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L Rensing

Publications and source records attributed to L Rensing.

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

Heat shock effects on second messenger systems of Neurospora crassa.

Exposure of growing hyphae of Neurospora crassa to heat shock (44 degrees C) or ethanol (2.6 M) for 1 h induced a significant increase in the cAMP level, which reached a maximum approximately 2 min after the beginning of treatment and then decreased to control values despite continued heat or ethanol exposure. A 10-s heat shock or a 5-s ethanol shock also resulted in a transient cAMP increase 2 min after the pulse. Heat shock or ethanol treatment led to an increase in the amount of catalytic subunits of the cAMP-dependent protein kinase A in the nucleus almost synchronously with the increase of cAMP in the cytoplasm. The concentration of cGMP decreased a few seconds after the beginning of heat shock (44 degrees C) or ethanol treatment (2.6 M) to approximately 50% of the control level. Exposure to heat shock (44 degrees C, 1 h) led to an increase in the amount of inositol phosphates 0.5-2 min after the onset of heat shock. Thereafter, inositol phosphate levels dropped to control values despite continued heat exposure. Incubation of growing hyphae with cAMP or 8-Br-cAMP led to a two- to threefold increase of inositol phosphates 10-300 s after the beginning of incubation. Heat treatment furthermore caused a rapid release of calcium from vacuoles as determined by Fura-2 measurement of the calcium content released from isolated vacuoles. These heat-shock-dependent second messenger changes may play a role in the heat-shock-induced phase shifts of the circadian clock and heat-shock-induced conidiation.

Calcium↗
PubMed1998

Nuclear translocation of stress protein Hsc70 during S phase in rat C6 glioma cells.

The expression and the nuclear translocation of the constitutive heat shock protein 70 (Hsc70) were determined during the cell cycle in synchronized rat astrocytomic C6 glioma cells. Cells were first shifted to the G0 by serum starvation. Twelve hours after a subsequent growth stimulation by transfer to 20% newborn calf serum, about 50% of the cells entered S phase. Western blot analysis with different monoclonal antibodies showed that only the constitutively expressed and moderately stress-activated Hsc70 is induced during serum stimulation. Maximal cellular Hsc70 content (170% of the control) was observed in early to mid S phase followed by a drastic decline while cells pass through G2/M (20% of the control). Hsp70, the major heat-inducible heat shock protein in C6 cells, is not detected in either asynchronously proliferating, serum-starved or in serum-stimulated C6 cells. Analysis of the nuclear and cytoplasmic protein fractions showed a significant increase of Hsc70 translocation into the nucleus during early S phase. These results indicate a role for Hsc70 but not for Hsp70 in the process of S phase entry and/or progression in C6 cells under physiological conditions.

Animals↗
PubMed1997

Heat shock protein induction by certain chemical stressors is correlated with their cytotoxicity, lipophilicity and protein-denaturing capacity.

Seven agents were analyzed with respect to their ability to induce heat shock protein (HSP) synthesis in C6 rat glioma cells. Induction of HSP synthesis was correlated with cytotoxicity and lipophilicity of the substances. In addition to the first four n-alcohols (methanol, ethanol, propanol and butanol) and phenol, whose capacity to induce HSP was analyzed earlier (Neuhaus-Steinmetz et al., 1994. Mol. Pharmacol. 45, 36-41), isopropanol, 1,4-dinitrophenol (DNP), diethylstilbestrol (DES), carbonylcyanide-m-chlorophenylhydrazone (CCCP), rotenone, paracetamol and acetyl salicylic acid (ASA) induced HSP synthesis after a 1-h incubation at a substance-specific concentration. The maximal induction of HSPs was closely correlated with the cytotoxicity of all substances and occurred when cell viability was reduced to 75 +/- 11% of the controls. Cytotoxicity and the ability to induce HSP were correlated with the lipophilicity of the alcohols, phenol, rotenone and paracetamol. Calculation of the hypothetical membrane concentrations of these compounds yielded a nearly equal value (0.54 +/- 0.13 M), indicating that interaction of substances with lipophilic cellular compounds, such as membranes or lipophilic core regions of proteins, is a critical step leading to HSP induction. This assumption is supported by a correlation between HSP induction and protein denaturation by the different alcohols (Herskovits et al., 1970. J. Biol. Chem. 245, 2588-2598). We assume that the amount of misfolded proteins induced by these lipophilic agents is responsible for the induction of HSP synthesis. ASA, DNP and CCCP induced HSP at lower concentrations than substances with a similar lipophilicity, which may be due to effects which add to the misfolding of proteins or to other signal pathways.

