Setosphaeria turcica ATR turns off appressorium-mediated maize infection and triggers melanin-involved self-protection in response to genotoxic stress

Eukaryotic organisms activate conserved signalling networks to maintain genomic stability in response to DNA genotoxic stresses. However, the coordination of this response pathway in fungal pathogens remains largely unknown. In the present study, we investigated the mechanism by which the northern corn leaf blight pathogen Setosphaeria turcica controls maize infection and activates self-protection pathways in response to DNA genotoxic insults. Appressorium-mediated maize infection by S. turcica was blocked by the S-phase checkpoint. This repression was dependent on the checkpoint central kinase Ataxia Telangiectasia and Rad3 related (ATR), as inhibition of ATR activity or knockdown of the ATR gene recovered appressorium formation in the presence of genotoxic reagents. ATR promoted melanin biosynthesis in S. turcica as a defence response to stress. The melanin biosynthesis genes StPKS and StLac2 were induced by the ATR-mediated S-phase checkpoint. The responses to DNA genotoxic stress were conserved in a wide range of phytopathogenic fungi, including Cochliobolus heterostrophus, Cochliobolus carbonum, Alternaria solani, and Alternaria kikuchiana, which are known causal agents for plant diseases. We propose that in response to genotoxic stress, phytopathogenic fungi including S. turcica activate an ATR-dependent pathway to suppress appressorium-mediated infection and induce melanin-related self-protection in addition to conserved responses in eukaryotes.     

Effects of monensin on volatile fatty acid metabolism in periparturient dairy cows using compartmental analysis

Eight multiparous periparturient Holstein cows fitted with ruminal cannulas were used in a split plot design to evaluate effects of monensin on ruminal volatile fatty acid (VFA) metabolism. Diets were supplemented with 300 mg/day of monensin, or no monensin, both prepartum and postpartum. Isotopic tracers, Na-1-¹³C-acetate (Ac), Na-1-¹³C-propionate (Pr), or Na-1-¹³C-butyrate (Bu) were used as markers to describe VFA kinetics in the rumen. The Windows version of SAAM software (WinSAAM) was used to develop a steady state VFA model. A 9-compartment model was adequate to comprehensively describe ruminal VFA metabolism. The main VFA compartments consisted of Ac, Pr and Bu. The model estimated lower Bu and Ac interconversions with monensin, postpartum (Bu to Ac; 0.14 versus 0.12; P=0.04, and Ac to Bu; 0.32 versus 0.25; P=0.11) compared to when measured prepartum. Results demonstrate that dilution studies employing stable isotopes of VFA can be used to provide information on VFA metabolism of the periparturient dairy cow. A time frame of 320 min of labeled VFA infusion employing a single injection allows accurate quantification of VFA metabolism in the rumen. Compartmental kinetic analysis of major VFA in the rumen indicate that monensin reduced about 0.125 the portion of the Ac that contributes to Bu by reducing movements of Bu originated carbons to the Ac pool. Monensin may affect certain biochemical pathways of interconversion of Bu and Ac in the rumen. Propionate kinetic data suggests that Pr behaves as a single pool in the rumen. Monensin did not affect Pr production in the rumen suggesting that monensin improves the metabolic status of the transition cow in a way other than increasing Pr production in the rumen.     

In Vitro Lactate Metabolism by Ruminal Ingesta

Ruminal ingesta (300 ml) obtained from a fistulated cow fed alfalfa hay (H), 3.6 kg of grain mixture with corn silage fed ad libitum (S), 2.5:1 grain-alfalfa hay mixture (G), or a 2.5:1 grain-alfalfa hay mixture providing 545 g of sodium and calcium lactate daily (L) were incubated for 8 hr with nonpolymerized sodium lactate or 17% polymerized lactic acid neutralized to pH 6.7. Polymerization had no effect on the rate of lactate utilization. The initial rates of lactate metabolism for the H, G, S, and L ingesta were 0.72, 0.95, 1.8, and 3.4 meq per 100 ml of rumen fluid per hr, respectively. Lactate-2-(14)C was incubated for 4 hr with each type of ruminal ingesta. Of the label recovered in the volatile fatty acids (VFA), 74.1, 61.2, 49.3, and 38.9% was recovered in acetate, and 9.4, 19.8, 23.3, and 51.9% was recovered in propionate with H, G, S, and L ingesta, respectively. The balance of label was distributed between butyrate and valerate. The titratable VFA did not follow this pattern of production. With the hay ingesta, lactate metabolism resulted in a net loss of acetate and a large increase in butyrate. Little propionate was produced. The G, S, and L ingesta metabolized lactate to yield progressively more propionate and less butyrate. Evidence was gathered to suggest that acetate was the primary end product of lactate metabolism but that oxidation of lactate to pyruvate dictated the synthesis of butyrate from acetate to maintain an oxidation-reduction balance. It was noted that acetate and butyrate production from lactate was pH-dependent, with acetate production maximal at pH 7.4 and butyrate at 6.2. Propionate production was largely unaffected within this pH range.     

