Analysis of the topological changes induced on cells exposed to adhesive or mechanical stimuli.

Fluorescent probes are widely used to study cell structure and function. However, few reports were devoted to a quantitative analysis of the intracellular distribution of fluorescent markers. In the present work, we describe the topographical changes of surface and cytoskeletal markers on individual cells subjected to adhesive or mechanical interaction. Conjugates were prepared with a cytotoxic T-lymphocyte clone and target cells. Specific antigens, membrane phospholipids, surface glycoconjugates, and polymerized actin were labeled with fluorescent antibodies or biochemical probes. The analysis of fluorescence distributions in conjugates demonstrated a selective reorganization of the plasma membrane with a gathering of some molecular species in the intercellular adhesion area. Furthermore, individual phagocytic cells were sucked into glass micropipets, then stained with fluorescent phallacidin to analyze the effect of mechanical efforts on the cytoskeleton organization. The concentration of polymerized actin was found to be similar in mechanically-induced protrusions and whole cells. It is concluded that adhesive interactions may result in marked cell polarization and formation of membrane zones with a particular biochemical composition. The submembranar cytoskeleton might play a role in this process.     

Analysis of the dynamic strains in a fingertip exposed to vibrations: Correlation to the mechanical stimuli on mechanoreceptors

The reduction in vibrotactile sensitivity in the fingertip is assumed to be associated with the exposure of the tissues to repetitive, non-physiological strains during dynamic loading. Experimental results demonstrated that the magnitude of a vibration-induced temporary threshold shift is dependent upon the vibration frequency of both the exposure and testing stimuli. In the present study, the frequency-dependent strain imposed on cutaneous and subcutaneous tissues of the fingertip is analyzed theoretically using a finite element model. The proposed fingertip model is two-dimensional and includes major anatomical substructures: skin, subcutaneous tissue, bone, and nail. The soft tissues (skin and subcutaneous tissues) were assumed to be nonlinearly elastic and viscoelastic, while the bone and nail were considered as linearly elastic. Simulations were performed for the contact between the fingertip and a flat surface for four different pre-compressions (0.5, 1.0, 1.5, and 2.0 mm). The frequency-dependent distributions of the dynamic strain magnitudes in the soft tissues were investigated. The model predictions indicated that the vibration exposure at a frequency range from 63 to 250 Hz will induce excessive dynamic strain in the deep zone of the finger tissues, effectively inhibiting the high-frequency mechanoreceptors; while the vibration exposure at low frequency (less than 31.5 Hz) tends to induce excessive dynamic strain in superficial layer in the tissues, inhibiting the low-frequency mechanoreceptors. These theoretical predictions are consistent with the experimental observations in literature. The proposed model can be used to predict the responses of the soft tissues in different depths to vibration exposures, providing valuable information and data that are essential for improving vibrotactile perception tests.     

Stress proteins are not induced in mammalian cells exposed to radiofrequency or microwave radiation

The induction of stress proteins in HeLa and CHO cells was investigated following a 2 h exposure to radiofrequency (RF) or microwave radiation. Cells were exposed or sham exposed in vitro under isothermal (37 ± 0.2 °C) conditions. HeLa cells were exposed to 27- or 2450 MHz continuous wave (CW) radiation at a specific absorption rate (SAR) of 25 W/kg. CHO cells were exposed to CW 27 MHz radiation at a SAR of 100 W/kg. Parallel positive control studies included 2 h exposure of HeLa or CHO cells to 40 °C or to 45 μM cadmium sulfate. Stress protein induction was assayed 24 h after treatment by electrophoresis of whole-cell extracted protein labeled with [³⁵S]-methionine. Both cell types exhibited well-characterized responses to the positive control stresses. Under these exposure conditions, neither microwave nor RF radiation had a detectable effect on stress protein induction as determined by either comparison of RF-exposed cells with sham-exposed cells or comparison with heat-stressed or Cd⁺⁺ positive control cells. Bioelectromagnetics 18:499–505, 1997. © 1997 Wiley-Liss, Inc.     

