Effect of oleuropein and sodium chloride on viability and metabolism of Lactobacillus plantarum

 The effect of the addition of oleuropein (OLP) and NaCl on the growth and the DL-lactic acid production of Lactobacillus plantarum DSM 10492 has been investigated by using an unconventional medium. The growth of L. plantarum was not inhibited by the addition of increasing amounts of untreated OLP in the presence or absence of glucose. However, bacterial cells grew in quantity slightly with OLP alone. The increased addition of NaCl was associated with a delay in growth. Moreover, there was no growth with 8% NaCl. The addition of both NaCl and OLP resulted in growth inhibition, and the survival of cells decreased strongly. The main fermentation product was DL-lactic acid, but acetic acid was also detected after a prolonged incubation. L. plantarum produced DL-lactic acid in the presence of OLP alone but its formation decreased with increasing levels of OLP. On the other hand, heat-treated OLP had a bactericidal effect. Received: 16 October 1995/Received last revision: 5 February 1996/Accepted: 12 February 1996     

The effects of pantothenate deficiency and acetate addition on anaerobic batch fermentation of glucose by Saccharomyces cerevisiae

 Physiological effects of deficiency of pantothenate, a necessary precursor in the synthesis of coenzyme A, were studied using the yeast strain Saccharomyces cerevisiae CBS 8066. Cells were grown on defined media in anaerobic batch cultures with glucose (50 g/l) as the carbon and energy source. Batch cultures containing more than 60 μg/l pantothenate showed no significant differences with respect to growth rates and product yields. However, with an initial pantothenate concentration of 30 μg/l, the average glucose consumption rate was 50% lower than in rich medium and, at even lower concentrations of pantothenate, the culture did not consume all the glucose in the medium. Furthermore, pantothenate deficiency caused the acetate and pyruvate yields to increase and the biomass yield to decrease, compared to the yields in pantothenate-rich medium. The increased acetate formation could be counteracted by initial addition of acetate to the medium, and thereby the glycerol yield could be decreased. An initial addition of acetate of 1.6 g/l to pantothenate-deficient medium (30 μg/l) caused a 35% decrease in glycerol yield and a 6% increase in ethanol yield. Furthermore, the time required for complete conversion of the glucose decreased by 40%. Acetate addition affected the acetate and glycerol yields in a similar way in pantothenate-rich medium (1000 μg/l) also. Received: 27 December 1995/Received revision: 3 May 1996/Accepted: 9 May 1996     

Mutants blocked in penicillin biosynthesis show a deletion of the entire penicillin gene cluster at a specific site within a conserved hexanucleotide sequence

The organization of the genes of the penicillin cluster has been studied in three different mutants of P. chrysogenum impaired in penicillin biosynthesis. The three blocked mutants (derived from the parental strain P. chrysogenum Bb-1) lacked the genes pcbAB, pcbC and penDE of the penicillin biosynthetic pathway and were unable to form isopenicillin N synthase and isopenicillin N acyltransferase. All strains were identified as P. chrysogenum derivatives by fingerprinting analysis with (GTG)_n as a probe. The borders of the deleted region were cloned and sequenced, showing the same junction point in the three mutants. The deleted DNA region was found to be identical to that described in P. chrysogenum npe10. The frequent deletion of the pen gene cluster at this point may indicate that this cluster is located in an unstable genetic region, flanked by hot spots of recombination, that is easily lost by mutagen-induced recombination.     

Discrimination of respiratory dysfunction in yeast mutants by confocal microscopy, image, and flow cytometry

