Inhibition of restriction endonuclease cleavage due to site-specific chemical modification of the B-Z junction in supercoiled DNA

Structural distortions on the boundary between right-handed B and left-handed Z DNA segments in plasmid pRW751 (a derivative of pBR322 containing (dC-dG)13 and (dC-dG)16 segments) were studied by means of chemical probes. Samples of supercoiled DNA were treated with the respective chemical probe, linearized with EcoRI and inhibition of BamHI (whose recognition sequence GGATCC lies on the boundary between the (dC-dG)n segments and the pBR322 nucleotide sequence) cleavage was tested. Treatment with osmium tetroxide in the presence of pyridine or 2,2'-bipyridine, respectively, resulted in a strong inhibition of the BamHI cleavage at both restriction sites, provided the (dC-dG)n segments were in the left-handed form. In the presence of 2,2'-bipyridine submillimolar concentrations of OsO4 (at 26 degrees C) were sufficient to induce the inhibition of BamHI. Chloroacetaldehyde was used as a probe reacting selectively with atoms involved in the Watson-Crick hydrogen bonding. Similarly as in the case of osmium tetroxide treatment of pRW751 with this agent resulted in the inhibition of BamHI cleavage. It was concluded that the B-Z junction regions in pRW751 contain few solitary bases with disturbed hydrogen bonding or non-Watson-Crick base pairs.     

Enzyme distribution and transport activities in electrophoretically isolated fractions of the muscle sarcotubular system

A simple electrophoretic method is introduced allowing to isolate five fractions of skeletal muscle ST-system vesicles. In a previous study differences in lipid content, 3H-ouabain binding and in presence of triads in individual fractions (Lehotsky et. al. 1986) were analysed. In the present study biochemical characterization was extended, and (in accordance with previous results) major differences were observed to exist between fraction 1 and fractions 3 and 4. SDS-PAGE showed that fractions 3 and 4 were enriched in a protein with m.w. 100 kD, these fractions showing the highest specific activities of (Mg2+ + Ca2+)-ATPase and oxalate-supported Ca2+-uptake; activities of Mg2+-ATPase and surface membrane marker enzymes were the lowest in these fractions. On the other hand, in fraction 1 the highest activities of Mg2+-ATPase and marker enzymes of the surface membrane were observed together with a decreased content of the 100 kD protein and activities of Ca2+ transport. It could be concluded that the method is suitable to differentiate between relatively pure SR (fractions 3 and 4) and fractions rich in sarcolemma or T-tubules components (fractions 1 and 5).     

Serum catalase enzyme activity in liver diseases

Serum catalase activity was moderately increased in fatty liver, acute alcoholic hepatitis and in the decompensated form of cardiac circulatory failure. It showed significant increase in acute yellow atrophy and in toxic hepatitis while no changes were detected in liver cirrhosis and viral hepatitis. Serum catalase activity showed a good correlation (r = 0.820) with the serum glutamate dehydrogenase activity. In accordance with our results, the inexpensive assay of serum catalase activity is suggested for the detection of severe liver cell damage.     

Autoradiographic localization of high affinity uptake sites for 3H-D-aspartate in the rat olfactory bulb

In order to reveal excitatory amino acid-ergic neuronal connections in the rat olfactory bulb, uptake sites for the tritiated D-aspartic acid were analyzed by high resolution autoradiography. Light microscopy revealed both cellular and terminal-like uptake. Based on electron microscopy, overwhelming majority of the cellular uptake was assigned to glial cells. A fairly high number of labelled terminals appeared in the surroundings of the mitral cell somata, within the deepest portion of the external plexiform layer, in the internal plexiform layer and in the outer half of the granule cell layer. Labelled terminals synapsed onto likely granule cell dendrites or spines, at asymmetric membrane thickenings. These results suggest that, although the output neurons may not utilize glutamic or aspartic acid as their transmitters, these amino acids may, however, contribute to the bulbar neurotransmission, as mediator substances of a subgroup of centrifugal fibers to the olfactory bulb.     

