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G Schmitz

Publications and source records attributed to G Schmitz.

At least 199 records · Page 11Linked to original sources
PubMed1995

High-density lipoprotein and low-density lipoprotein-mediated signal transduction in cultured human skin fibroblasts.

The signalling mechanisms whereby high-density lipoproteins (HDL) and low-density lipoproteins (LDL) affect a number of cellular functions in fibroblasts are unclear. This study has analyzed the influence of HDL3 and LDL on the phosphatidylinositol specific phospholipase C pathway in human skin fibroblasts. Exposure of myo-[2-3H]-inositol prelabelled fibroblasts to HDL3 or LDL elicited major increases in IP1 and minor increases in IP2 and IP3 within 30 s. In fura-2 loaded suspended fibroblasts, HDL3 and LDL increased intracellular Ca2+ concentrations ([Ca2+]i) with comparable rapid, transient kinetics. The dose-profiles for HDL3- and LDL-induced increases in [Ca2+]i were also comparable, with half-maximally and maximally effective concentrations being approximately 15 micrograms/mL and approximately 50 micrograms/mL, respectively. HDL3- and LDL-induced increases in [Ca2+]i were diminished by approximately 60% (vs. control fibroblasts) in thapsigargin-pretreated fibroblasts, indicating that release of Ca2+ from intracellular pools is the major contributor toward lipoprotein-induced increases in [Ca2+]i. Pertussis toxin-pretreatment of cells completely abolished lipoprotein induced Ca(2+)-transient, indicating the involvement of a guanine nucleotide-binding protein in the signalling process. In [3H]-palmitic acid-prelabelled fibroblasts, both HDL3 and LDL were observed to stimulate production of DAG. Activation of protein kinase C (PKC) was analysed by determining the cytosol-to-membrane translocation of both enzymatic activity and immunoreactivity of specific PKC isoforms (alpha, delta, epsilon, and zeta). Stimulation with HDL3 and LDL evoked a rapid (within 2.5 min) translocation of PKC activity, with PKC alpha and PKC epsilon being the isoforms translocated. It is concluded that HDL3 and LDL acutely stimulate a phosphoinositide-specific phospholipase C pathway in human skin fibroblasts. However, the specific cell membrane events mediating this signal transduction remain to be further elucidated.

Calcium↗
PubMed1995

Development of a heterogeneous amperometric immunosensor for the determination of apolipoprotein E in serum.

Apolipoprotein (apo) E is a constituent of serum lipoproteins and can serve as a diagnostic parameter for the assessment of disorders of lipid metabolism. For the determination of apo E in serum a sandwich-type amperometric immunosensor using disposable membranes was developed. The best results were obtained by using a site-directed attachment of a monoclonal capture antibody to a hydrazide-functionalized membrane surface. Bound antigen was then determined with the aid of a polyclonal antibody labelled with alkaline phosphatase in conjunction with p-aminophenyl phosphate as substrate. In this approach a carbon working electrode (vs. Hg/HgCl2) was used, and enzymatically generated p-aminophenol could be monitored with a detection limit of 40 pmol and a linear range of 26-20000 nM. The sensor displayed a linear response from 50 to 1000 ng ml-1 apo E. In contrast, antibody coupling through primary amino groups led to a total loss of antigen binding capacity in this assay configuration. The approach with site-directed immobilization however, not only allowed the determination of apo E in serum, but also the determination of antibody cross-reactivity against the apolipoproteins AI, AII and B.

Aminophenols↗
PubMed1995

Treatment of severe localized scleroderma by plasmapheresis--report of three cases.

We report three patients with severe, localized scleroderma, and with elevated titres of antinuclear antibodies, who were treated by plasmapheresis in combination with systemic steroid therapy. The therapeutic effectiveness of plasmapheresis was assessed on the basis of improvement in cutaneous and joint lesions. In all cases, significant improvement occurred after 2 months of therapy. Thus, in addition to treating systemic sclerosis, plasmapheresis can also be recommended for treatment of severe cases of localized scleroderma with elevated titres of antinuclear antibodies and antibodies to ss-DNA.

Adult↗
PubMed1995

Failure of calcium antagonistic agents to prevent hepatotoxicity induced by diclofenac.

Diclofenac (0.5-2 mM) dose- and time-dependently reduces the viability of isolated hepatocytes. This effect cannot be counteracted by the calcium channel blockers diltiazem (0.05-0.1 mM) and verapamil (0.05-0.5 mM), the calmodulin antagonist calmidazolium (0.01 mM) or Quin 2-AM (0.1 mM), an intracellular calcium chelating agent. On the contrary, verapamil even accentuates the toxic effects of diclofenac. It is concluded from these results, that diclofenac causes cell damage by other mechanisms than calcium overload.

