PubMed Health⌕ Search

Biomedical subjects

K Wielckens

Publications and source records attributed to K Wielckens.

At least 19 recordsLinked to original sources
PubMed2000

Apolipoprotein E polymorphism: automated determination of apolipoprotein E2, E3, and E4 isoforms.

Apolipoprotein E (apo E) plays an essential role in lipoprotein metabolism, where it is involved in the clearance of chylomicrons and very low density lipoproteins. Apart from some rare variants, apo E exists in three common isoforms (E2, E3, and E4). The different isoforms have not only been associated with different plasma lipid levels but have also been correlated with certain pathological conditions, such as lipid disorders (dysbetalipoproteinemia, hypercholesterolemia), cardiovascular diseases, and Alzheimer's disease. Here we describe a rapid, automated test for the determination of the most frequent polymorphisms (E2, E3, and E4). This polymerase chain reaction-based test allows the reliable discrimination of all six genotypes. The assay has been developed especially for the nonspecialized routine clinical laboratory by employing an analyzer and chemistry often present in this type of laboratory. Because of its low costs and easy handling, the assay can be performed on a daily basis.

Apolipoprotein E2↗
PubMed2000

Haemochromatosis: automated detection of the two point mutations in the HFE gene: Cys282Tyr and His63Asp.

Hereditary haemochromatosis (HH) is one of the most common inherited diseases among Caucasians. Two mutations in the HFE gene have been implicated in HH: 80 to 90% of the patients with HH are homozygous for the point mutation CYS282Tyr, while the majority of the remaining patients displays either a compound heterozygosity for the mutation CYS282Tyr and the point mutation HIS63Asp, or are homozygous for HIS63Asp. Though the disease can be treated easily, symptoms are non-specific, and onset and severity are influenced by environmental factors, and therefore the disease can remain undetected until decades of iron overload lead to irreversible damage in a variety of organs, which may result in their failure. In order to detect patients with HH, simple and cost-effective tests are needed. We have developed a rapid, automated, PCR-based test which makes use of a diagnostic restriction site in each of two amplified fragments. The test employs off-the-shelf chemistry and uses the automated detection process of an immunoassay analyzer that is available in many clinical laboratories, thus avoiding an additional investment in a more specialized PCR analyzer. Because of its low costs and easy handling, the assay is particularly suited for the routine clinical laboratories.

Aspartic Acid↗
PubMed2000

Apolipoprotein B-100: employing the electrochemiluminescence technology of the Elecsys systems for the detection of the point mutation Arg(3500)Gln.

Apolipoprotein B-100 (apo B-100) plays an essential role in lipoprotein metabolism where it is involved in the clearance of LDL particles from the bloodstream. The mutation Arg3500Gln in the apo B-100 gene impairs the binding of the LDL particles to the LDL receptor, resulting in elevated LDL-cholesterol levels in the blood which, in turn, fuel the development of premature atherosclerosis. Here we describe a rapid, automated test for the detection of the most frequent mutation in the apo B-100 gene. This PCR-based test employs electrochemiluminescence as detection technology and allows the reliable discrimination of all genotypes. The assay has been especially developed for the non-specialized routine clinical chemistry laboratory by employing an analyzer and chemistry often present in this type of labof1tory. Because of its low costs and easy handling the assay can be performed on a daily basis.

Amino Acid Substitution↗
PubMed1999

Activated protein C (APC)-resistance: automated detection of the point mutation at position 1691 in the factor V gene.

