Chemically assisted microbial production of succinic acid by the yeast <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> grown on ethanol

A new two-step process of production of succinic acid (SA) has been developed, which includes the microbial synthesis of alpha-ketoglutaric acid by the yeast Yarrowia lipolytica (step 1) and subsequent oxidation of the acid by hydrogen peroxide to SA (step 2). The maximum concentration of SA and its yield were found to be 63.4 g l⁻¹ and 58% of the ethanol consumed, respectively. The purity of the SA isolated from the culture liquid filtrate reached 100%. The yield of SA was as high as 82% of its amount in the culture liquid filtrate. The quality of the SA produced by the invented method meets the biochemical grade definitions, as is evident from the respiratory and other relevant parameters of rat liver mitochondria upon the oxidation of this SA.     

The Diversity of Karyotypes and Genomes within Section Syllinum of the Genus Linum (Linaceae) Revealed by Molecular Cytogenetic Markers and RAPD Analysis

The wide variation in chromosome number found in species of the genus Linum (2n = 16, 18, 20, 26, 28, 30, 32, 36, 42, 72, 84) indicates that chromosomal mutations have played an important role in the speciation of this taxon. To contribute to a better understanding of the genetic diversity and species relationships in this genus, comparative studies of karyotypes and genomes of species within section Syllinum Griseb. (2n = 26, 28) were carried out. Elongated with 9-aminoacridine chromosomes of 10 species of section Syllinum were investigated by C- and DAPI/С-banding, CMA and Ag-NOR-staining, FISH with probes of rDNA and of telomere repeats. RAPD analysis was also performed. All the chromosome pairs in karyotypes of the studied species were identified. Chromosome DAPI/C-banding patterns of 28-chromosomal species were highly similar. Two of the species differed from the others in chromosomal location of rDNA sites. B chromosomes were revealed in all the 28-chromosomal species. Chromosomes of Linum nodiflorum L. (2n = 26) and the 28-chromosomal species were similar in DAPI/C-banding pattern and localization of several rDNA sites, but they differed in chromosomal size and number. The karyotype of L. nodiflorum was characterized by an intercalary site of telomere repeat, one additional 26S rDNA site and also by the absence of B chromosomes. Structural similarities between different chromosome pairs in karyotypes of the studied species were found indicating their tetraploid origin. RAPD analysis did not distinguish the species except L. nodiflorum. The species of section Syllinum probably originated from a common tetraploid ancestor. The 28-chromosomal species were closely related, but L. nodiflorum diverged significantly from the rest of the species probably due to chromosomal rearrangements occurring during evolution.     

Acidification Activates Toxoplasma gondii Motility and Egress by Enhancing Protein Secretion and Cytolytic Activity

Pathogenic microbes rely on environmental cues to initiate key events during infection such as differentiation, motility, egress and invasion of cells or tissues. Earlier investigations showed that an acidic environment activates motility of the protozoan parasite T. gondii. Conversely, potassium ions, which are abundant in the intracellular milieu that bathes immotile replicating parasites, suppress motility. Since motility is required for efficient parasite cell invasion and egress we sought to better understand its regulation by environmental cues. We found that low pH stimulates motility by triggering Ca²⁺-dependent secretion of apical micronemes, and that this cue is sufficient to overcome suppression by potassium ions and drive parasite motility, cell invasion and egress. We also discovered that acidification promotes membrane binding and cytolytic activity of perforin-like protein 1 (PLP1), a pore-forming protein required for efficient egress. Agents that neutralize pH reduce the efficiency of PLP1-dependent perforation of host membranes and compromise egress. Finally, although low pH stimulation of microneme secretion promotes cell invasion, it also causes PLP1-dependent damage to host cells, suggesting a mechanism by which neutral extracellular pH subdues PLP1 activity to allow cell invasion without overt damage to the target cell. These findings implicate acidification as a signal to activate microneme secretion and confine cytolytic activity to egress without compromising the viability of the next cell infected.     

Flavoprotein miniSOG as a genetically encoded photosensitizer for cancer cells

Background

Genetically encoded photosensitizers are a promising optogenetic instrument for light-induced production of reactive oxygen species in desired locations within cells in vitro or whole body in vivo. Only two such photosensitizers are currently known, GFP-like protein KillerRed and FMN-binding protein miniSOG. In this work we studied phototoxic effects of miniSOG in cancer cells.

Methods

HeLa Kyoto cell lines stably expressing miniSOG in different localizations, namely, plasma membrane, mitochondria or chromatin (fused with histone H2B) were created. Phototoxicity of miniSOG was tested on the cells in vitro and tumor xenografts in vivo.

Results

Blue light induced pronounced cell death in all three cell lines in a dose-dependent manner. Caspase 3 activation was characteristic of illuminated cells with mitochondria- and chromatin-localized miniSOG, but not with miniSOG in the plasma membrane. In addition, H2B-miniSOG-expressing cells demonstrated light-induced activation of DNA repair machinery, which indicates massive damage of genomic DNA. In contrast to these in vitro data, no detectable phototoxicity was observed on tumor xenografts with HeLa Kyoto cell lines expressing mitochondria- or chromatin-localized miniSOG.

Conclusions

miniSOG is an excellent genetically encoded photosensitizer for mammalian cells in vitro, but it is inferior to KillerRed in the HeLa tumor.

General significance

This is the first study to assess phototoxicity of miniSOG in cancer cells. The results suggest an effective ontogenetic tool and may be of interest for molecular and cell biology and biomedical applications.     