Actins↗
PubMed1997

Nuclear translocation of constitutive heat shock protein 70 during S phase in synchronous macroplasmodia of Physarum polycephalum.

The level of constitutive heat shock protein 70 (HSC70) in Physarum polycephalum was analyzed by means of Western blots during the synchronous cell cycle of macroplasmodia. Total amounts as well as nuclear and cytoplasmic contents were determined separately and evaluated densitometrically. A drastic increase of nuclear HSC70 was observed 10-40 min after the initiation of S phase (600% of the M phase value) and thereafter a slow decline toward the next M phase. Total HSC levels showed a slight (30%) increase during S phase whereas cytoplasmic HSC70 was about 30% lower during S phase compared to mitosis.

Animals↗
PubMed1997

Heat shock- and ethanol-induced ionic changes in C6 rat glioma cells determined by NMR and fluorescence spectroscopy.

The effects of two different stressors, heat shock (HS; 44 degrees C, 20 min) and ethanol (1.2 M, 60 min), on ion content and membrane potential were investigated in C6 rat glioma cells. Both treatments were previously shown to induce the HS response [26]. Intracellular pH (pH(i)), sodium ion concentration ([NA+]i), potassium ion concentration ([K+]i) and membrane potential were determined by means of continuous 31P and 23Na nuclear magnetic resonance (NMR), continuous fluorescence spectroscopy and 86Rb uptake. Lactate extrusion was determined in addition with respect to pH(i) regulation. The aim of this study was a detailed picture of HS and ethanol-induced ion changes in a single cell type, because stress-induced changes in the intracellular ionic balance may be important factors for determining proliferation, stress response and apoptosis. HS lowered the pH(i) from 7.38 +/- 0.04 to about 7.05 +/- 0.04. [Na+]i decreased during HS to 50% of the control and recovered to normal level 95 min after HS treatment. During HS, [K+]i remained constant but increased after HS. The membrane potential hyperpolarized from -83 mV to -125 mV and returned to initial values during HS treatment. Lactate extrusion increased 3-fold after HS. Ethanol (1.2 M) lowered the pH(i) from pH 7.38 +/- 0.04 to pH 7.0 +/- 0.04, but in contrast to heat strongly increased [Na]i. It hyperpolarized the membrane potential from -83 to -125 mV. Ethanol also increased lactate extrusion similar to HS. Also in contrast to the effect of HS, the potassium concentration decreased during ethanol treatment. The Na(+)-H+ exchanger monensin was used to overcome the apparent inhibition of the cellular Na(+)-H+ exchanger by HS. At normal pH(e) (7.4) monensin increased [Na+]i and pH(i) considerably. A subsequent HS reduced [Na+]i only minimally. Acidification of the cells by low pH(e) (6.2) prior to HS did not abolish the HS-induced drop of pH(i), indicating that the Na(+)-H+ exchanger was also inhibited at low pH(i). At low pH(e), monensin transports H+ into the cell. A subsequent HS decreased pH(i) only little, showing the importance of inhibition of the Na(+)-H+ exchanger for the HS-induced pH(i) decrease. 100 microM amiloride reduced pH(i) and [Na+]i in a similar way as HS, but did not change pH(i) and [Na+]i much during a HS. These results indicate that some of the HS-induced ionic changes are mediated by inhibition of the Na(+)-H+ exchanger, activation of Na(+)-K(+)-ATPase and changes of membrane conductance for ions.

