This study was designed to elucidate the strategies adopted by mudskippers to handle endogenous ammonia during aerial exposure in constant darkness. Under these conditions, specimens exhibited ...minimal locomotory activity, and the ammonia and urea excretion rates in both Periophthalmodon schlosseri and Boleophthalmus boddaerti decreased significantly. As a consequence, ammonia accumulation occurred in the tissues of both species of mudskipper. A significant increase in urea levels was found in the liver of P. schlosseri after 24h of aerial exposure, but no similar increase was seen in the tissues of B. boddaerti. It is unlikely that these two species of mudskipper detoxified ammonia to urea during aerial exposure since B. boddaerti does not possess a complete ornithine-urea cycle (OUC) and, although all the OUC enzymes were present in P. schlosseri, the activity of carbamoyl phosphate synthetase present in the liver mitochondria was too low to render the OUC functional for ammonia detoxification. Peritoneal injection of 15NH4Cl into P. schlosseri showed that this mudskipper was capable of incorporating some of the labelled ammonia into urea in its liver. However, aerial exposure did not affect this capability and did not induce detoxification of the accumulated ammonia to urea. Mudskippers exposed to terrestrial conditions and constant darkness did, however, show significant decreases in the total free amino acid content in the liver and blood, in the case of P. schlosseri and in the muscle of B. boddaerti. No changes in the alanine or glutamine content of the muscle were found in either species. Analyses of the balance between the reduction in nitrogenous excretion and the increase in nitrogenous accumulation further revealed that these two species of mudskipper were capable of reducing their protein and amino acid catabolic rates. Such adaptations constitute the most efficient way to avoid the build-up of internal ammonia, and would render unnecessary the detoxification of ammonia through energetically expensive pathways. This finding may be the first report of a teleost fish showing a reduction in proteolysis and amino acid catabolism in response to aerial exposure.
Two distinct patterns of microtubular sliding were observed in rat sperm flagellar axonemes. The particular pattern of sliding was determined by the extraction conditions used to prepare the sperm ...for axoneme disintegration. Sperm prepared by incubating concentrated suspensions of Triton X-100-extracted sperm at pH 9.0 disintegrated by extruding the doublets and outer dense fibers numbered 4 through 7 in response to Mg-ATP. Sperm prepared by incubating motile Triton X-100-extracted models at 37 degrees C for 1 to 3 hours extruded doublets and outer dense fibers 9, 1 and 2. Axonemes disintegrated by both regimens tended to have doublets 3 and 8 (with their corresponding outer dense fibers), as well as the central pair, in place. In numerous instances, the 3-central-8 complex with outer dense fibers 3 and 8 could be found isolated in midpiece sections prepared from both methods. The 3-central-8 partition was also sometimes seen in isolation in cross-sections of the principal piece where it remained attached to the fibrous sheath. The flagellar remnant produced by extrusion of fibers 4 through 7 under high pH conditions was generally straight or randomly curved. In contrast, the flagellar remnant produced by extrusion of the 9-1-2 bundle of fibers was most often curved into a hook in the midpiece region. While the hook-like configuration was not Ca(2+)-dependent, it may be based on a related mechanism. The sliding of the 9-1-2 group of fibers is a consequence of dynein-tubulin sliding between the 2 and 3 doublets. This sliding pattern appears to be preferentially activated in the motile sperm models in EGTA, but seldom if ever produced sliding in the high-pH-extracted models. We conclude that the 3-central pair-8 complex and associated outer dense fibers form an I-beam-like partition that does not participate in sliding, but acts as a structural foundation for organizing a planar beat. In addition, it is clear that preferential activation of certain dynein arms can be evoked, depending on the treatment regimen employed. This shows definitively that the types of microtubule sliding in the two bend directions are not identical.
