Intrinsic factors influencing the maintenance of contractile embryonic heart cells in vitro : II. Biochemical analysis of heart muscle conditioned medium

Seven-day embryonic heart cells are tested for their ability to condition their own medium by comparing cell responses at various inoculum levels. The data show that contractile activity, spreading on glass, and survival increase as inoculum level rises. The data also show that the cell death rate is inversely proportional to the rate at which cell spreading and contractile activity increase.     

Synchrony of cell spreading and contraction force as phagocytes engulf large pathogens

A simple micromechanical method has been used to directly measure the force of contraction in single mammalian phagocytes (blood granulocytes) during engulfment of large yeast pathogens. Both the time course of cell spreading over the yeast particle and increase in cell body contractile force were quantitated at three temperatures in the range of 23-35 degrees C. The surprising feature of the phagocyte response was that engulfment and cell body contraction occurred in a serial sequence: i.e., the phagocyte spread rapidly over the particle at a steady rate with no detectable cell body contraction; when spreading stopped, contraction force in the cell body then rose steadily to a plateau level that remained stationary until the next sequence of spreading and contraction. Both spreading and contraction exhibited abrupt start/stop kinetics. Also impressive, the cell contraction force stimulated by phagocytosis was quite large (approximately 10(-8) N)-two orders of magnitude larger than the force necessary to deform passive phagocytes to the same extent. If distributed uniformly over the cell cross section, the contraction force is equivalent to an average contractile stress of approximately 10(3) N/m2 (0.01 Atm). These physical measurements in situ set critical requirements for the mechanism of force generation in granulocytes, imply that a major increase in network cross-linking accompanies build- up in contractile force and that subsequent network dissolution is necessary for locomotion.     

Attachment, spreading and growth of VERO cells on microcarriers for the optimization of large scale cultures

The VERO cell attachment, spreading and growth were measured as a function of the substrate and temperature used for cell cultivation, the presence of fetal calf serum (FCS) in the medium and the initial cell inoculum used for cultivation on MCs. The data show that the cell attachment kinetics were comparable at RT or 37v°C, a higher rate of cell attachment occurred to MCs and the presence of FCS inhibited the cell attachment to glass or plastic but not to MCs. The cell spreading, in general higher at 37v°C, was dependent on the presence of FCS, comparable on glass or plastic substrate and lower on MCs. The spread of VERO cells over MCs was fully dependent on the presence of FCS and decreases progressively with a delayed addition of FCS into the medium. The cell detachment by trypsin was slower from MCs and the cells recovered showed lower viability and reattachment. Better results of detachment, viability and reattachment were obtained by treatment with the trypsin at pH of 8 instead of 7. The lower was the number of cells/MC for the initial inoculum, the higher was the percent of unoccupied MCs (with 1 cell/MC we had 35.6% of unoccupied MCs), which were shown to remain uncovered during the whole period of culture. With an initial inoculum of 4, 6 and 8 VERO cells/MC, respectively 46%, 76% and 83% of the MCs were totally covered by cells after 7 days, the cultures showing at this time, respectively, 5.1 2 10⁵, 8.8 2 10⁵ and 1.8 2 10⁶ cells/ml, which represented a biomass production of respectively 8.5x, 9.7x and 15.5x. When compared to 175 cm² T-flasks, using the same amount of medium, a VERO cell culture on 2 mg/ml of MCs offers about 10 times more available surface for cell growth and allowed the obtention of 7 times more cells. The optimization procedures concerning initial steps of VERO cell cultures, such as the attachment, spreading and growth as a function of parameters like initial cell inoculum and medium supplementation are of special interest mainly due to the perspective of a large use of VERO cell cultures for human viral vaccine production.     

