Test of a novel transparent floating carrier by recombinant cho cells expressing sIL6 receptor protein

We describe a carrier with some unusual properties.“Cellrafts” increase yields from adherent cells in conventional static T150 flasks, by floating multilayer strips of transparent film at the oxygen-rich surface of unstirred medium. This new technique allowed microscopic inspection of cell growth inside the carriers during bulk culture. Individual carriers could be picked out for subculture of selected colonies. A novel surface treatment by hypochlorite/uv allowed recycling of used carriers. Cellrafts' open-deck structure facilitated trypsinization with90% release as viable single cells from bulk carriers. Macro size (10mm by 1 mm) enables retention in flask by a coarse sieve insert in its neck, facilitating separation of product cells or media. Residual cells in carriers regenerated repeated harvests without need for reseeding. Carriers were tested with shear-sensitive CHO clones expressing soluble human IL6 receptor (sIL6R). Control was monolayer bulk culture on trays. Floating multilayer cultures remained viable longer than monolayers, had higher cellular activity of protein expression, and were less serum dependent (resembling cells on porous carriers). Purity and anti-sIL6R binding were identical to control product. Cellrafts were also tested in a small spinner vessel, but for litre batches this proved less convenient than in T-flasks. Though yields are low compared to well established porous carrier technology (spinner or packed bed) static transparent carriers might provide transitional scaleup from normal cytogenetics laboratory culture. This revised version was published online in August 2006 with corrections to the Cover Date.     

Gibberellin A3 stimulates adventitious rooting of cuttings from cherry (Prunus avium)

We have examined the effect of exogenous gibberellin A₃(GA₃) on adventitious rooting of Prunus avium(cherry) cultivars Stella, F12/1 and Charger. We show that GA₃pre-treatment of P. avium stock plants causes an increasein shoot growth rate and also improves the rooting of cuttings subsequentlytaken from the treated plants. Approximately 37% of cuttings from controlshootsrooted, whereas the percentage rooting could be increased to 80% or more withGA₃ pre-treatment. The number of roots per rooted cutting was alsoincreased by GA₃ pre-treatment. The stimulation of adventitiousrooting could be partially explained by the increase in shoot growth rate.Cultivar Charger responded better than the other cultivars to the lowest levelof GA₃ treatment. In vitro cultures of cultivarCharger were also treated with GA₃. However, the stimulation ofadventitious rooting was less marked than in the GA₃-treated stockplants: percentage rooting increased from 70% to 85%. The results are discussedin the context of 'rejuvenation' effects of GA₃.     

Do Movement Patterns Differ Between Laboratory and Field Suction Feeding Behaviors in a Mexican Cichlid?

Synopsis We analyzed feeding behavior of individuals of Herichthys minckleyi, the Cuatro Ciénegas cichlid, under laboratory conditions and freely behaving in their natural environment using high-speed video imaging. In a multivariate analysis of suction feeding behaviors there was no clear grouping of feeding events based on the environment, which suggests that most of the variability in the data was unrelated to differences between lab and field behaviors. In fact, the variability within an environment was far greater than the variability between the two environments. These results suggest that laboratory studies can accurately describe the kinematics of behaviors seen in the field. However, although lab based studies can quantify behaviors seen in the field, natural habitats are complex and provide individuals with the opportunity to exploit a wide range of food types and microhabitats, which may elicit behaviors not observed in the laboratory. However, feeding behaviors observed in the lab are representative of frequently used feeding behaviors in the field, at least for this species. Thus, we suggest that laboratory studies of feeding behavior, particularly those that test biomechanical or performance-based hypotheses can be extrapolated to natural environments.     

Clinical relevance of TLR2, TLR4, CD14 and FcgammaRIIA gene polymorphisms in Streptococcus pneumoniae infection

Streptococcus pneumoniae is the most common cause of community-acquired pneumonia and a major cause of morbidity and mortality throughout the world. It has been a major research priority to identify gene polymorphisms responsible for/associated with susceptibility and severity of S. pneumoniae infection to gain a better understanding of host genetic variants and their influence and clinical relevance to pneumococcal infections. In the present study, polymorphisms in several candidate genes, including TLR2-Arg/Gln753, TLR4-Asp/Gly299, TLR4-Thr/Ile399, CD14-159C/T and FcgammaRIIA-R/H131, were examined in 85 children with pneumococcal sepsis as an invasive pneumococcal disease and 409 healthy blood donors as controls. The prevalence of the TLR4-299/399 polymorphisms was significantly lower in the patient population than in controls (4 vs 11%; P<0.05; odds ratio (OR) 0.3; 95% confidence interval (CI) 0.1-1), while the prevalence of the CD14-159CC and FcgammaRIIA-R/R131 genotypes was significantly higher (35 vs 25%; P<0.05; OR 1.7; 95% CI 1-2.8 and 39 vs 21%; P<0.001; OR 2.5; 95% CI 1.4-4, respectively). Further, only 35% of patients carried either low-risk genotypes or protective genotypes in contrast to 61% of controls (P<0.0001; OR 2.8; 95% CI 1.7-4.6). We conclude that genetic variability in the TLR4, CD14 and FcgammaRIIA genes is associated with an increased risk of developing invasive disease in patients who are infected with S. pneumoniae.     

