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Biomedical subjects

J Stegemann

Publications and source records attributed to J Stegemann.

At least 55 records · Page 3Linked to original sources
PubMed1991

Changes in extracellular muscle volume affect heart rate and blood pressure responses to static exercise.

To investigate the effect of microgravity-induced peripheral extracellular fluid reductions on heart rate and blood pressure during isometric exercise, six healthy male subjects performed three calf ergometer tests with different extracellular volumes of working muscles. In all tests, body positions during exercise were identical (supine with the knee joint flexed to 90 degrees). After a pre-exercise period of 25 min, during which calf volumes were manipulated, subjects had to counteract an external force of 180 N for 5 min. During the pre-exercise period three different protocols were applied. Test A: Subjects rested in the exercise position; test B: Body position was the same as in A but calf volume was increased by venous congestion (cuffs inflated to 80 mm Hg); test C: Calf volumes were decreased by a negative hydrostatic pressure (calves about 40 cm above heart level with the subjects supine). To clamp the changed calf volumes in tests B and C, cuffs were inflated to 300 mm Hg 5 min before the onset on exercise. This occlusion was maintained until termination of exercise. Compared to tests A and B, the reduced volume of test C led to significant increases in heart rate and blood pressure during exercise. Oxygen uptake did not exceed resting levels in B and C until cuffs were deflated, indicating that exclusively calf muscles contributed to the neurogenic peripheral drive. It is concluded that changes in extracellular muscle volume have to be taken into account when comparing heart rate and blood pressure during 1g- and microgravity-exercise.

Adult↗
PubMed1990

Comparison of arterial, end-tidal and transcutaneous PCO2 during moderate exercise and external CO2 loading in humans.

Static relationships between arterial, transcutaneous and end-tidal PCO2 (PaCO2, PtcCO2, PetCO2) as well as the dynamic relationship between PetCO2 and PtcCO2 were studied during moderate bicycle ergometer exercise with and without external CO2 loading. The exercise pattern consisted of 5-min intervals of constant power at 40 W and 100 W and 900 s of randomised changes between these two power levels. The external CO2 loading was achieved by means of controlled variations of inspiratory gas compositions aimed at a constant PetCO2 of 6.5 kPa (49 mm Hg). The PetO2 was regulated at 17.3 kPa (130 mm Hg). Under steady-state conditions all PCO2 parameters showed close linear relationships. PaCO2/PtcCO2 was near to identity while the PetCO2 systematically overestimated changes in PaCO2l No relationship showed a significant influence of the exercise intensity. Transients of PtcCO2 are considerably slower than PetCO2 transients. The dynamic relationship between both parameters was found to be independent of whether internal or external CO2 loadings were applied. It is concluded that the combination of PetCO2 and PtcCO2 measurements allows an improved non-invasive assessment of PaCO2. While PetCO2 better reflects the transients, PtcCO2 can be employed to determine slow changes of the absolute PaCO2.

Adult↗
PubMed1990

Ventilatory effects of hypercapnic end-tidal PCO2 clamps during aerobic exercise of varying intensity.

Nine subjects performed a sequence of sustained and randomised changes between 40 W and 100 W on a cycle ergometer while the end-tidal PO2 was kept close to 17.3 kPa (130 mm Hg) by means of a dynamic forcing technique (reference experiment). In a second series inspiratory CO2 was additionally manipulated so as to hold end-tidal PCO2 (PETCO2) near 6.5 kPa (49 mm Hg; 'CO2-clamp' experiment). By this forcing PETCO2 oscillations were attenuated and more evenly distributed over the frequency range. Ventilation (VT) responded to this manoeuvre with an upward trend that could not be ascribed to a slow CO2-response component, changes in metabolic rate or a dissociation of end-tidal and arterial PCO2. VT differences between reference and CO2-clamp experiments were abolished within a 3-min period following the termination of the external CO2 control. The present results suggest that the CO2-H+ stimulus plays a major role in adjusting ventilation when exercise intensity is decreased. The underlying CO2 effect appears to be neither additive nor bi-directionally symmetrical.

