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Based on negative data, Douglas et al refuted earlier work by the O'Day laboratory on the role of pheromones in the sexual development of Dictyostelium discoideum. This letter addresses some of the issues with their study.

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Calmodulin (CaM) antagonists, trifluoperazine (TFP) or calmidazolium (R24571), dose-dependently inhibited cAMP and folic acid (FA) chemotaxis in Dictyostelium. Developing, starved, and refed cells were compared to determine if certain CaM-binding proteins (CaMBPs) and CaM-dependent phosphorylation events could be identified as potential downstream effectors. Recombinant CaM ([35S]VU-1-CaM) gel overlays coupled with cell fractionation revealed at least three dozen Ca(2+)-dependent and around 12 Ca(2+)-independent CaMBPs in Dictyostelium. The CaMBPs associated with early development were also found in experimentally starved cells (cAMP chemotaxis), but were different for the CaMBP population linked to growth-phase cells (FA chemotaxis). Probing Western blots with phosphoserine antibodies revealed several phosphoprotein bands that displayed increases when cAMP-responsive cells were treated with TFP. In FA-responsive cells, several but distinct phosphoproteins decreased when treated with TFP. These data show that unique CaMBPs are present in growing, FA-chemosensitive cells vs. starved cAMP-chemoresponsive cells that may be important for mediating CaM-dependent events during chemotaxis.

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The onset of maternal behavior is characterized by the action of certain hormones, neuropeptides and neurotransmitters and a concomitant increase in the expression of c-Fos in the medial preoptic area (MPOA) but the signaling events that lie between have not been characterized. Because several of these hormones, neuropeptides and neurotransmitters function by activating Ca(2+)/calmodulin (CaM) mediated signaling pathways, many of which can lead to c-Fos expression, the goal of the current work was to identify calmodulin binding proteins (CaMBPs) or specific CaM-dependent phosphoproteins that might be involved. Probing of SDS-PAGE gels of extracts from the hippocampus, parietal cortex, basolateral amygdala and MPOA with recombinant (35)S-VU1-calmodulin (CaM) revealed 30 Ca(2+)-dependent and 4-6 Ca(2+)-independent CaMBPs. Statistically significant maternal behavior-related decreases in four Ca(2+)-dependent CaMBPs ( approximately 31 kDa, 50% decrease; approximately 33 kDa, 32%; approximately 50 kDa, 35%; approximately 60 kDa, 33%) were observed specifically in the MPOA. Numerous proteins were phosphorylated in a Ca(2+) CaM-dependent manner with two (MWs approximately 61 Da, approximately 58 kDa) showing a lack of phosphophorylation only in the MPOA. The selective decrease in CaMBPs coupled with the absence of CaM-dependent phosphoproteins implies that changes in Ca(2+)/CaM-mediated signaling may mediate some of the MPOA-specific processes during the onset of maternal behavior in the rat.

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Western blot analyses reveal that calcineurin A (CNA), which is present in the hippocampus, basolateral amygdala, parietal cortex, and MPOA of virgin males and females, is undetectable only in the MPOA of primiparous females regardless of whether they had postpartum pup contact or not. In contrast, CNB was expressed at unchanging levels in the PC and MPOA. Similarly, G(alphao) and PKA(RI) were expressed at high levels in all of the brain regions of virgin males, virgin females, and primiparous females, supporting the concept that this loss of CNA is a specific event. Understanding how and why the expression of CNA, the sole neuronal Ca2+/CaM-dependent protein phosphatase, is down-regulated specifically in the MPOA of primiparous females may yield some insight into the signal transduction events that mediate the onset of mammalian maternal behavior.

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The optimal coordination between the new mammalian mother and her young involves a sequence of behaviors on the part of each that ensures that the young will be adequately cared for and show healthy physical, emotional, and social development. This coordination is accomplished by each member of the relationship having the appropriate sensitivities and responses to cues that characterize the other. Among many mammalian species, new mothers are attracted to their infants' odors and some recognize them based on their odors; they also respond to their infants' vocalizations, thermal properties, and touch qualities. Together these cues ensure that the mother will nurse and protect the offspring and provide them with the appropriate physical and stimulus environment in which to develop. The young, in turn, orient to the mother and show a suckling pattern that reflects a sensitivity to the mothers odor, touch, and temperature characteristics. This article explores the sensory, endocrine, and neural mechanisms that underlie this early mother-young relationship, from the perspective of, first, the mother and, then, the young, noting the parallels between them. It emphasizes the importance of learning and plasticity in the formation and maintenance of the mother-young relationship and mediation of these experience effects by the brain and its neurochemistry. Finally, it discusses ways in which the infants' early experiences with their mothers (or the absence of these experiences) may come to influence how they respond to their own infants when they grow up, providing a psychobiological mechanism for the inter-generational transmission of parenting styles and responsiveness.

