Optimisation of supercritical fluid extraction of indole alkaloids from Catharanthus roseus using experimental design methodology--comparison with other extraction techniques

Response surface modelling, using MODDE 6 software for Design of Experiments and Optimisation, was applied to optimise supercritical fluid extraction (SFE) conditions for the extraction of indole alkaloids from the dried leaves of Catharanthus roseus. The effects of pressure (200-400 bar), temperature (40-80 degrees C), modifier concentration (2.2-6.6 vol%) and dynamic extraction time (20-60 min) on the yield of alkaloids were evaluated. The extracts were analysed by high-performance liquid chromatography and the analytes were identified using ion trap-electrospray ionisation-mass spectrometry. The method was linear for alkaloid concentration in the range 0.18-31 microg/mL. The limits of detection and quantification for catharanthine, vindoline, vinblastine and vincristine were 0.2, 0.15, 0.1 and 0.08 microg/mL and 2.7, 2.0, 1.3 and 1.1 microg/g, respectively. The dry weight content of major alkaloids in the plants were compared using different extraction methods, i.e. SFE, Soxhlet extraction, solid-liquid extraction with sonication and hot water extraction at various temperatures. The extraction techniques were also compared in terms of reproducibility, selectivity and analyte recoveries. Relative standard deviations for the major alkaloids varied from 4.1 to 17.5% in different extraction methods. The best recoveries (100%) for catharanthine were obtained by SFE at 250 bar and 80 degrees C using 6.6 vol% methanol as modifier for 40 min, for vindoline by Soxhlet extraction using dichloromethane in a reflux for 16 h, and for 3',4'-anhydrovinblastine by solid-liquid extraction using a solution of 0.5 m sulphuric acid and methanol (3:1 v/v) in an ultrasonic bath for 3 h.     

Phospholipid-protein coatings for chiral capillary electrochromatography

A phospholipid-bovine serum albumin (BSA) coating was developed for chiral capillary electrochromatographic separation of d- and l-tryptophan. Temperature, liposome composition, and liposome-BSA mixing and extrusion were found to have critical effects on the chiral separation of d- and l-tryptophan in terms of resolution, separation efficiency, and migration times. A solution of 0.5mM phosphatidylcholine (PC)-1 mg/ml BSA performed better than a solution of 0.5mM PC/phosphatidylserine (PS) (80:20, mol%)-1 mg/ml BSA as capillary coating; baseline separation of the enantiomers with satisfactory resolution was then achieved. Temperature played a crucial role in the chiral separation, as demonstrated for phospholipid-coated capillaries immobilized with BSA and lysozyme. The d- and l-tryptophans showed a marked difference in separation efficiency on the PC-BSA-coated capillary; the theoretical plate number of l-tryptophan was above 500,000 m(-1), whereas that of d-tryptophan was only about 22,000 m(-1). Immobilized BSA (pI 4.7) showed better chiral separation selectivity for the enantiomers than did immobilized lysozyme (pI 10.5), alpha-chymotrypsin (pI 8.1-8.3), or avidin (pI 10.0-10.5); also resolution was better and analysis time was faster. Hydrophobic interactions played an important role in the BSA-immobilized phospholipid-coated capillaries. The importance of protein net charge and molar mass for its immobilization in phospholipid-coated capillaries is discussed.     

Screening for connexin 32 mutations in Charcot-Marie-Tooth disease families with possible X-linked inheritance

The X-linked dominant form of Charcot-Marie-Tooth disease (CMTX) is associated with mutations in a gene coding for the gap-junction protein connexin 32 (Cx32). We screened 32 CMT families with a pedigree pattern suggestive of X-linked inheritance for the presence of mutations in the coding region of Cx32 by direct sequencing. Five of the families had a CMT1 diagnosis, 24 had a CMT2 diagnosis and 3 patients had an unspecified CMT. Eight families with a Cx32 point mutation were detected. Five different mutations (four of them published previously) were found in six CMT2 families and one mutation was found in a sporadic CMT1 male patient. One of the mutations, Met194Val, is among the first described in the fourth transmembrane domain of Cx32. Two CMT2 families and the sporadic CMT1 patient had the same mutation, Arg22Gln. An additional, previously unpublished mutation, Arg75Trp, was found in a male patient with unspecified CMT, who subsequently was verified to have a variant Klinefelter syndrome with 48,XXYY karyotype. Our findings show the difficulty in distinguishing CMTX patients from CMT1 and CMT2 patients, and they emphasize the need for Cx32 mutation screening in families previously diagnosed with CMT2. Received: 29 November 1996 / Revised: 17 February 1997     

