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R L Dimond

Publications and source records attributed to R L Dimond.

At least 37 records · Page 2Linked to original sources
PubMed1985

Regulation of lysosomal alpha-mannosidase-1 synthesis during development in Dictyostelium discoideum.

The cellular specific activity of lysosomal alpha-mannosidase-1 increases dramatically during development in Dictyostelium discoideum. alpha-Mannosidase-1 is composed of two subunits (Mr = 58,000 and 60,000) which are derived from a common precursor polypeptide (Mr = 140,000). Using enzyme-specific monoclonal antibodies we have determined that throughout development (a) the relative rate of precursor biosynthesis closely parallels the rate of accumulation of cellular enzyme activity and (b) the newly synthesized precursor is efficiently processed to mature enzyme (t1/2 less than 10 min). This indicates that the developmental accumulation of alpha-mannosidase-1 activity is primarily controlled by de novo enzyme synthesis. Furthermore, the change in the relative rate of enzyme precursor synthesis can be accounted for by an increase in the cellular level of functional alpha-mannosidase-1 mRNA during development.

Dictyostelium↗
PubMed1985

Developmental changes in the modification of lysosomal enzymes in Dictyostelium discoideum.

Evidence has been found for a generalized change in the post-translational modification of lysosomal enzymes during development of Dictyostelium discoideum. The physical and antigenic properties of four developmentally regulated lysosomal enzymes, N-acetylglucosaminidase, beta-glucosidase, alpha-mannosidase, and acid phosphatase, have been examined throughout the life cycle. In vegetative cells, a single major isoelectric species is detected for each enzymatic activity on native nonequilibrium isoelectric focusing gels. Between 6 and 10 hr of development, all activities, including the preformed enzyme, become less negatively charged, resulting in a modest but reproducible shift in the isoelectric focusing pattern. This alteration is not detected by native gel electrophoresis at constant pH. As development continues, the specific activity of beta-glucosidase, alpha-mannosidase, and acid phosphatase continues to increase and coincidentally, new, less acidic isozymic bands of activity can be observed on both gel systems. Some of these new isozymes accumulate preferentially in anterior cells, while others accumulate preferentially in posterior cells of migrating slugs. N-Acetylglucosaminidase does not increase in specific activity late in development and no new isozymic species appear. Using a monoclonal antibody that reacts with sulfated N-linked oligosaccharides shared by vegetative lysosomal enzymes in D. discoideum, the antigenicity of the developmental isozymes has been characterized. All of the enzymatic activity present during vegetative growth and early development is immunoprecipitable. However, the less negatively charged isozymes that accumulate after aggregation are not recognized by the antibody. Nonantigenic acid phosphatase and alpha-mannosidase are found in both anterior and posterior cells from migrating pseudoplasmodia. Since each enzyme is coded by a single structural gene, these results suggest that the isozymes present late in development arise from the synthesis of the same polypeptides with altered post-translational modifications. The appearance of anterior and posterior specific isozymes is likely to be the result of cell type specific changes in the glycoprotein modification pathway for newly synthesized proteins.

Acetylglucosaminidase↗
PubMed1985

Pathways involved in targeting and secretion of a lysosomal enzyme in Dictyostelium discoideum.

In Dictyostelium discoideum, the lysosomal enzyme alpha-mannosidase is first synthesized as an N-glycosylated precursor of Mr 140,000. After a 20-30-min lag period, up to 30% of the precursor molecules are rapidly secreted, whereas the rest remain cellular and are proteolytically processed (t 1/2 = 8 min) to mature subunits of Mr 58,000 and 60,000. The secreted precursor is modified more extensively than the cellular form, as is revealed by differences in size, charge, and sensitivity to endoglycosidase H. Subcellular fractionation has shown that, following synthesis in the rough endoplasmic reticulum, the precursor is transported to a low density membrane fraction that contains Golgi membranes. Proteolytic processing takes place in these vesicles, since newly cleaved mature enzyme, but no precursor, co-fractionates with lysosomes. Under conditions that disrupt vesicular membranes, the precursor remains associated with the membrane fraction, whereas the newly processed mature enzyme is soluble. Proteolytic cleavage of the precursor thus coincides with the release of the mature enzyme into the lumen of a lysosomal compartment. These findings suggest a possible mechanism for lysosomal targeting that involves the specific association of enzyme precursors with Golgi membranes.

