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Biomedical subjects

R H Racusen

Publications and source records attributed to R H Racusen.

12 recordsLinked to original sources
PubMed1991

Regeneration of the root pole in surgically transected carrot embryos occurs by position-dependent, proximodistal replacement of missing tissues.

Torpedo-stage carrot embryos were surgically transected at various locations along the shoot-root axis and explants of the cotyledon-bearing shoot pole were sectioned and examined in order to provide a more detailed description of root pole regeneration. When excisions occurred at the sites of the future hypocotyl, the future radicle or the future root apical meristem, the regenerating axial tissues exhibited patterns of cellular organization that were nearly identical to those seen in unsevered controls. To accomplish this restoration, new cells, of the type normally found at each cutting site, were produced behind a regeneration dome that formed over the original surgical site. The regeneration dome was displaced by division and expansion-driven extension of the longitudinal axis, and cells in the renewed region quickly acquired individual anatomical traits and collective tissue morphologies that corresponded to those of cells in the analogous locations in intact embryos. Although no clear mechanism is implied, the results of these experiments suggest that interactions between cells near the surgical margin permit them to retain their sense of location within the original structure, and apprise them of the removal of their basipetally positioned neighbors. With varying-length remnants of the shoot serving as the only vestige of the original size and shape of the embryo, cells close to the site of excision were apparently reconfigured to commence ordered divisions that ultimately reconstituted the embryonic axis.

Microscopy, Electron↗
PubMed1990

Microscale, filtration-type binding assay for studying myosin-erythrocyte protein 4.1 interactions.

In vitro binding of skeletal muscle myosin and the erythrocyte cytoskeleton linker protein, band 4.1, was evaluated in a novel small-volume, filtration-based binding assay. The assay equipment consisted of a plastic grid containing several buffer-filled wells into which were placed small nylon screens. Myosin was covalently tethered to an agarose (Sepharose) support and aliquots of this resin were pipetted onto the surface of the submerged nylon screen. Following addition of radiolabeled protein 4.1, and an appropriate incubation period, the myosin-Sepharose beads and bound protein 4.1 were separated by wicking the buffer from beneath the nylon screen with a piece of filter paper. Nylon screens, with adherent resin beads, and the filter paper wicks were then counted to give the amounts of bound and free protein 4.1, respectively. This system proved to be a rapid, simple, and quantitative method for evaluating the behavior of a myosin binding protein under conditions in which free myosin would be prone to assemble into filaments. Moreover, since the assay separates bound and free components within a few seconds, it is well suited for the analysis of low-affinity interactions.

Cytoskeletal Proteins↗
PubMed1990

Microsurgery reveals regional capabilities for pattern reestablishment in somatic carrot embryos.

The extent to which regions of a somatic embryo were committed to a particular developmental fate was explored by surgically removing portions of somatic embryos and observing patterns of regeneration. Through a variety of excisions that resulted in tissue slices ranging from less than 10% to nearly 90% of the original embryo mass, we observed only a few cases where such isolates completely abandoned preexisting patterns of organized growth. Instead, most subcultured portions of the embryonic axis restored all, or part of, a missing complement of the organism. At the shoot apex, a single lost cotyledon was replaced by new cotyledonary structures, although these usually occurred as multiple pairs of cotyledons. If both cotyledons were removed, secondary axes, each with its own cotyledons, typically formed at the embryo midlength. When embryos were divided into shoot and root pieces, the shoot pole usually regenerated a new root, while the original root and rapidly elongated and matured days earlier than uncut controls. Surprisingly, cotyledon regeneration from excised root sections occurred at much greater frequency when the root piece comprised only 10-25% of the embryo mass; larger portions of the root pole rarely produced recognizable shoot structures. These studies indicate that several discrete regions of the embryo are committed to specific types of patterned growth, and that continuity between certain of these regions is required for the maintenance of axial polarity.

Cells, Cultured↗
PubMed1990

Positional cues and differential gene expression in somatic embryos of higher plants.

Much of the organization of higher vascular plants is determined during the formation of the embryo. In addition to the zygotic embryo which results from sexual fertilization in the ovule, many plants are capable of producing embryos from somatic cells. Of particular interest to plant developmental biologists is the phenomenon of somatic embryogenesis in cultures of the domesticated carrot which, because of its tractable nature in experimental manipulations, is presently regarded as a suitable model for studying pattern formation in plants. This short review considers the state of our knowledge concerning the origin and perception of positional information in plant embryos, and the temporal and spatial expression of genes. The available data provide a number of promising leads for cell-cell interactions in embryos, and there are some clear indications that the spatial distribution of certain gene products is correlated with changes in morphology. However, there is, as yet, insufficient evidence with which to forge a link between positional cues and the expression of genes which influence developmental transitions in embryos.

