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J Sjövall

Publications and source records attributed to J Sjövall.

At least 19 recordsLinked to original sources
PubMed2005

Vernix caseosa as a multi-component defence system based on polypeptides, lipids and their interactions.

Vernix caseosa is a white cream-like substance that covers the skin of the foetus and the newborn baby. Recently, we discovered antimicrobial peptides/proteins such as LL-37 in vernix, suggesting host defence functions of vernix. In a proteomic approach, we have continued to characterize proteins in vernix and have identified 20 proteins, plus additional variant forms. The novel proteins identified, considered to be involved in host defence, are cystatin A, UGRP-1, and calgranulin A, B and C. These proteins add protective functions to vernix such as antifungal activity, opsonizing capacity, protease inhibition and parasite inactivation. The composition of the lipids in vernix has also been characterized and among these compounds the free fatty acids were found to exhibit antimicrobial activity. Interestingly, the vernix lipids enhance the antimicrobial activity of LL-37 in vitro, indicating interactions between lipids and antimicrobial peptides in vernix. In conclusion, vernix is a balanced cream of compounds involved in host defence, protecting the foetus and newborn against infection.

Amino Acid Sequence↗
PubMed2001

Efficacy and uptake of ropivacaine and bupivacaine after single intra-articular injection in the knee joint.

The efficacy of ropivacaine 100 mg (5 mg ml(-1)), 150 mg (7.5 mg ml(-1)) and 200 mg (10 mg ml(-1)) and bupivacaine 100 mg (5 mg ml(-1)) given by intra-articular injection into the knee after the end of surgery was studied in 72 ASA I-II patients scheduled for elective knee arthroscopy under general anaesthesia in a randomized, double-blind study. Kapake (paracetamol 1 g and codeine 60 mg) was given as a supplementary analgesic. Pain scores were assessed 1-4 h after surgery and a verbal rating scale of overall pain severity was assessed on second postoperative day. Ropivacaine or bupivacaine concentrations were determined in peripheral venous plasma up to 3 h after injection in eight patients in each group. Verbal rating pain scores were lower with ropivacaine 150 mg compared with bupivacaine 100 mg (P<0.05). There was a tendency for lower analgesic consumption and pain scores with all doses of ropivacaine (not significant). The mean (SD) maximum total plasma concentrations of ropivacaine were 0.64 (0.25), 0.78 (0.43), and 1.29 (0.46) mg litre(-1) after 100, 150 and 200 mg. The corresponding unbound concentrations were 0.018 (0.009), 0.024 (0.020) and 0.047 (0.022) mg litre(-1). Both were proportional to the dose. The maximum total concentration after bupivacaine 100 mg was 0.57 (0.36) mg litre(-1). The time to reach maximum plasma concentration was similar for all doses and varied between 20 and 180 min. All concentrations were well below the threshold for systemic toxicity.

Adult↗
PubMed2001

Identification of unusual 7-oxygenated bile acid sulfates in a patient with Niemann-Pick disease, type C.

Niemann-Pick disease, type C, was diagnosed in a 3-month-old boy with hepatosplenomegaly, mild signs of cholestasis, hepatic inflammation and extramedullary erythropoiesis, together with chronic airway disease. He developed muscular hypotonia, psychomotor retardation, rickets, and signs of peripheral neuropathy. The patient was found to excrete abnormal amounts of unusual bile acids in urine at 3 and 5 months of age. These acids were shown to have a 3beta-hydroxy-Delta(5) structure and to carry an oxo or hydroxy group at C-7. They were sulfated at C-3 and nonamidated or conjugated with glycine or taurine at C-24. Part of the 7-hydroxy acids, presumably the 7beta-hydroxylated one, was also conjugated with N-acetylhexosamine, probably N-acetylglucosamine, at the 7-hydroxy group. Possible metabolic pathways for the formation of the 7-oxo and 7beta-hydroxycholenoic acids are discussed. Based on previous data concerning the effects of 3beta-hydroxy-Delta(5) bile acids on bile acid transport, it is suggested that the formation of such bile acids is responsible for the cholestasis in this patient.

