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G Schmitz

Publications and source records attributed to G Schmitz.

At least 163 records · Page 9Linked to original sources
PubMed1997

Molecular cloning and characterization of a novel putative carboxylesterase, present in human intestine and liver.

A full-length cDNA coding for a putative intestinal carboxylesterase (iCE) was isolated from a human small intestine cDNA library. The cDNA has an open reading frame of 559 amino acids with up to 65% homology to other carboxylesterases of different mammalian species. The deduced amino-acid sequence contains many structural features, that are highly conserved among all carboxylesterase isoenzymes, like the serine esterase active site, an ER-retention signal and one Asn-Xxx-Thr site for N-linked carbohydrate addition. Northern blot analysis revealed that the corresponding mRNA is 3.4-3.6 kb in size and is preferentially expressed in human intestine with a weak signal also in liver. Analysis of cells from the gastrointestinal tract unveiled site-specific, transcriptional regulation of iCE, with higher expression in small intestine and lower expression in colon and rectum. The high expression in small intestine is attributable to a higher expression in jejunum compared to duodenum and ileum.

Amino Acid Sequence↗
PubMed1997

Differentiation-dependent expression of a human carboxylesterase in monocytic cells and transcription factor binding to the promoter.

Carboxylesterases play an important role in defense and clearance mechanisms of the monocyte/macrophage system. During the differentiation process of cells from the monocytic cell line THP-1 we observed a transient transcriptional upregulation of a human carboxylesterase analyzed by means of Northern blots. In PMA-treated THP-1 cells we could detect three major transcription initiation sites as revealed by Nuclease Protection Assay carried out with two overlapping antisense RNA probes. We have recently cloned the carboxylesterase upstream sequence and showed its basal promoter activity in CHO cells. Using electrophoretic mobility shift analysis we demonstrated that the promoter region spanning base pairs -1 to -275, which contains several putative binding sites for transcription factors, is bound by nuclear factors Sp1 and IRBP but not by C/EBPs. Taken together these data indicate that carboxylesterase gene transcription in THP-1 cells starts at multiple initiation sites and that Sp1 and IRBP may be critical factors for modulating the differentiation-dependent transcription of this human carboxylesterase gene.

Animals↗
PubMed1997

Structural organization and characterization of the promoter region of a human carboxylesterase gene.

A gene encoding a human liver carboxylesterase has been isolated and characterized. Analysis of three overlapping genomic lambda clones revealed that the gene spans about 30 kb and is made of 14 exons being 39 to 379 bp in length. The encoded protein is 550 amino acids long and is highly homologous to carboxylesterases of various mammalian species. The transcription start site was determined by 5'-RACE PCR. An additional 900 bp of DNA from the 5' flanking region of the gene was cloned and sequenced in order to elucidate the structure of the promoter. In this sequence several possible binding sites for transcription factors have been identified, but no TATA-box was present. When different parts of the putative promoter region were ligated in front of the luciferase gene and the constructs were transfected into CHO cells, the reporter gene was effectively transcribed, as demonstrated by the expression of enzyme activity.

Amino Acid Sequence↗
PubMed1997

Analytical capillary isotachophoresis of human serum lipoproteins.

An analytical free flow capillary isotachophoresis (cITP) procedure for the detailed analysis of lipoproteins on commercially available capillary electrophoresis systems has been developed. The technique is based on the specific staining of lipoproteins with the fluorescent lipophilic dye 7-nitrobenz-2-oxa-1,3-diazole (NBD)-ceramide before separation. Prestained lipoprotein samples are applied between leading and terminating buffer and separated into 9 well-characterized subpopulations according to their electrophoretic mobility in the absence of any molecular sieve effect. High density lipoproteins are separated into three major subpopulations: (i) the fast migrating high density lipoprotein (HDL) subpopulation (alpha-HDL, containing mainly apolipoprotein (apo) A-I and phosphatidylcholine, (ii) the subpopulation with intermediate mobility, consisting of particles rich in cholesterol, apo A-II, apo E and C apolipoproteins, and (iii) the slow migrating HDL subpopulation (pre-beta-HDL), containing particles rich in apo A-I, apo A-IV. The majority of HDL-associated lecithin:cholesterol acyltransferase (LCAT) activity is also associated with the last subpopulation. The apo B-containing lipoproteins can be subdivided into three major functional groups. The first represents chylomicron derived particles and large triglyceride-rich very low density lipoproteins (VLDL). The second group consists of small VLDL and intermediate density lipoprotein (IDL) particles, and the third group represents the low density lipoproteins (LDL). Results obtained by the isotachophoretic lipoprotein analysis revealed a good correlation in the range of HDL with routinely used techniques, like lipoprotein electrophoresis, HDL-cholesterol analysis by a precipitation procedure or turbidimetric determination of apo A-I. Similar correlations with other analytical techniques were found for the quantitation of the apo B-containing lipoproteins. Advantages of the isotachophoretic separation compared to zone electrophoresis are the high resolution combined with small sample volumes. Moreover, lipoprotein analysis can be performed directly from whole serum, plasma, lymph and other biological fluids in a short time. With these characteristics analytical capillary isotachophoresis may be a helpful tool for a fast and reliable automated quantitation of lipoprotein subpopulations in the clinical laboratory.

