Membrane targeting of p21-activated kinase 1 (PAK1) induces neurite outgrowth from PC12 cells.

Rho-family GTPases regulate cytoskeletal dynamics in various cell types. p21-activated kinase 1 (PAK1) is one of the downstream effectors of Rac and Cdc42 which has been implicated as a mediator of polarized cytoskeletal changes in fibroblasts. We show here that the extension of neurites induced by nerve growth factor (NGF) in the neuronal cell line PC12 is inhibited by dominant-negative Rac2 and Cdc42, indicating that these GTPases are required components of the NGF signaling pathway. While cytoplasmically expressed PAK1 constructs do not cause efficient neurite outgrowth from PC12 cells, targeting of these constructs to the plasma membrane via a C-terminal isoprenylation sequence induced PC12 cells to extend neurites similar to those stimulated by NGF. This effect was independent of PAK1 ser/thr kinase activity but was dependent on structural domains within both the N- and C-terminal portions of the molecule. Using these regions of PAK1 as dominant-negative inhibitors, we were able to effectively inhibit normal neurite outgrowth stimulated by NGF. Taken together with the requirement for Rac and Cdc42 in neurite outgrowth, these data suggest that PAK(s) may be acting downstream of these GTPases in a signaling system which drives polarized outgrowth of the actin cytoskeleton in the developing neurite.     

The effect of temperature on adrenergic receptors of alveolar type II cells of a heterothermic marsupial

Fat-tailed dunnarts, Sminthopsis crassicaudata, survive dramatic changes in body temperature during torpor without experiencing surfactant dysfunction. Adrenergic factors regulate surfactant secretion through beta(2)-adrenergic receptors on alveolar type II cells. Temperature has no effect on the secretory response of dunnart type II cells to adrenergic stimulation. We hypothesise that during torpor, dunnart type II cells up-regulate the number of adrenergic receptors present on type II cells to enable stimulation at lower concentrations of agonist. Here, we isolated type II cells from warm-active (35 degrees C) and torpid (15 degrees C) dunnarts and examined the effects of an in vitro temperature change on the number and activity of adrenergic receptors. Torpor did not affect the beta-adrenergic receptor number. However, we observed a significant decrease in adrenergic receptor number when cells from warm-active animals were incubated at 15 degrees C and when cells from torpid animals were incubated at 37 degrees C. cAMP production was significantly higher in type II cells from torpid dunnarts than warm-active dunnarts and this may contribute, in part, to the temperature insensitivity we have previously observed in the adrenergic regulation of surfactant secretion.     

Combinatorial materials research applied to the development of new surface coatings IV. A high-throughput bacterial biofilm retention and retraction assay for screening fouling-release performance of coatings

Abstract

A high-throughput bacterial biofilm retention screening method has been augmented to facilitate the rapid analysis and down-selection of fouling-release coatings for identification of promising candidates. Coatings were cast in modified 24-well tissue culture plates and inoculated with the marine bacterium Cytophaga lytica for attachment and biofilm growth. Biofilms retained after rinsing with deionised water were dried at ambient laboratory conditions. During the drying process, retained biofilms retracted through a surface de-wetting phenomenon on the hydrophobic silicone surfaces. The retracted biofilms were stained with crystal violet, imaged, and analysed for percentage coverage. Two sets of experimental fouling-release coatings were analysed with the high-throughput biofilm retention and retraction assay (HTBRRA). The first set consisted of a series of model polysiloxane coatings that were systematically varied with respect to ratios of low and high MW silanol-terminated PDMS, level of cross-linker, and amount of silicone oil. The second set consisted of cross-linked PDMS-polyurethane coatings varied with respect to the MW of the PDMS and end group functionality. For the model polysiloxane coatings, HTBRRA results were compared to data obtained from field immersion testing at the Indian River Lagoon at the Florida Institute of Technology. The percentage coverage calculations of retracted biofilms correlated well to barnacle adhesion strength in the field (R² = 0.82) and accurately identified the best and poorest performing coating compositions. For the cross-linked PDMS-polyurethane coatings, the HTBRRA results were compared to combinatorial pseudobarnacle pull-off adhesion data and good agreement in performance was observed. Details of the developed assay and its implications in the rapid discovery of new fouling-release coatings are discussed.     

Lowe Syndrome protein OCRL1 supports maturation of polarized epithelial cells

Mutations in the inositol polyphosphate 5-phosphatase OCRL1 cause Lowe Syndrome, leading to cataracts, mental retardation and renal failure. We noted that cell types affected in Lowe Syndrome are highly polarized, and therefore we studied OCRL1 in epithelial cells as they mature from isolated individual cells into polarized sheets and cysts with extensive communication between neighbouring cells. We show that a proportion of OCRL1 targets intercellular junctions at the early stages of their formation, co-localizing both with adherens junctional components and with tight junctional components. Correlating with this distribution, OCRL1 forms complexes with junctional components α-catenin and zonula occludens (ZO)-1/2/3. Depletion of OCRL1 in epithelial cells growing as a sheet inhibits maturation; cells remain flat, fail to polarize apical markers and also show reduced proliferation. The effect on shape is reverted by re-expressed OCRL1 and requires the 5'-phosphatase domain, indicating that down-regulation of 5-phosphorylated inositides is necessary for epithelial development. The effect of OCRL1 in epithelial maturation is seen more strongly in 3-dimensional cultures, where epithelial cells lacking OCRL1 not only fail to form a central lumen, but also do not have the correct intracellular distribution of ZO-1, suggesting that OCRL1 functions early in the maturation of intercellular junctions when cells grow as cysts. A role of OCRL1 in junctions of polarized cells may explain the pattern of organs affected in Lowe Syndrome.     

