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K Swann

Publications and source records attributed to K Swann.

60 records · Page 4Linked to original sources
PubMed1989

Evidence for an inhibitory effect of protein kinase C on G-protein-mediated repetitive calcium transients in hamster eggs.

Hamster eggs undergo repetitive increases in cytoplasmic free calcium concentration ([Ca2+]i) at fertilization or after injecting guanosine-5'-0-(3-thiotriphosphate) (GTP[S]). We report the effects of protein kinase C (PKC) agonists and antagonists on these repetitive [Ca2+]i transients as measured by their associated membrane potential hyperpolarizing responses (HRs). Iontophoretic injection of GTP[S] into unfertilized eggs caused a series of repetitive HRs that declined in amplitude with time. Continuous injection of inositol 1,4,5-trisphosphate (InsP3) also caused a series of repetitive HRs, but these HRs declined in amplitude less markedly. GTP[S]-induced HRs were inhibited by the PKC agonists phorbol 12-myristate 13-acetate (TPA) (100 nM) and 1,2-dioctanoyl-glycerol (diC8) (250 microM). Conversely the PKC inhibitor sphingosine (10 microM) enhanced the number of large HRs after GTP[S] injection. TPA or sphingosine did not alter InsP3-induced HRs. We suggest that G-protein-mediated InsP3 production causes repetitive [Ca2+]i transients but that GTP[S] injection stimulates a negative feedback loop involving PKC. Adding TPA (100 nM) before insemination caused a reduction in the frequency of HRs at fertilization, but neither TPA nor sphingosine affected the frequency or size of HRs when they were added after the start of fertilization. Fertilizing sperm may stimulate G-protein-mediated InsP3 production in a way that precludes feedback inhibition by PKC.

Animals↗
PubMed1989

A new and rapid method for the selection and cloning of antigen-specific hybridomas with magnetic microspheres.

A new and highly selective procedure is described for the rapid selection and cloning of antibody-secreting hybridomas with antigen-coated magnetic beads. Immune splenocytes were fused to Sp2 myeloma cells with a PEG/DMSO mixture. Cells were cultured in HAT and screened for the presence of specific antibody after 7-10 days. Hybridomas from the positive colonies were mixed with antigen-coated magnetic polymer beads and antigen-specific cells separated on a magnet. Cloning was carried out directly by limiting dilution with the magnetic beads still bound on the cells. No obvious toxic effects were observed. The antibodies established by this technique were of a high affinity (greater than 10(9) l/mol) and were generated in 50% of the usual process time. The procedure described here should greatly facilitate the process of obtaining hybridomas and expand the range of monoclonal antibodies available.

Animals↗
PubMed1988

Activation of sea urchin eggs by inositol phosphates is independent of external calcium.

We investigated the contribution of external calcium ions to inositol phosphate-induced exocytosis in sea urchin eggs. We show that: (a) inositol phosphates activate eggs of the sea urchin species Lytechinus pictus and Lytechinus variegatus independently of external calcium ions; (b) the magnitude and duration of the inositol phosphate induced calcium changes are independent of external calcium; (c) in calcium-free seawater, increasing the volume of inositol trisphosphate solution injected decreased the extent of egg activation; (d) eggs in calcium-free sea water are more easily damaged by microinjection; microinjection of larger volumes increased leakage from eggs pre-loaded with fluorescent dye. We conclude that inositol phosphates do not require external calcium ions to activate sea urchin eggs. This is entirely consistent with their role as internal messengers at fertilization. The increased damage caused to eggs in calcium-free seawater injected with large volumes may allow the EGTA present in the seawater to enter the egg and chelate any calcium released by the inositol phosphates. This may explain the discrepancy between this and earlier reports.

Animals↗
PubMed1986

The part played by inositol trisphosphate and calcium in the propagation of the fertilization wave in sea urchin eggs.

Sea urchin egg activation at fertilization is progressive, beginning at the point of sperm entry and moving across the egg with a velocity of 5 microns/s. This activation wave (Kacser, H., 1955, J. Exp. Biol., 32:451-467) has been suggested to be the result of a progressive release of calcium from a store within the egg cytoplasm (Jaffe, L. F., 1983, Dev. Biol., 99:265-276). The progressive release of calcium may be due to the production of inositol trisphosphate (InsP3), a second messenger. We show here that a wave of calcium release crosses the Lytechinus pictus egg; the peak of the wave travels with a velocity of 5 microns/s; microinjection of InsP3 causes the release of calcium within the egg; calcium release (as judged by fertilization envelope elevation) is abolished by prior injection of the calcium chelator EGTA; neomycin, an inhibitor of InsP3 production, does not prevent the release of calcium in response to InsP3 but does abolish the wave of calcium release; the egg cytoplasm rapidly buffers microinjected calcium; the calcium concentration required to cause fertilization membrane elevation when microinjected is very similar to that required to stimulate the production of InsP3 in vitro; and the progressive fertilization membrane elevation seen after microinjection of calcium buffers appears to be due to diffusion of the buffer across the egg cytoplasm rather than to the induction of the activation wave. We conclude that InsP3 diffuses through the egg cytoplasm much more readily than calcium ions and that calcium-stimulated production of InsP3 and InsP3-induced calcium release from an internal store can account for the progressive release of calcium at fertilization.

Aequorin↗
PubMedDate not supplied

Stimulation of the Na/H exchanger of sea urchin eggs by phorbol ester.

On fertilization of a sea urchin egg, marked changes occur in the cytoplasmic concentration of calcium and hydrogen ions. These ionic signals represent the necessary and sufficient stimuli for the increased metabolism, protein synthesis and DNA synthesis that constitute egg activation. Cytoplasmic alkalinization, the major immediate cause of the increased rate of protein synthesis which occurs at fertilization, arises because the sperm-induced intracellular calcium transient activates a coupled flux of sodium ions and hydrogen ions across the oolemma. The experiments reported here suggest that the second messenger which links the activation of the Na/H exchange to the calcium transient may be a substance which stimulates protein kinase C8, as 12-O-tetradecanoyl phorbol acetate (TPA), a known activator of protein kinase C9, appears to stimulate protein synthesis by turning on the Na/H exchanger and causing a cytoplasmic alkalinization. Our data indicate that one consequence of treating other tissues with TPA, a tumour promoter, may be an increase in intracellular pH.

Animals↗