G-specific RNA-cleaving Conjugates of Short Peptides and Oligodeoxyribonucleotides

Abstract

Artificial ribonucleases, conjugates of short oligodeoxyribonucleotides and peptides built of arginine, leucine, proline, and serine, were synthesized and assessed in terms of ribonuclease activity and specificity of RNA cleavage. A specific group of the conjugates was identified that display T1-ribonuclease-like activity and cleave RNA predominantly at G-X sequences. Circular dichroism study of the structures of the most active conjugates, free peptide (LR)4G, and oligonucleotides revealed that conjugation of oligonucleotide to the peptide results in a specific peptide folding that possibly provides ribonuclease activity to the conjugate.     

Potato signal molecules that activate pectate lyase synthesis in Pectobacterium atrosepticum SCRI1043

A new type of plant-derived signal molecules that activate extracellular pectate lyase activity in phytopathogenic bacterium Pectobacterium atrosepticum SCRI1043 was revealed. These compounds were characterized and partially purified by means of several approaches including RT-PCR analysis, luminescence bioassay and HPLC fractionation. They were smaller than 1 kDa, thermoresistant, nonproteinaceous, hydrophilic, and slightly negatively charged molecules. Using gene expression analysis and bacterial biosensor assay the mode of activity of revealed compounds was studied. The possibility of their action through quorum sensing- and KdgR-mediated pathways was analyzed.     

Antiviral activity of a small-molecule inhibitor of arenavirus glycoprotein processing by the cellular site 1 protease

Arenaviruses merit interest as clinically important human pathogens and include several causative agents, chiefly Lassa virus (LASV), of hemorrhagic fever disease in humans. There are no licensed LASV vaccines, and current antiarenavirus therapy is limited to the use of ribavirin, which is only partially effective and is associated with significant side effects. The arenavirus glycoprotein (GP) precursor GPC is processed by the cellular site 1 protease (S1P) to generate the peripheral virion attachment protein GP1 and the fusion-active transmembrane protein GP2, which is critical for production of infectious progeny and virus propagation. Therefore, S1P-mediated processing of arenavirus GPC is a promising target for therapeutic intervention. To this end, we have evaluated the antiarenaviral activity of PF-429242, a recently described small-molecule inhibitor of S1P. PF-429242 efficiently prevented the processing of GPC from the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) and LASV, which correlated with the compound's potent antiviral activity against LCMV and LASV in cultured cells. In contrast, a recombinant LCMV expressing a GPC whose processing into GP1 and GP2 was mediated by furin, instead of S1P, was highly resistant to PF-429242 treatment. PF-429242 did not affect virus RNA replication or budding but had a modest effect on virus cell entry, indicating that the antiarenaviral activity of PF-429242 was mostly related to its ability to inhibit S1P-mediated processing of arenavirus GPC. Our findings support the feasibility of using small-molecule inhibitors of S1P-mediated processing of arenavirus GPC as a novel antiviral strategy.     

Fe(III) Oxide Reduction by a Gram-positive Thermophile: Physiological Mechanisms for Dissimilatory Reduction of Poorly Crystalline Fe(III) Oxide by a Thermophilic Gram-positive Bacterium Carboxydothermus ferrireducens

Physiological strategies driving the reduction of poorly crystalline Fe(III) oxide by the thermophilic Gram-positive dissimilatory Fe(III)-reducing bacterium C. ferrireducens were evaluated. Direct cell-to-mineral contact appears to be the major physiological strategy for ferrihydrite reduction. This strategy is promoted by cell surface-associated c-type cytochromes, and the extracellular electron transfer to ferrihydrite is linked to energy generation via a membrane-bound electron transport chain. The involvement of pili-like appendages in ferrihydrite reduction has been detected for the first time in a thermophilic microorganism. A supplementary strategy for the utilization of a siderophore (DFO) in dissimilatory ferrihydrite reduction has also been characterized.     

Radioprotective role of cyanobacterial phycobilisomes

Cyanobacteria are thought to be responsible for pioneering dioxygen production and the so-called “Great Oxygenation Event” that determined the formation of the ozone layer and the ionosphere restricting ionizing radiation levels reaching our planet, which increased biological diversity but also abolished the necessity of radioprotection. We speculated that ancient protection mechanisms could still be present in cyanobacteria and studied the effect of ionizing radiation and space flight during the Foton-M4 mission on Synechocystis sp. PCC6803. Spectral and functional characteristics of photosynthetic membranes revealed numerous similarities of the effects of α-particles and space flight, which both interrupted excitation energy transfer from phycobilisomes to the photosystems and significantly reduced the concentration of phycobiliproteins. Although photosynthetic activity was severely suppressed, the effect was reversible, and the cells could rapidly recover from the stress. We suggest that the actual existence and the uncoupling of phycobilisomes may play a specific role not only in photo-, but also in radioprotection, which could be crucial for the early evolution of Life on Earth.     

Synthesis of a quaternary polyprenyl ammonium salt

Reaction of primary C(55)-allylic alcohol moraprenol (WT(3)C(7-9)-OH, a polyprenol from mulberry leaves) with triethylamine in the presence of phosphorus oxychloride leads to a quaternary ammonium chloride with a good yield (72%) and high cis-stereoselectivity of the terminal isoprene unit. Cationic polyprenyl derivatives may be useful for transfection and immunological studies.