Comparison of genetic damage in Brazilian footwear-workers exposed to solvent-based or water-based adhesive

Research has shown that workers employed in footwear manufacture are at increased risk of some cancers, the strongest evidence being for nasal cancer and leukemia. Footwear-workers are routinely exposed to complex mixtures of solvents in degreasers, cleaners, primers, and adhesives used in the production process as toluene, n-hexane, acetone, and possibly dust particles, additives in shoe materials and degradation products of materials. The recognition of the potential health-hazards of solvent-based adhesives (SBAs) has lead to the development of adhesives with no organic solvents, the water-based adhesives (WBA). We investigated footwear-workers (all males) exposed to SBA (n=29) (for 3.98+/-4.13 years), and WBA (n=16), which had spent the six months previous to the study employed in an experimental section which used only water-based adhesives, although they had previously worked in sections which used solvent-based adhesives (for 5.80+/-4.03 years); 25 healthy subjects were used as controls. The Comet assay and the micronucleus test were used as endpoints, while the traditional parameters for assessing exposure to toluene in organic mixtures by measuring the concentration of urinary hippuric acid were also assessed. Our results showed a significantly lower mean concentration of hippuric acid in the control group than found in the SBA (P<0.001) and WBA (P<0.05) groups. The Comet assay results showed that there was a significant increase in the mean damage index for the SBA (P<0.001) group in comparison to the WBA group and control (P<0.05). For the micronucleus test in binucleated lymphocytes and exfoliated buccal cell, the three groups were not statistically different. Our study demonstrated that water-based adhesives are clearly a better option for safeguarding the health of footwear-workers, even with possibility of isocyanate presence, while the positive results observed in SBA group might be explained by chloroprene presence in the adhesive.     

Morphological changes in Escherichia coli cells exposed to low or high concentrations of hydrogen peroxide

Escherichia coli cells challenged with low or high concentrations of hydrogen peroxide are killed via two different mechanisms and respond with morphological changes which are also dependent on the extracellular concentration of the oxidant. Treatment with low concentrations (less than 2.5 mM) of H2O2 is followed by an extensive cell filamentation which is dependent on the level of H2O2 or the time of exposure. In particular, addition of 1.75 mM H2O2 results in a growth lag of approximately 90 min followed by partial increase in optical density, which was mainly due to the onset of the filamentous response. In fact, microscopic analysis of the samples obtained from cultures incubated with the oxidant for various time intervals has revealed that this change in morphology becomes apparent after 90 min of exposure to H2O2 and that the length of the filaments gradually increases following longer time intervals. Analysis of the ability of these cells to form colonies has indicated a loss in viability in the first 90 min of exposure followed by a gradual recovery in the number of cells capable of forming colonies. Measurement of lactate dehydrogenase in culture medium (as a marker for membrane damage) has revealed that a small amount of this enzyme was released from the cells at early times (less than 150 min) but not after longer incubation periods (300 min). Cells exposed to high concentrations of H2O2 (greater than 10 mM) do not filament and their loss of viability is associated with a marked reduction in cell volume. In fact, treatment with 17.5 mM H2O2 resulted in a time-dependent decrease of the optical density, clonogenicity, and cellular volume. In addition, these effects were paralleled by a significant release in the culture medium of lactate dehydrogenase thus suggesting that the reduced cell volume may be dependent on membrane damage followed by loss of intracellular material. This hypothesis is supported by preliminary results obtained in electron microscopy studies. In conclusion, this study further demonstrates that the response of E. coli to hydrogen peroxide is highly dependent on the concentration of H2O2 and further stresses the point that low or high concentrations of the oxidant result in the production of different species leading to cell death via two different mechanisms and/or capable of specifically affecting the cell shape.     

Decreased cAMP levels in human diploid cells exposed to cholinergic stimuli.

