Identification ofmyo-inositol monophosphates in mycelia ofPholiota nameko

Inositol monophosphates from the mycelia ofPholiota nameko were analyzed by gas chromatography. Mycelia were found to contain inositol-1-phosphate and inositol-2-phosphate isomers in a proportion of 7∶1, the amounts being 50 and 7.2 pmol/mg dry weight, respectively. We assume that inositol-2-phosphate is a hydrolysis product of the phytase reaction with phosphatase. It was also found that the amount of inositol monophosphates in the mycelia was affected by the concentration of inorganic phosphate in the medium.     

Immunohistochemical localization of the catecholamine-synthesizing enzymes,substance P and enkephalin in the human fetal sympathetic ganglion

Summary The human fetal sympathetic ganglia were studied using the indirect peroxidase-antiperoxidase PAP method for immunocytochemical demonstration of three catecholamine-synthesizing enzymes, tyrosine hydroxylase (TH), dopamine--hydroxylase (DBH) and phenylethanolamine-N-methyltransferase (PNMT) as well as the neuropeptides leucine (Leu⁵)-enkephalin and substance P. The neuroblasts of the ganglia showed intense peroxidase immunoreactivity for TH, moderate reaction to DBH, and no reaction to PNMT. The small intensely fluorescent (SIF) cells situated along the blood vessels also showed positive labelling for only two enzymes, TH and DBH. The immunocytochemical localization of these enzymes suggests that both neuroblasts and SIF cells synthesize noradrenalin. Neither the neuroblasts nor SIF cells showed a reaction to substance P, and only the SIF cells contained enkephalin-like immunoreactivity. The role of enkephalin in the noradrenalin-containing SIF cells is unknown, but may be related to neuromodulation of ganglionic transmission.     

Incorporation of axonally transported glycoproteins into axolemma during nerve regeneration

The insertion of axonally transported fucosyl glycoproteins into the axolemma of regenerating nerve sprouts was examined in rat sciatic motor axons at intervals after nerve crush. [(3)H]Fucose was injected into the lumbar ventral horns and the nerves were removed at intervals between 1 and 14 d after labeling. To follow the fate of the “pulse- labeled” glycoproteins, we examined the nerves by correlative radiometric and EM radioautographic approaches. The results showed, first, that rapidly transported [(3)H]fucosyl glycoproteins were inserted into the axolemma of regenerating sprouts as well as parent axons. At 1 d after delivery, in addition to the substantial mobile fraction of radioactivity still undergoing bidirectional transport within the axon, a fraction of label was already associated with the axolemma. Insertion of labeled glycoproteins into the sprout axolemma appeared to occur all along the length of the regenerating sprouts, not just in sprout terminals. Once inserted, labeled glycoproteins did not undergo extensive redistribution, nor did they appear in sprout regions that formed (as a result of continued outgrowth) after their insertion. The amount of radioactivity in the regenerating nerves decreased with time, in part as a result of removal of transported label by retrograde transport. By 7-14 d after labeling, radioautography showed that almost all the remaining radioactivity was associated with axolemma. The regenerating sprouts retained increased amounts of labeled glycoproteins; 7 or 14 d after labeling, the regenerating sprouts had over twice as much of radioactivity as comparable lengths of control nerves or parent axons. One role of fast axonal transport in nerve regeneration is the contribution to the regenerating sprout of glycoproteins inserted into the axolemma; these membrane elements are added both during longitudinal outgrowth and during lateral growth and maturation of the sprout.     

Interaction of ouabain with the Na(+) pump in intact epithelial cells

To determine the specificity and efficacy of [(3)H]ouabain binding as a quantitative measure of the Na(+) pump (Na(+), K(+)-ATPase) and as a marker for the localization of pumps involved in transepithelial Na(+)-transport, we analyzed the interaction of [(3)H]ouabain with its receptor in pig kidney epithelial (LLC-PK(1)) cells. When these epithelial cells are depleted of Na(+) and exposed to 2 muM [(3)H]ouabain in a Na(+)-free medium, binding is reduced by 90 percent. When depleted of K(+) and incubated in a K(+)- free medium, the ouabain binding rate is increase compared with that measured at 5 mM. This increase is only demonstable when Na(+) is present. The increased rate could be attributed to the predominance of the Na(+)-stimulated phosphorylated form of the pump, as K(+) is not readily available to stimulate dephosphorylation. However, some binding in the K(+)-free medium is attributable to pump turnover (and therefore, recycling of K(+)), because analysis of K(+)-washout kinetics demonstrated that addition of 2 muM ouabain to K(+)-depleted cells increased the rate of K(+) loss. These results indicate that in intact epithelial cells, unlike isolated membrane preparations, the most favorable condition for supporting ouabain binding occurs when the Na(+), K(+)-ATPase is operating in the Na(+)-pump mode or is phosphorylated in the presence of Na(+). When LLC-PK(1) cells were exposed to ouabain at 4 degrees C, binding was reduced by 97 percent. Upon rewarming, the rate of binding was greater than that obtained on cells kept at a constant 37 degrees C. However, even at this accelerated rate, the time to reach equilibrium was beyond what is required for cells, swollen by exposure to cold, to recover normal volume. Thus, results from studies that have attempted to use ouabain to eliminate the contribution of the conventional Na(+) pump to volume recovery must be reevaluated if the exposure to ouabain was done in the cold or under conditions in which the Na(+) pump is not operating.     

