Site-specific de-N-glycosylation of CD44 can activate hyaluronan binding, and CD44 activation states show distinct threshold densities for hyaluronan binding

CD44 is a cell surface receptor for the glycosaminoglycan hyaluronan (HA). Not all CD44-positive cells bind HA, and binding ability is strictly regulated. Three different HA binding states have been defined: inactive, inducible (by certain CD44-specific monoclonal antibodies), and constitutively active. The observation that sets of genetically related cell lines representing different HA binding states showed correlated differences in N-glycosylation of CD44, and that inhibition of N-glycosylation enhanced HA binding (Lesley et al., J. Exp. Med., 182: 431-437, 1995) led us to examine directly whether specific N-glycosylation site modifications were involved in regulating the HA binding function. CD44-negative, -active, and inducible cell lines were stably transfected with mutant constructs in which each of the five N-glycosylation sites of murine CD44 had been separately inactivated. Ability to bind soluble HA was examined over a range of CD44 expression levels. For the active cell line, AKR1, transfectants for all N-glycosylation mutants bound HA as well as did transfectants for wild type CD44. No inhibitory effects of inactivating specific N-glycosylation sites were observed. HA binding was activated when two of the mutant constructs were transfected into a novel CD44-negative inducible cell line. Inactivation of N-glycosylation sites at residues 25 or 120 converted the inducible cell line to constitutively active, whereas inactivation of other sites had little or no effect. Fusion proteins secreted from inactive, inducible, or active cell lines were purified, bound to beads, and assayed for HA binding activity by flow cytometric analysis. Fusion proteins derived from inactive, inducible, and constitutively active cells exhibited three distinguishable "threshold" densities required for HA binding ability. The results imply that the CD44 molecules produced in cells in these three activation states have intrinsic differences in HA binding function. Treatment of the fusion proteins with neuraminidase altered the HA binding state, and glycosylation mutations that affected the phenotype of the inducible cell line lowered the threshold required for HA binding of CD44-immunoglobulin fusion proteins derived from the inducible cell line. Thus, alterations of glycosylation of CD44 itself can affect HA binding ability as manifested by a change in HA binding state.     

Nightly Home Hemodialysis: Fifteen Months of Experience in Lynchburg,Virginia

What constitutes adequate dialysis has been debated in the nephrology literature over the past eight years. The mortality rate of patients on dialysis in the United States is about 20% per year. We believed that short and infrequent dialysis sessions contributed to poor outcomes. To improve the results, Lynchburg Nephrology started the nightly home hemodialysis (NHHD) program in September 1997. Ten patients were trained in the first 15 months of the program. Patients dialyzed 7 – 9 hours, 6 nights/week, using the Fresenius 2008H machine. A standard dialysis solution with 2.0 mEq/L potassium, calcium concentration of 3.0 – 3.5 mEq/L was used. Dialysis solution flow rates were 200 – 300 mL/min. Serum phosphate levels were maintained above 2.5 mg/dL by adding 0 – 45 mL Fleet's Phosphosoda to the bicarbonate bath. Patients had marked improvement in quality of life as measured with the SF-36. Blood pressure was better controlled with fewer medications. All phosphate binders were eliminated. Caloric intake and protein intake increased to normal levels as measured by three-day dietary histories pre-NHHD, and at 3, 6, and 12 months on NHHD. Epoetin alfa dosages were reduced by about 50%. Nightly home hemodialysis should be considered as a valuable modality option for end-stage renal disease patients; it is potentially superior to conventional thrice-weekly hemodialysis.     

Optimization of electrically conductive films: Poly(3-methylthiophene) or polypyrrole in Kapton

A method to generate conductive films composed of small amounts of conductive polymer absorbed into the surface of polyimide films has been optimized. Both pyrrole (PY) and 3-methylthiophene (3MT) were evaluated as precursors for the conductive phase. Predictive models were empirically derived for each precursor to describe the effects of polymerization variables on the conductivity of the films. The variables studied were found to be highly synergistic. An optimum set of conditions was found for each conductive polymer that produces the highest conductivity. Using p-3MT as the conductive phase, films with conductivity as high as 5.7 Ω⁻¹ cm⁻¹ can be produced, an improvement of four orders of magnitude over previously reported results with Kapton as a base polymer. The highest conductivity achieved using p-PY as the conductive phase was 0.041 Ω⁻¹ cm⁻¹, still a two order of magnitude improvement over previously reported results. Mean mechanical properties of the 3MT-treated films were not significantly lower than that for untreated Kapton. The conductivities of p-3MT/Kapton films tested over time under ambient temperature in air persist fairly well for 300 days. © 1997 John Wiley & Sons, Inc. J Appl Polym Sci 63: 821–834, 1997     

