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Biomedical subjects

R van Wijk

Publications and source records attributed to R van Wijk.

90 records · Page 5Linked to original sources
PubMed1972

Differential sensitivities of the two malate dehydrogenases and the maltose permease to the effect of glucose in Saccharomyces carlsbergensis.

In Saccharomyces carlsbergensis the two malate dehydrogenase activities, which are localized in different compartments of the cell, were found to differ in their response to glucose. The cytoplasmic malate dehydrogenase activity appears to be sensitive to inactivation by very low concentrations of glucose. The mitochondrial malate dehydrogenase activity is only repressed at a higher glucose concentration. Maltose permease is also sensitive to inactivation by glucose. Conditions were found such that the maltose permease was present while the cytoplasmic malate dehydrogenase was inactivated. The different sensitivities of the two malate dehydrogenases and maltose permease to the effect of glucose may explain the preferential use of glucose, maltose, and products of glucose metabolism (2- and 3-carbon skeletons) as carbon sources for growth in the order as mentioned.

Amino Acids↗
PubMedDate not supplied

Arsenite induced sensitization and self-tolerance of Reuber H35 hepatoma cells.

Our data show that a short incubation with arsenite (30-300 microM) induces a biphasic change in cellular sensitivity towards a second exposure to arsenite. A transient sensitization was followed by the development of self-tolerance. Sensitization was measured using the step-down protocol; i.e., application of a high dose of arsenite pretreatment (100 or 300 microM) followed immediately by incubation in a low dose of arsenite (1-30 microM), with extensive rinsing in between. Whereas no effect of 1 and 3 microM on cellular survival is observed without pretreatment, a large decrease in cell survival can be established when these low doses of arsenite are applied immediately after a 1 hr pretreatment with 100 or 300 microM arsenite. According to the step-down protocol, a high dose of toxic compounds is applied and is followed by prolonged incubation in a lower concentration of the initial toxic compound. This might be a more accurate model for studying the effects of toxic insults on cells and organisms in the manner in which they occur in their natural environment. The level of tolerance was determined by a 1 hr test treatment with 300 microM arsenite applied at different times after pretreatment. Using this fractionated treatment protocol, it was established that tolerance increases with the increasing time intervals between the sodium arsenite treatments, during the 6 hr studied. These observations suggest that sensitization gradually decreases, whereas tolerance develops. Furthermore, our data indicate that the condition of pretreatment determines the extent to which the early sensitivity increases, as well as the development of tolerance later on. A relatively high arsenite concentration leads to more sensitized cells, which are transformed into more tolerant cells in comparison with the effect of a lower arsenite concentration.

Animals↗
PubMedDate not supplied

Stressor-specific induction of heat shock proteins in rat hepatoma cells.

In order to determine whether induction of specific stress proteins is dependent on a given stressor and whether induction of these proteins is linked to survival, Reuber H35 rat hepatoma cells were exposed to five different environmental stressors (heat shock, arsenite, cadmium, dinitrophenol and ethanol). The effect of these stressors was studied on cell survival as well as on inhibition and recovery of protein synthesis and on induction of heat shock proteins (hsps). In this article, we present evidence that several well-known hsp-inducers fail to stimulate specific hsps in a degree that is comparable to the induction of these hsps by heat shock. Most evidently, hsp60 is not induced by cadmium-treatment, whereas hsp100 is hardly induced by sodium arsenite. Treatment with DNP only slightly induces hsp68 and hsp84, whereas no detectable induction of hsps is observed after treatment with ethanol. In contrast, treatment with cadmium raises the amount of hsp28 to a higher level as compared to heat shock. A comparison of the stressor-specific induction of major hsps was also made under conditions of similar impact on cellular physiology: (a) stressor conditions up to the critical point that cell death starts to occur, and (b) conditions of iso-survival (50%). We conclude that hsps cannot be simply used as a general risk-assessment tool, and that the validation of stressor-specific risk-assessment warrants further research with larger groups of proteins.