Arteries↗
PubMed1990

Reduction in extracellular muscle volume increases heart rate and blood pressure response to isometric exercise.

To investigate the effect of local dehydration on heart rate and blood pressure during static exercise, six healthy male subjects performed exercise of the calf muscles with different extracellular volumes of the working muscles. Exercise consisted of 5 min of static calf muscle contractions at about 10% of maximal voluntary contraction. The body position during exercise was identical in all tests, i.e. supine with the knee joint 90 degrees flexed. During a 25-min pre-exercise period three different protocols were employed to manipulate the calf volume. In test A the subjects rested in the exercise position; in test B the body position was the same as in A but calf volumes were increased by venous congestion [cuffs inflated to 10.67 kPa (80 mmHg)]; in test C the calf volumes were decreased by lifting the calves about 40 cm above heart level with the subjects supine. To clamp the changed calf volumes in tests B and C, cuffs were inflated to 300 mmHg 5 min before the onset of exercise. This occlusion was maintained for 1 min after the termination of exercise. Compared to tests A and B, the reduced volume of test C led to significant increases in heart rate and blood pressure during exercise. Oxygen uptake did not exceed resting levels in tests B and C until the cuffs were deflated, indicating that only calf muscles contributed to the neurogenic peripheral drive. It is concluded that extracellular muscle volume plays a significant role in adjusting heart rate and blood pressure during static exercise.

Blood Pressure↗
PubMed1990

Automated DNA sequencing of the human HPRT locus.

The complete sequence of 57 kb of the human HPRT locus has been determined using automated fluorescent DNA sequencing. The strategy employed increasingly directed sequencing methods: A randomly generated M13 library was sequenced to generate contiguous overlapping sets of sequences (contigs). M13 clones at the ends of these contigs were further sequenced using M13 (universal and reverse) and custom oligonucleotide primers to order the contigs and to complete the sequencing project. The human HPRT sequence includes 1676 bp 5' and 15,238 bp 3' to exons 1 and 9, respectively. The sequence contains 49 representatives of the Alu repeat, along with several other types of repetitive sequences. The Alu sequences exhibit a biased orientation, with those sequences in the first half of the locus oriented in the minus direction relative to transcription of the gene (3'----5' = 77%, P less than 0.005) and those sequences in the latter half of the locus oriented randomly (5'----3' = 67%, P less than 0.5). The development and performance of the sequencing strategy and the features of the human HPRT gene are presented.

Amino Acid Sequence↗
PubMed1990

Automated sequencing of fluorescently labelled DNA by chemical degradation.

A new general method for sequencing fluorescently labelled DNA by chemical degradation has been developed. It is based on the observation that fluorescein attached via a mercaptopropyl or aminopropyl linker arm to the 5'-phosphate of an oligonucleotide is stable during the reactions commonly used in chemical cleavage procedures. DNA to be degraded is first enzymatically synthesized in vitro by annealing and extending a fluorescently labelled primer thereby introducing the fluorescent label at the 5'-end of the fragment. The newly synthesized fluorescently labelled DNA is then chemically degraded using: (a) a set of four different cleavage reactions; or (b) only one reaction comprising methylation of G-residues followed by a partial cleavage with piperidine in the presence of sodium chloride. The fluorescent degradation products are loaded on either four lanes or one lane of the gel, respectively, and the emitted fluorescence detected online during electrophoresis. In the 'four reactions/four lanes' method 200-350 bp (base pairs) can be read from the labelled end. The 'one reaction/one lane' method, in which the nucleotide sequence is determined by measuring different signal intensities following the rule G greater than A greater than C greater than T, currently yields around 100-200 bp of sequence per sample.

Automation↗
PubMed1989

Direct genomic fluorescent on-line sequencing and analysis using in vitro amplification of DNA.