Cell Differentiation↗
PubMed1989

Erythrocyte protein 4.1 binds and regulates myosin.

Myosin was recently identified in erythrocytes and was shown to partition both with membrane and cytosolic fractions, suggesting that it may be loosely bound to membranes [Fowler, V. M., Davis, J. Q. & Bennett, V. (1985) J. Cell Biol. 100, 47-55, and Wong, A. J., Kiehart, D. P. & Pollard, T. D. (1985) J. Biol. Chem. 260, 46-49]; however, the molecular basis for this binding was unclear. The present studies employed immobilized monomeric myosin to examine the interaction of myosin with erythrocyte protein 4.1. In human erythrocytes, protein 4.1 binds to integral membrane proteins and mediates spectrin-actin assembly. Protein 4.1 binds to rabbit skeletal muscle myosin with a Kd = 140 nM and a stoichiometry consistent with 1:1 binding. Heavy meromyosin competes for protein 4.1 binding with Ki = 36-54 nM; however, the S1 fragment (the myosin head) competes less efficiently. Affinity chromatography of partial chymotryptic digests of protein 4.1 on immobilized myosin identified a 10-kDa domain of protein 4.1 as the myosin-binding site. In functional studies, protein 4.1 partially inhibited the actin-activated Mg2+-ATPase activity of rabbit skeletal muscle myosin with Ki = 51 nM. Liver cytosolic and erythrocyte myosins preactivated with myosin light-chain kinase were similarly inhibited by protein 4.1. These studies show that protein 4.1 binds, modulates, and thus may regulate myosin. This interaction might serve to generate the contractile forces involved in Mg2+-ATP-dependent shape changes in erythrocytes and may additionally serve as a model for myosin organization and regulation in non-muscle cells.

Animals↗
PubMed1988

Modifications of extracellular electric and ionic gradients preceding the transition from tip growth to isodiametric expansion in the apical cell of the fern gametophyte.

Fern (Onoclea sensibilis L.) gametophytes exposed to blue light are induced to undergo a morphological transition from a tip-growing filament to a planar prothallus. Extracellular measurements of electric currents and localized ion activities around the apical cell of 8 to 10 day-old gametophytes were made with a vibrating probe and ion selective electrodes. In darkness, we observed exit current densities of an average of 75 nanoamperes per square centimeter near the tip and 2 to 15 nanoamperes per square centimeter along the lateral walls of this cell. Measurements with ion selective electrodes for H(+), K(+), and Ca(2+) showed that this cell was bounded by a thin layer of medium that was depleted in K(+) and Ca(2+) and exhibited a lower pH than the bulk solution. Both the K(+) and Ca(2+) depletion zones and the zone of higher acidity were particularly pronounced at the tip end of the cell; the pH at 2 micrometers from the tip was nearly 0.5 units more acid than the bulk medium at pH 6. Disruption of steady state, external gradients with media that contained lower concentrations of H(+), K(+), Ca(2+), or Cl(-) produced certain differences in the rates of restoration of particular ion zones, raising the possibility that some of the ion migrations are interdependent. Within 15 minutes after irradiation with blue light, current leaving the tip declined to levels which were indistinguishable from those leaving the lateral walls and there was a rapid lowering in the rates of tip acidification and K(+) depletion near the tip. The rapid dissipation of both the longitudinally aligned electrical field and the tip-localized asymmetries in external cation distribution in blue light suggest that loss of electrical polarity in this tip growing cell may be an initial step in the chain of events which govern changes in cell shape.

Journal Article↗
PubMed1986

The role of electrical phenomena in tip growth, with special reference to the developmental plasticity of filamentous fern gametophytes.

