Michael F. Filosa

Ultrastructure of macrocyst formation in the cellular slime mold, Dictyostelium mucoroides: Extensive phagocytosis of amoebae by a specialized cell☆

Macrocyst differentiation in Dictyostelium mucoroides was carried out in shaking flasks. Observations of the development of macrocysts were made by light and electron microscopy. Macrocyst development begins with the clumping of stationary phase amoebae. Subsequently, the clumps become subdivided into smaller masses, each surrounded by a fibrillar sheath. At the center of each mass there arises a cytophagic cell which proceeds to engulf in turn all the cells in the mass. The engulfed cells (endocytes) undergo drastic changes in their fine structure and eventually are transformed into granules. During the early stages of engulfment the cytophagic cell has a single large nucleus, but by the time all the cells have been converted to endocytes the cytophagic cell has become multinucleate. The multinucleate condition persists in the granular stage. The thick cellulose wall surrounding each macrocyst is produced by the cytophagic cell soon after it has engulfed all the cells in the mass and before the granular stage.

Proteins immunoreactive with antibody against a human leptin fragment are found in serum and tissues of the sea lamprey, Petromyzon marinus L.

An affinity-purified, polyclonal antibody raised against a peptide corresponding to amino acids 137-156 at the carboxy terminus of human leptin (16 kD) was used to search for immunoreactive protein(s) in the lamprey, Petromyzon marinus. Immunoblots of serum from different phases of the life cycle showed the presence of a 65-kD immunoreactive protein in the larvae and all stages of metamorphosis but not in feeding juvenile and upstream migrant adults. Extracts of tissues known to store fat were also examined using the same antibody. Muscle and fat column from all phases tested (larvae, stage 2 and 4 metamorphosing animals, feeding juveniles and upstream migrants) showed 100- and 50-kD immunoreactive proteins. Extracts of nephric fold, the primary site of fat storage during metamorphosis, lacked the 100-kD protein but had the 50 kD; they also had a 16 kD immunoreactive protein not found in the other tissues. The immunoreactivity of the proteins of both serum and tissue extracts was blocked by pretreatment of the antibody with the leptin-derived antigen. The results indicate that P. marinus has proteins that share at least one epitope with mammalian leptin.

The Possible Involvement of Cyclic AMP and Volatile Substance (s) in the Development of a Macrocyst-Forming Strain of Dictyostelium mucoroides

A mutant MF1 previously isolated from Dictyostelium mucoroides‐7 (Dm7) formed macrocysts with or without light when plated on agar at high cell dinsities. At lower cell densities, however, the MF1 cells formed only fruiting bodies. This failure to form macrocysts was shown to be due to the subthreshfold concentration of a volatile substance(s) required for macrocyst formation. Although ammonia is a volatile substance produced by both the Dm7 and MF1 cells, no evidence of its involvement in macrocyst formation was obtained. Mixing the Dm7 and MF1 in a one‐to‐one ratio resulted only in fruiting body formation suggesting that the Dm7 cells produced a factor which allowed MF1 cells to form fruiting bodies. This factor may be cyclic AMP (cAMP) since addition of cAMP to the medium directed development of MF1 cells to fruiting body formation. The effect of cAMP was exhibited most conspicuously when MF1 cells were exposed at the aggregation stage. Based on these results it is suggested that developmental pathway of the D. mucoroides macrocystforming strain Dm7 and its mutant MF1 may be determined by the relative concentrations of the volatile, macrocyst‐inducing substance(s) and cAMP at the aggregation stage.

An albumin-like protein in the serum of non-parasitic brook lamprey (Lampetra appendix) is restricted to preadult phases of the life cycle in contrast to the parasitic species Petromyzon marinus

Previous work showed that the parasitic sea lamprey, Petromyzon marinus, has two different albumin-like serum proteins during the course of its life cycle. One of these, AS, is the predominant protein in the serum of larval and metamorphosing intervals but is absent in the upstream migration phase of the life cycle; the other, SDS-1, is found at its highest level in the upstream migration phase and is the predominant serum protein in this phase. The present investigation examines the nonparasitic brook lamprey Lampetra appendix for the presence of albumins. Using electrophoresis and antisera monospecific for serum proteins, it was found that L. appendix has a serum protein that is antigenically similar to the AS protein of P. marinus. This protein (LAS) makes up about 70% of the total serum protein of the Lampetra larva, is present in metamorphic stages 1,2,3,4 and 5 but is virtually absent from the remaining metamorphic stages and the adult. Furthermore, the adult of L. appendix has no serum protein that is antigenically similar to SDS-1 of the adult P. marinus; our study shows that, unlike P. marinus, there is no single protein that represents a major portion of the total serum protein content in the L. appendix adult.

An electrophoretic and immunoelectrophoretic study of serum proteins during the life cycle of the lamprey, Petromyzon marinus L.

