Diverse effects of fibronectin and laminin on phenotypic properties of cultured arterial smooth muscle cells.

Plasma fibronectin promotes modulation of rat arterial smooth muscle cells from a contractile to a synthetic phenotype during the first few days in primary culture. This process includes cell adhesion and spreading, loss of myofilaments, and formation of a widespread rough endoplasmic reticulum and a prominent Golgi complex. The structural reorganization is accompanied by activation of overall RNA and protein synthesis. Moreover, the cells gain the ability to replicate their DNA and divide in response to platelet-derived growth factor. Here, it is demonstrated that the power of fibronectin to bring about this change in the differentiated properties of the smooth muscle cells resides in a 105-kD cell-binding fragment, whereas a 70-kD collagen-binding fragment and a 31-kD heparin-binding fragment are inactive in this respect. Laminin, another adhesive glycoprotein and a component of the basement membrane that normally surrounds arterial smooth muscle, was contrarily found to maintain the cells in a contractile phenotype. However, with increasing time more and more cells went through the modulation into a synthetic phenotype. This "catch-up" was counteracted by a peptide that contained the cell-attachment sequence of fibronectin (Arg-Gly-Asp-Ser). Hence, it is possible that the delayed modulation on laminin was due to production of fibronectin by the cells themselves. In support of this notion, fibronectin isolated from smooth muscle cultures was found to be as effective as plasma fibronectin in stimulating the phenotypic modulation. Moreover, using a combination of chemical, immunochemical, and immunocytochemical methods, it was demonstrated that the cells secreted fibronectin as well as laminin at an increasing rate during the first 4 d in primary culture and, notably, cells cultured on laminin produced more fibronectin than cells cultured on fibronectin. Newly synthesized fibronectin was incorporated into a network of pericellular and intercellular fibrils, whereas laminin formed a more diffuse layer covering the cells in a basement membrane-like manner. Taken together, the findings suggest diverse roles for fibronectin and laminin in the control of the differentiated properties of arterial smooth muscle cells. They further indicate that the ability of arterial smooth muscle cells to produce fibronectin and laminin early in primary culture is not directly related to the phenotypic state as determined morphologically and by measurement of overall rates of RNA and protein synthesis. This may be due to the cells being able to sense the macromolecular composition of the pericellular matrix and to modify their secretory activity accordingly.(ABSTRACT TRUNCATED AT 400 WORDS)

Phycomyces: modification of spiral growth after mechanical conditioning of the cell wall.

Mature stage IVb Phycomyces sporangiophores show left-handed spiral growth; that is, viewed from above, the sporangium rotates clockwise. Mechanical conditioning of the cell wall by the Instron technique increases the ratio of the rotational to the elongational growth rate. This result is in agreement with the fibril reorientation model of spiral growth, which suggests that cell wall microfibrils, initially oriented in a nearly transverse right-handed direction in the upper region of the growing zone, are displaced during growth toward the longitudinal axis, causing the observed left-handed spiral growth.

Multiphase-field approach for multicomponent alloys with extrapolation scheme for numerical application.

A multiphase-field model previously proposed by the authors is reformulated in a thermodynamically consistent form and extended to multicomponent systems. The phase-field and diffusion equations, derived from a free energy functional, are compared to those postulated in the previous model in the limit of a binary alloy. The constraint of local quasiequilibrium, which is equivalent to the postulate of equal diffusion potentials for coexisting phases, is deduced from a variational principle. Solute partitioning and evaluation of the thermodynamic driving force for phase transformation are done by numerical minimization of the free energy of the multiphase system using the Calphad approach. A local extrapolation scheme which enhances the computational efficiency for complex numerical simulations of technical alloys is presented. It is shown that this extrapolation scheme, used in a "multibinary" approximation, reproduces the former model without restriction to dilute solutions.

Phycomyces: irregular growth patterns in stage IVb sporangiophores.

Net rotation and net elongation of a stage IVb Phycomyces growing zone were simultaneously measured minute by minute with a photographic apparatus coupled with a rotating stage. A direct correlation between a growth response and a twist response after either a light stimulus or a house stimulus was found. There were significant irregularities in growth rate in both the elongation and rotation that were not a result of measurement error; these irregularities were poorly, if at all, correlated. We believe that these fluctuations reflect the underlying molecular mechanism of cell wall synthesis.

Avoidance and rheotropic responses in phycomyces. Evidence for an 'avoidance gas" mechanism.

