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G Schmitz

Publications and source records attributed to G Schmitz.

At least 19 recordsLinked to original sources
PubMed1999

Lipopolysaccharide inhibits the expression of the scavenger receptor Cla-1 in human monocytes and macrophages.

Human Cla-1 is the likely homologue of the murine scavenger receptor class B type I (SR-BI). SR-BI mediates selective transfer of cholesterol to high-density lipoprotein (HDL) and the efflux of endogenously synthesized and plasma membrane sterols to HDL. HDL protects against atherosclerosis but also reduces endotoxic activity by complexation and neutralization of LPS. We found that Cla-1 is upregulated during phagocytic as well as dendritic differentiation of monocytes, indicating a function of this receptor for cholesterol homeostasis in phagocytes and antigen-presenting cells. Cla-1 expression is suppressed by the proinflammatory stimuli lipopolysaccharide, interferon-gamma, and tumor necrosis factor alpha in monocytes and macrophages. Downregulation of Cla-1 mRNA by LPS is likely due to a modification and subsequent destabilization of the mRNA. We propose that suppression of Cla-1 expression may help to stabilize the lipoprotein status in the blood compartment important for host defense.

CD36 Antigens↗
PubMed1999

The human apM-1, an adipocyte-specific gene linked to the family of TNF's and to genes expressed in activated T cells, is mapped to chromosome 1q21.3-q23, a susceptibility locus identified for familial combined hyperlipidaemia (FCH).

The human adipocyte-specific apM-1 gene encodes a secretory protein of the adipose tissue that has been suggested to play a role in the pathogenesis of obesity. The regulation of apM-1 was studied along adipocyte differentiation. While apM-1-mRNA and apM-1 protein were absent in preadipocytes and in 48 h differentiated adipocytes, they were found upregulated from day 4 to day 9 of adipocyte differentiation as shown by RNase protection assay and Western blot analysis. These data indicate that apM-1 may be a late marker of adipocyte differentiation. In human sera apM-1 protein is also detectable by Western blots using a polyclonal antibody raised against a synthetic peptide sequence of the human apM-1. The genomic structure of the human apM-1 gene together with a total of 2.7 kb of the 5'-flanking region with putative transcription factor binding sites is presented. Interestingly, sequence comparisons link the apM-1 gene to the family of TNF's and to genes expressed in activated T-cells. The chromosomal localization of apM-1 was investigated by FISH and mapped to human chromosome 1q21.3-1q23, a region that was identified as a susceptibility locus for Familial Combined Hyperlipidaemia (FCH) and polygenic NIDDM. These data and the chromosomal localization on chromosome 1q21.3-q23 raises the possibility that apM-1 as an adipocyte-specific secretory protein may play a role in the pathogenesis of FCH and associated insulin resistance. Exon- and intron-specific primer sequences are presented as a basis for mutation screening of patients affected with FCH.

3T3 Cells↗
PubMed1999

Genomic organization and chromosomal localization of the human CD163 (M130) gene: a member of the scavenger receptor cysteine-rich superfamily.

The human protein CD163 (M130) is a member of the scavenger receptor cysteine-rich (SRCR) superfamily, which is exclusively expressed by monocytes and macrophages. Here, we investigated the genomic organization and the chromosomal localization of the human CD163 gene. The CD163 gene is composed of 17 exons and 16 introns and spans over 35 kb. Each of its nine SRCR domains is encoded by a separate exon, which is similar to other members of the group B SRCR subfamily. Two cytoplasmic variants of CD163 arise from alternative splicing of intron 15, while a truncated and an extracellular variant results from alternative splicing of intron 5 or intron 7, respectively. Using fluorescence in situ hybridization we mapped this gene to the human chromosome 12p13. The transcription initiation sites of the CD163 gene were determined and the 5'-flanking region was sequenced. The nucleotide analysis revealed several putative binding sites for transcription factors, which have been shown to play an important role in myeloid specific gene expression. In addition, we identified a L1 element located 1.4 kb upstream of the major transcription initiation site.

