High accumulation of dehydrodiconiferyl alcohol-4-β-d-glucoside in free and immobilized Linum usitatissimum cell cultures

As flaxseed mainly accumulates lignans (secoisolariciresinol diglucoside and matairesinol), these compounds were barely or not detected in plant cell suspensions initiated from Linum usitatissimum. In contrast, these cell suspensions were shown to accumulate substantial amounts of a neolignan identified as dehydrodiconiferyl alcohol-4-β-d-glucoside (DCG) (up to 47.7 mg g⁻¹ DW). The formation of this pharmacologically active compound was evaluated as a function of cell growth and in relation to phytohormone balance of the culture media. After establishment of efficient culture conditions, production of DCG was investigated in immobilized plant cell suspensions initiated from plantlet roots of L. usitatissimum. The results indicate that immobilization enhances the DCG production up to 60.0 mg g⁻¹ DW but depresses the cell growth resulting in no improvement of the total DCG yield. Nevertheless, with immobilized cell suspensions, a release of DCG into the medium is observed allowing an easier recovery.     

Aquaporin-facilitated transmembrane diffusion of hydrogen peroxide

Background

Hydrogen peroxide (H₂O₂) is an important signaling compound that has recently been identified as a new substrate for several members of the aquaporin superfamily in various organisms. Evidence is emerging about the physiological significance of aquaporin-facilitated H₂O₂ diffusion.

Scope of review

This review summarizes current knowledge about aquaporin-facilitated H₂O₂ diffusion across cellular membranes. It focuses on physicochemical and experimental evidence demonstrating the involvement of aquaporins in the transport of this redox signaling compound and discusses the regulation and structural prerequisites of these channels to transmit this signal. It also provides perspectives about the potential importance of aquaporin-facilitated H₂O₂ diffusion processes and places this knowledge in the context of the current understanding of transmembrane redox signaling processes.

Major conclusions

Specific aquaporin isoforms facilitate the passive diffusion of H₂O₂ across biological membranes and control H₂O₂ membrane permeability and signaling in living organisms.

General significance

Redox signaling is a very important process regulating the physiology of cells and organisms in a similar way to the well-characterized hormonal and calcium signaling pathways. Efficient transmembrane diffusion of H₂O₂, a key molecule in the redox signaling network, requires aquaporins and makes these channels important players in this signaling process. Channel-mediated membrane transport allows the fine adjustment of H₂O₂ levels in the cytoplasm, intracellular organelles, the apoplast, and the extracellular space, which are essential for it to function as a signal molecule. This article is part of a Special Issue entitled Aquaporins.     

Integrin α8β1 regulates adhesion,migration and proliferation of human intestinal crypt cells via a predominant RhoA/ROCK‐dependent mechanism

Background. Integrins are transmembrane αβ heterodimer receptors that function as structural and functional bridges between the cytoskeleton and ECM (extracellular matrix) molecules. The RGD (arginine‐glycine‐aspartate tripeptide motif)‐dependent integrin α8β1 has been shown to be involved in various cell functions in neuronal and mesenchymal‐derived cell types. Its role in epithelial cells remains unknown. Results. Integrin α8β1 was found to be expressed in the crypt cell population of the human intestine but was absent from differentiating and mature epithelial cells of the villus. The function of α8β1 in epithelial crypt cells was investigated at the cellular level using normal HIECs (human intestinal epithelial cells). Specific knockdown of α8 subunit expression using an shRNA (small‐hairpin RNA) approach showed that α8β1 plays important roles in RGD‐dependent cell adhesion, migration and proliferation via a RhoA/ROCK (Rho‐associated kinase)‐dependent mechanism as demonstrated by active RhoA quantification and pharmacological inhibition of ROCK. Moreover, loss of α8β1, through RhoA/ROCK, impairs FA (focal adhesion) complex integrity as demonstrated by faulty vinculin recruitment. Conclusions. Integrin α8β1 is expressed in epithelial cells. In intestinal crypt cells, α8β1 is closely involved in the regulation of adhesion, migration and cell proliferation via a predominant RhoA/ROCK‐dependent mechanism. These results suggest an important role for this integrin in intestinal crypt cell homoeostasis.     

