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T G Clark

Publications and source records attributed to T G Clark.

45 records · Page 3Linked to original sources
PubMed1979

An actin filament matrix in hand-isolated nuclei of X. laevis oocytes.

The nuclear gel of Xenopus oocytes contains a meshwork of randomly oriented microfilaments which have been identified as F-actin by decoration with rabbit skeletal muscle myosin subfragment-1 (S-1). Nuclear gel preparations treated with S-1 differ in several respects from control preparations incubated in either aqueous medium alone, or medium containing BSA. Actin filaments in control preparations appear less well preserved than those in S-1 treated preparations of the nuclear gel. The nucleoli of control preparations are extremely dense, while those of S-1 treated preparations have a more open, granular appearance. Large granular aggregates, which are a prominent feature of the controls, are seen much less frequently in S-1-treated preparations of the nuclear gel. These morphological differences appear to be correlated with the binding of protein to F-actin, since nuclear gel preparations incubated in tropomyosin, which also binds to actin filaments, appear similar to those treated with S-1. Approximately 63% of the total nuclear actin exists in a globular state, while 37% is filamentous.

Actins↗
PubMed1978

Actin in Xenopus oocytes.

It has been found that a high-speed supernatant fraction from Xenopus oocytes extracted in the cold will form a clear, solid gel upon warming. Gel formation occurs within 60 min at 18 degrees-40 degrees C, and is, at least initially, temperature reversible. Gelation is strictly dependent upon the addition of sucrose to the extraction medium. When isolated in the presence of ATP, the gel consists principally of a 43,000-dalton protein which co-migrates with Xenopus skeletal muscle actin on SDS-polyacrylamide gels, and a prominent high molecular weight component of approx. 250,000 daltons. At least two minor components of intermediate molecular weight are also found associated with the gel in variable quantities. Actin has been identified as the major consituent of the gel by ultrastructural and immunological techniques, and comprises roughly 47% of protein in the complex. With time, the gel spontaneously contracts to form a small dense aggregate. Contraction requires ATP. In the absence of exogenous ATP, a polypeptide which co-migrates with the heavy chain of Xenopus skeletal muscle myosin becomes a prominent component of the gel. This polypeptide is virtually absent from gels which have contracted in ATP-containing extracts. It has also been found that Ca++ is required for gelation in oocyte extracts. At both low and high concentrations of Ca++ (defined as a ratio of Ca++/EGTA in the extraction medium), gelation is inhibited.

Actins↗
PubMed1978

Actin in Xenopus oocytes. II. Intracellular distribution and polymerizability.

The largest oocytes of Xenopus Laevis were broken open in the absence of shearing forces which might transfer actin from particulate to supernatant fractions. Particulate and postmitochondrial supernatant fractions were prepared by centrifugation. SDS-electrophoretic fractionation on polyacrylamide gels and quantitative scanning techniques were used to separate actin and to assay its amount in cellular fractions. The actin has been identified in electrophoretograms by its molecular weight and its binding to DNase I. oocytes contain 1.4-1.7 {um}g of actin per cell, of which up to 88 percent is recovered in the postmitochondrial supernate under a variety of conditions. In the soluble fraction, it represents about 8.8 percent of the total protein. Its concentration in native cytoplasm was directly assayed at 4.1 mg/ml. There is no detectable actin that can be transferred from the particulate to the soluble phase by neutral detergents or ionic conditions that would depolymerize muscle actin. Centrifugation of the soluble oocyte fractions showed that 75-95 percent of the actin can not be sedimented under forces that would pellet filamentous actin. Addition of potassium and magnesium to the cytoplasm, to concentrations that would polymerize muscle actin, does not increase the amount of sedimentable actin. Roughly one-third of the soluble actin is recovered from Sephadex columns at about the position of monomer. About two- thirds is in complexes of 100,000 daltons or greater.

Actins↗
PubMed1977

Diffusible and bound actin nuclei of Xenopus laevis oocytes.

