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Biomedical subjects

A Richter

Publications and source records attributed to A Richter.

At least 343 records · Page 19Linked to original sources
PubMed1990

Development of technology for linking photosensitizers to a model monoclonal antibody.

A procedure is described whereby the photosensitizer, benzoporphyrin derivative monoacid ring A (BPD-MA) was covalently linked to a model monoclonal antibody in a manner which is reproducible, quantifiable, and retains both the biological activity of the antibody and the cytotoxicity of the photosensitizer. Preliminary steps involved the linkage of BPD-MA to a modified polyvinyl alcohol (PVA) backbone, followed by conjugation to the antibody using heterobifunctional linking technology. Briefly, polyvinyl alcohol (MW ca. 10,000) was modified with 2-fluoro-1-methyl pyridinium toluene-4-sulfonate and 1,6-hexanediamine to produce side chains containing free amino groups. The free carboxyl group of BPD-MA was utilized to conjugate photosensitizer molecules to modified PVA using a standard carbodiimide reaction. Final linkage of the PVA-BPD to a model monoclonal antibody involved further substitution of the carrier with 3-mercaptopropionic acid and carbodiimide to introduce 3-4 sulfhydryl residues per carrier molecule, and introduction of sulfo-m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester residues to the monoclonal (3-4 residues/molecule). Conjugation was effected by reaction of the two species at pH 5.5 for 18 h. Detailed methodology and tests for efficacy of the procedure are provided.

Antibodies, Monoclonal↗
PubMed1990

Molecular structures of two human DNA topoisomerase I retrosequences.

We have isolated recombinant lambda-phage clones that contain sequences complementary to the 3' half of the cDNA encoding human topoisomerase I (hTOP1). These lambda clones belong to three distinct classes: class-I clones contain sequences from the active gene located on human chromosome 20. Class-II and class-III clones contain sequences corresponding to the cDNA encoding hTOP1 from nucleotide (nt) 2208 to 3434 and from nt 1639 to 3434, respectively. These sequences exhibit the characteristic features of retroposons or retrosequences. They are most likely derived from truncated mRNA transcripts of the active gene. We propose to designate the truncated hTOP1 sequence located on chromosome 1 as the pseudogene 1 (psi 1-hTOP1) and the sequence on chromosome 22 as the pseudogene 2 (psi 2-hTOP1). Pseudogene psi 1-hTOP1 has two unique properties: it is flanked by upstream sequences which display promoter activity in transient expression assays, and it contains an open reading frame which could code for the 211 C-terminal amino acids of hTOP1. Pseudogene psi 2-hTOP1 is located within an AluI repetitive element and is flanked on one side by a (CA)21 stretch.

Base Sequence↗
PubMed1990

Expression of the topoisomerase I gene in serum stimulated human fibroblasts.

We have determined the levels of mRNA coding for human type I DNA topoisomerase (EC 5.99.1.2.) in resting and proliferating human cells in culture. After addition of serum to growth arrested cells, we observed an continuous increase in the amount of topoisomerase I mRNA, starting after serum addition and reaching a maximum at 25 h after stimulation. At the end of the S-phase, a 6-fold higher amount of topoisomerase I mRNA was present in these cells. Nuclear run on transcription experiments showed, that the increase of topoisomerase I mRNA was preceded by a 3- to 4-fold increase in de novo mRNA synthesis. In contrast, during the same time period the amount of topoisomerase I increased only by a factor of 2, and the specific activity (enzymatic activity/mg protein) remained constant.

Animals↗
PubMed1990

Expression of the type I DNA topoisomerase gene in adenovirus-5 infected human cells.

The amount of topoisomerase I specific mRNA increases three- to fivefold during the early phase of infection of HeLa cells with adenovirus-5. The observed increase in specific mRNA is mainly due to an increased rate of transcription of the gene. In human 293 cells, which constitutively express the viral E1A and E1B genes, we determined an elevated level of topoisomerase I mRNA, comparable to the amount of mRNA present in HeLa cells early after infection with adenovirus. In contrasts, in HeLa cells infected with adenovirus dI312, a mutant were the E1A region had been deleted, the amount of topoisomerase I mRNA remained constant, unless the cells were superinfected with wild type virus. Our experiments indicate that the topoisomerase I gene is transactivated by an early adenovirus protein product coded by the E1A region. In contrast to the increase in mRNA synthesis, the amount of topoisomerase I protein and the topoisomerase I activity remain constant up to 24 hours after infection.

Adenoviruses, Human↗
PubMed1990

Nonlinear phenomenon of interfacial polarization immittance of a Pt electrode.

