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L Rensing

Publications and source records attributed to L Rensing.

At least 55 records · Page 3Linked to original sources

The locomotion, shape and pseudopodial dynamics of unstimulated Dictyostelium cells are not random.

The dynamic periphery of unstimulated, preaggregation, hunger-stage Dictyostelium discoideum amoebae was investigated by time-lapse videomicroscopy and digital image processing. Circular maps (i.e. of each of 360 radii around the cell transformed upon Cartesian coordinates) were constructed around the centroid of individual cell images and analysed in time series. This novel technique generated spatiotemporal structures of various degrees of order in the maps, which resemble classical wave interference patterns. The patterns thus demonstrate that cell movement is not random and that cells are intrinsically vibrating bodies, transited by self-organized, superpositioned, harmonic modes of rotating oscillatory waves (ROWS). These waves appear to depend upon spatiotemporal oscillations in the physicochemical reactions associated with actin polymerization, and they govern pseudopodial movements, cell shape and locomotion generally. ROWS in this case are unrelated to the cyclic-AMP-regulated oscillations, which characterize later, aggregative populations of Dictyostelium. However, the exposure of aggregation-stage cells to a pulse of the chemoattractant cyclic-AMP induces a characteristic sequence of changes in the global cellular concentration and spatiotemporal distribution of fibrillar (F-)actin. This reaction begins with what appears to be a phase resetting of ROWS and it may, therefore, underlie the cellular perception of and response to chemotactic signals. We also develop here an analytical mathematical description of ROWS, and use it to simulate cell movements accurately.

Actins↗

Effects of light on protein secretion in Neurospora crassa.

The relative concentrations of secreted proteins in liquid cultures of Neurospora crassa differ in constant darkness compared to constant light (2500 lx). Light reduces the concentrations of some polypeptides markedly and increases the concentrations of protein species of 67, 40, 18 and 13 kDa. The "blind" wc-2 mutant of Neurospora does not show light dependent differences in amounts of secreted proteins. One of the light-sensitive extracellular proteins is shown to be a protease of 17.5 kDa.

Culture Media↗

Light and the recovery from heat shock induce the synthesis of 38 kDa mitochondrial proteins in Neurospora crassa.

The effect of light on the protein synthesis pattern in the mitochondria of Neurospora crassa was examined by in vivo labelling with [35S]-methionine and two-dimensional gel electrophoresis. A brief 5-min illumination induced the rapid and transient synthesis of a 38-kDa protein. White collar-mutants were not stimulated to synthesize this protein by light. A protein of a similar molecular weight and isoelectrical point was synthesized during recovery from heat shock.

Autoradiography↗

Effects of heat shock on neuroblastoma (N1E 115) cell proliferation and differentiation.

Heat shock (44 degrees C) applied for only 15 min induced the development of neurites in neuroblastoma cells 3-6 days later. During the first day after heat shock a transient increase in the rate of cytokinesis together with a synchronizing effect was observed, which led to waves of cytokinesis 14.5 h apart. Individual cell cycles were determined and showed a lengthening in the minimal cell cycle duration and a decrease in the cell cycle variance after shock. Two to 3 days after heat shock the proliferation rate decreased and then recovered. During the 6 days after heat shock, total protein synthesis was lower compared to the untreated cultures. The synthesis of heat shock proteins (100, 90, 84, 70, 68 kDa and some of lower MW) reached a maximum 6 h after heat shock. Parallel changes in the phosphorylation state of proteins were observed in an in vitro assay. Four proteins (100, 89, 67, and 15 kDa) increased and two proteins (97, 73 kDa) decreased their phosphorylation state significantly. Six days after heat shock two proteins (89, 55 kDa) increased their phosphorylation state; the 55-kDa phosphoprotein was identified as tubulin. The effect of heat shock on the intracellular calcium level was determined by measuring Fura 2 fluorescence. Six hours after shock, the Ca2+ level increased to a maximum (about three times the control value) and then dropped during the following days below the control values. We conclude from these results that a decrease in the calcium level may be causally involved in the differentiation process. The calcium effect is probably mediated by changes in the activity of different kinases. This assumption is compatible with the results of experiments with cyclic nucleotides when 10(-5) M cAMP and cGMP were added to in vitro assays of protein phosphorylation. They had different stimulating effects in heat-shocked, differentiating, and growing (control) cells.

Animals↗

The effects of temperature on the circadian rhythms of flashing and glow in Gonyaulax polyedra: are the two rhythms controlled by two oscillators?