Animals↗
PubMedDate not supplied

Variations in some molecular events during the early phases of the Reuber H35 cell cycle. IV-regulation of tyrosine aminotransferase.

Tyrosine aminotransferase activity increased during conversion of serum depleted quiescent Reuber H35 rat hepatoma cells into the proliferative state. Increased activity coincides with the actual increase of cells into S phase. The rate of tyrosine aminotransferase synthesis along the cell cycle was studied. The rate of enzyme synthesis fluctuated through the cell cycle but could not explain the increase of specific activity. Apparently enzyme activity is predominantly regulated by a post-translational event. Intracellular levels of cyclic AMP and cyclic GMP were measured at various times of G1 and S phases. In the early part of the cell cycle tyrosine aminotransferase decreased while intracellular levels of cyclic AMP increased. At later stages cyclic AMP rises concurrently with increased rates of enzyme synthesis. Induction of tyrosine aminotransferase by N6,O2'-dibutyryladenosine 3', 5'-monophosphate (Bt2cAMP) was studied. Inducibility by Bt2cAMP fluctuated through the cell cycle. Alternation of positive and negative control of tyrosine aminotransferase synthesis was observed. In early serum induced cells, Bt2cAMP increased enzyme activity without any increased rate of enzyme synthesis, on the contrary, a decreased rate of synthesis was observed. The data support the view that alternation of positive and negative control of tyrosine aminotransferase synthesis and temporary post-translational control of enzyme activity determine the enzyme level during the transition of quiescent hepatoma cells into proliferation.

Animals↗
PubMedDate not supplied

Increase of epidermal growth factor-stimulated cell-cycle progression and induction of thermotolerance by heat shock: temperature and time relationship.

When quiescent confluent cultures were incubated at increased temperature and then incubated at 37 degrees C prior to a second increase of temperature (46 degrees C) it appeared that heat-induced morphological alteration and ability to proliferate could be influenced by the previous thermal history of the cells. Incubations for 20 min in a temperature range of 41-46 degrees C caused cells to develop thermo-tolerance within 3 h of incubation at 37 degrees C. Confluent quiescent Swiss mouse 3T3 cells were incubated at 41.8, 43.7 or 45.6 degrees C and then reincubated at 37 degrees C to determine the effects of heat shock on the mitogenic effects of epidermal growth factor (EGF). Preincubation at 43.7 degrees C or 45.6 degrees C enhanced stimulation of G1-S progression by EGF. Preincubation at 43.7 degrees C markedly increased the rate at which cells enter the S phase without changing the length of the lag phase. A comparison of the duration of incubation at 43.7 degrees C for potentiation of EGF-induced DNA synthesis and that for induction of thermotolerance showed that a similar time interval for induction of effect could be implied.

Animals↗
PubMedDate not supplied

Effect of external K+ on protein and DNA synthesis during and after heat shock in rat hepatoma cells.

The effects of extracellular K+ concentrations on protein and DNA synthesis after non-lethal heat shock were studied in the hepatoma cell lines Reuber H35 and HTC. Elevation of the extracellular K+ concentration by equimolar replacement of Na+ by K+ in growth media of Reuber H35 and HTC cells caused an increase of the intracellular K+ content in both cell lines. This property was subsequently used to study the effect of elevated intracellular K+ concentrations on protein and DNA synthesis after hyperthermic treatment at 42 degrees C for 30 min. In normal K+ medium, protein and DNA synthesis were inhibited rapidly after the start of the hyperthermic treatment in both Reuber H35 and HTC cells. Increasing the external K+ concentration of the medium did not influence the inhibition and subsequent recovery of protein synthesis after heat shock in both cell lines. In contrast, in media with elevated K+ concentrations, DNA synthesis after heat-shock was inhibited less in Reuber H35 cells than in cells incubated in normal K+ medium and, furthermore, showed no inhibition in HTC cells. The protective effect of external K+ on DNA synthesis after heat shock was maximal between 50 and 70 mM in the temperature range 42-44 degrees C.