Simultaneous butyrate oxidation by Syntrophomonas wolfei and catalytic olefin reduction in absence of interspecies hydrogen transfer

Methanogenesis by a Syntrophomonas wolfei/ Methanospirillum hungatei coculture was inhibited in presence of ethylene and the hydrogenation catalyst Pd-BaSO₄. However, butyrate oxidation by S. wolfei continued and ethylene was reduced to ethane. Per mol of butyrate oxidized, 2.4 mol acetate was produced and 0.8 mol ethylene was reduced. Acetylene, propylene and butene were less effective as H₂ acceptors than ethylene, and addition of bromoethanesulfonic acid was necessary to inhibit methanogenesis in the presence of the two longer-chain olefins. Other hydrogenation catalysts were less effective in the order Pd-charcoal < PE-asbestos < Pd-PEI beads < Pt-Al₂O₃, Pd-CaCO₃. Optimal ethylene hydrogenation was achieved with still incubation in presence of 7.2 mg Pd-BaSO₄ and 0.7 g sand per ml medium. The higher catabolic rate of S. wolfei in presence of the methanogen indicated that the biological H₂ removal mechanism was more efficient than the catalytic olefin reduction.Abbreviations BES bromoethane sulfonic acid - VFA volatile fatty acid     

Effect of volatile fatty acids on Shigella dysenteriae during anaerobic digestion of human night soil

Thein vitro toxic effect of different volatile fatty acids (VFA) on Shigella dysenteriae was studied in pure culture. Volatile fatty acids viz., acetate, propionate, butyrate, valerate, caproate and heptanoate, exerted pH dependent toxic effect on the pathogen, with minimum inhibitory concentration in the range of 10–3000 mg l⁻¹. The effect of high levels of VFA on S. dysenteriae was studied during anaerobic digestion of human night soil in an experimental digester with VFA level ≅ 9000 mg l⁻¹ and pH ≅ 6.5. Another digester, with VFA level ≅ 700 mg l⁻¹ and pH 7.4, served as the control. In the experimental digester, S. dysenteriae was completely eliminated within 18 days. In the control digester, a four-log reduction in pathogen count was achieved however the pathogen was not completely eliminated. T ₉₀ values for the experimental and control digesters were 2.2 and 3.7 days respectively. This revised version was published online in July 2006 with corrections to the Cover Date.     

Activation of the Budding Yeast Securin Pds1 But Not Rad53 Correlates with Double-Strand Break-Associated G2/M Cell Cycle Arrest in a mec1 Hypomorphic Mutant

Budding yeast Mec1, encoded by the yeast ATR/ATM homolog, negatively regulates cell cycle progression by activating Rad53 (Chk2) and Chk1, two parallel downstream checkpoint pathways. Chk1 phosphorylates Pds1 (securin), which prevents Pds1 degradation. We determined whether activation of both downstream pathways is required to establish G2 arrest in response to double-strand breaks (DSBs). In a hypomorphic mec1 mutant, Rad53 activation was not required to establish G₂ arrest triggered by a single HO endonuclease-generated DSB. However, Pds1 phosphorylation did correlate with G₂ arrest and mec1-21 pds1 cells did not arrest in G₂ after exposure to ionizing radiation. The G₂ checkpoint genes, CHK1 and PDS1, did confer radiation resistance in mec1-21, indicating that CHK1-mediated pathway is functional in the mec1 hypomorph. Thus, phosphorylation of Pds1 but not Rad53 correlates with G₂ arrest in response to DSBs in the mec1 hypomorphic mutant.     