Living yeast cells can be selectively stained with the lipophilic cationic cyanine dye DiOC6(3) in a mitochondrial membrane potential-dependent manner. Our study extends the use of flow cytometric analysis and sorting to DiOC6(3)-stained yeast cells. Experimental conditions were developed that prevented the toxic side effect of the probe and gave a quantitative correlation between fluorescence and mitochondrial membrane potential, without any staining of other membranes. The localization of the fluorochrome was checked by confocal microscopy and image cytometry. The mitochondrial membrane alterations were also tested through cardiolipin staining with nonyl acridine orange. Differences in light scattering and in fluorescence were detected in mutants (rho-, rho degrees, mit-, or pet-) and wild-type (rho+mit+) populations of yeast. The dye uptake of respiratory-deficient yeast strains was significantly reduced as compared to that of the wild-type. Application of an uncoupler (mCICCP), which collapsed the mitochondrial membrane potential (alphapsi(m)), led to a drastic reduction of the dye uptake. It was observed that a decrease in deltapsi(m), was usually correlated with a decrease in cardiolipin stainability by nonyl acridine orange (NAO). Quantitative flow cytometry is a fast and reproducible technique for rapid screening of yeast strains that might be suspected of respiratory dysfunction and/or mitochondrial structural changes. We give evidence that it is an adequate method to characterize and isolate respiratory mutants through sorting procedure, with selective enrichment of the population studied in respiring or non-respiring yeast cells. Confocal microscopy and image cytometry corroborate the flow cytometry results.     

Purification and characterization of Kurloff cell sialoglycoproteins with acid phosphatase activity

The majora2–6 sialoglycoproteins in detergent-extracts of Kurloff cells were purified by anion-exchange andSambucus nigra agglutinin-affinity chromatographies. The similar ultrastructural localisations of (1)S. nigra agglutinin-gold conjugates and (2) acid phosphatase activities on the Kurloff body and particularly on its myelin figures indicated that the majora2-6 sialoglycoproteins of the Kurloff cell had acid phosphatase activity. Two-dimensional electrophoresis showed that these tartrate-sensitive phosphatases corresponded to 2 acidic (pI 3.4–3.7) polypeptides of 36 and 34 kDa. Hydrolysis with peptide-N-glycosidases F gave a 33 kDa apoprotein rich in alanine, glutamic acid, tyrosine and lysin. A lectin-affinity study demonstrated that they contained hybrid type bisected and fucosylatedN-linked oligosaccharides. Cytotoxic properties were previously attributed to Kurloff cells and other studies suggested that not only acid phosphatases but alsoa2-6-linked sialic acid residues themselves may participate in natural killer activity.     

Thiol-induced oligomerization of α-lactalbumin at high pressure

Denaturation and aggregation of-lactalbumin at high pressure (up to 10 kbar, 1000 MPa) were studied by means of circular dichroism, gel-permeation chromatography, sodium dodecyl sulfate and gel electrophoresis. It was found that the unfolding of-lactalbumin at high pressure is reversible even in basic pH and at a protein concentration as large as 10%. In these conditions only a negligible fraction of the protein is denatured irreversibly and aggregates. The rate of aggregation of-lactalbumin at high pressure increases significantly in the presence of low-molecular reducing agents such as cysteine, 2-mercaptoethanol, and dithiothreitol. Maximal yield of-lactalbumin oligomerization (over 90%) was achieved in the presence of cysteine at the molar cysteine/protein ratioq=2 and atpH 8.5. Apparent molecular weight of the obtained oligomers was over 500 kDa. It was shown that the size distribution of oligomers can be modulated by varyingpH and reducing agent. The size distribution shifts in the direction of very large, poorly soluble particles whenpH decreases. Maximal content of the insoluble fraction (about 30%) can be reached at pH 5.5 when cysteine (q=2) is used as reducing agent. The oligomers of-lactalbumin are stabilized mainly by nonnative interchain disulfide bridges. Circular dichroism measurements point to an additional mechanism of cohesion of polypeptide chains in the oligomers, which is formation of intermolecular-sheets.     

