Production of ψ-linolenic acid by the fungus Mucor rouxii on cheap nitrogen and carbon sources

Summary The production of -linolenic acid (GLA) by the fungus Mucor rouxii CBS 416.77 was studied on low budget nitrogen and carbon sources, i.e. rape meal, cocos expeller and two types of yeast extract (nitrogen sources), and starch, starch hydrolysate, beet molasses and cocos expeller (carbon sources). As references, Difco yeast extract and glucose were used. In flask cultivations the three yeast extracts were fully interchangeable, while the Difco yeast extract (the most expensive of those tested) gave a higher productivity of GLA in fermentor cultures (14 mg·l^–1·h^–1). The yield of lipids and GLA were increased in the order yeast extract < rape meal < cocos expeller. Thus the amount of lipid increased from 0.56 to 2.8 g·l^–1, and that of GLA from 0.15 to 0.33 g·l^–1. Use of beet molasses or cocos expeller as carbon sources gave poor growth. Starch and starch hydrolysate resulted in better productivity of GLA than glucose (4.7 and 4.9 compared to 3.4 mg·l^–1·h^–1). Offsprint requests to: A.-M. Lindberg     

Visualization of poly(ADP-ribose) synthetase associated with polynucleosomes by immunoelectron microscopy

Antibodies showing a high specificity for poly(ADP ribose) synthetase have been purified. A fraction binding nonspecifically to histones present in antiserum and non-immune serum has been demonstrated by immunoblotting and separated by histone-Sepharose chromatography. The antibody without the nonspecific binding fraction was analyzed by Western blot with calf thymus protein extract and was found to react only with a band at 116 kDa. There was no reaction with purified topoisomerase I, this weak activity was copurified with poly(ADP-ribose) synthetase preparation. The specific IgG fraction has been used for the visualization of the interaction of poly(ADP-ribose) synthetase with chromatin by indirect gold-labelling. This immunomicroscopic study suggests that the synthetase is located in the inner part of polynucleosomes and would be associated preferentially with the core nucleosome.     

Antisera Against the Indolealkylamines: Tryptophan, 5-Hydroxytryptophan, 5-Hydroxytryptamine, 5-Methoxytryptophan, and 5-Methoxytryptamine Tested by an Enzyme-Linked Immunosorbent Assay Method

Antisera were raised against tryptophan, 5-hydroxytryptophan, 5-hydroxytryptamine, 5-methoxytryptophan, and 5-methoxytryptamine, by conjugating each molecule to bovine serum albumin and to human serum albumin via glutaraldehyde, in such a way as to preserve the original part. Antibody specificity was tested with the enzyme-linked immunosorbent assay method. The specificity of each anti-indolealkylamine-glutaraldehyde antibody was established with competition experiments by using an adsorbed immunogenic conjugate and indolealkylamines either free or conjugated with poly-L-lysine. The nonconjugated compounds were poorly recognized. In the same way, the nonreduced conjugates always appeared less immunoreactive than the reduced ones. Calculated from the specificity study of each antiserum, the cross-reactivity ratios were found to be smallest for the most immunoreactive conjugates. Thus, a specific immune response was defined for each compound belonging to the same metabolic pathway.     

An Electron Microscope Study of Intranuclear Inclusions in Mouse Liver and Hepatoma

An electron microscope study of intranuclear inclusions which occur in giant cells in a transplantable mouse hepatoma and in enlarged liver cells in mice fed a diet containing bentonite demonstrates that these inclusions are formed by invaginations of the nuclear envelope, and corroborates a previous histochemical study which revealed that the contents of the inclusions are of cytoplasmic origin. In the hepatoma cells the intranuclear inclusions are abundant, small, and situated close to the border of the nucleus, and there are wide openings from the cytoplasm into the invaginations whose contents include lipid droplets, ergastoplasm, and structurally normal mitochondria. In the enlarged liver cells the inclusions are fewer in number, generally much larger than those in the hepatoma, hence they extend deeper into the nucleus, and the interior is continuous with the cytoplasm through only a small opening. Some normal ergastoplasm is present within the inclusions but all other constituents are abnormal. Both normal and degenerating mitochondria occur in the cytoplasm but only degenerating ones are found within the inclusions. Both types of inclusions arise in greatly enlarged cells in which an attempt is made to maintain the normal nuclear surface/nuclear volume ratio by the development of the invaginations of the nuclear envelope.     

