Phagocytosis in Dictyostelium discoideum is inhibited by antibodies directed primarily against common carbohydrate epitopes of a major cell-surface plasma membrane glycoprotein

Using a water-soluble, reversible biotinylating reagent, we retrieved three surface-exposed proteins from a complex mixture of crude membrane proteins. The compound, sulfosuccinimidyl 2-(biotinamido)ethyl-1-3'-dithiopropionate (sulfo-NHS-SS-biotin), which has a cleavable disulfide bond, was used to label Dictyostelium discoideum amebae. Cells were lysed and a crude membrane preparation was isolated and solubilized with Triton X-100. Biotinylated molecules were bound to immobilized streptavidin and then eluted from the affinity matrix with dithiothreitol. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that out of the original complex mixture of detergent-solubilized membrane proteins, three major species at 130, 100, and 77 kDa were specifically bound and eluted with thiol reagents. These three proteins were glycoproteins (gp) since they bound concanavalin A. As demonstrated by one-dimensional peptide mapping, the retrieved gp130 and gp100 also were present in specialized plasma membrane subdomains called contact regions which are regions of cell-cell cohesion isolated from aggregated, developed amebae. This finding provides preliminary evidence that the two proteins may be involved in cell-cell interactions during both the vegetative and aggregation stages of the D. discoideum life cycle. The retrieved gp130 species has a relative mobility on SDS-gels similar to that of gp126, a surface-exposed glycoprotein. gp126 has been suggested to play roles both as a phagocytosis receptor and as a cohesion molecule (C.M. Chadwick, J.E. Ellison, and D.R. Garrod, (1984) Nature (London) 307, 646). To test if the retrieved gp130 was the same as gp126, a polyclonal antiserum was raised against gel-purified, endoglycosidase F-treated gp130. The immune serum recognized epitopes, apparently carbohydrates, present on many D. discoideum membrane proteins. Univalent IgG fragments from this antiserum inhibited phagocytosis, suggesting that anti-carbohydrate activity was responsible for the functional inhibition of phagocytosis.     

Infrequent translation of a nonsense codon is sufficient to decrease mRNA level

In many organisms nonsense mutations decrease the level of mRNA. In the case of mammalian cells, it is still controversial whether translation is required for this nonsense-mediated RNA decrease (NMD). Although previous analyzes have shown that conditions that impede translation termination at nonsense codons also prevent NMD, the residual level of termination was unknown in these experiments. Moreover, the conditions used to impede termination might also have interfered with NMD in other ways. Because of these uncertainties, we have tested the effects of limiting translation of a nonsense codon in a different way, using two mutations in the immunoglobulin mu heavy chain gene. For this purpose we exploited an exceptional nonsense mutation at codon 3, which efficiently terminates translation but nonetheless maintains a high level of mu mRNA. We have shown 1) that translation of Ter462 in the double mutant occurs at only approximately 4% the normal frequency, and 2) that Ter462 in cis with Ter3 can induce NMD. That is, translation of Ter462 at this low (4%) frequency is sufficient to induce NMD.     

Recent advances in mechanisms regulating glucose oxidation at the level of the pyruvate dehydrogenase complex by PDKs

The mitochondrial pyruvate dehydrogenase complex (PDC) catalyzes the oxidative decarboxylation of pyruvate, linking glycolysis to the tricarboxylic acid cycle and fatty acid (FA) synthesis. Knowledge of the mechanisms that regulate PDC activity is important, because PDC inactivation is crucial for glucose conservation when glucose is scarce, whereas adequate PDC activity is required to allow both ATP and FA production from glucose. The mechanisms that control mammalian PDC activity include its phosphorylation (inactivation) by a family of pyruvate dehydrogenase kinases (PDKs 1-4) and its dephosphorylation (activation, reactivation) by the pyruvate dehydrogenase phosphate phosphatases (PDPs 1 and 2). Isoform-specific differences in kinetic parameters, regulation, and phosphorylation site specificity of the PDKs introduce variations in the regulation of PDC activity in differing endocrine and metabolic states. In this review, we summarize recent significant advances in our knowledge of the mechanisms regulating PDC with emphasis on the PDKs, in particular PDK4, whose expression is linked with sustained changes in tissue lipid handling and which may represent an attractive target for pharmacological interventions aimed at modulating whole body glucose, lipid, and lactate homeostasis in disease states.     

