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.     

Aggressive behaviour of female prairie deer mice in laboratory populations

Prairie deer mice (Peromyscus maniculatus bairdii), living in asymptotic laboratory populations established two years earlier, were observed for agonistic responses to conspecific intruders. In the first experiment, intruders of six age-sex classes were placed into 10 of the populations for 10 min. The sex of the intruder did not influence the behaviour of the residents, but juveniles elicited more aggression than did adults. A second experiment revealed that female residents were responsible for almost all of the attacks upon juveniles. Experiment 3, in which the responses of pairs of deer mice to juvenile intruders were recorded, demonstrated that the aggressiveness of a female was enhanced by the presence of a male. In the final experiment, females were observed to be highly aggressive during the first few days after giving birth. The aggressive behaviour of the female deer mouse may have greater significance for population dynamics than that of the male.     

Effects of freeze-preservation on some pollen enzymes: II. Freezing and freeze-drying stresses

The isozymes of lily and corn pollen esterases and acid phosphatase were studied in relation to freeze-drying and vacuum-drying. Fresh samples of Lilium longiflorum L. and Zea mays L. pollen were frozen at rates ranging between 200 and 100 °C/min and freeze-dried at temperatures from 0 to ?70 °C for approximately 48 to 70 hr. Freeze-dried samples were rehydrated slowly (10% relative humidity) and rapidly (90% relative humidity). Vacuum-drying was performed at room temperature (22 °C).Soluble pollen enzymes were analyzed by disc electrophoresis on polyacrylamide gels stained with substrate specific reagents. The stained gels were evaluated by densitometry for migration rate, isozyme pattern, and relative activity. The numerical data generated in this manner were then statistically analyzed.The following conclusions resulted from this study: (i) freeze-drying and freeze-thawing treatments were comparable except for corn esterases; (ii) freeze-drying induced alterations in enzyme activity except for corn acid phosphatase; (iii) the freezing rate, the final freezing temperature, and exposure to various relative humidities produced few changes in freeze-dried material; (iv) freeze-drying was less detrimental than vacuum-drying to the enzyme characteristics of corn; (v) freeze-drying yielded higher viabilities than vacuum-drying; and (vi) acid phosphatase alterations appeared to be related to pollen viability in most cases.     

Half-lives of messenger RNA species during growth and differentiation of Dictyostelium discoideum

The half-lives of functional messenger RNAs were determined by a method employing the drugs actinomycin D and daunomycin for the inhibition of mRNA synthesis; the activity of extracted mRNAs was determined by an in vitro translation assay. Several controls indicated that this method yielded reliable values for mRNA half-lives; in particular, the declining rate of protein synthesis in the presence of the drugs is due predominantly to the decay of translatable mRNA. This method was used to determine the half-lives of two specific mRNAs—encoding actin and a protein of MW 51,000—as well as that of total cytoplasmic mRNA activity during growth and at several times in differentiation. The half-lives of at least these two mRNAs were shown to be distinctly different from that of the total mRNA population—about 4 hr. However, no significant change in any of these half-lives was observed between growing and developing cells. Therefore wholesale alterations in the degradation rates of total and at least specific messages do not appear to play a role in the regulation of gene expression during Dictyostelium development.     

Developmental changes in messenger RNAs and protein synthesis in Dictyostelium discoideum

The pattern of proteins synthesized at different stages of differentiation of the slime mold Dictyostelium discoideum was studied by two-dimensional polyacrylamide gel electrophoresis. Of the approximately 400 proteins detected during growth and/or development, synthesis of most continued throughout differentiation. Approximately 100 proteins show changes in their relative rates of synthesis. During the transition from growth to interphase, the major change observed is reduction in the relative rate of synthesis of about 8 proteins. Few further changes are noticeable until the stage of late cell aggregation, when production of about 40 new proteins begins and synthesis of about 10 is reduced considerably. Thereafter, there are few changes in the pattern of protein synthesis. Major changes in the relative rates of synthesis of a number of proteins are found during culmination, but few culmination-specific proteins are observed. In an attempt to understand the molecular basis for these changes, mRNA was isolated from different stages of differentiation and translated in an improved wheat germ cell-free system; the products were resolved on two-dimensional gels. The ratio of total translatable mRNA to total cellular RNA is constant throughout growth and differentiation. Messenger RNAs for many, but not all, developmentally regulated proteins can be identified by translation in cell-free systems. Actin is the major protein synthesized by vegetative cells and by early differentiating cells. The threefold increase in the relative rate of synthesis of actin during the first 2 hr of differentiation and the decrease which occurs thereafter can be accounted for by parallel changes in the amount of translatable actin mRNA. Most of the changes in the pattern of protein synthesis which occur during the late aggregation and culmination stages can also be accounted for by parallel increases or decreases in the amounts of translatable mRNAs encoding these proteins. It is concluded that mRNAs do not appear in a translatable form before synthesis of the homologous protein begins, and that regulation of protein synthesis during development is primarily at the levels of production or destruction of mRNA.     