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The role of protein kinase C (PKC) during fertilization in the model eukaryote Dictyostelium discoideum was studied. Inhibition of PKC activity using staurosporine, chelerythrine, and bisindoylmaleimide resulted in a dose-dependent decrease in gamete fusion without any detectable effect on cell morphology or growth. At 1.0 microM, staurosporine led to a greater than 90% inhibition of gamete fusion. In support of this, chelerythrine and bisindoylmaleimide at 10 microM inhibited gamete cell fusion by 98 and 99%, respectively. In all cases, subsequent removal of the inhibitor allowed for the completion of sexual development in a manner indistinguishable from untreated, control cultures. In contrast, the stimulation of PKC by the addition of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate at 5 nM resulted in a 56% enhancement of cell fusion. In order to identify PKC substrates that may regulate fertilization in D. discoideum, in vitro phosphorylation was carried out followed by SDS-PAGE. A number of proteins were phosphorylated, only one of which, a protein of about 50,000 M(r), appears to be a PKC substrate. In total, these results coupled with earlier work suggest that PKC functions as part of a calcium-mediated signaling pathway that regulates fertilization in D. discoideum, suggesting that the dual signaling pathway that regulates fertilization in higher eukaryotes may have evolved very early.

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Previous work has shown that GTPase function is essential for fertilization and cannibalistic phagocytosis during the sexual development of Dictyostelium discoideum. In this work, the importance of heterotrimeric G proteins during these events was established further using aluminum fluoride which inhibited both fertilization and cannibalistic phagocytosis, as well as the phagocytosis of bacteria by vegetative amoebae. Using distinct immune sera directed against the amino terminus and the carboxy terminus of mammalian G alpha s, we have provided unique evidence for a G alpha s subunit of approximately 55 kDa in D. discoideum (referred to as dG alpha s). Furthermore, this protein localizes to the membranes of fusing cells as well as to both vegetative and zygote giant cells, indicating that it might function during fertilization as well as during both vegetative (i.e., bacterial) and cannibalistic (i.e., amoebal) phagocytosis. During its down-regulation in nonphagocytic cells new isozymes of dG alpha s appear, suggesting that it may be posttranslationally modified. Having identified a putitive G alpha s homologue, this work has set the stage for further investigations into its function in Dictyostelium.

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The role of signal transduction during chemotaxis of Dictyostelium discoideum cells to cAMP and folic acid was investigated using a radial bioassay technique. The effects of signalling agonists were assessed by measuring the diameters of visible rings formed by the outward migration of amoebae up radial gradients of chemoattractant. This rapid and simple bioassay method yields chemotactic rates equivalent to more complex assay systems. In support of previous studies, chemotaxis toward both cAMP and folic acid was inhibited in a dose-dependent manner by LaCl3, EDTA, chlorotetracycline and A1F3, supporting the importance of calcium ions and G protein-mediated signalling in both chemotactic events. The work was extended by examining the effects of the protein tyrosine kinase inhibitor genistein. This agent inhibited chemotaxis to folate in a dose-dependent manner but had no observable effect on chemotaxis toward cAMP. The notion that phosphorylation of proteins on tyrosine residues is critical for chemotaxis to folic acid was supported by Western blotting experiments with monoclonal anti-phosphotyrosine antibodies which detected two candidate proteins of M(r) 52,000 and 38,000 in the membranes of folate-responsive amoebae. These two bands disappeared with starvation which leads to the loss of responsiveness of folic acid and the acquisition of responsiveness to cAMP. Time-lapse videomicrography also revealed some unique differences in chemotactic response. Starved cells responded to cAMP as individuals but feeding cells chemoattracted to folic acid on a populational basis. The ability to compare two different types of chemotaxis using a simple, rapid and accurate bioassay system should enhance future studies of chemotaxis in wild-type and mutant strains of D. discoideum.

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Studies with Li+, an inhibitor of phosphoinositide metabolism, demonstrated that an early response of Saccharomyces cerevisiae to a-factor pheromone was negatively affected by this cation. This was monitored by the production of beta-galactosidase from a reporter construct containing the promoter region of the yeast STE3 gene and the coding region of the E. coli LacZ gene. Growth and progression through the cell cycle were also affected by Li+ and analysis of budded/unbudded ratios revealed that Li+ caused yeast cells to arrest in G1. These data provide support for the role of inositol phosphates in the mating response and cell cycle of Saccharomyces cerevisiae.

