Saccharomyces cerevisiae gene expression changes during rotating wall vessel suspension culture.

This study utilizes Saccharomyces cerevisiae to study genetic responses to suspension culture. The suspension culture system used in this study is the high-aspect-ratio vessel, one type of the rotating wall vessel, that provides a high rate of gas exchange necessary for rapidly dividing cells. Cells were grown in the high-aspect-ratio vessel, and DNA microarray and metabolic analyses were used to determine the resulting changes in yeast gene expression. A significant number of genes were found to be up- or downregulated by at least twofold as a result of rotational growth. By using Gibbs promoter alignment, clusters of genes were examined for promoter elements mediating these genetic changes. Candidate binding motifs similar to the Rap1p binding site and the stress-responsive element were identified in the promoter regions of differentially regulated genes. This study shows that, as in higher order organisms, S. cerevisiae changes gene expression in response to rotational culture and also provides clues for investigations into the signaling pathways involved in gravitational response.     

Control of endothelial cell gene expression by flow

The vessel wall is constantly subjected to, and affected by, the stresses resulting from the hemodynamic stimuli of transmural pressure and flow. At the interface between blood and the vessel wall, the endothelial cell plays a crucial role in controlling vessel structure and function in response to changes in hemodynamic conditions. Using bovine aortic endothelium monolayers, we show that fluid shear stress causes simultaneous differential regulation of endothelial-derived products. We also report that the downregulation of endothelin-1 mRNA by flow is a reversible process, and through the use of uncharged dextran supplementation demonstrate it to be shear stress-rather than shear rate-dependent. Recent work on the effect of fluid shear stress on endothelial cell gene expression of a number of potent endothelial products is reviewed, including vasoactive substances, autocrine and paracrine growth factors, thrombosis/fibrinolysis modulators, chemotactic factors, surface receptors and immediate-early genes. The encountered patterns of gene expression responses are classified into three categories: a transient increase with return to baseline (type I), a sustained increase (type II) and a biphasic response consisting of an early transient increase of varying extent followed by a pronounced and sustained decrease (type III). The importance of the dynamic character of the flow stimulus and the magnitude dependence of the response are presented. Potential molecular mechanisms of shear-induced gene regulation, including putative shear stress response elements (SSRE), are discussed. These results suggest exquisite modulation of endothelial cell phenotype by local fluid shear stress and may offer insight into the mechanism of flow-dependent vascular remodeling and the observed propensity of atherosclerosis formation around bifurcations and areas of low shear stress.     

Rotating wall vessel as a newin vitro shear stress generation system: Application to rat coronary endothelial cell cultures

In this paper, we describe a simple new design for the application of controlled, top-hat profiled wall shear stress forces in a way that is independent of hydrostatic pressure and oxygen tension, based on a rotating wall vessel system. This system has been applied to the culture of rat coronary endothelial cells obtained with a Langendorff-derived procedure isolation. Endothelial cells are immunopurified on the basis of RECA expression, and conservation of endothelial phenotype has been assessed on the basis of morphology, RECA and von Willebrand factor expressions and diI-Ac-LDL uptake. Shear stress induced by the rotating wall vessel was measured using a mathematical formula specifically designed for this type of model, and its impact on coronary endothelial cells was evaluated. Shear stress produced cell orientation parallel to the flux direction, elevated NO production and decreased monocyte adhesion. Cells were kept viable and functional for at least 4 days under shear. This simple design allows the handling and management of numerous vials in parallel and appears to be suitable for large-scale studies of both the acute and chronic impact of modulation of the physico-chemical environment on endothelial cell physiology and function.     