The distribution of restriction enzyme sites in Escherichia coli.

A statistical analysis of physical map data for eight restriction enzymes covering nearly the entire genome of E. coli is presented. The methods of analysis are based on a top-down modeling approach which requires no knowledge of the statistical properties of the base sequence. For most enzymes, the distribution of mapped sites is found to be fairly homogeneous. Some heterogeneity in the distribution of sites is observed for the enzymes Pstl and HindIII. In addition, BamHI sites are found to be more evenly dispersed than we would expect for random placement and we speculate on a possible mechanism. A consistent departure from a uniform distribution, observed for each of the eight enzymes, is found to be due to a lack of closely spaced sites. We conclude from our analysis that this departure can be accounted for by deficiencies in the physical map data rather than non-random placement of actual restriction sites. Estimates of the numbers of sites missing from the map are given, based both on the map data itself and on the site frequencies in a sample of sequenced E. coli DNA. We conclude that 5 to 15% of the mapped sites represent multiple sites in the DNA sequence.     

Enhanced Production of Chikungunya Virus-Like Particles Using a High-pH Adapted Spodoptera frugiperda Insect Cell Line

Chikungunya virus-like particles (VLPs) have potential to be used as a prophylactic vaccine based on testing in multiple animal models and are currently being evaluated for human use in a Phase I clinical trial. The current method for producing these enveloped alphavirus VLPs by transient gene expression in mammalian cells presents challenges for scalable and robust industrial manufacturing, so the insect cell baculovirus expression vector system was evaluated as an alternative expression technology. Subsequent to recombinant baculovirus infection of Sf21 cells in standard culture media (pH 6.2–6.4), properly processed Chikungunya structural proteins were detected and assembled capsids were observed. However, an increase in culture pH to 6.6–6.8 was necessary to produce detectable concentrations of assembled VLPs. Since this elevated production pH exceeds the optimum for growth medium stability and Sf21 culture, medium modifications were made and a novel insect cell variant (SfBasic) was derived by exposure of Sf21 to elevated culture pH for a prolonged period of time. The high-pH adapted SfBasic insect cell line described herein is capable of maintaining normal cell growth into the typical mammalian cell culture pH range of 7.0–7.2 and produces 11-fold higher Chikungunya VLP yields relative to the parental Sf21 cell line. After scale-up into stirred tank bioreactors, SfBasic derived VLPs were chromatographically purified and shown to be similar in size and structure to a VLP standard derived from transient gene expression in HEK293 cells. Total serum anti-Chikungunya IgG and neutralizing titers from guinea pigs vaccinated with SfBasic derived VLPs or HEK293 derived VLPs were not significantly different with respect to production method, suggesting that this adapted insect cell line and production process could be useful for manufacturing Chikungunya VLPs for use as a vaccine. The adaptation of Sf21 to produce high levels of recombinant protein and VLPs in an elevated pH range may also have applications for other pH-sensitive protein or VLP targets.     

hobo transposable elements in Drosophila melanogaster and D. simulans.

Genomic patterns of occurrence of the transposable element hobo are polymorphic in the sibling species Drosophila melanogaster and D. simulans. Most tested strains of both species have apparently complete (3.0 kb) and smaller hobo elements (H lines), but in both species some strains completely lack such canonical hobo elements (E lines). The occurrence of H and E lines in D. simulans as well as in D. melanogaster implies that an hypothesis of recent introduction in the latter species is inadequate to explain the phylogenetic occurrence of hobo. Particular internally deleted elements, the approximately 1.5 kb Th1 and Th2 elements, are abundant in many lines of D. melanogaster, and an analogous 1.1 kb internally deleted element, h del sim, is abundant in most lines of D. simulans. Besides the canonical hobo sequences, both species (and their sibling species D. sechellia and D. mauritiana) have many hobo-hybridizing sequences per genome that do not appear to be closely related to the canonical hobo sequence.     

GL261 glioma tumor cells respond to ATP with an intracellular calcium rise and glutamate release

Necrotizing enterocolitis (NEC) is one of the most severe and unpredictable complications of prematurity. There are two possible mechanisms involved in the pathogenesis of NEC: individual inflammatory response and impaired blood flow in mesenteric vessels with secondary ischemia of the intestine. The aim of this study was to evaluate the possible relationship between polymorphisms: Il-1β 3953C>T, Il-6 ?174G>C and ?596G>A, TNFα ?308G>A, and 86 bp variable number tandem repeat polymorphism of interleukin-1 receptor antagonist (Il-1RN VNTR 86 bp) and three polymorphisms that may participate in arteries tension regulation and in consequence in intestine blood flow impairment: eNOS (894G>T and ?786T>C) and END-1 (5665G>T) and NEC in 100 infants born from singleton pregnancy, before 32 + 0 weeks of gestation, exposed to antenatal steroids therapy, and without congenital abnormalities. In study population, 22 (22%) newborns developed NEC. Surgery-requiring NEC was present in 7 children. Statistical analysis showed 20-fold higher prevalence of NEC in infants with the genotype TT [OR 20 (3.71–208.7); p = 0.0004] of eNOS 894G>T gene polymorphism. There was a higher prevalence of allele C carriers of eNOS 786T>C in patients with surgery-requiring NEC [OR 4.881 (1.33–21.99); p = 0.013]. Our investigation did not confirm any significant prevalence for NEC development in another studied genotypes/alleles. This study confirms the significant role of polymorphisms that play role in intestine blood flow. Identifying gene variants that increase the risk for NEC development may be useful in screening infants with inherent vulnerability and creating strategies for individualized care.