213 research outputs found
A new route for the synthesis of Streptococcus pneumoniae 19F and 19A capsular polysaccharide fragments avoiding the beta-mannosamine glycosylation step
No full textSUMMARY The recently described (Carbohydr. Res. 2008, 43, 2545-2556) b-D-MaNAcp- (1→4)-b-D-Glcp thiophenyl glycosyl donor 3 was used in a-glycosylation reactions of OH-2 and OH-3 of the suitably protected p-MeO-benzyl a-L-rhamnopyranoside acceptors 7 and 8. The glycosylation of axial OH-2 of 7 took place in high yield (76%) and with good stereoselectivity (a/b = 3.4) leading to the protected trisaccharide a-11, corresponding to the repeating unit of Streptococcus pneumoniae 19F. The same reaction on equatorial OH-3 of acceptor 8 gave the trisaccharide a-15, constituent of the repeating unit of S. pneumoniae 19A, but in lower yield (41%) and without stereoselection (a/b = 1:1.3). Utilizing the introduced orthogonal protection of OH-1 and OH-4’’, the trisaccharide a-11 was transformed into a trisaccharide building block suitable for the synthesis of its phosphorylated oligomers
Synthesis of fucose derivatives with thiol motifs towards suicide inhibition of helicobacter pylori
No full textThe syntheses of six thiol-exhibiting monosaccharides towards suicide inhibition of Helicobacter pylori are reported. Blood group Antigen Binding Adhesin (BabA), a bacterial membrane-bound lectin, binds to human ABO and Lewis b blood group structures displayed on the surface of host epithelial cells. Crystal structures of the carbohydrate-recognition domain revealed a conserved disulfide bonded loop that anchors a critical fucose residue in these blood group structures. Disruption of this loop by N-acetylcysteine results in reduced BabA-mediated adherence to human gastric tissue sections and attenuated virulence in Lewis b-expressing transgenic mice. With a view of creating specific inhibitors of the lectin, we designed and successfully synthesised six fucose-derived compounds with thiol motifs to engage in a thiol-disulfide exchange with this disulfide bond of BabA and form a glycan-lectin disulfide linkage. Branching and extending the fucose backbone with 2- and 3-carbon thiol motifs delivered a range of candidates to be tested for biological activity against BabA
Bedömning på systemnivå - En komparativ studie av stegsystemet i språk i den svenska skolan och språknivåer i Europarådets Common European Framework of Reference for Languages (CEFR)
No full textIn the early 1970’s, the Council of Europe commenced work that aimed at identifying levels of language learning in a transparent and generally applicable way. The project resulted in the definition of six cardinal levels of competence according to a descriptive model called the Common European Framework of Reference for Languages. This made a strong and lasting impact and the CEFR concept is now being employed both in Europe and beyond as an important tool in the communication about goals and activities in language teaching and assessment. The highly influential work inspired the Swedish National Agency for Education (Skolverket) to transpose the curricular goals for languages into an approximate CEFR model encompassing 7 levels, or stages (steg), which would thereby, among other things, enable comparison of features of language education across cultural and national boundaries. This article describes a previously unpublished study that investigated the conceptual relationship between the 7 stages of language ability laid out in Swedish school curricula and the 6-level CEFR system (Oscarson 2002). Against the background of the increasingly strong interest in the latter system, the purpose of the article is to update and review the analytical procedure and the results of this study. A text analytics method for stepwise comparison of the systems was devised and used in the alignment. Areas of competence were matched for content, and descriptions of abilities in the Swedish curricula close in meaning to descriptors in the CEFR system were identified. Conclusions were drawn about the interrelationships that exist between the systems and points on the two scales. The results indicate where any given stage in a Swedish language curriculum is located on the CEFR scale. The general conditions for the work undertaken, as well as possible further research, are discussed. Keywords: Common European Framework, CEFR, GERS, language assessment, language proficiency scales Mats Oscarson Professor emeritus Department of Pedagogical, Curricular and Professional Studies University of Gothenburg [email protected]
Genetic polymorphism of human drug metabolising enzymes : structural and functional studies
