β-Glucosidase activities and HCN liberation in unimbibed and imbibed seeds, and the induction of cocklebur seed germination by cyanogenic glycosides

In many seed species, the major source of HCN evolved during water imbibition is cyanogenic glycosides. The present investigation was performed to elucidate the role of endogenous cyanogenic glycosides in the control of seed germination and to examine the involvment of β-glucosidase in this process. All seed species used here contained some activities of β-glucosidase already in the dry state before imbibition. in the decreasing order of Malus pumila, Daucus carota, Hordeum vulgare, Chenopodium album and so on. β-Gluosidase activity in upper and lower seeds of cocklebur (Xanthium pennsylvanicum Wallr.) decreased with imbibition, and in lower seeds the activity disappeared when they germinated. On the contrary, in caryopses of rice (Oryza sativa L. cv. Sasanishiki) β-glucosidase increased during imbibition, and this increase continued even after germination. β-Glucosidase in cocklebur seeds was more active in the axial than in the cotyledonary tissue. Amygdalin, prunasin and linamarin could all serve as substrattes for the β-glucosidase(s) from both cocklebur and rice. Amygdalin, prunasin and linamarin as well as KCN, were effective in stimulating the germination of upper cocklebur seeds. The seeds evolved much more free HCN gas when they were exposed to the cyanogenic glycosides than when the glycosides were absent. Moreover, the application of the cyanogenic glycosides or of KCN caused accumulation of bound HCN in the seeds. Carbon monoxide, which stimulated cocklebur seed germination only slightly, did not cause accumulation of bound HCN. We suggest that a balance between the cytochrome and the alternative respiration pathways, which is adequate for germination (Esashi et al. 1987. Plant Cell Physiol. 28: 141–150), may be brought about by the action of endogenous HCN; a large portion of which is liberated from cyanogenic glycosides via the action of β-glucosidase. In addition to the partial suppression of the cytochrome path and unlike carbon monoxide, the HCN thus produced may act to supply cyanide group(s) to unknown compounds necessary for germination.     

Inhibition of ATP-regulated K+-channels by a photoactivatable ATP-analogue in mouse pancreatic β-cells

The effects of a photoactivatable (DMNPE-caged) ATP-analogue on ATP-regulated K⁺-channels (K_ATP-channel) in mouse pancreatic β-cells were investigated using the inside-out patch configuration of the patch-clamp technique. The caged precursor caused a concentration-dependent reduction of channel activity with a K_i of 17 μM; similar to the 11 μM obtained for standard Mg-ATP. The small difference in the blocking capacity between the precursor and ATP is probably the reason why no change in channel activity was observed upon photolysis of the caged molecule and liberation of ATP. It is suggested that the part of the ATP molecule involved in the blocking reaction of the K_ATP-channel is not sufficiently protected in DMNPE-caged ATP making this compound unsuitable for studying the rapid kinetics of ATP-induced K_ATP-channel inhibition.     

Characterization of high affinity GTPase activity correlated to β-adrenergic receptor stimulation of adenylyl cyclase in rat parotid membranes

β-Adrenergic receptor stimulation of adenylyl cyclase involves the activation of a GTP-binding regulatory protein (G-protein, termed here Gs). Inactivation of this G-protein is associated with the hydrolysis of bound GTP by an intrinsic high affinity GTPase activity. In the present study, we have characterized the GTPase activity in a Gs-enriched rat parotid gland membrane fraction. Two GTPase activities were resolved; a high affinity GTPase activity displaying Michaelis-Menten kinetics with increasing concentrations of GTP, and a low affinity GTPase activity which increased linearly with GTP concentrations up to 10 mM. The β-adrenergic agonist isoproterenol (10 μM) increased the V_max of the high affinity GTPase component approx. 50% from 90 to 140 pmol/mg protein per min, but did not change its K_m value (≈ 450 nM). Isoproterenol also stimulated adenylyl cyclase activity in parotid membranes both in the absence or presence of GTP. In the presence of a non-hydrolyzable GTP analogue, guanosine 5′-(3-O-thio)triphosphate (GTPγS), isoproterenol increased cAMP formation to the same extent as that observed with AlF₄^?. Cholera toxin treatment of parotid membranes led to the ADP-ribosylation of two proteins (≈ 45 and 51 kDa). Cholera toxin also specifically decreased the high affinity GTPase activity in membranes and increased cAMP formation induced by GTP in the absence or the presence of isoproterenol. These data demonstrate that the high affinity GTPase characterized here is the ‘turn-off’ step for the adenylyl cyclase activation seen following β-adrenergic stimulation of rat parotid glands.     

Formaldehyde-derived tetrahydroisoquinolines and tetrahydro-β-carbolines in human urine

Human urine samples were examined for the occurrence of formaldehyde-derived tetrahydroisoquinolines and tetrahydro-β-carbolines generated by condensation of the methanol oxidation product with biogenic amines. Positive results were obtained for the tryptamine condensation product 1,2,3,4-tetrahydro-β-carboline and the serotonine condensation product 6-hydroxy-1,2,3,4-tetrahydro-β-carboline as well as for the condensation products with tyramine, dopamine, adrenaline and noradrenaline 1,2,3,4-tetrahydroisoquinoline, 6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, N-methyl-4,6,7-trihydroxy-1,2,3,4-tetrahydroisoquinoline, 4,6,7-trihydroxy-1,2,3,4-tetrahydroisoquinoline, and the metabolite 6-methoxy-7-hydroxy-1,2,3,4-tetrahydroisoquinoline. Negative results were obtained for N-methyl-1,2,3,4-tetrahydroisoquinoline and 6,7-di-methoxy-1,2,3,4-tetrahydroisoquinoline, N-methyl-1,2,3,4-tetrahydro-β-carboline, 6-methyl-1,2,3,4-tetrahydro-β-carboline, and 6-methoxy-1,2,3,4-tetrahydro-β-carboline in samples of chronic alcoholics as well as in the urine of healthy volunteers. No correlation between alcohol ingestion or state of alcoholization could be demonstrated.     