Proteolytic cleavage of factor Va, caused by activated protein C, is an important mechanism in limiting clot formation in normal haemostasis. A single point mutation in the factor V gene has been demonstrated to cause resistance of factor Va to proteolytic cleavage by activated protein C. With an 8-fold increased risk of thrombosis and a 2 to 13% prevalence in the Caucasian population for the heterozygous state of this mutation, knowledge of the patient's genetic disposition is of great importance in conditions such as pregnancy, surgery, use of oral contraceptives and immobilization. Therefore we have developed an automated test for the detection of the factor V mutation. This PCR based test makes use of the disappearance of an Mnl 1 restriction site if the mutation is present. The assay has been developed for the widely used ES-systems of Boehringer Mannheim. The test discriminates between the heterozygous and the homozygous state. Because of its low costs and easy handling the assay can be used as a screening test and can be performed in routine clinical laboratories.

Activated Protein C Resistance↗
PubMed1999

[The Cologne Guidelines Conference: computer-assisted clinical practice guidelines in clinical diagnosis].

Eighteen clinical practice guidelines on interdisciplinary diagnostic issues were developed at the University Hospital of Cologne, Germany. The guideline committee is organized and directed by the quality control program, which also includes the local Cochrane initiative and a wide range of organizational topics. During guideline development, questions of differential diagnosis were addressed to the same extent as the organizational and financial realization. Broad consideration was given to medicolegal implications, but need for interspecialty cooperation was judged to be more critical and even more relevant in this regard. Guidelines were primarily developed as algorithms and translated to text versions secondarily. Critical steps in the decision tree were supported by rated literature and recommendations weighte by criteria as used in evidence-based medicine. For implementation, guidelines were presented to colleagues in a series of short lectures, as print versions containing all literature used in the developing process, and in hypertext format, which is accessible via intranet. Three levels of presentation were chosen in the html-version: algorithm, decision, and information. The former is due to orientation in the guideline, the second displays the binary question, and the latter makes the scientific background available, together with literature and links for more information. Efforts to check effectiveness are currently been made, questions of efficiency will be addressed in future.

Algorithms↗
PubMed1999

Activated protein C resistance: automated detection of the factor V Leiden mutation by mismatch hybridization.

BACKGROUND: A single point mutation in the factor V gene has been demonstrated to be the cause of factor Va resistance to proteolytic cleavage by activated protein C. Knowledge of the patient's genetic disposition is of great importance in situations such as pregnancy, surgery, use of oral contraceptives, and immobilization. METHODS: We have developed a rapid, automated test for the detection of the factor V mutation that makes use of differences in thermal stability between perfect-match and non-perfect-match hybrids. A DNA fragment spanning the mutation is amplified with a biotin-labeled primer. Ruthenium-labeled oligonucleotides, perfectly matching either the biotinylated wild-type strand or the biotinylated mutation strand, are added. Heating to 95 degrees C and subsequent cooling lead to the formation of double-stranded DNA. Under the conditions chosen, ruthenium-labeled oligonucleotides form stable, double-stranded DNA with the biotinylated strand only if both strands perfectly match each other. The ruthenium signal is measured on a modified Elecsys 1010 system (Roche Diagnostics). RESULTS: The ratio between the signals obtained with perfectly matching and non-perfectly matching oligonucleotides reflects the genetic status. Analyzed samples can be divided into three nonoverlapping groups based on these ratios. We confirmed the reliability of the method by analyzing several samples of known genetic status; the results were identical in every single instance. CONCLUSIONS: The test discriminates unambiguously between the heterozygous and the homozygous states. Because of its low costs and easy handling, the assay is suitable for use in routine laboratories of clinical chemistry.

Autoanalysis↗
PubMed1998

Mutation analysis of exon 3 of the LDL receptor gene in patients with severe hypercholesterolemia.

