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Biomedical subjects

H Probst

Publications and source records attributed to H Probst.

At least 55 records · Page 3Linked to original sources

[Conconi test on the bicycle ergometer].

Several recent papers on the Conconi procedure tested on an ergometric bicycle have shown that the deflection of the heart rate is rarely seen if the procedure follows the usual protocol where the work duration remains the same at each work level. According to the di Prampero formula, work can be expressed as the energy cost of running (C), multiplied with covered distance. Therefore it is important, as in the current 400 m-track Conconi test, to keep work and not time constant at each work level. Consequently we examined heart rate and lactate profile in four groups of athletes, in following an experimental design respecting this condition. In all ergometric test runs, we observed the sought after heart rate deflection with the following lactate values: Physical education students (female) heart rate 169 with 4.6 mmol/l lactate Physical education students (male) heart rate 169 with 5.03 mmol/l lactate Amateur cyclists heart rate 178 with 2.44 mmol/l lactate Professional cyclists heart rate 171 with 1.98 mmol/l lactate.

Adult↗

Expression of a P-glycoprotein gene is inducible in a multidrug-resistant human leukemia cell line.

A human T lymphoblastoid CCRF-CEM cell line exhibiting cross resistance to a variety of drugs was selected with increasing doses of actinomycin D. A subline, designated CCRF ACTD400+, was permanently cultured in the presence of 400 ng/ml Actinomycin D for several months. Using a fragment of the human mdr1 cDNA we found high expression of a 5 kb mRNA species which was not detectable in the sensitive parental CCRF-CEM cell line. The extent of the mdr-mRNA expression in resistant cells, however, depended on the presence or absence of actinomycin D in the culture medium: when the inhibitor was omitted, the expression decreased to about 60% after one month. In reverse, the steady state level of the P-glycoprotein mRNA increased about 2.5-fold within 72 h after the original dose of the drug was added again. In further experiments we recorded the actinomycin D or adriamycin dose response curves of the variously treated sublines by evaluation of [3H]uridine or [3H]thymidine incorporation, respectively, into acid insoluble material. Consistently, the drug sensitivity of the respective macromolecular synthesis was found to decrease with increasing mdr-mRNA levels.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

Oxygen dependent regulation of DNA synthesis and growth of Ehrlich ascites tumor cells in vitro and in vivo.

Ehrlich ascites cells were cultured under different O2 partial pressures from less than 0.1 ppm to 2 x 10(5) ppm. During the artificial hypoxia and following reoxygenation the DNA synthesis rate was measured and the relative frequency of replicon initiations was examined by analyzing the length distributions of replicative daughter strand DNA. These studies were complemented by evaluation of growth and cycling of the cells and by biochemical analyses. It was demonstrated that the reversible shut-down of clusters of replication units already described before (Probst, H., Gekeler, V., and Helftenbein, E. Exp. Cell Res., 154: 327-341, 1984) occurred between 0.25 and about 2.5 microM dissolved O2. Above 2.5 microM, a transition range to aerobiosis extended to about 16 microM O2. Below 0.25 microM O2, the cells suffered damage impairing the reversibility of the shutdown. The observed changes of growth and cycling correlated well with the respective changes of replication. Analogous oxygenation dependent regulatory events in replication were also observed during growth of the cells as an in vivo ascites tumor. Obviously, the particular oxygenation conditions in the peritoneal cavity strongly influence tumor growth via the oxygen dependent regulation of replication.

Animals↗

Synchronization of replicons in Ehrlich ascites cells.

Ehrlich ascites cells, in which replication units at the beginning of the S phase started and grew synchronously, were obtained by the following protocol: (1) selection of G1 cells by zonal centrifugation, (2) hypoxia for 12 h, (3) reaeration, (4) addition of cycloheximide (30 microM) within the first minute after reoxygenation. Studies on the effectiveness of the different steps revealed: (i) G1 cells reoxygenated after 12 h of hypoxia traverse two succeeding cell cycles highly synchronously. This was shown by monitoring the thymidine incorporation rate, the thymidine pulse-labeling index, and the mitotic index. (ii) Cycloheximide, like hypoxia, suppresses replicon initiation in Ehrlich ascites cells without interfering with DNA chain growth and DNA maturation. The reversibility of the suppression is less complete than in the case of hypoxia. This was shown by DNA fiber autoradiography and by analyzing the length distribution of pulse- or pulse/pulse-chase-labeled daughter DNA in alkaline sucrose gradients. The alkaline sedimentation patterns of daughter-strand DNA, pulse labeled immediately after the cycloheximide addition at the end of the elaborated protocol and 1 and 2 h later, indicated synchronous initiation and growth of a homogeneous population of DNA molecules to replicon-sized lengths.