Production of arachidonic acid metabolites by operationally defined macrophage subsets

The antitumor activity and arachidonic acid metabolism of operationally defined macrophage populations was examined. Macrophages from mice injected with (strain BCG) or with pyran-copolymer were cytotoxic for tumor cells. The major arachidonic acid metabolite of these cells was PGE₂. Neither resident nor elicited macrophages were cytotoxic. However, elicited macrophages as well as macrophages from BCG injected mice inhibited tumor cell growth. The production of arachidonic acid metabolites by elicited cells, while low initially, was followed by a rapid increase in PGE₂. The major metabolites of resident cells were PGE₂ and prostacyclin. The cAMP:cGMP ratio correlated with the metabolic activity of the cells.     

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.     

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.     

Metabolism of benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene in cultured human fetal aortic smooth muscle cells.

Cultured human fetal aortic smooth muscle cells derived from the abdominal aorta converted benzo[a]pyrene (BaP) and 7,12-dimethylbenz[a]anthracene (DMBA) $\text{via}$ cytochrome P-450-dependent monooxygenation to metabolites detectable by both a highly sensitive radiometric assay and high pressure liquid chromatography (HPLC). Cells incubated with ³H-BaP transformed this substrate primarily to phenols. ¹⁴C-DMBA was converted to metabolites that cochromatographed with 12-hydroxymethyl-7-methylbenz[a]anthracene, 7-hydroxymethyl-12-methylbenz-[a]anthracene, 7,12-dihydroxymethylbenz[a]anthracene, and trans-8,9-dihydrodiol-7,12-DMBA. Exposure of cells in culture to 13 μM 1,2-benz[a]anthracene resulted in increased oxidative metabolism of both BaP and DMBA. In the case of BaP, total phenol formation was increased, while with DMBA all metabilities detected by HPLC were increased. Support for the potential role of metabolism of polycyclic aromatic hydrocarbons by aortic smooth muscle cells in the etiology of atherosclerosis was obtained.     

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.     

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.     

Endocytosis in Chinese hamster fibroblasts : Inhibition by glucose

Endocytosis in Chinese hamster ovary fibroblasts was investigated by measuring the rate of uptake of ³H-sucrose, which is known to enter cells only by endocytosis. Serum, polyvinylpyrrolidone (PVP), adenosine triphosphate, insulin, and cyclic 3′,5′-adenosine monophosphate, all of which are known to increase the rate of endocytosis by other cell systems, had no effect on Chinese hamster fibroblasts. However, medium in which these cells had been maintained for several days, referred to as conditioned medium, had a profound effect on endocytosis. These cells endocytosed 3 to 5 times as rapidly in conditioned medium as in fresh medium. A logarithmic inhibition of this effect was observed with increasing -glucose concentrations, however, glucose-free medium did not produce as great an effect as conditioned medium. This suggests that these cells may endocytose in response to their nutritional requirements.     

Trall-following behaviour in two species of wolf spiders: Sensory and etho-ecological concomitants

The trail-following behaviour of male Lycosa rabida and L. punctulata (in response to draglines of females) was analysed by high-speed cinematography (36 to 180 frames/s). L. rabida exhibited two modes of following, while L. punctulata showed three modes. One mode, palpal-sliding, was common to both species. During all modes of trail-following both species utilized the medial surface of the palpal tarsus, the surface having the highest concentration of chemosensitive sensilla. Film analyses suggested that male L. rabida used mechanical cues more than did L. punctulata. A significantly higher number of chemosensitive sensilla in male L. punctulata was related to this species's greater reliance on chemical rather than mechanical cues, the reverse being true in L. rabida. Different micro-habitat preferences of the two species may have shaped the differential use of cues for trail-following.     

Synthesis of developmentally regulated proteins in Dictyostelium discoideum which are dependent on continued cell-cell interaction

In the previous paper we showed that the major changes in the pattern of protein synthesis during differentiation of Dictyostelium discoideum occur during the 4-hr period when the cells are forming tight, visible aggregates. During this time, synthesis of 10 discrete polypeptides made by preaggregation cells ceases or is reduced considerably, and synthesis of 40 new proteins is induced. Induction or cessation of synthesis of these proteins was parallelled by the appearance or disappearance of the corresponding messenger RNAs. In this paper we show that many of these changes are induced by continued cell-cell contact. None of these occurs in aggregation-competent cells kept in suspension culture, but changes do take place when such cells are allowed to form tight aggregates. Disaggregation of cells causes cessation of synthesis of “aggregation-stage” proteins and reinduction of synthesis of polypeptides characteristic of preaggregation cells.     

Inhibition of the development of Dictyostelium discoideum by isolated plasma membranes

The life cycle of Dictyostelium discoideum can be divided into two mutually exclusive phases: growth and development. A distinguishing characteristic of the two phases is the absence of intercellular communication during vegative growth, and the many forms of such interaction during development. We have investigated the role of the cell surface membrane during the aggregation and development of this organism. We have asked the question: Are there molecules on the cell surface which are necessary for aggregation, and if so, can they be isolated in a biologically active membrane preparation? Further, when do these molecules appear during normal development, and does the interaction between two neighboring cell surfaces signal the cell or affect their subsequent development in any way? We have been able to isolate a partially purified plasma membrane fraction which is capable of specifically blocking the aggregation of other cells. Additional characterization of this preparation suggests that isolated aggregation phase membranes display a new, or newly exposed, heat-stable component which is capable of interacting with vegetative cells in such a way as to halt development.