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Biomedical subjects

G Schmitz

Publications and source records attributed to G Schmitz.

At least 217 records · Page 12Linked to original sources
PubMed1993

Purification and characterisation of the restriction endonuclease ItaI from Ilyobacter tartaricus recognizing 5'-GC decreases NGC-3'.

ItaI, an isoschizomer of the subclass IIW [Kessler and Manta, Gene 92 (1990) 1-248] restriction endonuclease (ENase), Fnu4HI [Leung et al., Nucleic Acids Res. 6 (1979) 17-25], has been isolated from Ilyobacter tartaricus. The ENase has the five-base palindromic recognition sequence, 5'GC decreases NGC-3'. It cleaves behind the second nucleotide and produces a one-nt 5' overhang.

Deoxyribonucleases, Type II Site-Specific↗
PubMed1993

High-performance liquid chromatographic method for the routine determination of diclofenac and its hydroxy and methoxy metabolites from in vitro systems.

A rapid and sensitive high-performance liquid chromatographic method is presented for the determination of diclofenac and its hydroxylated and methoxylated metabolites. The procedure describes extraction of diclofenac and its metabolites from acidified incubation medium into tert.-butylmethyl ether. Separation is achieved with a C18 reversed-phase column and quantification by UV detection at 280 nm. The method employs an internal standard resulting in good accuracy and precision. The limit of detection is 5 ng/ml for diclofenac and 10 ng/ml for its metabolites. One analysis requires no more than twelve minutes so that the assay is very suitable for the determination of a large number of samples.

Animals↗
PubMed1993

Glucose-6-phosphate: a key compound in glycogenosis I and favism leading to hyper- or hypolipidaemia.

The glycogen storage disorders (GSD)-I, -III, -VI and -VIII are associated with hypertriglyceridaemia or mixed hyperlipidaemia which poses the question whether these patients have an increased risk for atherosclerosis. The atherogenicity of triglycerides has remained controversial, while increased plasma cholesterol levels are generally accepted as a significant risk factor for coronary heart disease. However, clinical data show that one has to differentiate between metabolic conditions where triglycerides are atherogenic and those which are not significantly related to early onset of atherosclerosis but may cause other disorders such as pancreatitis. Among the disorders of carbohydrate metabolism patients with diabetes mellitus frequently have enhanced plasma triglycerides associated with a higher risk for coronary heart disease, while patients with certain types of glycogen storage disease have high triglyceride levels but do not seem to have an enhanced risk for atherosclerosis. Here we have compared the biochemical abnormalities and the atherogenic risk of three different disorders of glucose metabolism including GSD-I (glucose-6-phosphatase deficiency), favism (glucose-6-phosphate dehydrogenase deficiency), and diabetes mellitus which are related to either hyper- or hypolipidaemia. The available data indicate that glucose-6-phosphate (Glc-6-P) is a central molecule in cellular glucose metabolism which critically influences pentose phosphate cycle activity and, via NADPH2-generation, regulates glutathione peroxidase activity for radical detoxification and also cholesterol and triglyceride synthesis. Radical detoxification is a major protective factor for cell membrane integrity and together with an appropriate renewal of membrane lipids may protect against the development of atherosclerosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Arteriosclerosis↗
PubMed1993

Fluorescence flow cytometry of human leukocytes in the detection of LDL receptor defects in the differential diagnosis of hypercholesterolemia.