In vitro amplification of genomic DNA and total RNA, as well as recombinant DNA, using one fluorescently labelled and one unlabelled primer during amplification, together with on-line analysis of the products on the EMBL fluorescent DNA sequencer, is described. Further is reported direct sequencing of fluorescently labelled amplified probes by solid-phase chemical degradation, without subcloning and purification steps involved. At present up to 350 bases in 4 hours are determined with this technique. The fluorescent dye and its bond to the oligonucleotide are stable during the amplification cycles, and do not interfere with the enzymatic polymerization. High sensitivity of the detection device, down to 10(-18) moles, corresponding to less than 10(6) molecules makes possible analyses of the non-radioactive amplified probes after only 10 amplification cycles, starting with about 5 x 10(4) copies of recombinant DNA.

Autoanalysis↗
PubMed1989

Automated Sanger DNA sequencing with one label in less than four lanes on gel.

Novel Sanger dideoxy sequencing with only one fluorescent dye label for the four bases of one clone and sequence determination in two lanes on polyacrylamide gel is presented, loading A greater than G in one lane and T greater than C in the other. Sequencing reactions for the two bases in each lane are carried out in one tube. At present the ratio of ddATP:ddGTP and ddTTP:ddCPT is set to 5:1 in the two tubes. Distinction between the two bases in one lane is done by comparing the different magnitudes of the peaks. This method increases the capacity since more clones may be run simultaneously on one gel, while keeping the reliability and simplicity that comes with the use of only one fluorescent dye for the four bases of one clone. At present about 200 bases are determined with the one-dye two-lane method on the EMBL's automated fluorescent DNA sequencer, using T7 DNA polymerase. The error rate in the deduced sequence is about 1%. The technique is used for the determination of overlaps in mapping projects. In principle, it is possible to determine the sequence with one dye in only one lane on the gel by choosing the proper ddNTP ratios for all four bases, carrying out reactions in one tube and applying the product in one lane, but the error rate for this one-lane method seems too high at present and further improvements in the uniformity of peaks obtainable with the T7 DNA polymerase or other enzymes are required.

Base Sequence↗
PubMed1988

Automated Sanger dideoxy sequencing reaction protocol.

The protocol for Sanger dideoxy chain termination reactions in DNA sequencing is tedious and prone to errors due to the repetitive character of the pipetting steps. An industrial robot, with the addition of a few simple parts, was programmed to automate the dideoxy sequencing reactions. The system is set up in a short time for routine operation and it is faster and more reliable than a human operator. It is flexible and allows variations and optimization of the standard procedure. Disposable microtiter plates at a controlled temperature are used. In one reaction cycle (about 50 min) up to 48 templates are processed. Up to 450 bases were resolved in automated DNA sequencing on samples prepared by the robot. The protocol is applicable to fluorescent as well as to radioactive labeling.

Autoanalysis↗
PubMed1988

T7 DNA polymerase in automated dideoxy sequencing.

T7 DNA polymerase with chemically inactivated 3'-5' exo-nuclease activity, as well as unmodified T7 DNA polymerase, were used for sequencing by the dideoxy method in an automated system with fluorescence labelled primer and on-line detection of laser-excited reaction products. An analysis of signal intensity variations in the C track revealed that low C signals were usually preceded by a T in the sequence. This effect was modified by surrounding nucleotides. Signal intensities were more uniform with T7 polymerase than with the Klenow fragment of DNA polymerase I. Some sequences ambiguous with the Klenow enzyme could easily be evaluated with the T7 enzyme. One sequence could only be read by the unmodified T7 polymerase, while both the Klenow fragment and the chemically modified T7 enzyme gave uninterpretable data.

Base Sequence↗
PubMed1988

Non-radioactive automated sequencing of oligonucleotides by chemical degradation.