Development and validation of a mastication simulator

More and more research are being done on food bolus formation during mastication. However, the process of bolus formation in the mouth is difficult to observe. A mastication simulator, the Artificial Masticatory Advanced Machine (AM2) was developed to overcome this difficulty and is described here. Different variables can be set such as the number of masticatory cycles, the amplitude of the mechanical movements simulating the vertical and lateral movements of the human lower jaw, the masticatory force, the temperature of the mastication chamber and the injection and the composition of saliva. The median sizes of the particles collected from the food boluses made by the AM2 were compared with those of human boluses obtained with peanuts and carrots as test foods. Our results showed that AM2 mimicked human masticatory behavior, producing a food bolus with similar granulometric characteristics.     

Genetic Resistance to Scrapie Infection in Experimentally Challenged Goats

In goats, several field studies have identified coding mutations of the gene encoding the prion protein (I/M₁₄₂, N/D₁₄₆, S/D₁₄₆, R/Q₂₁₁, and Q/K₂₂₂) that are associated with a lower risk of developing classical scrapie. However, the data related to the levels of resistance to transmissible spongiform encephalopathies (TSEs) of these different PRNP gene mutations are still considered insufficient for developing large-scale genetic selection against scrapie in this species. In this study, we inoculated wild-type (WT) PRNP (I₁₄₂R₁₅₄R₂₁₁Q₂₂₂) goats and homozygous and/or heterozygous I/M₁₄₂, R/H₁₅₄, R/Q₂₁₁, and Q/K₂₂₂ goats with a goat natural scrapie isolate by either the oral or the intracerebral (i.c.) route. Our results indicate that the I/M₁₄₂ PRNP polymorphism does not provide substantial resistance to scrapie infection following intracerebral or oral inoculation. They also demonstrate that H₁₅₄, Q₂₁₁, and K₂₂₂ PRNP allele carriers are all resistant to scrapie infection following oral exposure. However, in comparison to WT animals, the H₁₅₄ and Q₂₁₁ allele carriers displayed only moderate increases in the incubation period following i.c. challenge. After i.c. challenge, heterozygous K₂₂₂ and a small proportion of homozygous K₂₂₂ goats also developed the disease, but with incubation periods that were 4 to 5 times longer than those in WT animals. These results support the contention that the K₂₂₂ goat prion protein variant provides a strong but not absolutely protective effect against classical scrapie.     

Bactrocera invadens (Diptera: Tephritidae), a new invasive fruit fly pest for the Afrotropical region: host plant range and distribution in West and Central Africa

In 2003, the invasive fruit fly Bactrocera invadens Drew, Tsuruta & White (Diptera: Tephritidae) (Drew et al. 2005), of possible Sri Lankan origin, has been detected in the East and about 1 yr later in West Africa. In regular surveys in Benin and Cameroon covering 4 yr, samples from 117 plant species across 43 families have been obtained. Incubation of field-collected fruits demonstrate that in West and Central Africa (WCA) B. invadens is highly polyphagous, infesting wild and cultivated fruits of at least 46 species from 23 plant families with guava (Psidium spp.), mango (Mangifera spp.), and citrus (spp.), and the wild hosts tropical almond (Terminalia catappa L.), African wild mango (Irvingia gabonensis (Aubry-Lecomte) Baill.), and sheanut (Vitellaria paradoxa C.F.Gaertn.) showing the highest infestation index. B. invadens occurs in 22 countries of WCA with new records for Angola, Central African Republic, the Congo, DR Congo, Equatorial Guinea, Gabon, Gambia, Guinea Bissau, Mali, Mauritania, Niger, and Sierra Leone. Overall, the pest has spread across a North-South distance of ?5,000 km representing a contiguous area of >8.3 million km(2) within WCA. B. invadens has adapted to a wide range of ecological and climatic conditions extending from low land rainforest to dry savanna. Because of its highly destructive and invasive potential, B. invadens poses a serious threat to horticulture in Africa if left uncontrolled. Moreover, the presence of this quarantine pest causes considerable restrictions on international trade of affected crops.     

