Biomedical subjects

F A Wiegant

Publications and source records attributed to F A Wiegant.

28 records · Page 2Linked to original sources

PubMed1985

Localization of cell surface glycoproteins in membrane domains associated with the underlying filament network.

To visualize the localization of cell surface constituents in relation to the plasma membrane-associated filament network, we developed a method based on a combination of immunogold labeling and dry-cleaving. For labeling we used trinitrophenyl-derivatized ligand, anti-TNP antibodies, and protein A-coated colloidal gold. Dry-cleaving (Mesland, D. A. M., H. Spiele, and E. Roos, 1981, Exp. Cell Res., 132: 169-184) involves cleavage of lightly fixed critical point-dried cells by means of adhesive tape. Since cells cleave close to the cell surface, the remaining layer is thin enough to be examined in transmission electron microscopy. Using this method, we studied concanavalin A-binding constituents on the medium-facing surface of H35 hepatoma cells. The distribution of the gold particles, which was partly dispersed and partly patchy, coincided strikingly with membrane-associated filaments, and label was virtually absent from areas overlying openings in the filament network. In stereo pairs we observed the label to be localized to areas of somewhat enhanced electron density at the plane of the membrane. These areas were interconnected in a pattern congruent with the filament network. Preliminary observations on wheat germ agglutinin receptors on the hepatoma cells as well as concanavalin A receptors on isolated hepatocytes yielded comparable results. It thus appears that surface glycoproteins, although seemingly randomly distributed as observed in thin sections, may actually be localized to particular membrane domains associated with underlying filaments.

Actin Cytoskeleton↗

PubMed1985

Immunocytochemical demonstrations of cytoplasmic and cell-surface EGF receptors in A431 cells using cryo-ultramicrotomy, surface replication, freeze-etching and label fracture.

In this paper we describe the use of a number of complimentary methods to visualize cytoplasmic and cell-surface located epidermal growth factor (EGF) receptors in cultured A431 cells. Cryo-ultramicrotomy in combination with immuno-gold labelling will be shown to provide an excellent method in visualizing cytoplasmic located EGF receptors in addition to cell-surface located EGF receptors. An important aspect in this method involves the possible effects of the fixatives on antigenicity. Using radioactive labelled anti EGF receptor antibodies, it was shown that formaldehyde as a fixative had no significant effect on label-efficiency. The density and lateral distribution of EGF receptors at the cell surface has been studied by three methods, i.e. surface replication, freeze etching and label fracture, all methods in conjunction with immuno-gold labelling. These methods allow in principle a quantitation of the surface distribution of the EGF receptors. The surface-replication method involves, however, dehydration and critical-point drying steps, and using radioactive labelled anti EGF receptor antibodies it was shown that in particular OsO4 fixation and dehydration caused a significant loss of cell-associated antibodies. This disadvantage is overcome by freeze etching and the label-fracture method, and as such these techniques provide the best methods for quantitative analysis of the planar distribution of cell-surface located EGF-receptors.

Antigen-Antibody Reactions↗

PubMed1984

Thermotolerance in cultured hepatoma cells: cell viability, cell morphology, protein synthesis, and heat-shock proteins.

Heat treatment at 42 degrees C of cultured Reuber H35 rat hepatoma cells induced both a rapid decrease of the rate of protein synthesis and the rounding up of the cells. Reincubation at 37 degrees C resulted in a gradual flattening of the cells, resumption of protein synthesis, and the synthesis of heat-shock proteins. During the recovery period cells developed a resistance toward a treatment which otherwise should lead to heat-induced cell death. Thermotolerance measured in terms of cell survival was paralleled by thermal resistance of protein synthesis and the cellular ability to refrain from rounding up under heat stress.

Acclimatization↗

PubMed1982

Variations in some molecular events during the early phases of the Reuber H35 hepatoma cell cycle. III. Role of protein synthesis in the initiation of DNA synthesis and the mechanism of stimulation of protein synthesis by serum.

Induction of DNA synthesis by serum and amino acids has been investigated in cultured Reuber H35 hepatoma cells. Commitment of DNA synthesis was found to occur 6-8 hours before the actual start of this synthesis. The rate of initiation of DNA synthesis is proportional to the stimulation of protein synthesis by serum and/or amino acids. The increased protein synthesis is important for the proliferation only during the early period after serum addition. The withdrawal of serum and the inhibition by cycloheximide confirm this finding. Actinomycin D hardly influenced the early effect of serum on protein synthesis and it is concluded that the serum-stimulated protein synthesis is carried out on pre-existing mRNA's. The mechanism of stimulation of protein synthesis by serum has been studied by determination of the polyribosome size, the number of growing polypeptide chains, and the ribosomal transit time. The rate of the initiation of translation has been found to be specifically enhanced while the rate of elongation remained unchanged. Two-dimensional gel electrophoresis showed that the early stimulation of protein synthesis by serum involves all types of major cellular proteins, and no new proteins could be detected.

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

Heat shock response by cells treated with azetidine-2-carboxylic acid.

The purpose of this study is to reinvestigate the heat shock response in cells treated with the antimetabolite azetidine-2-carboxylic acid (azetidine), an analogue of proline. Previous studies could not clearly discriminate between the progressive thermosensitization caused by amino acid analogues and a parallel induction of thermotolerance by heat shock. Incubation of H35 cells with 2.5 mm azetidine causes an increasing thermosensitization which achieves a maximum after approximately 18-22 h. At this point, azetidine does not prevent the development of acute thermotolerance following a heat shock at 42.5 degrees C, or the simultaneous induction of chronic thermotolerance during mild hyperthermia at 38-41 degrees C. However, for both the acute and chronic heating conditions thermotolerance levels are reduced in proportion with azetidine-thermosensitization. Incorporation of azetidine causes an apparent downward temperature shift of approximately 1 degree C relative to the time-temperature relationships for normal, or following heat shocks, for thermotolerant cells. After 18 h of incubation with azetidine, protein synthesis is reduced by a factor of 4 and cells show a preferential synthesis of heat shock proteins (hsp). A heat shock then, although inducing thermotolerance, is not followed by any noticeable effect on the synthesis of hsps. It is shown that the combination of prolonged azetidine treatment and heat shock causes a persistent inhibition of protein synthesis. This is hypothesized to result in the development of hsp synthesis independent thermotolerance. Additional treatment following heat shock in azetidine-treated cells with the protein synthesis inhibitor cycloheximide does not affect the induction of thermotolerance. In contrast to the heat shock response, no thermotolerance induction is observed in azetidine-treated cells after an exposure to sodium arsenite.

Animals↗

PubMedDate not supplied

Calmodulin inhibitors potentiate hyperthermic cell killing.

The role of calmodulin (CaM) in cellular heat injury of neuroblastoma N2A and hepatoma H35 cells has been investigated, using specific calmodulin-inhibiting drugs (Trifluoperazine, Compound 48/80 and Calmidazolium). These CaM-specific drugs potentiate hyperthermia-induced cell killing, suggesting CaM to be involved in processes aimed on the repair of heat injury. The CaM inhibitors also prevent hyperthermia-induced cytoskeletal alterations in the cell types studied. The action of CaM inhibitors was dose dependent, and seems to be confined to the first period of the hyperthermic treatment. Neither production of heat shock proteins in heat-shocked cultures, nor the rate of protein synthesis in control cultures were affected by the CaM inhibitors. It was concluded that an inverse correlation exists between hyperthermic cell killing and cytoskeletal alterations. Activation of CaM is suggested to be a fundamental aspect of the cellular heat shock response.

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↗