Ionizing radiation induces a Yap1-dependent peroxide stress response in yeast

Repair of DNA damage is fundamental for cellular tolerance to ionizing radiation (IR) and many IR-induced DNA lesions are thought to occur as a result of oxidative stress. We investigated the physiological effects of IR in Saccharomyces cerevisiae by performing protein expression profiles in cells exposed to electron pulse irradiation. Transient induction of several antioxidant enzymes in wild-type cells, but not in cells lacking the oxidative stress regulator Yap1, indicated that IR exposure causes cellular oxidative stress. Yap1 activation involved oxidation to the intramolecular disulfide bond, a signature of activation by peroxide, and was dependent on the Yap1 peroxide sensor Orp1/Gpx3. H(2)O(2) was produced in the culture medium of irradiated cells and was both necessary and sufficient for IR-induced Yap1 activation. When IR was performed in the presence of N(2)O, obviating H(2)O(2) production and increasing hydroxyl radical ((*)OH) production, the Yap1 response was lost, indicating that Yap1 was unable to respond to (*)OH or (*)OH-induced damage. However, the Yap1 response to IR did not seem to be a primary determinant of cellular IR tolerance. Altogether, these data provide a molecular demonstration that cells experience in vivo peroxide stress during IR and indicate that the H(2)O(2) produced cannot account for IR toxicity.     

Identification of the dynein light chains required for human papillomavirus infection

Human papillomaviruses (HPVs) are a family of small non-enveloped DNA viruses. Some genital HPV types, including HPV type 16 (HPV16), are the causative agent for the development of cancer at the site of infection. HPVs encode two capsid proteins, L1 and L2. After endocytic cell entry and egress from endosomes, L2 accompanies the viral DNA to the nucleus where replication is initiated. For cytoplasmic transport, L2 interacts with the microtubule network via the motor protein complex dynein. We have performed yeast two-hybrid screening and identified the dynein light chain DYNLT1 (previously called Tctex1) as interaction partner of HPV16 L2. Using co-immunoprecipitation and immunofluorescence colocalization studies we confirmed the L2-DYNLT1 interaction in mammalian cells. Further studies revealed that DYNLT3, the second member of the Tctex-light chain family, also interacts with L2 in vitro and in vivo, whereas other constituents of the dynein complex were not found to associate with L2. Depletion of DYNLT1 and DYNLT3 by specific siRNAs or cytosolic delivery of light chain-specific antibodies inhibited infection of HPV16. Therefore, this work identified two host cell proteins involved in HPV16 infection that are most likely required for transport purposes towards the nucleus.     

NADH oxidase activity of Bacillus subtilis nitroreductase NfrA1: Insight into its biological role

NfrA1 nitroreductase from the Gram-positive bacterium Bacillus subtilis is a member of the NAD(P)H/FMN oxidoreductase family. Here, we investigated the reactivity, the structure and kinetics of NfrA1, which could provide insight into the unclear biological role of this enzyme. We could show that NfrA1 possesses an NADH oxidase activity that leads to high concentrations of oxygen peroxide and an NAD⁺ degrading activity leading to free nicotinamide. Finally, we showed that NfrA1 is able to rapidly scavenge H₂O₂ produced during the oxidative process or added exogenously.

Structured summary

MINT-7990140: nfrA1 (uniprotkb:P39605) and nfrA1 (uniprotkb:P39605) bind (MI:0407) by X-ray crystallography (MI:0114)     

Induction of control genes in intestinal gluconeogenesis is sequential during fasting and maximal in diabetes

We studied in rats the expression of genes involved in gluconeogenesis from glutamine and glycerol in the small intestine (SI) during fasting and diabetes. From Northern blot and enzymatic studies, we report that only phosphoenolpyruvate carboxykinase (PEPCK) activity is induced at 24 h of fasting, whereas glucose-6-phosphatase (G-6-Pase) activity is induced only from 48 h. Both genes then plateau, whereas glutaminase and glycerokinase strikingly rebound between 48 and 72 h. The two latter genes are fully expressed in streptozotocin-diabetic rats. From arteriovenous balance and isotopic techniques, we show that the SI does not release glucose at 24 h of fasting and that SI gluconeogenesis contributes to 35% of total glucose production in 72-h-fasted rats. The new findings are that 1) the SI can quantitatively account for up to one-third of glucose production in prolonged fasting; 2) the induction of PEPCK is not sufficient by itself to trigger SI gluconeogenesis; 3) G-6-Pase likely plays a crucial role in this process; and 4) glutaminase and glycerokinase may play a key potentiating role in the latest times of fasting and in diabetes.     

