Effects of platelet-activating factor, tumor necrosis factor, and interleukin-1alpha on the expression of apolipoprotein M in HepG2 cells

Apolipoprotein M (apoM) is a recently discovered human apolipoprotein predominantly present in high-density lipoprotein (HDL) in plasma, exclusively expressed in liver and in kidney. The function of apoM is yet unknown. The human apoM gene is located in the major histocompatibility complex class III region on chromosome 6. Because many genes located in this region are related to the immune response, we have investigated whether apoM might also be involved in the host inflammatory response. In this study we examined effects of the platelet-activating factor (PAF), tumor necrosis factor (TNF-alpha), and interleukin-1alpha (IL-1alpha) on apoM expression in a hepatoblastoma cell line, HepG2 cells. PAF significantly enhanced the apoM mRNA levels and the secretion of apoM in HepG2 cell cultures. The enhancement of apoM secretion is seen at a low concentration of PAF (2 ng/ml), whereas a high concentration of PAF increases both the apoM mRNA levels and apoM secretion. Neither TNF-alpha nor IL-1alpha influenced apoM mRNA level and secretion. Furthermore, Lexipafant, a PAF-receptor (PAF-R) antagonist significantly suppressed the mRNA level and the secretion of apoM in HepG2 cells in a dose-dependent manner. Neither PAF nor Lexipafant influenced the mRNA levels and the secretion of apoA-I, apoB and apoE in HepG2 cells, indicating that the effects of PAF or Lexipafant on the apoM production on hepatic cells are selective for apoM. The cellular mechanism of the effects of PAF or Lexipafant on apoM metabolism requires further investigations.     

Substrate-based design of reversible Pin1 inhibitors

Human Pin1, a peptidyl-prolyl cis/trans isomerase with high specificity to -Ser/Thr(PO(3)H(2))-Pro- motifs, is required for cell cycle progression. In an effort to design reversible Pin1 inhibitors by using a substrate structure based approach, a panel of peptides were applied to systematically analyze the minimal structural requirements for Pin1 substrate recognition. Pin1 catalysis (k(cat)/K(m) < 5 mM(-1) s(-1)) for Ala-Pro, Ser-Pro, and Ser(PO(3)H(2))-Pro was detected using direct UV-visible spectrophotometric detection of prolyl isomerization, while weak competitive inhibition of Pin1 by these dipeptides was observed (K(i) > 1 mM). Substrates with chain lengths extending from either the P2 to P1' or the P1 to P2' subsite gave k(cat)/K(m) values of 100 mM(-1) s(-1) for Ala-Ser(PO(3)H(2))-Pro and 38 mM(-1) s(-1) for Ser(PO(3)H(2))-Pro-Arg. For both Pin1 and its yeast homologue Ess1, the optimal subsite recognition elements comprise five amino acid residues with the essential Ser(PO(3)H(2)) in the middle position. The resulting substrate Ac-Ala-Ala-Ser(PO(3)H(2))-Pro-Arg-NH-4-nitroanilide possesses a very low cis/trans interconversion barrier in the presence of either Pin1 or Ess1, with k(cat)/K(m) = 9300 mM(-1) s(-1) and 12000 mM(-1) s(-1), respectively. The D-Ser(PO(3)H(2)) residue preceding proline could serve as a substrate-deactivating determinant without compromising ground state affinity. Similarly, substitution of the amide bond preceding proline with a thioxo amide bond produces a potent inhibitor. Pin1 is reversibly inhibited by such substrate analogue inhibitors with IC(50) values in the low micromolar range. The D-amino acid containing inhibitor also exhibits remarkable stability against phosphatase activity in cell lysate.     

Location of N-unsubstituted glucosamine residues in heparan sulfate

Functional properties of heparan sulfate (HS) are generally ascribed to the sulfation pattern of the polysaccharide. However, recently reported functional implications of rare N-unsubstituted glucosamine (GlcNH(2)) residues in native HS prompted our structural characterization of sequences around such residues. HS preparations were cleaved with nitrous acid at either N-sulfated or N-unsubstituted glucosamine units followed by reduction with NaB(3)H(4). The labeled products were characterized following complementary deamination steps. The proportion of GlcNH(2) units varied from 0.7-4% of total glucosamine in different HS preparations. The GlcNH(2) units occurred largely clustered at the polysaccharide-protein linkage region in intestinal HS, also more peripherally in aortic HS. They were preferentially located within N-acetylated domains, or in transition sequences between N-acetylated and N-sulfated domains, only 20-30% of the adjacent upstream and downstream disaccharide units being N-sulfated. The nearest downstream (toward the polysaccharide-protein linkage) hexuronic acid was invariably GlcUA, whereas the upstream neighbor could be either GlcUA or IdoUA. The highly sulfated but N-unsubstituted disaccharide unit, -IdoUA2S-GlcNH(2)6S-, was detected in human renal and porcine intestinal HS, but not in HS from human aorta. These results are interpreted in terms of a biosynthetic mechanism, whereby GlcNH(2) residues are formed through regulated, incomplete action of an N-deacetylase/N-sulfotransferase enzyme.     

