Analysis of the site-specific asparagine-linked glycosylation of recombinant human coagulation factor VLLa by glycosidase digestions, liquid chromatography, and mass spectrometry

The two asparagine-linked glycosylation sites of recombinant coagulation factor VIIa have been characterized by glycosidase digestions, size-exclusion chromatography (SEC), and mass spectrometry (MS). Nine structures were characterized as core fucosylated bi- and triantennary structures with 0-3 sialic-acid residues, which were alpha2-3 linked to galactose exclusively. Three of the structures had one or two galactose residues substituted by N-acetylgalactosamine. Significant differences were found between the oligosac-charide profiles for the two glycosylation sites in rFVIIa. At Asn322, the degree of sialylation was lower and higher amounts of structures containing N-acetylgalactosamine were found compared to Asn l45.     

Topological studies on the enzymes catalyzing the biosynthesis of Glc-P- dolichol and the triglucosyl cap of Glc3Man9GlcNAc2-P-P-dolichol in microsomal vesicles from pig brain: use of the processing glucosidases I/II as latency markers

In the current model for Glc3Man9GlcNAc2-P-P-Dol assembly, Man5GlcNAc2- P-P-Dol, Man-P-Dol, and Glc-P-Dol are synthesized on the cytoplasmic face of the ER and diffuse transversely to the lumenal leaflet where the synthesis of the lipid-bound precursor oligosaccharide is completed. To establish the topological sites of Glc-P-Dol synthesis and the lipid-mediated glucosyltransfer reactions involved in Glc3Man9GlcNAc2-P-P-Dol synthesis in ER vesicles from pig brain, the trypsin-sensitivity of Glc-P-Dol synthase activity and the Glc-P- Dol:Glc0-2Man9GlcNAc2-P-P-Dol glucosyltransferases (GlcTases) was examined in sealed microsomal vesicles. Since ER vesicles from brain do not contain glucose 6-phosphate (Glc 6-P) phosphatase activity, the latency of the lumenally oriented, processing glucosidase I/II activities was used to assess the intactness of the vesicle preparations. Comparative enzymatic studies with sealed ER vesicles from brain and kidney, a tissue that contains Glc 6-P phosphatase, demonstrate the reliability of using the processing glucosidase activities as latency markers for topological studies with microsomal vesicles from non-gluconeogenic tissues lacking Glc 6-P phosphatase. The results obtained from the trypsin-sensitivity assays with sealed microsomal vesicles from brain are consistent with a topological model in which Glc-P-Dol is synthesized on the cytoplasmic face of the ER, and subsequently utilized by the three Glc-P-Dol-mediated GlcTases after "flip-flopping" to the lumenal monolayer.      

3pK, a novel mitogen-activated protein (MAP) kinase-activated protein kinase, is targeted by three MAP kinase pathways.

Recently we have identified a mitogen-activated protein kinase (MAPK)-activated protein kinase, named 3pK (G. Sithanandam, F. Latif, U. Smola, R. A. Bernal, F.-M. Duh, H. Li, I. Kuzmin, V. Wixler, L. Geil, S. Shresta, P. A. Lloyd, S. Bader, Y. Sekido, K. D. Tartof, V. I. Kashuba, E. R. Zabarovsky, M. Dean, G. Klein, B. Zbar, M. I. Lerman, J. D. Minna, U. R. Rapp, and A. Allikmets, Mol. Cell. Biol. 16:868-876, 1996). In vitro characterization of the kinase revealed that 3pK is activated by ERK. It was further shown that 3pK is phosphorylated in vivo after stimulation of cells with serum. However, the in vivo relevance of this observation in terms of involvement of the Raf/MEK/ERK cascade has not been established. Here we show that 3pK is activated in vivo by the growth inducers serum and tetradecanoyl phorbol acetate in promyelocytic HL60 cells and transiently transfected embryonic kidney 293 cells. Activation of 3pK was Raf dependent and was mediated by the Raf/MEK/ERK kinase cascade. 3pK was also shown to be activated after stress stimulation of cells. In vitro studies with recombinant proteins demonstrate that in addition to ERK, members of other subgroups of the MAPK family, namely, p38RK and Jun-N-terminal kinases/stress-activated protein kinases, were also able to phosphorylate and activate 3pK. Cotransfection experiments as well as the use of a specific inhibitor of p38RK showed that these in vitro upstream activators also function in vivo, identifying 3pK as the first kinase to be activated through all three MAPK cascades. Thus, 3pK is a novel convergence point of different MAPK pathways and could function as an integrative element of signaling in both mitogen and stress responses.     