Orientation dependence and motional properties of spin labels in cardiac and skeletal muscle fibres

Orientation dependence and rotational motion of maleimide spin labels attached to the fast reacting thiol sites of myosin were studied in glycerinated cardiac and skeletal muscle fibres in rigor and in relaxing medium. The probe order in skeletal muscle was shown to be about one order of magnitude higher than that in cardiac muscle. In skeletal muscle in rigor the orientational order is static on the time scale of the saturation transfer electron paramagnetic resonance measurement (ST EPR, rotational correlation time of the label is greater than 1 ms), but in cardiac muscle fibres, a disorder was observed which was at least partly dynamical, the rotational correlation time being about 100 microseconds. In relaxing solution the degree of order of probe molecules in both types of muscle was strongly reduced at and above the resting length. The disorder was at least partly dynamical on the ST EPR time scale, the apparent rotational correlation times being 200 microseconds for skeletal muscle and 60 microseconds for cardiac muscle, respectively. According to the results of ST EPR the rotational behavior of cross-bridges was identical in cardiac and skeletal muscle in relaxing medium.     

The regulation of the cerebral circulation in long lasting bradycardia

In the course of long lasting bradycardia in elderly patients, cardiac output will regularly diminish, circulation will slow down and signs of cerebral insufficiency may become manifest. The changes of cerebral circulation and its regulation were studied in 10 patients 61-74 years of age, with restricted cerebral regulatory capacity, suffering from permanent bradycardia. Cerebral blood flow was measured by using the venous isotope dilution technique by double punctures of the internal jugular vein. Hemispheric cerebral blood flow, cerebral O2 consumption and cerebral vascular resistance were determined during bradycardia and after termination of bradycardia by pacemaker. During long lasting bradycardia, cerebral blood flow and cerebral O2 consumption decreased, cerebral vascular resistance was elevated. After pacemaker implantation, cerebral blood flow and O2 consumption increased and cerebral vascular resistance decreased, approaching the normal value. The symptoms of cerebral insufficiency disappeared on improvement of the cerebral circulation.     

Septal deafferentation produces continuous rhythmic slow activity (theta) in the rat hippocampus

Hippocampal EEG was recorded in behaving rats in which the brain stem afferents to the septal region were previously damaged. In these animals rhythmic slow activity (RSA or theta) was continuously present, including drinking and immobility. The average frequency of RSA, however, was significantly higher during running (8-9 Hz) than during drinking or awake motionless state (6-8 Hz). In normal rats irregular sharp waves, rather than RSA were present during drinking and immobility. The results suggest that brain stem afferents are necessary to suppress the rhythmic firing of septal "pacemaker" cells.     

Serum cortisol level changes in the maternal-fetoplacental unit between the 28th-40th weeks of pregnancy

Changes in the serum cortisol level of maternal venous, umbilical venous and umbilical arterial blood were studied separately between the 28th-36th weeks in cases of preterm deliveries (n = 74) and in the 40th week in cases of term delivery (n = 34). Results indicate that between the 28th-40th weeks of pregnancy the cortisol concentration increased only in the serum of the umbilical artery; the "umbilical arterial/umbilical venous concentration X100" quotient rose from a value of 86% measured at 28th-32nd weeks to 103% in the 40th week of pregnancy. Positive correlation was found between the cortisol concentration of the three samples. On the basis of these results the authors believe that fetal adreno-cortical activity increases before birth.     

The effect of acute and chronic ethanol administration on serum corticosterone concentration in rats

The effect of intraperitoneally (i.p.) and intragastrically (i.g.) administered ethanol solution, and the influence of voluntary ethanol uptake (20% v/v) on adrenocortical activity of adult male rats was studied. Both i.p. and i.g. ethanol administration resulted in a significant activation of adrenocortical mechanisms, while voluntary ethanol uptake failed to induce elevation of serum corticosterone concentration. No difference was found in blood ethanol concentration among these groups. The responsiveness of adrenocortical mechanisms was also tested in rats which were given the free choice between ethanol solution (5% v/v) and tap-water for three weeks. Unavoidable electric foot-shocks, as stressor, resulted in an elevation of serum corticosterone concentration in control animals, but this response was found to be significantly reduced in chronically ethanol drinking rats.     

The role of Ca2+ in hormonal imprinting of the Tetrahymena

It is known from model experiments on Tetrahymena that primary exposure to a hormone induces receptor formation or amplification, in other words a hormonal imprinting. Substances acting on the intracellular Ca2+ level of the Tetrahymena, such as TMB-8, EDTA, EGTA, NiCl2 and La(NO3)3, interfered with hormonal imprinting of the unicellular to different degrees, and some of them influenced hormone (insulin, TSH) binding also independently of imprinting. Interference with the intracellular Ca-metabolism generally influenced imprinting by insulin and TSH, which were mediated by different mechanisms, to dissimilar degrees, or in opposite directions. On combined application of the agents acting on Ca-metabolism, their effects were additive. It appears that intact Ca-mediation is an essential prerequisite for normal hormonal imprinting.