Inhibition of glucose phosphate isomerase by metabolic intermediates of fructose

1. Purified rabbit-muscle and -liver glucose phosphate isomerase, free of contaminating enzyme activities that could interfere with the assay procedures, were tested for inhibition by fructose, fructose 1-phosphate and fructose 1,6-diphosphate. 2. Fructose 1-phosphate and fructose 1,6-diphosphate are both competitive with fructose 6-phosphate in the enzymic reaction, the apparent K_i values being 1·37×10⁻³−1·67×10⁻³m for fructose 1-phosphate and 7·2×10⁻³−7·9×10⁻³m for fructose 1,6-diphosphate; fructose and inorganic phosphate were without effect. 3. The apparent K_m values for both liver and muscle enzymes at pH7·4 and 30° were 1·11×10⁻⁴−1·29×10⁻⁴m for fructose 6-phosphate, determined under the conditions in this paper. 4. In the reverse reaction, fructose, fructose 1-phosphate and fructose 1,6-diphosphate did not significantly inhibit the conversion of glucose 6-phosphate into fructose 6-phosphate. 5. The apparent K_m values for glucose 6-phosphate were in the range 5·6×10⁻⁴−8·5×10⁻⁴m. 6. The competitive inhibition of hepatic glucose phosphate isomerase by fructose 1-phosphate is discussed in relation to the mechanism of fructose-induced hypoglycaemia in hereditary fructose intolerance.     

SDSE: A software package to simulate the evolution of a pair of DNA sequences

An algorithm to simulate DNA sequence evolution under a generalstochastic model, including as particular cases all the previouslyused schemes of nucleotide substitution, is described. The simulationis carried out on finite, variable length, DNA sequences througha strict stochastic process, according to the particular substitutionrates imposed by each scheme. Five FORTRAN programs, runningon an IBM PC and compatibles, carry out all the tasks neededfor the simulation. They are menu driven and interfaced to thesystem through a principal menu. All sequence data files usedand generated by the SDSE package conform to the standard GenBankdatabase format, thus allowing the use of any sequence retrievedfrom this databank, as well as the application of other packagesto analyse, manipulate or retrieve simulated sequences. Received on August 23, 1988; accepted on November 15, 1988     

Investigation of the regulation of pigmentation in alpha-melanocyte-stimulating hormone responsive and unresponsive cultured B16 melanoma cells

In vitro melanocyte-stimulating hormone (MSH) stimulates melanogenesis in some, but not all, melanocytes and melanoma cells. In an attempt to explain this variation in response to alpha MSH, we examined cyclic adenosine monophosphate (cAMP) accumulation, tyrosinase activity, and melanin production in primary (1 degree) murine B16 melanoma cells and in two B16 cell lines (B16 F1 and B16 F10) that are known to respond to alpha MSH. In vivo all three B16 melanoma cell types produced pigmented tumours. In vitro alpha MSH increased tyrosinase activity and melanin content in the F1 and F10 cells but not in the B16 1 degree cells. alpha MSH, however, increased cAMP production in all three cell types, confirming that the inability of B16 1 degree cells to produce melanin in response to alpha MSH is not due to a lack of alpha MSH receptors or cAMP response to alpha MSH. Further, we present evidence for a separate pathway of melanogenesis that is independent of cAMP as calmodulin antagonists, which do not elevate cAMP, increased tyrosinase activity, and melanin production in both 1 degree and F1 cells.     

Isozyme differentiation among sibling species and among populations of the Echinogammarus berilloni-group (Crustacea, Amphipoda)

The sibling species of the Echinogammarus berilloni-group are endemic for the Iberian Peninsula and southern France. These species show wide morphological variability with some overlap in their dianostic characters making their distinction difficult. Reroductive isolation and enzmatic jivergence amon allopatric and sympatric populations of four species sharing the same chromosome number has been studied. The results show a clear genetic differentiation of E. longiserosus and E. calvus versus the other two species. However, E. margalefi and E. echinosetosus show no clear genetic differentiation between them, confirming their crose relationship. All four species often coexist in the same drainage system. Isozme analysis was employed to check the hypothesis Of Margalef that sympathy would occur age, long-term phenomena of speciation inside of a given basin with subsequent contact and overlap between the differentiated forms. Electrophoretic data were also used to determine whether one flow among gammarids populations exists. A model proosed by other authors according to which the heterozyosity decreases towards the headwaters foes not fit to the data we have obtained from E. calvus. Thus, populations of this species from sources and springs of the Duero basin show the hiFhest values of mean heterozygosity. The differentiation in this basin can be explained by drift. Migration between populations of different rivers is prevented by natural barriers. The lowest river stretches are without amhipods interrupting the gene flow amon populations. A correction between genetic and geographic fistances among subbasins and basins was found applying a double logarithmic model. A model of migration of E. calvus in the Duero basin is proposed on the basis of allelic frequencies and on the distribution of mean hetero-zygosities.     

