multiple time scales, ...most assessments of model performance do not discriminate different time scales. Spectral methods, such as wavelet analyses, present an alternative approach that enables the identification of the dominant time scales contributing to model performance in the frequency domain. In this study we used wavelet analyses to synthesize the performance of 21 ecosystem models at 9 eddy covariance towers as part of the North American Carbon Program's site‐level intercomparison. This study expands upon previous single‐site and single‐model analyses to determine what patterns of model error are consistent across a diverse range of models and sites. To assess the significance of model error at different time scales, a novel Monte Carlo approach was developed to incorporate flux observation error. Failing to account for observation error leads to a misidentification of the time scales that dominate model error. These analyses show that model error (1) is largest at the annual and 20–120 day scales, (2) has a clear peak at the diurnal scale, and (3) shows large variability among models in the 2–20 day scales. Errors at the annual scale were consistent across time, diurnal errors were predominantly during the growing season, and intermediate‐scale errors were largely event driven. Breaking spectra into discrete temporal bands revealed a significant model‐by‐band effect but also a nonsignificant model‐by‐site effect, which together suggest that individual models show consistency in their error patterns. Differences among models were related to model time step, soil hydrology, and the representation of photosynthesis and phenology but not the soil carbon or nitrogen cycles. These factors had the greatest impact on diurnal errors, were less important at annual scales, and had the least impact at intermediate time scales.
Key Points
Twenty‐one ecosystem models were tested in the frequency domain at nine flux towers
Model error is greatest at the annual and growing‐season diurnal timescales
There are large event‐driven errors and model differences at the synoptic scale
Determining whether a tumor exhibits microsatellite instability (MSI) is useful in identifying patients with hereditary non-polyposis colorectal cancer and sporadic gastrointestinal cancers with ...defective DNA mismatch repair (MMR). The assessment of MSI status aids in establishing a clinical prognosis and may be predictive of tumor response to chemotherapy. A reference panel of five markers was suggested for MSI analysis by a National Cancer Institute (NCI) workshop in 1997 that has helped to standardize testing. But this panel of markers has limitations resulting from the inclusion of dinucleotide markers, which are less sensitive and specific for detection of tumors with MMR deficiencies compared to other types of markers that are currently available. This study demonstrates that mononucleotides are the most sensitive and specific markers for detection of tumors with defects in MMR and identifies an optimal panel of markers for detection of MSI-H tumors. A set of 266 mono-, di-, tetra- and penta-nucleotide repeat microsatellite markers were used to screen for MSI in colorectal tumors. The best markers for detection of MSI-H tumors were selected for a MSI Multiplex System, which included five mononucleotide markers: BAT-25, BAT-26, NR-21, NR-24 and MONO-27. In addition, two pentanucleotide markers were added to identify sample mix-ups and/or contamination. We classified 153 colorectal tumors using the new MSI Multiplex System and compared the results to those obtained with a panel of 10 microsatellite markers combined with immunohistochemical (IHC) analysis. We observed 99% concordance between the two methods with nearly 100% accuracy in detection of MSI-H tumors. Approximately 5% of the MSI-H tumors had normal levels of four MMR proteins and as a result would have been misclassified based solely on IHC analysis, emphasizing the importance of performing MSI testing. The new MSI Multiplex System offers several distinct advantages over other methods of MSI testing in that it is both extremely sensitive and specific and amenable to high-throughput analysis. The MSI Multiplex System meets the new recommendations proposed at the recent 2002 NCI workshop on HNPCC and MSI testing and overcomes problems inherent to the original five-marker panel. The use of a single multiplex fluorescent MSI assay reduces the time and costs involved in MSI testing with increased reliability and accuracy and thus should facilitate widespread screening for microsatellite instability in tumors of patients with gastrointestinal cancers.