Analysis of Variance↗
PubMed1995

HDL-mediated efflux of intracellular cholesterol is impaired in fibroblasts from Tangier disease patients.

To further elucidate the cellular mechanisms leading to HDL deficiency in Tangier disease, HDL-mediated cholesterol efflux was studied in cultured skin fibroblasts from Tangier patients. Both Tangier and control fibroblasts show specific saturable binding of HDL3 to the cell membrane (Bmax = 70 and 52 ng/mg protein, respectively; Kd = 8.8 and 10.6 micrograms/mL, respectively). There was no appreciable uptake of HDL3 by Tangier and control fibroblasts, indicating that cholesterol efflux from fibroblasts occurs at the cell membrane. When cellular cholesterol was labeled to equilibrium by [14C]cholesterol incubation for 48 hours at 37 degrees C, HDL3-mediated cholesterol efflux from Tangier fibroblasts was only 50% of control fibroblasts. To define this abnormality in HDL3-mediated cholesterol efflux more precisely, several additional experiments were performed. First, membrane desorption of cholesterol was determined after cell membranes were labeled with [14C]cholesterol for 3 hours at 15 degrees C. With this labeling protocol, there was no difference in HDL3-mediated cholesterol efflux between control and Tangier fibroblasts. Second, efflux of newly synthesized sterols was determined after incorporation of the precursor [14C]mevalonolactone. Under these conditions, specific HDL3-mediated efflux of sterols was almost absent in Tangier fibroblasts. Third, cells were labeled by incubation with reconstituted [3H]cholesteryl-linoleate-LDL. Efflux of LDL-derived cholesterol was only slightly reduced for the first 4 hours of incubation. After 12 hours, there was no difference between control and Tangier cells. The combined data indicate that the reduced efflux of cholesterol from Tangier fibroblasts observed after homogeneous labeling is due to severely reduced efflux of newly synthesized sterol.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Membrane↗
PubMed1995

Activation of phosphatidylinositol-specific phospholipase C in response to HDL3 and LDL is markedly reduced in cultured fibroblasts from Tangier patients.

We compared HDL3- and LDL-induced signal transduction in normal and Tangier fibroblasts to elucidate whether impaired signal transduction responses to lipoproteins might contribute to disturbed cellular lipid and lipoprotein metabolism in Tangier disease, a rare autosomal disorder of cellular lipid and lipoprotein metabolism. In several cell types HDL and LDL activate a currently unknown isoform of phosphatidylinositol-specific phospholipase C (PI-PLC) that results in the generation of 1,2-diacylglycerol and inositol 1,4,5-trisphosphate. Compared with normal fibroblasts, Tangier fibroblasts stimulated with HDL3 or LDL resulted in a significantly reduced accumulation of inositol phosphates and 1,2-diacylglycerol formation. Furthermore, in Tangier fibroblasts both lipoproteins failed to mobilize calcium from internal pools, and the cytosol-to-membrane redistribution of protein kinase C (in both the alpha and epsilon isoforms) was markedly reduced. Thus, the data indicate an impaired PI-PLC activation in response to lipoproteins in Tangier fibroblasts.

Adult↗
PubMed1994

Improved separation of radioactively labelled cellular phospholipids by high-performance liquid chromatography.

An improved high-performance liquid chromatographic method for the separation and determination of radioactively labelled cellular phospholipids is described. The method is based on separation of phospholipids on a 250 x 4 mm I.D. LiChrospher DIOL 100 (5 microns) column, fitted with a 50 x 4 mm I.D. LiChrospher Si 60 (5 microns) precolumn and a gradient of 5% H3PO4 and acetonitrile. It allows the determination of small amounts of labelled phosphatidylcholine and sphingomyelin due to the sharp elution profile in spite of long retention times.

Cells, Cultured↗
PubMed1994

Genomic organization of the human lysosomal acid lipase gene (LIPA).

Defects in the human lysosomal acid lipase gene are responsible for cholesteryl ester storage disease (CESD) and Wolman disease. Exon skipping as the cause for CESD has been demonstrated. We present here a summary of the exon structure of the entire human lysosomal acid lipase gene consisting of 10 exons, together with the sizes of genomic EcoRI and SacI fragments hybridizing to each exon. In addition, the DNA sequence of the putative promoter region is presented. The EMBL accession numbers for adjacent intron sequences are given.

Base Sequence↗
PubMed1994

Analysis of lipoproteins with analytical capillary isotachophoresis.

