Response of mammalian cells to shear stress

Summary The influence of shear forces on adherent mammalian cells was investigated by means of a developed flow chamber. The viability of the cells decreased with increasing exposure level and duration. Additional, changes in the morphology of the cells due to the shear forces were observed. Offprint requests to: G. Kretzmer     

Chlamydia attachment to mammalian cells requires protein disulfide isomerase

For Chlamydia, an intracellular pathogen of humans, host cell invasion is obligatory for survival, growth and pathogenesis. At the molecular level, little is known about the binding and entry of Chlamydia into the mammalian host cell. Chlamydia are genetically intractable therefore experimental approaches targeting the host are often necessary. CHO6 is a mutagenized cell line resistant to attachment and infection by Chlamydia. In this study, CHO6 was shown using proteomic methods to have a defect in processing of the leader sequence for protein disulfide isomerase (PDI). Complementation by expression of full-length PDI restored C. trachomatis binding and infectivity in the CHO6 mutant cell line. The cell line was also resistant to diphtheria toxin and required complemented cell-surface PDI for toxin entry. These data demonstrate that native PDI at the cell surface is required for effective chlamydial attachment and infectivity.     

Specificity of attachment of certain enterobacteriaceae to mammalian cells.

The specificity of adherence of various Enterobacteriaceae to different mammalian cells was studied in vitro. 3H-Labelled organisms of the same species isolated from various clinical sources differed significantly in their abilities to adhere to the same mammalian cells. Bacteria frequently adhered better to cells derived from sites other than those analogous to their original source. Bacteria did not display consistently 'high' or 'low' attachment to a variety of human and tissue-cultured cells and little selective adherence was demonstrable.     

Covalent attachment of RNA to nascent DNA in mammalian cells

We have used the technique of phosphate transfer analysis to test for the presence of phosphodiester bonds linking ribonucleotides (on the 5′ side) to deoxyribonucleotides (on the 3′ side) in DNA newly synthesized within lysates or purified nuclei of mammalian cells. We have found that such covalent junctions between RNA and DNA are present at a frequency of one junction per newly synthesized DNA strand. The junctions are located close to the ends of the nascent DNA strands. The stretches of RNA at the junction are very short compared to the stretches of DNA. These properties are consistent with the conclusion by Reichard, Eliasson, and Söderman (1974) that short stretches of RNA are present on the 5′ ends of nascent DNA strands produced during replication of polyoma virus.     

Double-mode impedance analysis of epithelial cell monolayers cultured on shear wave resonators

The viscoelastic behavior of epithelial cells (MDCK-I and MDCK-II) grown on AT-cut quartz crystals with a fundamental resonance at 5 MHz was investigated by impedance spectroscopy. Using the electromechanical model recently derived by Martin et al. [(1991) Anal Chem 63: 2272 – 2281] for Newtonian liquids in contact with shear wave resonators we quantified the viscous damping arising from the adherent cells by fitting the impedance data with a modified Butterworth-Van Dyke circuit in the region of the resonance frequency. Impedance spectroscopy was additionally performed in the frequency range from 1 Hz to 1 MHz to scrutinize the passive electrical properties of the epithelial cell layers using an additional platinum electrode. These data allow one to document the cell layers' integrity as well as the electrode coverage. We were able to confirm that the presence of a cell-layer mainly increases damping of the shear wave and does not exhibit a pure mass-load behavior. These findings were supported by the discovery that the inductance L in the electromechanical model was less influenced by the cell-layer than the resistance R. The apparent cell-viscosities determined by our method are 0.097 poise for MDCK-I and 0.142 poise for MDCK-II cell-layers. These low apparent viscosities may be explained in terms of a considerable spacing between the cells immobilized via their focal contacts and the quartz surface. Received: 5 June 1996 / Accepted: 6 August 1996     

In vitro attachment of Treponema hyodysenteriae to mammalian epithelial cells.