Eight indoor-reared, crossbred sheep with no pre-exposure to
Fasciola hepatica were infected, by oral gavage, with 200 metacercarial cysts of the triclabendazole-susceptible, Cullompton isolate of
F. ...hepatica. Anthelmintic dosing occurred at 4 weeks post-infection using 15
mg/kg compound alpha. Two treated sheep per time period were euthanized at 24
h, 48
h and 72
h post-treatment with compound alpha. The two sheep from the control group were euthanized alongside the 24
h alpha-treated sheep. Juvenile flukes were recovered from each of the sheeps’ liver and processed for examination by electron microscopy. The surface morphology of the flukes’ tegument was assessed using scanning electron microscopy (SEM). The ultrastructure of the tegumental syncytium and underlying tegumental cells and connections and somatic musculature were investigated using transmission electron microscopy (TEM).
Both the SEM and TEM results revealed a level of disruption that increased with time, culminating at 72
h with extensive tegumental loss and substantial degeneration of the cell bodies. The effects of compound alpha on the surface morphology were not particularly apparent until 48
h post-treatment, when disruption included swelling and blebbing of the tegument. At 72
h post-treatment, SEM revealed loss of the entire syncytial layer over large areas of the flukes. In the areas where the syncytium was lost and the basal lamina exposed, lesions of varying sizes had developed, revealing underlying tissues. Though minor forms of disruption to the ultrastructure of the syncytium were observed using TEM 24
h post-treatment, it was at 48
h post-treatment that substantial stress responses occurred. They included the presence of autophagic vacuoles and ‘open’ bodies at the apex of the syncytium and swelling of the basal infolds. The mitochondria within the syncytium and tegumental cells became progressively more disrupted over the three time periods and, by 72
h post-treatment, they were frequently distorted and swollen in appearance, and contained severely swollen cristae. By 72
h, the number of secretory bodies, particularly T1 bodies, had become significantly depleted in their respective cell bodies, cytoplasmic processes and in the tegumental syncytium. Both the circular and longitudinal muscle bundles were severely disrupted 72
h post-treatment. They frequently contained a reduced number of muscle fibres and, in more severe instances, there was an absence of fibres altogether.
Major trauma in geriatric patients Champion, H R; Copes, W S; Buyer, D ...
American journal of public health (1971),
09/1989, Volume:
79, Issue:
9
Journal Article
Peer reviewed
Open access
Contemporary trauma to the elderly, its severity and associated mortality and morbidity in 111 United States and Canadian trauma centers are described. Three-thousand eight-hundred thirty-three ...(3,833) trauma patients age 65 years or older are compared to 42,944 injured patients under age 65. Although both groups had equivalent measures of injury severity, the older group had higher case fatality and complication rates and longer hospital stays. The results raise important questions regarding the triage, acute care, accurate prediction of outcome, and hospital reimbursement for the elderly injured patient, with implications for care evaluation, quality assurance, and the long-term viability of trauma centers and systems of care.
Sheep infected with the triclabendazole-susceptible Cullompton isolate of Fasciola hepatica were dosed with 15 mg/kg of compound alpha at 12 weeks postinfection. Adult flukes were recovered from the ...bile ducts at 24, 48, and 72 h post-treatment (p.t.). Changes to the surface morphology of the flukes were assessed using scanning electron microscopy (SEM). Flukes were still active at 24 h p.t. and displayed limited areas of disruption, which were restricted to the oral cone region. At 48 h p.t., a reduced level of motility was observed in ~50% of the flukes recovered. Swelling of the tegument was more widespread and was accompanied by blebbing and partial loss of the tegumental covering of the spines. By 72 h p.t., the reduction in motility was greater, and approximately one quarter of the flukes recovered were inactive. In the majority of the flukes examined, the midbody region was marked by a discoloration of the flukes' tissues. This was seen to be due to the loss of the tegumental syncytium. Sloughing extended into the tail region in some specimens and, in the more badly-affected specimens, the basal lamina was breached to expose the underlying musculature. Elsewhere on the body, the tegument that remained was relatively normal, although areas of swelling and blebbing were present. Overall, the results provided information on the time-scale of changes to the surface morphology of the fluke that underpin the efficacy of compound alpha.
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