Animals↗
PubMed1997

Stress response of lysosomal cysteine proteinases in rat C6 glioma cells.

Acid proteinases of C6 rat glioma cells were analyzed by means of gelatine polyacrylamide electrophoresis with respect to their responses to stress (heat shock and butanol). Proteinase activities on gelatine gels were characterized by their molecular masses. pH-optima, isoelectric points and reactions to inhibitors. Four bands of 25, 35 and 65/85 kDa most probably represent active and proforms as well as precursor complexes of lysosomal cysteine proteinases with pH optima between 4.0 and 5.0. The 25-kDa band seems to contain cathepsin L and B, the 35-kDa band proforms of cathepsin L and B and the 65/85-kDa bands possibly precursor complexes of cathepsin L and B. After 30-min heat shocks of different temperatures (40-50 degrees C), the 35-kDa activity increased, whereas the 65/85-kDa activity decreased after exposure to 42 and 44 degrees C, which also caused a strong increase in the level of the inducible heat shock protein of 68 kDa (HSP 68). The alterations of the proteinase activities and the increases of the HSP 68 levels occur at heat shock treatments that cause cell death in about 25-40% of the population as determined by Trypan blue staining. HSP 68 induction and proteinase activity changes were also observed 12 hr after a 1-hr treatment with different butanol concentrations (0.14-0.16 M). Kinetics of the response to a 30-min heat shock (44 degrees C) revealed a maximal decrease of the 35-kDa and a maximal increase of the 65/85-kDa activities after 12 hr recovery. When cells were exposed to repeated heat shocks (44 degrees C) at 12-hr intervals, the HSP 68 level further increased, whereas the 35-kDa and 65/85-kDa proteinase activities did not change. This result indicates a role of HSP 68 (or other HSPs) in the processing or stability of the putative cathepsin precursors (65/85-kDa complexes).

Animals↗
PubMed1997

Temperature compensation of the circadian period length--a special case among general homeostatic mechanisms of gene expression?

In Neurospora crassa, as well as in other organisms, the expression of housekeeping genes is transiently suppressed after exposure to higher temperatures (30-45 degrees C); expression is then reactivated and adapts after a few-hours to values closer to the initial rates. Adaptive mechanisms apparently exist in the processes of transcription, RNA processing, and translation and render protein synthesis rates temperature compensated. Heat shock proteins (HSPs) play an important role within these mechanisms ("acquired thermotolerance of protein synthesis"), but their function is as yet not exactly known. Adaptive mechanisms seem also to involve intracellular ion changes after exposure to moderate temperature elevation. The expression of heat shock genes is transiently enhanced after exposure to higher temperatures and also adapts after a few hours. The adaptation mechanism includes inactivation of the heat shock transcription factor (HSF) by means of phosphorylation changes and possibly by binding of a gene product (HSP70)-a mechanism representing a negative feedback control. These examples demonstrate the existence of general adaptive mechanisms at different levels of gene expression that may also be at work in the temperature compensation of clock gene expression. Apart from such adaptation processes, antagonistic reactions within the processes of gene expression and protein modification might be equally enhanced or suppressed by temperature changes, leaving the equilibrium unaffected or balanced (antagonistic balance, see Ruoff et al., this issue of Chronobiology International). This principle is shown to apply to the effect of temperature elevation on total protein synthesis and degradation. It may also apply to other antagonistic processes such as phosphorylation-dephosphorylation or monomer-dimer formation. The circadian clock mechanism is assumed to consist of several processes that can either adapt or produce a balance. Single amino acid changes in a clock protein are assumed to partially upset this adaptation or balance.

Circadian Rhythm↗
PubMed1997

Modeling temperature compensation in chemical and biological oscillators.

