Trehalase activity and cyclic AMP content during early development of Mucor rouxii spores

Incubation of Mucor rouxii sporangiospores in complex medium under aerobic conditions resulted in a transient 20-fold increase in trehalase activity. Maximum activity was reached after 15 min. Simultaneously, the cyclic AMP (cAMP) content increased approximately eightfold, reaching a maximum within 10 min. Increases in trehalase activity and cAMP content were also observed under anaerobic conditions (CO2). The extent of trehalase activation and the changes in cAMP content, during both aerobic and anaerobic incubation, varied with the medium used. Trehalase was activated in vitro by a cAMP- and ATP-dependent process. An even faster activation was obtained when cAMP was replaced by the catalytic subunit of beef heart protein kinase. The coincidence of, and the correlation between, increased cAMP contents and trehalase activities support the involvement of a cAMP-dependent phosphorylation in the in vivo regulation of trehalase activity.     

Chitinase activity in germinating cells ofMucor rouxii

Chitinase activity in germinating cells (4 h cultures) ofMucor rouxii was studied. The enzyme activity was recovered in a high speed supernatant of cell homogenates. No activity was detected in the mixed membrane fraction or in the cell walls. Maximum activity was observed at pH 7.6 and at 30–35°C using the chromogenic assay with chitin azure. The latter was digested by GS-chitinase in a manner dependent on substrate concentration and time of incubation. As with other chitinases, GS-chitinase was much more effective against nascent than against preformed chitin. The main product of nascent chitin digestion was diacetylchitobiose, although significant amounts of the trimer were also detected in the hydrolyzates. Allosamidin, an insect and fungal chitinase inhibitor, strongly inhibited hydrolysis of nascent chitin but not of chitin azure by GS-chitinase. The drug failed to inhibit the germination and the ensuing growth of the fungus. Results are discussed in terms of the possible role of GS-chitinase in germination.Abbreviations UDP-GlcNAc uridine 5-diphospho-N-acetylglucosamine - GlcNAc N-acetylglucosamine - GlcNAcP N-acetylglucosamine phosphate - PMB phosphate-magnesium buffer - C₂ N, N-diacetylchitobiose - C₃ N, N, N-triacetylchitotriose - RVB remazol brilliant blue - DPC descending paper chromatography - MU(Ch)₃ 4-methylumbelliferyl-N - N N-triacetylchitotriose     

Evidence that a glucose-mediated rise in cyclic AMP triggers germination ofPilobolus longipes spores

Spores ofPilobolus longipes incubated in phosphate buffer were activated within 5–10 min following the addition of either glucose or 6-deoxyglucose. Cyclic AMP content increased in response to glucose or 6-deoxyglucose, and the increase consistently preceded spore activation. Dibutyryl cyclic AMP also caused activation. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) did not cause activation, but, when added to spores with a suboptimal level of 6-deoxyglucose, it amplified the signal to produce a large increase in activation. IBMX increased intracellular cyclic AMP levels when it was applied with 6-deoxyglucose, but had no effect when it was applied alone. Phosphodiesterase activities in cell extracts from dormant and activated spores were not significantly different. These results indicate that the rise in cyclic AMP that follows exposure to glucose may play an important role in triggering spore germination.     

