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G Schmitz

Publications and source records attributed to G Schmitz.

At least 19 recordsLinked to original sources
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 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

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↗
PubMed1998

Nitrous oxide impairs the signaling of neutrophils downstream of receptors.

(1) Nitrous oxide has been shown to impair the oxidative function of neutrophils. (2) To characterize the type and the site of the drug interaction, receptor expression, cytosolic-free calcium, and H2O2 production of neutrophils were assessed using flow cytometry. (3) Nitrous oxide depresses receptor-dependent generation of H2O2 in a concentration-dependent manner. The response upon direct activation of protein kinase C (PKC) was unaffected. (4) No interference was found at the receptor sites, the linked G proteins, and the subsequent release of Ca2+, indicating a localization of the nitrous oxide interaction downstream of receptors and G proteins at or near to PKC.

Anesthetics, Inhalation↗
PubMed1998

Proof of hepatitis A virus negative-sense RNA by RNA/DNA-hybrid detection: a method for specific detection of both viral negative- and positive-strand RNA species.

The detection of hepatitis A virus (HAV) negative-strand RNA, which is synthesized during replication of the positive-strand RNA genome, proved to be difficult. We developed a method for the specific detection of HAV negative-strand RNA by RNA-DNA hybridization and luminescence detection using an anti-RNA:DNA hybrid antibody. This method, which is also applicable for the specific detection of positive-strand RNA, offers a simple, yet relatively rapid and certain means of detecting low amounts of RNA such as HAV negative-strand RNA. By using appropriate hybridization DNA probes, the method should be applicable for the detection of single-stranded RNA species of different viruses in general.

Animals↗
PubMed1998

Whole blood analysis of reticulated platelets: improvements of detection and assay stability.

The flow cytometric analysis of reticulated platelets based on the fluorescent derivatization of their RNA content is increasingly used for the diagnostic classification of patients with thrombocytopenia as well as the monitoring of thrombopoiesis recovering under therapy. Many different modifications of the analytical protocol have been published following the first description in 1990 but consensus on the method has not yet been established. We have now reevaluated the assay's methodology in order to optimize sensitivity and specificity and reduce the time length of incubation and washing procedures. In the modified experimental approach native whole blood is incubated for 15 min with an increased amount of thiazole orange (1 microg/ml) in the presence of phycoerythrin labeled antibodies directed against the constitutively surface expressed antigen GPlb. Data acquisition on the flow cytometer can be started immediately after stopping and stabilization of the reaction by paraformaldehyde fixation. Thiazole orange fluorescence was not significantly changed in thrombin-activated, degranulated platelets compared to resting platelets indicating no significant non-specific staining of platelet granules under the selected test conditions. In addition, experiments employing RNAse digestion demonstrated specificity of thiazole orange staining for platelet RNA.

Blood Cell Count↗
PubMed1998

Flow cytometric quantitation of immunofluorescence intensity: problems and perspectives. European Working Group on Clinical Cell Analysis.

Quantitation of immunofluorescence intensity serves to estimate the number of defined molecules expressed on or in cells. Clinical applications of this diagnostic tool are increasing, e.g., aberrant expression of various antigens (Ag) by leukemic blasts or lymphoma cells, intensity of CD38 expression by CD8+ T-lymphocytes to monitor activation status, and intensity of CD62P to detect platelet activation. In this report we discuss the quality-control measures required for quantitation of fluorescence intensity, and we review seven concepts that have been developed to quantify fluorescence intensity during the past 15 years. Initial work addressed the conversion of logarithmic channel numbers into units of relative fluorescence. The design and use of calibration beads labeled with predefined amounts of dye allowed instrument-independent expression of fluorescence intensity in units of molecules of equivalent soluble fluorochrome (MESF). This method was refined by the combined use of such standards with monoclonal antibodies (mAb) conjugated 1:1 with phycoerythrin (PE), allowing translation of fluorescence intensity into numbers of antibodies bound per cell. Alternatively, the use of 1:1 PE-conjugated mAb under the assumption that CD4+ lymphocytes reproducibly bind 50,000 CD4 mAb molecules was proposed to convert units of relative fluorescence intensity into units of antibodies bound per cell. The use of antibody-binding capacity as a surrogate marker for quantification of Ag expression was addressed more directly by the development of antibody-binding standards. The quantitative indirect immunofluorescence assay is based on beads labeled with various amounts of CD5 mAb that calibrate the binding of the secondary antibody in units of antibody-binding capacity. Alternatively, goat anti-mouse-labeled calibration beads have been developed. Published results obtained with the latter calibrators showed an unexpected inaccuracy. The different ways in which calibrators and cells under study bind mAb (i.e., Fab mediated versus Fc mediated) may have contributed to this variation. Recently, the use of stabilized cell populations expressing Ag in a specified range of concentrations has been proposed as an Ag-specific calibration system of mAb binding. We identify several issues on the level of instrumentation, reagents, and cells under study that should be solved to allow standardization of quantitative assessments of immunofluorescence intensity.