Alternative Splicing↗
PubMed1999

C(-260)-->T polymorphism in the promoter of the CD14 monocyte receptor gene as a risk factor for myocardial infarction.

BACKGROUND: The CD14 receptor of monocytes is an important mediator for the activation of monocytes/macrophages by endotoxins from the envelope of Gram-negative bacteria (lipopolysaccharides). We identified a polymorphism in the CD14 receptor and examined whether this genetic marker influenced the expression of the CD14 receptor on monocytes and affected the predisposition to myocardial infarction. METHODS AND RESULTS: We identified a C(-260)-->T nucleotide change, creating a HaeIII polymorphism in the promoter of the CD14 gene. The polymorphism was determined in 178 male patients <65 years old (cases; average age, 55.9+/-6.3 years) at the time of their first myocardial infarction and in 135 representative selected male control subjects (controls; average age, 55.2+/-11.5 years). The frequency of the T allele (absence of the cutting site) was 0.49 in cases and 0.35 in controls (P=0.0005; OR, 1.781; 95% CI, 1.286 to 2.465). Subsequently, we measured the expression of monocyte CD14 by flow cytometry in 18 volunteers with different CD14 genotypes. A significantly higher density of the CD14 receptor was shown in the T/T homozygotes than in the others (P=0.0028). CONCLUSIONS: A higher frequency of allele T(-260) in the promoter of the CD14 receptor gene was found in myocardial infarction survivors than in controls. At the same time, this variation was associated with a higher density of CD14 receptors in healthy volunteers. Therefore, we can conclude that in addition to the well-established risk factors, a genetically determined reaction of monocytes/macrophages to infectious stimuli could play an important role in the process of atherosclerosis.

Aged↗
PubMed1999

Molecular cloning of the human ATP-binding cassette transporter 1 (hABC1): evidence for sterol-dependent regulation in macrophages.

We have cloned the full-length cDNA for the human ATP binding cassette transporter 1 (hABC1). The 6603-bp open reading frame encodes a polypeptide of 2201 amino acids resulting in a deduced molecular weight of 220 kDa. The hABC1 cDNA is highly homologous (62%) to the human rim ABC transporter (ABCR). hABC1 is expressed in a variety of human tissues with highest expression levels found in placenta, liver, lung, adrenal glands, and fetal tissues. We demonstrate that the hABC1 expression is induced during differentiation of human monocytes into macrophages in vitro. In macrophages, both the hABC1 mRNA and protein expression are upregulated in the presence of acetylated low-density lipoprotein (AcLDL). The AcLDL-induced increase in hABC1 expression is reversed by cholesterol depletion mediated by the addition of high-density lipoprotein (HDL3). Our data, demonstrating sterol-dependent regulation of hABC1 in human monocytes/macrophages, suggest a novel role for this transporter molecule in membrane lipid transport.

ATP Binding Cassette Transporter 1↗
PubMed1999

The Lateral suppressor (Ls) gene of tomato encodes a new member of the VHIID protein family.

The ability of the shoot apical meristem to multiply and distribute its meristematic potential through the formation of axillary meristems is essential for the diversity of forms and growth habits of higher plants. In the lateral suppressor mutant of tomato the initiation of axillary meristems is prevented, thus offering the unique opportunity to study the molecular mechanisms underlying this important function of the shoot apical meristem. We report here the isolation of the Lateral suppressor gene by positional cloning and show that the mutant phenotype is caused by a complete loss of function of a new member of the VHIID family of plant regulatory proteins.

Amino Acid Sequence↗
PubMed1999

A new antigenic epitope localized within human kappa light chains specific for rheumatoid arthritis and systemic lupus erythematosus.