A non-radioactive sequencing of fluorescently labelled oligonucleotides by solid-phase chemical degradation is described. Although non-radioactive methods have been reported for the dideoxy chain termination technique, such a method has not yet been developed for the chemical degradation sequencing of DNA fragments. A 21-mer fluorescein labelled M13 sequencing primer was sequenced in an on-line automated system in about 30 minutes. The fluorescent dye and its bond to the oligonucleotide were stable during the chemical reactions used for the base specific degradations. As the sequence is determined on-line during electrophoresis, reloading and running 10 fragments simultaneously allows us to use one gel for sequencing of about 50 different oligonucleotides.

Automation↗
PubMed1988

The influence of muscle interstitial volume on K+-induced heart rate drive in rats.

During exercise heart rate is influenced by reflexogenic drives which are elicited by receptors situated in the interstitial space. Since the structure of interstitial tissue is complex (e.g. fixed negative charges of glycosaminoglycans), the situation in the immediate surrounding of the receptors might differ from the free fluid phases of blood or lymph in which the concentrations of stimulating substances are usually determined. Physiological variations of the interstitial structure may be due to changes in interstitial volume induced by exercise or the hydrostatic effects on body fluids. The objective of the present study was to investigate the effect of the interstitial volume on the relationship between heart rate and K+ stimuli applied through the muscle blood vessels. The calves of 12 male Wistar rats were artificially perfused and separated from the rest of the body with the sciatic nerve remaining intact. In these preparations the heart rate (HR) responses to low (4 mM) and high (8 mM) potassium concentrations were determined at different interstitial volumes. Expansion of the interstitial volume was obtained by reducing the colloid-osmotic pressure of the perfusate. The combination of intracellular oedema and mechanical limitation of total volume expansion (tapeing) was utilized to decrease the interstitial volume. When switching between the low and high potassium concentrations, significant heart rate responses could be observed only with reduced interstitial volume. It is suggested that the interstitial structure surrounding the muscular receptors modifies the relationship between heart rate response and the K+ stimuli determined in blood or lymph.

Animals↗
PubMed1988

Local fluid losses enhance heart rate drives in light to moderate exercise.

UNLABELLED: Dynamic calf muscle exercise of different intensities was performed at 90 degrees knee-joint flexion. To obtain different hydrostatic pressures and calf volumes, all exercise tests were conducted both in prone and supine position after 10 min of rest in the respective positions. Severe prone exercise was associated with increased anaerobic metabolism, reduced exercise tolerance time, and pain sensation. Despite these differences, maximal heart rates were nearly identical in both positions. During light to moderate exercise, heart rate was significantly increased in the prone position. In a steady-state exercise test this difference developed gradually during the first 10 min of exercise. Neither changes in muscle metabolism nor baroreceptor influence or pain sensation could account for this effect. DISCUSSION: 1) At high exercise intensities in the prone position, the decreased hydrostatic pressure reduces maximal exercise duration by virtue of reduced maximal muscle perfusion. 2) In steady-state prone exercise, the local fluid loss appears to increase peripheral heart rate drives.

Adult↗
PubMed1988

The Valsalva maneuver as an indirect, non-invasive indicator of central blood volume shift.

Our objective was to determine whether the Valsalva maneuver may be used as an indicator of fluid shift during spaceflights. The subjects, 21 healthy young men, conducted the maneuver against expiratory pressures of 20, 30, and 40 mm Hg, each lasting 30 s, at body positions of vertical, horizontal, and 6 degrees head down tilt (HDT). Heart rate was continuously recorded on a beat by beat basis together with the expiratory pressure. The increase in heart rate at equal intrathoracic pressures was maximal in the vertical position, significantly lower (p less than 0.001) in the horizontal position and lowest (p less than 0.05) in the 6 degrees HDT position. Obviously, the blood volume shift in the horizontal and 6 degrees HDT position partly compensated the impaired venous return during the Valsalva maneuver. This simple and non-invasive technique might be used to indicate the time course of blood volume shift during the initial stage of an orbital flight.

Adult↗