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A marine strain of Penicillium waksmanii Zaleski was isolated from a sample of seawater from shellfish-farming area in the Loire estuary (France). The in vitro marine culture showed an important antifungal activity. Bioassay-guided fractionation was used to purify the crude extract. Dereplication by electrospray-ion trap/mass spectrometry (ESI-IT/MS) afforded the identification of the antifungal compound, after a semi-purification consisting of two stages. A comparison of the ionic composition between the active and the non-active fractions allowed the detection of a monocharged ion at m/z 353 containing a chlorine atom, which could be attributed to the antifungal griseofulvin [C17H17ClO6+H]+. Multi-stage fragmentation (MSn) confirmed the identity of the m/z 353 ion of the antifungal fraction as griseofulvin. It is the first description of griseofulvin production by a strain of P. waksmanii and the first chemical study of a strain of this species isolated from marine temperate cold water.

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We have recently demonstrated that bovine adrenal medulla contains a soluble phospholipase A2 (PLA2), which is localized in the cytosol. In the present study, this PLA2 was purified 1,097-fold using sequential concanavalin A, hydrophobic interaction, anion exchange, gel filtration, and an additional anion exchange chromatography. The enzyme is activated over the range of 20-1,000 microM Ca2+ and has a pH optimum near 8.0. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protein has a molecular mass of 26 kDa and an isoelectric point of 4.6 as revealed by isoelectric focusing. The cytosolic PLA2 is not inhibited by NaCl, and the enzymatic activity is stimulated at low concentrations of Triton X-100 (0.01%) and deoxycholate (1 mM) but inhibited at higher concentrations (0.1% and 3 mM, respectively) of these detergents. Furthermore, heat treatment (57 degrees C, 5 min) reduced the enzymatic activity by 80%, whereas glycerol (30%) increased the activity. p-Bromophenacylbromide, a frequently used irreversible inhibitor of type II PLA2, has little effect until 100 microM, and 2-10 mM dithiothreitol totally inactivated the enzyme. The purified PLA2 displays a preference for phosphatidylcholine as a substrate but hydrolyzes phospholipid substrates with arachidonic acid or linoleic acid esterified at the sn-2 position to the same extent. It is concluded that the chromaffin cell cytosolic PLA2, which was isolated and characterized in this study, represents a type of PLA2 that has not been formerly reported in chromaffin cells. Additional research on the chromaffin cell cytosolic PLA2 will help to reveal whether the enzyme is important for exocytosis.

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Since phospholipase A2 (PLA2) is expected to play a role in the mechanism of exocytosis, the presence and subcellular localization of PLA2 in bovine adrenal medulla have been studied. The results of this study reveal that, although a large part of the PLA2 activity in chromaffin cells is due to a lysosomal PLA2, a cytoplasmic PLA2 is also present. This finding is supported by experiments in which the influence of pH, CaCl2 and NaCl on cytoplasmic PLA2 as well as the binding capacity to concanavalin A are investigated. According to these results the properties of a cytoplasmic PLA2 are clearly different from those reported by other authors for the lysosomal PLA2. For this reason, in chromaffin cells a PLA2 could be present which remains in the cytosol when the cell is in rest. Future experiments will have to prove whether this PLA2 becomes associated with the plasma membrane upon stimulation of the cell, thus mediating exocytosis.

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Pregnant rats received propranolol (5 or 10 mg/kg/day) from day 10 of gestation; controls were untreated. Lung wet:dry weight ratios were increased in treated fetuses delivered by hysterotomy at day 21; no difference was seen at birth after vaginal delivery. On subsequent days, treated pups exhibited higher wet:dry weight ratios, implying impaired postnatal lung water clearance. Surfactant pools were decreased proportionately at both doses. Ongoing surfactant synthesis was unaffected at either dose. Baseline secretion was reduced for those exposed to 10 mg/kg/day. Secretory response to beta-agonist stimulation was impaired in both treatment groups. Chronic beta-blockade alters fetal lung water clearance, surfactant stores, and secretory response to beta-agonists.

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Calmodulin-binding proteins in chromaffin granule membrane and chromaffin cell plasma membranes have been investigated and compared. Chromaffin granules were purified by centrifugation over a 1.7 M sucrose layer. Plasma membranes were obtained in a highly purified form by differential and isopycnic centrifugation. Enzymatic determinations of 5'-nucleotidase, a generally accepted plasma membrane marker, showed a 40-50-fold enrichment as compared to the cell homogenate. Marker enzyme studies demonstrated only minimal contamination by other subcellular organelles. After solubilization with Triton X-100, calmodulin-binding proteins were isolated from chromaffin granule membranes and plasma membranes by affinity chromatography on a calmodulin/Sepharose 4B column. On two-dimensional polyacrylamide gelelectrophoresis a prominent protein (Mr = 65,000, pI ranging from 5.1 to 6) consisting of multiple spots, was present in the calmodulin-binding fraction from chromaffin granule membranes as well as from plasma membranes. Besides this 65 kDa protein both fractions had at least four groups of proteins in common. Also, proteins typical for either preparation were observed. In the calmodulin-binding protein preparations from chromaffin granule membranes a prominent spot with Mr = 80,000 and a pH ranging from 5.0 to 5.7 was present. This protein was enzymatically and immunologically identified as dopamine-beta-monooxygenase.

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