Single-strand conformation polymorphism analysis was used to screen for mutations in exon 3 of the low density lipoprotein receptor gene in a group of 218 unrelated patients with severe hypercholesterolemia (low density lipoprotein cholesterol > 6.7 mmol/l) living in the Cologne area of Germany. Including the complementary primers the fragment studied had a length of 176 bp. An abnormal single-strand conformation polymorphism pattern was observed in eight patients, four of whom had an identical abnormal fragment pattern indicating that five different mutations were present. By direct DNA sequencing, the underlying mutations could be confirmed (Cys54-->Tyr, Trp66-->Gly, Glu80-->Lys, 2 bp insertion (AT between codon 44 and 45, 9 bp deletion (codons 65 to 67)). The analysis of the pathogenicity indicates that all mutations were causative for the low density lipoprotein cholesterol elevation. The Trp66-->Gly and Glu80-->Lys mutations were previously described in a French-Canadian population and in an English population, respectively. The 2 bp insertion was detected in four unrelated patients and is one of the most frequent mutations detected up to now in the German population.

Base Sequence↗
PubMed1992

Synthesis and cytotoxic activity of C-glycosidic nicotinamide riboside analogues.

The C-glycosidic nicotinamide riboside analogue (2) was prepared by reaction of ribonolactone 24 with the lithiated oxazoline 19 followed by triethylsilane reduction to 26 and deprotection. Selective phosphorylation to the pseudonucleotide 34 was effected via the isopropylidene compound 33. In contrast to the benzoic acid riboside (28) the benzamide riboside (2) showed extremely high cytotoxicity at nanomolar concentrations to S49.1 lymphoma cells but only slightly increased dexamethasone toxicity.

Antineoplastic Agents↗
PubMed1988

Glucocorticoid-induced lymphoma cell growth inhibition: the role of leukotriene B4.

Glucocorticoid-sensitive murine S49.1 lymphoma cells respond in a biphasic way to the steroid challenge. The first effect of corticosteroids is to induce a reversible growth inhibition, which is probably permissive for the following cytolysis. Distinct mechanisms for the two effects are likely. Since dilution of S49.1 lymphoma cultures resulted in a drastic reduction of the proliferation rate, which could be overcome by the addition of conditioned medium, the proliferation appears to depend on the presence of autocrine growth factors. Therefore, the cytostatic effect of corticosteroids could possibly be attributable to an interference with the production of endogenous growth factors. Analysis of the growth-promoting activity in culture supernatant showed that the critical growth factor in diluted cultures is an arachidonic acid metabolite, the leukotriene B4. The role of leukotriene B4 in S49.1 cell proliferation received further support from the finding that while nordihydroguaiaretic acid, an inhibitor of the lipoxygenase pathway which is necessary for leukotriene formation blocked lymphoma multiplication, indomethacin, an inhibitor of cyclooxygenase activity, did not affect proliferation. Quantitation of the leukotriene B4 content of dexamethasone-treated vs. untreated cultures revealed an almost complete inhibition of leukotriene production, pointing to the significance of this mechanism for the glucocorticoid-induced lymphoma growth inhibition. Moreover, these findings offer a new approach to increase the therapeutic effectiveness of glucocorticoid therapy of steroid-sensitive leukemias and lymphomas.

Animals↗
PubMed1987

Glucocorticoid-induced lymphoma cell death: the good and the evil.

Glucocorticoid hormones and their synthetic derivatives are widely used in therapy due to their strong anti-inflammatory and immunosuppressive potential. While the molecular basis of the anti-inflammatory action is to date at least partially understood, knowledge regarding the mechanism underlying glucocorticoid effects on the immune system is rather fragmentary. The immunosuppression could be attributed to at least two distinct processes: inhibition of the production of growth mediators and glucocorticoid-induced cell death. The mechanism of glucocorticoid-induced cell death can be divided into two steps, a reversible growth inhibition and cell lysis. The first step is characterized by many metabolic alterations typical of the catabolic potential of corticosteroids. After a delay of several hours activation of an endonuclease appears to initiate the lytic phase. By the action of this endonuclease the DNA is fragmented. In opposition to the chromatin damage, poly(ADP-ribosyl)ation is activated in order to stabilize the chromatin structure until the antagonistic potential is exhausted and the cells die. Therefore it can be speculated that the lethal event in glucocorticoid-induced cell death is a destruction of the regular chromatin structure.

