Repair of cisplatin-DNA adducts in mutants for genes controlling spontaneous and induced mutagenesis in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> yeast

Sensitivity to the lethal action of the anticancer substance cisplatin was studied in the yeast mutants him1, hsm2, hsm3, and hsm6, deficient for repair of spontaneous and induced mutations. The him1 and hsm3 mutants were as resistant to the agent under study as the wild-type strain. The survival of the double mutant rad2 hsm3 was higher than that of the single mutant rad2. The hsm2 and hsm6 mutants were more cisplatin-sensitive than the wild type. Cisplatin was shown to have high mutagenic and recombinogenic effects on yeast cells.     

Fungicidal activity of cellobiose lipids from culture broth of yeast <Emphasis Type="Italic">Cryptococcus humicola</Emphasis> and <Emphasis Type="Italic">Pseudozyma fusiformata</Emphasis>

Cellobiose lipids of yeast fungi Cryptococcus huminola and Pseudozyma fusiformata have similar fungicidal activities against different yeast, including pathogenic Cryptococcus and Candida species. Basidiomycetic yeast reveals maximum sensitivity to these preparations; e.g., cells of cryptococcus Filobasidiella neoformans almost completely die after 30-min incubation in a glycolipid solution at a concentration of 0.02 mg/ml. The same effect toward ascomycetous yeast, including pathogenic Candida species, is achieved only at five to eight times higher concentrations of glycolipids. The cellobiose lipid from P. fusiformata, which, unlike glycolipid from Cr. humicola, has hydroxycaproic acid residue as O-subtituent of cellobiose and additional 15-hydroxy group in aglycone, inhibits the growth of the studied mycelial fungi more efficiently than the cellobiose lipid from Cr. humicola.     

DNA-Based Identification of <Emphasis Type="Italic">Valeriana officinalis</Emphasis> s.l.: a Multiplexed Amplification Refractory Mutation System (MARMS) and High Resolution Melting Curve Analysis (HRMA)

The wide distribution of Valeriana officinalis as a herbal remedy as well as the considerably higher concentration of putative mutagenic valepotriate metabolites in other drug-delivering valerian species like Valeriana procera Kunth and Valeriana jatamansi Jones ex Roxb. illustrate the necessity of secure authentication of roots of Valeriana officinalis s.l., especially as the morphologically similar roots of the acutely toxic Veratrum album can be mistaken for those of Valeriana officinalis. We developed two DNA-based systems, a multiplex amplification refractory mutation system (MARMS), and a high-resolution melting curve analysis (HRMA) assay, both based on a sequence mutation within the atpB-rbcL region. With both methods, identification of Valeriana officinalis s.l. was possible. With the HRMA, the characteristic melting curve of 33 samples of Valeriana officinalis s.l. and of two commercial samples of Valerianae radix was distinct from the melting curves of all other Valeriana species (60 accessions), and from the closely related genera Centranthus and Valerianella. Since adulteration of Valeriana with toxic Veratrum species was reported previously, Veratrum primers were included in a multiplex PCR-HRM analysis. This system allowed the detection of a Veratrum admixture down to the level of 0.01 %. Although the advantages, in terms of sensitivity, specificity and practicality of the HRM for analysis of degraded plant material were superior to the MARMS assay, both methods are suitable for routine analysis. The results demonstrated the general ability of HRMA to detect specific (toxic) adulterations in drugs in a semiquantitative way.     

Lectin-Conjugated Clarithromycin and Acetohydroxamic Acid-Loaded PLGA Nanoparticles: a Novel Approach for Effective Treatment of <Emphasis Type="Italic">H. pylori</Emphasis>

Helicobacter pylori infection remains challenging as it mainly colonized beneath the deep gastric mucosa and adheres to epithelial cells of the stomach. Concanavalin-A (Con-A)-conjugated gastro-retentive poly (lactic-co-glycolic acid) (PLGA) nanoparticles of acetohydroxamic acid (AHA) and clarithromycin (CLR) were prepared and evaluated under in vitro conditions. Solvent evaporation method was employed for preparation of nanoparticles and characterized for particle size distribution, surface morphology, percent drug entrapment, and in vitro drug release in simulated gastric fluid. Optimized nanoparticles were conjugated with Con-A and further characterized for Con-A conjugation efficiency and mucoadhesion and tested for in vitro anti-H. pylori activity. The conjugation with Con-A further sustained the drug release over a period of 8 h when compared to non-conjugated nanoparticles of AHA and CLR. In vitro anti H. pylori study confirmed that Con-A-conjugated nanoparticles containing both drugs, i.e., CLR and AHA, had shown maximum zone of inhibition compared to other formulations. In a nut shell, results suggest that the developed systems could be used for better therapeutic activity against H. pylori infection.     