Animals↗
PubMed1998

Performance of calibration standards for antigen quantitation with flow cytometry.

In the frame of the activities initiated by the Task Force for Antigen Quantitation of the European Working Group on Clinical Cell Analysis (EWGCCA), an experiment was conducted to evaluate microbead standards used for quantitative flow cytometry (QFCM). An unified window of analysis (UWA) was established on three different instruments (EPICS XL [Coulter Corporation, Miami, FL], FACScan and FACS Calibur [Becton Dickinson, San Jose, CA]) with QC3 microbeads (FCSC, PR). By using this defined fluorescence intensity scale, the performance of several monoclonal antibodies directed to CD3, CD4, and CD8 (conjugated and unconjugated), from three manufacturers (BDIS, Coulter [Immunotech], and DAKO) was tested. In addition, the QIFI system (DAKO) and QuantiBRITE (BDIS), and a method of relative fluorescence intensity (RFI, method of Giorgi), were compared. mAbs reacting with three more antigens, CD16, CD19, and CD38 were tested on the FACScan instrument. Quantitation was carried out using a single batch of cryopreserved peripheral blood leukocytes, and all tests were performed as single color analyses. Significant correlations were observed between the antibody-binding capacity (ABC) values of the same CD antigen measured with various calibrators and with antibodies differing in respect to vendor, labeling and possible epitope recognition. Despite the significant correlations, the ABC values of most monoclonal antibodies differed by 20-40% when determined by the different fluorochrome conjugates and different calibrators. The results of this study indicate that, at the present stage of QFCM consistent ABC values may be attained between laboratories provided that a specific calibration system is used including specific calibrators, reagents, and protocols.

Antibodies, Monoclonal↗
PubMed1998

Identification and characterization of the human adipocyte apM-1 promoter.

The human adipocyte-specific apM-1 gene encodes a secretory protein of the adipose tissue and seems to play a role in the pathogenesis of obesity. A 1.3 kb amount of the proximal promoter region has been cloned and analyzed for the presence of putative transcription factor binding sites. Several binding sites known to be involved in adipogenesis and regulation of adipocyte-specific genes (C/EBP, SREBP) are present. No TATA box, but a classical CCAAT box could be identified. To confirm functionality and cell specificity of the 1.3 kb promoter, a series of 5'-deleted fragments were ligated in front of the luciferase gene and the constructs were transfected into 3T3-L1 adipocytes. The reporter gene was effectively transcribed, as demonstrated by the expression of enzyme activity. The 5'-end of the human cDNA was completed by 5'-RACE-PCR. Several alternative transcription start sites were detected by RNase protection assay and primer extension analysis. In addition, an exon/intron boundary was mapped at the extreme 5'-end of the cDNA sequence. Genomic Southern blotting suggests that the human apM-1 gene is a single copy gene.

Adipocytes↗
PubMed1998

Increased bioavailability of nitric oxide after lipid-lowering therapy in hypercholesterolemic patients: a randomized, placebo-controlled, double-blind study.

BACKGROUND: Impaired endothelium-dependent vasodilation is an early sign of atherosclerosis in hypercholesterolemic patients. We hypothesized that lipid-lowering therapy can improve endothelial function and that this effect is mainly mediated by increased bioavailability of nitric oxide (NO). METHODS AND RESULTS: In a randomized, double-blind, placebo-controlled trial, we studied 29 patients (age, 50+/-12 years) with hypercholesterolemia (LDL cholesterol > or = 160 mg/dL) randomly assigned to receive either fluvastatin (40 mg twice daily; 17 patients) or placebo (12 patients). Forearm blood flow was measured by plethysmography before and after 24 weeks of treatment. Endothelium-dependent vasodilation was assessed by intra-arterial infusion of acetylcholine (ACh; 3, 12, 24, and 48 microg/min) and basal NO synthesis rate by intra-arterial infusion of NG-monomethyl-L-arginine (L-NMMA; 1, 2, and 4 micromol/min). Simultaneous intra-arterial infusion of L-NMMA (4 micromol/min) and ACh (12, 24, and 48 microg/min) was used to test whether any increase in endothelium-dependent vasodilation after lipid-lowering therapy could be blocked by this NO synthase inhibitor. Endothelium-dependent vasodilation improved significantly after 24 weeks of lipid-lowering therapy compared with before therapy (ACh 24 microg/min: 240+/-34% before versus 347+/-50% after therapy; P< or =0.01) and placebo (changes between after and before therapy with ACh 24 microg/min: 108+/-39% for fluvastatin versus -26+/-32% for placebo; P< or =0.05). This improvement in endothelium-dependent vasodilation could be blocked by simultaneous administration of L-NMMA (ACh 24 microg/min plus L-NMMA 4 micromol/min: 170+/-69% before versus 219+/-47% after treatment; P=NS). CONCLUSIONS: Lipid-lowering therapy with fluvastatin can improve disturbed endothelial function in hypercholesterolemic patients compared with placebo. This improvement is mediated by increased bioavailability of NO.

Acetylcholine↗