Optimization of the spore production of Penicillium roqueforti in solid substrate fermentation on buckwheat seeds

Summary The effect of substrate (buckwheat seeds) pretreatment on the growth and the sporulation behaviour of Penicillium roqueforti is presented. When a saccharifying enzyme (-amylase) is added to a medium which exhibits a low water content (0.46 g water/g initial dry matter, IDM), a more rapid internal colonization of the seeds occurs, but the final spore production does not increase and remains close to 8.10⁹ spores/g dry matter (DM) at 500 h. No carbon source limitation is then observed. The addition of casein hydrolysate to this medium gives rise to a great increase of the sporulation, since 14.5 10⁹ spores/g DM are obtained after 600 h. This result is attained by a better spore yield from the mycelium, the substrate colonization being unchanged. High water content (0.60 g water/g IDM) of buckwheat seeds induces a shorter cultivation time along with a higher biomass production. However, the spore content of the medium remains close to the low water content one, but 60% total spores are external against 30% to 35% in the other media.     

Induction of IL 2 responsiveness in a murine IL 3-dependent cell line

A mouse IL 3-dependent cell line, FD.C/1, can be induced to IL 2 growth responsiveness by culture in IL 2-conditioned culture medium. The IL 2-dependent cell lines derived by this procedure have been designated FD.C/2 cells. Once established, the FD.C/2 cells respond to human, rat, and mouse IL 2. When cultured with murine IL 3, FD.C/2 cells did not proliferate, appearing to accumulate in the G1 phase of the cell cycle. Other human and mouse lymphokines failed to stimulate FD.C/2 cell growth. The growth dependence of FD.C/2 cells on IL 2 could not be reversed to IL 3. Cell lines derived by these procedures could provide in vitro models for hemopoietic differentiative events.     

Endocytosis of an antibody ricin A-chain conjugate (immuno-A-toxin) adsorbed on colloidal gold. Effects of ammonium chloride and monensin

An immunotoxin (IT) formed by a specific antibody coupled to the ricin A chain was adsorbed on colloidal gold particles (IT-Au). Binding and internalization of IT-Au in human lymphoblastic CEM cells were studied using electron microscopy. IT-Au showed specific cytotoxic activity toward the target cells. After 1 h at 4 degrees C, IT-Au were linked diffusely to the plasma membrane with 45% of the particles regrouped in clusters. Upon transfer to 37 degrees C, the particles carrying the ligand were regrouped more frequently and internalized into the cell by endocytosis through smooth microinvaginations or coated pits of the plasma membrane. After 15 min, IT-Au was observed in endocytic vacuoles, or receptosomes, in tubular structure near the Golgi apparatus and in lysosomes. Entry of IT-Au into lysosomes was rapid (around 50% of intracellular IT-Au particles after 30 min). NH4Cl or monensin, well-known potentiators of immunotoxin activity, when present in incubation medium, altered neither the processes nor the rate of IT-Au endocytosis. In the presence of either of these substances, IT-Au accumulated in the normal or often enlarged endocytic vacuoles, and entry into the lysosomes was slowed down (50% of particles after 2 h 15 min). We conclude that this intense slowing-down in the speed of IT-Au transportation into lysosomes and the functional modifications of these organelles help to explain the increased efficacy of immunotoxins in the presence of potentiators.     

In vitro translation of human pheochromocytoma messenger RNAs: characterization of tyrosine-hydroxylase and dopamine-beta-hydroxylase

mRNAs extracted from human pheochromocytoma were translated in vitro in a lysate of a rabbit reticulocytes. Two enzymes of the biosynthetic pathway of the catecholamines, tyrosine-hydroxylase (TH) and dopamine-beta-hydroxylase (DBH), were characterized as translation products after immunoprecipitation by specific antisera and electrophoretic analysis. The precursor of TH is a polypeptide having a molecular mass of 62,000 identical to that found for the mature protein. The molecular mass of the precursor of DBH 73,000 while that of the mature form is 79,000. TH and DBH have been translated from mRNAs having sedimentation coefficients of 22S and 25S, respectively.     

The endogenous tripeptide Tyr-Gly-Gly as a possible metabolite of opioid peptides in rat brain: identification, regional distribution, effects of lesions and formation in depolarized slices

Using a sensitive radioimmunoassay, the tripeptide Tyr-Gly-Gly (YGG) which corresponds to the N-terminal sequence of opioid peptides was detected in rat brain and identified by HPLC. Its regional distribution paralleled that of (Met5)enkephalin (YGGFM), a marker of enkephalin neurons. Ablation of these neurons in the striato-pallidal pathway by intrastriatal kainate, induced a significant decrease in YGG levels in caudateputamen and globus pallidus (-49%), consistent with the hypothesis that YGG originates from enkephalin neurons. When pallidal slices were incubated under various conditions, YGG was mainly found in the incubation medium indicating a predominantly extracellular localization. Depolarization of these slices by a K+-stimulus elicited a release of YGGFM accompanied by a marked increase in YGG levels. Bestatin and amastatin further enhanced YGG levels, reflecting the participation of aminopeptidases in the metabolism of the tripeptide and its precursor. Captopril, an inhibitor of the angiotensin-converting enzyme (ACE) showed no effect on the recovery of YGGFM and YGG. In contrast, the formation of YGG was completely prevented by Thiorphan (IC50 value = 9 nM) and phosphoramidon, two inhibitors of "enkephalinase" (EC 3.4.24.11; membrane metallo-endopeptidase), thus identifying the latter as the YGG-forming enzyme. The K+-induced increase in YGG + YGGFM levels in medium containing bestatin exceeded by about 60% the amount of YGGFM released from tissues, suggesting that YGG was mainly formed by extracellular hydrolysis of the various opioid fragments of the proenkephalin molecule. In vivo, YGG levels of cerebral regions were also markedly reduced in rats treated with acetorphan, a parenterally active "enkephalinase" inhibitor. All data suggest that YGG levels constitute an index of opioid peptide release.     

Spore production ofPenicillium roqueforti in fermentors filled with buckwheat seeds: batch and semi-continuous cultivation

Summary The behaviour of a drum fermentor and a column fermentor during the sporulation ofPenicillium roqueforti on buckwheat seeds is presented. The main problem encountered during the course of a cultivation is the free water released (about 0.1 ml/g dry matter) which must be removed from the medium. The rotation of the drum fermentor may disturb the growth and the sporulation. The column fermentor thus represents the best way to perform batch cultivation of the fungus: 10⁹ external spores/g dry matter are obtained.Semi-continous cultivation, with sequential emptying and filling, is performed in 1-liter bottles. This kind of cultivation may give a maximal average productivity close to 9.2·10⁶ external spores/g dry matter per hour. A drum fermentor, rotading only when emptying and filling, could represent an alternative to perform this kind of cultivation.     

Copurification of tyrosine hydroxylase from rat pheochromocytoma by protein kinase

Rat pheochromocytoma contains a protein kinase activity which remains associated with tyrosine hydroxylase (TH) during its purification. The incorporation of phosphate in TH is observed after incubation of TH with labelled ATP and magnesium without the need for an exogenous protein kinase. This Ca2+ and cAMP-independent kinase activity is different from previously described TH phosphorylating kinases from rat pheochromocytoma and other tissues.