Gene expression and survival changes in Saccharomyces cerevisiae during suspension culture

This study explores the connection between changes in gene expression and the genes that determine strain survival during suspension culture, using the model eukaryotic organism, Saccharomyces cerevisiae. The Saccharomyces cerevisiae homozygous diploid deletion pool (HDDP), and the BY4743 parental strain were grown for 18 h in a rotating wall vessel (RWV), a suspension culture device optimized to minimize the delivered shear. In addition to the reduced shear conditions, the RWVs were also placed in a static position or in a shaker in order to change the amount of shear stress on the cells. Using simple linear regression, it was found that there were 140 differentially expressed genes for which >70% of the variation can be explained by shear stress alone. A significant number of these genes are involved in catalytic activity. In the HDDP, shear stress was associated with significant survival changes in 15 deletion strains (R(2>) > 0.7) Interestingly, both analyses uncovered changes in the ribosomal protein machinery. Comparing the changes in gene expression and strain survival under the different shear conditions allows for the insights into the molecular mechanisms behind the cells response to shear stress. This in turn can provide information for the optimization of suspension culture.     

Shear stress effects on plant cell suspension cultures in a rotating wall vessel bioreactor

A rotating wall vessel, designed for growth of mammalian cells under microgravity, was used to study shear effects on Taxus cuspidata plant suspension cell cultures. Shear stress, as quantified by defined shear fields of Couette viscometers, improved specific cell growth rates and was detrimental to volumetric product formation rates. Received 5 January 1998/ Accepted in revised form 8 December 1998     

Whole-Cell Mechanosensitive Currents in Rat Ventricular Myocytes Activated by Direct Stimulation

Mechanosensitive channels may have a significant role in the development of cardiac arrhythmia following infarction, but the data on mechanical responses at the cellular level are limited. Mechanosensitivity is a ubiquitous property of cells, and although the structure of bacteriological mechanosensitive ion channels is becoming known by cloning, the structure and force transduction pathway in eukaryotes remains elusive. Isolated adult rat ventricular myocytes were voltage clamped and stimulated with a mechanical probe. The probe was set in sinusoidal motion (either in, or normal to, the plane of the cell membrane), and then slowly lowered onto the cell. The sinusoidal frequency was held constant at 1 Hz but the stimulation amplitude was increased and the probe gradually lowered until a mechanically sensitive whole cell current was seen, which usually followed several minutes of stimulation. The whole cell mechanosensitive current in rat cells had two components: (i) a brief large inward current spike current; (ii) a more sustained smaller inward current. The presence of the initial sharp inward current suggests that some structure within the cell either relaxes or is broken, exposing the mechanosensitive element(s) to stress. Metabolic changes induced by continued stress prior to the mechanosensitive response may weaken the elements that break producing the spike, or simple stress-induced fracture of the cytoskeleton itself may occur.     

Mechanisms of blood flow-induced vascular enlargement

Chronic changes in wall shear stress lead to vascular remodeling, characterized by increased vascular wall diameter and thickness, to restore wall shear stress values to baseline. Release of nitric oxide from endothelial cells exposed to excessive shear is a fundamental step in the remodeling process, and potentially triggers a cascade of events, including growth factor induction and matrix metalloproteinase activation, that together contribute to restructuralization of the vessel wall. Understanding these processes could help explain how changes in blood vessel wall structure occur in the context of atherosclerosis or aortic aneurisms.     

Novel quantitative biosystem for modeling physiological fluid shear stress on cells

The response of microbes to changes in the mechanical force of fluid shear has important implications for pathogens, which experience wide fluctuations in fluid shear in vivo during infection. However, the majority of studies have not cultured microbes under physiological fluid shear conditions within a range commonly encountered by microbes during host-pathogen interactions. Here we describe a convenient batch culture biosystem in which (i) the levels of fluid shear force can be varied within physiologically relevant ranges and quantified via mathematical models and (ii) large numbers of cells can be planktonically grown and harvested to examine the effect of fluid shear levels on microbial genomic and phenotypic responses. A quantitative model based on numerical simulations and in situ imaging analysis was developed to calculate the fluid shear imparted by spherical beads of different sizes on bacterial cell cultures grown in a rotating wall vessel (RWV) bioreactor. To demonstrate the application of this model, we subjected cultures of the bacterial pathogen Salmonella enterica serovar Typhimurium to three physiologically-relevant fluid shear ranges during growth in the RVW and demonstrated a progressive relationship between the applied fluid shear and the bacterial genetic and phenotypic responses. By applying this model to different cell types, including other bacterial pathogens, entire classes of genes and proteins involved in cellular interactions may be discovered that have not previously been identified during growth under conventional culture conditions, leading to new targets for vaccine and therapeutic development.     