The interaction between Treponema hyodysenteriae and isolated swine intestinal epithelial cells or mouse adrenal cells in culture was examined. Studies were performed in which treponemes were incubated with each type of anomal cell in an atmosphere of 5% CO2 in air. Coincubation was terminated at various time intervals, and the percentage of treponemal attachment evaluated by light microscopy. The extent of attachment was dependent on both incubation time and temperature. The mechanism of attachment to the animal cell surface was examined by scanning electron microscopy. Interaction of the parasite with the host cell did not appear to alter cellular morphology or result in changes of the cell surface at the site of attachment. Preference for a cellular site of attachment was not found.     

The response of mammalian cells to double-stranded RNA

Double-stranded RNA (dsRNA) has long been recognized as a central component of the interferon (IFN) system. It was originally characterized as a key mediator of IFN induction in response to virus infection. Subsequently, it was identified as a prime activator of the antiviral response. In recent years the discovery of the RNA interference (RNAi) pathway in mammals has renewed interest in dsRNA-mediated cellular responses. This has coincided with the identification of key components of the IFN induction pathway. Here, we present an overview of the current knowledge of dsRNA-mediated pathways in mammalian cells and introduce a link between these pathways and application of RNAi.     

Proteolytic response to oxidative stress in mammalian cells

Oxidative stress in mammalian cells is an inevitable consequence of their aerobic metabolism. The production of reactive oxygen and nitric oxide species causes oxidative modifications of proteins often combined with a loss of their biological function. Like most partially denatured proteins, moderately oxidized proteins are more sensitive to proteolytic attack by proteases. The diverse cellular proteolytic systems are an important secondary defense against oxidative stress by degrading oxidized and damaged proteins, thereby preventing their intracellular accumulation. In mammalian cells, a range of proteases exists which are distributed throughout the cell. In this review we summarize the function of the cytosolic (proteasome and calpains), the lysosomal, the mitochondrial and the nuclear proteolytic pathways in response to oxidative stress. Particular emphasis is given to the proteasomal system, since this pathway appears to be the most important proteolytic system involved in the removal of oxidatively modified or damaged proteins.     

Heterogeneous response of microvascular endothelial cells to shear stress

We investigated changes in calcium concentration in cultured bovine aortic endothelial cells (BAECs) and rat adrenomedulary endothelial cells (RAMECs, microvascular) in response to different levels of shear stress. In BAECs, the onset of shear stress elicited a transient increase in intracellular calcium concentration that was spatially uniform, synchronous, and dose dependent. In contrast, the response of RAMECs was heterogeneous in time and space. Shear stress induced calcium waves that originated from one or several cells and propagated to neighboring cells. The number and size of the responding groups of cells did not depend on the magnitude of shear stress or the magnitude of the calcium change in the responding cells. The initiation and the propagation of calcium waves in RAMECs were significantly suppressed under conditions in which either purinergic receptors were blocked by suramin or extracellular ATP was degraded by apyrase. Exogenously applied ATP produced similarly heterogeneous responses. The number of responding cells was dependent on ATP concentration, but the magnitude of the calcium change was not. Our data suggest that shear stress stimulates RAMECs to release ATP, causing the increase in intracellular calcium concentration via purinergic receptors in cells that are heterogeneously sensitive to ATP. The propagation of the calcium signal is also mediated by ATP, and the spatial pattern suggests a locally elevated ATP concentration in the vicinity of the initially responding cells.     

Fluorescence based measurements of Fusobacterium nucleatum coaggregation and of fusobacterial attachment to mammalian cells

Fusobacterium nucleatum is a common oral anaerobe associated with gingivitis, periodontal disease and preterm deliveries. Coaggregation among oral bacteria is considered to be a significant factor in dental plaque development. Adhesion to host cells was suggested to be important for the F. nucleatum virulence associated with oral inflammation and with preterm births. An uncharacterized fusobacterial galactose inhibitible adhesin mediates coaggregation of F. nucleatum 12230 and F. nucleatum PK1594 with the periodontal pathogen Porphyromonas gingivalis. This adhesin is also involved with the attachment of both fusobacterial strains to host cells. However, it has been suggested that additional unidentified fusobacterial adhesins are involved in F. nucleatum virulence associated with preterm births. In this study, a fluorescence-based high throughput sensitive and reproducible method was developed for measuring bacterial coaggregation and bacterial attachment to mammalian cells. Using this method we found that coaggregation of F. nucleatum 4H with P. gingivalis and its attachment to murine macrophages is less inhibitible by galactose than that of F. nucleatum PK1594. These findings suggest that F. nucleatum 4H can serve as a model organism for identifying nongalactose inhibitible F. nucleatum adhesins considered to be involved in fusobacterial attachment to mammalian cells.     

