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Biomedical subjects

A Krug

Publications and source records attributed to A Krug.

At least 37 records · Page 2Linked to original sources
PubMed1998

Spontaneous and cationic lipid-mediated uptake of antisense oligonucleotides in human monocytes and lymphocytes.

Monocytes are important target cells for anti-inflammatory antisense strategies. However, monocytes are characterized by strong phagocytic and catalytic activity which may limit the effect of antisense oligonucleotides. Intracellular distribution of oligonucleotides in monocytes and the effect of cationic lipids on oligonucleotide uptake in monocytes and other leukocytes have not been evaluated. We investigated cationic lipid-mediated uptake of oligonucleotides in human monocytes and lymphocyte subpopulations. Incorporation of oligonucleotides was quantified by flow cytometry and by confocal microscopy. In the absence of cationic lipids, nearly 100% of monocytes and of B lymphocytes incorporated oligonucleotides compared with only 12% of natural killer cells and 1% of T lymphocytes. The amount of oligonucleotide uptake per cell, as determined by mean fluoresence intensity of positive cells, was four times higher in monocytes than in B lymphocytes. Cationic lipids, which form complexes with oligonucleotides, markedly enhanced the amount of oligonucleotide uptake in all cell types and were most effective at a ratio of 1.1 of positive-to-negative molar charges. In monocytes, oligonucleotides incorporated spontaneously (without a lipid carrier) were trapped in cytoplasmic vesicles. In contrast, cationic lipid-mediated uptake of fluorescence-labeled oligonucleotides resulted in cytoplasmic and nuclear staining. We conclude that 1) monocyte and lymphocyte subpopulations differ in the degree of spontaneous oligonucleotide uptake, and 2) lipofectin both quantitatively and qualitatively affects this uptake. Our results may explain the necessary role of cationic lipids in most antisense models with leukocytes as target cells.

Flow Cytometry↗
PubMed1996

Specific suppression of human tumor necrosis factor-alpha synthesis by antisense oligodeoxynucleotides.

Recent clinical studies using neutralizing antibodies point to a key role for tumor necrosis factor-alpha (TNF-alpha) in chronic inflammatory diseases. Antisense technique is a recent approach aiming at inhibition of single proteins. Previously, we described nonspecific induction of TNF by phosphorothioate oligonucleotides. In this study, we established an in vitro model that allows specific inhibition of TNF synthesis, bypassing TNF induction. Freshly isolated human monocytes were incubated with oligonucleotides and the cationic lipid lipofectin in different ratios. TNF synthesis was stimulated with lipopolysaccharide and quantified by a specific radioimmunoassay (RIA). Among all sequences tested, one of the antisense oligonucleotides complementary to the translation initiation region of TNF mRNA (5'-CAT GCT TTC AGT CAT-3') revealed highest efficacy. At 2 microM, the antisense oligonucleotide inhibited TNF synthesis by up to 79%. A concentration as low as 250 nM of the antisense oligonucleotide was effective. Scrambled controls and controls with different, defined degrees of mismatches confirmed a sequence-specific action. Examination with confocal fluorescence microscopy showed a marked difference comparing lipofectin-mediated vs. spontaneous uptake. This study defines criteria that from the prerequisite necessary for design and application of antisense oligonucleotides against TNF in vivo.

Fluorescent Dyes↗
PubMed1996

Oligodeoxynucleotides enhance lipopolysaccharide-stimulated synthesis of tumor necrosis factor: dependence on phosphorothioate modification and reversal by heparin.