Extracellular matrix controls myosin light chain phosphorylation and cell contractility through modulation of cell shape and cytoskeletal prestress

The mechanism by which vascular smooth muscle (VSM) cells modulate their contractility in response to structural cues from extracellular matrix remains poorly understood. When pulmonary VSM cells were cultured on increasing densities of immobilized fibronectin (FN), cell spreading, myosin light chain (MLC) phosphorylation, cytoskeletal prestress (isometric tension in the cell before vasoagonist stimulation), and the active contractile response to the vasoconstrictor endothelin-1 all increased in parallel. In contrast, MLC phosphorylation did not increase when suspended cells were allowed to bind FN-coated microbeads (4.5-microm diameter) or cultured on micrometer-sized (30 x 30 microm) FN islands surrounded by nonadhesive regions that support integrin binding but prevent cell spreading. Cell spreading and MLC phosphorylation also both decreased in parallel when the mechanical compliance of flexible FN substrates was raised. MLC phosphorylation was inhibited independently of cell shape when cytoskeletal prestress was dissipated using a myosin ATPase inhibitor in fully spread cells, whereas it increased to maximal levels when microtubules were disrupted using nocodazole in cells adherent to FN but not in suspended cells. These data demonstrate that changes in cell-extracellular matrix (ECM) interactions modulate smooth muscle cell contractility at the level of biochemical signal transduction and suggest that the mechanism underlying this regulation may involve physical interplay between ECM and the cytoskeleton, such that cell spreading and generation of cytoskeletal tension feed back to promote MLC phosphorylation and further increase tension generation.     

Structure-function correlation in airway smooth muscle adapted to different lengths

Airway smooth muscle is able to adapt and maintain a nearly constant maximal force generation over a large length range. This implies that a fixed filament lattice such as that found in striated muscle may not exist in this tissue and that plastic remodeling of its contractile and cytoskeletal filaments may be involved in the process of length adaptation that optimizes contractile filament overlap. Here, we show that isometric force produced by airway smooth muscle is independent of muscle length over a twofold length change; cell cross-sectional area was inversely proportional to cell length, implying that the cell volume was conserved at different lengths; shortening velocity and myosin filament density varied similarly to length change: increased by 69.4% ± 5.7 (SE) and 76.0% ± 9.8, respectively, for a 100% increase in cell length. Muscle power output, ATPase rate, and myosin filament density also have the same dependence on muscle cell length: increased by 35.4% ± 6.7, 34.6% ± 3.4, and 35.6% ± 10.6, respectively, for a 50% increase in cell length. The data can be explained by a model in which additional contractile units containing myosin filaments are formed and placed in series with existing contractile units when the muscle is adapted at a longer length. muscle contraction; myosin filaments; ATPase activity; electron microscopy     

Vitex glabrata R.Br.悬浮培养细胞的生长条件优化及20-羟基蜕皮激素的生产

研究了培养基、植物生长调节素以及接种量对Vitex glabrata R.Br.悬浮培养细胞的生长情况以及对20-羟基蜕皮激素形成的作用。当细胞在添加有2.0mg/LBAP(6-苯甲酸嘌呤)和1.0mg/L2,4-D的Gamborg’s B5培养基中培养时细胞生长和20-羟基蜕皮激素的形成达到了最高水平。当接种量为20%PCV(积压细胞体积)时观察到了20-羟基蜕皮激素的最高产量,大约是1.1mg/(L.d)。实验数据也表明当接种量增加到20%PCV时,产量提高了7倍。     

Induction of spreading during fibroblast movement

This paper describes the phenomenon of retraction-induced spreading of embryonic chick heart fibroblasts moving in culture. Measurable criteria of cell spreading (increase in area of the spreading lamella, and total spread area of the cell) are found to change predictably with retraction of a portion of the cell margin. Ruffling activity was found to increase. The leading lamella of a spread fibroblast ordinarily advances slowly, with an average area increase of approximately 21 mu2m/min. A 10- to 30-fold increase in spreading occurs within 8 s after onset of retraction at the trailing edge and then decreases slightly so that by 1 min the increase in spreading is five to tenfold. During this period, there is a linear relationship between area increase at the leading edge and area decrease at the trailing edge. During the next 10--15 min, spreading gradually decreases to normal. Although the relationship between area spreading and area retracting of fibroblasts at different phases of movement is not significantly linear, it is highly correlated (Table II). These results suggest that the rate of fibroblast spreading may be inversely related to the degree of spreading of the cell as a whole.     