No full textThere is a pronounced interindividual variability in the levels and activity of many drug metabolising enzymes, which might cause differences in the sensitivity to and the toxicity of many clinically used drugs as well as environmental compounds such as nicotine and precarcinogens. In the present investigation the molecular genetic basis for some of the differences in the CYP2A6, CYP2D6, CYP2E1 and GSTM1 enzymes have been elucidated.Cytochrome P450 2D6 (CYP2D6) exhibits a marked interindividual and interethnic variability, with many Asian and black African populations have a generally reduced CYP2D6 activity compared to Caucasians. In Chinese, who are known to having reduced capacity for metabolism of CYP2D6 substrates, we have identified and characterised the CYP2D6*10 allele. This allele was shown to carry two missense mutations, P34S and S486T, and heterologous expression in COS-1 cells revealed that the P34S substitution yields an unstable enzyme that IS rapidly degraded. Indeed CYP2D6*10 was found to be the most common CYP2D6 allele among Chinese. Among black African populations, it has been shown that the presence of the CYP2D6*17 allele correlates well to reduced in vivo activity. The three missense mutations found in this allele, T107I, R296C and S486T, were introduced in all eight possible combinations into a CYP2D6 cDNA and were expressed in yeast. This revealed that a combination of the T107I and R296C substitutions yielded an enzyme with a 5-fold higher apparent Km for the substrates, whereas no effect was seen when the amino acid substitutions were introduced separately. In a Saudi Arabian population, we found a low frequency of these reduced activity alleles and inactive alleles but an Egli frequency of the CYP2D6 gene duplication which is consistent with earlier phenotyping studies in this population.The expression of ethanol-inducible P450 2E1 (CYP2E1) is also known to exhibit striking interindividual variability. In order to elucidate possible genetic causes for this variation, we screened all exons for mutations using genomic DNA from almost 200 unrelated individuals. Two novel alleles were found, yielding enzymes with the amino add substitutions R76H and V3891, respectively. These alleles were however very rare, implicating a selective pressure for active CYP2E1 enzyme, most likely because of an important endogenous function. Sequencing and characterisation of the 5' flanking region of the CYP2E1 gene revealed however the presence of a polymorphic repeat region, with an allele frequency of 23 % in a Chinese population, but only 1 % in Swedes. This polymorphism might be of importance for interindividual differences in the regulation of the CYP2E1 gene.Cytochrome P450 2A6 (CYP2A6) is the major human nicotine C-oxidase. In fact, data has been presented where a correlation was seen between the number of defective CYP2A6*2 and CYP2A6*3 alleles in an individual and the risk of becoming a smoker as well as the number of cigarettes being smoked. The method used for CYP2A6 genotyping has, however, been found to give erroneous results with respect to the coumarin hydroxylase phenotype, a probe drug for the CYP2A6 enzyme. Improved genotyping methods were developed which could correctly predict the CYP2A6 phenotype. We could not, however, detect any CYP2A6*3 alleles in contrast to the 2-28 % previously reported and propose that a previously unknown common gene conversion event in the 3' flanking region of the CYP2A6 gene is the reason for this discrepancy. Furthermore, we found that the CYP2A6 gene deletion was due to an unequal cross-over event between the CYP2A6 gene and the related CYP2A7 gene. A PCR-based genotyping method was developed which showed a frequency of the deletion allele to be 15 % in a Chinese population, but only 1 % in Europeans.Finally, the molecular mechanism behind the ultrarapid glutathione S-transferase M1 (GSTM1) activity found in certain individuals was studied. Southern blotting and PCR analysis revealed a novel GSTM1 allele with a duplication of the active GSTM1 gene, which most likely occurred through an unequal cross-over event. A quantitative PCR method was developed which accurately quantified the number of GSTM1 genes.In summary, several novel cytochrome P450 and GSTM1 alleles have been identified and characterised. These are of putative importance for explanation of impaired drug metabolism and possibly for smoking behaviour and genetically determined sensitivity to carcinogens. These findings form a basis for future molecular epidemiological studies and for use in patients as a tool for individualised drug therapy resulting in less side effects and better efficacy.List of scientific papersI. Johansson I, Oscarson M, Yue QY, Bertilsson L, Sjoqvist F, Ingelman-Sundberg M (1994). Genetic analysis of the Chinese cytochrome P4502D locus: characterization of variant CYP2D6 genes present in subjects with diminished capacity for debrisoquine hydroxylation. Mol Pharmacol. 