BACKGROUND: Specific inhibition of target proteins by antisense oligodeoxynucleotides is an extensively studied experimental approach. This technique is currently being tested in clinical trials applying phosphorothioate-modified oligonucleotides as therapeutic agents. These polyanionic molecules, however, may also exert non-antisense-mediated effects. MATERIALS AND METHODS: We examined the influence of oligonucleotides on lipopolysaccharide (LPS)-stimulated tumor necrosis factor alpha (TNF alpha) synthesis in freshly isolated human peripheral blood mononuclear cells. Oligonucleotides (18 mer) with different degrees of phosphorothioate modification were studied. RESULTS: The addition of phosphorothioate oligonucleotides (5 microM) caused amplification of TNF synthesis of up to 410% compared with the control with LPS alone. Without LPS stimulation, phosphorothioate oligonucleotides did not induce TNF production. We demonstrate that the enhancement of LPS-stimulated TNF production by phosphorothioate oligonucleotides does not rely on the intracellular presence of oligonucleotides and is not mediated by LPS contamination. Partially phosphorothioate-modified oligonucleotides and unmodified oligonucleotides did not increase TNF synthesis. High concentrations of the polyanion heparin reversed the oligonucleotide-induced enhancement of TNF synthesis. CONCLUSIONS: The data suggest that amplification of TNF synthesis may be caused by binding of the polyanionic phosphorothioate oligonucleotide to cationic sites on the cell surface. Such binding sites have been proposed for polyanionic glycoaminoglycans of the extracellular matrix, which have also been described to augment LPS-stimulated TNF synthesis. The present results are relevant to all in vitro studies attempting to influence protein synthesis in monocytes by using phosphorothioate oligonucleotides. The significance of our findings for in vivo applications of phosphorothioates in situations where there is a stimulus for TNF synthesis, such as in sepsis, should be elucidated.

Animals↗
PubMed1994

Paraffin ingestion--the problem.

Paraffin ingestion is the commonest cause of accidental childhood poisoning in South Africa. Children from the lower socio-economic group are affected most. They drink paraffin in the summer months from bottles or intermediate containers, mistaking it for water or cold-drink. The children are predominantly male with a mean age of 24 months. The clinical picture is one of respiratory distress with a hospital case fatality rate of 0.74%. The use of paraffin as a source of household energy in South Africa is on the increase. Based on a modernisation index it would seem that this trend will continue into the next century. It can therefore be expected that the number of cases of paraffin ingestion will steadily increase if no active steps are taken to address the problem. Prevention should entail a wide spectrum of measures, the basis of which should be a child-resistant container. An effective durable, low-cost child-resistant container which is easy to pour from should be made available by petroleum companies and/or entrepreneurs and distributed through their network. This should be combined with health education on the danger of paraffin. Health care workers and administrators should be made more aware of the problem and become involved in health education and prevention. Further research should be undertaken on the effect a change in the colour of paraffin and the use of child-resistant caps would have on the incidence of paraffin ingestion in South Africa.

Accidents, Home↗
PubMed1994

The impact of child-resistant containers on the incidence of paraffin (kerosene) ingestion in children.

The commonest cause of accidental poisoning in the South African black paediatric population is paraffin ingestion. In this intervention study a specifically designed child-resistant container (CRC) was introduced to evaluate whether its use would decrease the incidence of paraffin ingestion. CRCs were distributed to 20,000 households in the study area (Gelukspan district). No CRCs were distributed in the control area (Lehurutshe district). Health education about paraffin poisoning prevention was given in both the control and the study areas. The monthly incidence rates of paraffin ingestion were monitored during the 14-month intervention period after the distribution and were compared with the pre-intervention incidence rates in the study and control areas. The main finding was that the incidence of paraffin ingestion dropped by 47% in the study area during the intervention period. The circumstances surrounding the cases of paraffin ingestion that still occurred in the study and control areas were investigated by means of a questionnaire. We recommend that paraffin be sold in CRCs, and suggestions are made for improving health education to prevent paraffin poisoning.

Accidents, Home↗
PubMed1993

[Not Available].

Explore the source record for details and available documents.

Equipment and Supplies↗
PubMed1992

Colorimetric determination of free and total cholesterol by flow injection analysis with a fiber optic detector.

A flow injection method for the determination of total and free cholesterol is presented. Cholesterol esterase and cholesterol oxidase are immobilized on aminoalkyl glass beads. The beads are packed into a tubular glass reactor. The cholesterol esters traversing through the esterase reactor are cleaved to cholesterol and fatty acids. The oxidase reactor converts cholesterol to cholest-4-en-3-one and hydrogen peroxide is generated. The sample stream is merged with reagent streams consisting of a peroxidase solution and a solution of 2,2'-azino-bis-(3-ethyl-benzthiazoline-6-sulfonic acid) diammonium salt, and a hydrogen peroxide-dependent color reaction takes place in a short coiled reactor. The signal is monitored by means of fiber optic instrumentation. Cholesterol concentration can be related to the absorption of the oxidized dye form at a wavelength of 425 nm. The working range is 0.5-0.8 mmol l-1, and the sample throughputs are 60 and 30 h-1 for free and total cholesterol, respectively.