Polyethylene glycol-stabilized lipid disks as model membranes in interaction studies based on electrokinetic capillary chromatography and quartz crystal microbalance

Distearoylphosphatidylcholine (DSPC)/cholesterol/distearoylphosphatidylethanolamine (DSPE)–polyethylene glycol 5000 [PEG(5000)] lipid disks, mimicking biological membranes, were used as pseudostationary phase in partial filling electrokinetic capillary chromatography (EKC) to study interactions between pharmaceuticals and lipid disks. Capillaries were coated either noncovalently with a poly(1-vinylpyrrolidone)-based copolymer or covalently with polyacrylamide to mask the negative charges of the fused-silica capillary wall and to minimize interactions between positively charged pharmaceuticals and capillary wall. Although the noncovalent copolymer coating method was faster, better stability of the covalent polyacrylamide coating at physiological pH 7.4 made it more reliable in partial filling EKC studies. Migration times of pharmaceuticals were proportional to the amount of lipids in the pseudostationary phase, and partition coefficients were successfully determined. Because the capillary coatings almost totally suppressed the electroosmotic flow, it was not practical to use the EKC-based method for partition studies involving large molecules with low mobilities. Hence, the applicability of the biomembrane mimicking lipid disks for interactions studies with large molecules was verified by the quartz crystal microbalance technique. Biotinylated lipid disks were then immobilized on streptavidin-coated sensor chip surface, and interactions with a high-molecular-mass molecule, lysozyme, were studied. Cryo-transmission electron microscopy and asymmetrical flow field-flow fractionation were used to clarify the sizes of lipid disks used.     

Whole-genome SNP association analysis of reproduction traits in the Finnish Landrace pig breed

Background

Good genetic progress for pig reproduction traits has been achieved using a quantitative genetics-based multi-trait BLUP evaluation system. At present, whole-genome single nucleotide polymorphisms (SNP) panels provide a new tool for pig selection. The purpose of this study was to identify SNP associated with reproduction traits in the Finnish Landrace pig breed using the Illumina PorcineSNP60 BeadChip.

Methods

Association of each SNP with different traits was tested with a weighted linear model, using SNP genotype as a covariate and animal as a random variable. Deregressed estimated breeding values of the progeny tested boars were used as the dependent variable and weights were based on their reliabilities. Statistical significance of the associations was based on Bonferroni-corrected P-values.

Results

Deregressed estimated breeding values were available for 328 genotyped boars. Of the 62 163 SNP in the chip, 57 868 SNP had a call rate > 0.9 and 7 632 SNP were monomorphic. Statistically significant results (P-value < 2.0E-06) were obtained for total number of piglets born in first and later parities and piglet mortality between birth and weaning in later parity, and suggestive associations (P-value < 4.0E-06) for piglet mortality between birth and weaning in first parity, number of stillborn piglets in later parity, first farrowing interval and second farrowing interval. Two of the statistically significant regions for total number of piglets born in first and later parities are located on chromosome 9 around 95 and 79 Mb. The estimated SNP effect in these regions was approximately one piglet between the two homozygote classes. By combining the two most significant SNP in these regions, favourable double homozygote animals are expected to have 1.3 piglets (P-value = 1.69E-08) more than unfavourable double homozygote animals. A region on chromosome 9 (66 Mb) was statistically significant for piglet mortality between birth and weaning in later parity (0.44 piglets between homozygotes, P-value = 6.94E-08).

Conclusions

Three separate regions on chromosome 9 gave significant results for litter size and pig mortality. The frequencies of favourable alleles of the significant SNP are moderate in the Finnish Landrace population and these SNP are thus valuable candidates for possible marker-assisted selection.     

