Recombinant Aspergillus β-galactosidases as a robust glycomic and biotechnological tool

Galactosidases are widespread enzymes that are used for manifold applications, including production of prebiotics, biosynthesis of different transgalactosylated products, improving lactose tolerance and in various analytical approaches. The nature of these applications often require galactosidases to be present in a purified form with clearly defined properties, including precisely determined substrate specificities, low sensitivity to inhibitors, and high efficiency and stability under distinct conditions. In this study, we present the recombinant expression and purification of two previously uncharacterized β-galactosidases from Aspergillus nidulans as well as one β-galactosidase from Aspergillus niger. All enzymes were active toward p-nitrophenyl-β-d-galactopyranoside as substrate and displayed similar temperature and pH optima. The purified recombinant galactosidases digested various complex substrates containing terminal galactose β-1,4 linked to either N-acetylglucosamine or fucose, such as N-glycans derived from bovine fibrin and Caenorhabditis elegans. In our comparative study of the recombinant galactosidases with the commercially available galactosidase from Aspergillus oryzae, all enzymes also displayed various degrees of activity toward complex oligosaccharides containing β-1,3-linked terminal galactose residues. All recombinant enzymes were found to be robust in the presence of various organic solvents, temperature variations, and freeze/thaw cycles and were also tested for their ability to synthesize galactooligosaccharides. Furthermore, the use of fermentors considerably increased the yield of recombinant galactosidases. Taken together, we demonstrate that purified recombinant galactosidases from A. niger and from A. nidulans are suitable for various glycobiological and biotechnological applications.     

Neddylation and deneddylation of CUL-3 is required to target MEI-1/Katanin for degradation at the meiosis-to-mitosis transition in C. elegans

BACKGROUND: SCF (Skp1-Cullin-F-box) complexes are a major class of E3 ligases that are required to selectively target substrates for ubiquitin-dependent degradation by the 26S proteasome. Conjugation of the ubiquitin-like protein Nedd8 to the cullin subunit (neddylation) positively regulates activity of SCF complexes, most likely by increasing their affinity for the E2 conjugated to ubiquitin. The Nedd8 conjugation pathway is required in C. elegans embryos for the ubiquitin-mediated degradation of the microtubule-severing protein MEI-1/Katanin at the meiosis-to-mitosis transition. Genetic experiments suggest that this pathway controls the activity of a CUL-3-based E3 ligase. Counteracting the Nedd8 pathway, the COP9/signalosome has been shown to promote deneddylation of the cullin subunit. However, little is known about the role of neddylation and deneddylation for E3 ligase activity in vivo. RESULTS: Here, we identified and characterized the COP9/signalosome in C. elegans and showed that it promotes deneddylation of CUL-3, a critical target of the Nedd8 conjugation pathway. As in other species, the C. elegans signalosome is a macromolecular complex containing at least six subunits that localizes in the nucleus and the cytoplasm. Reducing COP9/signalosome function by RNAi results in a failure to degrade MEI-1, leading to severe defects in microtubule-dependent processes during the first mitotic division. Intriguingly, reducing COP9/signalosome function suppresses a partial defect in the neddylation pathway; this suppression suggests that deneddylation and neddylation antagonize each other. CONCLUSIONS: We conclude that both neddylation and deneddylation of CUL-3 is required for MEI-1 degradation and propose that cycles of CUL-3 neddylation and deneddylation are necessary for its ligase activity in vivo.     

Fragment-Hopping-Based Discovery of a Novel Chemical Series of Proto-Oncogene PIM-1 Kinase Inhibitors

A new chemical series, triazolo[4,5-b]pyridines, has been identified as an inhibitor of PIM-1 by a chemotype hopping strategy based on a chemically feasible fragment database. In this case, structure-based virtual screening and in silico chemogenomics provide added value to the previously reported strategy of prioritizing among proposed novel scaffolds. Pairwise comparison between compound 3, recently discontinued from Phase I clinical trials, and molecule 8, bearing the selected novel scaffold, shows that the primary activities are similar (IC₅₀ in the 20 to 150 nM range). At the same time, some ADME properties (for example, an increase of more than 45% in metabolic stability in human liver microsomes) and the off-target selectivity (for example, an increase of more than 2 log units in IC₅₀ vs. FLT3) are improved, and the intellectual property (IP) position is enhanced. The discovery of a reliable starting point that fulfills critical criteria for a plausible medicinal chemistry project is demonstrated in this prospective study.     

Advances in zymography techniques and patents regarding protease analysis

Detection of enzymatic activity on gel electrophoresis, namely zymography, is a technique that has received increasing attention in the last 10 years, according to the number of articles published. A growing amount of enzymes, mainly proteases, are now routinely detected by zymography. Detailed analytical studies are beginning to be published, as well as new patents have been developed. This new article updates the information covered in our last review, condensing the recent publications dealing with the identification of proteolytic enzymes in electrophoretic gel supports and its variations. The new advances of this method are basically focused towards two dimensional zymography and transfer zymography. Though comparatively fewer patents have been published, they basically coincide in the study of matrix metalloproteases. The tendency is foreseen to be very productive in the area of zymoproteomics, combining electrophoresis and mass spectrometry for the analysis of proteases.     

Luteinizing hormone and androstendione are independent predictors of ovulation after laparoscopic ovarian drilling: a retrospective cohort study

Background  

Our objective was to investigate luteinizing hormone, follicle-stimulating hormone, testosterone, and androstenedione as predicitve markers for ovulation after laparoscopic ovarian drilling.     