Developmental changes in the spatial expression of genes involved in myosin function in Dictyostelium

We analyzed the spatial expression patterns of the genes involved in myosin function by in situ hybridization at the tipped aggregate and early culmination stages of Dictyostelium. Myosin heavy chain II mRNA was enriched in the anterior prestalk region of the tipped aggregates, whereas it disappeared from there and began to appear in both upper and lower cups of the early culminants. Similarly, mRNAs for essential light chain, regulatory light chain, myosin light chain kinase A, and myosin heavy chain kinase C were enriched in the prestalk region of the tipped aggregates. However, expression of these genes was distinctively regulated in the early culminants. These findings suggest the existence of mechanisms responsible for the expression of particular genes.     

Identification of a membrane glycoprotein found primarily in the prelysosomal endosome compartment

Cells contain an intracellular compartment that serves as both the "prelysosomal" delivery site for newly synthesized lysosomal enzymes by the mannose 6-phosphate (Man6P) receptor and as a station along the endocytic pathway to lysosomes. We have obtained mAbs to a approximately 57-kD membrane glycoprotein, (called here plgp57), found predominantly in this prelysosomal endosome compartment. This conclusion is supported by the following results: (a) plgp57 was primarily found in a population of late endosomes that were located just distal to the 20 degrees C block site in the endocytic pathway to lysosomes (approximately 83% of the prelysosomes were positive for plgp57 but less than 5% of the early endosomes had detectable amounts of this marker); (b) plgp57 and the cation-independent (CI) Man6P receptor were located in many of the same intracellular vesicles; (c) plgp57 was found in the membranes of an acidic compartment; (d) immunoelectron microscopy showed that plgp57 was located in characteristic multilamellar- and multivesicular-type vacuoles believed to be prelysosomal endosomes; and (e) cell fractionation studies demonstrated that plgp57 was predominantly found in low density organelles which comigrated with late endosomes and CI Man6P receptors, and only approximately 10-15% of the antigen was found in high density fractions containing the majority of secondary lysosomes. These results indicate that plgp57 is a novel marker for a unique prelysosomal endosome compartment that is the site of confluence of the endocytic and biosynthetic pathways to lysosomes.     

Biosynthesis of 117 antigen: a cell cohesion molecule in Dictyostelium discoideum

117 antigen is involved in the process of intercellular cohesion in Dictyostelium discoideum [Brodie et al., 1983]. The antigen, a 69- and 72-kDa doublet, was found to arise from a 60- and 62-kDa precursor. The mature antigen contains N-linked oligosaccharides that are sulfated and fucosylated [Sadeghi et al., 1987]. These oligosaccharide chains are resistant to endoglycosidase H digestion. 117 antigen also contains a post-translationally added carbohydrate-containing modification(s). Unlike the N-linked oligosaccharide, this carbohydrate moiety is sensitive to periodate oxidation. 117 antigen is developmentally regulated, and the changes in rate of 117 antigen synthesis reflect changes in the cellular levels of its mRNA. 117 mRNA accumulates in starving cells and reaches its maximum when cells become aggregation competent. The mRNA levels then decline, and by the time the slug structure is formed, no 117 mRNA is present. 117 mRNA reaccumulates for a brief period during early culmination and then returns to an undetectable level.     

Developmental regulation of a prestalk- and stalk-enriched protein in Dictyostelium discoideum

Abstract. Monoclonal antibodies reactive with proteins specifically present either in the prespore cells or the prestalk cells of Dictyostelium discoideum were obtained. Four of them recognized prespore-enriched proteins, as shown by both immunoblotting assays and immunofluorescent staining. The other monoclonal antibody ( mab150 ) produced more than 10 protein bands when reacted with both prespore and prestalk cell extracts in immunoblotting assays. However, a protein band with molecular weight 35 000 (st35) was specifically detected in prestalk cells as well as mature stalk cells. St35 was solubilized from the Triton X-100 insoluble fraction of mature stalks by sodium dodecyl sulfate (SDS). The purified sample gave a single spot on two-dimensional gel electrophoresis, with pI of 5.0. During development, st35 first appeared at the tipped aggregate stage and accumulated up to stalk-cell formation without modification. The protein was not lost even when slugs were disaggregated. The importance of the tipped aggregate stage for prestalk differentiation as well as prespore differentiation is discussed.     

Developmental regulation of a stalk cell differentiation-inducing factor in Dictyostelium discoideum

Previous work has shown that cells developing at high density release a low-molecular-weight factor that can induce isolated Dictyostelium discoideum amoebae of strain V12M2 to differentiate into stalk cells in the presence of cyclic AMP. We now show that this differentiation-inducing factor, called DIF, can be extracted from cells during normal development and that its production is strongly developmentally regulated. DIF is not detectable in vegetative cells but rises dramatically after aggregation to reach a peak during slug migration. DIF levels are very low in two mutants defective in aggregation. The postaggregative synthesis of DIF is stimulated by the addition of extracellular cyclic AMP. We propose that DIF is a morphogen controlling prestalk cell differentiation.     