Human B cell hybridomas were established to define new autoantigens of importance for autoimmune diseases such as rheumatoid arthritis (RA). One lgG1, lambda monoclonal antibody (FKN-E12), was derived from synovial B lymphocytes of a patient with sero-negative RA. The purified lg was used to select specifically binding peptides from a random peptide phage display library. Only one epitope with the heptamer sequence HLTFGPG was detected and named RASFp1. Very similar and partly identical sequences are found in the variable region of lg kappa light chains in position 96-101, at the junction of framework 2 and the J-region. The antibody FKN-E12 was shown to detect the epitope RASFp1 also on human lgG kappa chains, but only in a specific conformation. The aim of the present study was to analyse human sera from patients with autoimmune diseases, non-autoimmune inflammatory diseases and healthy blood donors for the presence of lgG binding to RASFp1. For this purpose a 15-mer-peptide was synthesized containing RASFp1 within Vk-derived flanking regions, and an ELISA assay established. Sera of 142 individuals were studied. Only <5% of the control sera including sera from patients with non-autoimmune inflammations were positive. In contrast, 45% of sera from patients with RA or SLE contained RASFp1-binding antibodies. Within the 40 RA sera analysed so far, rheumatoid factors and RASFp1-binding antibodies have shown no correlation with each other.

Amino Acid Sequence↗
PubMed1999

Isolation and characterization of two human monoclonal anti-phospholipid IgG from patients with autoimmune disease.

The antigenic specificity of anti-phospholipid antibodies (APA) is a matter of intensive investigation. To further characterize these antibodies, we attempted to isolate human monoclonal APA. B-cells of patients with at least one positive test for antibodies against cardiolipin, phosphatidylserine, beta2-glycoprotein I (beta2-GPI) or the lupus anti-coagulant were immortalized by transformation with Epstein-Barr virus and screened for production of specific IgG. Positive pools were fused with a heteromyeloma cell line and APA-secreting clones were isolated by standard procedures. Two monoclonal APA, HL-5B from a 51-year-old man with primary anti-phospholipid syndrome and recurrent cerebral microinfarctions, and RR-7F from a 48-year-old women with systemic lupus erythematosus but no evidence for thrombotic events were obtained. HL-5B is of the IgG2 subtype with lambda light chains, while RR-7F is IgG2 with kappa light chains. Both monoclonals show reactivity against cardiolipin and phosphatidylserine but lack reactivity against beta2-GPI or lupus anti-coagulant activity. To yield the same OD in the cardiolipin and phosphatidylserine ELISAs RR-7F must be used in an approximately 10-fold higher concentration than HL-5B, indicating a lower affinity towards these antigens. Interestingly, both mAPA can bind to cardiolipin in the absence of beta2-GPI. They do not cross-react with dsDNA but show reactivity against oxidized low-density lipoproteins. Analysis of the heavy chain mRNA of HL-5B and RR-7F showed that both are members of the VH3 family. While HL-5B shows extensive somatic mutations in the CDR1 and 2 regions, indicating that it was derived by a T cell-dependent antigen driven process, RR-7F is apparently germline encoded. The two monoclonal APA can be used as tools in further structural and functional analyses.

Amino Acid Sequence↗
PubMed1999

A more mature phenotype of blood mononuclear phagocytes is induced by fluvastatin treatment in hypercholesterolemic patients with coronary heart disease.

