CHO immobilization in alginate/poly-l-lysine microcapsules: an understanding of potential and limitations

Microencapsulation offers a unique potential for high cell density, high productivity mammalian cell cultures. However, for successful exploitation there is the need for microcapsules of defined size, properties and mechanical stability. Four types of alginate/poly-l-Lysine microcapsules, containing recombinant CHO cells, have been investigated: (a) 800 μm liquid core microcapsules, (b) 500 μm liquid core microcapsules, (c) 880 μm liquid core microcapsules with a double PLL membrane and (d) 740 μm semi-liquid core microcapsules. With encapsulated cells a reduced growth rate was observed, however this was accompanied by a 2–3 fold higher specific production rate of the recombinant protein. Interestingly, the maximal intracapsular cell concentration was only 8.7 × 10⁷ cell mL^-1, corresponding to a colonization of 20% of the microcapsule volume. The low level of colonization is unlikely to be due to diffusional limitations since reduction of microcapsule size had no effect. Measurement of cell leaching and mechanical properties showed that liquid core microcapsules are not suitable for continuous long-term cultures (>1 month). By contrast semi-liquid core microcapsules were stable over long periods with a constant level of cell colonization (ϕ = 3%). This indicates that the alginate in the core plays a predominant role in determining the level of microcapsule colonization. This was confirmed by experiments showing reduced growth rates of batch suspension cultures of CHO cells in medium containing dissolved alginate. Removal of this alginate would therefore be expected to increase microcapsule colonization.     

Hydrophobic plastics films synthesis by mean of agaroid acylation with lauroyl chloride in the N,N-dimethylacetamide homogeneous system

The synthesis of hydrophobic plastic films was performed by acylation of agaroids with lauroyl chloride in the N,N-dimethylacetamide homogeneous system. All the plastic films were characterized by FT-IR spectroscopy and their degrees of substitution (DS) was deduced from their ¹H-NMR spectra. In addition, thermomechanical feature of plastic films were analyzed and compared to those obtained from other kinds of hydrophobic plastic films. Latin square design of experiments helped us to determine optimized experimental conditions and identify the most important factors. Hence, mild conditions of acylation (90∘C, 1 eq/OH of lauroyl chloride, 1 eq/OH of 4-dimethylaminopyridine, 5,min) led to the production of highly substituted plastic films (DS = 3.62; maximum 4) with a high weight yield (211%) displaying mechanical properties close to polyethylene low density.     

Clostridium taeniosporum spore ribbon-like appendage structure, composition and genes

Clostridium taeniosporum spores have about 12 large, flat, ribbon-like appendages attached through a common trunk at one spore pole to a previously unknown surface layer outside the coat that is proposed to be called the 'encasement'. Appendages are about 4.5 microm long, 0.5 microm wide and 30 nm thick and taper at the attachment end into a semicircle that is twisted relative to the flat ribbon. Individual fibrils, about 45 nm in length with spherical heads and long thin tails, form a hair-like nap, visible along the appendage edge. Four appendage proteins have been detected: a paralogous pair of 29 kDa (designated P29a and P29b), a glycoprotein of about 37 kDa (designated GP85) and an orthologue of the Bacillus spore morphogenetic protein SpoVM. The P29 proteins consist of duplicated regions and each region includes a domain of unknown function 11. The GP85 glycoprotein contains a collagen-like region. The genes for P29a and b, GP85 and possibly related proteins are closely linked on two small chromosome fragments. Putative sigma(K)-dependent promoters upstream of the P29a and b genes indicate that they likely are expressed late in the mother cell, consistent with their deposition into the layer external to the coat.     

The Mcf1 toxin induces apoptosis via the mitochondrial pathway and apoptosis is attenuated by mutation of the BH3-like domain

Photorhabdus are Gram-negative, nematode-vectored bacteria that produce toxins to kill their insect hosts. The expression of one of these, Makes caterpillars floppy 1 (Mcf1), is sufficient to allow Escherichia coli to survive within, and kill, caterpillars which are otherwise able to clear E. coli infection. Mcf1 treated caterpillars show rapid loss of body turgor (the 'floppy' phenotype) and death is associated with massive apoptosis of both the midgut epithelium and insect phagocytes. Mammalian tissue culture cells treated with Mcf1 also display key features of apoptosis including zeiosis, apoptotic nuclear morphology, DNA laddering, activation of the effector caspase-3 and PARP cleavage. As Mcf1 carries a single BH3-like domain, here we investigate the hypothesis that this toxin promotes apoptosis via the mitochondrial pathway by mimicking a BH3 domain-only protein. Consistent with this hypothesis, a double mutant within the BH3-like domain causes a dramatic decline in apoptosis. Mcf1 also alters mitochondrial membrane potential and triggers the release of cytochrome c. Cells overexpressing Bcl-x(L), an anti-apoptotic Bcl-2 family member, are resistant to Mcf1-mediated apoptosis, as are cells deficient in Bax. In addition, translocation of Bax to the mitochondrion is observed in response to Mcf1 treatment. Together, these results show that Mcf1 mediates apoptosis via the mitochondrial pathway, and are consistent with the hypothesis that the BH3-like domain in Mcf1 is a functional requirement for the pro-apoptotic activity of Mcf1.     