Investigating the effects of reduced size on the properties of ferroelectrics

A series of experiments has been undertaken to understand more about the fundamental origin of the thickness-induced permittivity collapse often observed in conventional thin film ferroelectric heterostructures. The various experiments are discussed, highlighting the eventual need to examine permittivity collapse in thin film single crystal material. It has been seen that dielectric collapse is not a direct consequence of reduced size, and neither is it a consequence of unavoidable physics associated with the ferroelectric-electrode boundary. Research on three-dimensional shape-constrained ferroelectrics, emphasizing self-assembled structures based on nanoporous alumina templates and on FIB-milled single crystals, is also presented, and appears to represent an exciting area for ongoing research.     

Controlled immobilisation of active enzymes on the cowpea mosaic virus capsid

Immobilisation of horseradish peroxidase (HRP) and glucose oxidase (GOX) via covalent attachment of modified enzyme carbohydrate to the exterior of the cowpea mosaic virus (CPMV) capsid gave high retention of enzymatic activity. The number of enzymes bound per virus was determined to be about eleven for HRP and 2-3 for GOX. This illustrates that relatively large biomacromolecules can be readily coupled to the virus surface using simple conjugation strategies. Virus-biomacromolecule hybrids have great potential for uses in catalysis, diagnostic assays or biosensors.     

An improved method for specific-target preamplification PCR analysis of single blastocysts useful for embryo sexing and high-throughput gene expression analysis

Gene expression analysis in preimplantation embryos has been used for answering fundamental questions related to development, prediction of pregnancy outcome, and other topics. Limited amounts of mRNA in preimplantation embryos hinders progress in studying the preimplantation embryo. Here, a method was developed involving direct synthesis and specific-target preamplification (STA) of cDNA for gene expression analysis in single blastocysts. Effective cell lysis and genomic DNA removal steps were incorporated into the method. In addition, conditions for real-time PCR of cDNA generated from these processes were improved. By using this system, reliable embryo sexing results based on expression of sex-chromosome linked genes was demonstrated. Calibration curve analysis of PCR results using the Fluidigm Biomark microfluidic platform (Fluidigm, South San Francisco, CA) was performed to evaluate 96 STA cDNA from single blastocysts. In total, 93.75% of the genes were validated. Robust amplification was detected even when STA cDNA from a single blastocyst was diluted 1,024-fold. Further analysis showed that within-assay variation increased when cycle threshold values exceeded 18. Overall, STA quantitative real-time PCR analysis was shown to be useful for analysis of gene expression of multiple specific targets in single blastocysts.     

Responses of Herbivore and Predatory Mites to Tomato Plants Exposed to Jasmonic Acid Seed Treatment

Jasmonic acid (JA) signalling can influence plant defense and the production of plant volatiles that mediate interactions with insects. Here, we tested whether a JA seed treatment could alter direct and indirect defenses. First, oviposition levels of herbivorous mites, Tetranychus urticae, on JA seed-treated and control tomato plants were compared. They were not significantly different on tomato cv. ‘Moneymaker’, however, there was a significant reduction in oviposition on treated plants in additional experiments with cv. ‘Carousel’. Second, responses of predatory mites, Phytoseiulus persimilis, were assessed in a Y-tube olfactometer. Volatiles from JA seed-treated tomato cv. ‘Moneymaker’ plants were significantly more attractive than volatiles from control plants. Volatiles collected from plants were analysed by GC/MS, and samples from JA seed-treated plants contained more methyl salicylate and (E,E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene (TMTT) than samples from control plants. Our results indicate that JA seed treatment can make tomato plants more attractive to predatory mites, but that direct effects on herbivorous mites are variable and cultivar dependent.     

The Use of Accurate Pore Pressure Monitoring for Risk Reduction in Tailings Dams

Mine Water and the Environment - Simply monitoring movement of the tailings dam wall does not address the cause of tailings dam failures and will therefore never be an effective method to reduce or...