All physicochemical and biological oscillators maintain a balance between destabilizing reactions (as, for example, intrinsic autocatalytic or amplifying reactions) and stabilizing processes. These two groups of processes tend to influence the period in opposite directions and may lead to temperature compensation whenever their overall influence balances. This principle of "antagonistic balance" has been tested for several chemical and biological oscillators. The Goodwin negative feedback oscillator appears of particular interest for modeling the circadian clocks in Neurospora and Drosophila and their temperature compensation. Remarkably, the Goodwin oscillator not only gives qualitative, correct phase response curves for temperature steps and temperature pulses, but also simulates the temperature behavior of Neurospora frq and Drosophila per mutants almost quantitatively. The Goodwin oscillator predicts that circadian periods are strongly dependent on the turnover of the clock mRNA or clock protein. A more rapid turnover of clock mRNA or clock protein results, in short, a slower turnover in longer period lengths.

Animals↗
PubMed1996

Heat shock protein synthesis is affected by intracellular pH: inhibition by monensin-induced alkalosis in C6 rat glioma cells.

The effect of intracellular pH (pHi) on heat shock protein (HSP) synthesis was investigated in C6 rat glioma cells. pHi changes were analysed by means of fluorescence spectroscopy in a perfused monitoring system allowing continuous measurements before, during and after treatments. HSP induction was determined by means of Western blots and autoradiographs. A 20 min heat shock (HS) of 44 degrees C decreased the pHi from 7.36 to 7.05 during exposure [17] and elicited the synthesis of heat shock proteins 2-8 h later. A pHi decrease, brought about by low extracellular pH (pHe) of 4.5 and 5.0 or 5.5, induced HSP synthesis after 1 h or 3 h, respectively. During these treatments, pHi decreased to values significantly lower than that caused by HS. Three h exposure to pHe 6.2, however, was not inductive. These results indicate that the heat shock-induced pHi decrease alone is not sufficient to stimulate HSP synthesis. In order to investigate the effect of alkaline pHi on the induction of HSP by heat, pHi was increased prior to HS treatments. Preincubation of cells at pHe ranging from 6.8 to 8.0 had little effect on pHi and on HSP synthesis. A shift of pHi to more alkaline values was achieved by adding the H+/Na+ exchanger monensin at alkaline pHe. Twenty microM monensin raised the pHi and inhibited the HSP induction depending on the pHe values: as pHe was increased from pH 7.2 to 8.0 HSP synthesis was increasingly inhibited. Monensin also diminished the HS-induced drop of pHi particularly at higher pHe. The result showed that neither a lower pHi nor a drop of pHi during HS is a necessary prerequisite for the induction, whereas alkalosis inhibits the synthesis of HSP.

Alkalosis↗
PubMed1996

Heat shock proteins and circadian rhythms.

Significant circadian rhythms in heat shock gene expression were observed in a prokaryotic species (Synechocystis). In eukaryotes, in contrast, several heat shock genes (constitutive and inducible) were shown to be constantly expressed. A few cases of circadian expression of heat shock proteins (HSPs), however, have been reported. Significant circadian changes of thermotolerance were observed in yeast and several plant species. Higher thermotolerance can be attributed to a higher abundance of HSPs, but also to other adaptive mechanisms. Zeitgeber effects of temperature changes can be explained on the basis of their direct effects on the state variables of the clock gene (per,frq) expression and its negative feedback loop. Effects of increased HSP concentrations, as observed after heat shock, but also after light and serotonin (5HT), appear possible, in particular with respect to nuclear localization of the clock (PER) protein, but these effects have not been documented yet. Thus, the role of HSPs in the circadian clock system is little understood and, from our point of view, deserves more attention.

Animals↗
PubMed1995

The effects of temperature change on the circadian clock of Neurospora.