Baroreflex mechanisms regulating mean level of SNA differ from those regulating the timing and entrainment of the sympathetic discharges in rabbits

The arterial baroreflex pathway provides the fundamental basis for the short-term control of blood pressure via the rapid regulation of the mean level of sympathetic nerve activity (SNA) in response to changes in blood pressure. A central tenet in the generation and regulation of bursts of SNA is that input from the arterial baroreceptors also regulates the timing of the bursts of sympathetic activity. With the use of an implantable telemetry-based amplifier, renal SNA was recorded in intact and arterial baroreceptor-denervated (SAD) conscious rabbits. Data were collected continuously while animals were in their home cage. Mean levels of SNA were not different between SAD and baroreceptor-intact animals. Whereas SNA was unresponsive to changes in blood pressure in SAD rabbits, the timing of the bursts of SNA relative to the arterial pulse wave was maintained (time between the diastolic pressure and the next maximum SNA voltage averaged 107+/-12 ms SAD vs. 105+/-7 ms intact). Transfer function analysis between blood pressure and SNA indicates the average gain at the heart rate frequency was not altered by SAD, indicating strong coupling between the cardiac cycle and SNA bursts in SAD animals. Further experiments in anesthetized rabbits showed that this entrainment is lost immediately after performing baroreceptor denervation surgery and remained absent while the animal was under anesthesia but returned within 20 min of turning off the anesthesia. We propose that this finding indicates the regulation of the mean level of SNA requires the majority of input from baroreceptors to be functional; however, the regulation of the timing of the bursts in the conscious animal requires only minimal input, such as a sensitive trigger mechanism. This observation has important implications for understanding the origin and regulation of SNA.     

Tumor promoters and Kirsten sarcoma virus increase synthesis of a secreted glycoprotein by regulating levels of translatable mRNA

We have studied the effect of tumor promoters and viral transformation on the synthesis and secretion of a glycoprotein of molecular weight 35,000 by mouse fibroblasts in culture. The secretion of this protein (known as MEP) in large amounts by transformed cells has been previously described (Gottesman, 1978). We show in this paper that NIH/3T3 cells either transformed by the Kirsten sarcoma virus or treated with biologically active phorbol esters synthesize and secrete increased amounts of MEP. This is due to increased levels of translatable RNA for MEP, since translation in a reticulocyte lysate of total RNA purified from virus-transformed cells or from normal cells treated for 6 hr with 4-o-tetradecanoyl phorbol-13-acetate (TPA) reveals a dramatic increase in the synthesis in vitro of MEP when compared to untreated cells. Our evidence indicates that a tumor virus and a tumor promoter both regulate expression of a transformation-related function at a pretranslational level.     

Tactile stimulation-induced rapid elevation of the synaptophysin mRNA expression level in rat somatosensory cortex

Synaptophysin is an integral membrane protein abundant in the synaptic vesicle and is found in nerve terminals throughout the brain. It was recently suggested that synaptophysin is also involved in the modulation of activity-dependent synapse formation. In this study, we examined at the individual level whether tactile stimulation selectively influenced the synaptophysin mRNA expression level in the somatosensory cortex of rats. Anesthetized rats were caressed on the back by an experimenter's palms for 20 min and the mRNA expression levels in the somatosensory and the visual cortices 5 min afterwards were determined using quantitative PCR methodology. The synaptophysin mRNA expression level was selectively higher in the experimental group than in the control group in the somatosensory cortex but not in the visual cortex. This suggests that the mRNA expression level of synaptophysin induced by neuronal activity is related to the regulation of synapse formation or remodeling or both.     

Developmental costs of rapid growth in a damselfly

Abstract.  1. Developmental costs of rapid growth in terms of increased fluctuating asymmetry are expected to contribute to the widespread occurrence of growth rates below the physiological maximum, but have rarely been demonstrated. Here, these costs are studied for the first time in an invertebrate, the damselfly Lestes viridis , using a rearing experiment where early- and late-hatched larvae of both sexes were reared at decreasing or permanent water levels.
2. Late-hatched animals were more asymmetrical than early-hatched animals except for males in the drying treatment. Also, females were more asymmetrical than males except in early-hatched animals in the drying treatment.
3. The data presented suggest that in females but not in males treatment groups with higher growth rates have more asymmetrical wings. However, at the individual level no relationship between growth rate and asymmetry was present.
4. Possible reasons why the suggested trade-off between growth and developmental instability was not present at the individual level, and at the group level only in females, are discussed.