Mitochondrial gene divergence of Colombian Drosophila pseudoobscura

Isolated populations of drosophila pseudoobscura, separated from North American populations by about 2,400 km, were found in Colombia in 1960. We compared for sequences of the small ribosomal RNA (srRNA) gene on the mitochondria between North American and Colombian D. pseudoobscura in order to clarify the age of the Colombian isolates. The North American populations were not genetically different from each other but were genetically different from the Colombian populations. The Mexican strains represent the area from which the Colombian founders might have come. The estimated net nucleotide divergence between Mexican and Colombian D. pseudoobscura indicates that the Colombian population is not an ancient lineage. Phylogenies using both distance and parsimony methodologies reinforced this conclusion. The Colombian samples group together with both methods but, according to the bootstrap analysis, not significantly. It appears that the populations have not been separated long enough for their DNA sequences to show much divergence.      

Pyridoxal 5'-phosphate as a 31P reporter observing functional changes in the active site of Escherichia coli maltodextrin phosphorylase after site-directed mutagenesis.

Changes in the active site of Escherichia coli maltodextrin phosphorylase created by substituting residues Lys533, Arg534, Tyr538, and Glu637 were monitored in the absence and presence of arsenate as substrate analogue using pyridoxal-P as 31P NMR reporter. The chemical shift of the cofactor phosphate group of wild-type E. coli phosphorylase is pH dependent with an apparent pK of 5.6 and limiting delta values of 0.71 and 3.6 ppm for the low- and high-pH values, respectively. The apparent pK value of 5.6 indicates that the phosphate group of the cofactor is in hydrogen bond linkage to Lys533. In all mutant enzymes in which the enzymatic activity was significantly reduced, effects on the 31P chemical shift pattern of pyridoxal-P were observed. The K533S, R534Q, E637D, and E637Q mutant enzymes show 0.6, 0.01, 0.2, or 0.1% residual activity, and the apparent pK values of the cofactor phosphate transition of E637D and E637Q mutant enzymes are altered. The Y538F mutant enzyme is a remarkable exception, displaying 12% activity and an environment of the cofactor quite similar to that in wild-type enzyme. This finding suggests that Tyr538, although involved in substrate binding and specificity, is not functionally essential. One crucial aspect of catalysis is the close contact of the phosphates of pyridoxal-P and of substrate rendered by a cluster of positively charged amino acids, Lys533, Lys539, and Arg534. The similar apparent pK values of wild-type and K533S mutant phosphorylase suggest that the cofactor phosphate and the hydroxyl group of Ser533 are linked by a hydrogen bond.(ABSTRACT TRUNCATED AT 250 WORDS)     

Escherichia coli maltodextrin phosphorylase: contribution of active site residues glutamate-637 and tyrosine-538 to the phosphorolytic cleavage of alpha-glucans

The role of Escherichia coli maltodextrin phosphorylase (EC 2.4.1.1) active site residues Glu637 and Tyr538 which line the sugar-phosphate contact region of the enzyme was investigated by site-directed mutagenesis. Substitution of Glu637 by an Asp or Gln residue reduced kcat to approximately 0.2% of wild-type activity, while the Km values were affected to a minor extent. This indicated participation of Glu637 in transition-state binding rather than in ground-state binding. 31P NMR analysis of the ionization state of enzyme-bound pyridoxal phosphate suggested that Glu637 is also involved in modulation of the protonation state of the coenzyme phosphate observed during catalysis. Despite loss of proposed hydrogen-bonded substrate contacts, the Tyr538Phe mutant enzyme retained more than 10% activity; the apparent affinity of all substrates was slightly decreased. Mutations at either site affected the error rate of the enzyme (ratio of hydrolysis/phosphorolysis). Besides a role in substrate binding, the hydrogen-bond network of Tyr538 supports the exclusion of water from the active site.     