Mucor rouxii ultrastructure: cyclic AMP and actin cytoskeleton

Summary. A comparative analysis of the effect of two compounds, dibutyryl–cyclic-AMP (dbcAMP) and latrunculin B, on the morphology and ultrastructure of the dimorphic fungus Mucor rouxii under aerobic growth conditions is presented. dbcAMP acts through the sustained activation of protein kinase A, and latrunculin B through the disruption of the actin cytoskeleton. Upon addition of these compounds to the growth medium at any stage of the germination process, cells lost polarised growth and switched to isodiametric growth. The effect was reversible. The morphologies, visualised by light microscopy or scanning electron microscopy (SEM), were alike. A switch from a rough to a smooth surface was observed by SEM when cells were repolarised by removal of the added compound. Ultrastructural changes under both conditions, as observed by transmission electron microscopy, were similar, the main feature being the enlargement of the cell wall, with irregular depositions, and detachment from the cell membrane. dbcAMP-treated cells showed a decrease in the number of glycogen granules compared with control and latrunculin B-treated cells. F-actin staining with fluorescein isothiocyanate–phalloidin showed that both dbcAMP- and latrunculin B-treated cells displayed a much lower fluorescence than control cells, with only a few pale plaques. The results suggest that the sustained activation of protein kinase A, which impairs polarised growth, might exert its effect through a modification of actin cytoskeleton organisation, very probably also involving an integrinlike pathway, as judged by the cell wall detachment and loss of cell adhesiveness of the dbcAMP-treated isodiametric cells. Correspondence and reprints: Departamento de Química Biológica, Pabellón 2, Piso 4, Ciudad Universitaria, Universidad de Buenos Aires, 1428 Buenos Aires, Argentina.     

Effects of cyclic nucleotides upon the germination ofMucor racemosus sporangiospores

This study describes the effects of exogenous cyclic nucleotides upon the germination of sporangiospores ofMucor racemosus in a defined medium containing a variety of carbon and nitrogen sources. Adenosine 3′5′-cyclic monophosphate (cAMP) enhanced germination, with maximal stimulation at concentrations greater than 5 mm.N⁶-Monobutyryl cAMP, 8-bromo-cAMP, and adenosine also stimulated germination but 5′-adenosine monophosphate (AMP) did not.N⁶,O^2′-Dibutyryl cAMP blocked spore germination in cellobiose, maltose, galactose, 2-ketoglutarate, leucine, and lysine, a result that was also obtained when sporangiospores were incubated in a glucose medium without an exogenous nitrogen source. In glucose, dibutyryl cAMP retarded but did not arrest germination. Dibutyryl cAMP effected rapid and synchronous germination in alanine, aspartate, glutamate, and glutamine media. We conclude that cAMP nonspecifically stimulated the rate of sporangiospore germination while dibutyryl cAMP has a specific stimulatory effect on the initiation of germination ofMucor sporangiospores.     

Nikkomycin-resistant mutants ofMucor rouxii: physiological and biochemical properties

We isolated three nikkomycin-resistant mutants of the dimorphic fungusM. rouxii which were physiologically characterized regarding their response to yeast-phase inducing conditions and their sensitivity to bacilysin. Mutant strains G21 and G23, showed a qualitatively normal, though delayed, dimorphic transition and partial cross-resistance to bacilysin. Mutant strain G27 showed an altered dimorphism, producing a high proportion (50%) of hyphal cells, and a wild-type sensitivity to bacilysin. Cell-free extracts from this mutant exhibited an activity of both basal and protease-activated chitin synthetase which was overexpressed as compared with the parental strain and mutants G21 and G23. Results are discussed in terms of the different genetic background of the mutants.Abbreviations NTG N-methyl-N-nitro-N-nitrosoguanidine - UDP-GlcNAc uridine 5-diphospho-N-acetylglucosamine - GlcNAc N-acetylglucosamine     

High calcium content in Streptomyces spores and its release as an early event during spore germination.

The metal ion content of spores of five Streptomyces species was studied. A general feature of this study was the finding of a very high calcium content (1.1 to 2.1% of the dry weight). Accumulation of calcium occurred preferentially during the sporulation process. Spore calcium was located in the integument fraction, and more than 95% of the calcium was removed from intact spores by ethylene glycol-bis(beta-aminoethyl ether)-N,N-tetraacetic acid. Several divalent cations (Mg2+, Mn2+, Zn2+, and Fe2+) which induced darkening of spores and loss of heat resistance also caused the release of calcium from spores. In addition, darkening of spores was blocked by metabolic inhibitors, whereas calcium excretion was not affected. Two different categories of events in the initiation of germination may be differentiated; first, calcium release from spores which is not energy dependent and is a consequence of triggering of germination by some divalent cations, and second, some other events including loss of heat resistance, loss of spore refractility, and a decrease in absorbance, with at least one energy-dependent step.     

