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Biomedical subjects

K R Robinson

Publications and source records attributed to K R Robinson.

At least 55 records · Page 3Linked to original sources

The responses of pollen to applied electrical fields.

The growing pollen tubes of tomato and tobacco were exposed to electrical fields and the effect on the direction of growth was measured. They responded by turning toward the positive electrode (anode) and gave a detectable response in fields as small as 0.1 mV/tube diameter. The sites of germination of tobacco pollen grains were also affected by applied fields; the pollen tubes tended to emerge from the anodal side. This effect was detectable at a field of 0.4 mV/grain diameter.

Electricity↗

An endogenous sodium current may mediate wound healing in Xenopus neurulae.

We have studied healing of Xenopus neurulae in order to investigate the role of endogenous sodium-carried electric currents in wound closure. Transected embryos healed completely within 7 hr in an artificial pond water medium. Wound closure was biphasic, with a rapid purse string-like contraction that was independent of sodium concentration followed by a slower sodium-dependent phase. The initial contraction was reversibly inhibited by cytochalasin B. Healing was prevented when neurulae were wounded and left to heal in sodium-free medium. Healing was also prevented in the presence of amiloride, benzamil, or ouabain, drugs that inhibit sodium flux through the epithelium. The transepithelial potential measured in intact neurulae fell rapidly and reversibly by 70% in response to 10 microM amiloride. Currents measured leaving the wound also decreased by 70% following amiloride addition. Our results indicate that at least one phase of wound healing in Xenopus neurulae is dependent upon an endogenous sodium-carried electric current. We hypothesize that the current may act by guiding epithelial cells to the wound site.

Amiloride↗

A refined technique to apply electrical currents to callus cultures.

A simple, inexpensive technique is described which can be used to pass small electrical currents of a few microamperes through callus cultures under sterile conditions without contaminating the tissue or the medium with electrode products. Application of 1 or 2 microamperes currents of either polarity stimulated growth as well as shoot regeneration in tobacco (Nicotiana tabacum var. virginica) callus cultures, indicating that these effects are due to the electrical fields set up by the exogenous currents, rather than by electrode products.

Journal Article↗

A rise in cytosolic calcium is not necessary for maturation of Xenopus laevis oocytes.

Cytoplasmic free calcium levels during progesterone-induced meiotic maturation in Xenopus laevis oocytes were measured using the photoprotein aequorin. The resting level of [Ca2+]i was 92.6 +/- 30 nM. No significant changes were observed after progesterone addition, although a large pulse of [Ca2+]i was observed upon activation of matured oocytes. These findings are discussed in terms of the role of calcium in maturation and it is concluded that calcium is not the second messenger for progesterone. This conclusion is further supported by the finding that 100 microM TMB-8, a blocker of intracellular calcium release, had no effect on progesterone-induced maturation.

Aequorin↗

Calcium does not act as a second messenger for adrenergic and cholinergic agonists in corneal epithelial cells.

The role of changes in intracellular [Ca2+]i as a second messenger in response to either adrenergic or cholinergic agonists was determined in isolated bovine corneal epithelial cells. [Ca2+]i was measured in suspensions of cells loaded with either of the fluorescent indicators quin2 or indo-1, as well as in single cells loaded with fura-2. Fluorescence from the cell suspensions was measured in a spectrofluorometer while single cell fluorescence was measured using a modified fluorescence microscope with a photon counting photometer. Cells were loaded with these dyes by incubation in Ringer's (pH 8.1) containing 2-50 microM of the acetoxymethyl ester of the indicator. Fluorescence was measured before and after exposure to either, one of the adrenergic agonists isoproterenol, phenylephrine or epinephrine, or the cholinergic agonist carbachol. The resting [Ca2+]i level from the quin2 experiments was 115 nM +/- 41 nM (SEM) (n = 23) whereas with fura-2 it was 71 +/- 10 nM (n = 30). In no case did we see any change in [Ca2+]i within 15 min after addition of any agonist but we were able to observe increased calcium when 0.5 microM ionomycin was added to either the same or untreated cells. The disparity in the resting levels determined by the two methods may result from various calibration problems. Our results indicate that changes in [Ca2+]i have no second messenger role in response to these agonists.

Aminoquinolines↗

Maturation of Xenopus oocytes is not accompanied by electrode-detectable calcium changes.

Ca2+-sensitive microelectrodes were used to study changes in cytoplasmic free calcium during progesterone-induced meiotic maturation in Xenopus laevis oocytes. In contrast to previous reports, no changes were detected during the maturation process, although a large activation pulse of Ca2+ was seen when in vitro matured eggs were parthenogenetically activated. The resting level of Ca2+ in the oocytes was 0.14 microM +/- 0.05.

Animals↗

Cytochalasin treatment disrupts the endogenous currents associated with cell polarization in fucoid zygotes: studies of the role of F-actin in embryogenesis.