Circadian rhythms of flashing and glow were recorded simultaneously in Gonyaulax polyedra by determining maximum and minimum light emission at each measured interval of 28 sec. In constant light, the two rhythms in some cases showed different period lengths (tau), the glow rhythm being up to 1 hr shorter than the flashing rhythm. Lower temperatures shortened the tau of the glow rhythm more than that of the flashing rhythm. The amplitude of the flashing rhythm decreased when the temperature was increased from 15 degrees C to 25 degrees C, whereas that of the glow rhythm was increased. These results may indicate that the two rhythms are controlled by two separate oscillators.

Animals↗

cis,cis-cyclohexane 1,3,5-triol polyphosphates release calcium from Neurospora crassa via an unspecific Ins 1,4,5-P3 receptor.

We investigated the effects of new inositol 1,4,5-trisphosphate analogues on the release of Ca2+ from isolated vacuoles of Neurospora crassa. Tri-O-butyryl-inositol 1,4,5-trisphosphate and a set of cis,cis-cyclohexane 1,3,5-triol bis-(CHT-P2) and trisphosphates (CHT-P3) gave an increase in free Ca2+ as measured directly with fura-2, a Ca2(+)-chelator. However, inositol 1,4-bisphosphate, 6-O-palmitoyl-inositol 4,5-bisphosphate and trans-cyclohexane 1,2-diol bisphosphate (trans CHD-P2) did not induce Ca2(+)-release. These results suggest that the 1,5-bisphosphate position in inositol 1,4,5-trisphosphate (Ins 1,4,5-P3) is the only essential arrangement for receptor binding to vacuoles of Neurospora crassa. The structures of these analogues are discussed on the basis of a general concept for the design of new Ins 1,4,5-P3 analogues.

Benzofurans↗

On the role of Ca2(+)-calmodulin-dependent and cAMP-dependent protein phosphorylation in the circadian rhythm of Neurospora crassa.

Pulses of some Ca2+ channel blockers (dantrolene, Co2+, nifedipine) and calmodulin inhibitors (chlorpromazine) lead to medium (maximally 5-9 h) phase shifts of the circadian conidiation rhythm of Neurospora crassa. Pulses of high Ca2+, or of low Ca2+, a Ca2+ ionophore (A23187) together with Ca2+, and other Ca2+ channel blockers (La3+, diltiazem), however, caused only minor phase shifts. The effect of these substances (A 23187) and of different temperatures on the Ca2+ release from isolated vacuoles was analyzed by using the fluorescent dye Fura-2. A 23187 and higher temperatures increased the release drastically, whereas dantrolene decreased the permeation of Ca2+ (Cornelius et al., 1989). Pulses of 8-PCTP-cAMP, IBMX and of the cAMP antagonist RP-cAMPS, also caused medium (maximally 6-9 h) phase shifts of the conidiation rhythm. The phase response curve of the agonist was almost 180 degrees out of phase with the antagonist PRC. In spite of some variability in the PRCs of these series of experiments all showed maximal shifts during ct 0-12. The variability of the response may be due to circadian changes in the activity of phosphodiesterases: After adding cAMP to mycelial extracts HPLC analysis of cAMP metabolites showed significant differences during a circadian period with a maximum at ct 0. Protein phosphorylation was tested mainly in an in vitro phosphorylation system (with 35S-thio gamma-ATP). The results showed circadian rhythmic changes predominantly in proteins of 47/48 kDa. Substances and treatments causing phase-shifts of the conidiation rhythm also caused changes in the phosphorylation of these proteins: an increase was observed when Ca2+ or cAMP were added, whereas a decrease occurred upon addition of a calmodulin inhibitor (TFP) or pretreatment of the mycelia with higher (42 degrees C) temperatures. Altogether, the results indicate that Ca2(+)-calmodulin-dependent and cAMP-dependent processes play an important, but perhaps not essential, role in the clock mechanism of Neurospora. Ca2+ calmodulin and the phosphorylation state of the 47/48-kDa proteins may have controlling or essential functions for this mechanism.

Calcium↗

A purified cellular extract accelerates the cell cycle in Physarum polycephalum.

Plasmodia of the myxomycete Physarum polycephalum (strain Cl) were collected at different times during the cell cycle and extracts were prepared from homogenates using a buffer optimized for microinjection into plasmodial veins. These extracts were injected into plasmodia during the first 3 h of the cell cycle. The time of the following mitosis was monitored and compared with that of the buffer-injected controls. Extracts of plasmodia homogenized 45 min before late telophase accelerated the onset of mitosis in the injected plasmodium up to 70 min, i.e., an advance of 10-14% compared to the 8- to 10-h cell cycle duration of the controls. The accelerating activity vanished completely after heating, freezing, or protease digestion, thus indicating the peptide nature of the active agent. Purification of the active compound by means of gel filtration revealed a molecular mass of about 2500 Da. The active portion of the extract was further fractionated by HPLC and the activity determined in a single peak.