The phase resetting of the circadian oscillatory system by pulses of increased temperature (zeitgebers) and the temperature compensation of its period length during longer exposures are major features of the system, but are not well understood in molecular terms. In Neurospora crassa, the effects of pulses of increased temperature on the circadian rhythm of conidiation were determined and possible inputs to the oscillatory system tested, including changes in cyclic 3',5'-adenosine monophosphate (cAMP), inositol 1,4,5-trisphosphate and H+ concentrations, as well as changes of phosphorylation, synthesis and degradation of proteins. Following the kinetics of these parameters during exposure to increased temperature showed transient changes. Experimental manipulation of cAMP, Ca2+ and H+ levels, and of the synthesis and, possibly, degradation of proteins, resulted in phase shifts of the oscillatory system. It is assumed that the temperature signal affects the oscillator(s) by multiple pathways and shifts the whole state of the oscillatory system. Second messenger levels, protein synthesis and protein degradation show adaptation to longer exposures to elevated temperature which may be involved in the temperature compensation of the period length. The temperature compensation is also proposed to involve a shift in the state of all or most oscillator variables.

Calcium↗
PubMed1995

Close Correlation between Heat Shock Response and Cytotoxicity in Neurospora crassa Treated with Aliphatic Alcohols and Phenols.

In Neurospora crassa the aliphatic alcohols methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, isobutanol, ethylene glycol, glycerol, and allyl alcohol and the phenolic compounds phenol, hydroquinone, resorcinol, pyrogallol, phloroglucinol, sodium salicylate, and acetylsalicylic acid were analyzed with respect to their capacities to induce heat shock proteins (HSP) and to inhibit protein synthesis. Both the alcohols and phenols showed the greatest levels of HSP induction at concentrations which inhibited the overall protein synthesis by about 50%. The abilities of the different alcohols to induce the heat shock response are proportional to their lipophilicities: the lipophilic alcohol isobutanol is maximally inductive at about 0.6 M, whereas the least lipophilic alcohol, methanol, causes maximal induction at 5.7 M. The phenols, in general, show a higher capability to induce the heat shock response. The concentrations for maximal induction range between 25 mM (sodium salicylate) and 100 mM (resorcinol). Glycerol (4.1 M) shifted the concentration necessary for maximal HSP induction by hydroquinone from 50 to 200 mM. The results reveal that the induction of HSP occurs under conditions which considerably constrain cell metabolism. The heat shock response, therefore, does not represent a sensitive marker for toxicity tests but provides a good estimate for the extent of cell damage.

Journal Article↗
PubMed1995

[Stress proteins: their growing significance in medicine].

In response to a variety of "emergencies", cells produce several stress proteins (heat shock proteins, HSP) that provide them with higher resistance and are able to repair protein damage. Their expression is regulated by heat shock transcription factors. Stress proteins are arousing growing interest in medicine, as major antigens in some infections and certain autoimmune diseases and also because of their possible involvement in the development and therapy of cancer. Elevated levels of stress proteins protect tissues and organs against ischemic injury and reduce infarct size. Stress proteins may also serve as a marker in diagnostic studies and in toxicology.

Animals↗
PubMed1994

Heat shock inhibits and activates different protein degradation pathways and proteinase activities in Neurospora crassa.

In Neurospora crassa, heat shock treatment inhibits proteolytic activity. ATP-independent proteinases were analysed after polyacrylamide gel electrophoresis using renaturing gelatine gels. Proteinases of 24, 29, and 130 kDa were shown to be inhibited by heat shock and were further characterized as to their properties. A major part of the heat shock-induced inhibition is probably due to suppression of de novo synthesis of proteinases as deduced from experiments with cycloheximide. During several hours of recovery from heat shock, the inhibition of overall protein degradation and ATP-independent proteinases is reversed. Azocasein assays as well as pulse-chase experiments further showed that ATP-dependent protein degradation is only slightly affected by heat shock. Two ATP-binding proteinases of about 60 and 160 kDa even show an increased activity after heat shock. The degradation rate of heat shock proteins is inhibited by heat shock treatment, indicating that they are degraded by ATP-independent proteinases. Western blot analysis of a approximately 40-kDa degradation product of HSP70 containing its amino terminal portion revealed a reduction in the amount of this peptide after heat shock.

