Substratum-dependent and region-specific control of attachment and proliferation of gastrointestinal epithelial cells in primary serum-free culture

A system for the primary serum-free culture of fetal rat gastrointestinal epithelial cells was used to examine the role of the extracellular matrix (ECM) in the attachment and proliferation of these epithelial cells. Forestomach epithelial cells (FSEC) were able to attach to and proliferate on plastic dishes without a substratum, while glandular stomach epithelial cells (GSEC) and duodenal epithelial cells (DEC) were unable to do so. The presence of a substratum promoted the attachment and proliferation of these epithelial cells. The effects of various components of the ECM differed depending on the type of cell. FSEC attached most efficiently to a substratum of fibronectin, while GSEC did so to laminin. DEC attached more efficiently to type I collagen and fibronectin than to any other substratum. FSEC proliferated most rapidly on laminin, while GSEC and DEC did so on collagen gels. These substrata induced the most efficient attachment and proliferation of FSEC, and they were effective in promoting the attachment and proliferation of GSEC and DEC in decreasing order of efficiency, indicating the existence of a head-to-tail gradient in the response of epithelial cells to substrata. The expression of c-myc mRNA in these cells differed depending upon the substratum on which they were cultured and the mRNA level was well correlated with the extent of the cell proliferation, indicating that the cell proliferation is mediated by c-myc gene expression, which is regulated by cell-ECM interactions. The results of the present study demonstrate that proliferation of gastrointestinal epithelial cells is regulated region-specifically not only by soluble factors but also by insoluble components of the ECM.     

Value of ultrasound in differential diagnosis of pediatric hip joint effusion (Perthes disease, C. fugax, epiphysiolysis coapitis femoris

Migrating cells form dynamic and highly regulated adhesive interactions with their environment. In particular, integrin-mediated adhesions to the extracellular matrix (ECM) play a central role in cell migration. This review focuses on recent advances in understanding the adhesive mechanisms that regulate cell detachment at the rear of migrating fibroblasts and neutrophils. The contribution of several key adhesive regulators is discussed, including myosin mediated cell contractility, tyrosine phosphorylation, rho, calcium fluxes, and calpain. A challenge for future investigation will be to determine how adhesive events are spatially and temporally coordinated to promote productive directional cell movements.     

Adhesive properties of the upper surface of fibroblasts and epithelium in cultures

Adhesive properties of the upper surface of cultured normal and neoplastic epithelial cells and of fibroblasts were studied. It was shown that the attachment of prelabeled homo- and heterologous cells introduced into epithelial sheets were very low as compared with the free substratum and dense cultures of fibroblasts. The upper surface of cells of cultured anaplastic hepatoma 22a was adhesive for prelabeled cells. Morphological characteristics of the studied cultures were compared. It was found that low adhesiveness of the upper surface of epithelial sheets correlated with the formation of firm intercellular contacts which remained unbroken during migration into the wound. It is suggested that when epithelial cells make contacts with each other, their surface is subdivided into two types of regions: areas of low adhesiveness (the upper surface), and those of firm intercellular contacts. This may cause the formation of monolayer epithelial sheet.     

Childbearing, reproductive control, aging women, and health care: the projected ethical debates

We have examined the effects of an integrin-binding competitor, echistatin, in solution on adhesion and migration of rat microvessel endothelial cells on fibronectin in vitro. A biphasic dependence of cell motility on fibronectin surface density was observed, with a peak random motility coefficient of about 8 x 10(-9) cm2/sec occurring below 0.3 microgram/cm2 fibronectin. In the presence of echistatin at 0.5 microM, the peak random motility coefficient was similar but occurred at the significantly greater fibronectin surface density of 1.2 micrograms/cm2. Hence, the same concentration of this soluble integrin-binding competitor inhibited migration on low fibronectin densities but enhanced migration on high fibronectin densities. At the same time, echistatin decreased adhesiveness on all fibronectin surface densities. When motility was correlated explicitly with adhesiveness, a single biphasic relationship was obtained for both absence and presence of echistatin with peak motility occurring in both cases at identical adhesiveness. Both the inhibiting and enhancing effects of the soluble integrin-binding competitor on motility are predictable from its effect on adhesion, consistent with the theoretical models of Lauffenburger (15) and DiMilla et al. (3).     