Interaction between checkpoint genes <Emphasis Type="Italic">RAD9, RAD17, RAD24</Emphasis>, and <Emphasis Type="Italic">RAD53</Emphasis> involved in the determination of yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> sensitivity to ionizing radiation

Mechanisms for genetic control of cell division cycle (checkpoint control) have been studied in most detail in yeast Saccharomyces cerevisiae. To clarify the role of checkpoint genes RAD9, RAD17, RAD24, and RAD53 in cell radioresistance, double mutants were analyzed for cell sensitivity to ionizing radiation. Double mutants carrying mutations in combination with mutation rad9Δ were shown to manifest the epistatic type of interaction. Our results suggest that checkpoint genes RAD9, RAD17, RAD24, and RAD53 belong to a single epistatic group designated RAD9 and govern the same pathway. Genes RAD9 and RAD53 have a positive effect on sensitivity to γ-radiation, whereas RAD17 and RAD24 have a negative effect. Interactions between mutations may differ when considering their sensitivity to γ-radiation and UV light; mutations rad9Δ and rad24Δ were shown to manifest the additive effect in the first case and epistatic effect in the second.     

Immunohistochemical Cross-Reactivity Between <Emphasis Type="Italic">Paracoccidioides</Emphasis> sp. from Dolphins and <Emphasis Type="Italic">Histoplasma capsulatum</Emphasis>

Paracoccidioidomycosis ceti is a cutaneous disease of cetaceans caused by uncultivated Paracoccidioides brasiliensis or Paracoccidioides spp. Serological cross-reactions between paracoccidioidomycosis ceti and paracoccidioidomycosis, paracoccidioidomycosis and histoplasmosis, and paracoccidioidomycosis and coccidioidomycosis have been reported before. The present study aimed to detect immunohistochemical cross-reaction between antibodies to Paracoccidioides sp. and Histoplasma capsulatum, and vice versa. Thirty murine sera, obtained from experimental infections of 6 isolates of H. capsulatum, were reacted with paraffin-embedded yeast-form cells of Paracoccidioides sp. derived from a case of paracoccidioidomycosis ceti in Japan. The murine sera were also reacted with human isolates of H. capsulatum yeast cells, with P. brasiliensis yeast cells, and with fungal cells of Coccidioides posadasii. Three dolphins’ sera from cases of paracoccidioidomycosis ceti, two human sera from patients with paracoccidioidomycosis, and a serum from a healthy person with a history of coccidioidomycosis were used in order to determine that the tested fungal cells reacted properly. Sera derived from mice infected with an isolate of H. capsulatum reacted positively against yeast cells of Paracoccidioides sp., yeast cells of P. brasiliensis, and fungal cells of C. posadasii, while those derived from other strains were negative. The present study recorded for the first time the cross-reaction between the yeast cells of H. capsulatum and antibodies against Paracoccidioides spp., the yeast cells of Paracoccidioides sp. and antibodies against H. capsulatum, the yeast cells of Paracoccidioides sp. and antibodies against Coccidioides sp., and fungal cells of C. posadasii and antibodies against Paracoccidioides spp.     