A comparative analysis of the attachment of Leptospira interrogans and L. borgpetersenii to mammalian cells

Leptospirosis, the world's most ubiquitous zoonosis, is caused by pathogenic Leptospira. As microbe-host interactions are specific in pathogenesis, it is likely that there are several molecules mediating the attachment of the Leptospira to mammalian cells. In this study, we analysed the attachment of Leptospira interrogans serovar Portlandvere and Leptospira borgpetersenii serovar Jules to untreated HEp-2 cells or HEp-2 cells treated with the various enzymes, lectins or sugars and to integrins αVβ3 and α5β1, relative to control wells. We found that both serovars bound equally well to HEp-2 cells; however, serovar Jules showed a higher level of attachment to integrins. Both serovars showed an increase in attachment to HEp-2 cells coated with lectins peanut agglutinin, Ulex europaeus agglutinin, soybean agglutinin and Erythrina cristagalli agglutinin (p < 0.05); in the case of Concanavalin A, Jules showed an increase, while Portlandvere showed a significant decrease in attachment. Trypsinizing monolayers resulted in a decrease in attachment for both serovars, while when chondroitinase, neuraminidase and heparinase were used an increase in attachment was recorded. Leptospires coated with sugars showed a decrease in attachment. These results show that serovar Jules' general greater affinity for the mediators examined may suggest a greater potential for virulence over serovar Portlandvere.     

Cold shock response in mammalian cells

Compared to bacteria and plants, the cold shock response has attracted little attention in mammals except in some areas such as adaptive thermogenesis, cold tolerance, storage of cells and organs, and recently, treatment of brain damage and protein production. At the cellular level, some responses of mammalian cells are similar to microorganisms; cold stress changes the lipid composition of cellular membranes, and suppresses the rate of protein synthesis and cell proliferation. Although previous studies have mostly dealt with temperatures below 20 degrees C, mild hypothermia (32 degrees C) can change the cell's response to subsequent stresses as exemplified by APG-1, a member of the HSP110 family. Furthermore, 32 degrees C induces expression of CIRP (cold-inducible RNA-binding protein), the first cold shock protein identified in mammalian cells, without recovery at 37 degrees C. Remniscent of HSP, CIRP is also expressed at 37 degrees C and developmentary regulated, possibly working as an RNA chaperone. Mammalian cells are metabolically active at 32 degrees C, and cells may survive and respond to stresses with different strategies from those at 37 degrees C. Cellular and molecular biology of mammalian cells at 32 degrees C is a new area expected to have considerable implications for medical sciences and possibly biotechnology.     

Influence of shear stress on adherent mammalian cells during division

Summary The influence of shear stress (0 to 4.5 N m^–2) on morphology and adherence of anchorage-dependent baby hamster kidney (BHK 21 cl3) cells during cell division was investigated by means of a time lapse film. With increasing shear stress level divided cells needed more time for spreading.     

Analysis of the attachment of replicating DNA to a nuclear matrix in mammalian interphase nuclei.

The attachment of replicating DNA to a rapidly sedimenting nuclear structure was investigated by digestion with various nucleases. When DNA was gradually removed by DNase I, pulse label incorporated during either 1 min or during 1 hour in the presence of arabinosylcytosine, remained preferentially attached to the nuclear structure. Single strand specific digestion by nuclease S1 or staphylococcal nuclease at low concentrations caused a release of about 30% of the pulse label, without significantly affecting the attachment of randomly labelled DNA. The released material had a low sedimentation coefficient and contained most of the Okasaki fragments. The remaining pulse label was less accessible to further digestion by double strand specific nuclease activity than the bulk DNA. The results suggest that an attachment of the replication fork to the nuclear structure occurs at sites behind but close to the branch point.     

