Human acid beta-glucosidase: affinity purification of the normal placental and Gaucher disease splenic enzymes on N-alkyl-deoxynojirimycin-sepharose

Two sepharose-bound 1-deoxynojirimycin N-alkyl derivatives, N-(9-carboxynonyl)- and N-(11-carboxyundecyl)-deoxynojirimycin, were used for the affinity purification of acid beta-glucosidase (beta-Glc) from normal and type-1 Ashkenazi Jewish Gaucher disease (AJGD) sources. The capacities of these nondegradable inhibitor supports were 0.5 and 0.75 mg of normal beta-Glc/ml of settled gel, respectively. The purified normal enzyme (14-18% yield) had a specific activity of 1.6 X 10(6) nmol/h/mg protein and was homogeneous as evidenced by a single protein species of Mr = 67,000 on sodium dodecylsulfate-polyacrylamide gel electrophoresis and reverse phase high-performance liquid chromatography (HPLC). Microsequencing demonstrated a single N terminus, and the sequence of the first 22 N-terminal amino acids was colinear with that predicted from the beta-Glc cDNA. Amino acid composition analyses of beta-Glc revealed a high content (35%) of hydrophobic amino acids. The N-decyl-deoxynojirimycin support facilitated the purification of the residual enzyme from type-1 AJGD spleen to about 7,500-fold in four steps with a yield of about 11%. These new affinity supports provided improved stability, capacity and/or specificity compared to other affinity or HPLC methods for purifying this lysosomal glycosidase.     

Edge effects influence the composition and density of reef residents on subtidal restored oyster reefs

Within estuarine and coastal ecosystems globally, extensive habitat degradation and loss threaten critical ecosystem functions and necessitate widescale restoration efforts. There is abundant evidence that ecological processes and species interactions can vary with habitat characteristics, which has important implications for the design and implementation of restoration efforts aimed at enhancing specific ecosystem functions and services. We conducted an experiment examining how habitat characteristics (presence; edge vs. interior) influence the communities of resident fish and mobile invertebrates on restored oyster (Crassostrea virginica) reefs. Similar to previous studies, we found that restored reefs altered community composition and augmented total abundance and biomass relative to unstructured sand habitat. Community composition and biomass also differed between the edge and interior of individual reefs as a result of species-specific patterns over small spatial scales. These patterns were only weakly linked to oyster density, suggesting that other factors that vary between edge and interior (e.g. predator access or species interactions) are likely more important for community structure on oyster reefs. Fine-scale information on resident species' use of oyster reefs will help facilitate restoration by allowing decision makers to optimize the amount of edge versus interior habitat. To improve the prediction of faunal use and benefits from habitat restoration, we recommend investigations into the mechanisms shaping edge and interior preferences on oyster reefs.     

Estimating factors influencing the detection probability of semiaquatic freshwater snails using quadrat survey methods

The effective management and conservation of fishery resources requires knowledge of their spatial distribution and notably of their critical life history stages. Predictive modelling of the European hake (Merluccius merluccius L., 1758) distribution was developed in the south-central Mediterranean Sea by means of historical fisheries-independent databases available in the region. The study area included the international waters of the south-central Mediterranean Sea and the territorial waters of Italy, Malta, Tunisia and Libya. Distribution maps of predicted population abundance index, and probabilistic occurrence of recruits and large adults were obtained by means of generalized additive models using depth and seafloor characteristics as predictors. Presence/absence data of the two life stages was obtained using threshold values applied to the mean weight of the survey catches. Modelling results largely matched previously reported knowledge on habitat preference of the species and its critical life phases. Hake recruits showed an occurrence peak at 200 m depth with preference for soft bottoms. Large adults preferred deeper and harder bottom substrates. Prediction maps allowed to improve our knowledge on the distributional patterns of one of the most important shared stocks in the south-central Mediterranean. This knowledge is essential for an appropriate development of regional-spatial-based management plans.     

First-trimester prenatal diagnosis of Tay-Sachs disease.

The prenatal diagnosis of Tay-Sachs disease was made in two at-risk fetuses by the analysis of chorionic villi obtained at 9 and 11 menstrual weeks, respectively. The diagnoses were based on the absence of beta-hexosaminidase A activity as determined by: (1) specific enzyme assays, (2) anion-exchange chromatography, and (3) cellulose acetate gel electrophoresis. The enzymatic diagnoses were confirmed on fetal tissue as well as by ultrastructural demonstration of the first-trimester fetal neuropathology. Optimal assay conditions for beta-hexosaminidase A in chorionic villi were established for the prenatal diagnosis of Tay-Sachs disease. Importantly, it was noted that a small amount of decidua or maternal blood could lead to misdiagnosis. Thus, extreme care must be taken in the preparation of chorionic villi for Tay-Sachs as well as other prenatal metabolic diagnoses.     

Human acid beta-glucosidase. Use of inhibitory and activating monoclonal antibodies to investigate the enzyme's catalytic mechanism and saposin A and C binding sites.