Modulation of contraction by gelation/solation in a reconstituted motile model

The actin-based cytoskeleton is a dynamic component of living cells with major structural and contractile properties involved in fundamental cellular processes. The action of actin-binding proteins can decrease or increase the gel structure. Changes in the actin-based cytoskeleton have long been thought to modulate the myosin II-based contractions involved in these cellular processes, but there has been some debate concerning whether maximal gelation increases or decreases contractile activity. To address this question, we have examined how contractile activity is modulated by the extent of actin gelation. The model system consists of physiologically relevant concentrations and molar ratios of actin filaments (whose lengths are controlled by gelsolin), the actin-cross-linking protein filamin, and smooth muscle myosin II. This system has been studied at the macroscopic and light microscopic levels to relate the gel structure to the rate of contraction. We present results which show that while a minimal amount of structure is necessary to transmit the contractile force, increasing the gel structure inhibits the rate of contraction, despite an increase in the actin-activated Mg(2+)-ATPase activity of myosin. Decreasing the total myosin concentration also inhibits the rate of contraction. Application of cytochalasin D to one side of the contractile network increases the rate of contraction and also induces movement comparable to flare streaming observed in isolated amoeba cytoplasm. These results are interpreted relative to current models of the relationship between the state of gelation and contraction and to the potential effects of such a relationship in the living cell.     

Changes in alpha-actinin localization and myofibrillogenesis in rat cardiomyocytes under cultivation

By indirect immunofluorescence with monoclonal anti-alpha-actinin antibodies the localization of this contractile protein was studied in ventricular cardiac myocytes from newborn 2-4-day old rats in the course of their cultivation. In freshly isolated heart muscle cells a predominant longitudinal orientation of myofibrils was observed; in some cells on the periphery of cytoplasm the contours of Z-lines are indistinct. During cell spreading, in the areas of intercalated discs, growing processes were observed mostly containing no contractile structures at earlier stages of cultivation. On days 3 to 14, the cytoplasmic processes and ruffles are filled with developing myofibrils. The cultures are heterogeneous in the morphology of contractile apparatus of individual cells. In most cardiomyocytes mature myofibrils are well-developed in the central part of the cytoplasm, whereas in its peripheral areas non-myofibrillar stress-fiber-like structures and bundles with continuous distribution of alpha-actinin frequently connected to myofibrils are more typical. In the areas of active myofibrillogenesis, located mainly on the cell periphery, numerous alpha-actinin dots are observed; most of them are arranged linearly and periodically at a distance of 0.3-1.5 microns and seem to be structural precursors of Z-lines. The data obtained show that the cultures of mammalian cardiac cells may be a convenient object for studying myofibrillogenesis in the course of cardiomyogenic differentiation.     

Cell spreading factor. Occurrence and specificity of action.

A factor required for spreading of substratum-attached baby hamster kidney cells (BHK), Chinese hamster ovary (CHO) cells, HeLa cells, and L cells has been isolated and purified from fetal calf serum. A similar factor has also been found in calf, porcine, human, rabbit, and chicken sera. The spreading factor was active when adsorbed to the substratum and prior adsorption of other proteins prevented cell spreading, regardless of the addition of spreading factor or unfractionated serum to the incubation medium. Antibody against the fetal calf spreading factor inhibited the spreading activity associated with unfractionated fetal calf serum and also the spreading activity associated with calf serum and porcine serum. In model system studies it was found that antibody against BHK cell surfaces induced cell spreading when the antibody was adsorbed to the substratum; when it was present in the incubation medium as well as on the substratum, cell spreading was not observed. The data are discussed in terms of the hypothesis that there is a specific serum factor which adsorbs to the substratum surface and is thereby activated, and which then forms the target for certain cell surface receptors. Interaction between adsorbed-activated factor and cell surface receptors leads to cell spreading.     