If a mature sporangiophore is placed next to a barrier that is moving in a clockwise direction, it grows both away from the barrier and into the wind; the wind is generated by the moving barrier itself. When the barrier is moving in a counterclockwise direction, the sporangiophore grows towards both the barrier and the wind. The net direction of growth appears to be the vector sum of the rheotropic response and the avoidance aiming error and does not involve the classic stationary-barrier avoidance response. Our experiments all support the suggestion that the avoidance response, the rheotropic response and the variety of reported wind responses can be explained by the presence of a self-emitted, growth-simulating avoidance gas. We present data that suggest that it is the direction of the net flux (mass transfer) of this gas that determines both the direction and the magnitude of the sporangiophore growth. We further suggest that the region of the cell wall showing maximum mass transfer will show a minimum growth rate, i.e., the direction of growth will always be in the direction of maximum transfer. If water is the avoidance gas, then it would follow that the total hydration of the cell wall in an aqueous salt solution should result in cell wall softening; cell wall softening has been correlated directly to cell wall growth. Using the Instron technique, we now show that submerging the entire sporangiophore in an aqueous salt solution for 4 min causes an increase in cell wall extensibility.

Phycomyces: detailed analysis of the anemogeotropic response.

Stage IVb sporangiophores of Phycomyces grow into the wind--the anemotropic response--and away from gravity--the geotropic response. A procedure has been designed to measure the equilibrium bend angle that results when the two stimuli are given simultaneously over a long period of time. This angle will be referred to as the anemogeotropic equilibrium angle. This measurement of a sensory response is analogous to the photogeotropic equilibrium angle in which the variable stimulus is light instead of wind. We have found that the anemogeotropic angle, measured relative to the vertical, increases with both increasing wind speed and increasing relative humidity of the wind stimulus. This finding is new and argues against a major prediction of the mass transfer model that anemogeotropism and relative humidity are inversely related. Data from these anemogeotropic experiments further suggest that the self-emitted gas responsible for both the anemotropic response and the avoidance response is water.

Oxaliplatin pharmacokinetics during a four-hour infusion.

A new platin compound, oxaliplatin, has significant activity in advanced colorectal carcinomas. However, its pharmacokinetics have not been characterized adequately yet. This study extensively analyzes the pharmacokinetics of both ultrafiltrable (free) and protein-bound platin in 13 patients receiving 130 mg/m2 oxaliplatin as a 4-h infusion in combination with 375 mg/m2 5-fluorouracil as a 24-h infusion for advanced colorectal carcinomas. The interpatient variability was very low for all parameters analyzed. The levels of free platin decreased triphasically, with a mean terminal half-life of 27.3+/-10.6 h. The area under the time-concentration curve was 20.17+/-6.97 microg.h/ml and the total and renal clearances amounted to 222+/-65 and 121+/-56 ml/min, respectively. The values for the volume of distribution and for the maximum concentration at the end of infusion were 349+/-132 liters and 1612+/-553 ng/ml, respectively. On the basis of the simulation of the plasma levels and the urinary excretion of platin following the long-term administration of oxaliplatin as a constant-rate and a chronomodulated infusion, additional analyses are warranted to fully characterize the pharmacokinetics of the drug in these settings.

Transcellular labeling of taste bud cells by carbocyanine dye (DiI) applied to peripheral nerves in the barbels of the catfish, Ictalurus punctatus.

In order to study the pattern of innervation of taste buds and the surrounding epithelium, the carbocyanine dye diI was applied to the nerve stump in isolated, paraformaldehyde-fixed barbels obtained from channel catfish, Ictalurus punctatus. After a diffusion period of 7-41 days, the barbels were sectioned on a vibratome and examined with epifluorescence. Labeled axons were observed up to 1 cm from the site of application. Frequently, a fascicle of labeled axons turned outward toward the epithelium to innervate taste buds or to end apparently as free endings within the epithelium. Within 2-3 mm of the dye-application site, many taste buds contained one or at most 5-10, labeled spindle-shaped, presumed receptor, cells. In taste buds containing multiple labeled cells, the cells usually were arranged as intertwined pairs or triplets rather than being homogeneously distributed within the taste bud. In a few cases, labeled basal cells could be discerned among the labeled axons of the basal plexus. The cells of the taste bud apparently were labeled by transcellular passage of the dye from the nerve fibers into the cells. The limited number of labeled cells within each taste bud may indicate a special relationship between these cells and the nerve fibers innervating them.

Peripheral peptidergic fibers of the trigeminal nerve in the olfactory bulb of the rat.