A putative <Emphasis Type="Italic">NEM1</Emphasis> homologue regulates lipid droplet biogenesis <Emphasis Type="Italic">via PAH1</Emphasis> in <Emphasis Type="Italic">Tetrahymena thermophila</Emphasis>

Nuclear envelope morphology protein 1 (NEM1) along with a phosphatidate phosphatase (PAH1) regulates lipid homeostasis and membrane biogenesis in yeast and mammals. We investigated four putative NEM1 homologues (TtNEM1A, TtNEM1B, TtNEM1C and TtNEM1D) in the Tetrahymena thermophila genome. Disruption of TtNEM1B, TtNEM1C or TtNEM1D did not compromise normal cell growth. In contrast, we were unable to generate knockout strain of TtNEM1A under the same conditions, indicating that TtNEM1A is essential for Tetrahymena growth. Interestingly, loss of TtNEM1B but not TtNEM1C or TtNEM1D caused a reduction in lipid droplet number. Similar to yeast and mammals, TtNem1B of Tetrahymena exerts its function via Pah1, since we found that PAH1 overexpression rescued loss of Nem1 function. However, unlike NEM1 in other organisms, TtNEM1B does not regulate ER/nuclear morphology. Similarly, neither TtNEM1C nor TtNEM1D is required to maintain normal ER morphology. While Tetrahymena PAH1 was shown to functionally replace yeast PAH1 earlier, we observed that Tetrahymena NEM1 homologues did not functionally replace yeast NEM1. Overall, our results suggest the presence of a conserved cascade for regulation of lipid homeostasis and membrane biogenesis in Tetrahymena. Our results also suggest a Nem1-independent function of Pah1 in the regulation of ER morphology in Tetrahymena.     

Dynamic variation of toxic and non-toxic <Emphasis Type="Italic">Microcystis</Emphasis> proportion in the eutrophic Daechung Reservoir in Korea

This study was conducted to determine the environmental factors affecting the level of potentially toxic Microcystis. The long-term tendencies of temperature, precipitation, and water quality factors were analyzed to determine the environmental characteristics of the Daechung Reservoir in Korea, and water samples were directly collected to analyze the dynamics of toxic and non-toxic Microcystis at weekly intervals from May to October 2012. Microcystis was the dominant genus during the study period, and it was composed of potentially toxic and non-toxic Microcystis. The fraction of potentially toxic Microcystis ranged from 6.0% to 61.1%. The amount of toxic Microcystis was highly related to the intracellular microcystin concentration (r = 0.760, P < 0.01). Therefore, the fraction of potentially toxic Microcystis is an important concern in Microcystis blooming because the intracellular microcystin concentration may reflect microcystin levels in the water. The prevalence of potentially toxic Microcystis was highly related to water temperature in Daechung Reservoir (r = 0.585, P < 0.01). Thus, temperature increase during Microcystis blooming may lead to more frequent toxic Microcystis blooms in eutrophic water bodies.     

Molecular phylogeny of pectinase genes <Emphasis Type="Italic">PGU</Emphasis> in the yeast genus <Emphasis Type="Italic">Saccharomyces</Emphasis>

Using yeast genome databases and literature data, phylogenetic analysis of pectinase PGU genes from 112 Saccharomyces strains assigned to the biological species S. arboricola, S. bayanus (var. uvarum), S. cariocanus, S. cerevisiae, S. kudriavzevii, S. mikatae, S. paradoxus, and the hybrid taxon S. pastorianus (syn. S. carlsbergensis) was carried out. A superfamily of divergent PGU genes was found. Natural interspecies transfer of the PGU gene both from S. cerevisiae to S. bayanus and from S. paradoxus to S. cerevisiae may, however, occur. Within the Saccharomyces species, identity of the PGU nucleotide sequences was 98.8–100% for S. cerevisiae, 86.1–95.7% for S. bayanus (var. uvarum), 94–98.3% for S. kudriavzevii, and 96.8–100% for S. paradoxus/S. cariocanus. For the first time, a family of polymeric PGU1b, PGU2b, PGU3b and PGU4b genes is documented for the yeast S. bayanus var. uvarum, a variety important for winemaking.     

Microbioluminescent study of the general toxicity and mutagenicity of pollutants

Bacterial bioluminescence was applied to detection of general toxicity (MIT test) and genotoxicity (SOS-lux test) of some chemicals, seawater, and fresh water. The SOS-induced luminescence of E. coli WP2s (cda::luxCDABE) cells was higher than in E. coli C 600 (cda::luxCDABE) at 37°C and pH 6.5. The mutagenic effect of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), mitomycin C, and hydrogen peroxide determined from the induction of E. coli WP2s cell luminescence was detected at lower concentrations than in the assessment of reversion frequencies. General toxicity was demonstrated by using luminescence inhibition for hydrogen peroxide, Zn²⁺, and Cd²⁺ at low concentrations. Regions of the Krasnodar Krai where sea and fresh waters exerted toxic action on luminescence were determined by the microbioluminescent method.     

