Effect of stimulus strength and adaptation on the thermotactic response of Dictyostelium discoideum pseudoplasmodia

The thermotactic responses of Dictyostelium discoideum strain HL50 and mutants derived from this strain have been characterized by curves of stimulus-strength vs response. With gradient midpoint temperatures of 16 and 24 °C, these curves are typical of those of a single response, i.e., the strength of the response increases with increasing stimulus strength until at some strength the response saturates. However, with a gradient midpoint temperature close to the transition from negative to positive thermotaxis, the sign of the thermotactic response depends on gradient strength. These observations support the hypothesis that the transduction pathways for positive and negative thermotaxis act concurrently and contain separable elements. An investigation of the adaptation of thermotaxis indicated that the stimulus-strength-dependence and midpoint-temperature-dependence of both thermosensory responses was altered by shifting the growth and development temperature.     

Separation of enzymatic activities in Dictyostelium discoideum by high-pressure gel permeation chromatography.

High-pressure gel permeation chromatography was used to separate the cyclic AMP phosphodiesterase and ATP pyrophosphohydrolase activities of Dictyostelium discoideum. Two types of column packings, with different functional groups on the silica-bonded carbon side chains, were used to separate the two activities in approximately the same amount of time and with the same elution pattern. Recovery of both activities was enhanced when acetate, rather than sulfate, was the mobile phase. This recovery of activity following chromatography at high pressure demonstrates that high-pressure gel permeation chromatography can be used for the purification of enzymes.     

The glycoproteins of Dictyostelium discoideum. Changes during development.

The glycoproteins of D. discoideum have been analyzed by direct binding of radio-iodinated lectins to SDS gels of the successive developmental stages. Compared with the total pattern of proteins, many changes are found in the glycoproteins during development. WGA reacts with few gel bands from the vegetative cells and most of these, including a very intense band at the top of the gel, are lost during the first few hours of development. Approximately half-way through the developmental cycle at least 14 new glycoproteins reacting with WGA begin to appear and progressively accumulate. In contrast, ConA labels many glycoproteins over the complete molecular weight range and most are unaffected during development. Lectins which bind fucose label a single component at the top of the gel of vegetative cells and this decreases rapidly as development begins. No other reactive gel bands are revealed by fucose-binding lectins until the final stages of spore and stalk formation, when four high molecular weight glycoproteins are detected. Lectins specific for terminal galactose residues and for N-acetyl-galactosamine, including the intrinsic lectins produced by D. discoideum during its development, failed to reveal any reactive glycoproteins.     

The effects of cell density and starvation on early developmental events in Dictyostelium discoideum

We show that removal of yeast extract and trypticase from growth medium is sufficient for induction of several key events which occur during the early stages of Dictyostelium differentiation: run-off of polysomes, the earliest known change in macromolecular metabolism; appearance of the cell surface cAMP receptor; and aggregation itself. Starvation of glucose has little effect on these parameters. These results are consistent with those of other investigators who showed that starvation only of amino acids will induce other activities associated with cAMP-mediated cell signaling and cell-cell adhesion. We show, in contrast, that other factors are involved in the increase in the relative rates of synthesis of three polypeptides very early in differentiation: actin, and two proteins (“45-min” proteins) which are synthesized only during the period of 45–90 min. The induction of synthesis of these three proteins and presumably, of their mRNAs, is not the result of starvation for glucose or amino acids but is the result of plating cells at high density. The increases in the synthesis of these proteins are dependent on the density at which cells are plated and do not occur at a density 75-fold lower than the density used in standard experiments. Cells growing at high density or near stationary phase do not show the induction of increased synthesis of actin or the “45-min” proteins. These experiments suggest that these early developmental changes may be dependent on a threshold level of a diffusible factor excreted early in development.     

cAMP regulation of cell differentiation in Dictyostelium discoideum and the role of the cAMP receptor