Biological Transport↗
PubMed1985

alpha-Mannosidase-1 mutants of Dictyostelium discoideum: early aggregation-essential genes regulate enzyme precursor synthesis, modification, and processing.

The lysosomal enzyme alpha-mannosidase-1 is one of the earliest developmentally controlled gene products in Dictyostelium discoideum. Although this enzyme is synthesized throughout the first 20 h of development, it is not required for complete morphogenesis, since structural gene (manA) mutants lacking activity develop normally. We isolated six strains deficient in alpha-mannosidase-1 activity which, unlike structural gene mutants, fail to aggregate. Fruiting revertants of these strains accumulate wild-type levels of alpha-mannosidase-1 activity, suggesting that both the enzymatic and morphological defects are caused by single mutations in nonstructural genes essential for early development. Direct genetic evidence for mutations outside of the structural gene was obtained by complementation analysis. We used alpha-mannosidase-1-specific monoclonal antibodies to analyze the biochemical defects in these mad (alpha-mannosidase-1-deficient) mutants. All mad mutants show a significantly reduced relative rate of enzyme precursor biosynthesis. The mad-404 mutation results in a complete lack of precursor biosynthesis, as well as a lack of functional alpha-mannosidase-1 mRNA. In some cases, however, the enzymatic defect results from improper post-translational modification which affects precursor processing. We conclude that a small number of aggregation-essential genes are involved in regulating the synthesis, modification, and processing of alpha-mannosidase-1 during development.

Dictyostelium↗
PubMed1985

Lysosomal enzyme inactivation associated with defects in post-translational modification during development in Dictyostelium discoideum.

The developmental accumulation of lysosomal alpha-mannosidase-1 activity in Dictyostelium discoideum is controlled at the level of de novo enzyme precursor biosynthesis. Aggregation-deficient mutants are defective with regard to the accumulation of alpha-mannosidase-1 activity beyond 8-16 h of development. We used enzyme-specific monoclonal antibodies to show that the activity defect in aggregation-deficient strains is not due to a lack of alpha-mannosidase-1-precursor synthesis or processing, or to preferential degradation of the mature enzyme protein. Instead, the defect is a result of enzyme inactivation: cells of aggregation-deficient strains contain significant amounts of inactive alpha-mannosidase-1 protein late in development. The alpha-mannosidase-1 inactivation phenotype is associated with a more general defect in lysosomal enzyme modification. A change in the post-translational modification system occurs during normal slime-mold development, as shown by differences in enzyme isoelectric point, antigenicity, and thermolability. We found that this change in modification does not occur in mutant strains blocked early in development. We propose a model in which pleiotropic mutations in early aggregation-essential genes can indirectly affect the accumulation of alpha-mannosidase-1 activity by preventing the expression of a developmentally controlled change in the post-translational modification system, a change which is required for the stability of several lysosomal enzymes late in development.

Enzyme Activation↗
PubMed1985

Mule spinner's disease.

Mule spinner's disease represents the occurrence of scrotal cancer in cotton textile workers exposed to mineral oils on a long-term basis while working on a machine called "the mule." We describe a 66-year-old cotton textile worker who had frequent scrotal contact with mineral oils and later had multiple squamous cell carcinomas of the scrotum develop. He also had a variety of other keratotic lesions develop on the scrotum that histopathologically demonstrated features of verruca, lichenoid keratoses, and squamous cell carcinomas in situ. We have termed these lesions mule spinner's keratoses.

Aged↗
PubMed1984

Antigenic determinants shared by lysosomal proteins of Dictyostelium discoideum. Characterization using monoclonal antibodies and isolation of mutations affecting the determinant.

