Aspergillus nidulans UDP-glucose-4-epimerase UgeA has multiple roles in wall architecture,hyphal morphogenesis,and asexual development

Aspergillus nidulans UDP-glucose-4-epimerase UgeA interconverts UDP-glucose and UDP-galactose and participates in galactose metabolism. The sugar moiety of UDP-galactose is predominantly found as galactopyranose (Galp, the six-membered ring form), which is the substrate for UDP-galactopyranose mutase (encoded by ugmA) to generate UDP-galactofuranose (Galf, the five-membered ring form) that is found in fungal walls. In A. fumigatus, Galf residues appear to be important for virulence. The A. nidulans ugeAΔ strain is viable, and has defects including wide, slow growing, highly branched hyphae and reduced conidiation that resemble the ugmAΔ strain. As for the ugmAΔ strain, ugeAΔ colonies had substantially reduced sporulation but normal spore viability. Conidia of the ugeAΔ strain could not form colonies on galactose as a sole carbon source, however they produced short, multinucleate germlings suggesting they ceased to grow from starvation. UgeA purified from an expression plasmid had a relative molecular weight of 40.6 kDa, and showed in vitro UDP-glucose-4-epimerase activity. Transmission electron microscope cross-sections of wildtype, ugeAΔ, and ugmAΔ hyphae showed they had similar cytoplasmic contents but the walls of each strain were different in appearance and thickness. Both deletion strains showed increased substrate adhesion. Localization of UgeA-GFP and UgmA-GFP was cytoplasmic, and was similar on glucose and galactose. Neither gene product had a longitudinal polarized distribution. Localization of a UgmA-mRFP in a strain that resembled the ugmAΔ strain was cytoplasmic and lacked a longitudinal polarized distribution. The roles of UgeA in A. nidulans growth and morphogenesis are consistent with the importance of Galf, and are related but not identical to the roles of UgmA.     

Effect of low pH on organization of the actin cytoskeleton in Saccharomyces cerevisiae

Cell growth in the yeast Saccharomyces cerevisiae depends on polarization of the actin cytoskeleton. In this study, we investigated how the cell regulates the distribution of actin in response to low pH conditions, focusing on the role of mitogen-activated protein kinases, Hog1 and Slt2. Changing the extracellular pH from 6.0 to 3.0 caused a transient depolarization of the actin cytoskeleton. Actin cables were no longer visible, and actin patches appeared randomly distributed after 30 min at pH 3.0. The deletion strain hog1Δ did not show this low-pH phenotype, suggesting that Hog1 is involved in depolarization of the actin cytoskeleton in response to low-pH stress. Yeast cells incubated at pH 3.0 also showed markedly increased endocytosis compared with the control at neutral pH, as indicated by the uptake of Lucifer Yellow (LY). Both the hog1Δ and slt2Δ mutants took up LY into the vacuole to a similar extent as the wild-type strain. In addition, cells grown at pH 3.0 showed a 2-fold increase in phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2) levels, as did the hog1Δ or slt2Δ cells. Efficient uptake of LY and actin repolarization at pH 3.0 might therefore require activation of PI(4,5)P2 synthesis.     

Intracellular aspartic proteinase Apr1p of Candida albicans is required for morphological transition under nitrogen-limited conditions but not for macrophage killing

Vacuolar hydrolases have been thoroughly characterized in Saccharomyces cerevisiae, but their homologues in the fungal pathogen Candida albicans have received less attention. The genes APR1 and CPY1 of C. albicans encode putative vacuolar aspartic proteinase and serine carboxypeptidase, respectively. We examined properties of apr1Δ and cpy1Δ mutants, showing that Cpy1p molecular species detected in cell lysates of apr1Δ and its parental strain did not differ in molar mass. Processing of Cpy1p precursor is apparently independent of Apr1p. This is in contrast to S. cerevisiae, where vacuolar aspartic proteinase Pep4p is known to participate in the activation of other vacuolar hydrolases including serine carboxypeptidase. We also found that both apr1Δ and cpy1Δ strains are able to form hyphae in nutrient-rich filamentation media. However, proline as a sole nitrogen source induced filamentation only in cpy1Δ and its parental strain, but not in apr1Δ. This indicates the importance of Apr1p for the morphological transition under nitrogen-limited conditions. Despite that, the ability of apr1Δ to kill murine macrophages was not reduced under the conditions tested.     