An analytical free flow capillary isotachophoresis procedure, with a discontinuous electrolyte system, for the detailed analysis of lipoproteins in human body fluids has been developed. The technique is based on prestaining whole serum lipoproteins with a lipophilic dye before separation. Human serum lipoproteins are separated into 14 well-characterized subfractions according to their electrophoretic mobility. High density lipoproteins (fraction 1 to 6) are separated into three major subpopulations, the fast migrating high density lipoprotein (HDL) subpopulation, containing mainly apo AI and phosphatidylcholine, the subpopulation with intermediate mobility, consisting of particles rich in apo AII, apo E, and C apolipoproteins, and the slowly migrating HDL subfraction, containing mainly particles rich in apo AI, apo AIV, and lecithin: cholesterol acyltransferase (LCAT) activity. The apo B containing lipoproteins (fraction 7 to 14) can be subdivided into four 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, anf the third and fourth group represent the low density lipoproteins. The isotachophoretic analysis of human serum samples obtained from patients with hyperlipoproteinemias is compatible with the classification according to the Frederickson phenotypes and reflects the respective biochemical abnormalities. Furthermore, several genetic disorders of lipid and lipoprotein metabolism like HDL deficiency syndromes, familial LCAT deficiency, Fish eye disease, hypobetalipoproteinemia and abetalipoproteinemia can be well characterized by analytical capillary isotachophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)

Apolipoproteins↗
PubMed1994

Lipid-lowering therapy--implications for the prevention of atherosclerosis.

Lipid-lowering therapy has been advocated as a means to decrease the incidence of cardiovascular disease. The basis for this are results of animal studies, epidemiologic surveys, and intervention studies in man. There have been recommendations by national and international advisory boards regarding the aims and means of such interventional strategies. These have not been unanimously accepted, however. The present article will review the evidence on which intervention strategies may be based and the current status of the scientific discussion. The implications for future strategies to prevent atherosclerosis are discussed.

Adult↗
PubMed1994

Sequence analysis of the 3'-terminal half of RNA 1 of wheat spindle streak mosaic virus.

cDNA complementary to the 3'-terminal half of RNA 1 of wheat spindle streak mosaic virus (WSSMV) from Southern France has been cloned and sequenced. One large open reading frame (ORF) of 4410 nucleotides and a nontranslated region (NTR) of 213 nucleotides at the 3'-end excluding the poly(A)-tail were found. Because of the amino acid sequence homology to the polyprotein of barley yellow mosaic virus (BaYMV) RNA 1, the encoded polyprotein of the sequenced region of WSSMV is supposed to comprise the C-terminal part of the putative cytoplasmic inclusion (CI) protein, the nuclear inclusion a (NIa) proteinase, the (NIb) RNA-polymerase and the capsid protein. The first 19 N-terminal amino acids of the capsid protein were determined by direct sequencing of proteins of purified WSSMV particles and confirmed this hypothesis. The deduced capsid protein has 294 amino acids and shows 74% identity with the BaYMV capsid protein sequence. This high sequence homology with BaYMV, in addition to the significant identities with barley mild mosaic virus (BaMMV, 35%) and its marginal homology to capsid protein sequences of aphid and mite-borne potyviruses (22-24%), supports the classification of WSSMV as a distinct member of the genus Baymovirus, family Potyviridae.

Amino Acid Sequence↗
PubMed1994

A self-stabilizing Ac derivative and its potential for transposon tagging.

With the goal of developing a self-stabilizing transposon tagging system, a derivative of the maize transposable element Ac (Ds303) harbouring a deletion between bp 246 and 736 has been introduced into tomato (Lycopersicon esculentum) by Agrobacterium tumefaciens-mediated transformation. The deletion removes the major transcription start site, 84 bp of the putative Ac promoter and part of the untranslated leader. Transpositions from the T-DNA, where Ds303 was inserted between the mannopine synthase 1' promoter and the GUS gene, were observed in four independent transgenic plants analysed. After transposition the transposed Ds303 elements were stably integrated in the genome and could not transactivate a tester Ds element. However, somatic and germinal transpositions of the transposed Ds303 elements occurred in the presence of a transposase source.

Base Sequence↗
PubMed1994

Minimal length requirement of the single-stranded tails for ligation-independent cloning (LIC) of PCR products.