Hyperosmosis enhances radiation and hydroxyurea resistance of Schizosaccharomyces pombe checkpoint mutants through the spindle checkpoint and delayed cytokinesis

The DNA damage and stress response pathways interact to regulate cellular responses to genotoxins and environmental stresses. How these pathways interact in Schizosaccharomyces pombe is not well understood. We demonstrate that osmotic stress suppresses the DNA damage sensitivity of checkpoint mutants, and that this occurs through three distinct cell cycle delays. A delay in G2/M is dependent on Srk1. Progression through mitosis is halted by the Mad2‐dependent spindle checkpoint. Finally, cytokinesis is impaired by modulating Cdc25 expression. These three delays, imposed by osmotic stress, together compensate for the loss of checkpoint signalling.     

Bioinformatics and metabolic flux analysis highlight a new mechanism involved in lactate oxidation in Clostridium tyrobutyricum

Climate change and environmental issues compel us to find alternatives to the production of molecules of interest from petrochemistry. This study aims at understanding the production of butyrate, hydrogen, and CO₂ from the oxidation of lactate with acetate in Clostridium tyrobutyricum and thus proposes an alternative carbon source to glucose. This specie is known to produce more butyrate than the other butyrate-producing clostridia species due to a lack of solvent genesis phase. The recent discoveries on flavin-based electron bifurcation and confurcation mechanism as a mode of energy conservation led us to suggest a new metabolic scheme for the formation of butyrate from lactate-acetate co-metabolism. While searching for genes encoding for EtfAB complexes and neighboring genes in the genome of C. tyrobutyricum, we identified a cluster of genes involved in butyrate formation and another cluster involved in lactate oxidation homologous to Acetobacterium woodii. A phylogenetic approach encompassing other butyrate-producing and/or lactate-oxidizing species based on EtfAB complexes confirmed these results. A metabolic scheme on the production of butyrate, hydrogen, and CO₂ from the lactate-acetate co-metabolism in C. tyrobutyricum was constructed and then confirmed with data of steady-state continuous culture. This in silico metabolic carbon flux analysis model showed the coherence of the scheme from the carbon recovery, the cofactor ratio, and the ATP yield. This study improves our understanding of the lactate oxidation metabolic pathways and the role of acetate and intracellular redox balance, and paves the way for the production of molecules of interest as butyrate and hydrogen with C. tyrobutyricum.

     

Betaine increases net portal absorption of volatile fatty acids in Iberian pigs

Betaine is an osmolyte with the potential to increase volatile fatty acids (VFAs) production and hence improve intestinal health.The present study investigated how betaine affects portal and arterial concentrations and net portal absorption (NPA) of VFA in growing Iberian pigs. Eight 30 kg BW Iberian growing barrows with indwelling catheters in portal vein, ileal vein and carotid artery were randomly assigned to a control diet or a diet supplemented with 0.5% betaine. Para-aminohippuric acid was infused into the ileal vein as a marker to determine portal blood flow using the dilution method. Blood samples were simultaneously taken from the carotid artery and portal vein at −60, 60, 120, 180, 240, 300 and 360 min after feeding 1 200 g of the diet. The NPA of VFA (acetate, propionate, butyrate, valerate, isobutyrate and caproate) was determined by multiplying the porto-arterial plasma concentration differences by portal plasma flow. Betaine increased NPA of acetate (1.44 fold; P < 0.001) and total VFA (0.55 fold; P < 0.001) while decreased NPA of propionate (−0.38 fold; P < 0.05) and valerate (−1.46 fold; P < 0.05) compared with control pigs. Estimated heat production potentially derived from NPA of VFA accounted for 0.20–0.27 of metabolizable energy for maintenance. Acetate and propionate accounted for most of the total VFA estimated heat production (0.83–0.89). Regarding bacterial communities, betaine apparently did not change the DNA abundance of fecal total bacteria, Lactobacillus, Bifidobacterium, Enterobacteriaceae, Bacteroides and the Clostridium clusters I, IV and XIV. In conclusion, betaine increased portal appearance and NPA of VFA, contributing to cover maintenance energy requirements.     

Diversity of human colonic butyrate‐producing bacteria revealed by analysis of the butyryl‐CoA:acetate CoA‐transferase gene

