Hydrogen peroxide-induced oxidative damages in <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

Oxidative stress causes damage to proteins, lipids and nucleic acids, and thereby compromises cell viability. Some of the oxidative stress markers in an eukaryotic model organism, fission yeast Schizosaccharomyces pombe, were evaluated in this study. Intracellular oxidation, protein carbonyls, lipid peroxidation and reduced glutathione (GSH) levels were investigated in H₂O₂-treated and non-treated control cells. It was observed that increased H₂O₂ concentration proportionally lowered the cell number and increased the intracellular oxidation, lipid peroxidation and protein carbonyl levels in S. pombe. A dose-dependent decrease in GSH level was also detected. The fission yeast S. pombe is best known for its contribution to understanding of eukaryotic cell cycle control. S. pombe displays a different physiology from Saccharomyces cerevisiae in several ways and is thus probably more closely related to higher eukaryotes. The purpose of this study was to provide some data about the effects of hydrogen peroxide on the proteins and lipids in the fission yeast. The data obtained here is expected to constitute a basis for the further studies on redox balance and related processes in yeast and mammalian cells.     

A replication-time-controlling sequence element in <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

Eukaryotic replication origins are highly variable in their activity and replication timing. The nature and role of cis-acting regulatory sequences that control chromosomal replication timing is not well defined. In the fission yeast, Schizosaccharomyces pombe, a 200-bp late-replication-enforcing element (LRE), has been shown to enforce late replication of ARS elements in plasmids. Here, we show that a short (133-bp) fragment of the LRE (shLRE) is required for causing late replication of adjoining origins in its native as well as in an ectopic early-replicating chromosomal location. Active from both sides of an early-replicating origin, the shLRE is a bona fide cis-acting regulatory element that imposes late replication timing in the chromosome.     

The model unicellular eukaryote, <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

The fission yeast Schizosaccharomyces pombe has long been a model organism for studies of eukaryotic cells, winning renown especially for studies of the cell cycle. Now that its genome has been sequenced, S. pombe is ready to assume its rightful place in the pantheon of small eukaryotic giants.     

The <Emphasis Type="Italic">Lactococcus lactis</Emphasis> FabF fatty acid synthetic enzyme can functionally replace both the FabB and FabF proteins of <Emphasis Type="Italic">Escherichia coli</Emphasis> and the FabH protein of <Emphasis Type="Italic">Lactococcus lactis</Emphasis>

The genome of Lactococcus lactis encodes a single long chain 3-ketoacyl-acyl carrier protein synthase. This is in contrast to its close relative, Enterococcus faecalis, and to Escherichia coli, both of which have two such enzymes. In E. faecalis and E. coli, one of the two long chain synthases (FabO and FabB, respectively) has a role in unsaturated fatty acid synthesis that cannot be satisfied by FabF, the other long chain synthase. Since L. lactis has only a single long chain 3-ketoacyl-acyl carrier protein synthase (annotated as FabF), it seemed likely that this enzyme must function both in unsaturated fatty acid synthesis and in elongation of short chain acyl carrier protein substrates to the C18 fatty acids found in the cellular phospholipids. We report that this is the case. Expression of L. lactis FabF can functionally replace both FabB and FabF in E. coli, although it does not restore thermal regulation of phospholipid fatty acid composition to E. coli fabF mutant strains. The lack of thermal regulation was predictable because wild-type L. lactis was found not to show any significant change in fatty acid composition with growth temperature. We also report that overproduction of L. lactis FabF allows growth of an L. lactis mutant strain that lacks the FabH short chain 3-ketoacyl-acyl carrier protein synthase. The strain tested was a derivative (called the ∆fabH bypass strain) of the original fabH deletion strain that had acquired the ability to grow when supplemented with octanoate. Upon introduction of a FabF overexpression plasmid into this strain, growth proceeded normally in the absence of fatty acid supplementation. Moreover, this strain had a normal rate of fatty acid synthesis and a normal fatty acid composition. Both the ∆fabH bypass strain that overproduced FabF and the wild type strain incorporated much less exogenous octanoate into long chain phospholipid fatty acids than did the ∆fabH bypass strain. Incorporation of octanoate and decanoate labeled with deuterium showed that these acids were incorporated intact as the distal methyl and methylene groups of the long chain fatty acids.     