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Dictyostelium discoideum spores can be activated to initiate germination either endogenously via a diffusible autoactivator, or exogenously via heat. Following activation, three successive stages of germination occur, the lag stage, spore swelling and amoebal emergence. A previous study [Lydan M. A. and Cotter D. A. (1994) FEBS Lett. 115, 137-142] has shown that spore swelling is dependent on the activity of calmodulin. In this study, the calmodulin antagonists trifluoperazine and calmidazolium inhibited autoactivation, but had no effect upon heat activation. These agents also inhibited amoebal emergence following either form of activation. The effects caused by the anti-calmodulin agents were specific to an inhibition of calmodulin function since agents which modulate the activity of protein kinase C had no effect upon spore germination. A calcium-dependent calmodulin-binding protein of about 64,000 M(r) may be associated with the process of autoactivation since it was only seen in those spores which respond to the autoactivator. Overall, this study provides evidence to show that calmodulin plays a regulatory role during autoactivation and amoebal emergence during spore germination in D. discoideum and provides evidence for the calmodulin-dependent mechanisms which mediate each of these phases of germination.

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Extensive protein phosphorylation occurs during all phases of spore germination in Dictyostelium discoideum. The developmental changes were prevented when germination was inhibited by inhibitors of calmodulin function. In addition, differences in patterns of phosphorylation were detected based upon the method of spore activation. Several phosphoproteins were lost in heat activated as compared to autoactivated spores. In spite of the fact that several aspects (i.e. autoactivation, emergence) are calmodulin-dependent, there was no evidence that calcium- or calmodulin-dependent protein kinase activity is present during any phase of spore germination. This suggests that other CaM-dependent processes mediate the diverse aspects of spore germination in D. discoideum.

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After fertilization, the zygote giant cell of Dictyostelium discoideum chemoattracts and subsequently engulfs hundreds of amoebae of the same species and strains from which it was derived. A pharmacological approach indicates that, while it may have some role, calcium is not the trigger for this cannibalistic phagocytic process. Of several agents that perturb intracellular calcium levels [A23187, LaCl, 8-diethylamino-octyl-3,4,5-trimethoxylbenzoate (TMB-8), and chlorotetracycline], only A23187 had an effect in reducing amoebal ingestion. In keeping with this, agents which interfered with downstream effectors of calcium function did not alter sexual phagocytosis. Calmidazolium and trifluoperazine, which inhibit calmodulin function, were ineffective, as were a protein kinase C inhibitor (staurosporine) and activator (phorbol 12-myristate 13-acetate). On the other hand, the nucleotide analogues GTP gamma S and GDP beta S both inhibited sexual phagocytosis indicating a role for GTP-binding protein activity at some stage in the process. Sub-fractionation of cells from non-phagocytic and phagocytic stage cell cultures followed by immunolocalization after SDS-PAGE and western blotting revealed a number of GTP-binding proteins in both the cell membrane and intracellular membrane fractions that might function during the events of sexual phagocytosis.

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A unique aspect of phagocytosis during the sexual cycle of Dictyostelium discoideum is the ability of the zygote giant cell (ZGC) to attract and engulf hundreds of amoebae of the same species. The work presented here is one of two initial attempts to understand the signal transduction pathways that are involved during this event. Our data indicate that the uptake of amoebae by the ZGCs is negatively modulated by 5'-AMP and adenosine (ADO). The hierarchical inhibition of phagocytosis by the ADO analogues, N-ethylcarboxyadiadenosine and phenyl isopropyl adenosine, argue that the phagocytosis of amoebae by ZGCs is mediated by an ADO receptor, which is similar to the purinogenic class of receptor, A2. As with the A2 receptor, stimulation of cells by ADO binding causes an up-regulation of cAMP synthesis thereby increasing the intracellular levels of cAMP. During the phagocytic phase of sexual development, 5'-AMP and ADO undergo marked and successive increases in amounts supporting their natural role in modulating phagocytosis. This negative modulation of phagocytosis by ADO is similar to that found in mammalian macrophages and may represent an evolutionary precursor to that regulatory process. Furthermore, the slowing of phagocytosis has important implications to sexual development of D. discoideum.

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Many calmodulin-dependent biological phenomena are regulated by calmodulin-dependent phosphorylation and dephosphorylation. In Dictyostelium discoideum, cell and pronuclear fusion during fertilization are both calmodulin-dependent biomembrane fusion events. Calmodulin-dependent phosphorylation and dephosphorylation activity was present in sexually developing D. discoideum cells suggesting it has a role during fertilization. In support of this, a discrete calmodulin-independent kinase was present only during cell and pronuclear fusion.