Prospective Neural Areas and Their Morphogenetic Movements during Neural Plate Formation of Xenopus Embryos. I. Development of Vegetal Half Embryos and Chimera Embryos

Development of animal cap-less Xenopus gastrulae was examined. In vegetal halves from which the animal cap was removed 0.6 mm above the blastopore, an apparently normal array of craniocaudal structures developed. Histological examination showed differentiation of central nervous system (CNS) structures in the cap-less embryos, but differentiation of sensory organs, such as a lens and ear vesicle in only a few embryos. Only the dorsal midline of the embryos was covered with epidermis, and its lateral-ventral areas consisted of bare endoderm and mesoderm. The development of animal cap was also investigated by exchanging the animal cap of X. laevis embryos with that of X. borealis embryos, which can be distinguished by quinacrine fluorescence staining. The central nervous system of chimera embryos consisted mainly of X. laevis cells stained homogeneously with quinacrine but a small number of punctately-stained X. borealis cells was in the anterior tip of the forebrain. Cells of the lens and ear vesicle were punctately stained. More than two-thirds of the epidermal area consisted of punctately-stained cells and only the dorsal midline of the posterior head- and trunk-epidermis consisted of homogeneously-stained cells.
Areas of the prospective central nervous system and their movement during embryogenesis of Xenopus are discussed.     

Saccharomyces cerevisiae Mutants Affected in Vacuole Assembly or Vacuolar H+-ATPase are Hypersensitive to Lead (Pb) Toxicity

Lead is an important environmental pollutant. The role of vacuole, in Pb detoxification, was studied using a vacuolar protein sorting mutant strain (vps16Δ), belonging to class C mutants. Cells disrupted in VPS16 gene, did not display a detectable vacuolar-like structure. Based on the loss of cell proliferation capacity, it was found that cells from vps16Δ mutant exhibited a hypersensitivity to Pb-induced toxicity, compared to wild type (WT) strain. The function of vacuolar H⁺-ATPase (V-ATPase), in Pb detoxification, was evaluated using mutants with structurally normal vacuoles but defective in subunits of catalytic (vma1Δ or vma2Δ) or membrane domain (vph1Δ or vma3Δ) of V-ATPase. All mutants tested, lacking a functional V-ATPase, displayed an increased susceptibility to Pb, comparatively to cells from WT strain. Modification of vacuolar morphology, in Pb-exposed cells, was visualized using a Vma2p-GFP strain. The treatment of yeast cells with Pb originated the fusion of the medium size vacuolar lobes into one enlarged vacuole. In conclusion, it was found that vacuole plays an important role in the detoxification of Pb in Saccharomyces cerevisiae; in addition, a functional V-ATPase was required for Pb compartmentalization.     

Evidence for adenylate cyclase as a scaffold protein for Ras2–Ira interaction in Saccharomyces cerevisie

Data in literature suggest that budding yeast adenylate cyclase forms a membrane-associated complex with the upstream components of the cAMP/PKA pathway. Here we provide evidences that adenylate cyclase (Cyr1p) acts as a scaffold protein keeping Ras2 available for its regulatory factors. We show that in a strain with deletion of the CYR1 gene (cyr1Δ pde2Δ msn2Δ msn4Δ) the basal Ras2-GTP level is very high and this is independent on the lack of feedback inhibition that could result from the absence of adenylate cyclase activity. Moreover, strains effected either in the intrinsic adenylate cyclase activity (fil1 strain) or in the stimulation of adenylate cyclase activity by active G-proteins (lcr1 strain) had a normal basal and glucose-induced Ras2-GTP level, indicating that adenylate cyclase activity does not influence the Ras2 activation state and suggesting that Cyr1 protein is required for the proper interaction between Ras2 and the Ira proteins. We also provide evidence that the two Ras-binding sites mapped on Cyr1p are required for the signalling complex assembly. In fact, we show that the cyr1Δ strain expressing CYR1 alleles lacking either the LRR region or the C-terminal domain still have a high basal and glucose-induced Ras2-GTP level. In contrast, a mutant expressing a Cyr1 protein only missing the N-terminal domain showed a normal Ras2 activation pattern. Likewise, the Ras2-GTP levels are comparable in the wild type strain and the srv2Δ strain, supporting the hypothesis that Cap is not essential for the Ras-adenylate cyclase interaction.     