A flow-cytometric method with fluorescence-labeled monoclonal antibodies (MABs) against the low density lipoprotein (LDL) receptor (C7A MAB) or 3,3'-dioctadecylindocarbocyanin-iodide (DiI) LDL has been developed that allows the quantification of LDL receptors on leukocytes and the identification of patients with familial hypercholesterolemia (FH) within 48 hours. Leukocytes were isolated from 10 mL anticoagulated blood by density gradient centrifugation. To induce maximal expression of LDL receptors, mononuclear cells were preincubated with either phytohemagglutinine (PHA) or lipoprotein-deficient serum (LPDS). LPDS-treated monocytes provided a more homogeneous cell population with regard to LDL receptor activity than did the PHA-treated lymphocytes; they also provided a greater discrimination between the fluorescence of the receptor probes and cellular autofluorescence. The C7A MAB was able to compete for DiI LDL binding by about 40%. In competition with unlabeled LDL, DiI LDL revealed linear binding, indicating an affinity similar to native LDL. The binding characteristics of DiI LDL were also similar to 125I-LDL binding. LDL isolated from familial defective apolipoprotein B-100 was not able to compete for DiI LDL binding on monocytes, whereas native LDL reduced it by about 80%. In monocytes from FH heterozygous patients, the cellular mean fluorescence using either C7A MAB or DiI LDL at 4 degrees C was 30% to 70%; in FH homozygotes, cellular mean fluorescence was less than 20% of that in monocytes from normal individuals. In patients with familial defective apolipoprotein B-100 antibody binding was normal, but one patient's own LDL failed to compete with normal DiI LDL for 4 degrees C binding on U937 test monocytes. Patient monocytes having internalization defects showed normal 4 degrees C DiI LDL binding, but at 20 degrees C cell-associated fluorescence was reduced by about 40%. In our study 384 hypercholesterolemic patients (preselected according to serum cholesterol levels, clinical symptoms, and family history) were analyzed for LDL receptor expression using the C7A MAB-based assay. In 71.8% of the patients with cholesterol levels higher than 300 mg/dL, an LDL receptor deficiency was observed. Apolipoprotein E isoforms and lipoprotein[a] were found to be independent from the LDL receptor status. In some patients with high cholesterol levels but normal LDL receptor expression with the C7A MAB assay, LDL receptor defects could be diagnosed when either reduced binding or internalization of DiI LDL or familial defective apolipoprotein B-100 was detected.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗
PubMed1993

Effects of purinergic agents on human mononuclear phagocytes are differentiation dependent. Implications for atherogenesis.

The differentiation-dependent expression of purinergic receptors for metabolically stable analogues of adenosine and ATP was studied in human mononuclear phagocytes (MNPs). Ligands of these receptors are able to modulate cellular cholesterol metabolism. In addition, the intracellular signal transduction pathways of the purinergic receptor system were examined. ATP gamma S, the metabolic stable analogue of ATP, was used as a P2 ligand, and 2-p-(2-carboxyethyl)phenylethylamino-5'-N-ethylcarboxamido adenosine (CGS 21680) and 5'-(N-ethylcarboxamido)adenosine (NECA) were used as P1 ligands in binding studies. Binding of [35S]ATP gamma S to MNPs at 4 degrees C revealed saturable low-affinity binding sites with a Kd of 868 +/- 52 nmol/L and Bmax of 7.3 +/- 0.4 pmol per 10(6) cells in 1-day cultured human MNPs and a Kd of 780 +/- 30 nmol/L and Bmax of 14.0 +/- 0.8 pmol per 10(6) cells in 7-day cultured human MNPs. The characterization of the P1 receptors on 1- and 7-day cultured human MNPs showed that they are expressed only on 7-day cultured human MNPs. The specific binding curve of the adenosine A2 receptor agonist [3H]CGS 21680 was biphasic, with a Kd1 of 33 +/- 15 nmol/L and a Kd2 of 90 +/- 10 nmol/L and with Bmax1 of 0.19 +/- 0.06 pmol per 10(6) cells and Bmax2 of 0.41 +/- 0.09 pmol per 10(6) cells, whereas NECA did not exhibit specific binding. The typical agonists for probing A1 receptor subtypes did not bind to 1- and 7-day cultured human MNPs, indicating that only A2 receptors are expressed on 7-day cultured human MNPs. ATP gamma S enhanced [Ca2+]i in 1- and 7-day cultured human MNPs in a concentration-dependent manner, whereas the P1 ligands, adenosine and CGS 21680, induced Ca2+ flux only in 7-day cultured MNPs. All three drugs increased intracellular cAMP levels in 7-day cultured human MNPs at a concentration of 10(-5) mol/L, whereas no effect was observed in 1-day cultured human MNPs. The uptake of fluorescently labeled acetylated low-density lipoprotein (LDL) in 7-day cultured human MNPs was inhibited by adenosine, CGS 21680, ATP, and ATP gamma S. No significant influence of these compounds was measured on the uptake of LDL, acetylated LDL, and high-density lipoprotein, in 1-day cultured MNPs. Our investigations indicate that the expression of P2y and A2 receptors is increased during differentiation of blood monocytes to macrophages.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗
PubMed1993

High density lipoprotein deficiency with xanthomas. A defect in reverse cholesterol transport caused by a point mutation in the apolipoprotein A-I gene.