We determined the distribution of F-actin in fucoid (Pelvetia, Fucus) embryos with nitrobenzoxadiazole-phallacidin, and studied the effect of cytochalasin upon the endogenous currents associated with cell polarization by using the vibrating probe. F-actin is not localized at the presumptive rhizoid immediately after experimental induction of the polar axis with a light gradient; however, a preferential distribution of F-actin develops at the presumptive rhizoid by the time the position of the polar axis is fixed. F-actin continues to be localized at the tip of the rhizoid after germination, except during cytokinesis, when the furrow is the only brightly staining region of the embryo. Incubation with cytochalasin can result in either an enhanced or a diminished pool of F-actin in the embryonic cortex (see Results). Cytochalasin D (100 micrograms/ml) significantly reduces the inward current at the rhizoid pole (n = 11) after a 2.5-h incubation. This drop is concentration dependent and occurs within approximately 30 min at 100 micrograms/ml and approximately 60 min at 10 micrograms/ml. Cytochalasin treatment eliminates the pulsatile component of the current. Preliminary results suggest that 100 micrograms/ml cytochalasin D prevents development of inward current at the presumptive rhizoid but does not completely delocalize this locus if added after photopolarization. We conclude that microfilaments are required for the establishment and maintenance of the pattern of endogenous currents observed during early embryogenesis. This suggests a new model for axis formation and fixation.

Actins↗

Intracellular pH and ribosomal protein S6 phosphorylation: role in translational control in Xenopus oocytes.

The induction of amphibian oocyte maturation with progesterone as well as the activation of sea urchin eggs at the time of fertilization result in increased protein synthesis. The increase in both cases involves the recruitment of maternal mRNA onto polysomes. Further, it has been reported that sea urchin eggs, like full-grown Xenopus oocytes, contain no spare translational capacity based on the observation that injected heterologous mRNA is translated only at the expense of endogenous messages. The nature of the limiting component defined by such experiments is not known, but two factors which have been proposed to play a role in regulating protein synthesis are ribosomal protein S6 phosphorylation and intracellular pH. In the current paper, we review the literature and present new evidence on the roles intracellular pH and S6 phosphorylation have in regulating protein synthesis in Xenopus oocytes. We report that pHi does not increase between stage 3 and stage 6, yet the protein synthetic rate increases at least eight fold during the same period. Hence, we conclude that increasing pHi is not a prerequisite for increasing protein synthesis. Moreover, we present three arguments against increased ribosomal protein S6 phosphorylation being sufficient or necessary for increased protein synthesis S6 phosphorylation being sufficient or necessary for increased protein synthesis in Xenopus oocytes. First, the level of S6 phosphorylation does not increase between stages 4 and 6, a period exhibiting a two to three fold increase in protein synthesis. Second, the injection of globin mRNA into stage-4 oocytes increases total protein synthesis two to three fold, but has no effect on S6 phosphorylation. Third, when the injection of globin mRNA into stage-4 oocytes is followed by an injection of MPF, a dramatic increase in S6 phosphorylation is seen, but total protein synthesis is not further stimulated.

Adenosine Triphosphate↗

Effects of calcium on the steady outward currents at the equator of the rat lens.

The relationships between calcium and the steady outward currents at the equator of the rat lens were studied using the vibrating probe technique. In a calcium-free medium, the current was greatly increased and it returned to its original level when calcium was restored to the medium. The Ca-free effect was not observed in Na-free medium. Iodoacetate (IAA) inhibited the initial current, but a current then returned which is referred to as a secondary current. The secondary current was not observed in a Ca-free medium and, therefore, it is thought to be a calcium-dependent potassium current. These responses are consistent with effects on potassium efflux measured by others and lend support to the interpretation that the outward currents observed at the equator of the rat lens are potassium currents. The currents are partially inhibited but not abolished in Na-free bathing medium. This is consistent with the view that the inward currents at the optical poles may be related to the influx of sodium.

Animals↗

Electrical polarity in embryos of wild carrot precedes cotyledon differentiation.

Endogenous electrical currents traverse embryos of a higher plant, the wild carrot Daucus carota L. Current enters the apical pole and leaves the region near the presumptive radicle in the radially symmetric globular embryo. Current also enters the exposed surfaces of incipient globular embryos. This electrical polarity precedes differentiation of vascular tissue and cotyledon development. Localized current is observed at both growing ends of the embryos in subsequent stages of embryogenesis. Inward current is found at the cotyledons; outward current is found at the radicle/root. Exogenous indole-3-acetic acid (3 muM) reversibly inhibits these currents. Little current traverses the surface of intermediate regions of the embryo. The ionic gradients generated by these currents may be important in accumulation of metabolites and in other developmental processes within the embryo.

Journal Article↗

Self-generated electrical currents through Xenopus neurulae.

An extracellular vibrating electrode has been used to investigate epithelial current production by Xenopus neurulae. Xenopus neurulae, in 5% DeBoer (DB) solution, stages 14-22, generated an endogenous current which left the blastopores of the embryos, and entered all other locations. The inward current declined near the mid line of the neural groove in stage 15 embryos. The inward current was abolished or drastically reduced by exchange of 5% DB with Na+-free (choline- or bis(2-hydroxyethyl)dimethylammonium (BDAC)-substituted) 5% DB, 5.5 mM-KCl, 1 mM-verapamil in 5% DB, 50 or 500 microM-ouabain in 5% DB, or 1 microM-amiloride in 5% DB. Verapamil (10 microM) in 5% DB, 5.5 mM-NaCl and 1 mM-CoCl2 in 5% DB had little or no effect on the magnitude observed. The current also was present in 5% DB containing Li+ instead of Na+; current carried by Li+ was abolished by amiloride. This is consistent with a Na+ current, similar to that of short-circuited adult frog skin.