Comparison of L-lactate dehydrogenases of different origins produced in the cells of yeast <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

The expression of L-lactate dehydrogenase genes ldh1 (Bos taurus), ldhA (Homo sapiens), ldhA (Rhizopus oryzae), ldh1 (Lactobacillus plantarum), and ldh1 (Lactobacillus pentosus) in the cells of yeast Schizosaccharomyces pombe VKPM U-3106 has been investigated. The catalytic characteristics of the enzymes encoded by these genes have been compared, and the intensity of lactic acid synthesis by the recombinant strains obtained has been evaluated. The enzymatic activity of L-lactate dehydrogenases from L. plantarum and L. pentosus was the highest (approximately 2 to 2.5 times higher than that of the mammalian enzymes), and these enzymes therefore appear to have the highest potential for the development of lactic-acid producing strains of yeast S. pombe.     

The antibacterial agent,moxifloxacin inhibits virulence factors of <Emphasis Type="Italic">Candida albicans</Emphasis> through multitargeting

Fluoroquinolines are broad spectrum fourth generation antibiotics. Some of the Fluoroquinolines exhibit antifungal activity. We are reporting the potential mechanism of action of a fluoroquinoline antibiotic, moxifloxacin on the growth, morphogenesis and biofilm formation of the human pathogen Candida albicans. Moxifloxacin was found to be Candidacidal in nature. Moxifloxacin seems to inhibit the yeast to Hyphal morphogenesis by affecting signaling pathways. It arrested the cell cycle of C. albicans at S phase. Docking of moxifloxacin with predicted structure of C. albicans DNA Topoisomerase II suggests that moxifloxacin may bind and inhibit the activity of DNA Topoisomerase II in C. albicans. Moxifloxacin could be used as a dual purpose antibiotic for treating mixed infections caused by bacteria as well as C. albicans. In addition chances of developing moxifloxacin resistance in C. albicans are less considering the fact that moxifloxacin may target multiple steps in yeast to hyphal transition in C. albicans.     

Construction of flavocytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>2</Subscript>-overproducing strains of the thermotolerant methylotrophic yeast <Emphasis Type="Italic">Hansenula polymorpha</Emphasis> (<Emphasis Type="Italic">Pichia angusta</Emphasis>)

L-Lactate cytochrome c oxidoreductase (flavocytochrome b ₂, FC b ₂) from the thermotolerant methylotrophic yeast Hansenula polymorpha (Pichia angusta) is, unlike the enzyme form baker’s yeast, a thermostable enzyme potentially important for bioanalytical technologies for highly selective assays of L-lactate in biological fluids and foods. This paper describes the construction of flavocytochrome b ₂ producers with over-expression of the H. polymorpha CYB2 gene, encoding FC b ₂. The HpCYB2 gene under the control of the strong H. polymorpha alcohol oxidase promoter in a plasmid for multicopy integration was transformed into the recipient strain H. polymorpha C-105 (grc1 catX), impaired in glucose repression and devoid of catalase activity. A method was developed for preliminary screening of the transformants with increased FC b ₂ activity in permeabilized yeast cells. The optimal cultivation conditions providing for the maximal yield of the target enzyme were found. The constructed strain is a promising FC b ₂ producer characterized by a sixfold increased (to 3 μmol min^?1 mg^?1 protein in cell-free extract) activity of the enzyme.     

Genetic relationship and biological status of the industrially important yeast <Emphasis Type="Italic">Saccharomyces eubayanus</Emphasis> Sampaio et al.

The genomes of the recently discovered yeast Saccharomyces eubayanus and traditional S. cerevisiae are known to be found in the yeast S. pastorianus (syn. S. carlsbergensis), which are essential for brewing. The cryotolerant yeast S. bayanus var. uvarum is of great importance for production of some wines. Based on ascospore viability and meiotic recombination of the control parental markers in hybrids, we have shown that there is no complete interspecies post-zygotic isolation between the yeasts S. eubayanus, S. bayanus var. bayanus and S. bayanus var. uvarum. The genetic data presented indicate that all of the three taxa belong to the same species.