46(3): 452-459. https://pubmed.ncbi.nlm.nih.gov/95021124II. Oscarson M, Hidestrand M, Johansson I, Ingelman-Sundberg M (1997). A combination of mutations in the CYP2D6*17 (CYP2D6Z) allele causes alterations in enzyme function. Mol Pharmacol. 52(6): 1034-1040. https://pubmed.ncbi.nlm.nih.gov/98086431III. McLellan RA, Oscarson M, Seidegard J, Evans DA, Ingelman-Sundberg M (1997). Frequent occurrence of CYP2D6 gene duplication in Saudi Arabians. Pharmacogenetics. 7(3): 187-191. https://pubmed.ncbi.nlm.nih.gov/97385644IV. Hu Y, Oscarson M, Johansson I, Yue QY, Dahl ML, Tabone M, Arinco S, Albano E, Ingelman-Sundberg M (1997). Genetic polymorphism of human CYP2E1: characterization of two variant alleles. Mol Pharmacol. 51(3): 370-376. https://pubmed.ncbi.nlm.nih.gov/97211623V. Hu Y, Hakkola J, Oscarson M, Ingelman-Sundberg M (1999). Structural and functional characterization of the 5-flanking region of the rat and human cytochrome P450 2E1 genes: identification of a polymorphic repeat in the human gene. Biochem Biophys Res Commun. 263(2): 286-293. https://pubmed.ncbi.nlm.nih.gov/99423457VI. Oscarson M, Gullsten H, Rautio A, Bernal ML, Sinues B, Dahl ML, Stengard JH, Pelkonen O, Raunio H, Ingelman-Sundberg M (1998). Genotyping of human cytochrome P450 2A6 (CYP2A6), a nicotine C-oxidase. FEBS Lett. 438(3): 201-205. https://pubmed.ncbi.nlm.nih.gov/99043247VII. Oscarson M, McLellan RA, Gullsten H, Yue QY, Lang MA, Bernal ML, Sinues B, Hirvonen A, Raunio H, Pelkonen O, Ingelman-Sundberg M (1999). Characterisation and PCR-based detection of a CYP2A6 gene deletion found at a high frequency in a Chinese population. FEBS Lett. 448(1): 105-110. https://pubmed.ncbi.nlm.nih.gov/99231771VIII. Oscarson M, McLellan RA, Gullsten H, Agundez JA, Benitez J, Rautio A, Raunio H, Pelkonen O, Ingelman-Sundberg M (1999). Identification and characterisation of novel polymorphisms in the CYP2A locus: implications for nicotine metabolism. FEBS Lett. 460(2): 321-327. https://pubmed.ncbi.nlm.nih.gov/20012952</p
Molecular thermodynamic model for the solubility of noble gases in water
No full textThe thermodn. model based on the distributions of mol. populations among energy levels was employed for the anal. of the soly. of noble gases in water at different temps. The soly. is expressed in polynomial form. The apparent thermodn. quantities are obtained from the given expression. The whole system is considered as the convoluted ensemble (gc*c)e formed by a grand canonical ensemble, gce, and a canonical ensemble, ce, the latter corresponding to the solvent. The statistical distribution is described by a convoluted partition function, (GC*C)PF, which is the product of a grand canonical partition function, GCPF, and a canonical partition function, CPF. The apparent thermodn. functions can be decompd. into the contributions of the sep. partition functions. In particular, the apparent enthalpy {-ΔHapp}T = -ΔH° - nwCp,wT is the sum of the enthalpy change due to the reaction between gas and water, -ΔH°, and the heat absorbed by the water mols. involved in the reaction ΔHw = NwCp,wT. The enthalpy term ΔHw, which varies linearly with the temp., was calcd. by using the relation of thermal equiv. diln. valid for the canonical ensemble. By plotting the apparent enthalpy{-ΔHapp}T vs. T, the value nw can be obtained from the slope of the line. Sets of data from different sources were analyzed and yield congruent values of -ΔH° and nw. The values nw ranging from 1.5 for helium to 3.3 for xenon clearly depend on the size of the atoms of the noble gas and can be related to the formation of a cavity of water mols. in the solvent
Exploring Cryptococcus neoformans capsule structure and assembly with a hydroxylamine-armed fluorescent probe
No full textChemical biology is an emerging field that enables the study and manipulation of biological systems with probes whose reactivities provide structural insights. The opportunistic fungal pathogen Cryptococcus neoformans possesses a polysaccharide capsule that is a major virulence factor, but is challenging to study. We report here the synthesis of a hydroxylamine-armed fluorescent probe that reacts with reducing glycans and its application to study the architecture of the C. neoformans capsule under a variety of conditions. The probe signal localized intracellularly and at the cell wall-membrane interface, implying the presence of reducing-end glycans at this location where the capsule is attached to the cell body. In contrast, no fluorescence signal was detected in the capsule body. We observed vesicle-like structures containing the reducing-end probe, both intra- and extracellularly, consistent with the