The ligation-independent cloning of PCR products (LIC-PCR) is a versatile and highly efficient cloning procedure resulting in recombinant clones only. Recombinants are generated between PCR products and a PCR-amplified vector through defined complementary single-stranded (ss) ends artificially generated with T4 DNA polymerase. This procedure does not require restriction enzymes, alkaline phosphatase, or DNA ligase. The primers used for amplification contain an additional 12-nucleotide sequence at their 5' ends that is complementary in the vector- and insert-specific primers. The (3'-->5') exonuclease activity of T4 DNA polymerase is used in combination with a predetermined dNTP (dGTP for the inserts and dCTP for the vector) to specifically remove 12 nucleotides from each 3' end of the PCR fragments. Because of the complementarity of the ends that are generated, circularization can occur between vector and insert. The recombinant molecules do not require in vitro ligation for efficient bacterial transformation. To make this technique widely applicable, we have simplified the handling of the PCR fragments prior to LIC. The PCR products do not need further purification following the T4 DNA polymerase treatment. Incubation of vector and insert PCR fragments for as little as 5 min is sufficient for a high yield of recombinants. Comparison of the transformation efficiencies using different-length LIC tails revealed that using 12-nucleotide cohesive ends produced four times more transformants than were obtained with the LIC with 10-nucleotide cohesive ends. When the LIC tails were 8 nucleotides long, no transformants were obtained. PCR fragment purification, T4 DNA polymerase treatment, and LIC is complete in < 1 hr.

Base Sequence↗
PubMed1994

Isolation of region specific single-copy probes from human chromosome 1q23-->1q25.

Single-copy sequences of chromosome bands 1q23-->1q25 were enriched by subtractive hybridization of digoxigenin oligonucleotide-labeled DNA from a somatic cell hybrid containing human chromosome 1 with DNA from a somatic cell hybrid containing a deleted human chromosome 1, del(1)(q23q25). These sequences were purified with an immobilized anti-digoxigenin antibody, amplified by polymerase chain reaction (PCR) and cloned. The majority of clones contained single-copy sequences, thus allowing direct screening of human genomic DNA. With pools of probes complex hybridization patterns as well as differences in hybridization patterns between genomes were observed in Southern blotting experiments. Nine clones containing independent single-copy sequences from chromosome bands 1q23-->1q25 were isolated and sequenced to generate sequence-tagged sites (STSs). Using PCR, region specific genomic clones containing large inserts were identified in human genomic lambda phage and cosmid libraries. Thus, region specific genomic probes generated by genomic difference cloning are useful for identifying deletions and rearrangements, for generating STSs and for isolating genomic clones containing large inserts of a particular chromosomal region.

Base Sequence↗
PubMed1994

Purification, cloning, and expression of a human enzyme with acyl coenzyme A: cholesterol acyltransferase activity, which is identical to liver carboxylesterase.

An enzyme with acyl coenzyme A:cholesterol acyltransferase (ACAT) activity was isolated from porcine liver, and sequences derived from trypsinized peptides indicated homology to liver carboxylesterase. By use of degenerate primers, human cDNA clones were identified, which were identical to human liver carboxylesterase. Expression of the full-length cDNA in Chinese hamster ovary (CHO) cells led to an approximately threefold increase in cellular ACAT activity. This was accompanied by an approximately 20-fold increase of cellular cholesteryl ester content. By light and electron microscopy, recombinant CHO cells contained numerous lipid droplets that were not present in control CHO cells. Expression of an antisense cDNA in HepG2 cells reduced cellular ACAT activity by 35% compared with control. To further investigate the role of the enzyme in cellular cholesterol homeostasis, regulation of the mRNA was investigated in 7-day cultured human mononuclear phagocytes (MNPs). When these cells were incubated in lipoprotein-deficient serum for 18 hours, the mRNA for ACAT/carboxylesterase was almost not detectable on Northern blots, whereas after incubation with acetylated low-density lipoproteins, a strong hybridization signal was obtained. This is evidence that the mRNA of ACAT/carboxylesterase is induced by cholesterol loading. It is concluded from the data presented that ACAT/carboxylesterase is relevant for cellular cholesterol esterification in vivo. The regulation in MNPs indicates that the enzyme is also involved in foam cell formation during early atherogenesis.

Amino Acid Sequence↗
PubMed1994

[Classification of hyperlipoproteinemias and interpretation of laboratory parameters].

Hyperlipidemias are grouped according to their lipid levels in hypercholesterolemia, hypertriglyceridemias, and mixed hyperlipidemias. Among these, hypercholesterolemia has the strongest correlation to the risk for coronary heart disease. Based on the results from epidemiologic studies and intervention trials several expert panels have defined cholesterol values with low and high cardiovascular risk and cholesterol target levels for treatment. In the presence of hyperlipidemia additional parameters like LDL cholesterol, HDL cholesterol, and Lp(a) may be determined depending on the individual patient's risk profile. For classification of familial lipid disorders, analysis of relatives and the determination of special parameters may be necessary. These include apolipoproteins (concentration, isoforms, mutants), enzyme activities (lipases, LCAT), LDL receptor activity, oratypical lipoproteins.

Apolipoproteins↗