Monocytes are recruited as the principal inflammatory cells into the atherosclerotic lesion. In a previous study we demonstrated that a low HDL-cholesterol and the apo E4 allele are associated with an increased proportion of blood monocytes that are characterized by a high expression of Fcgamma-RIIIa (CD16), a dim expression of the lipopolysaccharide (LPS) receptor (CD14) and a high expression of beta1- and beta2-integrins (Rothe et al. Arterioscler Thromb Vasc Cell Biol 1996;16:1437-1447). In this study, 79 hypercholesterolemic patients were treated either with the HMG CoA reductase inhibitor fluvastatin in combination with diet or with placebo and diet in a double-blind and randomized multicenter study, and monitored for the potential effects on the phenotype of peripheral blood monocytes. At baseline, in the whole group of hypercholesterolemic patients the population size of these more mature monocytes (CD14dimCD16+) was positively correlated to triglyceride (P = 0.003) and total serum cholesterol levels (P = 0.012) confirming our previous study. Fluvastatin treatment for 52 weeks was associated with a 24.2% reduction in LDL-cholesterol (P < 0.001) as well as a 40.7% decrease in the expression density of CD14 on all monocytes (P = 0.027). A 24.5% decrease (P < 0.001) of the population of less differentiated CD14brightCD16- monocytes and an 83.1% increase (P = 0.029) of the population of more differentiated CD14dimCD16+ monocytes further confirmed this modification of the phenotype of peripheral blood monocytes. The positive pre-study correlation of the CD14dimCD16+ monocyte subset to the serum cholesterol concentration, but inverse changes of both parameters under fluvastatin therapy, in conclusion indicate that fluvastatin exerts an as yet uncharacterized immunomodulatory effect on either monocyte maturation and differentiation, or extravasation which may also depend on the endothelial phenotype that is independent of the change in serum lipids.

Adult↗
PubMed1999

Genetic control of branching in Arabidopsis and tomato.

The patterns of axillary bud formation and the growth characteristics of side-shoots determine to a large extent the form of plants. Characterization of mutants in the monopodial plant Arabidopsis thaliana and in the sympodial tomato, as well as cloning of some of the respective genes, contributes to a better understanding of side-shoot development. Genes have been identified that influence the initiation of axillary meristems and the pattern of their subsequent development.

Arabidopsis↗
PubMed1999

The gene encoding ATP-binding cassette transporter 1 is mutated in Tangier disease.

Tangier disease (TD) is an autosomal recessive disorder of lipid metabolism. It is characterized by absence of plasma high-density lipoprotein (HDL) and deposition of cholesteryl esters in the reticulo-endothelial system with splenomegaly and enlargement of tonsils and lymph nodes. Although low HDL cholesterol is associated with an increased risk for coronary artery disease, this condition is not consistently found in TD pedigrees. Metabolic studies in TD patients have revealed a rapid catabolism of HDL and its precursors. In contrast to normal mononuclear phagocytes (MNP), MNP from TD individuals degrade internalized HDL in unusual lysosomes, indicating a defect in cellular lipid metabolism. HDL-mediated cholesterol efflux and intracellular lipid trafficking and turnover are abnormal in TD fibroblasts, which have a reduced in vitro growth rate. The TD locus has been mapped to chromosome 9q31. Here we present evidence that TD is caused by mutations in ABC1, encoding a member of the ATP-binding cassette (ABC) transporter family, located on chromosome 9q22-31. We have analysed five kindreds with TD and identified seven different mutations, including three that are expected to impair the function of the gene product. The identification of ABC1 as the TD locus has implications for the understanding of cellular HDL metabolism and reverse cholesterol transport, and its association with premature cardiovascular disease.

ATP Binding Cassette Transporter 1↗
PubMed1999

Transplant characteristics: minimal residual disease and impaired megakaryocytic colony growth as sensitive parameters for predicting relapse in acute myeloid leukemia.