Effects of doxorubicin, 4'-epirubicin, and antioxidant enzymes on the contractility of isolated cardiomyocytes

The purpose of this study was to determine the acute effects of doxorubicin and its less cardiotoxic epimer, 4'-epirubicin, on the contractile response of isolated myocytes, and to assess similarities or differences with respect to active oxygen-derived mechanisms. Calcium-tolerant myocytes from rat ventricle were field stimulated at 1.0 Hz, and the maximum extent of cell shortening, peak shortening velocity, and peak relaxation velocity of single twitches were measured by video edge detection. The contractile responses of the myocytes to the two anthracyclines were approximately equal. Exposure of the cells to 10 microM of either anthracycline for 20 min decreased all indices of contractility by 28% (p < 0.05). The active oxygen scavengers, superoxide dismutase and catalase, distinguished the extent to which active oxygen was involved in modifying cellular contractility. Paradoxically, superoxide dismutase alone (10 U/mL) decreased contractility by 21%. Nevertheless, superoxide dismutase (10 U/mL) prevented the decreases in contractility produced by doxorubicin. In contrast, superoxide dismutase only mildly (32%) protected against 4'-epirubicin. Catalase (10 U/mL), however, provided substantial (82-93%) protection against both anthracyclines. Hydrogen peroxide therefore, and presumably hydroxyl radicals, were involved in mediating the decreases in contractility from both doxorubicin and 4'-epirubicin. These results show that an acute exposure to clinically relevant concentrations of these anthracyclines significantly depresses myocyte contractility and that, in this respect, 4'-epirubicin is as potentially cardiotoxic as doxorubicin. The results with antioxidant enzymes also strongly support a free radical mechanism for the toxicity of doxorubicin and 4'-epirubicin to cardiomyocytes.     

Cell to substratum adhesion is involved in v-Src-induced cellular protein tyrosine phosphorylation: implication for the adhesion-regulated protein tyrosine phosphatase activity

Protein tyrosine phosphorylation accompanies the integrin-mediated cell to substratum adhesion, and is essential for the progression of G1/S phase of the cell-cycle in normal fibroblasts. To examine how cellular protein tyrosine phosphatase (PTPase) activity is involved in regulating the adhesion-dependent protein tyrosine phosphorylation, we employed fibroblast cells bearing an active form of a protein tyrosine kinase (PTK), v-Src. We found that the v-Src induced tyrosine phosphorylation in certain proteins such as tensin, talin, p120, p80/85 (cortactin) and paxillin was greatly reduced when the cell to substratum adhesion was lost. Readhesion of the cells onto fibronectin restored these phosphorylation events, while this was inhibited by the addition of RGD peptide. The kinase activity of the v-Src was unchanged by the loss of cell to substratum adhesion. On the other hand, treatment with a protein tyrosine phosphatase inhibitor vanadate caused much the same increase in the v-Src-mediated cellular tyrosine phosphorylation between cells adhered to the culture environments and cells kept in suspension. These data suggest that PTPase(s) appears to be more critical than the v-Src PTK in determining the cell adhesion-dependent protein tyrosine phosphorylation. Moreover, most of the protein tyrosine phosphorylations that are mediated by the v-Src but still dependent on the cell adhesion were indeed greatly reduced during an anchorage-independent growth of v-Src cells. Thus our data collectively indicate that the v-Src induced high level of tyrosine phosphorylation in certain types of proteins are still under the control of the integrin(s) or the cell adhesion to culture substratum, and most of these adhesion-regulated high levels of tyrosine phosphorylations are not essential for the transformed phenotype.     

The Spectrum of Hot Water: Rotational Transitions and Difference Bands in the (020), (100), and (001) Vibrational States

Diatoms are unicellular microalgae encased in a siliceous cell wall, or frustule. Pennate diatoms, which possess bilateral symmetry, attach to the substratum at a slit in the frustule called the raphe. These diatoms not only adhere, but glide across surfaces whilst maintaining their attachment, secreting a sticky mucilage that forms a trail behind the gliding cells. We have raised monoclonal antibodies to the major cell surface proteoglycans of the marine raphid diatom Stauroneis decipiens Hustedt. The antibody StF.H4 binds to the cell surface, in the raphe and to adhesive trails and inhibits the ability of living diatoms to adhere to the substratum and to glide. Moreover, StF.H4 binds to a periodate-insensitive epitope on four frustule-associated proteoglycans (relative molecular masses 87, 112, and > 200 kDa). Another monoclonal antibody, StF.D5, binds to a carbohydrate epitope on the same set of proteoglycans, although the antibody binds only to the outer surface of the frustule and does not inhibit cell motility and adhesion.     