Bile Acids and Salts↗
PubMed2000

Ring opening of benzo[a]pyrene in the germ-free rat is a novel pathway for formation of potentially genotoxic metabolites.

The metabolism of benzo[a]pyrene (BP) is known to lead to a large number of oxygenated compounds, some of which can bind covalently to DNA. We have studied the integrated metabolism of BP in vivo in germ-free rats given (14)C-labeled BP. Urinary metabolites were separated into groups according to acidity using lipophilic ion exchangers. The groups were analyzed by mass spectrometry and were further fractionated by high-performance liquid chromatography. The fraction of urinary metabolites previously shown to contain N-acetylcysteine and glucuronic acid conjugates was found to contain derivatives of 7-oxo-benz[d]anthracene-3,4-dicarboxylic acid as major components. These compounds, which were identified by mass spectrometry and NMR, accounted for about 30% of the total metabolites in urine, demonstrating that, surprisingly, ring opening is a major pathway for metabolism of BP in the germ-free rat. The dicarboxylic acid may be excreted in urine as an ester glucuronide. By using the single cell gel electrophoresis or COMET assay, we were able to demonstrate that the anhydride of 7-oxo-benz[d]anthracene-3, 4-dicarboxylic acid was an efficient inducer of DNA damage. Taken together, these results indicate that the novel ring opening metabolic pathway may provide alternative mechanisms for the toxicity of BP.

Animals↗
PubMed2000

Docosahexaenoic acid, a ligand for the retinoid X receptor in mouse brain.

The retinoid X receptor (RXR) is a nuclear receptor that functions as a ligand-activated transcription factor. Little is known about the ligands that activate RXR in vivo. Here, we identified a factor in brain tissue from adult mice that activates RXR in cell-based assays. Purification and analysis of the factor by mass spectrometry revealed that it is docosahexaenoic acid (DHA), a long-chain polyunsaturated fatty acid that is highly enriched in the adult mammalian brain. Previous work has shown that DHA is essential for brain maturation, and deficiency of DHA in both rodents and humans leads to impaired spatial learning and other abnormalities. These data suggest that DHA may influence neural function through activation of an RXR signaling pathway.

Animals↗
PubMed2000

Validation of an analytical procedure to measure trace amounts of neurosteroids in brain tissue by gas chromatography-mass spectrometry.

A selective and extremely sensitive procedure has been developed and optimized, using high-performance liquid chromatography (HPLC), specific derivatization and gas chromatography-mass spectrometry (GC-MS), to simultaneously quantify very small amounts of different neurosteroids from rat brain. Unconjugated and sulfated steroids in brain extracts were separated by solid-phase extraction. The unconjugated fraction was further purified by HPLC, the steroids being collected in a single fraction, and the sulfated fraction was solvolyzed. All steroids were derivatized with heptafluorobutyric acid anhydride and analyzed by GC-MS (electron impact ionization) using selected-ion monitoring. High sensitivity and accuracy were obtained for all steroids. The detection limits were 1 pg for pregnenolone (PREG), dehydroepiandrosterone (DHEA) and their sulfate esters PREG-S and DHEA-S, 2 pg for progesterone (PROG) and 5 pg for 3alpha,5alpha-tetrahydroprogesterone (3alpha,5alpha-THP). In a pilot study on a rat brain, the concentrations of PREG-S and DHEA-S were 8.26+/-0.80 and 2.47+/-0.27 ng/g, respectively. Those of PREG, DHEA and PROG were 4.17+/-0.22, 0.45+/-0.02 and 1.95+/-0.10 ng/g, respectively. Good linearity and accuracy were observed for each steroid. The methodology validated here, allows femtomoles of neurosteroids, including the sulfates, found in small brain samples (at least equal to 10 mg) to be quantified simultaneously.

Animals↗
PubMed2000

Analysis of oxosteroids by nano-electrospray mass spectrometry of their oximes.