Three complementary techniques for the clarification of temperature effect on low-density lipoprotein–chondroitin-6-sulfate interaction

A rigorous processing of adsorption data from quartz crystal microbalance technology was successfully combined with the data obtained by partial filling affinity capillary electrophoresis and molecular dynamics for the clarification of the temperature effect on the interaction of a major glycosaminoglycan chain chondroitin-6-sulfate (C6S) of proteoglycans with low-density lipoprotein (LDL) and with a peptide fragment of apolipoprotein B-100 (residues 3359–3377 of LDL, PPBS). Two experimental techniques and computational atomistic methods demonstrated a nonlinear pattern of the affinity of C6S at temperatures above 38.0 °C to both LDL and PPBS. The temperature affects the interaction of C6S with LDL and PPBS by influencing the structural behavior of glycosaminoglycan C6S and/or that of LDL.     

Swedish population substructure revealed by genome-wide single nucleotide polymorphism data

The use of genome-wide single nucleotide polymorphism (SNP) data has recently proven useful in the study of human population structure. We have studied the internal genetic structure of the Swedish population using more than 350,000 SNPs from 1525 Swedes from all over the country genotyped on the Illumina HumanHap550 array. We have also compared them to 3212 worldwide reference samples, including Finns, northern Germans, British and Russians, based on the more than 29,000 SNPs that overlap between the Illumina and Affymetrix 250K Sty arrays. The Swedes - especially southern Swedes - were genetically close to the Germans and British, while their genetic distance to Finns was substantially longer. The overall structure within Sweden appeared clinal, and the substructure in the southern and middle parts was subtle. In contrast, the northern part of Sweden, Norrland, exhibited pronounced genetic differences both within the area and relative to the rest of the country. These distinctive genetic features of Norrland probably result mainly from isolation by distance and genetic drift caused by low population density. The internal structure within Sweden (F_ST = 0.0005 between provinces) was stronger than that in many Central European populations, although smaller than what has been observed for instance in Finland; importantly, it is of the magnitude that may hamper association studies with a moderate number of markers if cases and controls are not properly matched geographically. Overall, our results underline the potential of genome-wide data in analyzing substructure in populations that might otherwise appear relatively homogeneous, such as the Swedes.     

Identification and characterization of a lipopolysaccharide α,2,3-sialyltransferase from the human pathogen Helicobacter bizzozeronii

Terminal sialic acid in the lipopolysaccharides (LPSs) of mucosal pathogens is an important virulence factor. Here we report the characterization of a Helicobacter sialyltransferase involved in the biosynthesis of sialylated LPS in Helicobacter bizzozeronii, the only non-pylori gastric Helicobacter species isolated from humans thus far. Starting from the genome sequences of canine and human strains, we identified potential sialyltransferases downstream of three genes involved in the biosynthesis of N-acetylneuraminic acid. One of these candidates showed monofunctional α,2,3-sialyltransferase activity with a preference for N-acetyllactosamine as a substrate. The LPSs from different strains were shown by SDS-PAGE and high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) to contain sialic acid after neuraminidase treatment. The expression of this sialyltransferase and sialyl-LPS appeared to be a phase-variable characteristic common to both human and canine H. bizzozeronii strains. The sialylation site of the LPSs of two H. bizzozeronii strains was determined to be NeuAc-Hex-HexNAc, suggesting terminal 3'-sialyl-LacNAc. Moreover, serological typing revealed the possible presence of sialyl-Lewis X in two additional strains, indicating that H. bizzozeronii could also mimic the surface glycans of mammalian cells. The expression of sialyl-glycans may influence the adaptation process of H. bizzozeronii during the host jump from dogs to humans.     

A longitudinal study of <Emphasis Type="Italic">Campylobacter</Emphasis> distribution in a turkey production chain

Background  

Campylobacter is the most common cause of bacterial enteritis worldwide. Handling and eating of contaminated poultry meat has considered as one of the risk factors for human campylobacteriosis. Campylobacter contamination can occur at all stages of a poultry production cycle. The objective of this study was to determine the occurrence of Campylobacter during a complete turkey production cycle which lasts for 1,5 years of time. For detection of Campylobacter, a conventional culture method was compared with a PCR method. Campylobacter isolates from different types of samples have been identified to the species level by a multiplex PCR assay.