4-Chloro-7-nitrobenzofurazan↗
PubMed1997

Cytokine removal and cardiovascular hemodynamics in septic patients with continuous venovenous hemofiltration.

OBJECTIVES: To determine whether continuous venovenous hemofiltration leads to extraction of tumor necrosis factor alpha (TNF alpha) and cytokines from the circulation of critically ill patients with sepsis and acute renal failure and to quantitate the clearance and the removal rate of these cytokines and their effect on serum cytokine concentrations. DESIGN: Prospective, controlled study in patients with continuous venovenous hemofiltration (24 1/24 h) using a polysulphone membrane in patients with acute renal failure. PATIENTS: 33 ventilated patients with acute renal failure of septic (n = 18) and cardiovascular origin (n = 15) were studied. INTERVENTIONS: Hemodynamic monitoring and collection of blood and ultrafiltrate samples before and during the first 72 h of continuous hemofiltration. MEASUREMENTS AND MAIN RESULTS: Cardiovascular hemodynamics (Swan-Ganz catheter), Acute Physiology and Chronic Health Evaluation II score, creatinine, electrolytes, and blood urea nitrogen were recorded daily. Cytokines (TNF alpha, TNF alpha-RII, interleukin (IL) 1beta, IL1RA, IL2, IL2R, IL6, IL6R, IL8, IL10) were measured in prefilter blood and in ultrafiltrate immediately preceding and 12, 24, 48, and 72 h after initiating continuous venovenous hemofiltration (CVVH). Septic patients showed elevated cardiovascular values for cardiac output (7.2 +/- 2.1 l/min), cardiac index (4.2 +/- 1.3 l/min per m2), and stroke volume (67 +/- 23 ml) and reduced values for systemic vascular resistance (540 +/- 299 dyn x s x cm(-5)). All hemodynamic values normalized within the first 24 h after initiating CVVH treatment. TNF alpha was 1833 +/- 1217 pg/ml in septic patients and 42.9 +/- 6.3 pg/ml in nonseptic patients (p < 0.05) prior to CVVH. TNF alpha was detected in ultrafiltrate but did not decrease in blood during treatment with CVVH. There was no difference in IL 1beta between septic (3.8 +/- 1.9 pg/ml) and nonseptic patients (1.7 +/- 0.5 pg/ml). No significant elimination of cytokines was achieved in the present study by CVVH treatment. CONCLUSIONS: These findings demonstrate that CVVH can remove TNF alpha and special cytokines from the circulation of critically ill patients. Cardiovascular hemodynamics seemed to improve in septic patients after induction of hemofiltration treatment, although there was no evidence that extracorporeal removal of cytokines achieved a reduction in blood levels. The study indicates that low volume continuous hemofiltration with polysulphone membranes in patients with acute renal failure is not able to induce significant removal of cytokines.

APACHE↗
PubMed1997

Altered mononuclear phagocyte differentiation associated with genetic defects of the lysosomal acid lipase.