Occurrence of cyclic AMP and related enzymes during germination of Pinus pinea seeds

The occurrence of cAMP, adenylate cyclase and cAMP phosphodiesterase has been tested in Pinus pinea seed during germination. The study has been carried out on dormant and imbibed seeds, seedlings, endospermic residues, roots and cotyledons. cAMP has been detected by the protein binding method and its occurrence has been verified by HPLC detections. cAMP phosphodiesterase shows a very high activity at acidic pH, while being completely inactive at pH 7.4. At this pH value, well detectable levels of adenylate cyclase have been observed. Therefore, the classical pathway of synthesis and breakdown of cAMP, already accepted for animal and bacterial cells, seems to be operating in Pinus pinea plant too.     

Change in Bacillus anthracoides spores and their content of dipicolinic acid during germination]

The content of dipicolinic acid (DPA) was assayed in the spores of Bacillus anthracoides 96 during various stages of its growth. The content of DPA was ca. 10.7 per cent of the dry biomass weight in the seven-day-old culture containing 96 to 99 per cent of the spores in a "starvation" medium. The morphology of the culture was modified, and the content of DPA in the spores fell to 3.6 per cent half an hour after the inoculation into the medium favourable for the growth (MPA). During the following one to four hours of the germination, the refraction index of the spores and the content of DPA in them decreased (the content of DPA to 2 per cent).     

RGD-containing peptides and cyclic AMP have antagonistic roles in the morphology of Mucor rouxii

Summary. The tripeptide Arg-Gly-Asp (RGD) (1 mM) as well as the polymer ProNectin F (20 nM) added to culture medium of the fungus Mucor rouxii (defined medium) produced a delay in the switch from isodiametric growth to tip growth; at the time of germination the mother cell had a 4.6 times larger volume with 3.6 times more germ tubes per cell than control germinating sporangiospores. Disruption of the actin network with 2 µg of cytochalasin A per ml blocked the switch to tip growth; the effect was analogous to the one of 150 µM dibutyryl–cyclic AMP (cAMP), which we previously described to promote isodiametric growth via protein kinase A. 150 µM dibutyryl-cAMP antagonises partially the effect of 1 mM RGD; the cells still emit several germ tubes per mother cell but their number is smaller and the volume of the cell at germ tube emission is larger than with RGD alone. At higher concentrations the dibutyryl-cAMP overrides completely the effect of RGD. Our results suggest that M. rouxii has an RGD recognition system and demonstrate that RGD-containing peptides have a profound effect on the isotropic stage of growth and on the establishment of cell polarity and that cAMP analogues can override this effect.Correspondence and reprints: Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Pabell'n 2, Ciudad Universitaria, Universidad de Buenos Aires, 1428 Buenos Aires, ArgentinaReceived January 29, 2003; accepted March 10, 2003; published online September 23, 2003     

Changes in the cyclic AMP content during growth and development of Acetabularia.

The 3',5'-adenosine monophosphate (cyclic-AMP) content of the unicellular alga Acetabularia has been examined at various developmental stages. It has been found that very young algae, less than 10mm in length, have a high cAMP content [more than 7 pmoles per 100 mg wet weight (WW)], but that with the growth of the algae, the cAMP content decreases rapidly, reaching the low level of 0.5--1.0 pmoles per 100mg WW. The cAMP content remains at this level until cap differentiation, after which an increase in cAMP content accompanies cap enlargement. It has been shown that these results are unlikely to be affected by changes in the cAMP content induced by variations in circadian rhythm. Treatment with theophylline (2.10(-3) M), a phosphodieterase inhibitor, results in an increase in the cAMP content and delays growth and cap formation. Experiments on the effects of theophylline upon the circadian rhythm of oxygen evolution have shown that the continuous presence of theophylline in the culture medium does not induce a phase shift in the rhythm. The cAMP content of anucleate Acetabularia shows development stage variations parallel to that of the whole algae.     