Effect of hydrocortisone on catecholamines and the enzymes synthesizing them in the developing sympathetic ganglion

Summary Newborn rats were daily injected with 0.2 mg hydrocortisone acetate for seven days. They were killed 1, 7 or 21 days after the last injection, together with untreated controls. Hydrocortisone caused a great increase in the number of the small, intensely fluorescent (SIF) cells and the appearance of similar small cells with intense immunohistochemical reactions for tyrosine hydroxylase (TH), dopamine--hydroxylase (DBH) and phenylethanolamine (noradrenaline)N-methyltransferase (PNMT) in the superior cervical ganglion. At the same time, the adrenaline content and the PNMT activity of the ganglion greatly increased, while no significant changes were observed in the dopamine or noradrenaline content or TH or DBH activity. All these changes essentially disappeared after a recovery period of seven or 21 days.It is concluded that hydrocortisone causes a temporary increase in the number of SIF cells by causing a synthesis of TH, DBH and PNMT in previously existing small, non-fluorescent cells, which start to synthesize and store adrenaline, thus becoming intensely fluorescent SIF cells. These SIF cells are different from the normal SIF cells of the same ganglion, most of which appear at a later stage of postnatal development when response to hydrocortisone is lost, which contain TH but neither DBH nor PNMT, and which permanently remain in the ganglion.     

Immunohistochemical localization of three catecholamine synthesizing enzymes: aspects on methodology

Summary Three enzymes in the catecholamine synthesis—dopa decarboxylase (DDC), dopamine--oxidase (DBH) and phenylethanolamine-N-methyltransferase (PNMT)—have been isolated. Subsequently antibodies to these enzymes were prepared and used in immunohistochemical studies mainly with the aim to elucidate methodological problems. The indirect immunofluorescent technique was used throughout the study.It was found that cryostat sectioning followed by fixation in acetone or alcohol, a standard procedure in immunohistochemistry, was successful only with antibodies to the granule bound enzyme DBH. With antibodies to DDC and PNMT, two cytoplasmic enzymes, on the other hand, the results were hampered by diffusion artefacts. These drawbacks could be prevented by a brief aldehyde fixation, preferably by perfusion before cryostat sectioning. The best results were obtained with formalin followed by hydroxyadipaldehyde and acrolein. However, after glutaraldehyde fixation no specific fluorescence at all was observed.Freezing of fresh adrenals, followed by freeze-drying, treatment with formaldehyde vapours and paraffin embedding was tested but consistent results were only obtained with antibodies to PNMT.A new instrument, the Vibratome^®, which allows sectioning of unembedded fixed or unfixed tissue, was used and successful results were obtained with all three antibodies. Furthermore, the possibility with this instrument to combine the immunohistochemical technique e.g. with the formaldehyde fluorescence method for visualization of monoamines is demonstrated. It is emphasized that the Vibratome ^® technique may be a valuable tool for immunohistochemical studies on the central nervous system.     

Monoamine-synthesizing enzymes in central dopaminergic, noradrenergic and serotonergic neurons. Immunocytochemical localization by light and electron microscopy.

The monoamine-synthesizing enzymes tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH) and tryptophan hydroxylase (TrH) were immunocytochemical localized in dopaminergic, noradrenergic and serotonergic neurons of rat brain by light and electron microscopy. In dopaminergic and serotonergic neurons, the respective synthesizing enzymes. TH and TrH, were distributed throughout the cytoplasm of the neuronal perikarya, dendrites, axons and terminals. The most selective accumulation of reaction product for the specific enzyme was associated: (a) in perikarya with endoplasmic reticulum, Golgi apparatus and microtubules, (b) in processes with microtubules, and (c) in terminals with dense granules or clear vesicles. The labeled terminals were characterized by their content of labeled organelles and the absence of synaptic junctions. In noradrenergic neurons, both TH and DBH were localized in the perikarya, similar to TH in dopamine neurons. TH and DBH differed in their localization within proximal axons and dendrites in that TH was associated with microtubules but DBH was not. These results provide ultrastructural evidence to suggest that monoamines may be: (a) synthesized by enzymes which are associated with different organelles depending on the portion of the neuron and the type of enzyme; (b) synthesized in both axons and dendrites and (c) released from terminals without postsynaptic membrane specializations.     

Selective overproduction of 5-enol-pyruvylshikimic acid 3-phosphate synthase in a plant cell culture which tolerates high doses of the herbicide glyphosate

Cultured cells of the higher plant Corydalis sempervirens Pers. which had been adapted to growing in the presence of 5 mM glyphosate (N-[phosphonomethyl]-glycine), a herbicide and a potent specific inhibitor of the shikimate pathway enzyme 5-enol-pyruvylshikimate-3-phosphate (EPSP) synthase, had a nearly 40-fold increased level of the extractable activity of EPSP synthase. Activities of five other shikimate pathway enzymes were, however, similar in the adapted and nonadapted cells, and the concentrations of the free aromatic amino acids in the two cell lines were also similar. EPSP synthases purified from glyphosate-adapted, as well as nonadapted cells, had identical physical, kinetic, and immunological properties, which indicated that the glyphosate-sensitive enzyme was overproduced in the adapted culture. Overproduction of EPSP synthase in the adapted culture was unequivocally established by two-dimensional polyacrylamide gel electrophoresis, as well as by one-dimensional sodium dodecyl sulfate-gradient gel electrophoresis and quantitation of EPSP protein by immunoassay after transfer to nitrocellulose membranes. While about 0.06% of the total soluble protein from nonadapted cells was EPSP synthase protein, the proportion was 2.6% in the adapted cells. In vivo pulse-labeling experiments with [35S]methionine established that the adapted cells have an increased rate of EPSP synthase protein synthesis.