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The development changes in GTP-binding proteins and the regulation of their appearance by calcium ions were investigated during early sexual development in Dictyostelium discoideum. GTP gamma S strongly inhibited gamete cell fusion, while GDP beta S slightly augmented it, suggesting that G-proteins have a critical role in cell fusion. A 52-kDa protein recognized by an anti-GTP-binding site-specific immune serum, was abundant during calcium-dependent early sexual development but decreased in amount concomitant with cell fusion. This protein remained at high levels in Ca(2+)-deficient cultures, suggesting that its down-regulation is linked to the events of sexual development. Analysis of substrates for cholera and pertussis toxin-mediated [32P]ADP-ribosylation in D. discoideum extracts determined that the 52-kDa protein is a G-alpha subunit similar to mammalian Gs. The 52-kDa protein was also detected in vegetative, asexual amoebae, but diminished rapidly within the first 2 h of starvation. Together these data indicate that the 52-kDa protein functions during the growth phase and is lost upon entry into either the sexual or asexual developmental programs. The amounts of several lower molecular weight GTP-binding proteins, ranging from 21- to 28 kDa, increased during the stage of zygote differentiation and their increases were calcium dependent. These data provide the first analysis of G-proteins during sexual development of D. discoideum and lay the foundation for continued analysis of the signal transduction events mediating cell fusion and zygote differentiation.

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The calcium-dependent regulatory protein calmodulin (CaM) mediates diverse cellular functions via a large number of calmodulin-binding and -dependent proteins (CaMBPs). The use of [35S]calmodulin, labeled during its expression (VU-1-CaM) in Escherichia coli, visualized over 25 CaMBPs in Dictyostelium discoideum. Seven, with M(r)s of 155,000, 91,000, 85,000, 48,000, 46,000, 38,000, and 28,000, were present only during sexual development. In addition, intracellular calmodulin levels were low during gamete formation but rose during cell fusion in response to the presence of extracellular calcium. Thus, calmodulin appears to mediate gamete formation and fusion through two distinct mechanisms: first, via unique developmentally regulated CaMBPs, and, second, via the regulation of intracellular calmodulin levels. The identification of the CaMBP spectrin in sexually developing Dictyostelium cells suggests that this cytoskeleton/plasma membrane, crosslinking protein may function during biomembrane fusion in D. discoideum as it does in other organisms.

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Sexual development in Dictyostelium discoideum has many unique features making it an attractive eukaryotic model system for the study of biomembrane fusion and intercellular communication. The work presented here provides primary biochemical evidence for two distinct phases during early sexual development that appear to be defined by calcium-dependent gamete cell fusion. In addition, we introduce a novel procedure for the enrichment of zygote giant cells and use this method to define certain wheat-germ agglutinin binding glycoproteins which are specifically located in zygote giant cells and others which are markers for surrounding amoebae in the second phase of development. In addition, a G protein which is present in high amounts early in development is unique to giant cells in the second phase, suggesting a role in phagocytosis. Finally, alkaline phosphatase activity was found to mark the first phase of sexual development, suggesting a role in cell fusion. This contrasts with the patterns of alpha-mannosidase and beta-glucosidase activity that increase late in the second developmental phase, where they likely function in endocyte digestion during the cytophagic period. The developmental significance of these findings is discussed.

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To determine which glycoproteins may be critical to sexual development in Dictyostelium discoideum, cell samples from different developmental stages were separated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis and blotted to nitrocellulose. Concanavalin A (ConA) and wheat germ agglutinin (WGA) binding proteins were visualized on the blots using an immunochemical procedure employing peroxidase-antiperoxidase. ConA labelled at least 28 proteins, but only one band showed calcium-dependent changes in its expression. WGA bound at least 30 proteins and changes in several bands were observed that did not occur in calcium-deficient controls. Two WGA-binding glycoproteins which migrated at 200 and 166 kilodaltons (kDa), respectively, showed developmental changes associated with the time of cell fusion. One WGA-binding and one ConA-binding glycoprotein migrating at 130 and 126 kDa, respectively, appeared later during sexual development, in association with the phase of zygote differentiation. Several WGA- and ConA-binding glycoproteins decreased during sexual development, but were not affected by the absence of calcium ions. Tunicamycin (1 microgram/mL) inhibited cell fusion when added to sexual cultures prior to the appearance of the 166-kDa glycoprotein gp166. The effects of this inhibitor on development support the importance of glycoproteins to cell fusion during sexual development in D. discoideum.

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Biotin-dependent enzymes are involved in carboxylation, decarboxylation and transcarboxylation reactions. Here, we have used sodium dodecyl sulfate polyacrylamide gel electrophoresis and electroblotting followed by probing with avidin to identify biotin-containing polypeptides in Dictyostelium discoideum. Twenty biotinyl polypeptides were visualized, with a 23 kDa protein appearing transiently. Based upon the molecular mobility of the biotinyl polypeptides, D. discoideum may contain the biotin-dependent enzymes acetyl CoA carboxylase, proprionyl CoA carboxylase, pyruvate carboxylase, and 3-methylcrotonyl CoA carboxylase.

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