Ammonium uptake in carrot cell structures is influenced by pH-dependent cell aggregation

Semicontinuously grown wild carrot ( Daucus carota L.) cells were used in an investigation of the effect of culture medium pH on ammonium uptake in suspension cultures as a first step in exploring the relationship between pH and anthocyanin biosynthesis. In contrast to published data showing decreasing uptake rates with decreasing culture medium pH, ammonium-limited, semicontinuous carrot cell cultures showed a 25% greater ammonium uptake rate at pH 4.5 than at pH 5.5. When cells that had been grown semicontinuously in medium with a pH of 4.5 or 5.5 were grown in batch cultures at pH 4.5, 5.5 or 6.5 the ammonium uptake rates were those of the semicontinuous cultures, indicating that the pH of the batch culture medium had no effect on ammonium uptake rates over 7 days. The cell culture was composed of very small aggregates when it was grown semicontinuously in medium at pH 4.5, but was composed of large aggregates when it was grown semicontinuously in medium at pH 5.5. The aggregation/disaggregation of the cells was pH dependent, as changing the pH of the semicontinuous culture medium altered the extent of the aggregation. We conclude that the change in culture medium pH caused the cells to aggregate or disaggregate which in turn decreased or increased the rate of ammonium uptake from the medium.     

Goldfish cones secrete a two-repeat interphotoreceptor retinoid-binding protein

Vitamin A and fatty acids are critical to photoreceptor structure, function, and development. The transport of these nutrients between the pigment epithelium and neural retina is mediated by interphotoreceptor retinoid-binding protein (IRBP). IRBP, a 133-kDa (human) glycolipoprotein, is the major protein component of the extracellular matrix separating these two cell layers. In amphibians and mammals, IRBP consists of four homologous repeats of about 300 amino acids which form two retinol and four fatty acid-binding sites. Here we show that IRBP in teleosts is a simpler protein composed of only two repeats. Western blot analysis shows that goldfish IRBP is half the size (70 kDa) of IRBP in higher vertebrates. Metabolic labeling studies employing Brefeldin A taken together with in situ hybridization studies and the presence of a signal peptide show that goldfish IRBP is secreted by the cone photoreceptors. The translated amino acid sequence has a calculated molecular weight of 66.7 kDa. The primary structure consists of only two homologous repeats with a similarity score of 52.5%. The last repeats of human and goldfish IRBPs are 69.1% similar with hydrophobic regions being the most similar. These data suggest that two repeats were lost during the evolution of the ray-finned fish (Actinopterygii), or that the IRBP gene duplicated between the emergence of bony fish (Osteichthyes) and amphibians. Acquisition of a multirepeat structure may reflect evolutionary pressure to efficiently transport higher levels of hydrophobic molecules within a finite space. Quadruplication of an ancestral IRBP gene may have been an important event in the evolution of photoreceptors in higher vertebrates. Correspondence to: F. Gonzalez-Fernandez     

Generation of a membrane-bound, oligomerized pre-pore complex is necessary for pore formation by Clostridium septicum alpha toxin

Low-temperature inhibition of the cytolytic activity of alpha toxin has facilitated the identification of an important step in the cytolytic mechanism of this toxin. When alpha toxin-dependent haemolysis was measured on erythrocytes at various temperatures it was clear that at temperatures ≤15°C the haemolysis rate was significantly inhibited with little or no haemolysis occurring at 4°C. Alpha toxin appeared to bind to and oligomerize on erythrocyte membranes with similar kinetics at 4°C and 37°C. The slight differences in these two processes at 4°C and 37°C could not account for the loss of cytolytic activity at low temperature. At 4°C alpha toxin neither stimulated potassium release from erythrocytes nor formed pores in planar membranes. In contrast, at temperatures ≥25°C both processes proceeded rapidly. Pores that were opened in osmotically stabilized erythrocytes could not be closed by low temperature. Therefore, low temperature appeared to prevent the oligomerized complex from forming a pore in the membrane. These data support the hypothesis that alpha toxin oligomerizes into a membrane-bound, pre-pore complex prior to formation of a pore in a lipid bilayer.     

Homology modeling of histidine-containing phosphocarrier protein and eosinophil-derived neurotoxin: Construction of models and comparison with experiment

Homology modeling methods have been used to construct models of two proteins—the histidine-containing phosphocarrier protein (HPr) from Mycoplasma capricolum and human eosinophil-derived neurotoxin (EDN). Comparison of the models with the subsequently determined X-ray crystal structures indicates that the core regions of both proteins are reasonably well reproduced, although the template structures are closer to the X-ray structures in these regions—possible enhancements are discussed. The conformations of most of the side chains in the core of HPr are well reproduced in the modeled structure. As expected, the conformations of surface side chains in this protein differ significantly from the X-ray structure. The loop regions of EDN were incorrectly modeled—reasons for this and possible enhancements are discussed. © 1995 Wiley-Liss, Inc.     