Multiparameter flow cytometry reveals a complex heterogeneity of mononuclear phagocyte differentiation within the peripheral blood compartment. In this study, the relation of abnormal cellular lipid metabolism to the phenotype of peripheral blood mononuclear phagocytes, which finally may be related to atherogenesis, was analyzed using recently characterized autosomal recessive defects of lysosomal acid lipase (LAL) expression as model system. The reduction of LAL activity in nine heterozygote, disease free carriers of mutations from two cholesteryl ester storage disease (CESD) pedigrees and the family of a patient with Wolman disease was associated with an increased fraction of monocytes which expressed CD56 (N-CAM) (4.1 +/- 2.7% of monocytes, compared to 2.2 +/- 0.5% in ten controls, P < 0.05), an antigen characteristic of immature myeloid cells, suggesting an increased turnover of monocytes. Furthermore, a trend was observed towards an enhanced blood pool of more mature mononuclear phagocytes which show decreased expression of the 55 kD lipopolysaccharide receptor (CD14) together with either expression of the Fc-gamma-receptor III (CD16) or a high expression of CD33. A similar phenotype of peripheral mononuclear phagocytes was observed in the two CESD patients analyzed. In conclusion, our data suggest that these monogenetic defects of lysosomal lipoprotein metabolism are associated with complex alterations of mononuclear phagocyte differentiation and extravasation.

Adolescent↗
PubMed1997

A novel variant of lysosomal acid lipase in cholesteryl ester storage disease associated with mild phenotype and improvement on lovastatin.

Cholesterol ester storage disease (CESD) is a rare congenital disorder of lipid metabolism, with mutation of the lysosomal acid lipase gene, causing chronic liver disease, usually before adolescence. We here describe three adult siblings with CESD diagnosed by light microscopic demonstration of excessive lysosomal storage of lipids with accumulation of foamy cells in liver biopsies and by a decrease in acid lipase activity (2-3% of controls). One patient (male, 46a) had extensive liver fibrosis, another (female, 58a) had cirrhosis of the liver. The third patient had died from variceal haemorrhage (female, 56a). Using sequence analysis of RT-PCR products of LAL mRNA, the patients were identified as compound heterozygotes for a G-->A substitution at position -1 of the exon 8 splice donor site and a point mutation at the second allele, resulting in a His108-->Pro shift. In two patients, therapy with lovastatin was initiated, which led to normalisation of serum cholesterol and triglyceride levels. After 12 months, liver biopsy demonstrated a significant decrease in vacuolisation of hepatocytes, with fewer and smaller droplets. Semi-automated computer-assisted image analysis of electron microscopic sections demonstrated a decrease in the hepatocellular lysosomal area from 20.5+/-7.1% to 11.7+/-6.5% (p<0.05) and 41.7+/-5.1% to 33.4+/-4.4% (p<0.01). We conclude that in two siblings with a novel LAL variant and mild phenotype of CESD, lovastatin decreased both serum lipid concentrations and hepatocellular lysosomal content.

Anticholesteremic Agents↗
PubMed1997

Predictors of blood loss after coronary artery bypass grafting.

OBJECTIVE: To assess the predictive value of variables possibly associated with blood loss after coronary artery bypass grafting (CABG). DESIGN: A prospective study. SETTING: A university hospital. PARTICIPANTS: Eighty-nine patients scheduled for elective CABG. INTERVENTIONS: Blood samples were drawn before and after surgery. Chest tube drainage was measured hourly until removal of drains. MEASUREMENTS AND MAIN RESULTS: Activation of coagulation and fibrinolysis, routine clotting tests, and expression of platelet surface antigens were analyzed using flow cytometry. A significant correlation was found among blood loss and activated partial thromboplastin time, fibrinogen, prothrombin fragment 1 + 2, D-dimers, platelet count, GPIb and P-selectin expression on platelets, use of internal thoracic artery, cross-clamp time, and thrombin-antithrombin III complex. In a multiple regression model, glycoprotein (GP) Ib expression on platelets, platelet count, use of internal thoracic artery, and D-dimers were significantly associated with blood loss. Logistic regression analysis showed that GPIb and D-dimers predicted an increased blood loss with a positive predictive value of 73% and a negative predictive value of 91%. CONCLUSIONS: Postoperative D-dimers and GPIb expression may be useful to exclude nonsurgical causes in bleeding patients after CABG.

Aged↗
PubMed1997

Enhanced expression of HLA-DR, Fc gamma receptor 1 (CD64) and leukocyte common antigen (CD45) indicate activation of monocytes in regenerative training periods of endurance athletes.