Lake Michigan mottled sculpin, Cottus bairdi, exhibit a naturally occurring and unconditioned orienting response that can be triggered by both live prey and chemically inert vibrating spheres, even ...in blinded animals. CoCl(2)-induced reductions of the orienting response demonstrate that the lateral line is required for this behavior in the absence of non-mechanosensory cues (such as vision), but shed no light on the relative contributions of superficial and canal neuromasts to this behavior. To determine the relative roles of these two subsystems, we measured the frequency with which mottled sculpin oriented towards a small vibrating sphere before and after two treatments: (i) immersion of fish in a solution of gentamicin, an aminoglycoside antibiotic that damages hair cells in canal, but not superficial, neuromasts; and (ii) scraping the skin of the fish, which damages the superficial, but not the canal, neuromasts. To ensure that both superficial and canal neuromasts were adequately stimulated, we tested at different vibration frequencies (10 and 50 Hz) near or at the best frequency for each type of neuromast. At both test frequencies, response rates before treatment were greater than 70 % and were significantly greater than 'spontaneous' response frequencies measured in the absence of sphere vibration. Response rates fell to spontaneous levels after 1 day of gentamicin treatment and did not return to pre-treatment levels for 10-15 days. In contrast, response rates stayed approximately the same after superficial neuromasts had been damaged by skin abrasion. Scanning electron microscopy confirmed hair cell damage (loss of apical cilia) in canal, but not superficial, neuromasts of gentamicin-treated animals after as little as 24 h of treatment. The sensory epithelium of canal neuromasts gradually returned to normal, following a time course similar to behavioral loss and recovery of the orienting response, whereas that of superficial neuromasts appeared normal throughout the entire period. This study shows that the orienting response of the mottled sculpin is mediated by canal neuromasts.
Apicomplexan parasites possess a plastid-like organelle called the apicoplast. Most proteins in the Toxoplasma gondii apicoplast are encoded in the nucleus and imported post-translationally. T. ...gondii apicoplast proteins often have a long N-terminal extension that directs the protein to the apicoplast. It can be modeled as a bipartite targeting sequence that contains a signal sequence and a plastid transit peptide. We identified two nuclearly encoded predicted plastid proteins and made fusions with green fluorescent protein to study protein domains required for apicoplast targeting. The N-terminal 42 amino acids of the apicoplast ribosomal protein S9 directs secretion of green fluorescent protein, indicating that targeting to the apicoplast proceeds through the secretory system. Large sections of the S9 predicted transit sequence can be deleted with no apparent impact on the ability to direct green fluorescent protein to the apicoplast. The predicted transit peptide domain of the S9 targeting sequence directs protein to the mitochondrion in vivo. The transit peptide can also direct import of green fluorescent protein into chloroplasts in vitro. These data substantiate the model that protein targeting to the apicoplast involves two distinct mechanisms: the first involving the secretory system and the second sharing features with typical chloroplast protein import.
Protein phosphorylation and dephosphorylation systems modulate many cellular activities and have recently been implicated in the in vitro transport of newly synthesized proteins. Here we show that ...polarized transport from the Golgi to the plasma membrane in intact MDCK cells is regulated by phosphorylation-dephosphorylation. Transport is inhibited by the phosphatase inhibitor okadaic acid and is stimulated by the kinase inhibitor staurosporine. Stimulation of apical transport exceeds stimulation of basolateral transport by up to 5-fold. We also find that the G protein activator aluminum fluoride, which stimulates transport to the surface at low fluoride concentrations as previously reported, inhibits transport at higher concentrations. In the nonpolarized fibroblast cell line CV-1, neither staurosporine nor aluminum fluoride stimulates transport to the cell surface. Our results suggest that the phosphorylation-dephosphorylation system, like the G protein, may be involved in the specialized sorting process characteristic of polarized cells. We show some evidence that these two mechanisms of regulation may act through common intermediates.