Dose escalation during consolidation therapy of de novo AML, including myeloablative chemotherapy supported with autologous peripheral blood stem cell transplantation (aPBSCT), continuously improved outcome. Therefore, quality control of transplants is getting increasing interest. We studied leukapheresis products (LPs), consecutively collected during postremission treatment of 20 patients with de novo AML for minimal residual disease (MRD) by 5-parametric flow cytometry and for myelodysplasia (MDS)-associated alterations by paired lineage-selected colony assays for colony-forming units-megakaryocytes (CFU-mega) and burst-granulocytes-monocytes colony-forming units (CFU) to evaluate the predictive value of these transplant-associated parameters on outcome. We defined the leukemia-associated immunophenotype at diagnosis and studied the impact of MRD detection in LPs collected after double induction with TAD (thioguanine, daunorubicin, cytarabine) and HAM (mitoxantrone, high-dose cytarabine, n=18 patients) and TAD consolidation treatment (n=20 patients) on outcome after aPBSCT. The level of MRD in the transplants correlated with the relapse-free survival (RFS) using a cut-off level of 1 x 10(-3) residual leukemic cells. The median RFS was 6 months for the group with > or = 1 x 10(-3) residual leukemic cells and has not been reached in the group with low MRD levels (< 1 x 10(-3)). By using the same cut-off level a weak correlation could also be demonstrated between MRD in the pregraft bone marrow and RFS (P = 0.04). Quantitatively abnormal megakaryocytic colony growth in the back-up LPs collected after double induction and in the transplant LPs was characterized by the ratio CFU-mega/CFU. In the group of relapsing patients the ratio CFU-mega/CFU was significantly lower than in the group of patients with CCR (P = 0.004), both in the back-ups and in the transplants. All patients with CFU-mega/CFU ratios < 0.12 relapsed, five of seven patients developed MDS before progressing to full leukemic relapse. Using the optimized cut-off level for the ratio CFU-mega/CFU (< vs > or = 0.12), seven of 10 relapsing patients (70%) could be identified to be at risk of relapse, whereas MRD in the transplants identified only 50% of the relapses and MRD in the pregraft bone marrow 25%. In conclusion, the study could identify two pretransplant risk factors predicting relapse in patients with AML receiving aPBSCT in first CR: MRD in transplants as well as MDS-like alterations within the transplants. These results may have multifold implications on the design of risk-adapted chemotherapy as well as on purging techniques and may contribute to a better understanding of leukemogenesis.

Acute Disease↗
PubMed1999

Frequency and significance of the A-->G (-3826) polymorphism in the promoter of the gene for uncoupling protein-1 with regard to metabolic parameters and adipocyte transcription factor binding in a large population-based Caucasian cohort.

BACKGROUND: The recently described A-->G (-3826) point mutation within the distal region of the UCP-1 promoter is possibly involved in the development of obesity, diabetes and related metabolic disorders. It was the aim of this study to examine the allelic frequency and the prevalence of the three UCP-1 genotypes in a broad caucasian cohort and to investigate the significance of this polymorphism for obesity and diabetes. METHODS: 1020 subjects were randomly chosen from 6450 participants in the Diabetomobile Study. The UCP-1 genotype was determined by genomic PCR and Bcl-I-RFLP analysis in 1020 subjects and tested for association with a variety of metabolic parameters. In addition, the influence of this mutation on adipocyte nuclear factor binding was investigated by electrophoretic mobility shift assays (EMSA). RESULTS: The genotype frequencies in 1020 subjects were: AA genotype, 57.0%; AG genotype, 35.4%; GG genotype, 7.6%; with allelic frequencies of 0.75 for allele A and 0.25 for allele G. No significant differences between the genotypes and age, gender, BMI, leptin, glucose, fasting insulin, C-peptide, HbA1c, diabetes manifestation, total cholesterol, and HDL cholesterol were found. Analysis of the Trp64Arg polymorphism of the beta3-adrenergic receptor in a subgroup of 343 subjects revealed no additive effect to the UCP-1 polymorphism. An yet unknown adipocyte-specific factor of nuclear extracts from 3T3-L1 adipocytes during differentiation is able to bind specifically to the distal UCP-1 promoter region and this binding ability can not be abolished by the mutation. CONCLUSIONS: We determined the genotype and allelic frequency of the UCP-1 promoter polymorphism in the largest known population-based study. The results from genotyping demonstrate clearly that this polymorphism does not play a major role in the pathogenesis obesity and diabetes. A yet unknown adipocyte derived and differentiation-dependent regulated transcription factor is able to bind to the distal UCP-1 promoter surrounding -3826 bp. This binding is not affected by presence of the mutation.