DNA polymerases and DNA ligases have been studied during development of the amphibian, axolotl. Three forms of DNA polymerase, I, II, and III, with sedimentation coefficients in sucrose of 9, 6, and 3.1 S, respectively, have been found in the axolotl egg. The activity of these three DNA polymerases is unchanged during early embryonic development. The activity of DNA polymerase III then increases significantly, beginning at the tailbud stage, while the activity of DNA polymerase II increases at the larval stage. DNA polymerase I does not show significant variations during this time. On the basis of their catalytic properties, it appears that DNA polymerases I and II are α-type DNA polymerases whereas DNA polymerase III is a β-type enzyme. Two different DNA ligases are found in the axolotl, one showing a sedimentation coefficient in sucrose of 8.2 S (heavy form) and the other, 6 S (light form). The 6 S enzyme is the major DNA ligase activity found in the egg before and after fertilization. Its activity then decreases during embryonic development. It can be observed again, as the only DNA ligase activity, in some adult tissues. The 8.2 S enzyme appears during the first division cycle of the fertilized egg, is present at all stages of embryonic development, and is absent from the adult tissues tested. Properties of the two DNA ligases at different stages of embryonic development have also been compared.     

Conformational behaviour of the architectural units of peptides and proteins. Assessment of current understanding by ab initio quantum mechanical methods and refinement of the dipeptide energy surface.

The conformational energy surfaces of analogues of the dipeptide unit of polypeptides and proteins are calculated by ab initio methods using extended basis sets.The calculations are not particularly sensitive to the choice of (extended) basis set.The calculations are shown to support a particular empirical method parameterized with respect to crystal data. Non-hydrogen bonded conformations agree to within 3 kcal mol^?1, even for conformations in which quite considerable degrees of atomic overlap occur.Hydrogen bonded conformations, are, however, in less satisfactory agreement and it is the ab initio calculations which appear to be at fault.A simple correction is applied to the ab initio energy for hydrogen bonded conformations, and with the use of the empirical energy surface a full quantum mechanical conformational energy map is interpolated for the alanyl dipeptide.The effect of flexibility in the peptide backbone is taken into account, and supports recent empirical findings that distortions in valence angles must be considered in calculations of the conformational behaviour of peptides.     

Effect of extracellular Ca2+ on the morphogenesis of Dictyostelium discoideum.

Effect of extracellular Ca²⁺ on the morphogenesis of the cellular slime mold Dictyostelium discoideum was examined on agar plate. The concentration of Ca²⁺ in agar plate was controlled by keeping the concentration of a chelating reagent EGTA constant and varying the concentration of total calcium. From experiments in which EGTA concentration was kept at 2.0 × 10^?3 M, it was found that by decreasing Ca²⁺ concentration the morphogenesis was modified so that development of the aggregating amebae into fruiting bodies was accelerated and the period of migrating slugs was shortened. Below 1.0 × 10^?3 M of Ca²⁺ concentration, the total number of aggregates initially increased with decreasing Ca²⁺ concentration, reached a maximum at about 3.0 × 10^?7 M of Ca²⁺ concentration and hereafter decreased with decreasing Ca²⁺ concentration. The number of mature fruiting bodies obtained at 36 h period after starvation depends on Ca²⁺ concentration and the total number of aggregates. The cell aggregation initiated at the same time period after starvation even at an extreme case of 1.0 × 10^?8 M of Ca²⁺ concentration as under enough Ca²⁺ supply, while the formation of mature fruiting body was seriously inhibited. These observation suggested that the cAMP-mediated cell aggregation in D. discoideum is a Ca²⁺-independent phenomena, although extracellular Ca²⁺ is necessary for the normal development of the aggregated amebae.     