Fluid shear stress and the vascular endothelium: for better and for worse

As blood flows, the vascular wall is constantly subjected to physical forces, which regulate important physiological blood vessel responses, as well as being implicated in the development of arterial wall pathologies. Changes in blood flow, thus generating altered hemodynamic forces are responsible for acute vessel tone regulation, the development of blood vessel structure during embryogenesis and early growth, as well as chronic remodeling and generation of adult blood vessels. The complex interaction of biomechanical forces, and more specifically shear stress, derived by the flow of blood and the vascular endothelium raise many yet to be answered questions:How are mechanical forces transduced by endothelial cells into a biological response, and is there a "shear stress receptor"?Are "mechanical receptors" and the final signaling pathways they evoke similar to other stimulus-response transduction systems?How do vascular endothelial cells differ in their response to physiological or pathological shear stresses?Can shear stress receptors or shear stress responsive genes serve as novel targets for the design of diagnostic and therapeutic modalities for cardiovascular pathologies?The current review attempts to bring together recent findings on the in vivo and in vitro responses of the vascular endothelium to shear stress and to address some of the questions raised above.     

Symbiosis Between Methanogenic Archaea and δ-Proteobacteria as the Origin of Eukaryotes: The Syntrophic Hypothesis

We present a novel hypothesis for the origin of the eukaryotic cell, or eukaryogenesis, based on a metabolic symbiosis (syntrophy) between a methanogenic archaeon (methanobacterial-like) and a δ-proteobacterium (an ancestral sulfate-reducing myxobacterium). This syntrophic symbiosis was originally mediated by interspecies H₂ transfer in anaerobic, possibly moderately thermophilic, environments. During eukaryogenesis, progressive cellular and genomic cointegration of both types of prokaryotic partners occurred. Initially, the establishment of permanent consortia, accompanied by extensive membrane development and close cell–cell interactions, led to a highly evolved symbiotic structure already endowed with some primitive eukaryotic features, such as a complex membrane system defining a protonuclear space (corresponding to the archaeal cytoplasm), and a protoplasmic region (derived from fusion of the surrounding bacterial cells). Simultaneously, bacterial-to-archaeal preferential gene transfer and eventual replacement took place. Bacterial genome extinction was thus accomplished by gradual transfer to the archaeal host, where genes adapted to a new genetic environment. Emerging eukaryotes would have inherited archaeal genome organization and dynamics and, consequently, most DNA-processing information systems. Conversely, primordial genes for social and developmental behavior would have been provided by the ancient myxobacterial symbiont. Metabolism would have been issued mainly from the versatile bacterial organotrophy, and progressively, methanogenesis was lost. Received: 5 January 1998 / Accepted: 18 March 1998     

Expression of renal cell protein markers is dependent on initial mechanical culture conditions.

The rotating wall vessel is optimized for suspension culture, with laminar flow and adequate nutrient delivery, but minimal shear. However, higher shears may occur in vivo. During rotating wall vessel cultivation of human renal cells, size and density of glass-coated microcarrier beads were changed to modulate initial shear. Renal-specific proteins were assayed after 2 days. Flow cytometry antibody binding analysis of vitamin D receptor demonstrated peak expression at intermediate shears, with 30% reduction outside this range. Activity of cathepsin C showed the inverse pattern, lowest at midshear, with twofold increases at either extreme. Dipeptidyl-peptidase IV had no shear dependence, suggesting that the other results are specific, not universal, changes in membrane trafficking or protein synthesis. On addition of dextran, which changes medium density and viscosity but not shear, vitamin D receptor assay showed no differences from controls. Neither cell cycle, apoptosis/necrosis indexes, nor lactate dehydrogenase release varied between experiments, confirming that the changes are primary, not secondary to cell cycling or membrane damage. This study provides direct evidence that mechanical culture conditions modulate protein expression in suspension culture.     