Changes in the content of cyclic AMP and cyclic GMP during the development of Xenopus laevis.

The levels of the three major DNA-dependent RNA polymerases (enzymes I, II and III) present in the dimorphic fungus Mucor rouxii have been investigated during the transition from yeast-like cells to mycelial growth. Increases in the specific activity of crude extracts were observed at 2 h and at 6 h after induction of mycelium formation by aeration of yeast-like cells. These increases could be attributed to changes in the specific activities of enzymes I and II. Alterations were also found in the relative amounts of enzymes I and II: prior to aeration, 31% of the total polymerase activity of crude extracts was present as enzyme I; after 2 h of aeration, the specific activity of this enzyme doubled and the relative amount increased to 64% of the total activity. After 6 h of aeration, the relative amounts of enzymes I and II were 25 and 65%, respectively, and the specific activity of enzyme II had nearly doubled. The amounts and specific activities of enzyme III did not change significantly during the transition.     

Adenylate cyclase activity and cyclic AMP content in brain tissue of dogs during clinical death and in the post-resuscitation period

The cAMP levels and adenylate cyclase activity have been studied in the grey brain substance and striatum system of dogs during circulation arrest due to electrotrauma of different duration (1-2, 15, 45 min) and in postresuscitation period in animals recovered after 15-min clinical death. Adenylate cyclase is strongly activated and cAMP levels are increased in the brain areas under study during complete brain ischemia. The cAMP levels in the grey substance and in striatum system are reduced considerably compared to the control, accounting for 12-20 on days 2-5 of postresuscitation period in animals with neurologic deficit. Adenylate cyclase and phosphodiesterase enzyme activity is twice higher in the striatum system and 50% lower in the grey brain substance than the baseline. The disturbances in cyclic nucleotide exchange, along with other factors seem to play an important role in the pathogenesis of postresuscitation encephalopathy.     

Polyamine oxidase activity and polyamine content in maize during seed germination

Polyamine oxidase (PAO, EC 1.5.3.3) activity and polyamine content in the cell wall and soluble fractions obtained from embryos, endosperms and shoots and roots of etiolated or green seedlings of maize ( Zea mays L. cv. WF9) during the first 7 days of germination were investigated. Polyamine content was also determined in the trichloroacetic acid-soluble (free polyamines) and trichloroacetic acid insoluble (bound polyamines) fraction obtained from the same tissues. PAO activity, determined by the radiometric method based on the recovery of the labelled reaction product 1-pyrroline, was mostly localized in the cell wall fraction. The activity was very low in embryos and endosperms and present in traces in roots. In etiolated shoots PAO activity increased sharply, while in green shoots it was low and increased slowly. No polyamines were found in the cell wall fraction and only putrescine was detected in the soluble fraction, with the exception of the embryo, where spermidine and spermine were also present. In the TCA-soluble fraction of embryos, putrescine increased during imbibition, while spermidine and spermine decreased; in the endosperm no relevant changes in polyamines occurred. In the same fraction of green and etiolated seedlings, putrescine increased, giving a peak at days 3–5, while spermidine decreased to very low levels. The amount of bound polyamines was 1–4% of the free ones. The pattern of PAO activity seems to be unrelated to endogenous free polyamine content, which is the same in shoots and roots of etiolated and green seedlings. Enzyme activity, very low in ungerminated seeds, increased continuously during the progression of germination, especially in etiolated shoots, indicating a possible involvement in cell wall formation.     