Study of the in vitro bioactivation of albendazole in human liver microsomes and hepatoma cell lines

The metabolism of albendazole (ABZ), a benzimidazole anthelminthic, was studied in either microsomal preparations of human liver biopsies or cultured human hepatoma cell lines. Metabolites were analyzed by HPLC. Our data show that microsomes from human biopsies and two human cell lines, HepG2 and Hep3B, oxidize the drug to the sulfoxide very efficiently, whereas the third cell line tested, SK-HEP-1, does not. Both cytochrome P-450 dependent monooxygenases and favin-containing monooxygenases appear to be involved in human ABZ metabolism. Using the cell line displaying the highest ABZ-metabolizing activity, HepG2, the cytotoxic and the inducing effects of the parent drug ABZ and of two primary metabolites, the sulfoxide and the sulfone were studied. These three chemicals provoked a rise in mitotic index resulting from cell division blockage at the prophase or at the metaphase (ABZ metabolites) stage, and ABZ was more cytotoxic than its metabolites. With regard to enzyme-inducing effects, our data clearly demonstrate that the sulfoxide and, to a lesser degree, the sulfone are potent inducers of some drug metabolizing enzymes (i.e., cytochrome P-488 dependent monooxygenases and UDP glucuronyltransferase), whereas ABZ fails to increase and even slightly decreases these enzymatic activities. In conclusion, the HepG2 human hepatoma cell line appears to be suitable for the study of many parameters of metabolism and action of ABZ and other structurally related compounds in humans.Abbreviations ABZ albendazole - B[a]P benzo[a]pyrene - HPLC high-performance liquid chromatography - MC 3-methylcholanthrene - MFO mixed-function oxidase - UDPGT UDP-glucuronyltransferase     

Fragmentation of bovine chromogranin A by plasma kallikrein

Chromogranin A has been reported to be processed in vivo by an as yet undefined proteinase(s) suggesting that it is a precursor of biologically active peptides such as pancreastatin. In this study, plasma kallikrein was used as a model proteinase to identify the cleavage sites exposed in bovine parathyroid chromogranin A. Purified bovine parathyroid chromogranin A was digested with human plasma kallikrein. The proteolytic fragments produced were isolated by HPLC and chemically characterized by amino acid composition and sequence analysis. The combined results indicate that the enzyme has preference for specific single Arg residues, cutting C-terminal to this amino acid, although certain pairs of basic sites were also cleaved. The characterized fragments were released in a selective manner from the whole molecule with rapid production of the fragments covering positions 1-247 and 352-358.     

Localization of calcium and microfilament changes in mechanically stressed cells

We combined fluorescence labeling, digital image processing, and micromanipulation to investigate the intracellular events induced by inflicting a mechanical stress on rat basophilic leukemia cells. Our findings were as follows:

1. Most cells displayed a localized calcium rise in response to micropipet aspiration. This represented an average threefold increase as compared to resting level, and it was observed during the first 10 s following aspiration. A slow return to initial level occurred within about 3 min. Further, this calcium rise involved a mobilization of intracellular stores, since it was not prevented by adding a calcium chelator into the extracellular medium.
2. All micropipet-aspirated cells displayed a local accumulation of microfilaments, with a preferential localization in the cell protrusions or near the pipet tips.
3. No absolute correlation was found between the localization of calcium rise and cytoskeletal accumulation.
4. Cell deformability was decreased when intracellular calcium was maintained at a constant (high or low) level with ionomycin and/or EGTA.

It is concluded that cells have a general ability to respond to mechanical stimulation by a coordinated set of events. More parameters must be studied before the mechanisms of cell shape regulation are fully understood.     

Conformational studies of an undecapeptide reproducing the consensus sequence around the cleavage site of the RXVRG endoprotease from Xenopus laevis skin

Two synthetic fragments, corresponding to the 4–9 and 4–14 sequences of a tetradecapeptide used as a model to test the RXVRG-endoprotease activity from Xenopus laevis skin, have been studied by two-dimensional nmr spectroscopies, correlated spectroscopy, and nuclear Overhauser effect (NOE) spectroscopy. Both peptides wore the 5–9 consensus sequence found in several hormonal precursors. The nmr data for the 4–9 hexapeptide did not indicate any particular organization, either in water or in dimethylsulfoxide (DMSO), whereas, the 4–14 undecapeptide, a substrate for the RXVRG endoprotease, showed, in DMSO solution, significant trends of structural organization involving the amino acids pertaining to the consensus domain. From variations of integrated NOE peaks with temperature, the appearent interproton correlation times τ_c were estimated and the maxima observed with Va17, the central residue in the consensus sequence. A defined tertiary structure in that domain was also supported by medium-and long-range NOEs between As6 and Arg8, Glu4 and Gly9, and by the likely involvement of Arg8 and Gly9 NHs in intramolecular hydrogen bonds. Most of these observations could be rationalized by an equilibrium between a 5–3 β-turn and a 9 → 4 H-bonded loop. The predominance of one rotamer for the Cα-Cβ bond was established in four residues. Finally, the average ? and ψ angles were derived from two models taking, or not, into account variations in the correlation times along the sequence. This allowed us to discuss the artifacts generated by using an average correlation time through the whole molecule. © 1994 John Wiley & Sons, Inc.