High-performance liquid chromatographic assay of metabolites of thioguanine and mercaptopurine in capillary blood

The main metabolites of the cytotoxic drugs thioguanine (6TG) and mercaptopurine (6MP) can be measured conveniently in red blood cells (RBC). Isolation of RBC, however, is laborious and requires some milliliters of blood. This HPLC assay allows measurements of thiopurine metabolites in very small blood samples obtained from the finger-tip. The metabolites, derivatives of 6TG and methylmercaptopurine (6MeMP), were extracted and hydrolized with perchloric acid to liberate the corresponding base. 6MeMP is completely transformed under these conditions to 4-amino-5-(methylthio)carbonyl imidazole. The chromatographic separation of 6TG and this imidazole was performed in a single run under isocratic conditions within 10 min using a 70 mm column. The quantification limit was 0.5 nmol/ml for 6TG and 3 nmol/ml blood for 6MeMP. The accuracy was 83% for 6TG (CV=3%) over the concentration range of 0.5-20 nmol/ml blood and 102% (CV=4%) for 6MeMP over the range of 3-150 nmol/ml blood. The intra-assay CV ranged from 5.4 to 7.4% for 6TG and from 6.2 to 10.6% for 6MeMP. The inter-assay CV was 7.5 and 9.5% in a pooled blood sample. The levels in RBC in whole blood were nearly coincident with those obtained in separated RBC, isolation of RBC therefore is not necessary for these measurements, if the drugs are given per os in the day before blood sampling. The concentration of 6MeMP nucleotides is more dependent on the given 6MP dose than the concentration of 6TG nucleotides. Intraindividual variations were small at unchanged drug doses, interindividual metabolite concentrations were highly variable.     

Validation of a method for quantification of ketobemidone in human plasma with liquid chromatography-tandem mass spectrometry

A liquid chromatography tandem mass spectrometry (LC-MS-MS) method for determination of the analgesic aminophenol ketobemidone in human plasma is presented. Two preparation methods for plasma samples containing ketobemidone were compared, liquid-liquid extraction (LLE) and solid-phase extraction (SPE). Both methods showed good precision (n=10), 1.7% and 2.9%, respectively (0.04 micro M) and 1.1% and 2.5%, respectively (0.14 micro M). The accuracy was 98% and 103%, respectively (0.04 micro M) and 105% and 99%, respectively (0.14 micro M). Ketobemidone could be quantified at 0.43 nM, with a relative standard deviation of 17.5% (n=19) using LLE and 18.6% (n=10) using SPE. This level was an order of magnitude lower than earlier reported quantification limits. Quantitative data from plasma samples analyzed with LC-MS-MS were in good agreement with those obtained by gas chromatography with chemical ionization mass spectrometry (GC-CI/MS). This indicates that LC-MS-MS is a good alternative method to GC-MS as it is more sensitive and time-consuming derivatization can be avoided.     

Multiplexed genotyping with sequence-tagged molecular inversion probes

We report on the development of molecular inversion probe (MIP) genotyping, an efficient technology for large-scale single nucleotide polymorphism (SNP) analysis. This technique uses MIPs to produce inverted sequences, which undergo a unimolecular rearrangement and are then amplified by PCR using common primers and analyzed using universal sequence tag DNA microarrays, resulting in highly specific genotyping. With this technology, multiplex analysis of more than 1,000 probes in a single tube can be done using standard laboratory equipment. Genotypes are generated with a high call rate (95%) and high accuracy (>99%) as determined by independent sequencing.     

Trace element distribution in heart tissue sections studied by nuclear microscopy is changed in coxsackie virus B3 myocarditis in methyl mercury-exposed mice