Polyclonal antisera raised against a single lysosomal enzyme from Dictyostelium discoideum will cross-react with other lysosomal proteins due to the presence of common post-translational modification antigens. We have now isolated hybridoma cell lines secreting monoclonal antibodies that also recognize a shared lysosomal determinant. Although the same proteins are recognized by the monoclonal and polyclonal antibodies, the actual determinants differ. Also, the polyclonal serum contains antibodies which recognize at least one determinant not recognized by the monoclonal antibodies. All of the antigenic proteins are minor cell proteins but represent a majority of the proteins secreted from the cells during axenic growth. Anode-directed nonequilibrium two-dimensional gel electrophoresis of cellular and secreted protein indicates that the antigenic proteins have extremely acidic pI values; moreover, most are both sulfated and phosphorylated. Several sulfated polysaccharides, including dextran sulfate, chondroitin sulfate, and heparin inhibit binding of the monoclonal but not the polyclonal antibodies, indicating that the shared determinant recognized by the monoclonal antibodies may involve sulfated oligosaccharides. As a step in analyzing the function of the post-translational modification of lysosomal enzymes, we have screened a mutagenized population of amoeba for those lacking the antigenic determinant recognized by one of the monoclonal antibodies. Five cell lines have been isolated that completely lack the determinant. They appear to fall into at least three different classes based upon physical properties of the enzymes. One strain lacks any detectable sulfation of glycoproteins. Several of the strains are capable of forming fruiting bodies indicating that the antigenic determinant plays no crucial role in morphogenesis under laboratory conditions.

Antibodies, Monoclonal↗
PubMed1984

Sulfated oligosaccharides block antibodies to many Dictyostelium discoideum acid hydrolases.

The lysosomal hydrolases of the cellular slime mold, Dictyostelium discoideum, possess a common posttranslational modification which is extremely antigenic in rabbits and mice. Rabbit antisera and mouse monoclonal antibodies that recognize this determinant cross-react with a group of at least 40-50 highly negatively charged proteins which include most or all of the lysosomal enzymes. (Knecht, D. A., Dimond, R. L., Wheeler, S., and Loomis, W. F. (1984) J. Biol. Chem. 259, 10633-10640). The present study demonstrates that the determinant is found on certain N-linked oligosaccharides derived from one of these proteins. An esterified sulfate is absolutely required for antigenicity.

Antibodies, Monoclonal↗
PubMed1984

Visualization of antigenic proteins on Western blots.

A new technique for the detection of antibodies bound to proteins blotted onto nitrocellulose paper was developed. The method is rapid, sensitive, and does not require radioactive probes. Proteins transferred to nitrocellulose paper are first reacted with primary antibody followed by reaction with an alkaline phosphatase conjugated second antibody. The phosphatase activity is then visualized using an agar gel impregnated with the histochemical phosphatase stain 5-bromo-4-chloro-3-indolyl phosphate (BCIP) (J. P. Horwitz, J. Chua, M. Noel, J. T. Donatti, and J. Freisler (1966) J. Med. Chem. 9, 447; Sigma Chemical Co., Technical bulletin No. 710-EP (1978]. Antigen-antibody complexes give rise to sharp, permanent blue stained bands both on the nitrocellulose paper and in the agar overlay gel. This procedure allows detection of bands containing less than 20 ng of protein.