Carbachol at a concentration of 1 micron caused very significant decreases in PGE1 stimulated cAMP levels in three human diploid lung fibroblasts, WI-38, MRC-5, and IMR-90. Detailed studies with WI-38 cells showed that carbachol acted at low concentrations and very rapidly, cAMP levels being reduced by 50% in about 1 minute. The effect was moderately reduced but still very significant in Ca++ free medium containing 10(-5)M EGTA. Acetylcholine (in the presence of physostigmine), pilocarpine, and muscarine lowered cAMP levels at concentrations of 1 or 10 micron. The effects of carbachol were exquisitely sensitive to atropine but were unaffected by hexomethonium or d-tubocurarine. These data suggested that the cholinergic response of WI-38 cells was of the muscarinic type.     

Effect of ion composition on the changes in membrane potential induced with several stimuli in rat mast cells

We studied, in different ionic conditions, the effect of various agents on the membrane potential of rat peritoneal mast cells using the fluorescent probe bisoxonol. Ouabain and ionophore A23187 lead to a fast depolarization of the plasma membrane of mast cells, while compound 48/80 and thapsigargin induced membrane hyperpolarization, which was more pronounced in the case of compound 48/80. When using compound 48/80, the amount of gramicidin necessary to depolarize the cells was twice the amount required in resting cells, which indicates that compound 48/80 increases considerably the activity of the Na⁺/K⁺ pump. On the other hand, the ionophore A23187 elicited a clear depolarization which was oblated in the absence of intracellular calcium. The increase in the osmolarity of the medium causes a depolarization in the plasma membrane of mast cells. Hypertonicity-stimulated depolarization is inhibited by removing sodium and potassium. © 1994 Wiley-Liss, Inc.     

Ultrastructural changes in Mycobacterium rubrum cells exposed to sulfochloranthine

The object of this work was to study the effect of sulfochlorantine (SCA), a new disinfecting compound containing chlorine, on Mycobacterium rubrum cells. The sensitivity of the cells to this agent was studied as well as their ultrastructure at the doses 0.01, 0.025, 0.05 and 0.1%. Even at high doses, the cells did not die off immediately when their growth and division stopped. Changes in the ultrastructural organization of the cells depended on the concentration of SCA. The structure of the nucleoid, membranes and ribosomes was damaged. Considerable changes were found in the formation of cross septa. The structure of the cells wall was modified to a lesser degree. The results are consistent with the biochemical data available.     

High-density lipoproteins attenuate interleukin-6 production in endothelial cells exposed to pro-inflammatory stimuli

The purpose of the present study was to investigate the ability of high-density lipoproteins (HDL) to attenuate endothelial dysfunction, by assessing down-regulation of cytokine-induced interleukin-6 (IL-6) production in cultured endothelial cells, and measuring plasma IL-6 levels in three groups of healthy individuals with low, average, or high plasma HDL-cholesterol. Human plasma HDL caused a concentration-dependent inhibition of TNFalpha-induced IL-6 production in human endothelial cells (by 58.5+/-1.5% at 2 mg of HDL-protein/ml). Reconstituted HDL made with apolipoprotein A-I (apoA-I) and phosphatidylcholine were as effective as plasma HDL, while lipid-free apoA-I or phosphatidylcholine liposomes had no effect. HDL attenuated IL-6 mRNA levels, an effect which occurs through inhibition of p38 MAP kinase. The median plasma IL-6 concentration was significantly higher in subjects with low HDL-cholesterol (2.54 pg/ml) compared with those with average or high HDL-cholesterol (1.31 pg/ml and 1.47 pg/ml, respectively). When all subjects were considered together, a lower HDL-cholesterol was the strongest independent predictor of higher IL-6 (F=25.38, P<0.001). By inhibiting IL-6 production and lowering plasma IL-6 concentration, HDL may limit the pro-atherogenic effects of both acute and chronic inflammatory states, of which IL-6 is a key orchestrator.     

Phosphopeptide analysis of phenylalanine hydroxylase isolated from liver cells exposed to hormonal stimuli.