Activation of protein kinase C modulates cell-cell and cell-substratum adhesion of a human colorectal carcinoma cell line and restores 'normal' epithelial morphology

Abnormal cell adhesion is an important contributing factor in invasion and metastasis. Here, we show that morphologically 'normal' cell-cell and cell-substratum adhesion can be restored to a poorly differentiated carcinoma cell line by activation of protein kinase C (PKC). This cell line, VACO 10MS, grows as multicellular aggregates loosely attached to the substratum. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA, 7.5 nM) induces rapid adhesive changes with 2 components. First, within 15 min of TPA the cells become closely apposed, an event resembling the 'compaction' seen in the mouse early embryo. Next, over 2 hr, the cells spread, forming a monolayer. We show that compaction depends on extracellular calcium, E-cadherin-mediated adhesion and F-actin but not on protein synthesis, microtubules or substratum adhesion. By contrast, cell spreading is independent of cadherin and extracellular Ca2+ but involves the formation of focal contacts containing alpkha(v) integrin. TPA treatment causes rapid translocation of PKC-alpha to the insoluble fraction. During compaction, actin- and PKC-alpha-containing lamellae form over the entire aggregate surface, those adjacent to the substratum appearing to initiate spreading. Compaction does not involve increased phosphorylation of the cadherin/catenin complex. We conclude that activation of PKC-alpha restores 'normal' morphology to these poorly differentiated cells. Our results are of general interest in relation to the regulation of cell adhesion and, through further investigation, may lead to identification of novel targets for therapeutic suppression of invasion and metastasis.     

The fibroblast-populated collagen microsphere assay of cell traction force--Part 2: Measurement of the cell traction parameter

In Part 1 of this work, we formulated and analyzed a mathematical model for our fibroblast-populated collagen microsphere (FPCM) assay of cell traction forces (Moon and Tranquillo, 1993). In this assay, the FPCM diameter decreases with time as the cells compact the gel by exerting traction on collagen fibrils. In Part 1 we demonstrated that the diameter reduction profiles for varied initial cell concentration and varied initial FPCM diameter are qualitatively consistent with the model predictions. We show here in Part 2 how predictions of a model similar to that of Part 1, along with the determination of the growth parameters of the cells and the viscoelastic parameters of the gel, allow us to estimate the magnitude of a cell traction parameter, the desired objective index of cell traction forces. The model is based on a monophasic continuum-mechanical theory of cell-extracellular matrix (ECM) mechanical interactions, with a species conservation equation for cells (1), a mass conservation equation for ECM (2), and a mechanical force balance for the cell/ECM composite (3). Using a constant-stress rheometer and a fluids spectrometer in creep and oscillatory shear modes, respectively, we establish and characterize the linear viscoelastic regime for the reconstituted type 1 collagen gel used in our FPCM traction assay and in other assays of cell-collagen mechanical interactions. Creep tests are performed on collagen gel specimens in a state resembling that in our FPCM traction assay (initially uncompacted, and therefore nearly isotropic and at a relatively low collagen concentration of 2.1 mg/ml), yielding measurements of the zero shear viscosity, mu 0 7.4 x 10(6) Poise), and the steady-state creep compliance, J0e. The shear modulus, G (155 dynes/cm2), is then determined from the inverse of J0e in the linear viscoelastic regime. Oscillatory shear tests are performed in strain sweep mode, indicating linear viscoelastic behavior up to shear strains of approximately 10 percent. We discuss the estimation of Poisson's ratio, v, which along with G and mu 0 specifies the assumed isotropic, linear viscoelastic stress tensor for the cell/collagen gel composite which appears in (3). The proliferation rate of fibroblasts in free floating collagen gel (appearing in (1)) is characterized by direct cell counting, yielding an estimate of the first-order growth rate constant, k (5.3 x 10(-6) s-1). These independently measured and estimated parameter values allow us to estimate that the cell traction parameter, tau 0, defined in the active stress tensor which also appears in (3), is in the range of 0.00007-0.0002 dyne.cm4/mg collagen.cell.(ABSTRACT TRUNCATED AT 400 WORDS)     