Several criteria have been used to identify actin in hand-isolated nuclei of Xenopus laevis oocytes; these include co-migration with actin on SDS-polyacrylamide gels, immunological cross-reactivity with antiserum against actin, binding to DNAase I and peptide mapping on SDS gels. The use of hand-isolated nuclei precludes the possibility of contamination from cytoplasmic actin or the leakage of significant amounts of actin from nuclei during isolation. Actin constitutes roughly 6% of the total nuclear protein. Approximately 75% of the actin is diffusible under the conditions of nuclear isolation used. About 25%, however, is stably associated with an insoluble nuclear gel, in which chromosomes, nucleoli and other nuclear granules are embedded. Actin is the single most promient component of the nuclear gel, comprising roughly 16% of the total protein of the complex. The possible significance of diffusible and bound actin in these nuclei is discussed.

Actins↗
PubMedDate not supplied

Icthyophthirius multifiliis has membrane-associated immobilization antigens.

Sera from fish that survive infections with the ciliated protozoon, Ichthyophthirius multifiliis, immobilize the parasite in vitro. In order to identify cell surface antigens involved in the immobilization response, integral membrane proteins were extracted from tomites with Triton X-114 and used to immunize rabbits. The rabbit antisera immobilized the parasite in vitro and antigens were localized to cell and ciliary plasma membranes by indirect immunofluorescent microscopy. The membrane protein fractions from both whole cells and tomite cilia were characterized by 1- and 2-dimensional SDS-PAGE. A 43,000-dalton (D) glycoprotein with an isoelectric point of 7.0 is the predominant protein in these fractions, comprising 12% and 60% of the total protein of whole cell and ciliary membranes, respectively. Western blot analysis of ciliary proteins with immune rabbit sera indicated that the 43,000-D glycoprotein is the principal antigen.

Animals↗
PubMedDate not supplied

Serotypic variation among isolates of Ichthyophthirius multifiliis based on immobilization.

Efforts have been made to determine whether surface antigens could be used as biochemical markers to define strain differences in the parasitic ciliate Ichthyophthirius multifiliis. In previous studies, a wild-type isolate designated G1 was found to have surface proteins analogous to the immobilization antigens of Paramecium and Tetrahymena; rabbit antiserum against this strain immobilizes homologous cells in vitro. It has now been shown for two additional Ichthyophthirius isolates (designated G1.1 and G2) that immobilization antigens are both present and serologically distinct. Proteins of similar size, which cross-react in Western blots with rabbit antisera against immobilization antigens of the G1 strain, are nevertheless found in the G1.1 and G2 isolates. As shown by Southern blotting analysis, the G1.1 and G2 strains also contain genomic DNA sequences which hybridize with an immobilization antigen cDNA from G1 when probed under conditions of reduced stringency. The serotypic differences in immobilization between I. multifiliis isolates appear to be stable over time and provide a means of discriminating strains. In addition to providing a basis for comparative studies, the work described here has implications for the development of vaccines against this important fish parasite.

Animals↗
PubMedDate not supplied

The I-antigens of Ichthyophthirius multifiliis are GPI-anchored proteins.

The parasitic ciliate Ichthyophthirius multifiliis has abundant surface membrane proteins (i-antigens) that when clustered, trigger rapid, premature exit from the host. Similar antigens are present in free-living ciliates and are GPI-anchored in both Paramecium and Tetrahymena. Although transmembrane signalling through GPI-anchored proteins has been well-documented in metazoan cells, comparable phenomena have yet to be described in protists. Since premature exit of Ichthyophthirius is likely to involve a transmembrane signalling event, we sought to determine whether i-antigens are GPI-anchored in these cells as well. Based on their solubility properties in Triton X-114, the i-antigens of Ichthyophthirius are amphiphilic in nature and partition with the detergent phase. Nevertheless, following treatment of detergent lysates with phospholipase C, the same proteins become hydrophilic. Concomitantly, they are recognized by antibodies against a cross-reacting determinant exposed on virtually all GPI-anchored proteins following cleavage with phospholipase C. Finally, when expressed in recombinant form in Tetrahymena thermophila, full-length i-antigens are restricted to the membrane, while those lacking hydrophobic C-termini are secreted from the cell. Taken together, these observations argue strongly that the i-antigens of Ichthyophthirius multifiliis are, in fact, GPI-anchored proteins.

Amino Acid Sequence↗