Antibodies, Monoclonal↗
PubMed1984

Accumulation of alpha-mannosidase-1 in Dictyostelium discoideum requires many developmentally essential genes.

alpha-Mannosidase-1, one of the earliest known developmentally controlled gene products in the cellular slime mold Dictyostelium discoideum, accumulates intracellularly during both axenic growth and development. The accumulation of alpha-mannosidase-1 activity prematurely ceases in all of 125 randomly isolated aggregation-deficient mutants at discrete times in development resulting in significantly reduced levels of cellular enzyme activity. This suggests that, unlike other developmentally controlled enzymes in this organism, the continued accumulation of alpha-mannosidase-1 activity is controlled by a large number of genes essential for early development. alpha-Mannosidase-1 misregulation and the aggregation-deficient phenotype are caused by the same mutation since (1) morphological revertants exhibit a coreversion to both fruiting ability and wild-type alpha-mannosidase-1 accumulation and (2) normal enzyme accumulation depends on the ability to aggregate and ultimately fruit in a conditional aggregation-deficient mutant. This type of regulation does not appear to be due to differences in enzyme secretion or changes in the overall rate of total protein synthesis. Aggregation-deficient mutants continue to synthesize protein beyond the time in development at which alpha-mannosidase-1 accumulation ceases. Our studies indicate that most of the 50-125 genes required for aggregation in Dictyostelium are also required for the normal accumulation of alpha-mannosidase-1 activity.

Dictyostelium↗
PubMed1983

Synthesis of related forms of the lysosomal enzyme alpha-mannosidase in Dictyostelium discoideum.

Purified lysosomal alpha-mannosidase from the cellular slime mold, Dictyostelium discoideum, is composed of two subunits of Mr = 58,000 and 60,000 as revealed by polyacrylamide gel electrophoresis under denaturing conditions. The pattern of peptide fragments produced when these two species are digested with proteases indicates that they are related but not identical. Using monoclonal antibodies prepared against purified alpha-mannosidase, we have analyzed the different forms of the enzyme synthesized in vivo. In addition to two bands that co-migrate with the pure enzyme, a large Mr (140,000) species is found in immunoprecipitated [35S] methionine-labeled extracts of cellular and secreted proteins. The precipitation of all three bands is inhibited by preadsorption of the antibodies with pure enzyme and all three proteins are absent in extracts of an alpha-mannosidase structural gene mutant. One-dimensional peptide maps indicate that all sequences present in the smaller species are found in the Mr = 140,000 form. In addition, the large form accounts for about 20% of the extracellular alpha-mannosidase activity secreted by amoebae during growth in axenic culture. These results lead to the conclusion that alpha-mannosidase is first synthesized as a large enzymatically active precursor which is modified and proteolytically cleaved to form the smaller subunits.

Dictyostelium↗
PubMed1983

Functional heterogeneity of monoclonal antibodies obtained using different screening assays.

Two alternate screening methods have enabled the detection of monoclonal antibodies with different specificities toward the lysosomal enzyme alpha-mannosidase of Dictyostelium discoideum. Spleen/myeloma hybrid cell cultures were screened for antibody production by separate assays: an indirect enzyme-linked immunoadsorbent assay (ELISA) based on the antibody binding to enzyme adsorbed on plastic, and a direct assay of the antibodies' ability to precipitate enzyme activity with fixed Staphylococcus aureus cells (Pansorbin). Fourteen stable antibody-producing cell lines resulted from a single fusion; these fell into three distinct classes based on their screening characteristics. A group of eight were positive in both assays, and these immunoprecipitated a 140,000 Mr precursor form of alpha-mannosidase in addition to the 58,000 and 60,000 Mr mature enzyme subunits from [35S]methionine-labeled total secreted protein preparations. Two of the antibodies were positive only in the immunoprecipitation assay; these failed to precipitate the 140,000 Mr precursor. The third class consisted of four antibodies that were positive only in the ELISA method. These exclusively recognized an altered conformation of the enzyme (precursor and mature forms) that was immobilized either on plastic or on nitrocellulose paper. In addition, only members of this class were able to bind to immobilized fragments of protease-treated enzyme. The implications of these findings for the general design of monoclonal antibody screenings and for the alternative structures of this enzyme are discussed.

Animals↗
PubMed1983

Secretory mutants in the cellular slime mold Dictyostelium discoideum.

