Influence of intracellular nucleotide and nucleotide sugar contents on recombinant interferon-gamma glycosylation during batch and fed-batch cultures of CHO cells

Both the macroheterogeneity of recombinant human IFN-gamma produced by CHO cells and intracellular levels of nucleotides and sugar nucleotides, have been characterized during batch and fed-batch cultures carried out in different media. Whereas PF-BDM medium was capable to maintain a high percentage of the doubly- glycosylated glycoforms all over the process, mono-glycosylated and non-glycosylated forms increased during the batch culture using SF-RPMI medium. Intracellular level of UTP was higher in PF-BDM all over the batch culture compared to the SF-RPMI process. UDP-Gal accumulated only during the culture performed in PF-BDM medium, probably as a consequence of the reduced UDP-Glc synthesis flux in SF-RPMI medium. When the recombinant CHO cells were cultivated in fed-batch mode, the UTP level remained at a relatively high value in serum-containing RPMI and its titer increased during the fed-phase indicating an excess of biosynthesis. Besides, an accumulation of UDP-Gal occurred as well. Those results all together indicate that UTP and UDP-Glc syntheses in CHO cells cultivated in SF-RPMI medium in batch process, could be limiting during the glycosylation processes of the recombinant IFN-gamma. At last, the determination of the energetic status of the cells over the three studied processes suggested that a relationship between the adenylate energy charge and the glycosylation macroheterogeneity of the recombinant IFN-gamma may exist.     

Effect of rumen fill on intake of fresh perennial ryegrass in young and mature dairy cows grazing or zero-grazing fresh perennial ryegrass

Rumen fill may be a strong intake constraint for dairy cows fed on pasture, even though pasture is highly digestible in the grasslands of temperate climates. This constraint may also depend on the cows' maturity. Moreover, indoor feeding of fresh herbage may not always be a good model for the study of intake regulation at grazing. To test these hypotheses, four mature (6.3 ± 0.72 year old) and four young (3.8 ± 0.20 year old) dairy cows were offered fresh perennial ryegrass indoors or at grazing. The impact of rumen fill on intake was evaluated by addition of rumen inert bulk (RIB; coconut fiber, 15 l) compared to a control. The experimental design was a double 4 × 4 Latin square with four 14-day periods and a 2 × 2 factorial arrangement of two feeding methods (indoor feeding v. grazing), combined with the addition, or not, of RIB (RIB v. control), repeated for four mature and four young cows. Digestibility of offered herbage was 0.81. The average ytterbium measured dry matter intake (Yb DMI) was 19.0 and 15.5 kg/day for mature and young cows respectively (P = 0.019). The effect of RIB on predicted Yb DMI interacted with feeding method and cow age (P = 0.043). The presence of RIB decreased Yb DMI by 4.4 kg/day in mature cows at grazing and by 3.4 kg/day in young cows indoors, whereas it did not affect the Yb DMI of mature cows indoors or grazing young cows. Both grazing and young age constituted a clear constraint on the feeding behavior of the cows. Grazing cows had fewer ingestion and rumination sequences, which were longer and less evenly distributed throughout the day and night. Young cows had lower intake rates that were less adaptable to the feeding method and the presence of RIB. Mature cows clearly decreased their daily intake rate at grazing compared to indoor feeding, and with RIB compared to control, whereas the intake rate of young cows did not vary. These results indicate that rumen fill can represent a constraint on intake in grazing cows, even when highly digestible perennial ryegrass is offered. The study also shows that the impact of RIB on intake is highly dependent upon other constraints applied to the chewing behavior, which in this experiment were methods of offering herbage and cow age.     

Expression and purification of a recombinant Fip-fve protein from Flammulina velutipes in baculovirus-infected insect cells

Aims: To develop an efficient and facile expression system supply of high purity and stable activity of rFip-fve for oral administration, medicinal study and applications. Methods and Results: A recombinant virus that contained the chimera gene, encoding a bombyxin signal peptide sequence fused to a Fip-fve-6His sequence, was constructed. The rFip-fve was purified from the supernatant of the infected Sf21 cells using a nickel-chelated affinity column, and was verified by Western blot and MALDI-MS (matrix-assisted laser desorption ionization mass spectrometry) analyses. Results showed that a glycosylated mature rFip-fve was produced and secreted into the infected cell supernatant. The immunomodulatory activity of rFip-fve was evaluated by measuring the amount of interleukin-2 released from murine splenocytes. Conclusions: A reliable scheme to express and purify active rFip-fve in a baculovirus/insect cell system for medicinal applications and genetic study is a feasible means of solving potential problems related to the production and activity of rFip-fve protein. Significance and Impact of the Study: The rFip-fve expressed in insect cells was processed and modified in a manner more similar to that of its native counterpart than that in bacterial cells. Therefore, the potential applications of rFip-fve that is generated in Sf21 cells can be more effectively evaluated that produced in Escherichia coli.     