Axons immunoreactive for calcitonin gene-related peptide (CGRP) and substance P are present in the olfactory nerve, although few, if any, olfactory receptor cells contain immunocytochemically detectable levels of these peptides. The possible trigeminal origin of these fibers was tested by performing unilateral stereotaxic lesions of the ophthalmic division of the trigeminal nerve, followed 2-25 days later by immunocytochemistry for CGRP and substance P. As reported previously, free nerve endings immunoreactive for both peptides were found transversing the nasal epithelium on the unlesioned side. Also on the unlesioned side, peptidergic axons, immunoreactive for both CGRP and substance P, could be traced from the olfactory nerve into the glomerular layer throughout the olfactory bulb, but especially into its rostral third. Ipsilateral to the trigeminal ganglion lesion, such peptide-immunoreactive fibers were absent or markedly reduced in the bulb, nerve, and epithelium. These results indicate that the peripheral branches of the ophthalmic branch of the trigeminal nerve enter the olfactory bulb along with the olfactory nerve and terminate in the glomerular layer along with the olfactory axons. Ultrastructural analysis of the CGRP-immunoreactive terminals in the glomeruli reveal vesicle-filled axonal processes terminating in the absence of obvious pre- or postsynaptic specializations. Whether the trigeminal fibers in the bulb are functional, e.g., convey information to the olfactory bulb via an axon reflex, or relay information from the olfactory bulb to the brainstem trigeminal nuclei is unclear.

Kainate-activated cobalt uptake in the primary gustatory nucleus in goldfish: visualization of the morphology and distribution of cells expressing AMPA/kainate receptors in the vagal lobe.

Gustatory afferent fibers of the vagus nerve that innervate taste buds of the oropharynx of the goldfish, Carassius auratus, project to the vagal lobe, which is a laminated gustatory nucleus in the dorsal medulla. As in the mammalian gustatory system, responses by second-order cells in the goldfish medulla are mediated by N-methyl-D-aspartate (NMDA) and non-NMDA ionotropic glutamate receptors. We utilized a cobalt uptake technique to label vagal lobe neurons that possess cobalt-permeable ionotropic glutamate receptors. Vagal lobe slices were bathed in kainate (40 microM) or glutamate (0.5 or 1 mM) in the presence of CoCl(2), which can pass into cells through the ligand-gated cation channels of non-NMDA receptors made up of certain subunit combinations. Cobalt-filled cells and dendrites were observed in slices that were activated by kainate or glutamate, but not in control slices that were bathed in CoCl(2) alone, nor in slices that were bathed with the non-NMDA receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (10 microM) in addition to an agonist. Likewise, simple depolarization of the cells with KCl failed to induce cobalt loading. Cobalt-filled round unipolar cells, elongate or globular bipolar cells, and multipolar cells with elongate or polygonal perikarya were distributed throughout the cell layers in the sensory zone of the vagal lobe. Numerous labeled neurons had dendrites spanning layers IV and VI, the two principal layers of primary afferent input. Apical and basal dendrites often extended radially through neighboring laminae, but many cells also extended dendrites tangential to the lamination of the sensory zone. In the motor layer, cell bodies and proximal dendrites of small, multipolar neurons, and large motoneurons were regularly loaded with cobalt.

"Type III" cells of rat taste buds: immunohistochemical and ultrastructural studies of neuron-specific enolase, protein gene product 9.5, and serotonin.

Taste buds contain a variety of morphological and histochemical types of elongate cells. Serotonin, neuron-specific enolase (NSE), ubiquitin carboxyl terminal hydrolase (PGP 9.5), and neural cell adhesion molecule (N-CAM) all have been described as being present in the morphologically defined Type III taste cells in rats. In order to determine whether these substances coexist in a single cell, we undertook immunohistochemical and ultrastructural analysis of taste buds in rats. Double-label studies show that PGP 9.5 and NSE always colocalize. In contrast, PGP 9.5 and serotonin seldom colocalize. Further, whereas the serotonin-immunoreactive cells are always slender and elongate, the PGP 9.5/NSE population comprise two morphological types--one slender, the other broader and pyriform. Although gustducin-immunoreactive taste cells appear similar in overall shape to the pyriform PGP 9.5/NSE population, gustducin never colocalizes with PGP 9.5 or NSE. The serotonin-immunoreactive taste cells have an invaginated nucleus, synaptic contacts with nerve fibers, and taper apically to a single, large microvillus. These are all characteristics of Type III taste cells described previously in rabbits (Murray [1973] Ultrastructure of Sensory Organs I. Amsterdam: North Holland. p 1-81). PGP 9.5-immunoreactive taste cells exhibit two morphological varieties. One type is similar to the serotonin-immunoreactive population, containing an invaginated nucleus, synapses with nerve fibers, and a single large microvillus. The other type of PGP 9.5-immunoreactive taste cell has a large round nucleus and the apical end of the cell tapers to a tuft of short microvilli, which are characteristics of Type II taste cells. Thus, in rats, some Type III cells accumulate serotonin but do not express PGP 9.5, whereas others express PGP 9.5 but do not accumulate amines. Similarly, Type II taste cells come in at least two varieties: those immunoreactive for gustducin and those immunoreactive for PGP 9.5.