Butyrate‐producing bacteria play an important role in the human colon, supplying energy to the gut epithelium and regulating host cell responses. In order to explore the diversity and culturability of this functional group, we designed degenerate primers to amplify butyryl‐CoA:acetate CoA‐transferase sequences from faecal samples provided by 10 healthy volunteers. Eighty‐eight per cent of amplified sequences showed > 98% DNA sequence identity to CoA‐transferases from cultured butyrate‐producing bacteria, and these fell into 12 operational taxonomic units (OTUs). The four most prevalent OTUs corresponded to Eubacterium rectale, Roseburia faecis, Eubacterium hallii and an unnamed cultured species SS2/1. The remaining 12% of sequences, however, belonged to 20 OTUs that are assumed to come from uncultured butyrate‐producing strains. Samples taken after ingestion of inulin showed significant (P = 0.019) increases in Faecalibacterium prausnitzii. Because several of the dominant butyrate producers differ in their DNA % G+C content, analysis of thermal melt curves obtained for PCR amplicons of the butyryl‐CoA:acetate CoA‐transferase gene provides a convenient and rapid qualitative assessment of the major butyrate producing groups present in a given sample. This type of analysis therefore provides an excellent source of information on functionally important groups within the colonic microbial community.     

Reconstruction of the yeast Snf1 kinase regulatory network reveals its role as a global energy regulator

Highly conserved among eukaryotic cells, the AMP‐activated kinase (AMPK) is a central regulator of carbon metabolism. To map the complete network of interactions around AMPK in yeast (Snf1) and to evaluate the role of its regulatory subunit Snf4, we measured global mRNA, protein and metabolite levels in wild type, Δsnf1, Δsnf4, and Δsnf1Δsnf4 knockout strains. Using four newly developed computational tools, including novel DOGMA sub‐network analysis, we showed the benefits of three‐level ome‐data integration to uncover the global Snf1 kinase role in yeast. We for the first time identified Snf1's global regulation on gene and protein expression levels, and showed that yeast Snf1 has a far more extensive function in controlling energy metabolism than reported earlier. Additionally, we identified complementary roles of Snf1 and Snf4. Similar to the function of AMPK in humans, our findings showed that Snf1 is a low‐energy checkpoint and that yeast can be used more extensively as a model system for studying the molecular mechanisms underlying the global regulation of AMPK in mammals, failure of which leads to metabolic diseases.     

Complexity of roles and regulation of the PMK1-MAPK pathway in mycelium development,conidiation and appressorium formation in Magnaporthe oryzae

MST50, MST11, MST7, PMK1 and GAS1/GAS2 genes are the important components in the PMK1-MAPK signal transduction pathway in fungi. Mutants with deletion of these five genes of Magnaporthe oryzae, a pathogen of the rice blast, were constructed. A cDNA array containing 4108 unique genes of M. oryzae was developed and used to analyze the gene expression profiles of these mutants against the wild type to dissect the gene expression regulation networks responsible for conidiation and appressorium formation. With this approach, differentially regulated genes by these five components were identified. The vast majority of the regulated genes were mutant-specific, while only a small proportion were in common for all of the mutants, suggesting that each of these genes has its own regulon. Functional groups and expression patterns of the regulated genes showed that (1) gene members in the PMK1-MAPK pathway are associated with multiple signaling pathways; (2) the regulation of PMK1-mediated signaling pathways is very complex and likely involved in other signaling networks; (3) glucose metabolism and signals are required in mycelium development; and (4) appressorium formation likely shares the mechanisms responsible for sexual conjugation and meiosis, which is affected by carbohydrate metabolism.     

Microbial transformation of ginsenosides Rb1, Rb3 and Rc by Fusarium sacchari

Aims: This study examined the transformation pathways of ginsenosides G‐Rb₁, G‐Rb₃, and G‐Rc by the fungus Fusarium sacchari. Methods and Results: Ginsenosides G‐Rb₁, G‐Rb₃ and G‐Rc were isolated from leaves of Radix notoginseng, and their structural identification was confirmed using NMR. Transformation of G‐Rb₁, G‐Rb₃ and G‐Rc by Fusarium sacchari was respectively experimented. Kinetic evolutions of G‐Rb₁, G‐Rb₃ and G‐Rc and their metabolites during the cell incubation were monitored by HPLC analysis. High‐performance liquid chromatography (HPLC) was used for monitoring the transformation kinetics of bioactive compounds during F. sacchari metabolism. Conclusions: Ginsenoside C‐K was transformed by F. sacchari from G‐Rb₁ via G‐Rd or via G‐F₂, or from G‐Rb₁ via firstly Rd and then G‐F₂, and C‐Mx was transformed by F. sacchari or directly from Rb₃, or from Rb₃ via Gy‐IX, while G‐Mc was transformed by F. sacchari directly from G‐Rc. Furthermore, C‐K could be also formed from G‐Rc via notoginsenoside Fe (N‐Fe). Significance and Impact of the Study: The results showed an important practical application in the preparation of ginsenoside C‐K. As our precious research indicated C‐K possessed much more antitumor activities than C‐Mx and G‐Mc, so according to the transformation pathways proposed by this work, the production of antitumor compound C‐K may be performed by biotransformation of G‐Rb₁ previously isolated from PNLS.     