Dietary cyclic fatty acids derived from linolenic acid do not exhibit intrinsic toxicity in the rat during gestation

Heating oils and fats may lead to cyclization of polyunsaturated fatty acids, especially those showing multiple double bonds like linolenic acid. Cyclohexenyl and cyclopentenyl fatty acids are subsequently present in some edible oils and these were suspected to induce metabolic disorders. When fed during gestation in the rat, cyclic fatty acids were historically reported to induce high mortality of the neonates. Nevertheless, none of these studies have been performed with cyclic fatty acids fed as triacylglycerols, limiting the nutritional value of the conclusions. Therefore, we assessed the toxicity of a diet containing 0.7% of cyclic fatty acids fed as triacylglycerols during gestation and the first days of life in the rat. In this work, we report no deleterious effect of cyclic fatty acids in the mothers and neonates. However, cyclic fatty acids induced a tremendous insulinopenia in the mothers and pups that was associated with the reduction of food intake in the gestating females. Such a finding may be a plausible explanation for the adverse effects of cyclic fatty acids observed previously with higher doses of cyclic fatty acids. Based on present data, on previous ones showing elimination of cyclic fatty acids, and considering their low amounts in the diet, we suggest that cyclic fatty acids formed from cyclization of linolenic acid are not a major concern for human safety.     

ARCHIPELAGO: a dedicated resource for exploiting past, present, and future genomic data on disease resistance regulation in rice

Large amounts of expression data dealing with biotic stresses in rice have been produced in the past 5 years. Here, we extensively review approximately 70 publications and gather together information on more than 2,500 genes of the rice defense arsenal. This information was integrated into the OryGenesDB database. Several genes (e.g., metallothioneins and PBZ1) appear to be hallmarks of rice-pathogen interactions. Cross-referencing this information with the rice kinome highlighted some defense genes and kinases as possible central nodes of regulation. Cross referencing defense gene expression and quantitative trait loci (QTL) information identified some candidate genes for QTL. Overall, pathogenesis-related genes and disease regulators were found to be statistically associated with disease QTL. At the genomic level, we observed that some regions are richer than others and that some chromosomes (e.g., 11 and 12), which contain a lot of resistance gene analogs, have a low content of defense genes. Finally, we show that classical defense genes and defense-related genes such as resistance genes are preferentially organized in clusters. These clusters are not always coregulated and individual paralogs can show specific expression patterns. Thus, the rice defense arsenal has an ARCHIPELAGO-like genome structure at the macro and micro level. This resource opens new possibilities for marker-assisted selection and QTL cloning.     

MAP20, a microtubule-associated protein in the secondary cell walls of hybrid aspen, is a target of the cellulose synthesis inhibitor 2,6-dichlorobenzonitrile

We have identified a gene, denoted PttMAP20, which is strongly up-regulated during secondary cell wall synthesis and tightly coregulated with the secondary wall-associated CESA genes in hybrid aspen (Populus tremula × tremuloides). Immunolocalization studies with affinity-purified antibodies specific for PttMAP20 revealed that the protein is found in all cell types in developing xylem and that it is most abundant in cells forming secondary cell walls. This PttMAP20 protein sequence contains a highly conserved TPX2 domain first identified in a microtubule-associated protein (MAP) in Xenopus laevis. Overexpression of PttMAP20 in Arabidopsis (Arabidopsis thaliana) leads to helical twisting of epidermal cells, frequently associated with MAPs. In addition, a PttMAP20-yellow fluorescent protein fusion protein expressed in tobacco (Nicotiana tabacum) leaves localizes to microtubules in leaf epidermal pavement cells. Recombinant PttMAP20 expressed in Escherichia coli also binds specifically to in vitro-assembled, taxol-stabilized bovine microtubules. Finally, the herbicide 2,6-dichlorobenzonitrile, which inhibits cellulose synthesis in plants, was found to bind specifically to PttMAP20. Together with the known function of cortical microtubules in orienting cellulose microfibrils, these observations suggest that PttMAP20 has a role in cellulose biosynthesis.     

