An AvaII polymorphism in the human apolipoprotein C-II gene.
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Apolipoprotein C-II↗
Biomedical subjects
J Geisel
Publications and source records attributed to J Geisel.
Screening for mutations in the LDL receptor gene and apolipoprotein B-100 gene in 218 patients with severe hypercholesterolemia.
A group of 218 patients with severe hypercholesterolemia (LDL cholesterol > 260 mg/dl) living in the Cologne area were screened for mutations in the LDL receptor gene and apolipoprotein B-100 gene. In the LDL receptor gene Southern blotting was used for detection of major DNA rearrangements and the single-strand conformation polymorphism (SSCP) method was used to screen for micro-deletions and insertions and single base alterations. The Arg3500-->Glu mutation, which is the only relevant mutation in the apolipoprotein B-100 gene causing hypercholesterolemia, was detected by a modified PCR and restriction enzyme digestion. Three different major rearrangements, all of which were deletion, were found in the LDL receptor gene. The SSCP screening was started with exon 4. In 20 cases an abnormal fragment pattern was observed. The apolipoprotein B-100 mutation was detected in 15 patients. In summary, by the combined analysis of major rearrangements, micro-deletions, insertions and single base alterations in the LDL receptor gene and the Arg3500-->Glu mutation in the apolipoprotein B-100 gene, mutations causing or probably causing hypercholesterolemia could be detected in 38 of the 218 studied patients. The expansion of SSCP screening to other exons of the LDL receptor gene will greatly increase the identification of mutations causing hypercholesterolemia.
Apolipoprotein B-100↗
Structure of the human apolipoprotein C-II gene promoter.
To localize transcriptional active cis elements and to study the effect of a single base transition at -86 derived from a patient (PS) with an inherited severe form of hypertriglyceridemia a reporter gene transfection assay with apo C-II promoter fragments was performed. Sequences from -170 to -140, -140 to -59 and -59 to -39 were transcriptional active. The A to G transition at -86 reduced promoter activity to 25% (pC2PS-13). A nuclear protein bound sequence specific to a DNA fragment from -102 to -70, but disappeared when the point mutation at -86 was introduced. We found that one of these cis elements enhances promoter activity as a response of cAMP elevation. Apo CII mRNA level increases after incubation of HepG2 cells with 10 microM forskolin within 2 hours as measured by northern blot analyses. Incubation of transfected cells (pC2W) with forskolin [10 microM] or a cAMP analogue resulted in an 6-fold increase of luciferase activity within 6 hours and a subsequent decrease. The results suggest that several positive acting elements are located between -170 and -36 and that one of them may be responsible for promoter activation by cAMP. The reduction of promoter activity by the point mutation may be a consequence of impaired protein-DNA interaction.
Apolipoprotein C-II↗
A single nucleotide substitution in the promoter region of the apolipoprotein C-II gene identified in individuals with chylomicronemia.
Apolipoprotein (apo) C-II plays a major role as a cofactor for lipoprotein lipase, the enzyme involved in the hydrolysis of triglyceride-rich particles. We identified in two relatives of a family (mother and son) massive hypertriglyceridemia with chylomicronemia. In these individuals apoC-II was not measurable in plasma by radial immunodiffusion. On isoelectric focusing of very low density apolipoproteins, trace amounts of apoC-II became obvious in the regular position. By sequencing, no abnormalities in the exons or neighboring intron sequences were detected. However, three alterations in the DNA sequence were found upstream from the transcription initiation site. Two variations could be explained by differences in previously published DNA sequences. The third variation (A-->G; position -86; Das et al. 1987. J. Biol. Chem. 262: 4787-4793) was present only in the homozygous form in the two hypertriglyceridemic probands. In 46 hypertriglyceridemic individuals outside the family, this mutation was not found. In electrophoretic mobility shift experiments with nuclear extracts from HepG2 cells, the 31 bp DNA fragment carrying the A-->G substitution resulted in a markedly diminished protein binding compared with the wildtype DNA fragment. In promoter reporter gene assays, the activity of the basal promoter was reduced in the case of the A-->G substitution and the deletion of the bases -91 to -58. The pedigree analysis and the experimental results are evidence that this is the first mutation in the apolipoprotein C-II gene where a single nucleotide substitution diminishes the binding of a transcription factor to a positive cis-acting clement in the promoter resulting in a depletion of apolipoprotein C-II in plasma.
Adolescent↗
The role of ABO blood groups in infections induced by Staphylococcus saprophyticus and Pseudomonas aeruginosa.