<Emphasis Type="Italic">fabC</Emphasis> of <Emphasis Type="Italic">Streptomyces lydicus</Emphasis> involvement in the biosynthesis of streptolydigin

Streptolydigin, a secondary metabolite produced by Streptomyces lydicus, is a potent inhibitor of bacterial RNA polymerases. It has been suggested that streptolydigin biosynthesis is associated with polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS). Thus, there is great interest in understanding the role of fatty acid biosynthesis in the biosynthesis of streptolydigin. In this paper, we cloned a type II fatty acid synthase (FAS II) gene cluster of fabDHCF from the genome of S. lydicus and constructed the SlyfabCF-disrupted mutant. Sequence analysis showed that SlyfabDHCF is 3.7 kb in length and encodes four separated proteins with conserved motifs and active residues, as shown in the FAS II of other bacteria. The SlyfabCF disruption inhibited streptolydigin biosynthesis and retarded mycelial growth, which were likely caused by the inhibition of fatty acid synthesis. Streptolydigin was not detected in the culture of the mutant strain by liquid chromatography–mass spectrometry. Meanwhile, the streptolol moiety of streptolydigin accumulated in cultures. As encoded by fabCF, acyl carrier protein (ACP) and β-ketoacyl-ACP synthase II are required for streptolydigin biosynthesis and likely involved in the step between PKS and NRPS. Our results provide the first genetic and metabolic evidence that SlyfabCF is shared by fatty acid synthesis and antibiotic streptolydigin synthesis.     

Changes in fatty acids and sterols during batch growth of <Emphasis Type="Italic">Pavlova viridis</Emphasis> in photobioreactor

Changes in the composition of fatty acids and sterols of Pavlova viridis cultured in an air-lift photobioreactor were studied using gas chromatography-mass spectrometry (GC-MS). The results show that radical changes in fatty acid and sterol contents and compositions occurred during growth phase transitions: the total lipid increased along with the culture age, from 166.4 mg g⁻¹ (late exponential phase) to 232.7 mg g⁻¹ (linear phase), and increased further to be 235.1 mg g⁻¹ in the stationary phase. Polyunsaturated fatty acids (PUFAs), especially eicosapentaenoic acid (EPA), decreased along with the culture time, PUFAs, and EPA contents maximized in the late exponential phase to become 46.2 mg g⁻¹ and 22.1 mg g⁻¹ respectively; there was no significant change in docosahexaenoic acid (DHA) content during the whole growth phase, although it reached the peak in the linear phase with 3.5 mg g⁻¹. As for the sterols, two unique sterols with two hydroxyl groups, termed pavlovols, were observed. 4α,24-Dimethylcholestan-3β,4β-diol, one of the pavlovols, increased almost 2-fold from the late exponential phase (2.5 mg g⁻¹) to the stationary phase (4.3 mg g⁻¹). On the contrary, the contents of stigmasterol and sitosterol decreased with culture age, with the maximum content of 2.4 mg g⁻¹ and 3.1 mg g⁻¹, both obtained in the late exponential phase, respectively. The results indicate that growth phase control could be used as a methodology to optimize the total lipid, EPA, PUFA, and sterol contents with the potential for both aquaculture feeds and nutraceutical applications, especially for further research into unique pavlovols.     

Oleic acid prevents apoptotic cell death induced by <Emphasis Type="Italic">trans</Emphasis>10, <Emphasis Type="Italic">cis</Emphasis>12 isomer of conjugated linoleic acid via p38 MAP kinase dependent pathway