The effect of inoculum size on the growth of cell and root cultures ofHyoscyamus muticus: Implications for reactor inoculation

Cell suspensions inoculated at low cell concentrations displayed a typical growth reduction, whereas root cultures displayed an improvement in growth. Specific growth rate ofHyoscyamus muticus cell suspensions decreased from 0.25 to 0.12 d⁻¹ as inoculum concentration was reduced from 4.0 to 0.02 g fresh weight per liter. In contrast, roots show an increase in growth rate from 0.24 to 0.43 d⁻¹. These contrasting growth patterns can be explained as the result of: a) the high specific surface area of cells as compared to roots and, b) the differentiated structure of roots. The dispersed nature of cell suspensions makes them more prone to leakage of key growth factors/cellular contents to medium. The results of this work indicate that cell cultures require substantially higher inoculum concentrations. In contrast, roots can be inoculated at very low concentrations. These facts imply that whereas seed vessels must be employed by cell suspensions, their use for root cultures is a compromise between an easier handling of an entwined root mass and the reduction of the contamination risk of large medium volumes.     

Coordinate regulation of contractile protein synthesis during myoblast differentiation

The synthesis of contractile proteins has been studied during the differentiation of quail skeletal muscle myoblasts in culture. Myoblast differentiation was synchronized by transferring secondary cultures of rapidly dividing myoblasts into medium lacking cell division-promoting factors. Cultures at various stages of differentiation were then pulse-labeled with 35S-methionine, and cell extracts were resolved by electrophoresis on two-dimensional gels. Incorporation into specific proteins was quantitated by autoradiography and fluorography using a scanning densitometer. Contractile proteins synthesized by muscle cultures were identified by their co-electrophoresis on two-dimensional gels with contracile proteins purified from quail breast muscle. Our results show that the synthesis of myosin heavy chain, two myosin light chains, two subunits of troponin and two subunits of tropomyosin is first detected at the time of myoblast fusion and then rapidly increase at least 500 fold to maximum rates which remain constant in muscle fibers. Both the kinetics of activation and the molar rates of synthesis of these contractile proteins are virtually identical. Muscle-specific actin (alpha) synthesis also increases at the time of myoblast fusion, but this actin (alpha) is synthesized at 3 times the rate of other contractile proteins. The synthesis of 30 other muscle cell proteins was quantitated, and most of these are shown to follow different patterns of regulation. From these results, we conclude that the contractile proteins are regulated coordinately during myoblast differentiation.     

Mathematical description of geometric and kinematic aspects of smooth muscle plasticity and some related morphometrics.

Despite considerable investigation, the mechanisms underlying the functional properties of smooth muscle are poorly understood. This can be attributed, at least in part, to a lack of knowledge about the structure and organization of the contractile apparatus inside the muscle cell. Recent observations of the plasticity of smooth muscle and of morphometry of the cell have provided enough information for us to propose a quantitative, although highly simplified, model for the geometric arrangement of contractile units and their collective kinematic functions in smooth muscle, particularly airway smooth muscle. We propose that, to a considerable extent, contractile machinery restructures upon activation of the muscle and adapts to cell geometry at the time of activation. We assume that, under steady-state conditions, the geometric arrangement of contractile units and the filaments within these units determines the kinematic characteristics of the muscle. The model successfully predicts the results of experiments on airway smooth muscle plasticity relating to maximal force generation, maximal velocity of shortening, and the variation of compliance with adapted length. The model is also concordant with morphometric observations that show an increase in myosin filament density when muscle is adapted to a longer length. The model provides a framework for design of experiments to quantitatively test various aspects of smooth muscle plasticity in terms of geometric arrangement of contractile units and the muscle's mechanical properties.     