Animals↗

The cellular mechanism of circadian rhythms--a view on evidence, hypotheses and problems.

A stable period length is a characteristic property of circadian oscillations. The question about whether higher frequency oscillators (0.5-8 hr) contribute to or establish the stable circadian periodicity cannot be answered at present. A sequential coupling of quantal subcycles appears possible on the basis of known "ultradian" oscillations. There is, however, no supporting evidence for such a concept. Phase response curves of the circadian clock derived from various perturbing pulses allow qualitative conclusions concerning the perturbed clock process. Deductions from computer simulations also allow conclusions about the phase of this oscillatory process. The distinction between processes (a) essential to the clock mechanism, (b) maintaining and controlling the clock (inputs) and (c) depending on the clock (outputs) on the basis of "oscillatory" and "change of psi or tau after perturbation" seems to be useful but not stringent. Protein synthesis may be an essential or input process. Oscillatory changes of this process may be due to periodic translational control or RNA-supply. Circadian changes in protein concentration and/or activity may depend on periodic synthesis, proteolysis, covalent modifications or aggregations. Specific essential proteins have not been identified conclusively. The large overlap between the group of agents and treatments that phase shift the clock and the group that induces stress proteins suggest that the latter may play a role in the controlling (input) or essential domain. The role of membranes in the clock mechanism is not clear: concepts assuming an essential function are based on circumstantial evidence. The membrane potential as well as Ca2+ may be involved in either input or essential function. Ca(2+)-calmodulin may also be important as concluded from inhibitor experiments. It is tempting to assume that a calmodulin-dependent kinase is part of a periodic protein phosphorylation process, yet it is not clear whether the periodic protein phosphorylation that has been observed is essential or is just another output process.

Animals↗

Circadian rhythms of cell cycle processes in the marine dinoflagellate Gonyaulax polyedra.

The circadian expression of several growth properties was examined in the dinoflagellate Gonyaulax polyedra under constant light and light-dark conditions. The cell concentration, mean cell volume and rate of DNA synthesis varied in a circadian rhythm, with the primary maximum of cytokinesis and DNA synthesis at about dawn. High rates of cell mortality also occurred during phases related to events of cytokinesis, and may be important in the expression of the other rhythms and in "red tide" generation. Flow-cytofluorimetric analysis indicated that cells of a population contain either a relatively high or a low amount of DNA, but the proportion of cells in each of these classes and the absolute amount of DNA in each cell varied rhythmically depending on the circadian time. This DNA-distribution pattern was unlike the usual G1-S-G2+M pattern typical of eukaryotic cell populations. Isotopically labelled thymidine, used as a marker of DNA synthesis, was continuously incorporated; but the incorporation rate fluctuated in a regular pattern that repeated each circadian period.

Animals↗

The cytoplasmic pH, ATP content and total protein synthesis rate during heat-shock protein inducing treatments in yeast.

In S. cerevisiae the induction of heat-shock protein (HSP) synthesis is accompanied by a decrease in the cytoplasmic and vacuolar pH as determined by means of [31P]NMR spectroscopy. The relationship of HSP synthesis and acidification of the cytoplasmic pH is dose-dependent under a variety of treatments (temperature increases (23-32 degrees C), addition of 2,4-dinitrophenol (greater than 1 mM), sodium arsenite (greater than 3.75 X 10(-5) M) or sodium cyanide (greater than 10 mM]. Changes in the intracellular pH occur within 5 min after treatment, attain a maximum within 30 min and are subsequently stable. HSPs 98, 85 and 70 show maximum synthesis rates 1-2 h after a 40 degrees C heat shock. The synthesis rates then decline. HSPs 56, 44 and 33 reveal a smaller and slower increase and almost no decrease in the synthesis rate within 4 h at 40 degrees C. The similar dose dependencies of HSP synthesis and cytoplasmic pH. as well as the immediate response of the pH, can also be demonstrated in the mitochondrial mutant of S. cerevisiae (Q0). This result indicates that the heat-shock response is mainly independent of intact oxidative phosphorylation. No correlation was observed between HSP synthesis rate and total intracellular ATP content.

2,4-Dinitrophenol↗

Possible link between circadian rhythm and heat shock response in Neurospora crassa.

3-h pulses of elevated temperatures (30 degrees C, 35 degrees C, 40 degrees C) phase shift the circadian conidiation rhythm of Neurospora crassa. The phase and amplitude of the phase response curves (PRC) were measured in wild type (frq+) and frequency mutants (frq 1, frq 7). The dose dependence of the phase shifts was compared to the dose dependence of total protein synthesis inhibition and heat shock protein induction in the three strains. All processes showed an almost linear dependence on temperature. These experiments and other available data suggest that a temperature increase may act on the circadian rhythm by a process belonging to the heat shock response.