When the mudskipper Periophthalmodon schlosseri was exposed to terrestrial conditions under a 12h:12h dark:light regime the fish could be very active, and levels of total free amino acids increased ...significantly in the muscle and plasma. Alanine levels increased threefold in the muscle, fourfold in the liver and twofold in the plasma. Similar phenomena were not observed in the more aquatic mudskipper, Boleophthalmus boddaerti. From these results, we concluded that P. schlosseri was capable of partial catabolism of certain amino acids to support activity on land. The amino groups of these amino acids were transferred directly or indirectly to pyruvate to form alanine. The resulting carbon chain was fed into the Krebs cycle and partially oxidized to malate, which could replenish pyruvate through the function of malic enzyme. This favourable ATP yield from partial amino acid catabolism was not accompanied by a net release of ammonia. Such an adaptation would be advantageous to P. schlosseri confronted with the problem of ammonia excretion during aerial exposure. Indeed, when P. schlosseri were forced to exercise on land after 24 h of aerial exposure, the alanine level in the muscles increased significantly, with no apparent change in glycogen content. In addition, there was no significant change in the ATP level and energy charge of the muscle. In contrast, when B. boddaerti were exercised on land, glycogen levels in the muscles decreased significantly and lactate levels increased. In addition, muscle energy charge was not maintained and the ATP level decreased significantly. Hence, it was concluded that when P. schlosseri were active on land, they were capable of using certain amino acids as a metabolic fuel, and avoided ammonia toxicity through partial amino acid catabolism. Such a strategy is the most cost-effective way of slowing down internal ammonia build-up without involving energy-expensive ammonia detoxification pathways. Furthermore, an examination of the balance between nitrogenous excretion and accumulation in a 70 g P. schlosseri revealed that degradation of amino acids in general was likely to be suppressed to slow down the build-up of ammonia internally. It is possible that such a strategy may be widely adopted, especially by obligatory air-breathing fishes, to avoid ammonia intoxication during aerial exposure.
Several studies have suggested that chondrocytes must have the capacity to internalize and degrade extracellular hyaluronan. In the present study we show direct evidence that hyaluronan is, in fact, ...endocytosed by chondrocytes and that the endocytosis is mediated via cell surface CD44/hyaluronan receptors. Cultures of bovine articular chondrocytes as well as rat chondrosarcoma chondrocytes were incubated with either fluorescein- or 3H-labeled hyaluronan. Intense binding and accumulation of labeled hyaluronan was visualized by fluorescence microscopy or bright-field/dark-field microscopy following autoradiography. Cell surface hyaluronan was removed with either trypsin or Streptomyces hyaluronidase in order to distinguish and quantify intracellular endocytosed hyaluronan. Labeled hyaluronan was visualized within small discrete intracellular vesicles distributed throughout the cytoplasm. Binding and endocytosis of fluorescein- or 3H-labeled hyaluronan was totally blocked by the addition of excess unlabeled hyaluronan or hyaluronan hexasaccharides, competitive inhibitors of hyaluronan/hyaluronan receptor interactions. Binding and endocytosis was also blocked by the addition of anti-CD44 monoclonal antibodies. Characterization of endocytosed 3H-labeled hyaluronan demonstrated that a significant portion of the hyaluronan was degraded by both the bovine articular and rat chondrosarcoma chondrocytes. Interestingly, a higher proportion of bound hyaluronan was internalized by the bovine chondrocytes. Therefore, hyaluronan receptor-mediated endocytosis and degradation of hyaluronan may provide a critical link to the maintenance and homeostasis of cartilage tissue.