Coupled influence of substratum hydrophilicity and surfactant on epithelial cell adhesion

The influence of substratum surface hydrophilicity and of a surfactant on human epithelial cell adhesion and protein adsorption was investigated. Therefore, tissue culture grade polystyrene (TCPS) and bacteriological grade polystyrene (BGPS) substrata were treated with different media, with or without Pluronic F68 [a poly(ethylene oxide) and poly(propylene oxide) triblock copolymer surfactant], and with or without type I collagen as a typical extracellular matrix protein. The conditioned substrata were submitted to XPS analysis and assayed for cell adhesion by inoculating Hep G2 cells in a chemically defined nutritive medium. The presence of collagen at the substratum surface is required to obtain attachment and spreading of Hep G2 cells. With PS substrata, treating with a solution of collagen does not promote cell adhesion if the solution contains Pluronic; XPS data show that this is due either to prevention of collagen adsorption or to its desorption by rinsing. With less hydrophobic TCPS substrata, the presence of Pluronic in the conditioning solution does not preclude cell adhesion, nor collagen adsorption. The effect of BGPS and TCPS substrata on Hep G2 cell adhesion is thus mediated by the presence of a surfactant that affects the adsorption of collagen.     

NG2 proteoglycan and the actin-binding protein fascin define separate populations of actin-containing filopodia and lamellipodia during cell spreading and migration

The transmembrane proteoglycan NG2 is able to interact both with components of the extracellular matrix and with the actin cytoskeleton. An examination of the distribution of NG2 during cell spreading suggests that NG2 can associate with two distinct types of actin-containing cytoskeletal structures, depending on the nature of the stimulus derived from the substratum. On fibronectin-coated dishes, cell surface NG2 associates exclusively with stress fibers developing within the cell. On poly-L-lysine-coated dishes, cell surface NG2 is associated with radial processes extending from the cell periphery. Spreading on fibronectin/poly-L-lysine mixtures, as well as on matrix components such as laminin, tenascin, and type VI collagen, produces cells with mosaic characteristics, i.e., NG2 is associated with both types of structures. NG2-positive radial processes are distinct from a second population of radial structures that contain fascin. NG2-positive extensions appear to be individual self-contained units (filopodia), whereas fascin is associated with actin ribs within sheets of membrane (lamellipodia). NG2- and fascin-positive structures are often localized to opposite poles of spreading cells, suggesting a possible role for the two classes of cellular extensions in the establishment of cell polarity during morphogenesis or migration. Time lapse imaging confirms the presence of lamellipodia on the leading edges of migrating cells, while numerous filopodia are present on trailing edges.     

Osteoblast hydraulic conductivity is regulated by calcitonin and parathyroid hormone

Many aspects of cell social behavior, including aspects of tumor invasiveness, embryonic development, and wound healing, can be explained by the principle of contact inhibition (CI) of cell movement. CI refers to the tendency of fibroblasts cultured on a plane substratum to cease movement on contacting other fibroblasts. A problem in studying collisions between cells on a flat substratum is that it is difficult to control the specific regions of the cell that come in contact. In this study we used grooved micromachined titanium substrata to produce collisions between the following cell combinations: fibroblast/fibroblast, fibroblast/epithelium, and epithelium/epithelium. The cells were oriented by the substratum so that the leading lamellae of the cells confronted each other. Cell behaviors before and after contact were observed and recorded using time-lapse cinemicrography employing Nomarski reflected light differential interference microscopy. Electron-microscopy sections were prepared from areas where cell interactions occurred. Fibroblasts (F) moved significantly faster and more persistently on grooved than on smooth surfaces, but the speed of epithelial (E)-cell locomotion was not significantly altered. The grooves, however, guided the direction of locomotion for both cell types. When cultured on grooved surfaces in such a manner that the F and E cells collided head-on, the F, but not the E cells, frequently demonstrated contact inhibition of movement. However, after such collisions, significantly more F continued to invade the E sheet than were observed after F-E collisions on smooth surfaces. After F-F collisions on grooved surfaces, most cells moved to the sides of the grooves and continued in their original directions, while on smooth surfaces they moved off in various different directions. A possible explanation of these observations is that a grooved surface produces and maintains F polarity so that the direction of locomotion is less readily altered by cell-cell interactions.     