Description of a computerized, automated method to measure the interfacial polarization immittance of a Pt electrode in nonlinear range is presented. The classical three-electrode setup is used for measurements in conjunction with a special purpose software implemented on a Unix computer using C language. A collection of data at very low frequencies (below 2 Hz) and at high input intensities with various dc biases imposed on the input are presented to show the behavior of the interface in nonlinear range. The instrument also provides on-line harmonic analysis of the output signal, by calculating the first four Fourier series coefficients, in response to a pure sinusoidal input.

Electrodes↗
PubMed1990

Characteristics of heart rate fluctuations and respiratory movements during orienting, passive avoidance and flight-fight behaviour in rabbits.

In the present study different heart rate patterns were demonstrated to accompany flight-fight behaviour, orienting behaviour and passive avoidance in rabbits. Flight-fight behaviour was characterized by markedly increased heart rate and diminished overall heart rate variability. The effect was mediated by vagal inhibition and beta-adrenergic activation in a type-specific relation. Orienting behaviour was accompanied by a smaller heart rate increase and the exaggeration of slow heart rate fluctuations. The latter effect was absent during beta-adrenergic blockade suggesting a behaviourally provoked beta-adrenergic activation. Single beta-adrenergic blockade did not change the characteristics of the heart rate fluctuations at rest. During passive avoidance a vagally mediated heart rate deceleration was followed by a slow heart rate return toward the initial heart rate level. This level was not reached during beta-adrenergic blockade. The enhanced overall heart rate variability during passive avoidance was mainly caused by strengthened respiratory-induced heart rate fluctuations and, furthermore, by exaggerated slow rhythmical heart rate fluctuations. The latter effect was not observed during beta-adrenergic blockade and is referred to as an orienting component within passive avoidance. Three individual behavioural types may be differentiated in rabbits 'Weisses Gross-Silber' by stable behavioural characteristics i.e. spontaneous motor activities, preferred postures at rest and coping behaviour. The results of the present study suggest that different neurovegetative reaction types, i.e. dominating beta-adrenergic or vagal activation are correlated with stable behavioural characteristics, especially in terms of preferring active or passive coping behaviour, respectively.

Animals↗
PubMed1990

A recombinant topoisomerase I used for autoantibody detection in sera from patients with systemic sclerosis.

We report the expression of a cDNA clone encoding 695 carboxyl-terminal amino acids of human DNA topoisomerase I (topoI) in Escherichia coli. More than 96% of the anti-HeLa topoI-positive sera from patients with a connective tissue disease displayed also an immunoreactivity with this recombinant protein (the HTopoA protein). Sera from patients with a definite diagnosis systemic sclerosis and reacting with HeLa topoI, all reacted with the HTopoA protein as well. Sera from patients with systemic sclerosis that did not contain anti-topoI antibodies (about 30% of the systemic sclerosis sera), as concluded from HeLa immunoblot, displayed also no immunoreactivity with our recombinant antigen. By expressing different fragments of HTopoA, we were able to assign at least three different autoimmune epitope regions on the HTopoA protein and we show that over a period of 5 years the amount of anti-topoI antibodies against these regions may fluctuate.

Amino Acid Sequence↗
PubMed1990

Heart rate fluctuations in rabbits during different behavioural states.

Heart rate fluctuations (HRF) were investigated in 18 adult female rabbits (Weisses Grosssilber) in chronical experiments at rest, during motor activity, passive and active avoidance. Pharmacological blockades of the neurovegetative transmission were used to determine the characteristics of neurovegetative mediation during the behavioural states examined. Mean heart rate was always increased during vagal blockade and decreased during beta-adrenergic blockade. At rest the HRF in rabbits are mainly vagally mediated. Vagal blockade reduced the overall heart rate variability by more than 50%. Vagal blockade did not abolish respiratory heart rate fluctuations. If the mean respiratory frequency is greater than half of the mean heart rate frequently occurring in rabbits, the respiratory HRF are generated not synchronously with respiration but in a slower frequency range according to the physiologic effect of "cardiac aliasing". The movement related heart rate increase was absent if the movements were performed during combined beta adrenergic and vagal blockade. The heart rate reaction during passive avoidance was characterized by a heart rate deceleration abolished by vagal blockade. Respiratory HR were increased. Furthermore, the heart rate was returned to the previous mean level and the slow HRF not induced by respiration increased, mediated by beta adrenergic activation. Vagal blockade and beta adrenergic blockade diminished the heart rate acceleration and reduced the overall heart rate variability by more than 50% during active avoidance. Combined vagal and beta-adrenergic blockade fully abolished the heart rate acceleration effect during active avoidance. The ratio of the vagal to sympathetic reaction components differed in behavioural types.

Adrenergic alpha-Antagonists↗
PubMed1989

Degree of biotinylation in nucleic acids estimated by a gel retardation assay.