Animals↗

Determination of aztreonam in faeces of human volunteers: a comparison of reversed-phase high pressure liquid chromatography and bioassay.

Aztreonam was given orally to eight healthy volunteers in two different dosage regimens. To determine the concentration of aztreonam in faecal specimens a method was established using reversed-phase high pressure liquid chromatography (HPLC). Comparison of microbiological and HPLC assays showed a linear relationship between both techniques which, however, was not 1:1. The microbiological assay tended to yield higher aztreonam levels than the HPLC method. The HPLC assay for aztreonam proved to be five-fold less sensitive than the bioassay; however, since in most cases high aztreonam levels were found in the faeces of the test subjects, this was not a disadvantage. The HPLC assay was much easier to perform than the bioassay and yielded easily quantifiable results in a wider concentration range. In the stool specimens of one volunteer no aztreonam could be detected by either method, indicating inactivation and/or degradation of the compound.

Adolescent↗

Application of hypoxia-induced shut down of replicon initiation to the analysis of replication intermediates in Ehrlich ascites cells.

Cultured Ehrlich ascites cells were subjected to hypoxic conditions for about 2 h, then reaerated or allowed to remain hypoxic. The newly formed DNA of hypoxic or reaerated cells was labeled with [3H]thymidine using different pulse and pulse/pulse-chase protocols. The chain length distribution of the labeled DNA molecules was analysed by sedimentation after lysing the cells on the top of alkaline sucrose gradients. The results indicated that the hypoxia effectively and reversibly suppressed the initiation of new replication units. Initiation, growth and integration of Okazaki pieces into active replicons was not noticeably affected. In marked contrast to aerobic cells, the use of hypoxic cells allows the separation of Okazaki pieces as a distinct class of pulse labeled short DNA chains. Short daughter DNA of very recently initiated replicons did not interfere at pulse times shorter than 4 min. For examination of the newly initiated replicons it seems favourable to trigger a burst of initiations by reaeration.

Animals↗

DNA regions associated with the nuclear matrix of Ehrlich ascites cells expose single-stranded sites after deproteinization.

Ehrlich ascites cells were pulse-labeled with [3H]thymidine and subjected to prolonged labeling with [14C]thymidine. The isolated nuclei were digested with the restriction endonuclease BspRI and then processed to yield a 'matrix fraction' and a 'non-matrix fraction'. The DNA fragments purified from these fractions and from whole digested nuclei were examined for nitrocellulose-binding sites before and after digestion with single-strand-specific (S1) nuclease. Both, pulse-labeled and long-time-labeled fragments, isolated from the matrix fraction, exhibited a significantly increased content of nitrocellulose-binding sites. The major portion of these sites were rendered non-binding by digestion with single-strand-specific nuclease and consisted most probably of structures exposing relatively small stretches of non-base-paired DNA. The nature of the minor portion of binding sites which was insensitive to single-strand-specific nuclease is not clear. Both types of binding sites are possible candidates for mediating the attachment of DNA to the nuclear matrix.

Animals↗

Oxygen dependence of nuclear DNA replication in Ehrlich ascites cells.