Circadian Rhythm↗

Similar dose response of heat shock protein synthesis and intracellular pH change in yeast.

In Saccharomyces cerevisiae both the induction of heat shock proteins (98, 85, 70 kD) and the intracellular pH, determined by means of 31P-NMR spectroscopy, show a similar dose response to increasing temperature or concentrations of 2,4-dinitrophenol (DNP). Temperature increases from 23 degrees to 32 degrees C or more, or concentrations of DNP higher than 1 mM cause a significant increase in the synthesis rate of heat shock proteins and a significant decrease of the intracellular pH. A similar correlation is found in a mitochondrial mutant (Q) defective in oxidative phosphorylation. Intracellular signal transduction may thus involve H+-concentration changes independent of intact oxidative phosphorylation.

Dinitrobenzenes↗

Circadian rhythm of total protein synthesis in the cytoplasm and chloroplasts of Gonyaulax polyedra.

Protein synthesis of Gonyaulax polyedra was analyzed by means of electron microscopic autoradiographs under constant conditions at different times of the 24-hr cycle. Circadian rhythmic changes in the synthesis rate of total protein were determined in the cytoplasm and chloroplasts of growing cells. Three independent series of experiments in constant light showed a maximum of grains per unit area during the 'subjective' dark phase (= phase that corresponds to the dark phase during a 12:12 hr LD cycle) in both compartments. Minimum and maximum grain number are different by a factor of 5-10. The maximum of total protein synthesis coincided with the maximum phase shift by cycloheximide pulses (1) suggesting protein species within the total pool involved in the mechanism of the circadian clock. A similar rhythm of lower amplitude was observed in the mitochondria, but this rhythm cannot with certainty be attributed to these organelles. In a slowly growing culture a rhythm of total protein synthesis was observed that showed a smaller amplitude and a different phasing.

Animals↗

On the role of energy metabolism in Neurospora circadian clock function.

Neurospora crassa (bdA) mycelia were kept in liquid culture. Without rhythmic conidiation the levels of adenine nucleotides undergo circadian changes in constant darkness. Maxima occur 12-17 hr and 33-35 hr after initiation of the rhythm, i.e., at CT 0-6 hr. Pulses of metabolic inhibitors such as vanadate (Na3Vo4), molybdate (Na2MoO4 : 2 H2O), N-ethylmaleimide (NEM), azide (NaN3), cyanide (NaCN) and oligomycin phase shift the circadian conidiation rhythm of Neurospora crassa. Maximal advance phase shifts are observed at about CT 6 with all inhibitors. Pulses of N,N'dicyclohexylcarbodiimide (DCCD) and light phase shift the conidiation rhythm following a phase response curve different from those of the other agents (maximal advance at about CT 18-24). The phase shifts with DCCD and light are significantly larger in the wild type compared to the mitochrondrial mutant poky. Such differences are not found in PRCs of the protein synthesis inhibitor cycloheximide. [31P] NMR spectra of wild type Neurospora crassa and the clock mutants frq 1 and frq 7 which differ in their circadian period lengths did not reveal differences in the concentrations of adenine nucleotides, pyridine nucleotides or sugar phosphates. Starvation causes drastic changes of the levels of adenine nucleotides, phosphate and mobile polyphosphate without effecting phase or period length of the circadian rhythm.

Adenine Nucleotides↗

Are membrane properties essential for the circadian rhythm of Gonyaulax?

Membrane properties of whole cells of Gonyaulax polyedra were measured by fluorescence polarization. Circadian changes of fluorescence polarization exist in exponentially growing cultures. They show an amplitude larger than that of stationary cultures, indicating that a part of the change is due to or amplified by an ongoing cell cycle. Measurements of parameters of the circadian glow rhythm were analyzed for possible correlation with the membrane data. Considerable differences (Q10 = 2.5-3.0) in fluorescence polarization were found in cultures kept at different temperatures ranging from 15 to 27.5 degrees C. The free-running period length at different temperatures, on the other hand, differed only slightly (Q10 = 0.9-1.1). Stationary cultures showed higher fluorescence polarization compared with growing cultures, whereas the free-running period lengths did not differ in cultures of various densities and growth rates. Temperature steps of different sign changed the fluorescence polarization slightly in different directions. The phase shift of 4-h pulses (-5, -9, +7 degrees C) resulted in maximal phase advances of 4, 6, and 2 h, respectively. The phasing of the phase-response curves was identical in all these experiments, a finding not to be expected if the pulses act via the measured membrane properties. Pulses of drugs that change the fluorescence polarization (e.g., chlorpromazine and lidocaine) did not or only slightly phase-shift the circadian rhythm.

Animals↗