Dieter Techel

Inositol trisphosphate induces calcium release from Neurospora crassa vacuoles.

Inositol 1,4,5-trisphosphate is known to release calcium ions from intracellular stores thought to be parts of endoplasmic reticulum in animal cells. In Neurospora crassa, however, inositol 1,4,5-trisphosphate acts on vacuoles stimulating a calcium efflux with a Km of 5.28 microM. The calcium release is inhibited effectively by dantrolene. These results were obtained by applying two independent methods, measuring calcium binding to fura-2 and loading vacuoles with 45Ca.

Comparison of salt- and heat-induced alterations of protein synthesis in the cyanobacterium Synechocystis sp. PCC 6803

Protein synthesis of the cyanobacterium Synechocystis spec. PCC 6803 decreases after a 684 mM NaCl salt shock. Qualitative changes were observed during the shock and the subsequent adaptation process using one-dimensional polyacrylamide electrophoresis. Proteins of apparent molecular masses of 13.0, 14.2, 16.6, 20.0, 21.0, 23.0, 33.0, 47.0, 52.0, 65.0 and 72.0 kDa are synthesized at enhanced rates after salt stress. The proteins of 14.2, 21.1 and 52.0 kDa are transiently induced during the first hours of the adaptation phase, while the other proteins are also synthesized at enhanced rates in salt-adapted cells. The proteins of 14.2, 23.0, 33.0 and 65.0 kDa are also induced by heat shock (43°C). Heat shock proteins of about 88.0, 75.0, 58.0, 17.5 and 13.8 kDa, in contrast, are induced by heat shock but not by salt. Two-dimensional polyacrylamide electrophoresis showed that the induced salt and heat shock proteins in some cases consisted of isoforms of different isoelectric points.

On the role of Ca2+-calmodulin-dependent and cAMP-dependent protein phosphorylation in the circadian rhythm ofNeurospora crassa

SummaryPulses of some Ca2+ channel blockers (dantrolene, Co2+, nifedipine) and calmodulin inhibitors (chlorpromazine) lead to medium (maximally 5–9 h) phase shifts of the circadian conidiation rhythm ofNeurospora crassa. Pulses of high Ca2+, or of low Ca2+, a Ca2+ ionophore (A23187) together with Ca2+, and other Ca2+ channel blockers (La3+, diltiazem), however, caused only minor phase shifts. The effect of these substances (A 23187) and of different temperatures on the Ca2+ release from isolated vacuoles was analyzed by using the fluorescent dye Fura-2. A 23187 and higher temperatures increased the release drastically, whereas dantrolene decreased the permeation of Ca2+ (Cornelius et al., 1989).Pulses of 8-PCTP-cAMP, IBMX and of the cAMP antagonist RP-cAMPS, also caused medium (maximally 6–9 h) phase shifts of the conidiation rhythm. The phase response curve of the agonist was almost 180° out of phase with the antagonist PRC. In spite of some variability in the PRCs of these series of experiments all showed maximal shifts during ct 0–12. The variability of the response may be due to circadian changes in the activity of phosphodiesterases: After adding cAMP to mycelial extracts HPLC analysis of cAMP metabolites showed significant differences during a circadian period with a maximum at ct 0.Protein phosphorylation was tested mainly in an in vitro phosphorylation system (with35S-thio γ-ATP). The results showed circadian rhythmic changes predominantly in proteins of 47/48 kDa. Substances and treatments causing phase-shifts of the conidiation rhythm also caused changes in the phosphorylation of these proteins: an increase was observed when Ca2+ or cAMP were added, whereas a decrease occurred upon addition of a calmodulin inhibitor (TFP) or pretreatment of the mycelia with higher (42° C) temperatures.Altogether, the results indicate that Ca2+-calmodulin-dependent and cAMP-dependent processes play an important, but perhaps not essential, role in the clock mechanism ofNeurospora. Ca2+ calmodulin and the phosphorylation state of the 47/48-kDa proteins may have controlling or essential functions for this mechanism.