Adipocytes↗
PubMed1999

Growth and cell cycle abnormalities of fibroblasts from Tangier disease patients.

We have investigated the abnormal proliferation and morphology of fibroblasts from patients with Tangier disease (TD), a high density lipoprotein (HDL) deficiency syndrome that is characterized by impairment of HDL3-mediated lipid efflux and Gi-protein-mediated signaling via phosphatidylinositol-specific phospholipase C (PI-PLC) and phospholipase D (PLD). TD fibroblasts displayed a 30% to 50% reduced in vitro growth rate and a 1.6-fold increased cell surface area. The response to different mitogens was diminished, and asynchronously growing TD fibroblasts showed 4.4+/-0.3% S-phase and 19.1+/-0.5% G2/M-phase cells compared with 9.7+/-0.6% and 7.8+/-0.5%, respectively, in controls. Monensin, but not brefeldin A, induced an S- and G2/M-phase distribution in control cells similar to that found in TD fibroblasts. This effect of monensin was accompanied by an increase of ceramide levels in controls, whereas TD fibroblasts already had a 2.5-fold increased basal ceramide concentration. Incubation of control cells with C2 ceramide and threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) mimicked the effect of monensin on the cell cycle. The inhibition of neither Gi protein function by pertussis toxin nor PLD by butanol resulted in a G2/M-phase arrest. Propranolol, known to increase phosphatidic acid levels, was ineffective in reversing the G2/M-phase arrest in TD fibroblasts. In addition, cDNA sequences and mRNA expression of the participants of PI-PLC or PLD signaling, ie, G-protein subunits alphai1, alphai2, and alphai3; phosphatidylinositol transfer proteins-alpha and -beta; and ADP ribosylation factors 1 and 3 were found to be normal. Thus, growth and cell cycle abnormalities in TD fibroblasts are likely to be related to impaired Golgi function and sphingolipid signaling rather than inoperative G-protein signal transduction. Because PDMP was also found to decrease HDL3-mediated lipid efflux in control but not TD fibroblasts, similar pathways seem to be involved in the disturbances of lipid transport and growth retardation.

ADP-Ribosylation Factors↗
PubMed1999

Transcription factors Sp1 and AP-2 mediate induction of acid sphingomyelinase during monocytic differentiation.

Cells from the human monocytic leukemia cell line THP-1 differentiate towards a macrophage-like phenotype when stimulated with phorbol 12-myristate -13- acetate (PMA), 1,25-dihydroxy-vitamin D3, and various other agents. We demonstrate here that the expression of the lysosomal enzyme acid sphingomyelinase (ASM; E.C. 3.1.4.12) is induced during this process and is strongly elevated in differentiated THP-1 cells, as well as in differentiated human mononuclear phagocytes. Using Northern blotting, RNase protection assay, and nuclear run-on analyses, we show that the up-regulation of ASM expression is regulated mainly at the level of transcription and that new protein synthesis is required for enhanced ASM activity. This cell-type specific induction by PMA treatment was further investigated with respect to transcriptional control. A series of 5' deletion derivatives of the upstream regulatory region were used in transient transfection assays to identify promoter elements required for basal and inducible gene expression. A PMA responsive element was localized to a region between -319 and -219 bp upstream of the initiation codon and co-transfections with transcription factor expression plasmids for AP-2 and Sp1 resulted in augmented ASM promoter activity, which was abolished when the binding sites for these two factors were deleted. Using electrophoretic mobility shift assays and supershift assays we demonstrate that this region is specifically bound by Sp1 and AP-2. These factors are present in nuclear extracts prepared from both induced and uninduced THP-1 cells. However, the intensity of the complex formed appeared to increase when nuclear extracts from PMA-treated cells were used. From these studies, we conclude that a concerted action of the transcription factors AP-2 and Sp1 is essential for the up -regulation of ASM expression during the process of macrophage differentiation.

Base Sequence↗