Amino acids and peptides. Part 39: a bivalent poly(ethylene glycol) hybrid containing an active site (RGD) and its synergistic site (PHSRN) of fibronectin

Fibronectin contains the active sequence Arg-Gly-Asp (RGD), along with its synergic site Pro-His-Ser-Arg-Asn (PHSRN). However, the PHSRN peptide does not show synergic activity when it is mixed with the RGD peptide, indicating that a spatial array between RGD and PHSRN in fibronectin may be necessary for synergic activity. Here, we have used an amino acid type poly(ethylene glycol) derivative (aaPEG) to design a bivalent PEG hybrid of fibronectin active peptides. We prepared the aaPEG hybrid peptides PHSRN-aaPEG, aaPEG-RGD, and PHSRN-aaPEG-RGD, and tested their biological activity. Whereas aaPEG-RGD promoted cell spreading activity, PHSRN-aaPEG had no activity. The PHSRN-aaPEG-RGD hybrid strongly promoted cell spreading compared with aaPEG-RGD. These results suggest that the PHSRN sequence in the PHSRN-aaPEG-RGD molecule synergistically enhances the cell spreading activity of the RGD sequence, and that the bivalent aaPEG hybrid method may be useful for conjugating functionally active peptides.     

Cholinergic mechanisms of the hypothalamus medial preoptic area in control of thermoregulation and of wakefulness-sleep states in pigeons

The data obtained show that cholinergic mechanisms of the medial preoptic area of hypothalamus participate in control of wakefulness-sleep states and thermoregulation parameters in pigeons. Muscarinic and nicotinic cholinergic receptors are established to be involved in the wakefulness maintenance. The muscarinic cholinergic receptor activation of the medial preoptic area is accompanied by an elevation of the brain temperature, by development of peripheral vasoconstriction, and by an in increase in level of the muscle contractile activity. During the nicotinic cholinergic receptor activation of the area, a decrease in the brain temperature and an increase in level of the muscle contractile activity are found. A comparative analysis of experiments and early investigation suggests that during the cholinergic receptors activation changes in the brain temperature of pigeons depend on type of the cholinergic receptors but not on their localization in the preoptic area of hypothalamus.     

Contractile and Mechanical Properties of Epithelia with Perturbed Actomyosin Dynamics

Mechanics has an important role during morphogenesis, both in the generation of forces driving cell shape changes and in determining the effective material properties of cells and tissues. Drosophila dorsal closure has emerged as a reference model system for investigating the interplay between tissue mechanics and cellular activity. During dorsal closure, the amnioserosa generates one of the major forces that drive closure through the apical contraction of its constituent cells. We combined quantitation of live data, genetic and mechanical perturbation and cell biology, to investigate how mechanical properties and contraction rate emerge from cytoskeletal activity. We found that a decrease in Myosin phosphorylation induces a fluidization of amnioserosa cells which become more compliant. Conversely, an increase in Myosin phosphorylation and an increase in actin linear polymerization induce a solidification of cells. Contrary to expectation, these two perturbations have an opposite effect on the strain rate of cells during DC. While an increase in actin polymerization increases the contraction rate of amnioserosa cells, an increase in Myosin phosphorylation gives rise to cells that contract very slowly. The quantification of how the perturbation induced by laser ablation decays throughout the tissue revealed that the tissue in these two mutant backgrounds reacts very differently. We suggest that the differences in the strain rate of cells in situations where Myosin activity or actin polymerization is increased arise from changes in how the contractile forces are transmitted and coordinated across the tissue through ECadherin-mediated adhesion. Altogether, our results show that there is an optimal level of Myosin activity to generate efficient contraction and suggest that the architecture of the actin cytoskeleton and the dynamics of adhesion complexes are important parameters for the emergence of coordinated activity throughout the tissue.     

Requirement for diacylglycerol and protein kinase C in HeLa cell-substratum adhesion and their feedback amplification of arachidonic acid production for optimum cell spreading.