A method for the analysis of neutral oxosteroids by electrospray mass spectrometry is described. The oxosteroids are converted into their oximes by treatment with hydroxyammonium chloride in aqueous methanol. Intense peaks corresponding to protonated oxime molecules are observed in nano-electrospray mass spectra. The detection limits for the oximes of progesterone, pregnenolone and dehydroepiandrosterone were 2.5, 5 and 25 pg/microL, respectively, approximately 20 times lower than for the underivatised steroids. The signal intensities were proportional to the concentration of the steroids in the range of 500 to 2.5 pg/microL. Fragmentation by collision-induced dissociation (CID) was studied using oximes of 28 model steroids carrying an oxo group at C-3, C-17 or C-20. Some of the steroid oximes were labelled with deuterium or (15)N. Fragment ions were observed which yielded useful structural information. Upon CID, protonated oximes of 3-oxo-Delta(4)-steroids produced abundant ions by cleavage through the B-ring and by loss of the side chain, while protonated oximes of saturated 3-oxosteroids did not give abundant ions by cleavage through the B-ring. Protonated oximes of 20-oxosteroids unsubstituted at C-21, C-17 or C-16 produced a characteristic ion at m/z 86 containing the side chain, C-16 and C-17. Protonated oximes of steroids containing only a 17-oxo group gave fewer ions of diagnostic value. Coupled with the selective isolation of steroid oximes from a biological matrix this method of derivatisation and CID may be used for the analysis of neutral oxosteroids in biological samples.

Dehydroepiandrosterone↗
PubMed2000

De novo sequencing of proteolytic peptides by a combination of C-terminal derivatization and nano-electrospray/collision-induced dissociation mass spectrometry.

A series of synthetic peptides (3-15 residues), C-terminally derivatized with 4-aminonaphthalenesulfonic acid (ansa), have been analyzed on a hybrid magnetic sector-orthogonal acceleration time-of-flight tandem mass spectrometer, fitted with a nano-electrospray (nano-ES) interface. Deprotonated molecules generated by negative-ion ES were subjected to collision-induced dissociation (CID) using either methane or xenon as the collision gas, at a collision energy of 400 eV (laboratory frame of reference). As a consequence of charge localization on the sulfonate group, only C-terminal fragment ions were formed, presumably by charge-remote fragmentation mechanisms. Interpretable CID spectra were obtained from fmol amounts of the small peptides (up to 6 residues), whereas low pmol amounts were required for the larger peptides. CID spectra were also recorded of derivatized, previously noncharacterised peptides obtained by proteolysis of cytosolic hamster liver aldehyde dehydrogenase. Interpretation of these CID spectra was based on rules established for the fragmentation of the synthetic peptides. This study shows that derivatization with ansa may be useful in the de novo sequencing of peptides.

Aldehyde Dehydrogenase↗
PubMed2000

Bile acids and progesterone metabolites in intrahepatic cholestasis of pregnancy.

The pathogenesis of intrahepatic cholestasis of pregnancy (ICP) can be related to abnormalities in the metabolism and disposition of sex hormones and/or bile acids, determined by a genetic predisposition interacting with environmental factors. The total amount of oestrogens and progesterone circulating in the blood or excreted in the urine of ICP patients is similar to normal pregnancies. Thus, the search for the cause has been focused on abnormal hormone metabolites. The cholestatic potential of some D-ring oestrogen metabolites is supported by experimental and clinical data. Similar observations with regard to bile acids and progesterone metabolites are still scarce. This article reviews current knowledge in this field, including our own data. Bile acid synthesis appears to be reduced in patients with ICP, in whom primary conjugated bile acids are retained in blood. The major bile acid in blood and urine of these patients is cholic acid instead of chenodeoxycholic acid present in normal pregnancies. Hydroxylation and sulfation of bile acids are enhanced, while glucuronidation appears to be of lesser importance. The synthesis of progesterone appears unimpaired, while the profiles of progesterone metabolites in plasma and urine are different from normal pregnancies, with a larger proportion of mono- and disulfated metabolites, mainly 3alpha,5alpha isomers. Glucuronidated metabolites, however, are unchanged. With the administration of ursodeoxycholic acid (UDCA) to patients with ICP, pruritus and serum liver values are improved, the concentration of bile acids in blood is diminished and the proportion of their conjugated metabolites returned to normal. Simultaneously, the concentration of sulfated progesterone metabolites in blood and their urinary excretion are reduced. The serum levels of bile acids and progesterone metabolites before UDCA administration and their decrease during treatment do not correlate with each other. We propose that patients with ICP have a selective defect in the secretion of sulfated progesterone metabolites into bile and speculate that this may be caused by genetic polymorphism of canalicular transporter(s) for steroid sulfates or their regulation. Interaction with oestrogen metabolites and/or some exogenous compounds may further enhance the process triggering ICP in genetically predisposed individuals.