ADP phosphorylation and glutamate oxidation in mitochondria isolated from Dictyostelium discoideum amoebae

Mitochondria have been isolated from $\text{D.}\text{discoideum}$ amoebae in which respiration is coupled to ADP phosphorylation. P:O ratios and respiratory control ratios have been obtained for a number of metabolites. In rat liver mitochondria, glutamate is oxidized almost exclusively by a respiration-dependent cyclic transamination pathway, in which glutamate is converted to aspartate. When $\text{D.}\text{discoideum}$ amoebae are incubated with glutamate alone, aspartate does not accumulate appreciably. Furthermore, when the mitochondria are incubated with glutamate plus malonate at a concentration sufficient to inhibit respiration, their utilization of glutamate is depressed only slightly. Thus, it appears that glutamate oxidation within the mitochondria of $\text{D.}\text{discoideum}$ amoebae does not, for the most part, proceed by the cyclic transamination pathway.     

The planar distributions of surface proteins and intramembrane particles in Acholeplasma laidlawii are differentially affected by the physical state of membrane lipids

We have studied the influence of changes in lipid organization on the planar distribution of two classes of membrane proteins: integral proteins which have amino groups exposed to labelling at the membrane surface by the biotin-avidin-ferritin procedure, and those proteins which penetrate the lipid bilayer sufficiently to be seen as intramembranous particles by freeze-fracture electron-microscopy.When the membranes are examined at temperatures below the lipid phase transition, the first class is dispersed and the second patched. At temperatures in the middle of the transition range, both classes are patched. At temperatures just above the phase transition the first class is dispersed and the second patched, and at temperatures well above the transition both classes are dispersed. Freeze-etch studies of avidin-ferritin-labeled membranes confirmed that the distribution seen by the labeling and the freeze-fracture techniques coexist in single membranes. Thus, there exist two distinct classes of membrane proteins with differential organizational responses to the lipid state.     

Analyses of cell surface and secreted proteins of primary cultures of mouse extraembryonic membranes

Procedures have been developed for primary culture of 13th day mouse parietal and visceral endoderm, yolk sac mesoderm, and amnion cells. We have analyzed cell surface and secreted proteins of these cultures by labeling the cells with radioactive iodine, glucosamine, or amino acids, and/or by immunofluorescence. Cell surface and secreted proteins of visceral endoderm, yolk sac mesoderm, and amnion cells resemble each other closely, whereas parietal endoderm cells are strikingly different. Unlike the other cell types, parietal endoderm cells synthesize and secrete substantial quantities of a protein tentatively identified as procollagen. These cells also secrete a number of other glycoproteins not observed in the media from the other cultures. It is proposed that the procollagen and one or more of the other unique, secreted glycoproteins are normally constituents of Reichert's membrane. Compared to the other cultures, parietal endoderm cells appear to be deficient in production of LETS protein. However, parietal endoderm—Reichert's membrane complexes analyzed by immunofluorescence directly after dissection from the uterus show an abundant association with LETS protein. It is not clear whether this LETS protein is actually synthesized by the parietal endoderm cells themselves. If so, it is possible that this protein is rapidly degraded after its secretion in parietal endoderm primary cultures. The studies reported here represent a first step in the characterization of cell surface properties of embryonic and extraembryonic cell types. The information already accumulated should be useful in investigations aimed at identification of cells derived from blastocysts and teratocarcinomas in vitro.     

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.     

Nuclear matrix as acceptor for cAMP-binding proteins

Using [³²P]-8-N₃-cAMP, a photoaffinity analog of cAMP, we have established that nuclear binding of cAMP is preferentially localized in the “nuclear matrix”. Two major radioactive bands corresponded to proteins of M_r 40 K and 50 K, and three minor bands to proteins of M_r 55, 150 and 200 K. Even though the molecular weight of the major nuclear binding proteins in the matrix are similar to those of the cytosolic cAMP binding proteins, the characteristics of the binding reaction in the nucleus were markedly different from those in the cytosol.     

Synthesis of 2,3,4-trihydroxy-L-phenylalanine from s-methyl-L-cysteine and pyrogallol by L-tyrosine phenol-lyase.

A trihydroxy derivative of phenylalanine was synthesized from S-methyl-L-cysteine and pyrogallol by the crystalline tyrosine phenollyase (L-tyrosine phenol-lyase (deaminating) EC 4. I. 99.2 formerly known as β-tyrosinase) from Escherichia intermedia. The product was isolated as its N-acetyl-triacetoxymethylester and identified as 2,3,4-trihydroxy-L-phenylalanine by the analyses of NMR, MS spectra and optical rotation.     

