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G Schmitz

Publications and source records attributed to G Schmitz.

At least 253 records · Page 14Linked to original sources
PubMed1988

Regulation of a transport operon promoter in Salmonella typhimurium: identification of sites essential for nitrogen regulation.

The promoter of nitrogen-regulated transport, argtr, has been mutationally altered in order to determine the features that are essential for its response to nitrogen availability. Deletions of all sequences upstream of position -44 or downstream of position +2 had no effect on nitrogen regulation of argTr. These deletions define a small region of 44 bp where all necessary features for nitrogen regulation are located. This region includes sequences highly homologous to the nif consensus promoter. Alteration of this particular sequence caused drastic changes in the response to changes of nitrogen availability, thus indicating that they are directly involved in regulation. This implies that the NtrC protein must also act within this small region of the promoter. The data are discussed in terms of current-hypotheses concerning nitrogen regulation. In addition, we have shown 1. that carbon regulation at this promoter must occur at a site upstream from the nitrogen promoter; 2. that nifA can replace ntrC in the regulation of argTr.

Arginine↗
PubMed1987

Nitrogen regulation of transport operons: analysis of promoters argTr and dhuA.

In Salmonella typhimurium the periplasmic permeases for histidine and for lysine-arginine-ornithine are regulated by nitrogen availability. The nature of the dhuA and argTr promoters of the operons coding for these permeases was analyzed by placing the galactokinase gene under their control (in vector pKO-1). argTr was found to respond to nitrogen regulation. We investigated the involvement of a mirror symmetry in argTr in its regulation by nitrogen. It had been postulated previously (Higgins and Ames 1982) that mirror symmetries might act as protein recognition sites important in regulation of gene expression. Here we demonstrate that the mirror symmetry in argTr is not involved in nitrogen control. Contrary to expectation, the galK gene was not regulated by nitrogen when it was placed under dhuA control. Here we propose a possible explanation for this finding.

Arginine↗
PubMed1987

Flow-cytometric determination of high-density-lipoprotein binding sites on human leukocytes.

In this method, leukocytes were isolated from 6 mL of EDTA-blood by density-gradient centrifugation and subsequently incubated with rhodamine isothiocyanate (RITC)-conjugated high-density lipoproteins (HDL). The receptor-bound conjugate particles were determined by fluorescent flow cytometry and compared with 125I-labeled HDL binding data for the same cells. Human granulocytes express the highest number of HDL binding sites (9.4 X 10(4)/cell), followed by monocytes (7.3 X 10(4)/cell) and lymphocytes (4.0 X 10(4)/cell). Compared with conventional analysis of binding of 125I-labeled HDL in tissue-culture dishes, the present determination revealed significantly lower values for nonspecific binding. In competition studies, the conjugate competes for the same binding sites as 125I-labeled HDL. With the use of tetranitromethane-treated HDL3, which fails to compete for the HDL receptor sites while nonspecific binding is not affected, we could clearly distinguish between 37 degrees C surface binding and specific 37 degrees C uptake of RITC-HDL3, confirming that the HDL receptor leads bound HDL particles into an intracellular pathway rather than acting as a "docking" type of receptor. Patients with familial dysbetalipoproteinemia showed a significantly higher number of HDL binding sites in the granulocyte population but normal in lymphocytes and monocytes, indicating increased uptake of cholesterol-containing lipoproteins. In patients with familial hypercholesterolemia, HDL binding was increased in all three cell types, indicating increased cholesterol uptake and increased cholesterol synthesis. The present method allows rapid determination of HDL binding sites in leukocytes from patients with various forms of hyper- and dyslipoproteinemias.

Binding, Competitive↗
PubMed1987

What's new in the pathology of atherosclerosis?

Intimal smooth muscle cell proliferation has been known to be the key event in the development of advanced lesions of atherosclerosis. Since the important role of macrophages in the lipoprotein metabolism has been detected, however, current interest focuses on the macrophage reaction in the arterial wall. Animal experiments have shown that blood monocytes become attached to certain endothelial areas and enter the intima, where they are transformed to macrophages. Subendothelial infiltration of monocytes is the earliest cellular event in the formation of fatty streaks. Transformed to macrophages, they incorporate LDL by receptor-mediated endocytosis and are thus transformed to foam cells. The majority of foam cells in the atherosclerotic plaque is derived from macrophages. Furthermore, the importance of macrophages in the regulation of the lipoprotein metabolism and cholesterol homeostasis is increasingly attributed to their secretory capacities. It has been shown in vitro that they can secrete apolipoprotein E which associates with cholesterol and HDL to form a lipoprotein complex, which targets resecreted cholesterol to the liver cells. Recently, apolipoprotein E secretion of macrophages has also been demonstrated immunohistologically in the human atherosclerotic plaque. Cell culture investigations revealed that macrophages secrete different growth factors for fibroblasts and smooth muscle cells. So they are probably able, among other factors, to initiate smooth muscle cell proliferation in the intima. While smooth muscle cell proliferation and matrix components in the arterial wall had occupied the center of interest in previous investigations, the current focus on the cellular reactions of endothelial cells and monocytes/macrophages, especially in the early stages of atherosclerotic plaque formation, seems well justified.