Molecular identification and functional characterization of a novel glutamate transporter in yeast and plant mitochondria

The genome of Saccharomyces cerevisiae encodes 35 members of the mitochondrial carrier family (MCF) and 58 MCF members are coded by the genome of Arabidopsis thaliana, most of which have been functionally characterized. Here two members of this family, Ymc2p from S. cerevisiae and BOU from Arabidopsis, have been thoroughly characterized. These proteins were overproduced in bacteria and reconstituted into liposomes. Their transport properties and kinetic parameters demonstrate that Ymc2p and BOU transport glutamate, and to a much lesser extent L-homocysteinesulfinate, but not other amino acids and many other tested metabolites. Transport catalyzed by both carriers was saturable, inhibited by mercuric chloride and dependent on the proton gradient across the proteoliposomal membrane. The growth phenotype of S. cerevisiae cells lacking the genes ymc2 and agc1, which encodes the only other S. cerevisiae carrier capable to transport glutamate besides aspartate, was fully complemented by expressing Ymc2p, Agc1p or BOU. Mitochondrial extracts derived from ymc2Δagc1Δ cells, reconstituted into liposomes, exhibited no glutamate transport at variance with wild-type, ymc2Δ and agc1Δ cells, showing that S. cerevisiae cells grown in the presence of acetate do not contain additional mitochondrial transporters for glutamate besides Ymc2p and Agc1p. Furthermore, mitochondria isolated from wild-type, ymc2Δ and agc1Δ strains, but not from the double mutant ymc2Δagc1Δ strain, swell in isosmotic ammonium glutamate showing that glutamate is transported by Ymc2p and Agc1p together with a H⁺. It is proposed that the function of Ymc2p and BOU is to transport glutamate across the mitochondrial inner membrane and thereby play a role in intermediary metabolism, C1 metabolism and mitochondrial protein synthesis.     

Going green in Cryptococcus neoformans: The recycling of a selectable drug marker

Cryptococcus neoformans is an opportunistic fungal pathogen that primarily affects immunocompromised individuals. Reverse genetics is commonly used to identify and characterize genes involved in a variety of cellular processes. In C. neoformans there is a limited set of positive selectable markers available to make gene deletions or other genetic manipulations. This has hampered the application of reverse genetics in this organism. We have adapted the Bacteriophage P1 Cre-loxP system for use in C. neoformans and successfully excised and reused the same drug marker, G418, to make two sequential gene deletions, lac1Δ and cap59Δ, in the same strain. This tool will allow investigators to make multiple sequential gene deletions in the same strain, which should facilitate the analysis of multigene families.     

Deletion of the ATP20 gene in Ustilago maydis produces an unstable dimer of F1FO-ATP synthase associated with a decrease in mitochondrial ATP synthesis and a high H2O2 production

The F₁F_O-ATP synthase uses the energy stored in the electrochemical proton gradient to synthesize ATP. This complex is found in the inner mitochondrial membrane as a monomer and dimer. The dimer shows higher ATPase activity than the monomer and is essential for cristae folding. The monomer-monomer interface is constituted by subunits a, i/j, e, g, and k. The role of the subunit g in a strict respiratory organism is unknown. A gene knockout was generated in Ustilago maydis to study the role of subunit g on mitochondrial metabolism and cristae architecture. Deletion of the ATP20 gene, encoding the g subunit, did not affect cell growth or glucose consumption, but biomass production was lower in the mutant strain (gΔ strain). Ultrastructure observations showed that mitochondrial size and cristae shape were similar in wild-type and gΔ strains. The mitochondrial membrane potential in both strains had a similar magnitude, but oxygen consumption was higher in the WT strain. ATP synthesis was 20 % lower in the gΔ strain. Additionally, the mutant strain expressed the alternative oxidase in the early stages of growth (exponential phase), probably as a response to ROS stress. Dimer from mutant strain was unstable to digitonin solubilization, avoiding its isolation and kinetic characterization. The isolated monomeric state activated by n-dodecyl-β-D-maltopyranoside showed similar kinetic constants to the monomer from the WT strain. A decrease in mitochondrial ATP synthesis and the presence of the AOX during the exponential growth phase suggests that deletion of the g gene induces ROS stress.     

The polarisome component SPA-2 localizes at the apex of Neurospora crassa and partially colocalizes with the Spitzenkörper

In fungal hyphae multiple protein complexes assemble at sites of apical growth to maintain cell polarity and promote nucleation of actin. Polarity allows the directional traffic of vesicles to the Spitzenkörper (Spk) prior to fusing with the plasma membrane to provide precursors and enzymes required for cell extension and nutrition. One of these complexes is the polarisome, which in Saccharomyces cerevisiae contains Spa2p, Pea2p, Bud6p/Aip3p and Bni1p. To investigate the localization and role of the polarisome during Spk establishment in Neurospora crassa we tagged SPA-2 with the green fluorescent protein (GFP) and examined growing cells by laser scanning confocal microscopy in elongating germ tubes and mature hyphae. SPA-2-GFP accumulated gradually at the apex of germ tubes, when a FM4-64 stained Spk was not still detectable. When the germlings reached about 40 μm in length, a FM4-64 stained Spk started to be apparent and from this point on SPA-2-GFP was observed in the apical region of both germ tubes and mature hyphae, as a hand fan shape with a brighter spot at the base. Fusion of the N. crassa SPA-2-GFP strain with a N. crassa strain expressing chitin synthase 1 (CHS-1) labeled with mCherryFP indicated only partial colocalization of the polarisome and the Spk core. N. crassa SPA-2-GFP was also found at the apex of forming branches but not in septa, suggesting that it participates only in areas of tip growth. A Δspa-2 strain displayed hyphae with uneven constrictions, apices with an unstable Spk, reduced growth rate and higher number of branches than the wild type strain, indicating that SPA-2 is required for the stability, behavior and morphology of the Spk and maintenance of regular apical growth in hyphae of N. crassa, although not for polarity or Spk establishment.     