Cell expansion in many plant structures, including algal rhizoids, fungal hyphae, root hairs, and pollen tubes, is restricted to their apical tips. Endogenous electric fields are seen to accompany polarized growth in all tip-growing cells studied to date. The extensive studies on absorptive tip-growing structures have established that positive currents enter their elongating tips, with a portion of the entry current being carried by a localized calcium influx into the extreme tip. The resulting tip-to-base gradient in calcium concentration appears to be responsible for maintaining polarized growth in these systems, although it is uncertain whether this calcium effect is mediated via either electrophoretic or cytoskeletal mechanisms. In contrast, the few electrical measurements made on photosynthetic cells suggest that the orientation of their transcellular fields is transiently or permanently reversed relative to the fields in absorptive structures. In darkness, microelectrode measurements indicate that the apical tip of the fern filament is 5 mV electronegative relative to the base of the apical cell. This cellular dipole is perceived with the vibrating probe as a focused outward current that departs from the tip region and a more diffuse inward current that enters the lateral sides of the apical cell. The tip current is predominantly composed of protons, as can be identified with various cation-selective electrodes. This proton current is thought to help maintain localized wall expansion in the filament tip. Blue light mediates the major morphogenetic transition in fern gametophytes, i.e. the transition from the tip-growing filament to the planar prothallus. All the above electrical and ionic parameters change in the few minutes of irradiation before the filament tip starts lateral swelling. The plasma membrane at the extreme tip begins to hyperpolarize within 3 s, while the basal region shows a delayed, but greater response. The cellular dipole that had existed in darkness is thus abolished in 10 to 15 min after the start of irradiation. With the vibrating probe a more diffuse pattern of positive currents is observed to emerge from the tip as well as the subapical regions of the apical cell. Simultaneously, proton efflux increases in the subapical region; the resulting decrease in cell wall pH should help plasticize the lateral walls, which may, in turn, facilitate the process of lateral swelling over the next few hours.(ABSTRACT TRUNCATED AT 400 WORDS)

Electric Conductivity↗
PubMed1983

Membrane-associated ATPases in isolated secretory vesicles.

Polysaccharide-containing vesicles were collected from secretory cells maintained in liquid culture. Characterization of membrane-associated nucleosidephosphatases revealed that the vesicles specifically hydrolyze ATP, have a pH optimum between 6.0 and 6.5, and are stimulated by inorganic cations, especially K(+). The ATPase activity in these vesicles was inhibited by orthovanadate and N,N'-dicyclohexylcarbodiimide; other inhibitors, such as oligomycin, sodium azide, and diethylstilbestrol were generally ineffective. Results from these studies are consistent with the notion that vesicles derived from the Golgi apparatus have partially differentiated into plasmalemma before they fuse with the plasma membrane.

Journal Article↗
PubMed1982

Electrical Changes in the Apical Cell of the Fern Gametophyte during Irradiation with Photomorphogenetically Active Light.

Electrophysiological procedures were used to evaluate cellular responses of fern (Onoclea sensibilis L.) gametophytes to photomorphogenetically active light. Red, far red, and blue light caused rapid changes in the membrane potential of the apical cell of the gametophyte filament; other cells in the filament were not similarly responsive. Measurements made with one electrode in the apical cell revealed that the membrane potential depolarized in red light and repolarized in far red light. Irradiation with blue light caused a hyperpolarization, the rapidity of which was dependent on a red light pretreatment. More refined measurements with one electrode in the tip of the apical cell and another in the base of the cell showed that both red and blue light treatments cause the apical cell to behave as a dipole. Because of the profound, long-term morphological changes that follow light irradiation in this organism, it was hoped to use it to elucidate the role that electrical parameters play in determining subsequent developmental events.

Journal Article↗
PubMed1980

Phytochrome Modifies Blue-light-induced Electrical Changes in Corn Coleoptiles.

Unilateral blue light administered to corn coleoptile segments produces no alteration of transmembrane potential on the light side, and only a small and slow hyperpolarization on the dark side. Red light causes a 5-15 millivolt depolarization in cells on the light side causes and somewhat smaller effects on the dark side. Blue given after red causes a rapid hyperpolarization on both sides of the coleoptile. The effect of the potentiating red preirradiation is probably due to phytochrome, being largely abolished by far-red given after red, but before the blue light. The effect of prior red irradiation decays in the dark, showing a half-time of about 45 minutes at room temperature. This rapid cooperativity between phytochrome and the phototropic pigment may indicate a common locale, possibly in a membrane.

Journal Article↗
PubMed1975

Role of Membrane-bound, Fixed-charge Changes in Phytochrome-mediated Mung Bean Root Tip Adherence Phenomenon.

The movement of cells and cell fragments in an electric field provided a means for determining the nature of cellular surface charges. We found that changes in ionic strength and particularly changes in Ca(2+) and H(+) in the bathing medium cause changes in the surface charges on the root cap cells in the absence of red light. Red light-induced charge changes are demonstrable only on root cap cells and are reversible with far red light. By osmotically separating the membrane from the wall, we demonstrated that both light-induced and ionically mediated charge changes are associated with the cell membrane and not the cell wall.

Journal Article↗