Of 14 identified epitopes on human GCase (acid beta-glucosidase), monoclonal antibodies (MCABs) recognizing 3 produced inhibition and 1 resulted in activation of GCase. MCABs F1 and F2 completely, and MCAB 61 partially (approximately 70%), inhibited GCase activity. Substrates and active site-directed inhibitors (specific sphingolipid and 5-amino-5-deoxyglucose derivatives) protected the enzyme from inhibition by MCAB F1 and F2, but not that by MCAB 61. Conduritol B epoxide did not protect GCase from the inhibition by these MCABs when covalently bound to the active site. These results indicated highly specific binding requirements of MCABs F1 and F2 for residues in a complex active site. In comparison, kinetic analyses using GCase transition state analogues, N-alkyl-glucosylamines, and MCAB 61 demonstrated that this MCAB "freezes" the conformation of the enzyme and inhibits GCase by preventing formation of a conformer needed for maximal catalytic rates. The activating MCAB 122 mimicked the effects of saposin C and competed with this natural activator for residues on the enzyme. Interaction of saposin A and saposin C or MCAB 122 with GCase produced a synergistic effect leading to a marked sensitization of the enzyme to these activators. No such synergism or additivity was found for the maximal catalytic rate since it could be achieved by saturating amounts of any one or combinations of these activators. In the presence of MCAB 61, only 15 to 25% of the maximal activation of GCase was obtained by saposin C or MCAB 122, indicating that the major activation effects of these effectors derived from an induction of a GCase conformational change. These results demonstrate that saposins A and C mediate their activating effects by binding to distinct sites on GCase. Furthermore, major components of the mechanisms for catalysis and saposin C activation are due to conformational changes during the transition state. These findings have implications for understanding the perturbations of GCase function due to the missense mutations which cause Gaucher disease.     

The killer shrimp,Dikerogammarus villosus,invading European Alpine Lakes: A single main source but independent founder events with an overall loss of genetic diversity

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CMT3 alters mitochondrial function in murine osteoclast lineage cells

Chemically modified tetracyclines (CMTs 1-10) were developed as non-antibiotic inhibitors of matrix metalloproteinases (MMPs). We previously demonstrated that MMP inhibition alone is insufficient to explain the pro-apoptotic action of CMTs in osteoclast lineage cells and we have explored additional mechanisms of action. We compared the characteristics of apoptosis in RAW264.7 murine monocyte and osteoclast cultures treated with pharmacologically relevant concentrations of CMT3 or the bisphosphonate alendronate, which induces osteoclast apoptosis through inhibition of farnesyl diphosphate synthase. CMT3 induced apoptosis rapidly (2-3 h), whereas alendronate-induced apoptosis was delayed (>12 h). CMT3-treated cells did not accumulate unprenylated Rap1A in contrast to cells treated with alendronate. Importantly, CMT3 induced a rapid loss of mitochondrial stability in RAW264.7 cells measured by loss of Mitotracker^® Red fluorescence, while bongkrekic acid protected polykaryons from CMT3-induced apoptosis. Modulation of mitochondrial function is therefore a significant early action of CMT3 that promotes apoptosis in osteoclast lineage cells.     

Do Arctic-nesting birds respond to earlier snowmelt? A multi-species study in north Yukon, Canada

Climate change has altered the timing of many ecological processes, especially in the Arctic. The initiation of nesting is a key signal of phenological changes in Arctic-nesting birds, and is possibly connected to the circumpolar trend of earlier snowmelt. We collected data on lay dates of 7 bird species, representing shorebirds, passerines, a bird of prey, and seabirds, nesting on Herschel Island, Yukon, Canada, in the years 1984–1986 and 2007–2009. Snowmelt was significantly earlier in the 2007–2009 period. Shorebirds and passerines showed trends to earlier lay dates in conjunction with earlier snowmelt; the other species did not. The strength of response in lay date was correlated with the general categories of foods known to be used by study species. However, six species showed a longer time interval between snowmelt and egg-laying in early compared to late springs, suggesting the need for further monitoring of how robust their responses to snowmelt are in the future.     

Synthesis, physicochemical and pharmacokinetic characterization of calcium uronates

Four calcium compounds containing uronic acids (D(+)-galacturonic and D(+)-glucuronic) in L:M ratio = 2 and 3 were isolated by applying novel (except for one complex) synthetic procedures. The compounds were characterized by elemental analysis, spectroscopic methods (diffuse reflectance and absorption UV-visible, IR, FIR), mass spectrometry, fast atom bombardment (FAB), thermal decomposition, thermogravimetry/derivative thermogravimetry (TG/DTG) data and differential scanning calorimetric studies (DSC). Two modes of water binding in the complexes, i.e., hydration and coordination-like, were established. Computer-aided analysis has shown that further investigations are needed in order to determine the applicability of calcium uronates as calcium carriers.