Biorheological views of endothelial cell responses to mechanical stimuli

Vascular endothelial cells are located at the innermost layer of the blood vessel wall and are always exposed to three different mechanical forces: shear stress due to blood flow, hydrostatic pressure due to blood pressure and cyclic stretch due to vessel deformation. It is well known that endothelial cells respond to these mechanical forces and change their shapes, cytoskeletal structures and functions. In this review, we would like to mainly focus on the effects of shear stress and hydrostatic pressure on endothelial cell morphology. After applying fluid shear stress, cultured endothelial cells show marked elongation and orientation in the flow direction. In addition, thick stress fibers of actin filaments appear and align along the cell long axis. Thus, endothelial cell morphology is closely related to the cytoskeletal structure. Further, the dynamic course of the morphological changes is shown and the related events such as changes in mechanical stiffness and functions are also summarized. When endothelial cells were exposed to hydrostatic pressure, they exhibited a marked elongation and orientation in a random direction, together with development of centrally located, thick stress fibers. Pressured endothelial cells also exhibited a multilayered structure with less expression of VE-cadherin unlike under control conditions. Simultaneous loading of hydrostatic pressure and shear stress inhibited endothelial cell multilayering and induced elongation and orientation of endothelial cells with well-developed VE-cadherin in a monolayer, which suggests that for a better understanding of vascular endothelial cell responses one has to take into consideration the combination of the different mechanical forces such as exist under in vivo mechanical conditions.     

DNA microarray study on gene expression profiles in co-cultured endothelial and smooth muscle cells in response to 4- and 24-h shear stress

Shear stress, a major hemodynamic force acting on the vessel wall, plays an important role in physiological processes such as cell growth, differentiation, remodelling, metabolism, morphology, and gene expression. We investigated the effect of shear stress on gene expression profiles in co-cultured vascular endothelial cells (ECs) and smooth muscle cells (SMCs). Human aortic ECs were cultured as a confluent monolayer on top of confluent human aortic SMCs, and the EC side of the co-culture was exposed to a laminar shear stress of 12 dyn/cm² for 4 or 24 h. After shearing, the ECs and SMCs were separated and RNA was extracted from the cells. The RNA samples were labelled and hybridized with cDNA array slides that contained 8694 genes. Statistical analysis showed that shear stress caused the differential expression (p ≤ 0.05) of a total of 1151 genes in ECs and SMCs. In the co-cultured ECs, shear stress caused the up-regulation of 403 genes and down-regulation of 470. In the co-cultured SMCs, shear stress caused the up-regulation of 152 genes and down-regulation of 126 genes. These results provide new information on the gene expression profile and its potential functional consequences in co-cultured ECs and SMCs exposed to a physiological level of laminar shear stress. Although the effects of shear stress on gene expression in monocultured and co-cultured EC are generally similar, the response of some genes to shear stress is opposite between these two types of culture (e.g., ICAM-1 is up-regulated in monoculture and down-regulated in co-culture), which strongly indicates that EC–SMC interactions affect EC responses to shear stress.     

The Role of Calcium in the Volume Regulation of Rat Lacrimal Acinar Cells

Earlier studies have suggested a role for Ca²⁺ in regulatory volume decrease (RVD) in response to hypotonic stress through the activation of Ca²⁺-dependent ion channels (Kotera & Brown, 1993; Park et al., 1994). The involvement of Ca²⁺ in regulating cell volume in rat lacrimal acinar cells was therefore examined using a video-imaging technique to measure cell volume. The trivalent cation Gd³⁺ inhibited RVD, suggesting that Ca²⁺ entry is important and may be via stretch-activated cation channels. However, Fura-2 loaded cells did not show an increase in [Ca²⁺] _i during exposure to hypotonic solutions. The absence of any changes in [Ca²⁺] _i resulted from the buffering of cytosolic Ca²⁺ by Fura-2 during hypotonic shock and therefore inhibition of RVD. The intracellular Ca²⁺ chelator, BAPTA, also inhibited the RVD response to hypotonic shock. An increase in [Ca²⁺] _i induced by either acetylcholine or ionomycin, was found to decrease cell volume under isotonic conditions in lacrimal acinar cells. Cell shrinkage was inhibited by tetraethylammonium ion, an inhibitor of Ca²⁺-activated K⁺ channels. On the basis of the presented data, we suggest an involvement of intracellular Ca²⁺ in controlling cell volume in lacrimal acinar cells. Received: 20 February 1998/Revised: 1 May 1998     