Activation of helper T cells by immune complexes.

Spleen cells from mice primed with dinitrophenylated human γ-globulin (DNP-HGG) did not mount a secondary anti-DNP response in diffusion chamber cultures upon stimulation with dinitrophenylated keyhole limpet hemocyanin (DNP-KLH). The same cells, however, responded to stimulation with DNP-KLH complexed with anti-KLH antibody of rabbit or mouse origin. There is an optimal antigen:antibody ratio at which the immune complexes (IC) must be formed for maximal activity. T cells are required for the immunogenic activity of IC, since T-cell-depleted cultures did not respond. It was found that IC made with carrier and anticarrier antibody stimulated the development of carrier-specific helper T cells in cultures of spleen cells, thymocytes, and nylon wool nonadherent spleen cells from nonimmune mice. In contrast, free carrier did not elicit helper T cells. IC made with carrier and the F(ab′)₂ fragment of anticarrier antibody were immunogenic, but those made with carrier and the Fab′ fragment of anticarrier antibody were not, suggesting that helper T-cell activation is triggered by crosslinking of antigen-specific surface receptors.     

Binding of radioiodinated human beta-endorphin to serum proteins from rats and humans, determined by several methods

Binding of immunoreactive radioiodinated human beta-endorphin (125I-beta-EP) to rat serum was demonstrated by gel filtration of 125I-beta-EP in pooled rat serum on Sephadex G-200. Two radioactive peaks associated with proteins eluted from the column. The first peak eluted at the void volume containing lipoproteins, alpha 2- and beta 2-macroglobulins, and the second peak at the fraction of albumin. Binding of 125I-beta-EP to albumin was directly proved by gel filtration of 125I-beta-EP in buffer containing 4% human serum albumin on Sephadex G-200. Equilibrium dialysis was not applicable to investigating the interaction of 125I-beta-EP with serum proteins, because of the intense nonspecific adsorption to the semipermeable membrane and the degradation of the peptide during dialysis. Therefore, in order to quantitatively evaluate the binding of 125I-beta-EP in sera from rats and humans, we utilized four other methods (ultrafiltration, charcoal adsorption, polyethylene glycol precipitation and equilibrium gel filtration). These methods corresponded well with each other and indicated 35-44% binding of 125I-beta-EP in rat serum. Binding of 125I-beta-EP in normal human serum was 36%, determined by ultrafiltration. Serum protein binding of 125I-beta-EP was concentration independent over the concentration range studied (1-1000 nM).     

Preparative separation of hemoglobins A and S by gel electrofocusing, using selective zone elution by gel transposition between suitable anolytes and catholytes.

Preparative electrofocusing on polyacrylamide gels has been limited, until recently, to excision of gel slices, diffusion, and collection of the slice diffusates. An advance was made by the introduction of a method of selective electrophoretic zone recovery by specific changes of anolyte (A. McCormick, L. E. M. Miles, and A. Chrambach, 1976, Anal. Biochem.75, 314–324). It was shown (a) that selective zone recovery could be achieved by transposition of the gels into either isoelectric ampholytes or charged buffers, (b) that it could be applied to the gram scale, and (c) that zone elution could proceed either continuously or discontinuously. The early study was, however, limited to a trivial model problem, the separation of hemoglobin from bovine serum albumin (BSA). The present study was an attempt to apply a similar selective zone recovery method to a more demanding separation problem, the separation of hemoglobin A from hemoglobin S as well as from other minor components contained in a sickle-trait human hemolysate. The study shows that selective electrophoretic zone elution from a electrofocusing gel 18 mm in diameter is capable of yielding hemoglobin A, separated from hemoglobin S, differing by only 0.2 pH units in isoelectric point. The recovery of hemoglobin A was 70%, with a load of 32 mg of hemoglobin mixture per gel, using discontinuous zone elution into a collection cup.