The arrangement of F-actin and microtubules during germination of Mucor rouxii sporangiospores

The distribution of F-actin microfilaments and microtubules was analyzed in germinating sporangiospores of Mucor rouxii by labeling with rhodamine-tagged phalloidin and by immunofluorescence microscopy. The transition from isodiametrical to apical growth was accompanied by a switch from uniform distribution of F-actin patches to a polarized accumulation of F-actin material at the germ tube tips. Immunoblotting of cell-free extracts of M. rouxii with a monoclonal anti-porcine -tubulin antibody (TU-01) disclosed two discrete bands of -tubulin suggesting the existence of two -tubulin genes in this fungus. Immunofluorescence microscopy of germinating cells stained with the same antibody revealed an elaborate network of cytoplasmic microtubules that persisted during the entire germination process and extended into the apex of the germ tube. Although their precise roles remain undetermined, the observed arrangement of cytoskeletal elements during germination is consistent with their presumed involvement in cell wall morphogenesis: the long axial microtubules serving as long-distance conveyors of wall-building vesicles to the apical region while the concentrated F-actin patches mark the participation of microfilaments in the zone of intense vesicle exocytosis at the hyphal apex.Abbreviations DAPI 4,6-diamidino-2-phenylindole - DTT dithiothreitol - EGTA Ethylene glycol-bis (beta-aminoethyl ether) - N,N,N,N tetraacetic acid - F-actin Filamentous actin - MES 2-(N0morpholino)-ethanesulfonic acid - PIPES Piperazine-N,N-bis-2-ethanesulfonic acid - PMSF Phenyl-methylsulphonyl fluoride - TBS Tris-buffered saline     

Ethylene requirement during germination of Botrytis cinerea spores

Application of 2,5-norbornadiene, a competitive inhibitor of ethylene, effectively inhibited the germination of Botrytis cinerea Pers. ex Fr. spores. The transfer of spores from 2,5-norbornadiene to air relieved inhibition by norbornadiene, indicating that its effects are non-toxic and reversible. Ethephon (2-chloroethylophosphonic acid), which stimulates spore germination of B. cinerea , does not affect germination in the presence of norbornadiene. However, ethephon appeared to be effective in relieving inhibition, when norbornadiene was removed from the atmosphere surrounding spores. The addition of ethylene to an atmosphere enriched with norbornadiene, counteracted the inhibition of spore germination. The inhibition of spore germination by 2,5-norbornadiene and the reversal of this effect by ethephon or ethylene, indicate that the action of ethylene is indispensable for germination of B. cinerea spores.     

Expression of proteins and protein kinase activity during germination of aerial spores of Streptomyces granaticolor

Dormant aerial spores of Streptomyces granaticolor contain pre-existing pool of mRNA and active ribosomes for rapid translation of proteins required for earlier steps of germination. Activated spores were labeled for 30 min with [35S]methionine/cysteine in the presence or absence of rifamycin (400 microg/ml) and resolved by two-dimensional electrophoresis. About 320 proteins were synthesized during the first 30 min of cultivation at the beginning of swelling, before the first DNA replication. Results from nine different experiments performed in the presence of rifamycin revealed 15 protein spots. Transition from dormant spores to swollen spores is not affected by the presence of rifamycin but further development of spores is stopped. To support existence of pre-existing pool of mRNA in spores, cell-free extract of spores (S30 fraction) was used for in vitro protein synthesis. These results indicate that RNA of spores possesses mRNA functionally competent and provides templates for protein synthesis. Cell-free extracts isolated from spores, activated spores, and during spore germination were further examined for in vitro protein phosphorylation. The analyses show that preparation from dormant spores catalyzes phosphorylation of only seven proteins. In the absence of phosphatase inhibitors, several proteins were partially dephosphorylated. The activation of spores leads to a reduction in phosphorylation activity. Results from in vitro phosphorylation reaction indicate that during germination phosphorylation/dephosphorylation of proteins is a complex function of developmental changes.