Differential localization of putative amino acid receptors in taste buds of the channel catfish, Ictalurus punctatus.

The taste system of catfish, having distinct taste receptor sites for L-alanine and L-arginine, is highly sensitive to amino acids. A previously described monoclonal antibody (G-10), which inhibits L-alanine binding to a partial membrane fraction (P2) derived from catfish (Ictalurus punctatus) taste epithelium, was found in Western blots to recognize a single band, at apparent MW of 113,000 D. This MW differs from the apparent MW for the presumed arginine receptor identified previously by PHA-E lectin affinity. In order to test whether PHA-E lectin actually reacts with the arginine-receptor, reconstituted membrane proteins partially purified by PHA-E affinity were used in artificial lipid bilayers. These reconstituted channels exhibited L-arginine-activated activity similar to that found in taste cell membranes. Accordingly, we utilized the PHA-E lectin and G-10 antibody as probes to differentially localize the L-alanine and L-arginine binding sites on the apical surface of catfish taste buds. Each probe labels numerous, small (0.5-1.0 micron) patches within the taste pore of each taste bud. This observation suggests that each bud is not tuned to a single taste substance, but contains putative receptor sites for both L-arginine and L-alanine. Further, analysis of double-labeled tissue reveals that the PHA-E and G-10 sites tend to be separate within each taste pore. These findings imply that in catfish, individual taste cells preferentially express receptors to either L-arginine or L-alanine. In addition, PHA-E binds to the apices of solitary chemoreceptor cells in the epithelium, indicating that this independent chemoreceptor system may utilize some receptor sites similar to those in taste buds.

Expression of the dopaminergic phenotype in the olfactory bulb: neither calcitonin gene-related peptide nor olfactory input is necessary.

In the olfactory bulb, expression of tyrosine hydroxylase (TH) in juxtaglomerular neurons is dependent on innervation by the olfactory nerve. The presence of the neuropeptide calcitonin gene-related peptide (CGRP) within the olfactory nerve has led to the hypothesis that CGRP is responsible for regulation of TH expression in the bulbar neurons. On the other hand, other investigators claim that olfactory receptors never produce CGRP and that functional contact with olfactory axons regulates production of TH by bulbar neurons. Two different experimental procedures were used to test whether either CGRP or contact with the olfactory nerve is essential for production of TH by bulbar neurons in vivo. The peptidergic innervation of the olfactory bulb was eliminated either by neonatal capsaicin treatment, or by stereotaxic, electrolytic lesions of the ophthalmic division of the trigeminal nerve. Both of the treatments leave the olfactory innervation of the bulb intact while eliminating the CGRP-immunoreactive fibers in the olfactory nerve and glomeruli. Subsequent immunocytochemistry reveals a normal complement of bulbar TH-immunoreactive juxtaglomerular neurons in the absence of peptidergic innervation. In order to test whether olfactory nerve input is necessary for expression of TH in vivo, the anlage of the olfactory bulb was removed from embryonic (E16) rat pups and transplanted into the anterior chamber. These ectopic olfactory bulbs, although devoid of olfactory nerve input, contain numerous TH-immunoreactive neurons. Thus olfactory nerve input is not necessary for expression of TH in bulbar neurons.

Phycomyces: distribution of growth velocities in the growing zone.

Distribution of growth velocities in the growing zone of stage IVb Phycomyces sporangiophores was measured by photographing the growing zone after dusting it with starch grains. When the entire growing zone is fully dark-adapted to red light and then subjected to a saturating white light stimulus, the entire growing zone increases in growth rate. When the growing zone is partially light-adapted, again the entire growing zone responds when subjected to a saturating white light stimulus but to a lesser degree than the fully dark-adapted sporangiophore. Phototropic mutants of class 1 and class 2 show a distribution of growth in the growing zone similar to wild type sporangiophores both during steady-state growth and during light-stimulated growth.

Phycomyces: MODIFICATION OF LIGHT-INDUCED SPIRAL GROWTH AFTER MECHANICAL CONDITIONING OF THE CELL WALL.