Platelet activation studied by fluorescence polarization

Platelet activation was elevated by changes in the fluorescence anisotropy of the sulfhydryl-reactive fluorescent probe, (5-[2-(iodoacetyl) aminacetyl]aminonaphthalene-1-sulfonic acid. The membrane-permeable fluorophore was shown to bind to a multitude of cytoplasmic and membrane proteins. Platelets were stimulated by addition of thrombin, arachidonic acid or ADP under conditions that did not induce aggregation. A sudden increase in the fluorescence anisotropy, r of moderate degree (25-33%) occurred during the first 60 s after exposure of platelets to the aggregating agents and was sustained during the entire period of observation (15-18 min). Phenylmethylsulfonyl thrombin was unable to produce these changes in fluorescence anisotropy. Preincubation of platelets with colchicine reduced r within 30-60 s after platelets were exposed to thrombin. These findings are interpreted as an indication of a general decrease in the 'motional freedom' of the fluorophores and indirectly their ligand molecules.     

The determination of the separation of tyrosine-99 and tyrosine-138 in calmodulin: Radiationless energy transfer

The average separation of the phenolic groups of tyrosine-99 and tyrosine-138 has been measured by radiationless energy transfer between each tyrosine and the nitro derivative of the second tyrosine. A separation of 16.7 ± 0.7 Å was found in the absence of Ca²⁺ and 15.5 ± 0.7 Å in the presence of Ca²⁺.     

Location of a binding site for 1-anilinonaphthalene-8-sulfonate on calmodulin

The quenching by radiationless energy transfer of the ultraviolet fluorescence of Tyr-99 and Tyr-138 by bound 1-anilinonaphthalene-8-sulfonate (1,8-ANS) has been employed to determine the separation of a hydrophobic binding site of 1,8-ANS from each of the tyrosines. The results suggest that the dominant binding site is located in the N-terminal region of domain III.     

Effect of inhibitors of the lipoxygenase pathway on mouse myoblast fusion

In this study we examined the effects of inhibitors of the lipoxygenase and cyclooxygenase pathways on mouse myoblast fusion. The fusion of cloned mouse myoblasts was markedly inhibited, in a dose-dependent manner, when cells were cultured in medium supplemented with either phenidone (1-phenyl-3-pyrazolidione) or BW755c (3-amino-1-(3-tri-fluoromethylphenyl)-2-pyrazoline), drugs which have been reported to inhibit lipoxygenase and cyclo-oxygenase activities. Fusion was also inhibited when these cells were cultured in medium supplemented with esculetin (6,7-dihydroxycoumarin) which has been reported to inhibit lipoxygenase activity. Removal of the above inhibitors resulted in a return to control levels of fusion. Fusion was not demonstrably inhibited with aspirin (acetylsalicylic acid) and only inhibited to a minor extent with indomethacin (1-(p-chlorobenzoyl)-5-methoxy-2-methylindole-3-acetic acid); both of these drugs are inhibitors of cyclo-exygenase activity.     

Characterization of cyclic AMP-binding proteins in rat Sertoli cells using a photoaffinity ligand

Summary Protein-bound cyclic AMP (cAMP) levels in cultured rat Sertoli cells have been determined after exposure to follicle-stimulating hormone (FSH) and agents which elevate intracellular cAMP or mimic cAMP action. Changes in the content of protein-bound cAMP were correlated with changes in receptor availability determined by measuring [³H] cAMP binding. Using the photoaffinity analog of cAMP, 8-N₃ [³²P] cAMP, two major cAMP-binding proteins in Sertoli cell cytosol, with molecular weights of 47 000 and 53 000 daltons, were identified as regulatory subunits of type I and type II cAMP-dependent protein kinases, respectively. Densitometric analysis of autoradiograms demonstrated differential activation of the two isozymes in response to treatment with FSH and other agents. Results of this study demonstrate the value of measuring changes in protein-bound cAMP and the utility of the photoaffinity labeling technique in correlating hormone-dependent processes in which activation of cAMP-dependent protein kinase occurs.     

An Automated Technique for the Radiographic Determination of Bone Age

We have developed an automated procedure for determining bone-age in the monkey, Macaca fascicularis. The status of epiphysial junctions and sesamoids were determined from radiographs. Epiphyses were scored as either open or closed, and sesamoids were scored as either present or absent. Bone-age was calculated by a computer program that stored and used a data base of probabilities for epiphysial closure and sesamoid appearance. The simplicity of scoring, combined with the speed and accuracy of the calculations, make this technique potentially adaptable for use in a variety of primate species.