Adenosine Triphosphate↗
PubMed1994

Cell movement and shape are non-random and determined by intracellular, oscillatory rotating waves in Dictyostelium amoebae.

We present evidence for a mechanism of eukaryotic cell movement. The pseudopodial dynamics and shape of Dictyostelium discoideum amoebae were investigated using computer-supported video microscopy. An examination of the cell periphery by the novel method of serial circular maps revealed explicit, classical wave patterns, which indicate the existence of intrinsic intracellular oscillations. The patterns are generated by the transit of self-organized, super-positioned, harmonic modes of rotating oscillatory waves (ROWS). These waves are probably associated with the dynamics of intracellular actin polymerisation and depolymerisation. A Karhunen-Loève expansion was conducted on one cell during 10 min of locomotion using points each 10 degrees around the cell's boundary. The results show that only 2-3 modes are necessary to describe the most essential features of cell movement and shape. Based on this analysis, a wave model was developed, which accurately simulates the dynamics of cell movement and shape during this time. The model was tested by reconstructing the cell's dynamical form by means of the Karhunen-Loève transform. No difference was detected between this reconstruction and the actual cell outline. Although cell movement and shape have hitherto been viewed as random, our results demonstrate that ROWS determine the spatio-temporal expression of pseudopodia, and consequently govern cell shape and movement, non-randomly.

Animals↗
PubMed1994

Heat shock response and cytotoxicity in C6 rat glioma cells: structure-activity relationship of different alcohols.

In C6 rat glioma cells, the n-alcohols methanol, ethanol, propanol, and butanol and the aromatic alcohol phenol all induce heat shock proteins (HSPs) of high molecular mass (68, 70, 90, and 110 kDa) when applied for 1 hr. The lowest alcohol concentrations that induce HSP synthesis cause about 20% cell death, as determined by neutral red assay. HSP induction thus occurs at alcohol concentrations close to the highest tolerable dose. The cytotoxicity and the potential of alcohols to induce the synthesis of HSPs increase with chain length and are correlated with the lipophilicity of the alcohols. A clear structure-activity relationship is observed for both parameters. A calculation of the putative membrane concentrations of these alcohols reveals that cytotoxic effects (50% cell death) occur at nearly the same membrane concentration (approximately 0.2 M). This also holds true for the lowest HSP 68-inducing alcohol concentrations, but at a lower concentration (approximately 0.12 M). The activities of major proteinases are affected by both heat shock and alcohols. The effects of alcohols also depend on the lipophilicity of the alcohols. Effective concentrations again are close to the highest tolerable dose. The stress reactions measured in terms of significant changes in HSP synthesis and proteinase activity provide information about the mechanisms by which toxic agents act on the cell.

Alcohols↗
PubMed1993

Formation of morphological differentiation patterns in the ascomycete Neurospora crassa.

Morphological differentiation patterns--among them concentric rings and radial zonations--can be induced in the band-mutant of Neurospora crassa by appropriate experimental conditions, in particular by a mere shift of certain salt concentrations in the medium. The role of initial experimental conditions is examined and, furthermore, the influences of artificially induced phase differences are analyzed with respect to pattern formation. While the concentric ring pattern is due to some (endogenous) circadian rhythmicity within every hypha, nothing is known about the underlying mechanism of radial zonation development. Various hypotheses were tested with the help of a cellular automaton model which mimics growth, branching and differentiation of a fungal mycelium. In particular, sufficient conditions are provided which imply the formation of radial spore zonations. These conditions postulate a rather homogeneous microscopic hyphal branching pattern and induction of spore differentiation by means of an activator-inhibitor system. Furthermore, a working hypothesis for the formation of spore patterns in Neurospora crassa is suggested which is based on an extracellular control of fungal differentiation.

Cell Differentiation↗