Pharmacological and pharmacokinetic characterization of 2-piperazine-alpha-isopropyl benzylamine derivatives as melanocortin-4 receptor antagonists

A series of 2-piperazine-alpha-isopropylbenzylamine derivatives were synthesized and characterized as melanocortin-4 receptor (MC4R) antagonists. Attaching an amino acid to benzylamines 7 significantly increased their binding affinity, and the resulting compounds 8-12 bound selectively to MC4R over other melanocortin receptor subtypes and behaved as functional antagonists. These compounds were also studied for their permeability using Caco-2 cell monolayers and metabolic stability in human liver microsomes. Most compounds exhibited low permeability and high efflux ratio possibly due to their high molecular weights. They also showed moderate metabolic stability which might be associated with their moderate to high lipophilicity. Pharmacokinetic properties of these MC4R antagonists, including brain penetration, were studied in mice after oral and intravenous administrations. Two compounds identified to possess high binding affinity and selectivity, 10d and 11d, were studied in a murine cachexia model. After intraperitoneal (ip) administration of 1mg/kg dose, mice treated with 10d had significantly more food intake and weight gain than the control animals, demonstrating efficacy by blocking the MC4 receptor. Similar in vivo effects were also observed when 11d was dosed orally at 20mg/kg. These results provide further evidence that a potent and selective MC4R antagonist has potential in the treatment of cancer cachexia.     

Tetrahydrobiopterin scavenges superoxide in dopaminergic neurons.

Increased oxidative stresses are implicated in the pathogenesis of Parkinson's disease, and dopaminergic neurons may be intrinsically susceptible to oxidative damage. However, the selective presence of tetrahydrobiopterin (BH(4)) makes dopaminergic neurons more resistant to oxidative stress caused by glutathione depletion. To further investigate the mechanisms of BH(4) protection, we examined the effects of BH(4) on superoxide levels in individual living mesencephalic neurons. Dopaminergic neurons have intrinsically lower levels of superoxide than nondopaminergic neurons. In addition, inhibiting BH(4) synthesis increased superoxide in dopaminergic neurons, while BH(4) supplementation decreased superoxide in nondopaminergic cells. BH(4) is also a cofactor in catecholamine and NO production. In order to exclude the possibility that the antioxidant effects of BH(4) are mediated by dopamine and NO, we used fibroblasts in which neither catecholamine nor NO production occurs. In fibroblasts, BH(4) decreased baseline reactive oxygen species, and attenuated reactive oxygen species increase by rotenone and antimycin A. Physiologic concentrations of BH(4) directly scavenged superoxide generated by potassium superoxide in vitro. We hypothesize that BH(4) protects dopaminergic neurons from ordinary oxidative stresses generated by dopamine and its metabolites and that environmental insults or genetic defects may disrupt this intrinsic capacity of dopaminergic neurons and contribute to their degeneration in Parkinson's disease.     

Whole chromosome loss and associated breakage–fusion–bridge cycles transform mouse tetraploid cells

Whole chromosome gains or losses (aneuploidy) are a hallmark of ~70% of human tumors. Modeling the consequences of aneuploidy has relied on perturbing spindle assembly checkpoint (SAC) components, but interpretations of these experiments are clouded by the multiple functions of these proteins. Here, we used a Cre recombinase‐mediated chromosome loss strategy to individually delete mouse chromosomes 9, 10, 12, or 14 in tetraploid immortalized murine embryonic fibroblasts. This methodology also involves the generation of a dicentric chromosome intermediate, which subsequently undergoes a series of breakage–fusion–bridge (BFB) cycles. While the aneuploid cells generally display a growth disadvantage in vitro, they grow significantly better in low adherence sphere‐forming conditions and three of the four lines are transformed in vivo, forming large and invasive tumors in immunocompromised mice. The aneuploid cells display increased chromosomal instability and DNA damage, a mutator phenotype associated with tumorigenesis in vivo. Thus, these studies demonstrate a causative role for whole chromosome loss and the associated BFB‐mediated instability in tumorigenesis and may shed light on the early consequences of aneuploidy in mammalian cells.     

Fixation and Spread of Somatic Mutations in Adult Human Colonic Epithelium

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