The aim of this study was to test the hypothesis whether endurance athletes have distinct expressions of functionally relevant surface antigens of monocytes. Under standardized experimental and laboratory conditions CD 14+ blood monocytes of 55 males (22 sedentary controls [CO], individual anaerobic threshold [IAT]: 2.2 +/- 1.2 W.kg-1, VO2 max: 47 +/- 12 ml.min-1.kg-1; 13 endurance athletes, training: 3-10 hours weekly [E1], IAT: 3.0 +/- 1.1 W.kg-1, VO2max 57 +/- 5 ml.min-1.kg-1; 20 endurance athletes training: > 10 hours weekly [E2], IAT: 3.7 +/- 1.3 W.kg-1, VO2max: 64 +/- 6 ml.min-1.kg-1) were investigated for relative surface receptor expression of CD45, CD64 and HLA-DR (direct immunofluorescence, flow cytometry). E1 and E2 showed significantly (ANOVA, Tukey's HSD test; each p < 0.01) higher relative receptor expressions for CD64 in comparison to CO (E1: +62/E2: +71%; percentage of increase compared to CO value), CD45 (+44/+32%) and HLA-DR (+22/+35%). Cell counts of four monocyte subpopulations (CD56+, CD14bright+CD16-, CD14bright+CD16dim+, CD14dim(+)-CD16bright+) did not show significant differences between the groups. These data suggest that in endurance athletes during regenerative training period circulating monocytes show an enhancement of functionally relevant surface receptors indicating an activation monocytes.

Adult↗
PubMed1997

Scavenging, signalling and adhesion coupling in macrophages: implications for atherogenesis.

Integrins are in physical association and functional cooperation with other membrane proteins that include receptors with scavenger functions, glycosyl-phosphatidylinositol-linked receptors, the family of integrin associated multimembrane spanning signalling proteins and possibly other less characterized proteins with a coupling or signalling function. Monocyte adhesion, migration and differentiation to phagocytically active scavenger cells are directly coupled processes, involving integrins as common transducers for a panel of integrin-linked specific receptors, which assemble a master cluster to coordinate adhesion, migration, scavenging and associated metabolic pathways of the lysosomal and secretory route, and also processes involved in host response. As macrophages represent highly heterogeneous cells that have major phenotypical and functional differences associated with specific patterns of integrin expression, the functional cooperation of integrins with scavenger receptors has to be related to specialized subsets of monocytes and macrophages as well as to ligand specific effects which mediate receptor coupling.

Animals↗
PubMed1997

Autoantibodies against the protease inhibitor calpastatin: a new risk factor for venous thrombosis?

Autoantibodies reactive against human calpastatin were detected by screening a cDNA expression library with the serum of a 53 year old white female patient with a history of venous thrombosis and suspected antiphospholipid syndrome. When further sera were analyzed it could be shown that > 90% of calpastatin autoantibodies, detected by Western blotting against the partial calpastatin clone, react with the C-terminal amino acids of the protein. Therefore, an ELISA based on a synthetic peptide containing the C-terminal 27 amino acids of calpastatin was developed and 205 healthy blood donors and 138 random sera from hospital patients were analyzed. A total of 11 sera (3.2%) were positive with no significant difference between the two groups (7/205 and 4/138). In 80 consecutive patients with a history of venous thrombosis 9 positive sera (11.3%; p < 0.01 vs. blood donors, p < 0.02 vs. hospital patients) were detected. Our results indicate that autoantibodies against calpastatin may constitute a so far unknown risk factor for venous thrombosis.

Adolescent↗
PubMed1997

A multicentre evaluation of the CA 15-3 assay, CA 19-9 assay and CA 125 II assay on the Bayer Immuno 1 System.