Animals↗
PubMed1987

Reduction of DNA-polymerase beta activity of CHO cells by single and combined heat treatments.

The effect of single and combined heat treatments on the activity of DNA polymerase beta was studied in CHO cells. The activity of polymerase beta was determined by measuring the amount of [3H]TTP incorporated into activated calf thymus DNA in the presence of aphidicolin, a specific inhibitor of DNA polymerase alpha. Biphasic response curves were obtained for all temperatures tested (40-46 degrees C) showing the sensitivity to decrease during heating. A constant activation energy of Ea = 120 +/- 10 kcal/mole was found for the initial heat sensitivity, whereas the Arrhenius plot for the final sensitivity is characterized by an inflection point at 43 degrees C with Ea = 360 +/- 40 kcal/mole or Ea = 130 +/- 20 kcal/mole for temperatures below or above 43 degrees C, respectively. The observed decrease of the polymerase activity is not due to a decrease in the number of active enzyme molecules but to a change in its affinity, since the inhibition is reversible when increasing concentrations of TTP are applied. When acute or chronic thermo-tolerance was induced by a priming heat treatment at 43 degrees C for 45 min followed by a time interval at 37 degrees C for 16 h or by a preincubation at 40 degrees C for 16 h, respectively, the thermal sensitivity of polymerase beta was lowered by a factor of up to 5. By contrast, pretreatment at a higher temperature followed by a lower temperature (step-down heating) did not alter the sensitivity of polymerase beta to the second treatment. The results indicate that heat-induced cell death cannot be the consequence of the reduction of the polymerase beta activity, confirming earlier studies on this subject.

Animals↗
PubMed1986

Glucocorticoid-induced cell death and poly[adenosine diphosphate(ADP)-ribosyl]ation: increased toxicity of dexamethasone on mouse S49.1 lymphoma cells with the poly(ADP-ribosyl)ation inhibitor benzamide.

Treatment of S49.1 mouse lymphoma cells with the synthetic glucocorticoid dexamethasone resulted in a delayed cell death. During the 24-h latency period, DNA, RNA, and protein synthesis fell to about 50%, 60%, and 30% of control values, respectively, without a change in ATP levels, the latter suggesting cellular integrity. The onset of cellular suicide was characterized by the occurrence of DNA strand breaks, finally leading to the total digestion of internucleosomal DNA. Concomitant with the appearance of DNA fragmentation, poly(ADP-ribosyl)ation was activated, a process probably involved in DNA repair. Activation of poly(ADP-ribose)synthetase was paralleled by a fall in the level of the substrate NAD. An antagonistic role of poly(ADP-ribosyl)ation in glucocorticoid-induced cell death was suggested by the observation that low concentrations of the potent poly(ADP-ribose)synthetase inhibitor benzamide enhanced the toxicity of dexamethasone several-fold and shortened the interval between steroid addition and the onset of cell death. In addition, the fall in NAD was prevented by benzamide. The antagonistic function of poly(ADP-ribosyl)ation in glucocorticoid-induced cell death is, therefore, comparable to the role of the poly(ADP-ribose)synthetase in cells treated with alkylating agents, suggesting involvement of a DNA repair phenomenon in opposition to the mechanism of glucocorticoid-induced cell death.

Adenosine Triphosphate↗
PubMed1985

Cellular recovery of dividing and confluent C3H10T1/2 cells from N-methyl-N'-nitro-N-nitrosoguanidine in the presence of ADP-ribosylation inhibitors.

The relationship between treatment with 3-methoxy-benzamide (MBA), a potent inhibitor of ADP-ribosylation reactions, and the response of C3H10T1/2 cells to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) has been examined. Quiescent cells effected potentially lethal damage repair (PLDR) over a 48-h period following MNNG and the repair was coincident with the removal of DNA strand breaks. MBA had no effect on PLDR but was very co-cytotoxic with MNNG in dividing cells. The presence of MBA caused the appearance of an additional number of DNA strand breaks following MNNG in both quiescent and dividing cells. These results suggest that ADP-ribosylation is required for normal cell cycle progression following DNA damage in dividing cells.