Animals↗
PubMedDate not supplied

Induction of thermotolerance and heat-shock protein synthesis during nutritional deprivation.

Under various conditions of heating, H35 cells were submitted to acute nutritional deprivation by omitting a number of substrates (L15D medium). At 37 degrees C cell death starts after a lag-period of 3-5 h. During hypothermia cell death is delayed, whereas during hyperthermia it is accelerated especially as a result of thermosensitization. In L15D the ATP level decreases approximately 3 times faster in combination with hyperthermia than at 37 degrees C. In non-thermotolerant cells thermosensization is very high at 41 degrees C and decreases with increasing temperature; in thermotolerant cells it is comparatively decreased at 41 degrees C and increased at 42.5 degrees C and above. In response to a heat shock of 30 min at 42.5 degrees C only 10% of the cell population expresses acute thermotolerance after incubation at 37 degrees C in L15D as compared to nearly 100% in complete medium (L15C). Chronic development of thermotolerance appears to be even more repressed in the presence of L15D, which partly explains the high thermosensitization at 41 degrees C. Changes in the rate of protein synthesis for combinations of nutritional deprivation and hyperthermia show a correlation with the cell survival data. Development of acute thermotolerance in L15D is accompanied by an increase in heat-shock protein synthesis relative to total protein. At 41 degrees C in L15D no heat-shock protein induction could be detected. Of the omitted substrates only glutamine can effectively abolish thermosensitization and the effects of L15D on protein and heat-shock protein synthesis depending on the condition of the cells, thermotolerant or non-thermotolerant, and to a different extent for the various proteins considered.

Adenosine Triphosphate↗
PubMedDate not supplied

Effects on the expression of heat shock proteins by step-down heating and hypothermia in rat hepatoma cells with a different degree of heat sensitivity.

Thermosensitization induced by pretreatment at supra- and subnormal temperatures, rate of protein synthesis and expression of the major heat shock proteins under such conditions was investigated in relation to intrinsic heat sensitivity of rat hepatoma cells, i.e. Reuber H35 and HTC. The high degree of heat susceptibility of H35 cells was reflected by a high degree of thermosensitization after pretreatment by heat (step-down heating) at temperatures of 42-44 degrees C for 30 min or cold for 16 h at temperatures ranging from 0 to 25 degrees C. Sensitization under step-down heating conditions was found to be paralleled by a delayed recovery of protein synthesis. Despite an increased relative rate, enhancement of the absolute rate of synthesis of the major heat shock proteins, HSP28, HSP60, HSP68, HSP70, HSP84 and HSP100, was less pronounced during step-down exposure. Comparable results were obtained during recovery of sensitized H35 cells at 37 degrees C after exposure to heat following pretreatment at 0 degrees C. Furthermore, clear differences in the regulation of the specific HSP synthesis, depending on the particular treatment protocol, were observed.

Animals↗
PubMedDate not supplied

Mild step-down heating causes increased levels of HSP68 and of HSP84 mRNA and enhances thermotolerance.

Mammalian cells exhibit increased sensitivity to hyperthermic temperatures of 38-42 degrees C after an acute high-temperature heat shock; this phenomenon is known as thermo-sensitization or the step-down heating effect. In order to determine whether the increase of heat shock mRNA after heat stress can be thermosensitized, we studied the induction of the mRNA of HSP68 and of HSP84 after application of step-down heating (SDH) in Reuber H35 rat hepatoma cells. SDH consisted of a pretreatment of 30 min at 41.5, 42.5 or 43.5 degrees C, followed by a continuous incubation at a lower hyperthermic temperature (40 or 41 degrees C). After mild pretreatment (30 min at 41.5 degrees C) the mRNA level of HSP68 was increased by subsequent incubation at 40 degrees C, although incubation at 40 degrees C alone had no effect. This increase was even more pronounced at 41 degrees C. An increase in the level of HSP84 mRNA was also observed after mild pretreatment (41.5 degrees C/30 min) followed by 41 degrees C post-incubation. Interestingly, an enhanced occurrence of thermotolerance was also observed upon application of mild step-down heating (42 degrees C/30 min-40 degrees C-43.5 degrees C/60 min). In contrast, cell cultures treated for 30 min at 43.5 degrees C (a temperature which induces an increase in HSP mRNA levels) showed an inhibited or delayed synthesis of HSP mRNA when post-treated at 40 or 41 degrees C. Under these conditions the development of thermotolerance did not take place either. With respect to the effect of step-down heating on HSP mRNA levels as well as on thermotolerance development, our data imply that a distinction should be made between 'mild' and 'severe' pretreatment temperature of the step-down heating protocol.