Animals↗
PubMed1987

Cell surface distribution and intracellular fate of human beta-very low density lipoprotein in cultured peritoneal mouse macrophages: a cytochemical and immunocytochemical study.

The receptor-mediated endocytosis pathway of colloidal gold labeled beta-very low density lipoprotein (beta-VLDL-Au) derived from patients with familial dysbetalipoproteinemia was analyzed at the ultrastructural level in macrophages. The results showed that beta-VLDL-Au complexes were specifically recognized by a cell surface receptor of the macrophages. beta-VLDL-Au particles once bound to the randomly distributed cell surface receptors clustered in coated pits and were taken up by coated vesicles. Subsequently, the beta-VLDL-Au particles passed through tubular structures and small endosomes before deposited into large electron lucent smooth surfaced endosomes. As revealed by ruthenium red and enzyme cytochemistry the endosomes appeared to be separated from the extracellular space and did not contain acid phosphatase. There were no clear signs of passage of beta-VLDL through the Golgi complex. The accumulation of many flocculated gold particles within Ac-Pase positive vesicles suggests that beta-VLDL once internalized by the macrophages is diverted into a degradative pathway. Incubation of beta-VLDL-loaded macrophages with the hydrophobic fluorescent dye nile red revealed numerous large fluorescent bodies within the cells indicating that the macrophages accumulate large amounts of lipid droplets with time. Additional studies large amounts of lipid droplets with time. Additional studies with native beta-VLDL in conjunction with postembedding immunocytochemical techniques were used to delineate further the intracellular pathway. Immunolabeling was carried out on thin sections of LR White embedded cells using affinity-purified polyclonal rabbit antibodies against apolipoprotein B with the protein A-gold or goat anti-rabbit IgG-gold technique. Indirect visualization of beta-VLDL by these immunocytochemical studies yielded results comparable to those with gold-labeled beta-VLDL. On the basis of both indirect immunocytochemical and direct cytochemical localization of beta-VLDL it is concluded that although colloidal gold labeling of beta-VLDL molecules unquestionably modifies their morphology, their function appears to be unaltered, at least with respect to the process of receptor-mediated endocytosis.

Animals↗
PubMed1987

Interaction of Tangier lipoproteins with cholesteryl ester-laden mouse peritoneal macrophages.

Cholesterol efflux was studied from cholesteryl esterladen mouse peritoneal macrophages in the presence of Tangier lipoproteins derived from fasting and postprandial sera of three patients homozygous for Tangier disease (analphalipoproteinemia). The d greater than 1.063 g/ml fractions isolated from fasting patients and 3 hr and 18 hr after an oral fat load were all effective in cellular cholesterol removal. By contrast, the d greater than 1.063 g/ml fractions isolated 6 hr and 12 hr after fat ingestion did not affect net removal of cellular cholesterol. The d greater than 1.21 g/ml protein fractions derived from fasting as well as postprandial sera were all effective in removing cholesterol. D 1.063-1.21 g/ml fractions from fasting Tangier patients contained HDLT. In the corresponding postprandial fractions, in addition to HDLT, apoB-100- and apoB-48-containing lipoproteins were present. Furthermore, the 6 hr and 12 hr postprandial Tangier HDL fractions contained apoB-immunoreactive proteins of lower molecular weight. The abnormal activity of the elastase/alpha 1-antitrypsin proteolytic system and the abnormal fibronectin concentration we found in Tangier plasma suggests a possible relationship to the in vivo degradation of apoB. The peculiar type of membrane-bound lipid droplets in Tangier splenic macrophages points to a lipoprotein source of lipid accumulation which possibly originates from the uptake of chylomicrons or chylomicron-derived particles. It is concluded that cholesteryl ester storage in Tangier macrophages results from an imbalance of cholesterol influx and efflux. In the absence of HDL, the net increase of cholesterol caused by abnormal lipoproteins in certain postprandial states cannot be fully compensated by effective efflux and ultimately leads to macrophage cholesteryl ester accumulation.

Adult↗
PubMed1986

Role of the high density lipoprotein-receptor cycle in macrophage-cholesterol metabolism.