Hydrodynamic shear stress and mass transport modulation of endothelial cell metabolism

Mammalian cells responds to physical forces by altering their growth rate, morphology, metabolism, and genetic expression. We have studied the mechanism by which these cells detect the presence of mechanical stress and convert this force into intracellular signals. As our model systems, we have studied cultured human endothelial cells, which line the blood vessels and forms the interface between the blood and the vessel wall. These cell responds within minutes to the initiation of flow by increasing their arachidonic acid metabolism and increasing the level of the intracellular second messengers inositol trisphosphate and calcium ion concentration. With continued exposure to arterial levels of wall shear stress for up to 24 h, endothelial cells increase the expression of tissue plasminogen activator (tPA) and tPA messenger RNA (mRNA) and decrease the expression of endothelin peptide and endothelin mRNA. Since the initiation of flow also causes enhanced convective mass transfer to the endothelial cell monolayer, we have investigated the role of enhanced convection of adenosine trisphosphate (ATP) to the cell surface in eliciting a cellular response by monitoring cytosolic calcium concentrations on the single cell level and by computing the concentration profile of ATP in a parallel-plate flow geometry. Our result demonstrate that endothelial cells respond in very specific ways to the initiation of flow and that mass transfer and fluid shear stress can both play a role in the modulation of intracellular signal transduction and metabolism.     

Genomic Organization Around the Centromeric End of the HLA Class I Region: Large-Scale Sequence Analysis

We previously sequenced two regions around the centromeric end of HLA class I and the boundary between class I and class III. In this paper we analyze the two regions of about 385 kb and confirm, giving a new line of evidence, that the following two pairs of the genomic segments were duplicated in evolution: (i) a 43-kb genomic segment including the HLA-B gene showing the highest polymorphism among the classical HLA class I loci (class Ia) and a 40-kb segment including the HLA-C locus showing the lowest polymorphism and (ii) a 52-kb segment including the MIC (MHC class I chain related gene) B and a 35-kb segment including MICA. We also found that repetitive elements such as SINEs, LINEs, and LTRs occupy as much as 47% of nucleotides in this 385-kb region. This unusually high content of repetitive elements indicates that repeat-mediated rearrangements have frequently occurred in the evolutionary history of the HLA class Ia region. Analysis of LINE compositions within the two pairs of duplicated segments revealed that (i) LINEs in these regions had been dispersed prior to both the duplication of the HLA-B and -C loci and the duplication of the MICB and MICA loci, and (ii) the divergence of the HLA-B and -C loci occurred prior to the duplication of the MICA and MICB loci. To find novel genes responsible for HLA class I-associated or other diseases, we performed computer analysis applying GenScan and GRAIL to GenBank's dbEST. As a result, at least five as yet uncharacterized genes were newly mapped on the HLA class I centromeric region studied. These novel genes should be analyzed further to determine their relationships to diseases associated with this region. Received: 16 June 1998 / Accepted: 18 August 1998     

Photofrin II Sensitized Modifications of Ion Transport Across the Plasma Membrane of an Epithelial Cell Line: I. Electrical Measurements at the Whole-Cell Level

Photofrin II is a photosensitizer frequently applied in photodynamic therapy. Light-induced tumor cell inactivation observed in the presence of this substance has been suggested to start with modifications at the level of cellular membranes. In the present study electrophysiological techniques are applied in order to investigate the action of photofrin II on functional properties of the plasma membrane of opossum kidney (OK) cells (as an epithelial model system) and of fibroblasts. Illumination of the cells in the presence of photofrin II (or Zn-phthalocyanine) leads to comparatively fast depolarization of the membrane potential. It is caused by a strong change of the membrane conductance which proceeds in two phases. Both phases contribute to a loss of ion selectivity of the plasma membrane between K⁺ and Na⁺. In the first phase, specific pathways for K⁺, which determine the resting potential under physiological conditions, are inactivated. The second phase is distinguished by a marked increase of a nonselective conductance. The increase of the latter — after light-induced initiation — continues in the dark. The conclusions are derived from light-induced, time-dependent changes of the membrane conductance and of the shape of the current-voltage relationship detected under different experimental conditions. Received: 26 May 1998/Revised: 8 September 1998     

What Amino Acid Properties Affect Protein Evolution?