A 7-yr-old girl with high density lipoprotein (HDL) deficiency and xanthomas has been identified in a Turkish kindred with repetitive consanguinity. She has severely reduced HDL-cholesterol and no apolipoprotein (apo) A-I. ApoA-II is reduced, whereas apoA-IV and apoC-III are normal. ApoB and low density lipoprotein (LDL)-cholesterol are increased. This is reflected in hypercholesterolemia. VLDL and IDL particles are low, and serum triglycerides are normal. The genetic defect could be identified as a base insertion into the third exon of the apoA-I gene. This leads to a nonsense peptide sequence beginning at amino acid 5 of the mature plasma protein and early termination of translation. The patient is homozygous for this mutation. Pedigree analysis indicated an autosomal dominant inheritance with no evidence of another genetic defect of lipoprotein metabolism in the kindred. In HDL deficiency, HDL binding to leukocytes was increased compared to normal. In the postprandial state, binding of labeled HDL3 to leukocytes is unchanged. This is in contrast to results with postprandially isolated leukocytes from controls or Tangier patients, which have a reduced binding capacity for HDL3. These results indicate that postprandial HDL precursors may compete the binding of labeled HDL3. The metabolic consequences of HDL deficiency were analyzed. There is only a small number of HDL-like particles containing apoA-II, apoA-IV, apoE, and lecithin/cholesteryl acyl transferase. The C-apolipoproteins were normal in the proband. Due to the lack of HDL they can only associate with apoB-containing particles, where they may interfere with cellular uptake. Thus, pure apoA-I deficiency leads to a complex metabolic derangement.

Amino Acid Sequence↗
PubMed1993

A splice junction mutation causes deletion of a 72-base exon from the mRNA for lysosomal acid lipase in a patient with cholesteryl ester storage disease.

The genetic defect leading to cholesteryl ester storage disease (CESD) has been determined in a 12-yr-old patient. Lysosomal acid lipase (LAL) activity in cultured skin fibroblasts was reduced to approximately 9% of control fibroblasts. Plasma cholesterol (255 mg/dl) and LDL-cholesterol (215 mg/dl) were elevated whereas HDL-cholesterol was reduced (19 mg/dl). Triglycerides were moderately elevated (141 mg/dl). There were no clinical abnormalities with the exception of hepatosplenomegaly. Both parents have reduced LAL activity in white blood cells. PCR analysis of the LAL mRNA from the propositus revealed a single slightly smaller mRNA species in skin fibroblasts as well as in leukocytes. The mother of the patient and his older brother had two mRNA species: one of normal size and one of the same size as the propositus. The father has a LAL mRNA of normal size only. Sequence analysis of a PCR-amplified cDNA fragment showed a 72-bp in-frame deletion resulting in the loss of the codons for amino acids 254-277. Analysis of genomic DNA revealed that the 72 bp represent an exon, indicating that the deletion in the mRNA is caused by defective splicing. Sequence analysis of the patient's genomic DNA revealed a G-->A substitution in the last nucleotide of the 72-bp exon in one of his alleles. The mutant allele was shown to cosegregate with the truncated mRNA in the pedigree, providing further evidence that the G-->A substitution causes aberrant splicing and exon skipping. No normal-sized mRNA is detectable in the propositus even though he is not homozygous for the splice site mutation. This can be only accounted for by assuming that he is a compound heterozygote with a null allele inherited from his father. In summary, the data presented provide evidence that deletion of the codons for amino acids 254-277 in the LAL mRNA in combination with a null allele cause the clinical expression of CESD in our patient.

Adolescent↗
PubMed1992

Structural and functional analysis of the Bz2 locus of Zea mays: characterization of overlapping transcripts.

Analysis of the transcription pattern of the Bz2 locus revealed that overlapping transcripts are derived from opposite DNA strands. The most abundant transcript (sense transcript) has an open reading frame coding for a protein of 241 amino acids, whilst in the antisense orientation no open reading frame has been detected; the antisense transcripts are detected only in those tissues that show high levels of sense transcript. Particle gun experiments indicate that the sense transcript is sufficient to provide the Bz2 function. The promoter driving the sense transcript contains the elements usually found in front of eukaryotic genes. In addition an element with similarity to the C1 and R binding sites identified in the Bz1 promoter is found. Further upstream in the promoter region a transposon-like insertion has been identified. This element has features similar to members of the Ac/Ds transposable element family. The putative Bz2 protein shows similarity to various other plant proteins and to an Escherichia coli protein. All related proteins have in common the fact that they are involved in stress responses.

Amino Acid Sequence↗
PubMed1992

The clinical relevance of IgG subclasses in acute leukemias: low serum IgG as a risk factor for early death due to infection in acute myelogenous leukemia.