Action Potentials↗

Endogenous electrical current leaves the limb and prelimb region of the Xenopus embryo.

The electrical current pattern around the developing Xenopus laevis embryo was mapped with a vibrating probe. Current (taken as the movement of positive charge) was found to leave the emerging hind limb bud and to enter the gill region of the stage 47 embryo. The magnitude of the current leaving the limb was about 7 microA/cm2 and the current entering the gill was about 60 microA/cm2. Other regions of smaller outward current were found between the limb and gill. At stage 43, prior to the appearance of the limb bud, a highly localized region of the outward current existed in the general area from which the bud would later emerge. The inward current was localized to the gill bud, as in the older embryo. The ionic basis of the currents could not be determined. In about one-third of the cases studied, the inward current was sodium sensitive since the removal of external sodium or the addition of amiloride reversably blocked the current. In the remaining cases, however, removal of sodium did not change (or else increased) the current. No other external ion (Ca2+, Mg2+, K+) could be identified as the current-carrying ion; the possibility of an outward movement of some anion such as Cl- or HCO-2 remains.

Amiloride↗

Internal pH of Xenopus oocytes: a study of the mechanism and role of pH changes during meiotic maturation.

The internal pH (pHi) of Xenopus laevis oocytes, as measured by the DMO method, covered a broad range of values from 7.06 +/- 0.01 to 7.93 +/- 0.01, with a mean value of 7.43 +/- 0.03. The pHi measured by DMO and microelectrodes was nearly identical in control and maturing oocytes from the same batch. The oocytes from most females elevated their pHi in response to progesterone, reaching a maximum elevation of 0.30 +/- 0.03 pH units above control values at 100% germinal vesicle breakdown (GVBD). However, some females were found to contain oocytes that already had an elevated pHi of 7.71 +/- 0.03 which did not significantly increase during maturation. Human chorionic gonadotrophin (hcG)-stimulated females had oocytes with slightly higher control pHi values than oocytes from nonstimulated females but still showed the same elevation in response to progesterone. Thus, the "stimulated" state of oocyte physiology as induced by hcG did not account for the variation in control pHi and responsiveness to progesterone. Other aspects of this variability are discussed. Elevating or lowering the external pH is shown to elevate and lower pHi, respectively, in a stable and predictable manner. Using this approach to change pHi we have found no effect of changes in pHi on the rate of protein synthesis in control and maturing oocytes. Similarly, pHi had only a slight facilitating effect on the rate of GVBD. A pH indicator gel was used to demonstrate that the pHi increase during oocyte maturation involved an acid efflux. We conclude that an elevated pHi is not necessary for oocyte maturation, yet the mechanism of the pHi elevation is discussed as a possible lead to events that are necessary.

Animals↗

Xenopus neural crest cell migration in an applied electrical field.

Xenopus neural crest cells migrated toward the cathode in an applied electrical field of 10 mV/mm or greater. This behavior was observed in relatively isolated cells, as well as in groups of neural crest cells; however, the velocity of directed migration usually declined when a cell made close contact with other cells. Melanocytes with a full complement of evenly distributed melanosomes did not migrate of their own accord, but could be distorted and pulled by unpigmented neural crest cells. Incompletely differentiated melanocytes and melanocytes with aggregated melanosomes displayed the same behavior as undifferentiated neural crest cells, that is, migration toward the cathode. An electrical field of 10 mV/mm corresponded to a voltage drop of less than 1 mV across the diameter of each cell; the outer epithelium of Xenopus embryos drives an endogenous transembryonic current that may produce voltage gradients of nearly this magnitude within high-resistance regions of the embryo. We, therefore, propose that electrical current produced by the skin battery present in these embryos may act as a vector to guide neural crest migration.

Animals↗

Ion currents and membrane domains in the cleaving Xenopus egg.

We used an extracellular vibrating probe to measure ion currents through the cleaving Xenopus laevis egg. Measurements indicate sharp membrane heterogeneities. Current leaves the first cleavage furrow after new, unpigmented membrane is inserted. This outward current may be carried by K+ efflux. No direct involvement of the Na+,K+-ATPase in the generation of this outward current is detected at first cleavage. Inward current enters the old, pigmented membrane; however, it does not enter uniformly. The inward current is largest at the old membrane bordering the new membrane. This suggests a heterogeneous ion channel distribution within the old membrane. Experiments suggest that the inward current may be carried by Na+ influx, Ca2+ influx, and Cl- efflux. No steady currents were detected during grey crescent formation, the surface contraction waves preceding cleavage, or with groove formation at the beginning of cleavage.

Animals↗