importance of vesicles in capsular assembly. Disrupting the capsule with DMSO, ultrasound, or mechanical shear stress resulted in capsule alterations that affected the binding of the probe, as reducing ends were exposed and cell membrane integrity was compromised. Unlike the polysaccharides in the assembled capsule, isolated exopolysaccharides contained reducing ends. The reactivity of the hydroxylamine-armed fluorescent probe suggests a model for capsule assembly whereby reducing ends localize to the cell wall surface, supporting previous findings suggesting that this is an initiation point for capsular assembly. We propose that chemical biology is a promising approach for studying the C. neoformans capsule and its associated polysaccharides to unravel their roles in fungal virulence
Isothermal titration calorimetric study defines the substrate binding residues of calreticulin
No full textEarlier we established using modeling studies the residues in calreticulin (CRT) important for sugar-binding (M. Kapoor, H. Srinivas, K. Eaazhisai, E. Gemma, L. Ellgaard, S. Oscarson, A. Helenius, A. Surolia, Interactions of substrate with calreticulin, an endoplasmic reticulum chaperone, J. Biol. Chem. 278 (8) (2003) 6194-6200). Here, we discuss the relative roles of Trp-319, Asp-317, and Asp-160 for sugar-binding by using site-directed mutagenesis and isothermal titration calorimetry (ITC). Residues corresponding to Asp-160 and Asp-317 in CNX play important role towards sugar-binding. From the present study we demonstrate that the residue Asp-160 is not involved in sugar-binding, while Asp-317 plays a crucial role. Further, it is also validated that cation-π interactions of the sugar with Trp-319 dictate sugar-binding in CRT. This study not only defines further the binding site of CRT but also highlights its subtle differences with that of calnexin
Isothermal titration calorimetric study defines the substrate binding residues of calreticulin
No full textEarlier we established using modeling studies the residues in calreticulin (CRT) important for sugar-binding (M. Kapoor, H. Srinivas, K. Eaazhisai, E. Gemma, L. Ellgaard, S. Oscarson, A. Helenius, A. Surolia, Interactions of substrate with calreticulin, an endoplasmic reticulum chaperone, J. Biol. Chem. 278 (8) (2003) 6194–6200). Here, we discuss the relative roles of Trp-319, Asp-317, and Asp-160 for sugar-binding by using site-directed mutagenesis and isothermal titration calorimetry (ITC). Residues corresponding to Asp-160 and Asp-317 in CNX play important role towards sugar-binding. From the present study we demonstrate that the residue Asp-160 is not involved in sugar-binding, while Asp-317 plays a crucial role. Further, it is also validated that cation–\pi interactions of the sugar with Trp-319 dictate sugar-binding in CRT. This study not only defines further the binding site of CRT but also highlights its subtle differences with that of calnexin
Synthetic Glycans Reveal Determinants of Antibody Functional Efficacy against a Fungal Pathogen
No full textAntibodies play a vital role in the immune response to infectious diseases and can be administered passively to protect patients. In the case of Cryptococcus neoformans, a WHO critical priority fungal pathogen, infection results in antibodies targeting capsular glucuron-oxylo-mannan (GXM). These antibodies yield protective, non-protective, and disease-enhancing outcomes when administered passively. However, it was unknown how these distinct antibodies recognized their antigens at the molecular level, leading to the hypothesis that they may target different GXM epitopes. To test this hypothesis, we constructed a microarray containing 26 glycans representative of those found in highly virulent cryptococcal strains and utilized it to study 16 well-characterized monoclonal antibodies. Notably, we found that protective and non-protective antibodies shared conserved reactivity to the M2 motif of GXM, irrespective of the strain used in infection or GXM-isolated to produce a conjugate vaccine. Here, only two antibodies, 12A1 and 18B7, exhibited diverse trivalent GXM motif reactivity. IgG antibodies associated with protective responses showed cross-reactivity to at least two GXM motifs. This molecular understanding of antibody binding epitopes was used to map the antigenic diversity of two Cryptococcus neoformans strains, which revealed the exceptional complexity of fungal capsular polysaccharides. A multi-GXM motif vaccine holds the potential to effectively address this antigenic diversity. Collectively, these findings underscore the context-dependent nature of antibody function and challenge the classification of anti-GXM epitopes as either "protective" or "non-protective"
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