Our previous study indicated that oleic acid prevented apoptotic cell death induced by trans10, cis12 (t10, c12)-conjugated linoleic acid in rat hepatoma dRLh-84 cells. The intracellular mechanism of action oleic acid is still unknown. Here, we showed that p38 mitogen-activated protein kinase (MAPK) inhibition using its specific inhibitor SB203580 cancelled the ameliorative effect of oleic acid on the cytotoxicity of t10, c12-conjugated linoleic acid. In addition, SubG1 cell population analysis showed that p38 MAPK played an essential role in the prevention of apoptotic cell death by oleic acid. In fact, p38 phosphorylation level was upregulated in cells treated with oleic acid irrespective of t10, c12-conjugated linoleic acid stimulation. Interestingly, t10, c12-conjugated linoleic acid increased intracellular triglyceride accumulation. However, oleic acid completely inhibited this effect. These observations indicated the involvement of blockade of a p38 MAPK pathway in the ameliorative effect of oleic acid on apoptosis induced by t10, c12-conjugated linoleic acid.     

Characterization of chromate-sensitive and-tolerant mutants of<Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

Stable chromium(VI)-sensitive and -tolerant mutants were obtained by induced mutagenesis of Schizosaccharomyces pombe lysine and leucine auxotrophic heterothallic strains 6chr+ and 9chr+. Eleven of them were selected for further studies. Fast transport of 51CrO4(2-) was detected in a representative sensitive mutant, chr-51S, while the tolerant mutant chr1-66T and the parental strain 6chr+ exhibited significantly lower 51CrO4(2-) uptake. The segregation of tetrads of three selected CrVI-tolerant mutants, chr1-66T, chr1-14T and chr2-04T, strongly indicated that tolerance was determined by single mutations. Random spore analysis proved that the mutations of chr1-66T and chr1-14T were allelic and the mutation of mutant chr2-04T was not allelic with the mutation of chr1-66T. Recombinants carrying the ura4D18 selective marker were created for transformation experiments. Two of them (chr1-661T and chr2-046T) can be used to clone and identify the genes responsible for their CrVI tolerance phenotype.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

An evaluation of the diet of <Emphasis Type="Italic">Mysis relicta</Emphasis> using gut contents and fatty acid profiles in lakes with and without the invader <Emphasis Type="Italic">Bythotrephes longimanus</Emphasis> (<Emphasis Type="Italic">Onychopoda,Cercopagidae</Emphasis>)

Diets of Mysis relicta from four lakes in central Ontario that had been invaded by Bythotrephes longimanus and three lakes that had not been invaded were investigated using gut content analysis and fatty acid (FA) composition. Gut content analysis of M. relicta revealed a high incidence of cannibalism in all lakes, and consumption of B. longimanus and native zooplanktivorous midges in the genus Chaoborus in lakes where these were present. Cladocera other than B. longimanus were present in the guts of all M. relicta examined except those from Bernard Lake, the lake with the most B. longimanus. In that lake, B. longimanus was the most frequent diet item. Copepod remains were found in 60–100% of M. relicta guts with the lowest frequency occurring in Bernard Lake. Fatty acids (FA) that contributed strongly to the variation in FA composition in M. relicta, as revealed by a principal component analysis, were C16:0 (palmitic acid), C16:1n7 (palmitoleic acid), C18:1n9c (oleic acid), C20:4n6 (arachidonic acid), C20:5n3 (eicosapentaenoic acid), and C22:6n3 (docosahexaenoic acid). Significant differences in FA amount and composition of M. relicta were found between invaded and non-invaded lakes, and among lakes within these groups. Generally, M. relicta in non-invaded lakes had higher concentrations of C16:0, C18:1n9c, C18:2n6c (linoleic acid), C18:3n3 (α-linolenic acid) and C20:4n6, while M. relicta in invaded lakes had higher concentrations of C22:6n3. Two of the non-invaded lakes had lower water transparency, as measured by Secchi depth, which may be the reason why mysids and abundant populations of Chaoborus spp. could be found in the water column during the day. However, differences in FA profiles and gut contents of M. relicta between invaded and non-invaded lakes are consistent with competition for Cladocera in the presence of the invader rather than pre-existing differences among lakes. We conclude that the diet of M. relicta is affected by the invasion of B. longimanus.     