Amino acid utilization by the ruminal bacterium Synergistes jonesii strain 78-1

The ruminal bacterium Synergistes jonesii strain 78-1, which is able to degrade the pyridinediol toxin in the plant Leucaena leucephala, was studied for its ability to utilise amino acids. The organism used arginine, histidine and glycine from a complex mixture of amino acids, and both arginine and histidine supported growth in a semi-defined medium. The products of (U-¹⁴C)-arginine metabolism were CO₂ acetate, butyrate, citrulline and ornithine. The labelling pattern of end products from (U-¹⁴C)-histidine metabolism differed in that carbon also flowed into formate and propionate. Arginine was catabolised by the arginine deiminase pathway which was characterised by the presence of arginine deiminase, ornithine transcarbamylase and carbamate kinase. This is the first report of a rumen bacterium that uses arginine and histidine as major energy yielding substrates.     

CDK4 inhibition restores G₁-S arrest in MYCN-amplified neuroblastoma cells in the context of doxorubicin-induced DNA damage

Relapse with drug-resistant disease is the main cause of death in MYCN-amplified neuroblastoma patients. MYCN-amplified neuroblastoma cells in vitro are characterized by a failure to arrest at the G?-S checkpoint after irradiation- or drug-induced DNA damage. We show that several MYCN-amplified cell lines harbor additional chromosomal aberrations targeting p53 and/or pRB pathway components, including CDK4/CCND1/MDM2 amplifications, p16INK4A/p14ARF deletions or TP53 mutations. Cells with these additional aberrations undergo significantly lower levels of cell death after doxorubicin treatment compared with MYCN-amplified cells, with no additional mutations in these pathways. In MYCN-amplified cells CDK4 expression is elevated, increasing the competition between CDK4 and CDK2 for binding p21. This results in insufficient p21 to inhibit CDK2, leading to high CDK4 and CDK2 kinase activity upon doxorubicin treatment. CDK4 inhibition by siRNAs, selective small compounds or p19^INK4D overexpression partly restored G?-S arrest, delayed S-phase progression and reduced cell viability upon doxorubicin treatment. Our results suggest a specific function of p19^INK4D, but not p16^INK4A, in sensitizing MYCN-amplified cells with a functional p53 pathway to doxorubicin-induced cell death. In summary, the CDK4/cyclin D-pRB axis is altered in MYCN-amplified cells to evade a G?-S arrest after doxorubicin-induced DNA damage. Additional chromosomal aberrations affecting the p53-p21 and CDK4-pRB axes compound the effects of MYCN on the G? checkpoint and reduce sensitivity to cell death after doxorubicin treatment. CDK4 inhibition partly restores G?-S arrest and sensitizes cells to doxorubicin-mediated cell death in MYCN-amplified cells with an intact p53 pathway.     

Mutations in the yeast cyclin-dependent kinase Cdc28 reveal a role in the spindle assembly checkpoint

Anaphase onset and mitotic exit are regulated by the spindle assembly or kinetochore checkpoint, which inhibits the anaphase-promoting complex (APC), preventing the degradation of anaphase inhibitors and mitotic cyclins. As a result, cells arrest with high cyclin-dependent kinase (CDK) activity due to the accumulation of cyclins. Aside from this, a clear-cut demonstration of a direct role for CDKs in the spindle checkpoint response has been elusive. Cdc28 is the main CDK driving the cell cycle in budding yeast. In this report, mutations in cdc28 are described that confer specific checkpoint defects, supersensitivity towards microtubule poisons and chromosome loss. Two alleles encode single mutations in the N and C terminal regions, respectively (R10G and R288G), and one allele specifies two mutations near the C terminus (F245L, I284T). These cdc28 mutants are unable to arrest or efficiently prevent sister chromatid separation during treatment with nocodazole. Genetic interactions with checkpoint and apc mutants suggest Cdc28 may regulate checkpoint arrest downstream of the MAD2 and BUB2 pathways. These studies identify a C-terminal domain of Cdc28 required for checkpoint arrest upon spindle damage that mediates chromosome stability during vegetative growth, suggesting that it has an essential surveillance function in the unperturbed cell cycle.Communicated by A. Aguilera