Methyl mercury (MeHg) has been shown to change Coxsackie virus type B3 (CB3) myocarditis in a direction compatible with the development of chronic disease. Murine models of CB3 myocarditis closely mimic the pathogenesis in humans. There are also indications that metals, such as mercury, and trace elements may interact and adversely affect viral replication and development of inflammatory lesions. The effects of low-dose MeHg exposure on myocardial trace element distribution, as determined by means of nuclear microscopy, was studied in CB3 myocarditis. Balb/c mice were fed a MeHg-containing diet (3.9 mg/kg diet) for 12 wk prior to infection. Areas of inflammatory lesions in the myocardium were identified by traditional histologic examination, and serial tissue sections in these selected areas were used for immune histology (macrophages), in situ hybridization of virus genomes, and nuclear microscopy of tissue trace element distribution. Areas with no inflammation or virus were compared with areas of ongoing inflammation and viral replication. In the inflammatory lesions of MeHg-exposed mice as compared to nonexposed mice, the myocardial contents of calcium (Ca), manganese (Mn), and iron (Fe) were significantly increased, whereas the zinc (Zn) content was decreased. The increased Ca and decreased Zn contents in the inflamed heart may partly explain a more severe disease in MeHg-exposed individuals. Although not significant in the present study, with a limited number of mice, the inflammatory and necrotic lesions in the ventricular myocardium on d 7 of the infection was increased by 50% (from 2.2% to 3.3% of the tissue section area) in MeHg-exposed mice and, also, there was a tendency of increased persistence of virus with MeHg exposure. No increased MeHg uptake, either in the inflammatory lesions or in the areas of noninflamed heart tissue in infected mice, could be detected. The present results indicate that a "competition" exists between potentially toxic heavy metals from the environment/diet and important trace elements in the body and that a disturbed trace element balance adversely influences the development of pathophysiologic changes in inflammatory heart disease.     

Flatworm phylogeny from partial 18S rDNA sequences

Partial 18S rDNA sequences from 29 flatworms and 2 outgroup taxa were used in a cladistic analysis of the Platyhelminthes. Support for the clades in the resulting single most parsimonious tree was estimated through bootstrap analysis, jack-knife analysis and decay indices. The Acoelomorpha (Acoela and Nemertodermatida) were absent from the most parsimonious tree. The Acoela and the Fecampiidae form a strongly supported clade, the sister group of which may be the Tricladida. There is some support for monophyly of the rhabdocoel taxon Dalyellioida, previously regarded as paraphyletic. The sister group of the Neodermata remains unresolved. This revised version was published online in July 2006 with corrections to the Cover Date.     

Growth and nitrogen utilization in seedlings of mountain birch (Betula pubescens ssp. tortuosa ) as affected by ultraviolet radiation (UV-A and UV-B) under laboratory and outdoor conditions

 Growth patterns and nitrogen economy were studied in pot-grown seedlings of mountain birch subjected to different ultraviolet radiation under both laboratory and outdoor conditions at Abisko in northern Sweden. In the laboratory, nutrient supply, temperature, humidity, ultraviolet radiation-A (UV-A, 320–400 nm) and B (UV-B, 280–320 nm) were controlled, while photosynthetically active radiation (PAR, 400–700 nm) and photoperiod varied naturally. Under outdoor conditions nutrient supply was controlled, and the irradiation treatments were ambient and above-ambient UV-B using additional fluorescent lamps. Mountain birch nitrogen economy was affected by increased ultraviolet radiation, as reflected by a changed relationship between plant growth and plant nitrogen both in the laboratory and outdoors. In the laboratory enhanced UV-A decreased leaf area per unit plant biomass (leaf area ratio) but increased biomass productivity, both per unit leaf area (leaf area productivity) and per unit leaf nitrogen (leaf nitrogen productivity). Low levels of UV-B affected growth patterns and nitrogen economy in a similar way to enhanced UV-A. High levels of UV-B clearly decreased relative growth rate and nitrogen productivity, as leaf area ratio, leaf area productivity and leaf nitrogen productivity were all decreased. Under outdoor conditions above-ambient levels of UV-B did not alter growth or biomass allocation traits of the seedlings, whilst nitrogen productivity was increased. Mountain birch seedlings originating from different mother trees varied significantly in their responses to different ultraviolet radiation. Received: 10 April 1997 / Accepted: 19 September 1997     

OxLDL-induced macrophage cytotoxicity is mediated by lysosomal rupture and modified by intralysosomal redox-active iron

Oxidized low density lipoprotein (oxLDL) is believed to play a central role in atherogenesis. LDL is oxidized in the arterial intima by mechanisms that are still only partially understood. OxLDL is then taken up by macrophages through scavenger receptor-mediated endocytosis, which then leads to cellular damage, including apoptosis. The complex mechanisms by which oxLDL induces cell injury are mostly unknown. This study has demonstrated that oxLDL-induced damage of macrophages is associated with iron-mediated intralysosomal oxidative reactions, which cause partial lysosomal rupture and ensuing apoptosis. This series of events can be prevented by pre-exposing cells to the iron-chelator, desferrioxamine (DFO), whereas it is augmented by pretreating the cells with a low molecular weight iron complex. Since both DFO and the iron complex would be taken up by endocytosis, and thus directed to the lysosomal compartment, the results suggest that the normal contents of lysosomal low molecular weight iron may play an important role in oxLDL-induced cell damage, presumably by catalyzing intralysosomal fragmentation of lipid peroxides and the formation of toxic aldehydes and oxygen-centered radicals.