Release of arachidonic acid (AA) and subsequent formation of a lipoxygenase (LOX) metabolite(s) is an obligatory signal to induce spreading of HeLa cells on a gelatin substratum (Chun and Jacobson, 1992). This study characterizes signaling pathways that follow the LOX metabolite(s) formation. Levels of diacylglycerol (DG) increase upon attachment and before cell spreading on a gelatin substratum. DG production and cell spreading are insignificant when phospholipase A2 (PLA2) or LOX is blocked. In contrast, when cells in suspension where PLA2 activity is not stimulated are treated with exogenous AA, DG production is turned on, and inhibition of LOX turns it off. This indicates that the formation of a LOX metabolite(s) from AA released during cell attachment induces the production of DG. Consistent with the DG production is the activation of protein kinase C (PKC) which, as with AA and DG, occurs upon attachment and before cell spreading. Inhibition of AA release and subsequent DG production blocks both PKC activation and cell spreading. Cell spreading is also blocked by the inhibition of PKC with calphostin C or sphingosine. The inhibition of cell spreading induced by blocking AA release is reversed by the direct activation of PKC with phorbol ester. However, the inhibition of cell spreading induced by PKC inhibition is not reversed by exogenously applied AA. In addition, inhibition of PKC does not block AA release and DG production. The data indicate that there is a sequence of events triggered by HeLa cell attachment to a gelatin substratum that leads to the initiation of cell spreading: AA release, a LOX metabolite(s) formation, DG production, and PKC activation. The data also provide evidence indicating that HeLa cell spreading is a cyclic feedback amplification process centered on the production of AA, which is the first messenger produced in the sequence of messengers initiating cell spreading. Both DG and PKC activity that are increased during HeLa cell attachment to a gelatin substratum appear to be involved. DG not only activates PKC, which is essential for cell spreading, but is also hydrolyzed to AA. PKC, which is initially activated as consequence of AA production, also increases more AA production by activating PLA2.     

Zyxin-mediated Actin Assembly Is Required for Efficient Wound Closure

Cytoskeletal regulation of cell adhesion is vital to the organization of multicellular structures. The focal adhesion protein zyxin emerged as a key regulator of actin assembly because zyxin recruits Enabled/vasodilator-stimulated phospho-proteins (Ena/VASP) to promote actin assembly. Zyxin also localizes to the sites of cell-cell adhesion and is thought to promote actin assembly with Ena/VASP. Using shRNA targeted to zyxin, we analyzed the roles of zyxin at adhesive contacts. In zyxin-deficient cells, the actin assembly at both focal adhesion and cell-cell adhesion was limited, but their migration rate was unchanged. Cell spreading on E-cadherin-coated surfaces and the formation of cell clusters were slower for zyxin-deficient cells than wild type cells. By ablating a single cell within a cell monolayer, we quantified the rate of wound closure driven by a contractile circumferential actin ring. Zyxin-deficient cells failed to recruit VASP to cell-cell junctions at the wound edge and had a slower wound closure rate than wild type cells. Our results suggest that, by recruiting VASP, zyxin regulates actin assembly at the sites of force-bearing cell-cell adhesion.     

Study of cardio- and hemodynamic reactions to administration of GABA into the neuronal structures of the ventrolateral area of the medulla oblongata of cats

The data obtained in acute experiments on cats have indicated that the most GABA-sensitive injected neuronal structures in the ventrolateral medulla appear to be located in the vicinity to the roots of the XII cranial nerves. GABA injected into these structures just rostrally to the roots induced hypotensive reactions due to heart rate and myocardial contractile activity decrease, besides inhibition of sympathetic vasoconstrictor influences. GABA injected into the neuronal structures in the caudal ventrolateral medulla induced hypertensive reactions, to a large extent due to increase in heart rate and contractile activity of myocardium.     

Anti-adhesive molecules of the extracellular matrix.

The prototype extracellular matrix glycoproteins had been identified on the basis of their activity in promoting cell adhesion and spreading. Recently, more and more evidence is accumulating that the reverse effect of extracellular matrix proteins, namely the inhibition of cell adhesion and spreading, may be equally important for proper cell function during morphogenesis and development. Several anti-adhesive proteins have been described and their mechanisms of action are being investigated.