Cholesterol↗
PubMed1991

Oligonucleotide cleavage by restriction endonucleases MvaI and EcoRII: a comprehensive study on the influence of structural parameters on the enzyme-substrate interaction.

To elucidate the mechanism of action of the restriction endonucleases--isoschizomers EcoRII and MvaI--a study was made of their interaction with a set of synthetic oligonucleotide duplexes containing a single 5'-d(CCA/TGG)-3' EcoRII (MvaI) recognition site. The substrates had varying length and structure of the nucleotide sequences flanking the recognition site. The structure of the flanking sequence is important for the cleavage by EcoRII and MvaI enzymes; there is a structure which was found to speed up the EcoRII and MvaI action. The cleavage of oligonucleotide duplexes by EcoRII enzyme does not go to completion. EcoRII endonuclease cleaved extended substrates less efficiently than short ones. Extension of the flanking sequences, with the same nucleotide surrounding of the recognition site, substantially altered the whole kinetic pattern of MvaI hydrolysis. This was not observed with EcoRII enzyme. The restriction endonuclease MvaI distinguished between dA and dT residues in the recognition site, which was reflected in the higher rate of hydrolysis of the dA-containing strand of the quasi-palindromic DNA duplex.

Base Sequence↗
PubMed1990

Synthesis of oligonucleotide probes containing 2'-deoxy-2'-fluoronucleosides for cleavage of RNA by RNase H.

Modified oligodeoxyribonucleotides containing 3'-terminal 2'-deoxy-2'-fluorouridine (UF) or 2'-fluorothymidine (TF) were successfully applied for specific RNA hydrolysis by RNase H from E. coli. The nonanucleotides d(CACCGCGCTF) and d(CACCGCGCUF) were synthesized using the phosphoramidite solid support method. The modified nucleosides were immobilized on the CPG support and provided the starting nucleoside residues. Model experiments were carried out using the 5S RNA from E. coli ribosomes and its 1-41 fragment. It was found that the use of this type of modified probes did not decrease neither the efficiency nor the specificity of the RNase H reaction.

Bacterial Proteins↗
PubMed1988

Mutagenic activity of acetonitrile and fumaronitrile in three short term assays with special reference to autoinduction.

Two aliphatic nitriles, acetonitrile and fumaronitrile were tested for their genotoxic potential in three mutagenicity test systems: the Salmonella/microsome-assay, an assay using Saccharomyces cerevisiae (strain D7), and the bone marrow micronucleus test. Both compounds were tested with and without metabolic activation in the yeast and the bacterial test systems using S9 preparations from phenobarbitone-pretreated and autoinduced rats. Autoinduction was performed by chronic (7 days) application of a dose equivalent to a 5% oral LD50-value of the respective compound. With yeast strain D7 both nitriles induced low levels of gene conversion in the presence of phenobarbitone-induced liver homogenate. An increase in the number of ile+-revertants was not detectable under any condition. Neither of the compounds showed mutagenic activity in the Ames test with or without metabolic activation. A weak positive effect of acetonitrile could be detected in the micronucleus test 24 h after i.p.-injection of the compound using a dose of 60% LD50. Fumaronitrile showed positive results with a 50% LD50 dose 48 h after administration to mice not preinduced. After 1 week of autoinduction these effects did not appear anymore, with the exception of acetonitrile 72 h after application of a dose amounting to 60% of the oral LD50-value.

Acetonitriles↗
PubMed1988

[Automated synthesis of oligodeoxyribonucleotides with terminal phosphate groups].

3'- and 5'-phosphorylated oligodeoxyribonucleotides have been synthesized on the "Victoria-4M" automatic synthesizer by phosphoramidite method. Two approaches have been suggested: introduction of a terminal ribo-unit as a potential source of phosphate group or the use of hydroxyl-containing polymer supports which loose the end product of the synthesis via beta-elimination reaction. Yields of oligonucleotides obtained according to both schemes proved to be almost identical. Using the first approach, oligonucleotides containing units with altered configuration of the sugar (xylo-thymidine and arabino-uridine) have been obtained.

Chemical Phenomena↗