Increasing ethanol titer and yield in a gpd1Δ gpd2Δ strain by simultaneous overexpression of GLT1 and STL1 in Saccharomyces cerevisiae

We have investigated whether simultaneous modification of cofactor metabolism and glycerol in a strain of Saccharomyces cerevisiae can eliminate glycerol synthesis during ethanol production. Two strains, S812 (gpd1Δ gpd2Δ PGK1p-GLT1) and LE17 (gpd1Δ gpd2Δ PGK1p-GLT1 PGKp-STL1) were generated that showed a 8 and 8.2 % increase in the ethanol yield, respectively, compared to the wild type KAM-2 strain. The ethanol titer was improved from 90.4 g/l for KAM-2 to 97.6 g/l for S812 and 97.8 g/l for LE17, respectively. These results provide a new insight into rationalization of metabolic engineering strategies for improvement of ethanol yield through elimination of glycerol production.     

Absence of superoxide dismutase activity causes nuclear DNA fragmentation during the aging process

Superoxide dismutases (SOD) serve as an important antioxidant defense mechanism in aerobic organisms, and deletion of these genes shortens the replicative life span in the budding yeast Saccharomyces cerevisiae. Even though involvement of superoxide dismutase enzymes in ROS scavenging and the aging process has been studied extensively in different organisms, analyses of DNA damages has not been performed for replicatively old superoxide dismutase deficient cells. In this study, we investigated the roles of SOD1, SOD2 and CCS1 genes in preserving genomic integrity in replicatively old yeast cells using the single cell comet assay. We observed that extend of DNA damage was not significantly different among the young cells of wild type, sod1Δ and sod2Δ strains. However, ccs1Δ mutants showed a 60% higher amount of DNA damage in the young stage compared to that of the wild type cells. The aging process increased the DNA damage rates 3-fold in the wild type and more than 5-fold in sod1Δ, sod2Δ, and ccs1Δ mutant cells. Furthermore, ROS levels of these strains showed a similar pattern to their DNA damage contents. Thus, our results confirm that cells accumulate DNA damages during the aging process and reveal that superoxide dismutase enzymes play a substantial role in preserving the genomic integrity in this process.     

Low doses of niclosamide and quinacrine combination yields synergistic effect in melanoma via activating autophagy-mediated p53-dependent apoptosis

Malignant melanoma is a highly aggressive, malignant, and drug-resistant tumor. It lacks an efficient treatment approach. In this study, we developed a novel anti-melanoma strategy by using anti-tapeworm drug niclosamide and anti-malarial drug quinacrine, and investigated the molecular mechanism by in vitro and in vivo assays. Meanwhile, other types of tumor cells, immortalized epithelial cells and bone marrow mesenchymal stem cells were used to evaluate the universal role of anti-cancer and safety of the strategy. The results showed, briefly, an exposure to niclosamide and quinacrine led to an increased apoptosis-related protein p53, cleaved caspase-3 and cleaved PARP and autophagy-related protein LC3B expression, and a decreased expression of autophagy-related protein p62, finally leading to cell apoptosis and autophage. After inhibiting autophagy by Baf-A1, flow cytometry and western blot showed that the expression of apoptosis-related proteins was down-regulated and the number of apoptotic cells decreased. Subsequently, in the siRNA-mediated p53 knockdown cells, the expression of apoptosis-related proteins and the number of apoptotic cells were also reduced, while the expression of autophagy-related proteins including LC3B, p62 did not change significantly. To sum up, we developed a new, safe strategy for melanoma treatment by using low doses of niclosamide and quinacrine to treat melanoma; and found a novel mechanism by which the combination application of low doses of niclosamide and quinacrine exerts an efficient anti-melanoma effect through activation of autophagy-mediated p53-dependent apoptosis. The novel strategy was verified to exert a universal anti-cancer role in other types of cancer.     