We studied 10 protein-coding mitochondrial genes from 19 mammalian species to evaluate the effects of 10 amino acid properties on the evolution of the genetic code, the amino acid composition of proteins, and the pattern of nonsynonymous substitutions. The 10 amino acid properties studied are the chemical composition of the side chain, two polarity measures, hydropathy, isoelectric point, volume, aromaticity, aliphaticity, hydrogenation, and hydroxythiolation. The genetic code appears to have evolved toward minimizing polarity and hydropathy but not the other seven properties. This can be explained by our finding that the presumably primitive amino acids differed much only in polarity and hydropathy, but little in the other properties. Only the chemical composition (C) and isoelectric point (IE) appear to have affected the amino acid composition of the proteins studied, that is, these proteins tend to have more amino acids with typical C and IE values, so that nonsynonymous mutations tend to result in small differences in C and IE. All properties, except for hydroxythiolation, affect the rate of nonsynonymous substitution, with the observed amino acid changes having only small differences in these properties, relative to the spectrum of all possible nonsynonymous mutations. Received: 2 January 1998 / Accepted: 25 April 1998     

The shear stress of it all: the cell membrane and mechanochemical transduction

As the inner lining of the vessel wall, vascular endothelial cells are poised to act as a signal transduction interface between haemodynamic forces and the underlying vascular smooth-muscle cells. Detailed analyses of fluid mechanics in atherosclerosis-susceptible regions of the vasculature reveal a strong correlation between endothelial cell dysfunction and areas of low mean shear stress and oscillatory flow with flow recirculation. Conversely, steady shear stress stimulates cellular responses that are essential for endothelial cell function and are atheroprotective. The molecular basis of shear-induced mechanochemical signal transduction and the endothelium's ability to discriminate between flow profiles remains largely unclear. Given that fluid shear stress does not involve a traditional receptor/ligand interaction, identification of the molecule(s) responsible for sensing fluid flow and mechanical force discrimination has been difficult. This review will provide an overview of the haemodynamic forces experienced by the vascular endothelium and its role in localizing atherosclerotic lesions within specific regions of the vasculature. Also reviewed are several recent lines of evidence suggesting that both changes in membrane microviscosity linked to heterotrimeric G proteins, and the transmission of tension across the cell membrane to the cell-cell junction where known shear-sensitive proteins are localized, may serve as the primary force-sensing elements of the cell.     

The Role of Interelement Selection in Saccharomyces cerevisiae Ty Element Evolution

Retrotransposons are mobile genetic elements that are ubiquitous components of eukaryotic genomes. The evolutionary success of retrotransposons is explained by their ability to replicate faster than the host genomes in which they reside. Elements with higher rates of genomic replication possess a selective advantage over less active elements. Retrotransposon populations, therefore, are shaped largely by selective forces acting at the genomic level between elements. To evaluate rigorously the effects of selective forces acting on retrotransposons, detailed information on the patterns of molecular variation within and between retrotransposon families is needed. The sequencing of the Saccharomyces cerevisiae genome, which includes the entire genomic complement of yeast retrotransposons, provides an unprecedented opportunity to access and analyze such data. In this study, we analyzed in detail the patterns of nucleotide variation within the open reading frames of two parental (Ty1 and Ty2) and one hybrid (Ty1/2) family of yeast retrotransposons. The pattern and distribution of nucleotide changes on the phylogenetic reconstructions of the three families of Ty elements reveal evidence of negative selection on both internal and external branches of the Ty phylogenies. These results indicate that most, if not all, Ty elements examined represent active or recently active retrotransposon lineages. We discuss the relevance of these findings with respect to the coevolutionary dynamic operating between genomic element populations and the host organisms in which they reside. Received: 5 November 1998 / Accepted: 17 March 1999