The analytical performance of the tumour markers CA 15-3 assay, CA 19-9 assay and Ca 125 II assay on the Bayer Immuno I System was studied according to a revised version of the ECCLS guidelines (Haeckel R. In: Evaluation methods in laboratory medicine, Weinheim, VCH Verlag 1993:47-69) in a multicentre evaluation involving five laboratories. Determination of the 3 analytes generated more than 6000 data. On the Bayer Immuno I System, the imprecisions of the CA 15-3 assay, CA 19-9 assay and CA 125 II assay were better than those found for comparison methods. The median recovery over all five laboratories of system assigned values in control sera was within the 1-s range for the three tumour marker assays. No deviation of linearity could be detected experimentally for all assays. Results for patients' samples showed acceptable agreement between the Bayer Immuno 1 system and several different comparison methods in most cases. One exception was the CA 15-3 assay in comparison with the MCA assay from Roche Diagnostic Systems, where the large difference in values is due to the use of different antibodies and calibrators in the two assays. No carry-over effects could be detected. The selective Bayer Immuno 1 system is fully automated; its practicability was rated as high.

Antigens, Tumor-Associated, Carbohydrate↗
PubMed1997

[Effect of processed blood volume, leukocyte count and concentration of CD34-positive cells in peripheral blood on efficiency of stem cell apheresis].

Despite many published studies no parameter could be identified yet to acceptably and individually predict collection results in stem cell apheresis. We analyzed leukocyte counts and processed blood volume, absolute and relative CD34+ cell counts, and overall collection efficiency in 120 patients with hematological and solid malignancies (354 leukaphereses using the Cobe Spectra cell separator, a median of 3 per patient, span 1-9). Stem cells were mobilized into peripheral blood by conventional chemotherapy followed by daily doses of G-CSF. CD34+ progenitor cell counts were monitored through multiparametric flow cytometry. Blood and collection flows varied in the range of 45-90 ml/min and 0.7-1.5 ml/min, respectively. CD34+ progenitor cells were enriched 38-fold in the apheresis product as compared to peripheral blood at a processed blood volume lower than one total blood volume. Efficiency continuously declined, on to a 25-fold concentration at a processed blood volume above the 3-fold total blood volume. Total collection efficiency, calculated from the absolute content of CD34+ progenitor cells in peripheral blood and apheresis concentrate (a parameter for progenitor cell mobilization during the apheresis), reached a plateau at a processed blood volume above the 3-fold total blood volume. However, variation among individual patients was high. The concentration rate of CD34+ cells at a leukocyte count below 5,000/microliter averaged 50 and declined continuously to 8 at leukocyte counts between 45,000 and 50,000/microliter. To summarize, in 70% of patients with leukocyte counts below 5,000/microliter and CD34+ progenitor cell counts above 10,000/ml, more than 1.5 x 10(6) progenitors per kg body weight could be collected in a single leukapheresis. According to the presented data, the variation in overall collection efficiency is mainly due to: 1) varying mobilization of progenitors during the apheresis procedures itself and 2) dependence on peripheral leukocyte counts.

Antigens, CD34↗
PubMed1997

[Specific Lp(a) apheresis for secondary prevention of arteriosclerosis].

Lipoprotein(a) (Lp(a)) is an independent risk factor for arteriosclerosis. It consists of the Lp(a)-specific apo(a) which is bound to the apo-B of an LDL particle by a disulfide bridge. Apo(a) is homologous to parts of the plasminogen molecule: It consists of one kringle 5 and 10-40 kringles 4 of the plasminogen molecule. Due to the lack of alternative drug treatment, 3 patients with early onset of arteriosclerosis, rapid progression, and elevated Lp(a) as their dominating risk factor were treated weekly with specific Lp(a)-aphereses. Since October 1992, we carried out 229 immunoadsorptions (IA) with specific columns containing anti-Lp(a) antibodies covalently bound to sepharose. To reduce Lp(a) from preapheresis values of 142 +/- 53 mg/dl to 25 +/- 11 mg/dl immediately after apheresis, we had to adsorb 1.4-3 patient's plasma volumes. Lp(a) rise to preapheresis values took 3-4 days. Protein reduction caused by loss of plasma during column changes remained tolerable (total protein before IA: 71 +/- 4 g/l, after IA: 56 +/- 4 g/l. Immediately after IA, these values were measured after the application of 991 +/- 207 ml of ACDB with 5,000 IU of heparin as anticoagulant. Hemoglobin remained unchanged (before IA: 13.4 +/- 1.4 g/dl, after IA: 13.6 +/- 1.5 g/dl). Side effects were mainly flush and tachycardia. They were seen especially when using new columns and plasma flow rates above 55 ml/min and were immediately reverted by interrupting the IA. Control angiography performed after 2 years in 2 patients showed no progression of disease, in the 3rd patient, the stress test showed a significant improvement as did clinical parameters. In our hands, IAs are a safe and efficient method for Lp(a) reduction and secondary prevention of myocardial infarction. Therapeutic efficiency should further be proven by a controlled trial.