Selective prediction of interaction sites in protein structures with THEMATICS

Background  

Methods are now available for the prediction of interaction sites in protein 3D structures. While many of these methods report high success rates for site prediction, often these predictions are not very selective and have low precision. Precision in site prediction is addressed using Theoretical Microscopic Titration Curves (THEMATICS), a simple computational method for the identification of active sites in enzymes. Recall and precision are measured and compared with other methods for the prediction of catalytic sites.     

Identification and expression analysis of genes associated with bovine blastocyst formation

Background  

Normal preimplantation embryo development encompasses a series of events including first cleavage division, activation of the embryonic genome, compaction and blastocyst formation.     

Antinociceptive efficacy of lacosamide in the monosodium iodoacetate rat model for osteoarthritis pain

The etiology of osteoarthritis is multifactorial, with inflammatory, metabolic, and mechanical causes. Pain in osteoarthritis is initiated by mild intra-articular inflammation and degeneration of articular cartilage and subchondral bone. The principle of treatment with acetaminophen or non-steroidal anti-inflammatory drugs is to reduce pain and improve joint function. Recently, animal models for osteoarthritic pain behavior have been established. The most frequently used rat model for analyzing properties of drugs on the pathology of osteoarthritis is the injection of the metabolic inhibitor monosodium iodoacetate into the joint, which inhibits the activity of glyceraldehyde-3-phosphate dehydrogenase in chondrocytes. Here, we characterize the effect on pain behavior of lacosamide, a member of a family of functionalized amino acids that are analogues of endogenous amino acids and D-serine, in the monosodium iodoacetate rat model for osteoarthritis in comparison to diclofenac and morphine. Lacosamide (3, 10, and 30 mg/kg) was able to reduce secondary mechanical allodynia and hyperalgesia similarly to morphine (3 mg/kg). In contrast, diclofenac (30 mg/kg) was only effective in reducing secondary mechanical hyperalgesia. During the first week, pain is induced mainly by inflammation in the iodoacetate model, but afterwards inflammation plays only a minor role in pain. Lacosamide was able to inhibit pain at days 3, 7 and 14 after induction of arthritis. This shows that lacosamide is able to reduce pain behavior induced by multiple mechanisms in animals.     

Relationships between tree component structure,topography and soils of a riverside forest,Rio Botucaraí, Southern Brazil

The relationships among floristic, structural and physiognomic variables of the tree component, flooding regime variations and soil fertility were investigated in a riparian forest fragment (ca. 43 ha) in the Rio Botucaraí watershed near the confluence with the Rio Jacuí, southern Brazil. All the trees with a dbh ≥ 15 cm were surveyed in 100 contiguous 10 × 10 m plots and soil chemical and textural variables were obtained from the analyses of 15 topsoil samples (0–20 cm depth) collected at different locations. The sample sites encompass all the topographic variation of the area. We used canonical correspondence analysis (CCA) to seek correlations between environmental variables and tree component distribution. The 1,547 surveyed individuals belonged to 30 species and 16 botanical families. The Shannon diversity index (H′) and the Pielou equability (J′) were 1.995 and 0.586 nats ind.⁻¹, respectively. Sebastiania commersoniana, Casearia sylvestris, Eugenia uniflora and Eugenia hyemalis presented the highest importance values. Species’ densities chiefly correlated with site elevation, flooding regime and soil chemical fertility. The analyses of ecological categories (EC) revealed that most species are heliophylous and typical of early successional stages. Richness and diversity (P < 0.001) variations from different topographic positions reflect that at the local scale, flooding was a limiting factor for the establishment of certain species suggesting that local processes control species richness and diversity.     

Oviposition Sites of the Cypress Seed Bug <Emphasis Type="Italic">Orsillus maculatus</Emphasis> and Response of the Egg Parasitoid <Emphasis Type="Italic">Telenomus</Emphasis> gr. <Emphasis Type="Italic">Floridanus</Emphasis>

Phytophagous insects have many strategies to escape parasitoids, for example by hiding eggs into plant tissues, but oviposition in holes made by another insect is rather scarce. The cypress seed bug Orsillus maculatus Fieber (Heteroptera: Lygaeidae) is strictly dependent on the availability of cones of Cupressus sempervirens L. to oviposit. Females lay eggs either in exit holes cut through the cone scale by emerging adults of the cypress seed chalcid, Megastigmus wachtli Seitner (Hymenoptera: Torymidae), or on the inner side of scales of partly open cones. A recently discovered egg parasitoid belonging to the genus Telenomus has been shown to attack bug eggs in both oviposition sites. In this paper we investigated the parasitoid performance according to oviposition sites. Field samplings were conducted in two evergreen cypress orchards located in the south of France. The distribution and condition of the egg patches were compared between the two locations and oviposition sites. Seed bugs preferred to oviposit in emergence holes of M. wachtli, and parasitoid performance was higher in eggs laid on cone scales. The chalcid emergence holes seemed to ensure bug eggs with enemy-free space. Oviposition site selection could be an adaptive strategy to escape parasitoid attack.