Modeling the inactivation kinetics of bacillus spores by glutaraldehyde

Aims: Our goal was to develop a mathematical kinetic model to predict the sporicidal activity of glutaraldehyde, which is an active ingredient frequently used in commercial products employed for liquid disinfection and decontamination. Methods and Results: We used our previously published data on spore inactivation by glutaraldehyde to develop a predictive model obtained by calculating multiple independent modifying functions. The model was then validated by comparing model predicted values to new experimental data. For model validation, quality‐controlled spores of Bacillus athrophaeus (previously and generally known as Bacillus subtilis globigii) were exposed under conditions where several physicochemical variables were modified simultaneously, and the spore surviving fractions were measured by titration. Conclusions: The model predicted within one order of magnitude variations in sporicidal effectiveness due to changes in main parameters (glutaraldehyde concentration, temperature or time‐duration of the treatment). Other parameters such pH, salinity and the effect of serum concentration were also addressed, albeit with less accuracy. Significance and Impact of the study: The model should be useful to quantitatively estimate the effectiveness of glutaraldehyde‐based disinfectants, decontaminants, and germicides under the described conditions, particularly when limited data are available or when spore virulence (like that of Bacillus anthracis) precludes extensive experimentation. A similar approach could predict the effectiveness of a variety of decontaminant and disinfecting agents.     

Social facilitation of fur rubbing behavior in white-faced capuchins

In their natural environment, capuchins select certain plants, containing secondary compounds with bactericide, insecticide or fungicide properties, to rub their pelage energetically (i.e. fur rubbing). Fur rubbing can be performed in solitary, or collectively in subgroups of variable size and composition, and most of the time fur rubbing happens in synchrony with other group members. The aim of this study is to understand the underlying mechanisms of this phenomenon, and, more particularly, to determine the processes involved in its synchronization. For this purpose, we designed a set of experiments where white-faced capuchins (Cebus capucinus) were presented with onions (Allium cepa) that they use to fur rub. We conducted a detailed kinetic study of fur rubbing behavior to determine if its synchronization is the consequence of simultaneous responses of different individuals to the same stimulus or if, on the contrary, there is a real collective phenomenon where individuals respond to conspecific behavior. Our results reveal that fur rubbing is a collective behavior with a mimetic underlying mechanism. If fur rubbing with onions (a plant with antifungal and repellent properties) allows capuchins to treat their fur against parasites or pathogens, its synchronization would optimize the treatment by acting as a group barrier to ectoparasite propagation.     

Synthesis and iron-binding properties of quinolobactin,a siderophore from a pyoverdine-deficient <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis>

Quinolobactin is a new siderophore produced by a pyoverdine deficient mutant of Pseudomonas fluorescens. A simple and efficient synthesis of quinolobactin is described, starting from xanthurenic acid. The protonation constants of quinolobactin were determined by potentiometric titrations as pK_a2=5.50 ± 0.07, pK_a1=10.30 ± 0.05. The equilibria of the metal complexes were studied by means of spectrophotometric and potentiometric titrations. The overall stability constants of the quinolobactin-Fe^III complexes was found to be log ₁₁₁=18.60 ± 0.10, log ₁₂₁=32.60 ± 0.20, log ₁₂₀=28.20 ± 0.25 resulting in a pFe^III value of 18.2 at pH 7.4. The UV-visible spectral parameters of the [FeL₂] are in agreement with a complex containing two ligands coordinated to one Fe³⁺ cation through the oxygen and nitrogen quinoline atoms.     

G(1)/S but not G(0)/G(1)cell fraction is related to 5-fluorouracil cytotoxicity

BACKGROUND: Bromodeoxyuridine (BrdU) cell cycle analysis using flow cytometry is of clinical interest for making treatment decisions or for predicting response and survival, through proliferation rate (labeling index or S-phase fraction) assessment or T(pot) calculation. Thymidylate synthase expression was tested in vitro, in vivo, and clinically as a prognostic factor for 5-fluorouracil (5FU) sensitivity. However, results were still controversial. Moreover, we had reported that 5FU sensitivity was related to the labeling index of untreated cell cultures. METHODS: We used six human cancer cell lines that exhibited a wide range of 5FU sensitivity. Cell cycle analysis was performed using flow cytometry monovariate propidium iodide (PI) analysis and bivariate distributions of BrdU incorporation versus DNA content. 5FU sensitivity was assayed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) colorimetric assay. RESULTS: In all cell lines, 5FU exposure resulted in a statistically significant G(1)/S accumulation. No statistically significant relationship was seen between G(0)/G(1) delay determined by monovariate analysis and 5FU sensitivity. However, 5FU sensitivity was statistically correlated to the labeling index and G(1)/S subpopulation assessed with bivariate analysis using BrdU incorporation versus DNA content. CONCLUSIONS: Cellular proliferation parameters using BrdU incorporation are more informative than PI for in vitro 5FU sensitivity. Because BrdU incorporation could be assessed clinically, it could also be informative for 5FU clinical response prediction.