Adult↗
PubMed1997

[Clinical and flow cytometry outcome of improved photopheresis methods in dermatologic patients].

In photopheresis, 8-methoxypsoralen (8-MOP) is added to a mononuclear cell concentrate and then activated by UVA light, thus forming covalent bonds between DNA strands. Infusion of these modified cells that are not able to replicate any more seems to lead to the elimination of pathogenic T-cell clones and clinical improvement in patients suffering from cutaneous T-cell lymphoma, graft versus host reactions, and various autoimmune diseases. The original method described by Edelson et al. (1987) was improved by the following modifications proposed by Andreu et al. (1994): i) by using a cell separator (COBE Spectra) that produced purer concentrate with less red blood cells absorbing UVA light. ii) By applying 8-MOP directly into the collection bag, the drug side effects due to the oral application of the thousandfold dose and varying serum levels were avoided. iii) By irradiating the concentrate after collection, all cells received the same irradiation dosage. We treated 2 men with cutaneous T-cell lymphoma, 2 women with atopic eczema and 1 man with severe pustular psoriasis with overall 123 extracorporeal photochemotherapy (ECPC) sessions. In addition to routine laboratory analysis, detailed characterization of lymphocyte subpopulations was carried out by flow cytometry to differentiate T-helper and T-suppressor cells and their activation (HLA-DR, CD 25), B, NK cells, and monocyte subpopulations. The following mediators soluble were analyzed as well: Il-2-receptor and as indicators of acute phase reaction Il-8, neopterin and Il-1 beta. We observed a good clinical improvement independent of no significant trend in the immunological phenotype of circulating blood leukocytes. Our results suggest that ECPC effects are not mediated by a systemic immune response or alternatively are not measured in the blood compartment.

Dermatitis, Atopic↗
PubMed1996

Isolation and characterization of a 14.5-kDa trichloroacetic-acid-soluble translational inhibitor protein from human monocytes that is upregulated upon cellular differentiation.

A trichloroacetic-acid-soluble 14.5-kDa protein (p14.5) has been isolated from human mononuclear phagocytes (MNP) by a combination of trichloroacetic acid extraction, preparative electrophoresis and hydrophobic affinity chromatography; five tryptic peptides were subjected to protein sequencing. The full-length cDNA of the protein was cloned and sequenced from a lambda gt11 human liver library. The cDNA showed a remarkable similarity to a rat protein preferentially expressed in hepatocytes and renal tubular epithelial cells. The encoded protein is 137 amino acids long and similar to members of a new hypothetical family of small proteins with presently unknown function, named YER057c/YJGF. Human recombinant p14.5 inhibits in vitro protein synthesis in a rabbit reticulocyte lysate system. Unlike other inhibitors of protein synthesis, p14.5 is not phosphorylated despite the presence of putative phosphorylation sites. The p14.5 mRNA is weakly expressed in freshly isolated monocytes but is significantly upregulated when these monocytes are subjected to differentiation. This is also reflected by a differentiation-dependent increase in the protein concentration as demonstrated by immunoblots from cytosolic fractions and fluorescence-activated flow cytometry of permeabilized cells. A differentiation-dependent mRNA and protein expression of p14.5 is further suggested by the observation of a low expression in a variety of liver and kidney tumor cells and a high expression in fully differentiated cells as assessed by immunohistochemistry and northern blots. The highest mRNA expression was found in hepatocytes and renal distal tubular epithelial cells and only weak expression was found in other human tissues as evaluated by northern blot analysis. The preferential localization of the immunoreaction product seemed to be cytoplasmatic but, in less differentiated cells, nuclear labeling was occasionally visible. Immunoblotting of subcellular fractions confirmed these data. The high degree of evolutionary conservation of p14.5, the considerable upregulation during cellular differentiation and its potential role as a translational inhibitor may reflect an involvement in basic cellular mechanisms, e.g. a differentiation-dependent regulation of protein synthesis in hepatocytes, renal tubular epithelial cells, smooth muscle cells and MNP.

Amino Acid Sequence↗