Multiple minute marker chromosomes derived from Y identified by FISH in an intersexual infant

Summary Chromosomal analysis in a child with ambiguous sex showed mosaicism of at least two cell lines with one or more marker chromosomes or none at all. They were shown to be derived from the Y chromosome by fluorescent in situ hybridisation (FISH) using different DNA probes that cover parts of the long and the short arm.     

Chirality evaluation in flavour and essential oil analysis

The simultaneous stereodifferentiation of all aromarelevant 4(5) alkylsubstituted γ(δ)-lactones is described, using enantioselective multidimensional gas chromatography (MDG), and the column combination OV 1701/octakis(3-O-butyryl-2,6-di-O-pentyl)-γ-cyclodextrin. The method is applicated to the lactone flavour compounds of fruits, indicating the advance to the analytical differentiation between “natural” and “nature-identical” aromas. Modified cyclodextrins are also proved to be powerful tools in the chirospecific CGC analysis of monoterpenoid constituents of essential oils. Optical purity control is discussed as an indicator for their natural origin.     

The potential to use QSAR to populate ecotoxicity characterisation factors for simplified LCIA and chemical prioritisation

Purpose

Today’s chemical society use and emit an enormous number of different, potentially ecotoxic, chemicals to the environment. The vast majority of substances do not have characterisation factors describing their ecotoxicity potential. A first stage, high throughput, screening tool is needed for prioritisation of which substances need further measures.

Methods

USEtox characterisation factors were calculated in this work based on data generated by quantitative structure-activity relationship (QSAR) models to expand substance coverage where characterisation factors were missing. Existing QSAR models for physico-chemical data and ecotoxicity were used, and to further fill data gaps, an algae QSAR model was developed. The existing USEtox characterisation factors were used as reference to evaluate the impact from the use of QSARs to generate input data to USEtox, with focus on ecotoxicity data. An inventory of chemicals that make up the Swedish societal stock of plastic additives, and their associated predicted emissions, was used as a case study to rank chemicals according to their ecotoxicity potential.

Results and discussion

For the 210 chemicals in the inventory, only 41 had characterisation factors in the USEtox database. With the use of QSAR generated substance data, an additional 89 characterisation factors could be calculated, substantially improving substance coverage in the ranking. The choice of QSAR model was shown to be important for the reliability of the results, but also with the best correlated model results, the discrepancies between characterisation factors based on estimated data and experimental data were very large.

Conclusions

The use of QSAR estimated data as basis for calculation of characterisation factors, and the further use of those factors for ranking based on ecotoxicity potential, was assessed as a feasible way to gather substance data for large datasets. However, further research and development of the guidance on how to make use of estimated data is needed to achieve improvement of the accuracy of the results.

     

Striking a new path in reducing cartilage breakdown: combination of antioxidative therapy and chondroanabolic stimulation after blunt cartilage trauma

Cartilage injury can trigger crucial pathomechanisms, including excessive cell death and expression of matrix‐destructive enzymes, which contribute to the progression of a post‐traumatic osteoarthritis (PTOA). With the intent to create a novel treatment strategy for alleviating trauma‐induced cartilage damage, we complemented a promising antioxidative approach based on cell and chondroprotective N‐acetyl cysteine (NAC) by chondroanabolic stimulation. Overall, three potential pro‐anabolic growth factors – IGF‐1, BMP7 and FGF18 – were tested comparatively with and without NAC in an ex vivo human cartilage trauma‐model. For that purpose, full‐thickness cartilage explants were subjected to a defined impact (0.59 J) and subsequently treated with the substances. Efficacy of the therapeutic approaches was evaluated by cell viability, as well as various catabolic and anabolic biomarkers, representing the present matrix turnover. Although monotherapy with NAC, FGF18 or BMP7 significantly prevented trauma‐induced cell dead and breakdown of type II collagen, combination of NAC and one of the growth factors did not yield significant benefit as compared to NAC alone. IGF‐1, which possessed only moderate cell protective and no chondroprotective qualities after cartilage trauma, even reduced NAC‐mediated cell and chondroprotection. Despite significant promotion of type II collagen expression by IGF‐1 and BMP7, addition of NAC completely suppressed this chondroanabolic effect. All in all, NAC and BMP7 emerged as best combination. As our findings indicate limited benefits of the simultaneous multidirectional therapy, a sequential application might circumvent adverse interferences, such as suppression of type II collagen biosynthesis, which was found to be reversed 7 days after NAC withdrawal.