Mature stage IVb Phycomyces sporangiophores show left-hand spiral growth; that is, viewed from above, the sporangium rotates clockwise. It has been shown that mechanical conditioning (strain-hardening) of the cell wall by the Instron technique increases the ratio of rotation to the elongation growth rate compared to nonmechanically conditioned controls. It is reported that the addition of a saturating light stimulus to these sporangiophores causes a decrease in the ratio of rotation to elongation growth rate. This result is in agreement with the fibril slippage model, i.e. the counterclockwise rotation of stage IVa is a result of parallel fibrils lying in a right-handed spiral configuration slipping by one another. It is suggested that a light stimulus added to a mechanically conditioned stage IVb sporangiophore activates one or more cell wall-loosening enzymes which act by decreasing the number of intermolecular bonds between parallel fibrils causing fibril slippage, resulting in counterclockwise rotation. It is precisely this counterclockwise contribution that decreases the rotation to elongation growth ratio of mechanically conditioned and then light-stimulated stage IVb sporangiophores.

Phycomyces: discovery of the aiming error in the avoidance response.

It has been determined experimentally that, when a stage IVb Phycomycea sporangiophore is subjected to an asymmetric avoidance stimulus (a single barrier), it uses its own net rotation to convert the spatial stimulus to a temporal one. This finding explains the sporangiophore's nonadaptation to a single-barrier stimulus and adaptation to a double-barrier stimulus.

Chloroquine and monensin inhibit induction of DNA synthesis in rat arterial smooth muscle cells stimulated with platelet-derived growth factor.

The weak base chloroquine and the Na+/H+ ionophore monensin were used to study the role of lysosomes in the induction of DNA synthesis by platelet-derived growth factor (PDGF) in rat arterial smooth muscle cells cultivated in vitro. The results show that PDGF initiates DNA synthesis in a defined, serum-free medium. This indicates that a single factor may control, directly or indirectly, the transition from the G0 to the G1 phase, the progress through the G1 phase, and the entrance into the S phase of the cell cycle. It is further demonstrated that PDGF has to be present throughout most of the prereplicative period (12-16 h) to induce DNA synthesis in the maximum number of cells, suggesting that one or more processes need to be stimulated continually or successively to push the cell into the S phase. Chloroquine and monensin inhibit induction of DNA replication by PDGF, with maximum effect at 50 microM and 5 microM, respectively. To be fully active, the drugs have to be added within 4-8 h after the growth factor, but a partial inhibition persists if they are added at any time during the prereplicative period. Both drugs reduce PDGF-stimulated RNA and protein synthesis, and suppress degradation of [3H]leucine-labeled cellular protein and [125I]-labeled PDGF. Fine-structurally, they give rise to an accumulation of lysosomes or prelysosomal vacuoles with inclusions of incompletely degraded material. These findings suggest that the mitogenic effect of PDGF is dependent on a normal function of lysosomes during the prereplicative phase, especially its first half (0-8 h).

Integrin-type fibronectin receptors of rat arterial smooth muscle cells: isolation, partial characterization and role in cytoskeletal organization and control of differentiated properties.

The spreading of freshly isolated rat arterial smooth muscle cells (SMCs) on a substrate of fibronectin (FN) is associated with marked changes in fine structure and function of the cells, collectively referred to as a modulation from a contractile to a synthetic phenotype. Recent studies have indicated that this process is mediated via an interaction between the minimal cell-attachment sequence of FN (RGDS) and cell surface receptors. Here, we report the isolation of such receptors by sequential chromatography on affinity columns of wheat germ agglutinin (WGA) and a 105-kDa cell-binding fragment of FN (105-kDa fragment). The receptor was composed of two proteins with electrophoretic mobilities in SDS-polyacrylamide gels of 160 and 115 kDa under nonreducing conditions and 150 and 130 kDa under reducing conditions. Immunoprecipitation of surface-labeled cells with a rabbit antiserum against the beta chain of the rat hepatocyte FN receptor similarly yielded two proteins of 160 and 115 kDa. In metabolically labeled cells an additional component of 105 kDa was precipitated, presumably representing a precursor of the 115-kDa protein. Immunocytochemical studies demonstrated that SMCs grown on laminin formed FN fibrils and actin filament bundles in close alignment with cell surface receptors after a few days of culture. In cells seeded on the 105-kDa fragment, the receptors were already arranged in parallel with actin filaments on the first day of culture. Later on, the cells secreted FN and laid down FN fibrils along the receptors on the cell surface and the actin filament bundles in the cytoplasm. Taken together, the findings indicate that arterial SMCs are equipped with FN receptors that belong to the integrin family of proteins and consists of alpha (160-kDa) and beta (115-kDa) subunits. The receptor complexes apparently play an important role in determining the differentiated characteristics of the cells, possibly by mediating a linkage between the extracellular matrix and the cytoskeleton.