Development of a bioreactor based on trypsin immobilized on monolithic support for the on-line digestion and identification of proteins.

The preparation and characterization of a new trypsin-based bioreactor is here described for on-line protein digestion and peptide analysis. Trypsin was immobilized on an epoxy-modified silica monolithic support with a single reaction step and the amount of immobilized enzyme was found to be 66.07 mg (+/-11.75 S.D.)/column (n = 6). The bioreactor was coupled through a switching valve to an analytical column for the on-line digestion, peptide separation and identification of test proteins by ESI-MS-MS. The influence of various parameters (flow rate, temperature, buffer pH and molarity, etc.) on enzymatic activity was investigated by an experimental design and the mostly significant factor was found to be the flow rate. The efficacy of the reported on-line bioreactor for tryptic mapping is reported for somatostatin and myoglobin, selected as model compounds. Tryptic peptide maps obtained by on-line digestion of myoglobin were compared to those obtained by traditional off-line digestion. Sequence coverage obtained with the on-line protocol (21 peptides, 75.16% coverage of myoglobin sequence) was found to be comparable to the one obtained with the off-line protocol (18 peptides, 76.47% coverage). Sensitivity for myoglobin digestion and identification was 0.1 mg/ml. The reproducibily of the peptide maps in terms of retention time was from 1.53 to 4.31%, R.S.D.

[Chorionic villi sampling. Results with the first 500 samples]. / Prélèvements de villosités choriales. Expérience des 500 premiers prélèvements.

531 chorionic villi forceps biopsies were carried out, to look for genetic diseases. Sampling was impossible in 24 cases (4.5%). The following indications were chosen: maternal age over 38 years: 361 cases; parental chromosome abnormality: 28 cases; previous history of chromosome abnormality: 21 cases; X-linked disease: 77 cases; metabolic disease: 32 cases; hemoglobinopathy: 7 cases; blood group determination: 5 cases. An elective abortion was carried out in 44 cases (8.7%), whereas 465 pregnancies were not interrupted. Full term has been reached in 327 pregnancies. The rate of miscarriage in this group is 3.9% (13 miscarriages out of 327 pregnancies followed). The results obtained at the Saint-Vincent-de-Paul Hospital show that the cytogenetic technique is reliable, and that the low risk of sampling allows its application in the diagnosis of chromosome anomalies.

Further observations on the in vitro metabolism of chenodeoxycholic acid and ursodeoxycholic acid.

Human intestinal flora from normal subjects was incubated with chenodeoxycholic acid (CDCA), ursodeoxycholic acid (UDCA) and 7-ketolithocholic acid (7-keto-LCA) for various periods of time and the metabolites thus formed were quantitatively determined by gas chromatography (GC) and identified by gas chromatography-mass spectrometry (GC-MS). Results showed that: a) the 7 alpha-dehydroxylation of CDCA produced more lithocholic acid more rapidly than the 7 beta-dehydroxylation of UDCA; b) the oxidation of CDCA produced more 7-keto-LCA more rapidly than the oxidation of UCDA; and c) the transformation of 7-keto-LCA produced CDCA and UDCA peaks after 8 h of incubation and a lithocholic peak at the end of 24 h of incubation after rising from almost 0% at 4 h. The presence of 3 alpha-hydroxy-delta 6-5 beta-cholenic acid, a hypothetical unsaturated intermediate in the 7-dehydroxylation of CDCA and UDCA, could not be detected by GC-MS.

New fatty acid esters of drimane sesquiterpenes from Lactarius uvidus.

This study of lipids of Lactarius uvidus has revealed the presence of new fatty acid esters of uvidin A [2a-d] and drimenol [6a-d]. Treatment of uvidin A [1] with base induced a Favorskii-like rearrangement leading to compounds with a new sesquiterpenoid skeleton. Uvidin A [1] showed insect antifeedant and cytotoxic activities.

Mass spectrometry of avermectins: structural determination of two new derivatives of Ivermectin B(1a).

Fragmentation pathways of Avermectins were studied by electron impact (EI), chemical ionisation (CI), electrospray ionisation (ESI) and by collision experiments. Structure characterisation was obtained using ESI combined with multi-stage (MS(n)) tandem mass spectrometry, analysis of homologues, and effects on fragment masses of H/D exchange. By these approaches the structures of two new derivatives of Avermectins were characterised.

The structures of new lanostane triterpenes from the fruiting bodies of Hebeloma senescens.

Three new lanostane triterpenes, hebelomic acids B[4], E[5], and F[6], were isolated from the inedible mushroom Hebeloma senescens. The latter two compounds are acyl derivatives of the new triterpene senescensol (12-deoxycrustulinol) [12]. The structures of compounds 4-6, including the absolute configuration of the 3-hydroxy-3-methylglutaric acid moiety, were established on the basis of spectral and chemical evidence.

Constituents of essential oil of Aframomum giganteum.

The composition of the essential oil of Aframomum giganteum was investigated by GC-MS. Forty seven terpenes were identified, the oil contained a high percentage of linalool and linalyl acetate which are mainly responsible for the smell of the A. giganteum stems. Fatty acids, paraffins, n-octanol, n-nonanol and guaiacol were also identified.

Prostaglandin E1 (PGE1) gastric tissue levels in the rat after truncal vagotomy and/or pre-hepatic portal hypertension.

Three groups of female Wistar rats were considered to study the pathophysiologic changes induced by truncal vagotomy and/or prehepatic portal hypertension. The first group was subjected to transabdominal bilateral truncal vagotomy (T.V.) and Heinecke-Mikulicz pyloroplasty, the second group to main portal vein partial stenosis (P.P.S.), and the third one simultaneously to T.V. plus P.P.S. The rats were sacrificed and the stomach homogenized. The separation and simultaneous measurement of gastric prostaglandin E1 (PGE1) resulted from high performance liquid chromatography (H.P.L.C.). Mass spectrometry (M.S.) was used for identification of several gastric PGE1 samples in purified extracts. Control rats were also sacrificed and examined. With regard to PGE1 gastric contents, the following results are reported: 1.2405 +/- 0.1356 micrograms/g (control rats), 0.786 +/- 0.1296 micrograms/g (T.V. rats), 1.8495 +/- 0.3433 micrograms/g (P.P.S. rats), and 1.2754 +/- 0.1154 micrograms/g (T.V. + P.P.S. rats). The interpretation of results is made at the moment on a hypothetical basis.

Characterization of Seven New Hydrocarbon Compounds Present in the Aroma of Virgin Olive Oils.

Seven isomeric hydrocarbons were detected in the volatile fraction of three samples of virgin olive oil. Chemical ionization mass spectrometry was used to assign the molecular formulas. Their structures were confirmed by comparison of retention time and mass spectral data with those of a synthetic sample obtained by pentene radical coupling. A final characterization of each chromatographic peak was done by means of a chiral capillary column to distinguish the optically active compounds from the isomers without chiral centers. For the quantitation the recovery factor in the oily matrix during the extraction of the volatile fraction was obtained using a related chemical, the commercially available beta-citronellene. On the basis of the previous literature and the present experiments a tentative rationalization of the involved biochemical pathway is proposed.