Differential expression of glucose-regulated (grp78) and heat-shock-inducible (hsp70) genes during asexual development of Neurospora crassa

The expression of a glucose-regulated gene (grp78) changes significantly during the vegetative life cycle of Neurospora crassa: the amounts of grp78 mRNA are low in dormant conidia, increase during germination and exponential growth, decline in young aerial hyphae and reach a maximum in late (15-18 h) aerial hyphae. Heat shock (30 min at 45 degrees C) elevated the mRNA level of this gene especially in early aerial hyphae, whereas no increase above the high constitutive amount was found after heat treatment of late aerial hyphae. The expression of the inducible hsp70 gene after heat shock also varied with the state of development and showed the highest inducibility in late aerial hyphae. Surface mycelium, from which aerial hyphae emerge, showed a similar increase in the amounts of both mRNA species. A developmental mutant (acon-2), which is defective in minor constriction budding of aerial hyphae, showed lower levels of con-2 mRNA as well as of grp78 and hsp70 mRNA (after heat shock) in late aerial hyphae. The acon-2 mutant did not form conidia at this stage. It is concluded that the high constitutive and inducible expression of stress genes in late aerial hyphae is due to a developmental activation of their transcription or, alternatively, to a lower degradation rate of their mRNA during this stage.

31P-NMR-studies on intracellular pH and metabolite concentrations in relation to the circadian rhythm, temperature and nutrition in Neurospora crassa

Perfused mycelia of Neurospora crassa were analyzed in vivo with 31P-NMR. Both the cytoplasmatic and the vacuolar pH and the concentrations of phosphate metabolites were followed up to 30 h under constant conditions. No circadian changes were detected. However, slight changes in the nutrition or oxygen supply induced distinct changes in the intracellular pH and in the concentrations of metabolites. An increase of temperature from 21 to 43 degrees C lowered the intracellular pH and the metabolite concentrations. Changes in the perfusion rate affect the temperature responses, probably due to different availability of carbon sources and exhaustion of acid catabolites.

Some starvation-induced proteins inNeurospora crassa are related to glucose-regulated proteins

Glucose-free medium induces the synthesis of at least 11 proteins (165, 94, 86, 80, 79, 78, 70, 65, 56, 35, 28 kDa) inNeurospora crassa within 1, 2, or 4 days. Other inducers of glucose-regulated proteins (GRPs) in mammalian cells, such as tunicamycin, β-mercaptoethanol, and A23187, stimulate the synthesis of some stress proteins (35 and 28 kDa) which are also induced by starvation. Two proteins were identified with properties similar to those of mammalian GRPs (94 and 78 kDa). These and some of the other starvation-induced polypeptides are induced also by heat shock. After the transfer of starving mycelia to a sucrose-containing medium, the synthesis rate of the most prominent starvation protein (86 kDa) is shifted back to a constitutive level. Two proteins (65 and 70 kDa) were induced after the transfer.

Heat shock induction of a 65 kDa ATP-binding proteinase in rat C6 glioma cells

ABSTRACTThe 45, 55, 65 and 100 kDa ATP-binding proteinases (ATP-BPases) of the heat-shocked (44 °C for 30 min, recovery for 12 h) rat C6 glioma cells were purified by DEAE-ionexchange and ATP-affinity chromatography. Their molecular masses, isoelectric points (pI), pH-optima and other properties were analyzed by native proteinase gels. It was shown that the 65 kDa ATP-BPase is specifically induced by heat shock and not detectable in control cells. Its N-terminal 1-9 amino acid sequence was determined by Edman degradation, but no homologies to other proteins in the protein data bases were found. 30 and 31 kDa proteinases can be cleaved from the 45, 55 and 65 kDa proteinases to which they are linked. A possible relationship of the heat-induced 65 kDa ATP-BPase with the ATP-dependent proteinases (ATP-DPases) in prokaryotes and eukaryotes is discussed.