Animals↗
PubMedDate not supplied

Time-temperature relationships for step-down heating in normal and thermotolerant cells.

Normal and thermotolerant H35 cells were submitted to step-down heating (SDH). SDH can significantly reduce the induction and expression of thermotolerance. For SDH a sensitizing treatment (ST) at 44.6 degrees C was followed by a test treatment (TT) at a lower hyperthermic temperature. The comparison between the thermotolerant and non-thermotolerant condition was based on isosurvival ST doses. For both conditions dose-effect relationships were obtained by plotting the ST-surviving fraction against the D0 of a TT. The TT was at either 41 or 42.5 degrees C, representing respectively, a permissive or a non-permissive condition for chronic induction of thermotolerance (CIT). The complex dose-effect relationships are partly exponential. In non-thermotolerant cells tested at 42.5 degrees C the dose-effect relationship between ST and TT is relatively weak. At 41 degrees C, however, the expression of CIT is strongly inhibited after a ST that kills < 20% of the cells. At higher ST doses the response is comparable with that at 42.5 degrees C. In thermotolerant cells a high degree of thermosensitization is also observed for relatively low ST doses, but in contrast with non-thermotolerant cells a stronger dose-effect relationship remains at the higher ST doses. Ultimately this results in a comparatively higher degree of thermosensitization that can be achieved in non-thermotolerant cells. For example, at an isosurviving fraction of 0.15 the reduction of D0 is non-thermotolerant cells at 42.5 degrees C is less than five times, whereas in thermotolerant cells, the D0 reduction is between 40 and 50 times. A similar reduction is found in non-thermotolerant cells tested at 41 degrees C. Subsequently, an isosurvival ST dose of about 40% was used in combination with a TT that was varied between 39 and 44 degrees C. D0's were plotted in an Arrhenius diagram to obtain a time-temperature relationship for the effect of SDH on thermotolerant and non-thermotolerant cells. The four plots are all biphasic with a downward inflection. Thermotolerance causes an upward shift of the inflection point of 2 degrees C relative to single-heated cells, whereas SDH causes a downward shift of 1 degree C in single-heated cells and of 2 degrees C in thermotolerant cells. For most of the temperature range, i.e. 39-43.5 degrees C, SDH decreases the activation energies.

Animals↗
PubMedDate not supplied

A mathematical model of the hsp70 regulation in the cell.

A mathematical model of the regulation process of the heat shock protein hsp70 in the cell is presented. The model describes the damaging effect of elevated temperature on proteins; the interaction of free hsp70 with injured proteins and its chaperone role in nascent protein translation; the relation between the amount of free hsp70 and the formation of the activated trimer form of the heat shock factor protein (HSF); the binding of activated HSF with the heat shock elements on the DNA; the transcription of mRNA of hsp70 and the synthesis of hsp70. The reaction of the model to a temporal rise in temperature shows an initial decline and a subsequent sharp rise to an ultimately increased level of free hsp70 in the cell. The response of the model to both a single and two consecutive heat shocks appears to closely resemble experimental data on hsp70 synthesis. This general agreement demonstrates the structure of the model to be sound and suitable as a basis for further modelling the complex tolerance mechanism of the cell.

Cell Survival↗