Bile Acids and Salts↗
PubMed1999

Nano-electrospray tandem mass spectrometry for the analysis of neurosteroid sulphates.

Neurosteroids are synthesised in the central and peripheral nervous system or are derived from peripheral sources, and act in the nervous system. In the present study we have evaluated the potential for using nano-electrospray (nano-ES) tandem mass spectrometry (MS/MS) for the structural analysis and detection of neurosteroids, in particular, steroid sulphates found in brain. Complete structural information can be obtained from 1 ng (3 pmol) of steroid sulphate, while fragment ions characteristic of the sulphate ester group can be obtained from only 3 pg (10 fmol) of sample. These values correspond to the expected quantities of steroid sulphates (e.g. pregnenolone sulphate) in about 100 mg and 300 microg of brain, respectively. Deuterated neurosteroid sulphates added to homogenised rat brain have been successfully analysed by nano-ES-MS/MS at a level of 50 pg/mg of brain.

Animals↗
PubMed1999

Characterization of conjugated metabolites of benzo[a]pyrene in germ-free rat urine by liquid chromatography/electrospray tandem mass spectrometry.

The characterization of conjugated metabolites of benzo[a]pyrene (BP) in the urine of male germ-free rats given a single intraperitoneal dose of [(14)C]BP is described. Urinary metabolites, constituting 9% of the administered radioactivity, were extracted on a Sep-Pak C(18) cartridge and separated by lipophilic ion-exchange chromatography into neutral and acidic fractions (fractions I-V). Metabolites in the latter fractions, constituting more than 80% of the urinary radioactivity, were characterized by reversed-phase HPLC and capillary column liquid chromatography/electrospray mass spectrometry (LC/ESMS) and tandem mass spectrometry (MS/MS). Relative quantities of BP metabolites were estimated from the distribution of radioactivity. Some coeluting compounds were semiquantified from the ion current chromatograms obtained in the capillary column LC/ESMS analyses. The major conjugated metabolites in fraction II, containing about 50% of the urinary radioactivity, consisted of three tetrahydrotrihydroxy-BP-S-N-acetylcysteines, the major isomer being 7,8,9,10-tetrahydro-8,9, 10-trihydroxy-BP-7-S-N-acetylcysteine, two dihydrotrihydroxy-BP-S-N-acetylcysteines, and a tetrahydrotetrahydroxy-BP-S-N-acetylcysteine. Fraction II also contained three apparently unconjugated compounds whose structures will be described elsewhere. Metabolites characterized in fractions III and IV, containing about 30% of the urinary radioactivity, included three BP-O,O'-disulfates, two monohydroxy-BP-O-sulfates, three dihydrodihydroxy-BP-O-sulfates, three BP-O,O'-diglucuronides, and a BP-O-sulfate-O'-glucuronide. Trace levels of a tetrahydrotrihydroxy-BP-S-glutathione conjugate were detected in fraction V.

Animals↗
PubMed1999

Paracrine glucocorticoid activity produced by mouse thymic epithelial cells.