Recently, considerable evidence has been accumulated showing that carbohydrate-containing blood group substances represent prime candidates for the specific interaction with microbial surface lectins in infectious diseases. Accordingly, clinical studies have proved that urinary tract infections by Staphylococcus saprophyticus and outer ear canal infections by Pseudomonas aeruginosa can be positively correlated with the patients blood group. Apparently, the blood group antigens (terminal carbohydrates) represent receptors recognized by S. saprophyticus and P. aeruginosa surface lectins.
ABO Blood-Group System↗
Up-regulation of keratin 17 expression in human HaCaT keratinocytes by interferon-gamma.
The immortalized human keratinocyte cell line HaCaT was used to assess the effect of interferon-gamma (IFN-gamma) on expression of keratin K17. Both IFN-gamma and K17 have been implicated in the pathophysiology of psoriasis. Western and quantitative enzyme-linked immunosorbent assay analyses demonstrated increasing induction of K17 protein by 48 h exposure to IFN-gamma at concentrations of 10, 50, and 250 U/ml. At 50 U/ml IFN-gamma, immunohistochemical analysis revealed numerous K17-positive foci, whereas in situ hybridization demonstrated K17 message in the majority of cells. In addition, at low (5 U/ml) concentrations of IFN-gamma, cell proliferation and protein synthesis decreased, as determined by 3H-thymidine labeling and 14C-amino acid uptake. These data suggest that aberrant K17 expression observed in psoriatic lesions may be a consequence of IFN-gamma overexpression, and that the HaCaT cell line may be a useful in vitro model system to elucidate the underlying mechanisms.
Antigens, Differentiation↗
Antiproliferative and cytotoxic profiles of antipsoriatic fumaric acid derivatives in keratinocyte cultures.
Oral administration with complex mixtures of fumaric acid derivatives is known to have antipsoriatic efficacy. The present studies aimed to clarify the mode of action and toxicity of the individual compounds. Hyperproliferative HaCaT keratinocytes in monolayer cultures were exposed to fumaric acid, dimethylfumarate, zinc monoethylfumarate, calcium monoethylfumarate and magnesium monoethylfumarate at concentrations between 0.4 microM and 960 microM for 48 h. Cell proliferation was studied by [3H]thymidine incorporation. In addition 14C-labelled amino acid uptake and total protein content were measured. Direct cytotoxicity was determined by the release of cytoplasmic lactate dehydrogenase (LDH) into the culture medium. The corresponding 50% inhibition concentrations (IC50) were calculated for DNA/protein synthesis: 2.3/2.5 microM (dimethylfumarate), 133/145 microM (zinc monoethylfumarate), 215/230 microM (calcium monoethylfumarate), 275/270 microM (magnesium monoethylfumarate), > 960/> 960 microM (fumaric acid). The total protein content was less sensitive. Antiproliferative activity was found for dimethylfumarate and to a lesser degree for calcium monoethylfumarate already at the subtoxic concentrations of 1.3 and 4 microM, respectively. In the case of magnesium monoethylfumarate, zinc monoethylfumarate and fumaric acid there was no such dissociation between their cytotoxic and antiproliferative potential. These data indicate that most of the antipsoriatic potential of fumaric therapies is due to the dimethylfumarate compound.
Cell Death↗
Endotoxin tolerance is associated with decreased prostaglandin H synthases-1 and -2.
Lipopolysaccharide (LPS) induces macrophage protein and eicosanoid synthesis. Previous studies have shown that LPS induction of eicosanoid metabolism is transcription dependent and that prostaglandin (PG) H synthase is the committed step in the conversion of arachidonic acid (AA) to thromboxane (Tx) B2. LPS tolerance induced by sublethal in vivo injections of LPS renders rats resistant to LPS in vivo and macrophages refractory to LPS-stimulated eicosanoid synthesis in vitro. This study examined potential activity and content changes in constitutive and mitogen inducible PGH synthase in LPS-stimulated control and tolerant macrophages. TxB2 levels were measured to evaluate basal PGH synthase activity and stimulation by Salmonella enteritidis LPS (50 micrograms/ml), calcium ionophore A-23187, and AA. All tolerant macrophage groups demonstrated decreased TxB2 synthesis. TxB2 synthesis stimulated by AA in tolerant cells was decreased by 70% (P < 0.05) compared with control macrophages. In subsequent studies changes in PGH synthase content were examined in rat peritoneal macrophages from tolerant and control rats incubated with and without LPS. Immunoblot analysis of PGH synthase-1 (constitutive) demonstrated no increase in cells stimulated with LPS compared with basal but was diminished by 62 +/- 9% (n = 4, P < 0.05) in tolerant macrophages compared with control cells. Immunoblot analysis of PGH synthase-2 (mitogen inducible) demonstrated induction in response to LPS that was maximal between 12 and 24 h. PGH synthase-2 was also induced in tolerant macrophages in response to LPS but was less than in control cells. The results demonstrate that endotoxin tolerance is associated with reduced activity and content of PGH synthase-1 and a decreased LPS induction of PGH synthase-2.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
[Effect of mistletoe lectin therapy on serum level of defined serum proteins (acute phase proteins) in tumor patients].