Identification of thermostable glyoxalase I in the fission yeast <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

Glyoxalase I is a ubiquitous enzyme that detoxifies methylglyoxal, which is derived from glycolysis but inhibits the growth of cells from microorganisms to mammals. Here, the structural gene for glyoxalase I (glo1⁺) from the fission yeast Schizosaccharomyces pombe was identified. Disruption of glo1⁺ enhanced susceptibility to methylglyoxal, while expression of glo1⁺ in a glo1 mutant of Saccharomyces cerevisiae restored tolerance to this aldehyde. The glo1⁺ gene product was purified. The glyoxalase I of S. pombe was a monomeric enzyme with a molecular weight of 34,000 and the k_cat/K_m value for methylglyoxal was 4.3×10⁷ M^–1 min^–1. Treatment of purified enzyme with EDTA in imidazole buffer completely abolished enzyme activity, whereas the EDTA-treated enzyme was reactivated by several divalent metal ions, such as Zn²⁺, Co²⁺, Ni²⁺ and Mn²⁺. The glyoxalase I of S. pombe exhibited fairly high thermal stability, and almost 100% activity was retained after incubating the enzyme at 60°C for 4 h.     

Neuro-and psychotropic activity of <Emphasis Type="Italic">N</Emphasis>-uronoylamino acids and <Emphasis Type="Italic">N</Emphasis>-uronoylpeptides

Peculiarities of the rat behavior were studied in a series of experimental stress models after a systemic administration of new N-uronoyl derivatives of amino acids. The psychotropic effect was shown to be determined by the nature of the amino acid fragment. N-(1,2:3,4-Di-O-isopropylidene-α-D-galactopyraneuronoyl)-glycylglycine exhibited an anxiolytic effect more pronounced than that of pyracetam, whereas N-(1,2:3,4-di-O-isopropilidene-α-D-galactopyranuronoyl)-glycylglutamic acid has antidepressant action stronger than that of amitriptyline. Mechanisms for the psychotropic effects of the examined derivatives are discussed.     

Anti-cyanobacterial fatty acids released from <Emphasis Type="Italic">Myriophyllum spicatum</Emphasis>

This study was carried out to identify unknown allelochemicals released from Myriophyllum spicatum and to investigate their anti-cyanobacterial effects. A series of analyses of culture solutions and methanol extracts of M. spicatum using gas chromatograph equipped with a mass selective detector revealed that M. spicatum released fatty acids, specifically, nonanoic, tetradecanoic, hexadecanoic, octadecanoic, and octadecenoic acids. Nonanoic, cis-6-octadecenoic, and cis-9-octadecenoic acids significantly inhibited growth of Microcystis aeruginosa, whereas tetradecanoic, hexadecanoic, and octadecanoic acids did not show any effect. When the inhibitory effect of nonanoic acid was compared with those of 4 polyphenols and eugeniin, which are anti-cyanobacterial compounds previously reported to be released by M. spicatum, nonanoic acid was found to be the most inhibitory to M. aeruginosa. These results indicate that not only polyphenols and eugeniin but also fatty acids such as nonanoic acid must be studied to reveal how M. spicatum exerts its allelopathic effect on M. aeruginosa.     

An efficient process for production and purification of hyaluronic acid from <Emphasis Type="Italic">Streptococcus </Emphasis><Emphasis Type="Italic">equi</Emphasis> subsp. <Emphasis Type="Italic">zooepidemicus</Emphasis>

Growth of Streptococcus zooepidemicus in a 10 l bioreactor with 50 g sucrose/l and 10 g casein hydrolysate/l gave 5–6 g hyaluronic acid/l after 24–28 h. Purification of hyaluronic acid gave a recovery of 65% with the final material having an Mr of ∼4 × 10⁶ Da with less than 0.1% protein.     

Recombinational repair in <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>: The role of mediator proteins

Repair of double-strand breaks (DSBs), which arise in DNA spontaneously or under the influence of external factors, is critical for cell survival. The evolutionarily conserved mechanism of error-free recombinational repair plays a major role in maintaining the genome integrity and has a number of similarities between lower eukaryotes and vertebrates. The review considers the currently available data on the mechanism of recombinational DSB repair in the fission yeast Schizosaccharomyces pombe and its differences from the corresponding mechanisms of Saccharomyces cerevisiae and higher eukaryotes.