Oxygen was excluded from cultured Ehrlich ascites cells for 5-7 h and then readmitted. During the anaerobic period and for about 1 h following reoxygenation the DNA synthesis of the cells was studied by determining the DNA synthesis rate from [3H] thymidine incorporation data, by evaluation of the thymidine (pulse labelling) index, by DNA fibre autoradiography, and by alkaline sucrose gradients in order to follow the maturation of the daughter chains. The DNA synthesis rate was found to decay to a few percent of the initial value within 5-7 h after deoxygenation. Immediately after reoxygenation it increased to exceed the control level within 0.5-1 h. The only partial process of the genome replication definitely responding to deoxygenation/reoxygenation was the initiation of new replicon units, while progress of the replication forks and maturation of the new daughter chains were not significantly affected. The coordination of replicon initiation within groups or clusters was maintained throughout. The interruption of replication at the level of initiation of clusters upon deoxygenation was interpreted as a regulatory response of the cells to ensure basic viability under unfavourable conditions.

Animals↗

Metabolism and non-random occurrence of nonnascent short chains in the DNA of Ehrlich ascites cells.

The DNA of Ehrlich ascites cells was labeled with radioactive thymidine using different labeling schedules: Incubation periods between 15 s and 4 h; pulse/pulse-chase experiments with pulses in the range of a few minutes; longtime incubation followed by a longtime chase (both in the range of 1 cell generation). From the purified DNA of the labeled cells a fraction (0.3-0.4%) of short chains was separated and partially fractionated by means of a hydroxyapatite thermochromatography procedure. The evaluation of the labelling patterns of the short chains indicated that less than 5% of them can be regarded as replication intermediates ('Okazaki pieces'). The rest, termed nonnascent pieces, exhibited a slow turnover. The life span of the nonnascent pieces was estimated to be about 1 cell generation. On helical DNA, nonnascent pieces were distributed in a non-random manner. Their preferential localisation was nearby sites which caused binding of the DNA, after purification, to nitrocellulose and which occurred about every 60-80 microns on the nuclear DNA of the cells.

Animals↗

Replicon initiation frequency and intracellular levels of ATP, ADP, AMP and of diadenosine 5',5'''-P1,P4-tetraphosphate in ehrlich ascites cells cultured aerobically and anaerobically.

When Ehrlich ascites cells were cultured for 2 h under oxygen-free atmosphere, a shut-down of initiation of new replication units was observed by chain length analysis of the nascent daughter strands and by DNA fibre autoradiography. The intracellular level of ATP, ADP and AMP remained virtually normal in the anaerobized cells, while that of diadenosine 5',5'''-P1,P4-tetraphosphate was found reduced by about two orders of magnitude. It is proposed that the ceasing of DNA synthesis after O2 removal is at actively controlled regulatory response of the cells in which diadenosine 5',5"'-P1,P4-tetraphosphate is probably involved.

Adenine Nucleotides↗

Isolation of replication forks from growing Ehrlich ascites cells.

A procedure is described which permits the large-scale isolation of essentially complete replications forks from the DNA of Ehrlich ascites cells. The whole nuclear DNA is first isolated by a method which involves minimal hydrodynamic shear. The DNA is then degraded by cryolysis, a freeze-thawing procedure, to a size providing the otherwise very labile forked structures with a sufficient resistance against shear forces. Finally, the Y-shaped structures of replicating DNA are separated by nitrocellulose column chromatography. When the newly formed strands of replicating DNA were density-labeled with 5-bromodeoxyuridine the DNA fraction isolated by this procedure banded in isopycnic CsCl gradients at a density expected for Y-shaped molecules with two light-heavy branches and one light-light branch and sedimented significantly faster than the corresponding bulk DNA fraction through neutral sucrose gradients. The forked molecules could be visualized by electron microscopy. The essential step of the procedure is the cryolysis which produces fragments from larger DNA structure essentially at random. When the cryolysis is omitted the forked structures are disrupted within the highly susceptible regions around the branching point.

Animals↗

Random fragmentation of replicative DNA structures of eucaryotes.

The expectation values of the sizes of the different types of fragments containing replication forks obtained by random fragmentation of the DNA of growing mammalian cells have been derived. Fragments containing a single fork are expected to have, on the average, three branches of the length of a mean linear fragment thus exceeding the molecular weight of the latter by a factor 3. This factor is about 2 for fragments containing two forks very recently started from a common initiation point, and about 4 for fragments containing two forks meeting each other at the end of adjacent replication units. Only forks very close to one another are expected in these latter fragment types. Therefore, they should represent the earliest or the latest stages of replicon action.

DNA↗