The influence of galactoside-specific mistletoe lectin (ML-1) administration on defined acute phase reactants in the serum of cancer patients (mammary carcinoma, n = 4; larynx carcinoma, n = 11; TNM-stages II to IV; after appropriate surgery, chemotherapy, radiation) was studied. Regular subcutaneous injections of the optimal doses of ML-1 (1 mg/kg body weight, twice a week) yielded statistically significant increases of certain acute phase reactants (C-reactive protein, haptoglobin, coeruloplasmin, C3-complement, albumin, immunoglobulin IgM) after four weeks of treatment. However, serum concentrations of transferrin, C4-complement and the immunoglobulins IgG and IgA were found within the biological range (means +/- 2 s). The increase of acute phase reactants after administration of ML-1 correlates positively with the activity of lymphatic cells (e.g. expression of IL-2 and HLA-DR receptors) in FACS (fluorescence-activated cell sorter) staining experiments and indicates the immunoreactive potency of this substance which may be speculated to be cytokine-induced.
Acute-Phase Proteins↗
[Long-term effects of low dosage fish oil on serum lipids and lipoproteins].
In a prospective and double-blind study, the long-term effects of low dose fish oil on serum lipids and lipoproteins was tested in patients with normal or moderately elevated serum lipids and compared to the effects of olive oil. The compliance to the study medication was evaluated by analysis of serum fatty acids and proved to be very good. Dietary supplementation with 9 g fish oil, respective 3.15 g n-3 fatty acids per day over one year resulted in a decrease of serum-triglycerides by 26% and increase of HDL-cholesterol by 26%. Treatment with 9 g olive oil resulted in an 18% increase of HDL-cholesterol. There was no effect on serum-triglyceride levels.
Adult↗
Humoral and cellular immunity in HIV positive and HIV negative Helicobacter pylori infected patients.
The prevalence of H. pylori associated gastritis seems to be different in HIV positive and HIV negative patients. Therefore a correlation to immunodeficiency can be postulated. The histology of gastritis, status of H. pylori infection and parameters of humoral and cellular immune response were investigated in 41 HIV positive and 47 HIV negative patients, who were subjected to upper endoscopy for the evaluation of gastrointestinal symptoms. In HIV positive patients 37% had active chronic gastritis against 62% of the HIV negative patients. In 73% of HIV positive cases of active chronic gastritis H. pylori was detected by bacteriological culture and/or Warthin-Starry stain. In HIV negative patients active chronic gastritis was always associated to H. pylori infection. Production of antibodies as measured by two commercially available ELISA tests was significant in HIV positive and HIV negative patients; both tests correlated well with H. pylori detection by culture or direct microscopy. Immunoglobulin class specific immunoblots corresponded to the ELISA results in HIV negative patients but to a lesser extent in the HIV positive group which was assumed to be related to unspecific polyclonal activation in these patients. Systemic cellular immunity was investigated by proliferation assays of peripheral blood mononuclear cells (PBMC). Proliferative response to the unspecific mitogen PHA was reduced in HIV positive patients. A sonicated H. pylori antigen failed to induce lymphocyte proliferation. The antimitogenic effect was also seen in case of coincubation with PHA. This observation was independent of H. pylori and HIV infection status. We conclude that in HIV positive as in HIV negative patients active chronic gastritis is predominantly related to H. pylori infection. The prevalence of H. pylori associated gastritis in HIV positive patients is significantly reduced (p < 0.025) compared to HIV negative controls. Decreased susceptibility to H. pylori infection in HIV positive patients may not be explained by the abnormal reactivity of their humoral or cellular immune response.
Adult↗
Selective generation of CD8+ T-cell clones from the peripheral blood of patients with cutaneous reactions to beta-lactam antibiotics.
