Lotus japonicus CASTOR and POLLUX Are Ion Channels Essential for Perinuclear Calcium Spiking in Legume Root Endosymbiosis

The mechanism underlying perinuclear calcium spiking induced during legume root endosymbioses is largely unknown. Lotus japonicus symbiosis-defective castor and pollux mutants are impaired in perinuclear calcium spiking. Homology modeling suggested that the related proteins CASTOR and POLLUX might be ion channels. Here, we show that CASTOR and POLLUX form two independent homocomplexes in planta. CASTOR reconstituted in planar lipid bilayers exhibited ion channel activity, and the channel characteristics were altered in a symbiosis-defective mutant carrying an amino acid replacement close to the selectivity filter. Permeability ratio determination and competition experiments reveled a weak preference of CASTOR for cations such as potassium over anions. POLLUX has an identical selectivity filter region and complemented a potassium transport–deficient yeast mutant, suggesting that POLLUX is also a potassium-permeable channel. Immunogold labeling localized the endogenous CASTOR protein to the nuclear envelope of Lotus root cells. Our data are consistent with a role of CASTOR and POLLUX in modulating the nuclear envelope membrane potential. They could either trigger the opening of calcium release channels or compensate the charge release during the calcium efflux as counter ion channels.     

Consumption of toxic cyanobacteria by Potamopyrgus antipodarum (Gastropoda, Prosobranchia) and consequences on life traits and microcystin accumulation

Among the wide range of toxins produced by cyanobacterial blooms, microcystins (MCs) are the most common and are known to accumulate in aquatic organisms. Freshwater gastropods are grazers and likely to ingest toxic cyanobacteria, particularly Planktothrix agardhii, one of the most common species in the northern hemisphere. The study examines (i) the ingestion of toxic P. agardhii by the prosobranch Potamopyrgus antipodarum, (ii) the kinetics of MC accumulation and depuration in snail tissues during and post-exposure, and (iii) the impact of MCs on their life traits (survival, growth and fecundity). We showed that P. antipodarum ingested 71% of cyanobacteria available during the first 24 h in the presence or not of non-toxic food, and accumulated 1.3% of ingested MCs during the 5-week intoxication period. Elimination of MCs was total after 3 weeks of depuration. A decrease of growth and fecundity was observed during the intoxication period, but it was reversible after the end of exposure. Results are discussed in terms of variation of the response between prosobranch and pulmonate gastropods to toxic cyanobacteria exposure, and the negative impact of toxic cyanobacteria on natural communities of freshwater gastropods.     

Automated region of interest retrieval and classification using spectral analysis

Efficient use of whole slide imaging in pathology needs automated region of interest (ROI) retrieval and classification, through the use of image analysis and data sorting tools. One possible method for data sorting uses Spectral Analysis for Dimensionality Reduction. We present some interesting results in the field of histopathology and cytohematology. In histopathology, we developed a Computer-Aided Diagnosis system applied to low-resolution images representing the totality of histological breast tumour sections. The images can be digitized directly at low resolution or be obtained from sub-sampled high-resolution virtual slides. Spectral Analysis is used (1) for image segmentation (stroma, tumour epithelium), by determining a "distance" between all the images of the database, (2) for choosing representative images and characteristic patterns of each histological type in order to index them, and (3) for visualizing images or features similar to a sample provided by the pathologist. In cytohematology, we studied a blood smear virtual slide acquired through high resolution oil scanning and Spectral Analysis is used to sort selected nucleated blood cell classes so that the pathologist may easily focus on specific classes whose morphology could then be studied more carefully or which can be analyzed through complementary instruments, like Multispectral Imaging or Raman MicroSpectroscopy.     

Intranasal HB-EGF administration favors adult SVZ cell mobilization to demyelinated lesions in mouse corpus callosum

In the adult rodent brain, the subventricular zone (SVZ) represents a special niche for neural stem cells; these cells proliferate and generate neural progenitors. Most of these migrate along the rostral migratory stream to the olfactory bulb, where they differentiate into interneurons. SVZ-derived progenitors can also be recruited spontaneously to damaged brain areas to replace lost cells, including oligodendrocytes in demyelinated lesions. In this study, we searched for factors able to enhance this spontaneous recruitment of endogenous progenitors. Previous studies have suggested that epidermal growth factor (EGF) could stimulate proliferation, migration, and glial differentiation of SVZ progenitors. In the present study we examined EGF influence on endogenous SVZ cell participation to brain repair in the context of demyelinated lesions. We induced a focal demyelinated lesion in the corpus callosum by lysolecithin injection and showed that intranasal heparin-binding epidermal growth factor (HB-EGF) administration induces a significant increase in SVZ cell proliferation together with a stronger SVZ cell mobilization toward the lesions. Besides, HB-EGF causes a shift of SVZ-derived progenitor cell differentiation toward the astrocytic lineage. However, due to the threefold increase in cell recruitment by EGF treatment, the absolute number of SVZ-derived oligodendrocytes in the lesion of treated mice is higher than in controls. These results suggest that enhancing SVZ cell proliferation could be part of future strategies to promote SVZ progenitor cell mobilization toward brain lesions.     