Animals↗
PubMed1984

NAD[S], an NAD analogue with reduced susceptibility to phosphodiesterase. Chemical synthesis and enzymic properties.

The chemical synthesis of adenosine(5') [alpha-thio]diphospho(5')ribofuranosyl-nicotinamide (NAD[S]) is described. The product occurs as a pair of diastereomers with different configuration at the sulfur-bearing phosphorus atom. The diastereomers were separated by high-performance liquid chromatography and their absolute configuration was determined after chemical degradation to the ADP[alpha S] diastereomers and chromatographic comparison with enzymically synthesized ADP[alpha S] diastereomers of known absolute configuration. Additional support for this assignment is based on different rates in the phosphodiesterase-catalyzed hydrolysis. Furthermore the synthesis of [14C]NAD[S] is described. The coenzyme activity of NAD[S] in the reaction with alcohol dehydrogenase from baker's yeast and lactate dehydrogenase from pig heart is very similar to that of beta-NAD. Also, NAD and NAD[S] serve equally well as substrates for NAD glycohydrolase from calf spleen. In contrast, no reaction was detected with NAD pyrophosphorylase, and hydrolysis of the separated NAD[S] diastereomers with snake venom phosphodiesterase showed a 26-fold and a 33-fold slower reaction rate than that of NAD. Nucleotide pyrophosphatase was less sensitive to the S substitution, hydrolyzing NAD[S] 14-times slower than NAD. Poly(ADP-ribose) polymerase from Ehrlich ascites tumor cell nuclei accepted NAD[S] as a substrate but the reaction was significantly slower and approached saturation at much lower values than with NAD. Alkaline hydrolysis of the products insoluble in trichloroacetic acid yielded AMP[S] as the main derivative. It is concluded that with NAD[S] as a substrate the nuclear acceptors were nearly exclusively mono(ADP-ribosyl) ated .

Alcohol Dehydrogenase↗
PubMed1984

DNA repair-associated ADP-ribosylation in vivo. Modification of histone H1 differs from that of the principal acceptor proteins.

ADP-ribosylation in vivo of histone H1 was studied in hepatoma cells (Yoshida AH 7974) after treatment with the alkylating agent dimethyl sulfate for 30 min and compared with that of other polypeptides. In unstimulated cells, histone H1 was only a minor acceptor (less than 4%) of total monomeric and polymeric ADP-ribosyl residues. Induction of DNA repair by dimethyl sulfate treatment increased total mono(ADP-ribosyl) protein conjugates 1.6-fold whereas histone H1-linked mono(ADP-ribosyl) groups were elevated greater than 30-fold, thus accounting for nearly one-fourth of the net increase in monomeric ADP-ribosyl residues. In contrast, histone H1-associated poly(ADP-ribosyl) residues comprised only 2% of the total increase in poly(ADP-ribose). The extent to which the histone H1 population became ADP-ribosylated was low even in dimethyl sulfate-treated cells. Less than 2% of the histone H1 molecules were mono(ADP-ribosyl)ated and only 0.003% carried poly(ADP-ribosyl) chains when an average chain length of 10 is assumed. The principal polypeptide acceptors of alkylation-induced ADP-ribosylation were concentrated in two peaks, one migrating close to the position of core histones H3/H2B and accepting most of the induced mono(ADP-ribosyl) and poly(ADP-ribosyl) residues. The other (Mr = 110,000-160,000) resembled auto-modified poly(ADP-ribose) polymerase. Our data demonstrate marked differences of alkylation-induced (ADP-ribosyl)n protein patterns to analyses performed in vitro.

Adenosine Diphosphate Ribose↗