Serum proteins of upstream migrants, parasitic adults, early and late metamorphosing animals, and ammocoetes of Petromyzon marinus L. were examined by polyacrylamide gel electrophoresis and crossed-immunoelectrophoresis. The electrophoretic patterns for two proteins, SDS-1 and CB-III, were found to change during the life cycle. quantitative measurements of these two proteins showed that they continued to increase until in the adult they together constituted approx. 85% of the total serum protein. Evidence was obtained for a protein present in ammocoetes and metamorphosing animals, but not in upstream migrants.

Immunocytochemical localization of thyroglobulin in the transforming endostyle of anadromous sea lampreys, Petromyzon marinus L., during metamorphosis.

Abstract Light and electron microscopic immunocytochemistry was used to detect the localization of thyroglobulin (TG) in the transforming endostyle of the anadromous sea lampreys, Petromyzon marinus L., during metamorphosis. TG is found within the transforming endostyle throughout metamorphosis (stages 1 to 7) and in early stages (1–2) staining for TG is similar to that observed in the endostyle of the ammocoete. By stage 3, most of the TG is found in large bodies that accumulate within type 3 cells. At stage 4 immunostaining for TG is found within the epithelial cells of the transforming endostyle and in the lumina of some of the first formed follicles. Most of the follicular lumina contain TG by the end of metamorphosis (stage 7). TG in the young parasitic adult lamprey is localized within the follicular lumina, colloid droplets, and occasionally in large bodies found in some of the follicular epithelial cells.

An electrophoretic and immunoelectrophoretic characterization of the serum proteins of the adult lamprey, Petromyzon marinus L.

1. Serum proteins of upstream migrants of Petromyzon marinus were separated electrophoretically on polyacrylamide and agarose as well as by crossed immunoelectrophoresis using anti-lamprey serum antiserum. 2. Some constituents of four of the five major bands separated by electrophoresis on polyacrylamide were located in the agarose and crossed-immunoelectrophoretic patterns. 3. Two serum proteins, SDS-1 and CB-III, were isolated. SDS-1 was found to be a glycoprotein; CB-III may be a lipoprotein. 4. Monospecific antisera were produced against SDS-1 and CB-III and were used in crossed immunoelectrophoresis to analyze lamprey serum. This method revealed that electrophoretically different forms of these two proteins exist in serum. 5. Immunoelectrophoretic methods demonstrated that three of the major bands produced by polyacrylamide gel electrophoresis of whole serum contained more than a single protein.

Light and electron microscopic immunocytochemical localization of thyroglobulin in the thyroid gland of the anadromous sea lamprey, Petromyzon marinus L., during its upstream migration

SummaryAntibodies made against thyroglobulin (TG) were used in an immunocytochemical study for the light and electron microscopic localization of TG in the thyroid gland of the anadromous sea lamprey, Petromyzon marinus, during its upstream migration. TG was found in the follicular lumen and in some colloid droplets within the follicular cells. Except for an immunoreactive product observed in a small portion of the interstitial connective tissue, the location of TG in the lamprey was similar to that in the thyroid of the rat.

Partial clone of the gene for AS protein of the lamprey Petromyzon marinus, a member of the albumin supergene family whose expression is restricted to the larval and metamorphic phases of the life cycle

AS was previously found to be a liver-synthesized serum protein that is found in the larval (ammocoete), metamorphosing, and juvenile individuals during the life cycle of Petromyzon marinus but not in the sexually mature upstream-migrant individuals (Filosa et al. [1982] Comp. Biochem. Physiol., 72B:521-530; [1986] Comp. Biochem. Physiol., 83B:143-149; Ito et al. [1988] J. Exp. Zool., 245:256-263). In the present work, a partial clone for the gene for the AS protein was isolated from a cDNA expression library made from ammocoete liver. Northern blots using this clone showed hybridization with mRNA from the intervals of the life cycle prior to the upstream-migration period but not from the upstream-migration period itself. The cloned DNA was sequenced and the deduced amino acid sequence was found to have 40% identity with an albumin (our SDS-1 protein) from the upstream migrants of P. marinus (Gray and Doolittle, [1992] Protein Sci., 1:289-302), which is homologous to mammalian serum albumin. Thus the lamprey has two genes, AS and SDS-1, that code for different but similar albumin-like proteins, which predominate at different phases in its life cycle. It is suggested that AS protein, because it is present only at the earlier phases of the life cycle and because its gene is transcribed only during this same period, may be an early version of the alpha-fetoprotein (AFP) of mammals that is found only in the embryonic, fetal, and neonatal phase of their life cycle.

Dimethyl sulfoxide inhibits capping of surface receptors

The microfilaments of cellular slime mold cells are known to be dislocated from their attachment to the plasma membrane by 10% dimethyl sulfoxide (DMSO). We report here that 10% DMSO inhibited capping of lentil lectin receptors on the surface of vegetative cells of the cellular slime mold, Dictyostelium mucoroides. When the DMSO was washed from the cells, capping took place. Capping of surface immunoglobulin of mouse lymphocytes was also reversibly inhibited by 10% DMSO.