Autophagic and apoptotic response to stress signals in mammalian cells

Autophagy is a highly conserved catabolic programme for degrading proteins and organelles. This process has been shown to act as a pro-survival or pro-death mechanism in different physiological and pathological conditions. Several stress stimuli can induce autophagy, such as nutrient deprivation or critical steps in development of lower and higher eukaryotes. Apoptosis is an orchestrated form of cell death in which cells are actively involved in their own demise. Again, stress is a positive regulator of apoptosis and, in particular, of its apoptosome-mediated mitochondrial pathway. Besides discussing the individual roles played by the key molecules involved in autophagy in mammals in response to stress signals, we discuss here the interrelations between autophagy and apoptosis under these conditions.     

Nonmonotonous metabolic response of mammalian cells and tissues to ionizing radiation

Changes in the activity of ornithindecarboxylase in various tissues and in the amount of catecholamine in rat hypothalamus by the action of acute and chronic ionizing radiation were studied. A nonmonotonous relationship between the metabolic parameters of animal tissues and cells and the radiation dose was revealed. It was assumed that the nonmonotonous character of the dose-response dependence results from the nonmonotonous time course of the metabolic response to irradiation. It was also assumed that living systems have the property of responding to stress agents by nonmonotonous changes in metabolism. In the case of acute irradiation, this response manifests itself as oscillations of metabolic parameters about the control. The oscillations occur with a particular amplitude and periods, which vary with radiation dose, and damp out with time. As a result, in a fixed time interval, the dose-response curve may be nonmonotonous. Reverse dose-response relationships are also possible. In the case of chronic irradiation, the metabolic and functional parameters oscillate throughout irradiation time, and a modification of the response occurs. A prolong exposure to ionizing radiation causes strong changes in the metabolism of lipids of cell membranes, organelles and chromatin, as well as in the functional properties of some mammalian cells and tissues. The necessity of constructing quantitative models for explaining the nonmonotonous dose-response dependence is discussed.     

The current status of the adaptive response to ionizing radiation in mammalian cells

The results of numerous studies indicate that cells can become refractory to the detrimental effect of ionizing radiation when previously exposed to a low, “adapting dose”;. This phenomenon has been termed an “adaptive response”; to ionizing radiation. It has been postulated that the induced radioresistance is due to the induction of DNA repair systems which efficiently protect the adapted cells from the effects of a subsequent, high “challenging dose”;. However, a direct proof of this hypothesis is still lacking. The analyzed endpoints include chromosomal aberrations, survival, mutations, genetic instability and DNA damage repair measured by the comet assay. Frequently contradictory results were published by different authors. For example some authors observed a reduced frequency of apoptosis in adapted cells, whereas others reported the opposite. The source of variablity of the adaptive response in human lymphocytes remains unresolved. While there is no doubt that an adapting dose can trigger some protecting mechanisms within the cell it appears that there is no single, universal mechanism of the adaptive response that is valid for all cell types and irradiation conditions.     

Effect of shear on the death of two strains of mammalian tissue cells

This study reports some findings on the death of mammalian cells occurring from fluid dynamic effects when they are pumped through capillaries. The cell strains used were human HeLa S3 and mouse L929, grown in monolayer culture. Cells were harvested and suspensions were pumped through various lengths and diameters of stainless steel capillary tubing. Viability of the cells was assayed by the dye exclusion test. Cell deaths occurred and could be correlated with either average wall shear or power dissipation within the capillary tube. L929 cells were found to be more sensitive than HeLa S3 cells at all shear rates tested.     

The role of deformability of endothelial cells in the arterial response to shear tension

Treatment of the femoral artery luminal surface with glutaraldehyde dimere or dithiosuccinimidyl propionate reduced or eliminated flow-induced dilation, the responses to acetylcholine and the ATP being preserved. The findings suggest that the endothelial cells perceive changes in shear stress and that the cell stiffness is a factor subject to the influence of the magnitude of flow-induced arterial dilation.