Enzyme activity measurements on isolated organs of Brachionus plicatilis (Rotifera)

A method is described by which the integument of Brachionus plicatilis, together with its intracellular lamina, is quickly dissolved before other parts or tissues of the animal are destroyed. After removing the integument several parts of the body can be separated and fractionated in a more or less intact state by centrifugation in a Percoll gradient. The measurement of enzyme activities has indicated that this procedure might provide a way of localizing enzymes within the rotifer body.     

β-Endorphin like immunoreactivity of brain,pituitary gland and plasma of rats submitted to postnatal protein malnutrition: Effect of behavioral training

Wistar-derived rats were raised and maintained either on a normal- (25% casein) or on a low-protein (8% casein) diet until the age of 100 to 114 days. Both diets were isocaloric and contained an adequate supply of salts and vitamins. There were gross differences in body, brain and pituitary weight between the two groups. In addition, the brain and pituitary content of β-endorphin like immunoreactivity was lower in the protein malnourished rats, and three different forms of training (50 tone-footshock shuttle avoidance trials; 50 tones alone (habituation); 50 footshocks alone) caused a depletion of brain β-endorphin like immunoreactivity in the normal, but not in the malnourished rats. Footshock stimulation caused, in addition, a pituitary decrease and a plasma increase of β-endorphin like immunoreactivity, also restricted to the normal diet group. Performance in the habituation and in the shuttle avoidance tasks was similar in the two groups, despite the different responsiveness of their brain and pituitary β-endorphin systems to training and/or stimulation. In view of the possible involvement of these systems in learning suggested by these and by previous data, it seems likely that the neurohumoral regulation of habituation and avoidance learning may be different in rats submitted to protein malnutrition when compared to controls.     

Biosynthesis, processing and release of pro-opiomelanocortin related peptides in the intermediate lobe of the pituitary gland of the frog (Rana ridibunda)

The biosynthesis of pro-opiomelanocortin (POMC) and related peptides by the intermediate lobe of the pituitary gland was studied in the frog Rana ridibunda using the pulse-chase technique. Analysis of radioactive proteins by dodecyl sulfate polyacrylamide gel electrophoresis showed that during pulse incubations a 36,000 dalton (36K) glycosylated prohormone was synthesized. It disappeared slowly during chase incubations, giving rise to another glycosylated protein (Mr 18K), identified as the N-terminal fragment of POMC. This latter protein was secreted to the incubation medium. High performance liquid chromatography analysis of peptides synthesized during chase incubations revealed the biosynthesis of two peptides related to gamma-MSH, three peptides related to alpha-MSH, one endorphin-related and one CLIP-related peptides. These newly synthesized peptides were slowly secreted to the incubation medium. Among the alpha-MSH related peptides, only the des-N alpha-acetyl alpha-MSH form of the peptide was found to be present within the cells, in contrast to the incubation medium where the presence of des-N alpha-acetyl alpha-MSH and a modified alpha-MSH was demonstrated.     

A 250-kilodalton cellular protein is induced by progestins in two human breast cancer cell lines MCF7 and T47D

We have studied the effect of R5020, a synthetic progestin, on the biosynthesis of cellular proteins extracted from the MCF7 and T47D human breast cancer cells, using gel electrophoresis. R5020 stimulates the synthesis, as measured after [35S]-methionine labelling, and the accumulation, as shown by silver staining, of a protein of molecular weight approximately equal to 250,000. The increase of the labelled 250-kilodalton protein was rapid (3 hours) and after 3 days this protein represented approximately equal to 6% of the total cellular proteins (approximately equal to 1 microgram/150,000 cells). The induction of the 250-kilodalton protein was obtained by physiologically active concentrations of several progestins and high concentrations of 5 alpha-dihydrotestosterone but not by estradiol or dexamethasone. It was inhibited by R486 , a progestin antagonist, but not by flutamide, an androgen antagonist. These results indicate a mediation by the progesterone receptor. The 250-kilodalton protein appears to be an excellent probe to study in cell culture the mechanism of action of progestin on human cells.     

Brain CCK receptors are structurally distinct from pancreas CCK receptors

Brain and pancreas cholecystokinin (CCK) receptors differ markedly in their selectivity for CCK analogs. To determine the size and subunit structure of the brain CCK receptor and compare it to that of the pancreas, 125I-CCK33 was covalently cross-linked with ultraviolet light to its receptor on mouse brain particles and purified pancreatic plasma membranes. When CCK was crosslinked to brain membranes, a single consistent major labeled protein band of Mr = 55,000 was observed in both the presence and the absence of DTT. These data with brain receptors contrast to results with pancreatic receptors where two bands of Mr = 120,000 and 80,000 are labeled in the absence and presence of DTT, respectively. These studies indicate, therefore, that the brain and pancreas CCK receptors are structurally and functionally distinct.