Strategies to Improve the Surface Plasmon Resonance-Based Immmunodetection of Bacterial Cells

We have made a comparison of (a) different surface chemistries of surface plasmon resonance (SPR) sensor chips (such as carboxymethylated dextran and carboxymethylated C1) and (b) of different assay formats (direct, sandwich and subtractive immunoassay) in order to improve the sensitivity of the determination of the model bacteria Acidovorax avenae subsp. citrulli (Aac). The use of the carboxymethylated sensor chip C1 resulted in a better sensitivity than that of carboxymethylated dextran CM5 in all the assay formats. The direct assay format, in turn, exhibits the best sensitivity. Thus, the combination of a carboxymethylated sensor chip C1 with the direct assay format resulted in the highest sensitivity for Aac, with a limit of detection of 1.6x106 CFU mL-1. This SPR immunosensor was applied to the detection of Aac in watermelon leaf extracts spiked with the bacteria, and the lower LOD is 2.2x107 CFU mL-1.

Growth, dye degradation and ligninolytic activity studies on Zimbabwean white rot fungi

White rot fungi were collected from Chirinda and Chimanimani hardwood forests in Zimbabwe and studied with respect to growth temperature optima and dye decolorization. Temperature optima were found to vary (between 25-37 degreesC) amongst the isolates. The isolates were screened for their ability to degrade the polymeric dyes; blue dextran and Poly R478 and the triphenylmethane dyes; cresol red, crystal violet and bromophenol blue. Semi-quantitative determination of the hydrolytic enzyme activities possessed by the white rot fungi was determined using the API ZYM system. Lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase activities in the fungi were also determined. No LiP was detected in any of the isolates but all isolates showed manganese peroxidase and laccase activities. Time related decolorization studies and optimum pH determinations for Poly R478 degradation by the isolates were carried out in liquid cultures. The most significant rates of Poly R478 decolorization in liquid cultures were found with the following isolates: Trametes cingulata, Trametes versicolor, Trametes pocas, DSPM95 (a species to be identified), Datronia concentrica and Pyenoporus sanguineus. (C) 2001 Elsevier Science Inc. All rights reserved.

Role of vascular endothelial growth factor and placental growth factors during retinal vascular development and hyaloid regression

PURPOSE. Vascular endothelial growth factor (VEGF)-A and placental growth factor (PIGF) are members of a large group of homologous peptides identified as the VEGF family. Although VEGF-A is known to act as a potent angiogenic peptide in the retina, the vasoactive function of PIGF in this tissue is less well defined. This study has sought to elucidate the expression patterns and modulatory role of these growth factors during retinal vascular development and hyaloid regression in the neonatal mouse. METHODS. C57BL6J mice were killed at postnatal days (P)1, P3, P5, P7, P9, and P11. The eyes were enucleated and processed for in situ hybridization and immunocytochemistry and the retinas extracted for total protein or RNA. Separate groups of neonatal mice were also injected intraperitoneally daily from P2 through P9 with either VEGF-neutralizing antibody, PIGF-neutralizing antibody, isotype immunoglobulin (Ig)-G, or phosphate-buffered saline (PBS). The mice were then perfused with fluorescein isothiocyanate (FITC)-dextran, and the eyes were subsequently embedded in paraffin wax or flat mounted. RESULTS. Quantitative (real-time) reverse transcription-polymerase chain reaction (RT-PCR) demonstrated similar expression patterns of VEGF-A and PIGF mRNA during neonatal retinal development, although the fluctuation between time periods was greater overall for VEGF-A. The localization of VEGF-A and PIGF in the retina, as revealed by in situ hybridization and immunohistochemistry, was also similar. Neutralization of VEGF-A caused a significant reduction in the hyaloid and retinal vasculature, whereas PIGF antibody treatment caused a marked persistence of the hyaloid without significantly affecting retinal vascular development. CONCLUSIONS. Although having similar expression patterns in the retina, these growth factors appear to have distinct modulatory influences during normal retinal vascular development and hyaloid regression.

Synergistic effects on gene delivery - co-formulation of small disulfide-linked dendritic polycations with Lipofectamine 2000 (TM)

This paper describes the application of gene delivery vectors based on connecting together two well-defined low-generation poly(L-lysine) (PLL) dendrons using a disulfide-containing linker unit. We report that the transfection ability of these vectors in their own right is relatively low, because the low-generation number limits the endosomal buffering capacity. Importantly, however, we demonstrate that when applied in combination with Lipofectamine 2000 (TM), a vector from the cationic lipid family, these small cationic additives significantly enhance the levels of gene delivery (up to four-fold). Notably, the cationic additives have no effect on the levels of transfection observed with a cationic polymer, such as DEAE dextran. We therefore argue that the synergistic effects observed with Lipofectamine 2000 (TM) arise as a result of combining the delivery advantages of two different classes of vector within a single formulation, with our dendritic additives providing a degree of pH buffering within the endosome. As such, the data we present indicate that small dendritic structures, although previously largely overlooked for gene delivery owing to their inability to transfect in their own right, may actually be useful well-defined additives to well-established vector systems in order to enhance the gene delivery payload.