The intracellular catabolism of lipids and the blood cholesterol level are known to have an essential impact on the onset and progress of atherosclerosis. As lipoprotein receptors can influence the co-operation of macrophages, high density lipoproteins and enzymes of lipid metabolism, the contribution of these receptors to transport and intracellular processing of cholesterol are investigated by means of biochemical analysis and electron microscopy. Taking the effect of second messenger systems into consideration, a model for impaired lipoprotein receptor function affecting the cholesterol metabolism is developed.

Animals↗
PubMed1986

Radiation stability of organic matter in liquid and frozen H2O, NH3 and water-ammonia mixtures.

The redox properties of irradiated liquid and frozen H2O, NH3 and H2O/NH3 mixtures at 298 K and 77 K, resp., towards some simple organic molecules have been checked by injecting carrierfree 11C atoms and analyzing their chemical state by means of radiochromatography. The reactions and the stability of organic products versus radiation dose (in this study by MeV protons) depend on temperature, phase state, mobility of radicals, their concentration and reactivity. Especially dangerous are the reactive OH and O2H radicals which oxidize organic material to inorganic CO2. Highest stability has been found at low temperatures (solid state, reduced mobility of radicals) and for systems containing H-donors (H2O/NH3 mixtures), which reduce the concentration of oxidizing radicals. The fact that many bodies in space consist of H2O-ice with NH3 and CH4 additives at temperatures between 10 and 150 K is promising in view of the survival of organic matter under high doses of radiation.

Ammonia↗
PubMed1985

Analytical capillary isotachophoresis: a routine technique for the analysis of lipoproteins and lipoprotein subfractions in whole serum.

A capillary isotachophoretic separation technique was developed for lipoproteins in native serum which, compared with previous electrophoretic techniques, has negligible molecular sieve effects, does not need gel casting, is suitable for whole serum and has a high discriminative power for lipoprotein subfractions. The technique is based on pre-staining whole serum lipoproteins for 30 min at 4 degrees C before separation of 0.5 microliter of the sample in a free-flow capillary system (0.5 mm I.D.) with discontinuous buffer system. In normolipidaemic sera, high-density (HDL) and low-density lipoproteins (VLDL) are separated into two major subpopulations according to their net electric mobility. The identification of these fractions was confirmed by substitution with ultracentrifugally isolated lipoproteins and by their complete absence from Tangier and abetalipoproteinaemic serum. Triglyceride-rich very low-density lipoproteins (VLDL) revealed a defined zone between the HDL and LDL subpopulations. Our preliminary results indicate that the separation of human whole serum lipoproteins by capillary isotachophoresis is a promising method for the determination of lipoprotein subfractions.

Abetalipoproteinemia↗
PubMed1985

Interaction of high density lipoproteins with cholesteryl ester-laden macrophages: biochemical and morphological characterization of cell surface receptor binding, endocytosis and resecretion of high density lipoproteins by macrophages.

Morphological and biochemical experiments were carried out to investigate the interaction of human serum high density lipoproteins (HDL) with mouse peritoneal macrophages. It is demonstrated that resident mouse peritoneal macrophages express HDL receptors. Subsequent to receptor-mediated binding, HDL are internalized and intracellularly transported into endosomes. These endosomes do not fuse with the lysosomal compartment but interact with the margin of intracellular plasma lipid droplets. Macrophages do not degrade, but rather resecrete internalized HDL particles as described for the transferrin-receptor pathway. HDL binding to freshly isolated macrophages is saturable at a concentration of approximately 320 ng HDL-protein/mg cell protein and a Scatchard plot indicates the presence of some 130 000-190 000 receptors/cell with a Kd of approximately 9 X 10(-7) M. Binding of HDL on the macrophage surface is significantly enhanced in cholesterol-laden macrophages, whereas the increase in the rate of uptake and secretion is less pronounced. Within the HDL fraction the HDL2 subclass showed higher binding, uptake and secretion activity as compared with HDL3. From these experimental data we postulate that cholesterol uptake from macrophages is mediated by HDL particles which interact with these cells via a receptor-mediated retroendocytosis pathway.

Animals↗
PubMed1985

Regulation of high density lipoprotein receptors in cultured macrophages: role of acyl-CoA:cholesterol acyltransferase.