Signalling by protein kinase C isoforms in the heart

Understanding transmembrane signalling process is one of the major challenge of the decade. In most tissues, since Fisher and Krebs's discovery in the 1950's, protein phosphorylation has been widely recognized as a key event of this cellular function. Indeed, binding of hormones or neurotransmitters to specific membrane receptors leads to the generation of cytosoluble second messengers which in turn activate a specific protein kinase. Numerous protein kinases have been so far identified and roughly classified into two groups, namely serine/threonine and tyrosine kinases on the basis of the target amino acid although some more recently discovered kinases like MEK (or MAP kinase kinase) phosphorylate both serine and tyrosine residues.Protein kinase C is a serine/threonine kinase that was first described by Takai et al. [1] as a Ca- and phospholipid-dependent protein kinase. Later on, Kuo et al. [2] found that PKC was expressed in most tissues including the heart. The field of investigation became more complicated when it was found that the kinase is not a single molecular entity and that several isoforms exist. At present, 12 PKC isoforms and other PKC-related kinases [3] were identified in mammalian tissues. These are classified into three groups. (1) the Ca-activated -, -,and -PKCs which display a Ca-binding site (C2); (2) the Ca-insensitive -, -, -, -, and -PKCs. The kinases that belong to both of these groups display two cystein-rich domains (C1) which bind phorbol esters (for recent review on PKC structure, see [4]). (3) The third group was named atypical PKCs and include , , and -PKCs that lack both the C2 and one cystein-rich domain. Consequently, these isoforms are Ca-insensitive and cannot be activated by phorbol esters [5]. In the heart. evidence that multiple PKC isoforms exist was first provided by Kosaka et al. [6] who identified by chromatography at least two PKC-related isoenzymes. Numerous studies were thus devoted to the biochemical characterization of these isoenzymes (see [7] for review on cardiac PKCs) as well as to the identification of their substrates.This overview aims at updating the present knowledge on the expression, activation and functions of PKC isoforms in cardiac cells. (Mol Cell Biochem 157: 65–72, 1996)     

Intrinsic fluorescence as a probe of structure-function relationships in Ca2+-transport ATPases

Applications of intrinsic fluorescence measurements in the study of Ca²⁺-transport ATPases are reviewed. Since the initial reports showing that the fluorescence emission was sensitive to Ca²⁺ binding, a substantial amount of work has focused on the use of both steady-state and time-resolved fluorescence spectroscopy to investigate structure-function relationships in sarcoplasmic reticulum and plasma membrane Ca²⁺-ATPases. These studies have revealed ligand-induced conformational changes, as well as provided information on protein-protein, protein-solvent and/or protein-lipid interactions in different functional states of these proteins. The main results of these studies, as well as possible future prospects are discussed.     

Molecular evolution of the N-formyl peptide and C5a receptors in non-human primates

N-formyl peptides (FMLP) and complement fragment C5a are neutrophil chemoattractants. In humans, a single-copy gene was identified for the C5a receptor, and the receptor for FMLP (FPR1) is encoded by a single gene that shows 53% amino acid similarity to the C5aR. Two other humanFPR1 homologues,FPR-like 1 (FPR2/FPRL1) andFPR-like 2 (FPRL2) have been cloned. The human C5aR, FPR1, FPRL1, and FPRL2 are physically linked. By direct sequencing or by sequencing plasmid clones we studied theC5aR andFPR genes from four non-human primates (chimpanzee, gorilla, orangutan, and macaque). The sequences showed 95%–99% similarity to the human homologues, with the major divergences observed in macaque. In these genes, the transmembrane and the cytoplasmic domains are highly conserved, while the highest divergence corresponded to the extracellular loops involved in ligand binding. Additionally, we constructed a physical map of these genes in non-human primates. In all species the four genes were physically linked and we defined the relative orientation of the four genes in primates:C5aR>FPR1>FPR2 (FPRL1)>FPRL2. The nucleotide sequence data reported in this paper have been submitted to the EMBL/GenBank nucleotide sequence databases and have assigned the accession numbers X97730 (PTC5aR), X97731 (MMC5aR), X97732 (PPC5aR), X97730 (GGC5aR), X97734 (MMFPR1), X97735 (PPFPR1), X97736 (GGFPR1), X97737 (MMFPRL1), X97738 (GGFPRL1), X97739 (PTFPRL1), X97740 (MMFPRL2), X97741 (PPFPRL2), X97742 (GGFPRL2), X97743 (PTFPRL2), X97744 (PPFPRL1), and X97745 (PTFPR1)