Spatial regulation improves antiparallel microtubule overlap during mitotic spindle assembly

The mitotic spindle plays an essential role in chromosome segregation during cell division. Spindle formation and proper function require that microtubules with opposite polarity overlap and interact. Previous computational simulations have demonstrated that these antiparallel interactions could be created by complexes combining plus- and minus-end-directed motors. The resulting spindles, however, exhibit sparse antiparallel microtubule overlap with motor complexes linking only a nominal number of antiparallel microtubules. Here we investigate the role that spatial differences in the regulation of microtubule interactions can have on spindle morphology. We show that the spatial regulation of microtubule catastrophe parameters can lead to significantly better spindle morphology and spindles with greater antiparallel MT overlap. We also demonstrate that antiparallel microtubule overlap can be increased by having new microtubules nucleated along the length of existing astral microtubules, but this increase negatively affects spindle morphology. Finally, we show that limiting the diffusion of motor complexes within the spindle region increases antiparallel microtubule interaction.     

A high-sensitivity method for detection and measurement of HMGB1 protein concentration by high-affinity binding to DNA hemicatenanes

Background

Protein HMGB1, an abundant nuclear non-histone protein that interacts with DNA and has an architectural function in chromatin, was strikingly shown some years ago to also possess an extracellular function as an alarmin and a mediator of inflammation. This extracellular function has since been actively studied, both from a fundamental point of view and in relation to the involvement of HMGB1 in inflammatory diseases. A prerequisite for such studies is the ability to detect HMGB1 in blood or other biological fluids and to accurately measure its concentration.

Methodology/Principal Findings

In addition to classical techniques (western blot, ELISA) that make use of specific anti-HMGB1 antibodies, we present here a new, extremely sensitive technique that is based on the fact that hemicatenated DNA loops (hcDNA) bind HMGB1 with extremely high affinity, higher than the affinity of specific antibodies, similar in that respect to DNA aptamers. DNA-protein complexes formed between HMGB1 and radiolabeled hcDNA are analyzed by electrophoresis on nondenaturing polyacrylamide gels using the band-shift assay method. In addition, using a simple and fast protocol to purify HMGB1 on the basis of its solubility in perchloric acid allowed us to increase the sensitivity by suppressing any nonspecific background. The technique can reliably detect HMGB1 at a concentration of 1 pg per microliter in complex fluids such as serum, and at much lower concentrations in less complex samples. It compares favorably with ELISA in terms of sensitivity and background, and is less prone to interference from masking proteins in serum.

Conclusion

The new technique, which illustrates the potential of DNA nanoobjects and aptamers to form high-affinity complexes with selected proteins, should provide a valuable tool to further investigate the extracellular functions of HMGB1 and its involvement in inflammatory pathologies.     

Nonsense-mediated mRNA decay impacts MSI-driven carcinogenesis and anti-tumor immunity in colorectal cancers

Nonsense-mediated mRNA Decay (NMD) degrades mutant mRNAs containing premature termination codon (PTC-mRNAs). Here we evaluate the consequence of NMD activity in colorectal cancers (CRCs) showing microsatellite instability (MSI) whose progression is associated with the accumulation of PTC-mRNAs encoding immunogenic proteins due to frameshift mutations in coding repeat sequences. Inhibition of UPF1, one of the major NMD factors, was achieved by siRNA in the HCT116 MSI CRC cell line and the resulting changes in gene expression were studied using expression microarrays. The impact of NMD activity was also investigated in primary MSI CRCs by quantifying the expression of several mRNAs relative to their mutational status and to endogenous UPF1 and UPF2 expression. Host immunity developed against MSI cancer cells was appreciated by quantifying the number of CD3epsilon-positive tumor-infiltrating lymphocytes (TILs). UPF1 silencing led to the up-regulation of 1251 genes in HCT116, among which a proportion of them (i.e. 38%) significantly higher than expected by chance contained a coding microsatellite (P<2x10(-16)). In MSI primary CRCs, UPF1 was significantly over-expressed compared to normal adjacent mucosa (P<0.002). Our data provided evidence for differential decay of PTC-mRNAs compared to wild-type that was positively correlated to UPF1 endogenous expression level (P = 0.02). A negative effect of UPF1 and UPF2 expression on the host's anti-tumor response was observed (P<0.01). Overall, our results show that NMD deeply influences MSI-driven tumorigenesis at the molecular level and indicate a functional negative impact of this system on anti-tumor immunity whose intensity has been recurrently shown to be an independent factor of favorable outcome in CRCs.