Origins of myo-inositol tetrakisphosphates in agonist-stimulated rat pancreatoma cells. Stimulation by bombesin of myo-inositol 1,3,4,5,6-pentakisphosphate breakdown to myo-inositol 3,4,5,6-tetrakisphosphate

In the rat pancreatoma cell line, AR4-2J, three inositol tetrakisphosphate isomers were identified, (1,3,4,6), (1,3,4,5), (3,4,5,6), which were increased during activation of phospholipase C by bombesin. Two other isomers were identified, (1,4,5,6) and a fifth isomer which was either (1,2,3,4) or (1,2,3,6), which have not previously been detected in any cell type. To study the metabolic interrelationships between these compounds and inositol 1,3,4,5,6-pentakisphosphate in the intact cell, their turnover was assessed under different protocols of [3H]myo-inositol labeling; the inositol phosphates were labeled to near steady state or under conditions where either rapidly or slowly turning over inositol polyphosphates were preferentially labeled. The relative specific radioactivities of inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate, inositol 1,3,4-trisphosphate, and inositol 1,3,4,6-tetrakisphosphate were very similar in bombesin-stimulated cells, consistent with the pathway for the conversion of inositol 1,4,5-trisphosphate to the other three inositol polyphosphates. Compared with these inositol phosphates, the turnover of inositol 1,3,4,5,6-pentakisphosphate was slow. An accumulation of radioactivity into inositol 1,3,4,5,6-pentakisphosphate was observed only under labeling conditions where its relative specific radioactivity was substantially below that of inositol 1,3,4,6-tetrakisphosphate. This indicated that the precursor for de novo synthesis of inositol 1,3,4,5,6-pentakisphosphate was inositol 1,3,4,6-tetrakisphosphate. Bombesin stimulated the net breakdown of inositol 1,3,4,5,6-pentakisphosphate and increased the level of inositol 3,4,5,6-tetrakisphosphate; the relative specific radioactivities of these two compounds were similar under all conditions. These data led to the novel proposal that inositol 3,4,5,6-tetrakisphosphate is the product of inositol 1,3,4,5,6-pentakisphosphate breakdown. This reaction was apparently stimulated by a regulated change in the enzyme(s) which interconvert inositol 1,3,4,5,6-pentakisphosphate and inositol 3,4,5,6-tetrakisphosphate.     

Antigen-dependent transition of IgE to a detergent-insoluble form is associated with reduced IgE receptor-dependent secretion from RBL-2H3 mast cells

In mast cells, basophils, and the RBL-2H3 tumor mast cell model, crosslinking cell surface IgE-receptor complexes by multivalent ligands activates a signal transduction pathway that leads to the secretion of histamine, serotonin, and other inflammatory mediators. Receptor crosslinking in RBL-2H3 cells also changes cell surface morphology and increases F-actin assembly. Previously, Robertson et al. demonstrated that crosslinked IgE-receptor complexes become associated with the Triton X-100-insoluble fraction (the "cytoskeleton") of RBL-2H3 cells and raised the possibility that receptor-cytoskeletal association may be a required step in the stimulation of secretion. The studies reported here confirm by flow cytometry that crosslinking cell surface IgE by antigen induces the association of the crosslinked complexes with the detergent-insoluble fraction. Dose-response studies, also reported here, indicate that the detergent insolubility of the complexes does not correlate with secretion. Thus, secretion increases with antigen concentration to a maximum beyond which more antigen causes less, not more, secretion. There is little residual detergent-insoluble IgE at the concentrations of antigen that promote optimal secretion, whereas the association of IgE with the detergent-insoluble fraction is maximal at the high antigen concentrations that result in reduced secretion. The addition of monovalent hapten to reduce the amount of crosslinking caused by high concentrations of antigen increases secretion and simultaneously reduces the association of IgE with the detergent-insoluble fraction. Dihydrocytochalasin B, an inhibitor of antigen-stimulated actin polymerization, also increases the rate and extent of secretion and simultaneously delays the association of crosslinked IgE-receptor complexes with the detergent-insoluble fraction. From these data, we propose that the association of crosslinked IgE receptors with the detergent-insoluble fraction of RBL-2H3 cells increases with increased receptor crosslinking, is enhanced by antigen-induced actin polymerization, and is more likely related to the termination than the stimulation of secretion. The ligand-induced conversion of receptors to a detergent-insoluble form is not restricted to mast cells but occurs in a variety of cell types. Its general function may be to limit the generation or transmission of transmembrane signals.     

Morphological changes induced by the calcium ionophore A23187 in rat basophilic leukemia (2H3) cells.

RBL-2H3 cells have been widely used to study histamine release in vitro. It was previously shown that these cells undergo striking morphological changes after IgE-mediated secretion. The present study was undertaken to examine if the morphological changes were dependent on activation of the Fc epsilon receptor. Therefore, the cells were stimulated to release histamine by two different mechanisms: activation of the Fc epsilon receptor by antigen and treatment with the calcium ionophore A23187. Cell surface and cytoskeletal changes were examined by fluorescence microscopy and scanning electron microscopy after either IgE- or ionophore-mediated histamine release. After exposure of the cells to either secretagogue, the cells spread over the surface of the culture dish and underwent rearrangement of the cytoskeleton. In addition, scanning electron microscopy revealed that deep ruffles developed on the surface of the cells undergoing IgE-mediated release. The surface changes were not as pronounced with the ionophore. The distribution of the cytoskeletal elements was examined by immunofluorescence using FITC-phalloidin and antibodies against vimentin and tubulin. In unstimulated cells actin was localized at the cell periphery, just under the plasma membrane. In the stimulated cells it was associated with the cell periphery and concentrated in the surface ruffles. As the stimulated cells spread, intermediate filaments and microtubules became distributed throughout the cell body, but there was no obvious association with the membrane ruffles. These morphological changes were dependent on the presence of extracellular calcium and on the concentration of ionophore or antigen, and were also correlated with the amount of histamine released. Additionally, IgE-mediated stimulation led to increased uptake of the soluble-phase tracer Lucifer yellow, whereas stimulation with the ionophore A23187 showed no increase in Lucifer yellow internalization. Ionophore A23187 produced changes similar but not identical to those seen in the RBL-2H3 cells after IgE-mediated histamine release. The differences may be owing to the involvement of the Fc epsilon receptor in IgE-mediated secretion.