A simple and nonradioactive gel retardation assay was established in order to separate weakly biotinylated nucleic acids according to the number of biotin molecules with which they are labeled. The method uses streptavidin to retard biotinylated RNA (Bio-RNA) in nondenaturing polyacrylamide gels; the streptavidin-Bio-RNA complexes are detected by silver staining. From the quantitative analysis of the band distribution, the average number of biotinylated nucleotides incorporated in a particular preparation may be calculated. The degree of biotinylation was analyzed with this method by studying the incorporation of Bio-4-UTP or Bio-11-UTP into RNA by in vitro transcription with T7 RNA polymerase. We found that both Bio-UTPs were incorporated into RNA by T7 polymerase three to four times less efficiently than unmodified UTP and that the average degree of biotinylation depended in a simple and calculable way on the Bio-UTP to total UTP ratio during transcription. Therefore, RNA transcripts with a defined average degree of biotinylation can be synthesized by T7 RNA polymerase simply by setting the Bio-UTP to total UTP ratio to the appropriate value. The gel retardation assay was also used to determine the average number and distribution of biotinyl residues in photobiotinylated RNA.

Bacterial Proteins↗
PubMed1989

Characterization of a 48-kDa nucleic-acid-binding fragment of nucleolin.

Nucleolin (C23 or 100 kDa) is an abundant single-stranded-nucleic-acid-binding nucleolar protein proposed to be involved in the early stages of ribosome assembly. A stable 48-kDa fragment of the protein was produced either by proteolytic activity present in nucleolar extracts or by added trypsin. The hydrodynamic and DNA-binding properties of the 48-kDa fragment were compared with the parent molecule. Protein sequencing indicated that the fragment begins at residue 282; amino acid composition of the fragment including 10-12 methylated arginine residues suggested that the fragment contains the entire COOH-terminal two-thirds of the protein. The 48-kDa fragment was more globular than nucleolin, as indicated by a lower frictional coefficient (1.3 vs. 2.0 for nucleolin) and a similar sedimentation coefficient (4.1-4.3S) in spite of the reduction in molecular mass. Although the 48-kDa fragment retained single-stranded-DNA-binding activity, the binding capacity and the ability to reassociate DNA were about fivefold and sixfold lower, respectively, than nucleolin. Similarly, tenfold higher concentrations of the 48-kDa fragment were required to form nucleoprotein aggregates. These results suggest that nucleolin contains a globular COOH-terminal domain for nucleic-acid binding and a NH2-terminal region which is involved in protein-protein interactions and modulating nucleic-acid-binding activity.

Amino Acid Sequence↗
PubMed1989

Localization of the active type I DNA topoisomerase gene on human chromosome 20q11.2-13.1, and two pseudogenes on chromosomes 1q23-24 and 22q11.2-13.1.

Different subfragments of a cDNA coding for DNA topoisomerase I were used as probes to determine the chromosomal localization of topoisomerase I sequences in human cells. Southern blotting of restricted DNA from a panel of rodent-human somatic cell hybrids revealed the localization of the complete gene on chromosome 20 and the presence of two truncated topoisomerase I pseudogene sequences on chromosomes 1 and 22. In situ chromosome hybridization experiments confirmed these results showing the location of the complete gene on band q11.2-13.1 of chromosome 20, and the location of the pseudogene sequences on band q23-24 of chromosome 1 and q11.2-13.1 of chromosome 22.

Blotting, Southern↗
PubMed1989

Simultaneous purification of DNA topoisomerase I and II from eukaryotic cells.

We have developed a procedure for the simultaneous purification of DNA topoisomerase I and II from calf thymus. Both enzymes were first extracted from isolated nucleoprotein complexes. After batchwise chromatography on hydroxylapatite the two enzyme activities were separated on a FPLC phenylsuperose column. The enzymes were further purified by a second chromatography on phenylsepharose (topo I) or FPLC Mono Q (topo II). The purification can be finished within three days, yielding 0.5-1.0 mg quantities of homogeneous, enzymatic active preparations of the two proteins from 200 g of starting material.

Animals↗
PubMed1989

[Detection certainty of the contamination of bull sperm with mycoplasma].

Tests for presence of mycoplasmas were conducted on 20 insemination bulls known as mycoplasma spreaders, with 5 sperm pellet batches of 20 pellets each being investigated for each animal. Mycoplasma contamination was positively recorded from 83 of the above 100 batches. Mycoplasma (M.) bovigenitalium, M. californicum. M. arginini, M. bovirhinis, and Acholeplasma laidlawii were typed by means of indirect immunofluorescent technique, which confirmed the presence also in the GDR of mycoplasma species described in the literature and detected in sperm samples. The solid culturing media used in the above tests, medium-B agar and cattle blood agar with staphylococcal nutrix, proved to be equally suitable for isolation of mycoplasma from sperm samples. Mycoplasma was positively identified in about one third of all pellets/batches tested. 3 pellets to one batch should be sufficient a random sample size from which to obtain information at least very close to real contamination.

Acholeplasma laidlawii↗