The presence, phenotype, and functional characteristics of peripheral blood penicillin-specific T lymphocytes in individuals with cutaneous allergic reactions to penicillin were investigated using in vitro long-term culture techniques. Peripheral blood mononuclear cells from two penicillin-allergic patients were stimulated in vitro with penicillin, and T-cell blasts were clonally expanded by limiting dilution. Seven T-cell clones were derived, all of which were CD3+ CD4- CD8+ HLA-DR+, and produced IL-2 and IFN-gamma upon stimulation. T-cell proliferation required the presence of antigen and autologous, but not allogeneic, antigen-presenting cells. In addition to the parent compound, the T-cell clones also developed a proliferative response to penicilloyl, the major metabolite of penicillin. The cloned T-cell lines were found to exhibit marked suppressor activity for Con A mitogenesis. The observed suppressor activity required cell-to-cell contact, as supernatants from these T-cell clones had no comparable inhibitory effect. These findings indicate that there is a predominance of penicillin-specific CD8+ T cells in the peripheral blood of individuals sensitized to beta-lactam antibiotics.
Anti-Bacterial Agents↗
Effects of two magainin peptides on eicosanoid release from rat peritoneal macrophages.
Magainins are novel polycationic peptides with broad-spectrum antimicrobial activity, including activity against gram-negative bacteria. Gram-negative bacteremia can elicit endotoxic shock that is associated with the increased formation of eicosanoids. Inhibition of eicosanoid synthesis has been shown to improve the outcome of experimental endotoxic shock. The aim of the present study was to test the in vitro effects of two magainin peptides, MSI-97 (M1) and MSI-98 (M2), on eicosanoid synthesis by rat peritoneal macrophages (M phi) stimulated by Salmonella enteritidis lipopolysaccharide (LPS; 50 micrograms/ml) and Salmonella minnesota lipid A (5 micrograms/ml) and to compare their effects on LPS reactivity with a metachromatic dye. M1 (100 micrograms/ml) significantly (P < 0.05) reduced LPS-stimulated synthesis of thromboxane B2 (TXB2), without changing 6-keto-prostaglandin F1 alpha in M phi. Similarly, M2 (10 micrograms/ml) significantly attenuated M phi synthesis of TXB2 stimulated by either LPS or lipid A. However, at a higher concentration (100 micrograms/ml), M2 but not M1 significantly augmented LPS-induced increases in TXB2 and 6-keto-prostaglandin F1 alpha. Polymyxin B (40 micrograms/ml) inhibited LPS production and lipid A-stimulated TXB2 production. M1 (100 micrograms/ml) and polymyxin B (10 and 40 micrograms/ml) also inhibited calcium ionophore A23187 (10 microM)-induced synthesis of TXB2. The lipid A moiety of LPS reacts with dimethylmethylene blue dye, providing a metachromatic assay of LPS. This metachromatic reaction with lipid A was significantly reduced by polymyxin B and M2 at all concentrations. M1 was effective only at the highest M1:lipid A concentration ratio (2:1). Thus, M1 and M2 share some similarities with polymyxin B in inhibiting lipid A reactivity with the dye, which suggests that these magainins may also bind to lipid A. However, M1 was devoid of any inhibitory effects on dye reactivity with S. enteritidis LPS and M2 was inhibitory at only one concentration ratio (1:5). In conclusion, the varied effects of the magainin peptides on LPS, lipid A, and M phi eicosanoid synthesis appear to depend on the type of peptide used and on its concentration.
Amino Acid Sequence↗
[Comparative studies on the immunoactive action of galactoside-specific mistletoe lectin. Pure substance compared to the standardized extract].
Comparative Studies on the Immunoactive Potency of Galactoside-specific Lectin from Mistletoe/Pure substance against standardized extract. Cellular and humoral aspects of the immunomodulating activity of the galactoside-specific lectin from mistletoe (ML-1) were investigated in cancer patients suffering from mammary carcinoma and compared to the immunoactive potency of a proprietary mistletoe extract standardized for ML-1 (ML-1 stand., Eurixor). Regular subcutaneous injections of the optimal dose of ML-1 and ML-1 stand. (1 ng/kg body weight; twice a week; for 4 weeks) yielded statistically significant increases of certain lymphocyte subsets (helper T-lymphocytes, natural killer (NK)-cells) which are generally believed to be involved in antitumor activity. Moreover, administration of either ML-1 preparation resulted in enhanced expression of interleukin (Il)-2 receptors on lymphatic cells and significantly increased serum levels of defined acute phase reactants (c-reactive protein, haptoglobin, C3 complement) as indicator of cellular and humoral activity. In vitro, exposition of human lymphocytes to ML-1 and ML-1 stand. resulted in enhanced expression of Il-2 receptors, which substantiated the capacity of both ML-1 preparations to affect immunological parameters within the host defense system. The effects of ML-1 and ML-1 stand. were comparable.