The association of personality characteristics with parenting problems among alcoholic couples

Phosphorylation of the regulatory light chain of myosin II (RMLC) at Serine 19 by a specific enzyme, MLC kinase, is believed to control the contractility of actomyosin in smooth muscle and vertebrate nonmuscle cells. To examine how such phosphorylation is regulated in space and time within cells during coordinated cell movements, including cell locomotion and cell division, we generated a phosphorylation-specific antibody. Motile fibroblasts with a polarized cell shape exhibit a bimodal distribution of phosphorylated myosin along the direction of cell movement. The level of myosin phosphorylation is high in an anterior region near membrane ruffles, as well as in a posterior region containing the nucleus, suggesting that the contractility of both ends is involved in cell locomotion. Phosphorylated myosin is also concentrated in cortical microfilament bundles, indicating that cortical filaments are under tension. The enrichment of phosphorylated myosin in the moving edge is shared with an epithelial cell sheet; peripheral microfilament bundles at the leading edge contain a higher level of phosphorylated myosin. On the other hand, the phosphorylation level of circumferential microfilament bundles in cell-cell contacts is low. These observations suggest that peripheral microfilaments at the edge are involved in force production to drive the cell margin forward while microfilaments in cell-cell contacts play a structural role. During cell division, both fibroblastic and epithelial cells exhibit an increased level of myosin phosphorylation upon cytokinesis, which is consistent with our previous biochemical study (Yamakita, Y., S. Yamashiro, and F. Matsumura. 1994. J. Cell Biol. 124:129-137). In the case of the NRK epithelial cells, phosphorylated myosin first appears in the midzones of the separating chromosomes during late anaphase, but apparently before the formation of cleavage furrows, suggesting that phosphorylation of RMLC is an initial signal for cytokinesis.     

Role of asparagine-linked oligosaccharides in protein folding, membrane targeting, and thyrotropin and autoantibody binding of the human thyrotropin receptor

The amino-terminal ectodomain of thyrotropin (TSH) receptor (TSHR) is heavily glycosylated with asparagine-linked (N-linked) oligosaccharides. The present studies were designed to evaluate how acquisition and processing of N-linked oligosaccharides play a role in the functional maturation of human TSHR. A glycosylation inhibitor tunicamycin, which inhibits the first step of N-linked glycosylation (acquisition of N-linked oligosaccharides), and a series of mutant Chinese hamster ovary (CHO)-Lec cells defective in the different steps of glycosylation processing were used. Inhibition of acquisition of N-linked oligosaccharides by tunicamycin treatment in CHO cells stably expressing TSHR produced nonglycosylated TSHR, which was totally nonfunctional. In contrast, all of the TSHRs synthesized in mutant CHO-Lec1, 2, and 8 cells (mannose-rich, sialic acid-deficient, and galactose-deficient oligosaccharides, respectively) bound TSH and produced cAMP in response to TSH with an affinity and an EC50 similar to those in TSHR expressed in parental CHO cells (CHO-TSHR; sialylated oligosaccharides). However, Lec1-TSHR and Lec2-TSHR were not efficiently expressed on the cell surface, whereas the expression levels of Lec8-TSHR and CHO-TSHR were essentially identical. All of the TSHRs expressed in CHO-Lec cells cleaved into two subunits. Finally, anti-TSHR autoantibodies from Graves' patients interacted with all of the TSHRs harboring different oligosaccharides to a similar extent. These data demonstrate that acquisition and processing of N-linked oligosaccharides of TSHR appear to be essential for correct folding in the endoplasmic reticulum and for cell surface targeting in the Golgi apparatus. We also show that complex type carbohydrates are not crucially involved in the interaction of TSHR with TSH and anti-TSHR autoantibodies.     