Deoxyribonucleic Acid synthesis in root cap cells of cultured roots of convolvulus

Isolated cultured roots of Convolvulus arvensis L. were incubated in 0.2 microcurie per milliliter methyl-³H-thymidine for 14 hours, for 64 hours, or for 14 hours followed by transfer to fresh nutrient medium without tritiated thymidine. Autoradiographs of serial, longitudinal sections of roots which were continuously incubated with tritiated thymidine showed that cells of the root cap columella did not undergo DNA synthesis after their formation from the root cap initials. In roots pulse-labeled with tritiated thymidine, the movement of labeled cells through the root cap columella was followed. Labeled cells were displaced at a constant rate of 72 microns per day over a period of 6 to 9 days before they were sloughed off from the root cap. The specialized role of the root cap cells in relation to their distinctive metabolism and longevity is discussed.     

Transcriptional response to mitochondrial NADH kinase deficiency in Saccharomyces cerevisiae

Yeast cells lacking the mitochondrial NADH kinase encoded by POS5 display increased sensitivity to hydrogen peroxide, a slow-growth phenotype, reduced mitochondrial function and increased levels of mitochondrial protein oxidation and mtDNA mutations. Here we examined gene expression in pos5Δ cells, comparing these data to those from cells containing deletions of superoxide dismutase-encoding genes SOD1 or SOD2. Surprisingly, stress–response genes were down-regulated in pos5Δ, sod1Δ and sod2Δ cells, implying that cells infer stress levels from mitochondrial activity rather than sensing reactive oxygen species directly. Additionally, pos5Δ, but not sod1 or sod2, cells displayed an anaerobic expression profile, indicating a defect in oxygen sensing that is specific to pos5, and is not a general stress–response. Finally, the pos5Δ expression profile is quite similar to the hap1Δ expression profile previously reported, which may indicate a shared mechanism.     

Effects of indole-3-acetic acid on <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> survival and on symbiotic nitrogen fixation and stem dry weight production

We evaluated the effects of the main auxin phytohormone, indole-3-acetic acid (IAA), on the central metabolism of Sinorhizobium meliloti 1021. We either treated S. meliloti 1021 wild-type cells with 0.5 mM IAA, 1021+, or use a derivative, RD64, of the same strain harboring an additional pathway for IAA biosynthesis (converting tryptophan into IAA via indoleacetamide). We assayed the activity of tricarboxylic acid cycle (TCA) key enzymes and found that activity of citrate synthase and α-ketoglutarate dehydrogenase were increased in both 1021+ and RD64 as compared to the wild-type strain. We also showed that the intracellular acetyl-CoA content was enhanced in both RD64 and 1021+ strains when compared to the control strain. The activity of key enzymes, utilizing acetyl-CoA for poly-β-hydroxybutyrate (PHB) biosynthesis, was also induced. The PHB level measured in these cells were significantly higher than that found in control cells. Moreover, 4-week-long survival experiments showed that 80% of 1021 cells died, whereas 50% of RD64 cells were viable. Medicago truncatula plants nodulated by RD64 (Mt-RD64) showed an induction of both acetylene reduction activity and stem dry weight production.     

Tel1 and Rif2 oppositely regulate telomere protection at uncapped telomeres in Saccharomyces cerevisiae

It has been well documented that Tel1 positively regulates telomere-end resection by promoting Mre11-Rad50-Xrs2(MRX) activity, while Rif2 negatively regulates telomere-end resection by inhibiting MRX activity. At uncapped telomeres, whether Tel1 or Rif2 plays any role remains largely unknown. In this work, we examined the roles of Tel1 and Rif2 at uncapped telomeres in yku70△ and/or cdc13-1 mutant cells cultured at non-permissive temperature. We found that deletion of TEL1 exacerbates the temperature sensitivity of both yku70△ and cdc13-1 cells. Further epistasis analysis indicated that MRX and Tel1 function in the same pathway in telomere protection. Consistently, TEL1 deletion increases accumulation of Exo1-dependent telomeric single-stranded DNA(ssDNA) at uncapped telomeres, which stimulates checkpoint-dependent cell cycle arrest. Moreover, TEL1 deletion in yku70△ cells facilitates Rad51-dependent Y0 recombination. In contrast, RIF2 deletion in yku70△ cells decreases the accumulation of telomeric ssDNA after 8 h of incubation at the non-permissive temperature of 37℃ and suppresses the temperature sensitivity of yku70△ cells, likely due to the increase of Mre11 association at telomeres.Collectively, our findings indicate that Tel1 and Rif2 regulate telomere protection at uncapped telomeres via their roles in balancing MRX activity in telomere resection.