Acute-Phase Proteins↗
Prostaglandins, thromboxanes, leukotrienes, and cytochrome P-450 metabolites of arachidonic acid.
Arachidonic acid is metabolized via three major enzymatic pathways: cyclooxygenase, lipoxygenase, and cytochrome P-450. The cyclooxygenase pathway gives rise to the prostaglandins and thromboxane A2. The lipoxygenase pathway produces the leukotrienes and 5-, 12-, and 15-hydroxyeicosatetraenoic acids. The cytochrome P-450 pathway metabolites are oxygenated metabolites of arachidonic acid. Synthesis of several of the cyclooxygenase and lipoxygenase metabolites are increased in circulatory shock. A potential role for several of these compounds in the pathophysiologic sequelae of endotoxic and septic shock has been implied from a combination of animal and human studies. There are no studies that have explored a potential role for cytochrome P-450 metabolites in the pathogenesis of circulatory shock.
Animals↗
Improved detection of familial defective apolipoprotein B-100 by restriction-site-introducing polymerase chain reaction.
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Apolipoprotein B-100↗
Familial defective apolipoprotein B-100 in 12 subjects and their kindred.
Twelve unrelated subjects with heterozygous familial defective apolipoprotein B-100 were identified in a group of 252 patients with type IIa hypercholesterolaemia. Approximately 5% of hypercholesterolaemia can be explained by this mutation in the collective studied. Familial defective apolipoprotein B-100 is therefore the most common known mutation causing primary hypercholesterolaemia. Family studies revealed an additional 14 affected subjects. All family members with the mutation had elevated cholesterol concentrations. In a normolipidaemic control group of 146 subjects the mutation was not present. In the affected individuals a variable expression of total cholesterol concentrations and atherosclerosis was observed. Plasma cholesterol ranged from 6.60 to 14.89 mmol/l with a mean of 9.43 mmol/l. Premature atherosclerosis was present in 4 patients, while one affected woman is now 92 years old and has no symptoms of coronary heart disease or peripheral atherosclerosis. Analysis of the haplotypes and genotypes by 3 biallelic and 1 multi-allelic DNA marker suggests that the disorder is caused in all affected patients by the same rare allele. The fact that the same mutant allele was also identified in other European populations and in a North American population of Caucasian origin argues for a common European origin of this mutation.
Adolescent↗
Endotoxin-induced arachidonic acid metabolism requires de novo protein synthesis and protein kinase C activation.
The mechanisms whereby bacterial endotoxins stimulate arachidonic acid metabolism in macrophages are uncertain. Both protein kinase C activation and de novo protein synthesis occur in macrophages in response to endotoxin. In this study we evaluated the time course and role of protein kinase C and de novo protein synthesis in endotoxin stimulated arachidonic acid metabolism in resident rat peritoneal macrophages. Thromboxane (TX) B2 was measured as the representative arachidonic acid metabolite synthesized in response to Salmonella enteritidis endotoxin, calcium ionophore A23187, or phorbol 12-myristate 13-acetate (PMA). The effect of inhibition of protein kinase C by 1-(5-isoquinolinsulfonyl)-2-methylpiperazine dihydrochloride (H-7) and staurosporine on endotoxin- and A23187-induced TXB2 synthesis was examined. The potential roles of transcriptional and translational events in endotoxin- and A23187-stimulated TXB2 synthesis were determined by utilizing the transcriptional inhibitors camptothecin (10 microM) or actinomycin D (0.08 microM), and the translational inhibitor cycloheximide (0.1 microM). Whereas, A23187 stimulated maximal TXB2 synthesis within 15 min, endotoxin showed a more prolonged time course with a 12-fold increase in TXB2 synthesis above basal levels after 3 h (P less than 0.05). PMA induced an approx. 8-fold increase above basal TXB2 levels that was blocked by inhibition of transcription with actinomycin D. H-7 (10 microM to 50 microM) inhibited endotoxin- and A23187-stimulated eicosanoid synthesis. Staurosporine (0.2 microM) produced a selective 66% inhibition of endotoxin, but not A23187-stimulated TXB2 synthesis. Endotoxin-induced TXB2 production was significantly (P less than 0.05) inhibited by staurosporine, camptothecin, actinomycin D or cycloheximide at intervals from 30 min prior to, through 60 min after endotoxin stimulation. These studies suggest a role for protein kinase C activation and de novo protein synthesis in endotoxin signal transduction events leading to increased macrophage arachidonic acid metabolism. These intracellular events are essential in sustaining the prolonged inflammatory response to endotoxin.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