In order to investigate the clinical significance of IgG subclass levels in adult patients treated for acute lymphocytic (ALL) or myelogenous leukemias (AML), patients were tested before and in regular intervals during and after chemotherapy. Ten patients treated for acute lymphocytic leukemia had normal total IgG and IgG subclass levels that decreased under chemotherapy. This decrease was not associated with septic complications. In contrast, total IgG and subclass levels increased in most patients with acute myelogenous leukemia during chemotherapy and bone marrow aplasia. Those patients with newly diagnosed AML and low total serum IgG before treatment with intensive remission induction chemotherapy had, however, an increased risk of early death due to septic complications.

Adult↗
PubMed1992

Conditioning of drug-induced immunomodulation in human volunteers: a European collaborative study.

Although several studies on conditioning of the immune system in animals have been published, no comparable data on human research have been available in the past. The present paper presents results of conditioning studies in volunteers performed in two research centres, namely the University of Trier (Germany) and the University of Utrecht (The Netherlands). After administration of a neutral stimulus (conditioned stimulus: CS), subjects were injected with epinephrine (unconditioned stimulus) for three or four days (depending on study). Subcutaneous injection of epinephrine caused a rapid enhancement of the activity of natural killer cells (NKCA) in venous blood, which was chosen as the unconditioned response. On the test trial, when saline instead of epinephrine was injected, the Trier group found a conditional enhancement of NKCA. No changes in NKCA were found in the control subjects, who received saline injections on all days along with the CS. The Utrecht group tried to replicate these results using a slightly different design. After obtaining non-confirmative results, the Utrecht experimenters tried to parallel the experimental settings of the Trier group as closely as possible. However, once again they failed to replicate the results of the Trier group. Possible reasons for the different results obtained in the two research groups are discussed.

Adult↗
PubMed1992

Sensitive chemiluminescent detection of digoxigenin-labeled nucleic acids: a fast and simple protocol and its applications.

A fast and simple protocol for the chemiluminescent detection of digoxigenin-labeled nucleic acids with anti-digoxigenin antibody Fab fragments coupled to alkaline phosphatase and 3-(4-methoxyspiro[1,2-dioxetane-3,2'-tricyclo-[3.3.1.1 (3,7)]decan]-4- yl)phenyl phosphate as substrate is described. The washing and blocking procedure was optimized to yield low background even on positively charged nylon membranes. The sensitivity of the system is equal or better than radioactive methods. Exposure to x-ray or Polaroid film for up to 30 minutes is sufficient for the detection of 70 femtograms of homologous DNA. Human single-copy genes are detected in Southern blots of as low as 0.3 microgram total placental DNA. Blots can be reprobed multiple times very easily. The advantages of the digoxigenin system are high sensitivity, absence of background and ease of reprobing and are illustrated by applications for single-copy gene detection in genomic blots of human DNA, Northern hybridizations to rare mRNA, detection of E. coli genes on blots of genomic digests after pulse field gel electrophoresis, as well as for nonradioactive DNA sequencing blots with digoxigenin-labeled primers.

Actins↗
PubMed1991

Clusterin (complement lysis inhibitor) forms a high density lipoprotein complex with apolipoprotein A-I in human plasma.

Clusterin/human complement lysis inhibitor (CLI) is incorporated stoichiometrically into the soluble terminal complement complex and inhibits the cytolytic reaction of purified complement components C5b-9 in vitro. Using an anti-clusterin affinity column, we found that an additional protein component with a molecular mass of 28-kDa co-purifies with clusterin from human plasma. We show by immunoblotting and amino acid sequencing that this component is apolipoprotein A-I (apoA-I). By using physiological salt buffers containing 0.5% Triton X-100, apoA-I is completely dissociated from clusterin bound to the antibody column. Free clusterin immobilized on the antibody-Sepharose selectively retains apoA-I from total human plasma. Delipidated apoA-I and to a lesser extent ultracentrifugation-purified high density lipoproteins (HDL) adsorbed to nitrocellulose also have a binding affinity for purified clusterin devoid of apoA-I. The isolated apoA-I-clusterin complex contains approximately 22% (w/w) lipids which are composed of 54% (mole/mol) total cholesterol (molar ratio of unesterified/esterified cholesterol, 0.58), 42% phospholipids, and 4% triglycerides. In agreement with the low lipid content, apoA-I-clusterin complexes are detected only in trace amounts in HDL fractions prepared by density ultracentrifugation. In free flow isotachophoresis, the purified apoA-I-clusterin complex has the same mobility as the native clusterin complex in human plasma and is found in the slow-migrating HDL fraction of fasting plasma. Our data indicate that clusterin circulates in plasma as a HDL complex, which may serve not only as an inhibitor of the lytic terminal complement cascade, but also as a regulator of lipid transport and local lipid redistribution.

Apolipoprotein A-I↗