Thyroid hormone regulates the obesity gene tub

Thyroid hormone T3/T4 is a major regulator of energy metabolism in vertebrates, and defects in thyroid status are frequently associated with changes in body weight. It is demonstrated here that thyroid hormone regulates in vivo and in vitro the tub gene, which when mutated in tubby mice causes obesity, insulin resistance and sensory deficits. Hypothyroidism in rats altered tub mRNA and protein in discrete brain areas. These changes could be attributed to thyroid hormone deficiency since T3/T4 treatment restored normal tub expression. T3 also upregulated tub mRNA within 4–6 h in neuronal cells in culture, suggesting that T3 is a positive regulator of tub gene expression. Thus, these results establish a novel pathway of T3 action and provide an important molecular link between thyroid status and the tubby-associated syndrome.     

Negative impacts of high temperatures on growth of black spruce forests intensify with the anticipated climate warming

An increasing number of studies conclude that water limitations and heat stress may hinder the capacity of black spruce (Picea mariana (Mill.) B.S.P.) trees, a dominant species of Canada's boreal forests, to grow and assimilate atmospheric carbon. However, there is currently no scientific consensus on the future of these forests over the next century in the context of widespread climate warming. The large spatial extent of black spruce forests across the Canadian boreal forest and associated variability in climate, demography, and site conditions pose challenges for projecting future climate change responses. Here we provide an evaluation of the impacts of climate warming and drying, as well as increasing [CO₂], on the aboveground productivity of black spruce forests across Canada south of 60°N for the period 1971 to 2100. We use a new extensive network of tree‐ring data obtained from Canada's National Forest Inventory, spatially explicit simulations of net primary productivity (NPP) and its drivers, and multivariate statistical modeling. We found that soil water availability is a significant driver of black spruce interannual variability in productivity across broad areas of the western to eastern Canadian boreal forest. Interannual variability in productivity was also found to be driven by autotrophic respiration in the warmest regions. In most regions, the impacts of soil water availability and respiration on interannual variability in productivity occurred during the phase of carbohydrate accumulation the year preceding tree‐ring formation. Results from projections suggest an increase in the importance of soil water availability and respiration as limiting factors on NPP over the next century due to warming, but this response may vary to the extent that other factors such as carbon dioxide fertilization, and respiration acclimation to high temperature, contribute to dampening these limitations.     

Cryopreservation of Alkaloid-Producing Cell Cultures of Periwinkle (Catharanthus roseus)

A procedure for cryogenic storage of alkaloid producing cell lines of periwinkle, Catharanthus roseus (L.) G. Don., has been developed. The procedure differs from established cryopreservation protocols in several aspects. Specifically, 4-day-old suspension subcultures of three cell lines were precultured in nutrient media supplemented with 1 molar sorbitol for 6 to 20 hours. The cells were then incubated in nutrient media with 1 molar sorbitol plus 5% DMSO in an ice bath for 1 hour and, thereafter, were frozen in this solution at a cooling rate of 0.5°C per minute to −40°C prior to immersion in liquid nitrogen (LN). After rapid thawing in a 40°C water bath, the regrowth of LN stored cells was achieved by transferring them without washing onto filter paper discs over nutrient media solidified with agar for a period of 4 to 5 hours. The filter paper discs with the cells were then transferred to fresh media of the same composition for regrowth. The viability immediately after thawing as evaluated by the 2,3,5-triphenyl tetrazolium chloride method was about 60% of controls. Suspension cultures established from LN stored cells retained the capability for alkaloid synthesis and accumulation.     

A colorimetric method for DNA hybridization.

A general method has been developed which allows crosslinks to be produced between proteins and single-stranded DNA. Such single-stranded DNA protein complexes have been tested for blot hybridization using two colorimetrically detectable enzymes, namely peroxidase and alkaline phosphatase, as the protein moiety of the probe. After hybridization and incubation with a substrate solution sequences complementary to the probe can be visualized directly without the need of tedious cytochemical sandwich methods. This procedure will detect target sequences, a few kilobases long, in the 1- to 5-pg range.     

Pericytes in the mature chorioallantoic membrane capillary plexus contain desmin and α-smooth muscle actin: relevance for non-sprouting angiogenesis

The chicken chorioallantoic membrane (CAM) is a frequently used tissue for studying vascular growth and remodeling, notably non-sprouting angiogenesis by formation of transluminal pillars. Vascular pericytes have received increasing attention in the field of angiogenesis research and appear important for pillar growth. Our earlier observation that desmin (DES), but not α-smooth muscle actin (αSMA) was expressed in pericytes of the mature CAM capillary plexus after E12 was confirmed by others. However, in different species or vascular beds, either marker or both have been used to identify pericytes, raising the questions if (1) expression of these cytoskeletal proteins really was mutually exclusive; or (2) different types of pericytes existed in the same vascular bed. Using triple labeling with fluorochrome-conjugated markers Sambucus nigra agglutinin, DES or αSMA, and DNA-specific YoPro-1, we report here for the first time a delicate filamentous, circumferentially oriented αSMA pattern in periendothelial cells of the mature CAM capillary plexus, quite different from the coarser, axially oriented DES pattern. A new method for automatic classification of DNA-staining pattern was applied to compare nuclei of DES- or αSMA-positive cells. It predicted colocalisation of both proteins in most capillary pericytes, which was confirmed by double immunostaining for DES and αSMA. We conclude that (1) in contrast to published work, DES and αSMA are not mutually exclusive in most pericytes; (2) different types of pericytes may co-exist in the same vascular bed; (3) on average, one pericyte is associated with two transluminal pillars; (4) a novel imaging modality may be useful for cell identification in angiogenesis research and elsewhere.