Previous data have suggested that glucocorticoids (GCs) are involved in the differentiation of thymocytes into mature T cells. In this report we demonstrate that the mouse thymic epithelial cells (TEC) express the cytochrome P450 hydroxylases Cyp11A1, Cyp21, and Cyp11B1. These enzymes, in combination with 3beta-hydroxysteroid dehydrogenase (3betaHSD), convert cholesterol into corticosterone, the major GC in rodents. In addition, when TEC were cocultured with 'reporter cells' containing the glucocorticoid receptor (GR) and a GR-dependent reporter gene, a specific induction of reporter gene activity was observed. Induction of reporter gene activity was blocked when the TEC and reporter cells were incubated in the presence of the Cyp11B1 inhibitor metyrapone or the 3betaHSD inhibitor trilostane, as well as by the GR antagonist RU486. Coculturing of TEC with thymocytes induced apoptosis in the latter, which was partially blocked by the enzyme inhibitors and RU486. We conclude that TEC secrete a GC hormone activity and suggest a paracrine role for this in thymocyte development.

3-Hydroxysteroid Dehydrogenases↗
PubMed1998

Dolichol-like lipids with stimulatory effect on DNA synthesis: substrates for protein dolichylation?

Substantial evidence has suggested that a nonsterol product of mevalonic acid (MVA) is essential for the initiation of DNA synthesis in mammalian cells. Several possible isoprenoid candidates have been suggested, but the identity of this compound still remains unknown. In this study we have isolated and purified MVA products from SV40-transformed human fibroblasts and identified fractions with a growth-stimulatory effect. The cells were labelled with [14C]MVA in the presence of inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. After lipid extraction, the [14C]MVA-labelled lipids were subjected to high performance liquid chromatography and size-exclusion chromatography, and the effect of the fractionated eluate on the DNA synthesis of arrested MVA-depleted target cells was tested. Thereby we found a fraction of [14C]MVA-labelled lipids with a substantial stimulatory effect on DNA synthesis. The chromatographic behavior suggested that the growth-stimulating fractions contained dolichol-20. This was confirmed by mass spectrometric analysis. Similar results were obtained when lipids from hepatocellular carcinoma cells and a sample from breast tumor were isolated and analyzed by the same procedure. The mechanisms by which these compounds induce DNA synthesis are unknown. Recent data obtained in our laboratory have provided evidence that dolichyl groups are covalently linked to tumor cell proteins, which implicates a new biological function for long-chain polyisoprenoid alcohols (Hjertman et al. [1997] FEBS Lett 416:235-238). In this study we demonstrate that tumor cells containing dolichol-like growth-stimulatory lipids also contained dolichylated proteins. This raises the question whether the growth-stimulatory dolichol-like lipids serve as substrates for the dolichylation reaction.

Cell Division↗
PubMed1998

Synthetic peptide-containing surfactants--evaluation of transmembrane versus amphipathic helices and surfactant protein C poly-valyl to poly-leucyl substitution.