The interaction of human serum high density lipoproteins (HDL) with mouse peritoneal macrophages and human blood monocytes was studied. Saturation curves for binding of apolipoprotein E-free [125I]HDL3 showed at least two components: non-specific binding and specific binding that saturated at approximately 40 micrograms HDL protein/ml. Scatchard analysis of specific binding of apo E-free [125I]-HDL3 to cultured macrophages yielded linear plots indicative of a single class of specific binding sites. Pretreatment of [125I]HDL3 with various apolipoprotein antibodies (anti apo A-I, anti apo A-II, anti apo C-II, anti apo C-III and anti apo E) and preincubation of the cells with anti-idiotype antibodies against apo A-I and apo A-II prior to the HDL binding studies revealed apolipoprotein A-I as the ligand involved in specific binding of HDL. Cellular cholesterol accumulation via incubation with acetylated LDL led to an increase in HDL binding sites as well as an increase in the activity of the cytoplasmic cholesterol esterifying enzyme acyl-CoA:cholesterol acyltransferase (ACAT). Incubation of the cholesterol-loaded cells in the presence of various ACAT inhibitors (Sandoz 58.035, Octimibate-Nattermann, progesterone) revealed a time- and dose-dependent amplification in HDL binding and HDL-mediated cholesterol efflux. It is concluded that the homeostasis of cellular cholesterol in macrophages is regulated in part by the number of HDL binding sites and that ACAT inhibitors enhance HDL-mediated cholesterol efflux from peripheral cells.

Animals↗
PubMed1985

Tangier disease: a disorder of intracellular membrane traffic.

The interaction of human high density lipoproteins (HDL) with isolated human monocytes from control and Tangier patients was studied in tissue culture experiments. It was observed that normal monocytes, similar to mouse peritoneal macrophages, bind HDL to a cell surface receptor, internalize the bound HDL particles, transport the internalized HDL intracellularly through the cytoplasmic compartment without significant degradation, and ultimately resecrete intact HDL. The cellular interaction of Tangier monocytes with normal HDL was markedly different from control monocytes. HDL binding to Tangier monocytes was moderately increased and cell-associated HDL radioactivity was 6- to 10-fold enhanced in Tangier monocytes. The bulk of the internalized HDL, however, was detected in secondary lysosomes. Only minor amounts of the internalized HDL were resecreted from the Tangier monocytes, and most of this was degraded. These data suggest that the cellular metabolism of HDL is abnormal in Tangier monocytes. It is postulated that Tangier disease may be a disorder of intracellular membrane traffic in which HDL is diverted into the lysosomal compartment and degraded instead of being secreted through its regular transcellular route.

Adolescent↗
PubMed1985

Cell-bound and secreted proteases of Serratia marcescens.

Exoprotease of Serratia marcescens ATCC 25419 is exceptional among members of the family Enterobacteriaceae in that it is secreted in large amounts by viable cells into the culture medium. Labeling of cells with radioactive amino acids revealed no intracellular protein that could be precipitated with antibodies raised against purified exoproteases. With substances known to interfere with the excretion of some proteins--tosyl-L-lysine chloromethyl ketone, phenethyl alcohol, procaine, and sodium azide--and with rifampin, an intracellular form (apparent molecular weight, 52,000) larger than the major exoform (molecular weight, 51,000) was identified. Moreover, the 52,000-molecular-weight form was the main protein in immunoprecipitates of a cysteine-auxotrophic mutant starved for cysteine. Beside the major exoform, protease I, two additional exoproteases, termed II and III, appeared in the medium of stationary cultures. They were precipitated by antibodies against protease I, were identical in the Ouchterlony double-diffusion assay, and exhibited only a small difference, if any at all, in the peptide pattern after partial hydrolysis with protease V8 of Staphylococcus aureus. The amino- and carboxy-terminal amino acid sequences of protease I and II were determined and found to be identical, NH2-Ala-Ala-Thr-Gly-Gly-Tyr-Asp-Ala-Val-Asp and Phe-Ile-Val-COOH, respectively. The microheterogeneity of the isolated exoforms revealed by anion-exchange chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis was also observed in samples pulse-labeled with radioactive amino acids. It remains to be determined whether the different protease forms are the result of processing (modification) reactions or whether they constitute isoenzymes encoded by very similar genes.

Amino Acid Sequence↗
PubMed1985

Receptor domains in the plasma membrane of cultured mouse peritoneal macrophages.

Using the platinum-carbon surface replication technique, the distribution of receptors for two colloidal gold labeled lipoproteins (acetylated low density lipoprotein and high density lipoprotein), iron-saturated transferrin and bovine serum albumin in the plasma membrane of cultured mouse peritoneal macrophages was mapped. The plasma membrane surface of cultured peritoneal macrophages exhibits three morphologically distinct regions which have shown differences in the distribution, density and dynamics of these receptors. The flat border of the plasma membrane surfaces are the domains that appear to have the highest concentration of transferrin receptors, whereas the intermediate regions contain most of the receptors for acetylated low density lipoprotein and high density lipoprotein. Bovine serum albumin receptors are concentrated at the edge of the cells and had a uniform distribution on the rest of the cell surface. The functional significance and the mechanisms by which these regional differences in the distribution of the receptors are generated and then maintained are not yet known.

Animals↗