The 43 kDa inositol polyphosphate 5-phosphatase (5-phosphatase) hydrolyses the signalling molecules inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4, ...5)P4) and thereby regulates cellular transformation. To investigate the role Ins(1,4,5)P3-mediated Ca2+ oscillations play in cellular transformation, we studied Ins(1,4,5)P3-mediated Ca2+ responses in cells underexpressing the 43 kDa 5-phosphatase. Chronic reduction in 43 kDa 5-phosphatase enzyme activity resulted in a 2.6-fold increase in the resting Ins(1,4,5)P3 concentration and a 4.1-fold increase in basal intracellular Ca2+. The increased Ins(1,4,5)P3 levels resulted in partial emptying (40%) of the Ins(1,4,5)P3-sensitive Ca2+ store, however, store-operated Ca2+ influx remained unchanged. In addition, Ins(1,4,5)P3 receptors were chronically down-regulated in unstimulated cells, as shown by a 53% reduction in 3HIns(1,4,5)P3 binding to microsomal receptor sites. Agonist stimulation with endothelin-1 resulted in the rapid rise and fall of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 levels, with no significant differences in the rates of hydrolysis of these second messengers in antisense- or vector-transfected cells. These studies indicate, in contrast to its predicted action, the 43 kDa 5-phosphatase does not metabolise Ins(1, 4,5)P3 and Ins(1,3,4,5)P4 post agonist stimulation. Cells with decreased 43 kDa 5-phosphatase activity exhibited spontaneous Ca2+ oscillations in the absence of any agonist stimulation, and increased sensitivity and amplitude of intracellular Ca2+ responses to both high and low dose endothelin-1 stimulation. We conclude the 43 kDa 5-phosphatase exerts a profound influence on Ins(1,4, 5)P3-induced Ca2+ spiking, both in the unstimulated cell and following agonist stimulation. We propose the enhanced Ca2+ oscillations may mediate cellular transformation in cells underexpressing the 43 kDa 5-phosphatase.
Previous studies have shown that the divalent-cation independent aggregation of simian virus 40-transformed 3T3 cells (SV-3T3) is mediated by the interaction of endogenous hyaluronate with binding ...sites on the cell surface. In the present study, the nature of the interaction of hyaluronate with the binding sites was characterized further by examining both the aggregation of SV-3T3 cells and the binding of exogenously added 3Hhyaluronate. The aggregation of SV-3T3 cells was found to be: (1) optimal at pH 7; (2) only slightly affected by variations in temperature between 0 and 40 degrees C; (3) more potently inhibited by the addition of high molecular weight preparations of hyaluronate than by lower molecular weight preparations; and (4) enhanced by increasing the ionic strength of the medium. With the exception of the effects of temperature, these characteristics correspond closely to those previously described for binding of 3Hhyaluronate. The effect of ionic strength on the binding of 3Hhyaluronate by SV-3T3 cells was found to correspond closely to its effect on lowering the viscosity of hyaluronate. Both effects (i.e. binding and reduction in viscosity) had similar dose-response curves for NaCl and MgCl2. This close correspondence between binding and the reduction of viscosity suggested that the effect of ionic strength on binding was due to a change in the structure of hyaluronate. Lowering the temperature (40 to 0 degrees C) also enhanced the binding of 3Hhyaluronate to SV-3T3 cells. However, this effect was blocked if the cells were first fixed with glutaraldehyde, suggesting that temperature mediates its effect through the binding sites. Additional experiments showed that the binding of 3Hhyaluronate by SV-3T3 cells could be prevented by preincubating the cells with several types of lectins.
Ammonia levels in various tissues of the marble goby Oxyeleotris marmoratus remained constant throughout a 72 h period of air exposure. The rate of ammonia excretion in these experimental fish ...decreased to approximately one-fifth of that of the submerged control. Ammonia was not converted to urea during air exposure because there were no significant increases in urea content in the tissues. Also, urea excretion rate was lowered to one-fiftieth that of the submerged fish. After 24 h of air exposure, there was a significant increase in muscle glutamine content, which peaked at 48 h. The increase in glutamine content could account for the decreases in the amounts of ammonia and urea excretion during air exposure. The specific activities of hepatic glutamate dehydrogenase (amination) and glutamine synthetase in these experimental fish increased threefold and thirtyfold, respectively, in comparison with the submerged controls. Thus, O. marmoratus appears to be the first known teleost that responds to air exposure by activating hepatic glutamine synthetase to detoxify internally produced ammonia.
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