Hormonal control of the phosphorylation of phenylalanine hydroxylase was studied by using rat liver cells incubated with [32P]Pi. After immunoprecipitation from cell extracts, the hydroxylase was subjected to proteinase digestion and subsequent sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. V8-proteinase digestion yielded one major 32P-labelled fragment, of approx. 9 kDa. Chymotrypsin digestion gave five 32P-labelled fragments ranging from approx. 39 kDa to approx. 10 kDa. Noradrenaline (10 microM) and glucagon (0.1 microM) enhanced the 32P content of all peptide fragments uniformly. Phorbol ester, in contrast with ionophore A23187, did not stimulate enzyme phosphorylation or enhance phenylalanine metabolism in liver cells. These results are discussed in relation to the nature of the protein kinase(s) that mediate phosphorylation of phenylalanine hydroxylase in liver cells.     

Cytomorphological changes in human tumor cells exposed to high-polar compounds

The effects of some high-polar compounds on the cytomorphological characteristics of cultured cells of synovial and osteogenic human sarcomas (Sa-2 and Sa-4) were studied. Incubation of Sa-2 cells with 1 or 2% dimethylsulfoxide (DMSO) or 100 mM dimethylformamide (DMF) during 4-6 days induced the cellular, structural and functional changes in Sa-2 cells which were considered as manifestations of pseudoepithelial differentiation. DMSO did not influence the activity of Sa-2 cell enzymes, while DMF suppressed their activity, mainly that of acid and alkaline phosphatases. DMSO and DMF induced in Sa-4 cultured the appearance of the cells which were similar cytomorphologically to normal osteoblasts. DMSO and DMF significantly depressed the activity of acid phosphatase in Sa-4 cells and transformed the positive reaction of alkaline phosphatase to the negative one. DMSO and DMF prolonged the time of the doubling of Sa-2 and Sa-4 cells. The effects of DMSO and DMF were reversible. Methylformamide (200 mM) and dimethyl acetamide (0,1 and 10 mM) did not induce analogous changes in human sarcoma cells.     

Mitochondrial changes during the apoptotic process of HeLa cells exposed to cisplatin.

HeLa cells undergo apoptosis after exposure to cisplatin. Since mitochondria have recently been proposed as a probable effector of this type of cell death, we performed an analysis using the fluorescent cation rhodamine 123, which is transported actively by this organelle. Cisplatin induces a decrease in the mitochondrial staining, as assessed by cytofluorometric analysis. Microscopic analysis demonstrated that this effect was accompanied by damage of the mitochondria. These features were not exclusive of cisplatin, as other antineoplasic agents (taxol, etoposide) elicited similar effects. These results point toward the notion of a general effect of antineoplasic drugs over the mitochondria during induction of apoptotic cell death.     

Metabolic changes in the isolated rat jejunum exposed to continuous light or darkness]

Rats were kept for about seven weeks in continuous light or darkness. The oxygen consumption and glucose consumption by the isolated small intestine were determined. Data were compared to those of control animals kept under a light-dark cycle. Continuous exposure to light resulted in a 21% increase in anaerobic glycolysis. Continuous exposure to darkness increased anaerobic glycolysis by 18 and 12% respectively.     

Structural and ultrastructural changes in Mycobacterium rubrum cells exposed to penicillin

The action of penicillin taken at subbacterioscopic doses on Mycobacterium rubrum cells causes changes in the size and shape of the cells, in the structure of the cell wall, in the intracellular membrane systems and in functions associated with them, and in the structure of nucleoids whose DNA packing becomes more loose. If the antibiotic is added at bacteriostatic doses, the size and shape of the cells do not change, but peptidoglycan precursors being synthesized are not incorporated into the polymer and accumulate in the periplasm. DNA overspiralization in nucleoids is a non-specific reaction, which indicates that DNA is physiologically passive. DNA is isolated with a membrane from the cytoplasm in certain cells. It is possible that the resistance of cells against penicillin is associated with the capability of DNA to become inactive in physiological terms.