Initial attachment of baby hamster kidney cells to an epoxy substratum. Ultrastructural analysis

In the presence of serum-containing medium, BHK cells attached and spread during a 1-h period onto a 3-5 nm thick serum layer absorbed on the substratum surface. The closest approach of the plasma membrane to the serum layer was observed to be about 9nm, which was determined by tilting the sectioned cells in a goniometer holder. Bundles of microfilaments or other cytoplasmic specializations were not observed in association with the regions of close contact. However, in the space between the plasma membrane and the adsorbed serum layer, a diffusely stained material could be visualized after fixation/staining by the tannic acid-glutaraldehyde technique. This technique also permitted increased clarity of visualization of trilaminar appearance of the plasma membrane. The distribution and mobility of anionic sites on the surfaces of attached and spreading cells was determined by labeling with polycationic ferritin. We observed movement of polycationic ferritin into large clusters on the cell surface, collapse of cell surface microextensions, and endocytosis, all of which were similar to our previous findings utilizing cells in suspension. However, the absolute amount of ferritin bound to the upper cell surface was less than that previously observed when suspended cells were put under similar labeling conditions. Also, polycationic ferritin did not appear to penetrate between the lower cell surface and the substratum.     

Assessing the validity of serodiagnostic test results

Abnormal adhesion of sickle cells to vascular endothelium may be a factor in the initiation of painful vaso-occlusive crisis. The sickle cell population contains an unusually large number of less dense reticulocytes that are known to be more adhesive than mature red cells, but there is contradictory evidence regarding the adhesiveness of dense sickle cells. We used a flow-based assay of adhesion to cultured human umbilical vein endothelial cells to test the properties of density fractions of sickle cells, prepared either by density gradient or by centrifugation of packed cells. We also examined the effects of incubating sickle cells with or without cyclical deoxygenation on their adhesion. After fractionation on a Percoll-isopaque gradient, the less dense 10% (reticulocyte-rich) cells and the most dense 10% cells adhered in greater number than the remainder (by about twofold). However, after centrifugation of packed cells, the less dense 10% were again more adhesive than the "middle" cells, but the most dense were not. Exposing sickle cells to constituents of the gradient had no consistent effect on adhesion, while centrifugal packing induced a degree of hemolysis, and tended to reduce adhesiveness of the dense fraction previously obtained from a gradient. Incubation in air at 37 degrees C for 15 hr reduced the number of reticulocytes and the adhesiveness of less dense sickle cells compared to those held at 4 degrees C. On the other hand, incubation at 37 degrees C for 15 hr with cyclical deoxygenation caused formation of dense cells and increased adhesiveness compared to incubation without cyclical deoxygenation. We conclude that young, less dense sickle cells are unusually adhesive, but that this adhesiveness is reduced during maturation. However, repeated sickling in vivo causes formation of an abnormally dense subpopulation of cells which either redevelop an increased tendency to adhere to endothelial cells or preserve their initial adhesiveness. Both adhesive cell populations may be implicated in promoting vascular obstruction.     

The lurcher mutation and ionotropic glutamate receptors: contributions to programmed neuronal death in vivo

Differentiation of trophoblast giant cells in the rodent placenta is accompanied by exit from the mitotic cell cycle and onset of endoreduplication. Commitment to giant cell differentiation is under developmental control, involving down-regulation of Id1 and Id2, concomitant with up-regulation of the basic helix-loop-helix factor Hxt and acquisition of increased adhesiveness. Endoreduplication disrupts the alternation of DNA synthesis and mitosis that maintains euploid DNA content during proliferation. To determine how the mammalian endocycle is regulated, we examined the expression of the cyclins and cyclin-dependent kinases during the transition from replication to endoreduplication in the Rcho-1 rat choriocarcinoma cell line. We cultured these cells under conditions that gave relatively synchronous endoreduplication. This allowed us to study the events that occur during the transition from the mitotic cycle to the first endocycle. With giant cell differentiation, the cells switched cyclin D isoform expression from D3 to D1 and altered several checkpoint functions, acquiring a relative insensitivity to DNA-damaging agents and a coincident serum independence. The initiation of S phase during endocycles appeared to involve cycles of synthesis of cyclins E and A, and termination of S was associated with abrupt loss of cyclin A and E. Both cyclins were absent from gap phase cells, suggesting that their degradation may be necessary to allow reinitiation of the endocycle. The arrest of the mitotic cycle at the onset of endoreduplication was associated with a failure to assemble cyclin B/p34(cdk1) complexes during the first endocycle. In subsequent endocycles, cyclin B expression was suppressed. Together these data suggest several points at which cell cycle regulation could be targeted to shift cells from a mitotic to an endoreduplicative cycle.