From dormant to germinating spores of Streptomyces coelicolor A3(2): new perspectives from the crp null mutant

The complete understanding of the morphological differentiation of streptomycetes is an ambitious challenge as diverse sensors and pathways sensitive to various environmental stimuli control the process. Germination occupies a particular position in the life cycle as the good achievement of the process depends on events occurring both during the preceding sporulation and during germination per se. The cyclic AMP receptor protein (crp) null mutant of Streptomyces coelicolor, affected in both sporulation and germination, was therefore presented as a privileged candidate to highlight new proteins involved in the shift from dormant to germinating spores. Our multidisciplinary approach-combining in vivo data, the analysis of spores morphological properties, and a proteome study-has shown that Crp is a central regulatory protein of the life cycle in S. coelicolor; and has identified spores proteins with statistically significant increased or decreased expression that should be listed as priority targets for further investigations on proteins that trigger both ends of the life cycle.     

Synthesis and antiviral evaluation of thieno[3,4-d]pyrimidine C-nucleoside analogues of 2′,3′-dideoxy- and 2′,3′-dideoxy-2′,3′-didehydro-adenosine and -inosine

Several thieno[3,4-d]pyrimidine derivatives, including four hitherto unknown 2′,3′-dideoxy- and 2′,3′-dideoxy-2′,3′-didehydro-C-nucleoside analogues of adenosine and inosine have been synthesized. When evaluated in cell culture experiments against human immunodeficiency virus, none of the tested compounds exhibited any significant antiviral effect, while two of them showed some cytotoxicity.     

Minimal cut sets in biochemical reaction networks

MOTIVATION: Structural studies of metabolic networks yield deeper insight into topology, functionality and capabilities of the metabolisms of different organisms. Here, we address the analysis of potential failure modes in metabolic networks whose occurrence will render the network structurally incapable of performing certain functions. Such studies will help to identify crucial parts in the network structure and to find suitable targets for repressing undesired metabolic functions. RESULTS: We introduce the concept of minimal cut sets for biochemical networks. A minimal cut set (MCS) is a minimal (irreducible) set of reactions in the network whose inactivation will definitely lead to a failure in certain network functions. We present an algorithm which enables the computation of the MCSs in a given network related to user-defined objective reactions. This algorithm operates on elementary modes. A number of potential applications are outlined, including network verifications, phenotype predictions, assessing structural robustness and fragility, metabolic flux analysis and target identification in drug discovery. Applications are illustrated by the MCSs in the central metabolism of Escherichia coli for growth on different substrates. AVAILABILITY: Computation and analysis of MCSs is an additional feature of the FluxAnalyzer (freely available for academic users upon request, special contracts for industrial companies; see web page below). Supplementary information: http://www.mpi-magdeburg.mpg.de/projects/fluxanalyzer     

Quail Egg Yolk: A Novel Cryoprotectant for the Freeze Preservation of Poitou Jackass Sperm

For many years, attempts have been made to establish a sperm bank for the Poitou jackass population which is threatened with extinction. Unfortunately, no cryopreservation technique has ever been described for spermatozoa of this species. In an attempt to find a suitable technique, we studied the relative effectiveness of chicken egg yolk and quail egg yolk in preserving the motility and characteristics of movement of Poitou jackass spermatozoa during the freezing–thawing process. Semen was diluted to 60 × 10⁶sperm/ml in a preservation medium containing 4% (v/v) glycerol with 0, 2, 5, 10, 15, or 20% (v/v) of chicken or quail egg yolk. The chemical composition of these two eggs was compared. Effects were assessed using an automated analyzer which measured curvilinear velocity (VCL), straight line velocity (VSL), and the velocity of the average path. Linearity was defined as VSL/VCL × 100. The amplitude of the lateral head displacement was also measured. It was found that after the freeze–thaw process, quail egg yolk improved the percentages of motile and progressively undulating spermatozoa and the movement characteristics compared with chicken egg yolk. The optimal concentration of quail egg yolk was 10%. The general composition of the two types of egg yolk were similar, but quail egg yolk contained significantly more phosphatidylcholine, less phosphatidylethanolamine, and a smaller ratio of polyunsaturated to saturated fatty acids than chicken egg yolk. The improvement of motility for frozen–thawed Poitou jackass spermatozoa using frozen–thawed quail egg yolk compared to chicken egg yolk may be due to the differences in composition of the two yolks.     

Essential oil and terpenoids of Mikania micrantha

Twenty-seven mainly terpenoid constituents were identified in the essential oil of Mikania micrantha whole plant. Higher terpenoids present in the same plant included two kaurene derivatives and taraxasterol.