Our initial studies have shown that the cellular slime mold Dictyostelium discoideum is a particularly suitable organism for the study of lysosomal enzyme secretion. During appropriate stages in the life cycle, secretion is prominent for a number of lysosomal enzymes. The methods described here have been developed to investigate various aspects of the secretion process. Moreover, our evidence that regulation of the secretory system is influenced by environmental changes and by cell differentiation indicates that this organism may be useful for studying the functional regulation of this organellar system. To a large degree these types of studies have been limited in the past due to the lack of an appropriate experimental system. The ability to isolate secretory mutants affecting the secretion of lysosomal enzymes adds another dimension to investigations using D. discoideum. In our initial attempts we have been successful in isolating a variety of different types of mutants that alter the secretion of one or more lysosomal enzymes. While the results are in agreement with the results of our physiological investigations, they also indicate much more heterogeneity in the lysosomal system than we had previously suspected. The indications that many of our secretory mutants may also affect modification of the enzymes is also intriguing. This observation may also help to explain the fact that many of these strains are defective in normal development. Together with the immunological methods available in this organism for studying posttranslational modification, the mutants may be valuable in deciphering the relationship between modification of lysosomal enzymes and their proper localization and secretion from the cell. Thus, Dictyostelium discoideum may become as useful for the study of some questions of cell biology as it has been for development.

Dictyostelium↗
PubMed1983

Heterokaryosis in Fusarium tricinctum and F. sporotrichioides.

Heterokaryons were formed in intra- and interspecific crosses between Fusarium sporotrichioides and F. tricinctum auxotrophs. Segregant homokaryons were evaluated for trichothecene toxin production in culture. Results were consistent with nuclear control of toxin synthesis. The sexual compatibility of auxotrophs and 30 additional F. tricinctum sensu Snyder & Hansen strains was tested. Perithecial production was restricted to crosses between Florida isolates pathogenic to English ivy (Hedera helix). The linkage of several auxotrophic markers was determined by analysis of progeny of certain crosses. No T-2 toxin was produced by sexually compatible F. tricinctum isolates.

Crosses, Genetic↗
PubMed1983

Febrile ulceronecrotic Mucha-Habermann's disease.

A patient with febrile ulceronecrotic Mucha-Habermann's disease manifested the characteristic features of this entity. These include a polymorphous eruption with histopathologic findings of Mucha-Habermann's disease, large ulceronecrotic skin lesions, intermittent high fever, and constitutional symptoms. The patient was unique in that he also had malabsorption and eosinophilia. This disease may represent a hypersensitivity reaction. To our knowledge, there are five previous cases of febrile ulceronecrotic Mucha-Habermann's disease reported in the world literature.

Eosinophilia↗
PubMed1982

Mycosis fungoides d'emblée: a rare presentation of cutaneous T-cell lymphoma.

A case of the d'emblée variant of mycosis fungoides is presented to confirm the validity of this rare variant of cutaneous T-cell lymphoma. The patient had rapidly progressing cutaneous tumors of mycosis fungoides with no internal organ or nodal involvement at the onset of the disease. This was confirmed at a laparotomy, which was done to remove a uterine leiomyoma. The classic light microscopy was confirmed by electron microscopy. Postmortem findings showed a remarkable degree of "epitheliotropism." The d'emblée form of mycosis fungoides has been disputed by some who argue that these cases represent a lymphoma that begins elsewhere and has secondary skin involvement. In this patient there was an opportunity, within a few weeks of the onset of the problem, to do an extensive staging work-up, including laparotomy. No evidence of internal lymphoma was found.

Adult↗
PubMed1982

Placental steroid deficiency: association with arylsulfatase A deficiency.

A family with an obstetric history consistent with placental sulfatase deficiency has X-linked ichthyosis. Steroid sulfatase deficiency was confirmed in placenta, leukocytes, and cultured skin fibroblasts of affected males; arylsulfatase A diminution was also observed in these tissues of both affected males and 2 generations of related females. No symptoms of metachromatic leukodystrophy are present in any family members. In this family, placental sulfatase deficiency, and arylsulfatase A pseudodeficiency are nonallelic.

Alleles↗