The complex biogeographic history of a widespread tropical tree species

Many tropical forest tree species have broad geographic ranges, and fossil records indicate that population disjunctions in some species were established millions of years ago. Here we relate biogeographic history to patterns of population differentiation, mutational and demographic processes in the widespread rainforest tree Symphonia globulifera using ribosomal (ITS) and chloroplast DNA sequences and nuclear microsatellite (nSSR) loci. Fossil records document sweepstakes dispersal origins of Neotropical S. globulifera populations from Africa during the Miocene. Despite historical long-distance gene flow, nSSR differentiation across 13 populations from Costa Rica, Panama, Ecuador (east and west of Andes) and French Guiana was pronounced (F(ST)= 0.14, R(ST)= 0.39, P < 0.001) and allele-size mutations contributed significantly (R(ST) > F(ST)) to the divergences between cis- and trans-Andean populations. Both DNA sequence and nSSR data reflect contrasting demographic histories in lower Mesoamerica and Amazonia. Amazon populations show weak phylogeographic structure and deviation from drift-mutation equilibrium indicating recent population expansion. In Mesoamerica, genetic drift was strong and contributed to marked differentiation among populations. The genetic structure of S. globulifera contains fingerprints of drift-dispersal processes and phylogeographic footprints of geological uplifts and sweepstakes dispersal.     

The effects of culture conditions on the glycosylation of secreted human placental alkaline phosphatase produced in Chinese hamster ovary cells

The effects of different culture conditions, suspension and microcarrier culture and temperature reduction on the structures of N-linked glycans attached to secreted human placental alkaline phosphatase (SEAP) were investigated for CHO cells grown in a controlled bioreactor. Both mass spectrometry and anion-exchange chromatography were used to probe the N-linked glycan structures and distribution. Complex-type glycans were the dominant structures with small amounts of high mannose glycans observed in suspension and reduced temperature cultures. Biantennary glycans were the most common structures detected by mass spectrometry, but triantennary and tetraantennary forms were also detected. The amount of sialic acid present was relatively low, approximately 0.4 mol sialic acid/mol SEAP for suspension cultures. Microcarrier cultures exhibited a decrease in productivity compared with suspension culture due to a decrease in both maximum viable cell density (15-20%) and specific productivity (30-50%). In contrast, a biphasic suspension culture in which the temperature was reduced at the beginning of the stationary phase from 37 to 33 degrees C, showed a 7% increase in maximum viable cell density, a 62% increase in integrated viable cell density, and a 133% increase in specific productivity, leading to greater than threefold increase in total productivity. Both microcarrier and reduced temperature cultures showed increased sialylation and decreased fucosylation when compared to suspension culture. Our results highlight the importance of glycoform analysis after process modification as even subtle changes (e.g., changing from one microcarrier to another) may affect glycan distributions.     

Coordinate induction of PPAR alpha and SREBP2 in multifunctional protein 2 deficient mice

Mice with inactivation of the D-specific multifunctional protein 2 (MFP2), a crucial enzyme of peroxisomal beta-oxidation, develop multiple pathologies in diverse tissues already starting in the postnatal period. Gene expression profiling performed on liver of 2-day-old pups revealed up-regulation of PPAR alpha responsive genes in knockout mice. Surprisingly, also genes involved in cholesterol biosynthesis were markedly induced. Real-time PCR confirmed the induction of PPAR alpha target genes and of HMGCR and SREBP2, both involved in cholesterol synthesis, in lactating and in adult MFP2 knockout mice. In accordance, the rate of cholesterol biosynthesis was significantly increased in liver of knockout mice but the hepatic cholesterol concentration was unaltered. In MFP2/PPAR alpha double knockout mice, up-regulations of SREBP2 and HMGCR were markedly attenuated. These data demonstrate a tight interrelationship between induction of PPAR alpha by endogenous ligands and up-regulation of genes of cholesterol biosynthesis through increased expression of SREBP2.     

Biotransformation of Natural and Synthetic Isoflavonoids by Two Recombinant Microbial Enzymes

Isolation and synthesis of isoflavonoids has become a frequent endeavor, due to their interesting biological activities. The introduction of hydroxyl groups into isoflavonoids by the use of enzymes represents an attractive alternative to conventional chemical synthesis. In this study, the capabilities of biphenyl-2,3-dioxygenase (BphA) and biphenyl-2,3-dihydrodiol 2,3-dehydrogenase (BphB) of Burkholderia sp. strain LB400 to biotransform 14 isoflavonoids synthesized in the laboratory were investigated by using recombinant Escherichia coli strains containing plasmid vectors expressing the bphA1A2A3A4 or bphA1A2A3A4B genes of strain LB400. The use of BphA and BphB allowed us to biotransform 7-hydroxy-8-methylisoflavone and 7-hydroxyisoflavone into 7,2′,3′-trihydroxy-8-methylisoflavone and 7,3′,4′-trihydroxyisoflavone, respectively. The compound 2′-fluoro-7-hydroxy-8-methylisoflavone was dihydroxylated by BphA at ortho-fluorinated and meta positions of ring B, with concomitant dehalogenation leading to 7,2′,3′,-trihydroxy-8-methylisoflavone. Daidzein (7,4′-dihydroxyisoflavone) was biotransformed by BphA, generating 7,2′,4′-trihydroxyisoflavone after dehydration. Biotransformation products were analyzed by gas chromatography-mass spectrometry and nuclear magnetic resonance techniques.