Pulmonary surfactant contains two hydrophobic proteins, SP-B and SP-C. With the aim of identifying synthetic SP-B and SP-C substitutes for replacement therapy of respiratory distress syndromes, we have studied two transmembrane peptides and two amphipathic peptides that are located in the plane of a phospholipid bilayer. One amphipathic peptide was designed by changing the amino acid sequence, but not the composition or size, of the 21-residue peptide KL4. This peptide, designated KL(2,3) from its spacing of nonpolar and polar residues, exhibited similar alpha-helical content as KL4 but was oriented along a phospholipid bilayer plane, in contrast to the transmembrane orientation of KL4 in the same environment. The second amphipathic peptide analyzed was succinyl-LLEKLLEWLK-amide (WMAP10). KL4 more efficiently accelerated the spreading of a mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (Pam2GroPCho)/phosphatidylglycerol (PtdGro)/palmitic acid (PamOH), 68:22:9 (by mass), at an air/water interface than did any of the amphipathic peptides. Similarly, KL4, but not KL(2,3), when present in an interfacial monolayer composed of Pam2GroPCho/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol, 7:3 (by mass), increased lipid insertion from subphase vesicles. An SP-C analogue, SP-C(Leu), with all helical valyl residues in native SP-C replaced with Leu and the palmitoylcysteines at positions 5 and 6 replaced with Ser, but otherwise with essentially the same primary structure as the native peptide, was analyzed. SP-C(Leu) exhibited similar alpha-helical content as native SP-C and a transmembrane orientation and, in contrast to poly-valyl-containing synthetic peptides, it folds into a helical conformation after acid-induced denaturation. SP-C(Leu) accelerated the spreading of Pam2GroPCho/PtdGro/PamOH, 68:22:9 (by mass), almost identically to native SP-C, and lowered the surface tension during rapid cyclic film compressions in a pulsating bubble surfactometer to near zero and 43 mN/m at minimum and maximum bubble size, respectively. Airway instillation of 2% (by mass) SP-C(Leu) combined with Pam2GroPCho/PtdGro/PamOH in preterm rabbit fetuses improved dynamic lung compliance by about 30% compared with untreated controls.

Animals↗
PubMed1998

Analysis of the major mercapturic acid pathway metabolites of benzo[a]pyrene found in rat urine by nano-electrospray mass spectrometry.

Nano-electrospray has been used in combination with high resolution and tandem mass spectrometry in the analysis of the major mercapturic acid pathway metabolites of benzo[a]pyrene (BP). Accurate mass measurements indicate that the [M-H]- ion of the major metabolite has a chemical formula C25H22NO6S, which corresponds to the deprotonated form of tetrahydro-trihydroxy-BP-S-N-acetylcysteine. Tandem mass spectrometry of this [M-H]- ion results in a collision induced dissociation spectrum identical to that of synthetic 7,8,9,10-tetrahydro-8,9,10-trihydroxy-BP-7-S-N-acetylcysteine.

Acetylcysteine↗
PubMed1998

Analysis of variant forms of porcine surfactant polypeptide-C by nano-electrospray mass spectrometry.

Electrospray (ES) mass spectrometry has been used to analyse preparations of porcine pulmonary surfactant polypeptide-C (SP-C). A number of variant forms of the native 35-residue dipalmitoylated peptide were detected including (a) C-terminally methylated SP-C, (b) C-terminally methylated and methionine oxidized SP-C, (c) N-terminally truncated, C-terminally methylated and methionine oxidized SP-c, (d) C-terminally elongated, C-terminally methylated and methionine oxidized SP-C, and (e) tripalmitoylated, C-terminally methylated and methionine oxidized SP-C. C-terminal methylation and methionine oxidation are probably a consequence of the sample handling procedure. The occurrence of the C-terminally elongated form of SP-C has implications for the in vivo processing of proSP-C and the Tandem mass spectrometry (MS/MS) was used to confirm the amino acid sequence of SP-C and the presence of palmitoyl groups covalently linked to the peptide. Some of the structures of the variant forms of SP-C were determined by MS/MS.

Amino Acid Sequence↗
PubMed1997

Evidence for protein dolichylation.

Labeling of human colon carcinoma cells with [3H]dol, followed by extensive delipidation and removal of dol-P oligosaccharides, showed that dol are bound to cellular proteins with sizes of 5, 10, 27, 75 and > 140 kDa. HPLC purification of proteolytic products of [3H]dol- and [35S]cys-labeled proteins revealed a hydrophobic peak containing both dol and cysteine. The dol/cys-labeled products were clearly separated from GG-cys, and exhibited a hydrophobicity between that of dol-P and dol. In another set of experiments delipidated proteins were treated with methyl iodide, which cleaves thioether bonds. After HPLC purification of released dol-like lipids, these were subjected to mass spectrometry. This demonstrated molecular ions with the same mass as that of dol. Taken together our data provide evidence for the existence of proteins covalently modified with dol.

Autoradiography↗