Incorporation of glycosylphosphatidylinositol-anchored granulocyte- macrophage colony-stimulating factor or CD40 ligand enhances immunogenicity of chimeric simian immunodeficiency virus-like particles

The rapid worldwide spread of human immunodeficiency virus (HIV) mandates the development of successful vaccination strategies. Since live attenuated HIV is not accepted as a vaccine due to safety concerns, virus-like particles (VLPs) offer an attractive safe alternative because they lack the viral genome yet they are perceived by the immune system as a virus particle. We hypothesized that adding immunostimulatory signals to VLPs would enhance their efficacy. To accomplish this we generated chimeric simian immunodeficiency virus (SIV) VLPs containing either glycosylphosphatidylinositol (GPI)-anchored granulocyte-macrophage colony-stimulating factor (GM-CSF) or CD40 ligand (CD40L) and investigated their biological activity and ability to enhance immune responses in vivo. Immunization of mice with chimeric SIV VLPs containing GM-CSF induced SIV Env-specific antibodies as well as neutralizing activity at significantly higher levels than those induced by standard SIV VLPs, SIV VLPs containing CD40L, or standard VLPs mixed with soluble GM-CSF. In addition, mice immunized with chimeric SIV VLPs containing either GM-CSF or CD40L showed significantly increased CD4(+)- and CD8(+)-T-cell responses to SIV Env, compared to standard SIV VLPs. Taken together, these results demonstrate that the incorporation of immunostimulatory molecules enhances humoral and cellular immune responses. We propose that anchoring immunostimulatory molecules into SIV VLPs can be a promising approach to augmenting the efficacy of VLP antigens.     

Imaging protein molecules using FRET and FLIM microscopy

F?rster (or fluorescence) resonance energy transfer (FRET) and fluorescence lifetime imaging (FLIM) have moved center stage and are increasingly forming part of multifaceted imaging approaches. They are complementary methodologies that can be applied to advanced quantitative analyses. The widening application of FRET and FLIM has been driven by the availability of suitable fluorophores, increasingly sophisticated microscopy systems, methodologies to correct spectral bleed-through, and the ease with which FRET can be combined with other techniques. FRET and FLIM have recently found use in several applications: in the analysis of protein-protein interactions with high spatial and temporal specificity (e.g. clustering), in the study of conformational changes, in the analysis of binding sequences, and in applications such as high-throughput screening.     

Fluorescence resonance energy transfer (FRET) microscopy imaging of live cell protein localizations

The current advances in fluorescence microscopy, coupled with the development of new fluorescent probes, make fluorescence resonance energy transfer (FRET) a powerful technique for studying molecular interactions inside living cells with improved spatial (angstrom) and temporal (nanosecond) resolution, distance range, and sensitivity and a broader range of biological applications.     

Electrocatalysis and simultaneous determination of catechol and quinol by poly(malachite green) coated multiwalled carbon nanotube film

Electrochemically active composite film that contains multiwalled carbon nanotubes (MWCNTs), Nafion (NF), and poly(malachite green) (PMG) has been synthesized on glassy carbon electrode (GCE), gold, and indium tin oxide (ITO) electrodes by potentiodynamic method. The presence of MWCNTs in the composite film (MWCNT–NF–PMG) enhances the surface coverage concentration (Γ) of PMG by fivefold. Similarly, an electrochemical quartz crystal microbalance study revealed enhancement in the deposition of PMG at MWCNT–NF film when compared with bare and only NF modified electrodes. The surface morphology of the composite film was studied using atomic force microscopy, which revealed that the PMG incorporated on MWCNT–NF film. The composite film exhibited enhanced electrocatalytic activity toward the mixture of biochemical compounds catechol and quinol. The electrocatalytic responses of analytes at MWCNT–NF–PMG composite film were measured using both cyclic voltammetry (CV) and differential pulse voltammetry (DPV). From electrocatalysis studies, well-separated voltammetric peaks were obtained at the composite film for catechol and quinol with a peak separation of 147 mV. The sensitivity values of the composite film toward catechol and quinol by the DPV technique were 0.4 and 3.2 mA mM⁻¹ cm⁻², respectively, which are higher than the values obtained by the CV technique. Similarly, the above-mentioned values are better than the previously reported electroanalytical values for the same analytes.     

Co-expression of multiple myosin heavy chain genes, in addition to a tissue-specific one, in extraocular musculature

We have investigated the developmental transitions of myosin heavy chain (MHC) gene expression in the rat extraocular musculature (EOM) at the mRNA level using S1-nuclease mapping techniques and at the protein level by polypeptide mapping and immunochemistry. We have isolated a genomic clone, designated lambda 10B3, corresponding to an MHC gene which is expressed in the EOM fibers (recti and oblique muscles) of the adult rat but not in hind limb muscles. Using cDNA and genomic probes for MHC genes expressed in skeletal (embryonic, neonatal, fast oxidative, fast glycolytic, and slow/cardiac beta-MHC), cardiac (alpha-MHC), and EOM (lambda 10B3) muscles, we demonstrate the concomitant expression at the mRNA level of at least six different MHC genes in adult EOM. Protein and immunochemical analyses confirm the presence of at least four different MHC types in EOM. Immunocytochemistry demonstrates that different myosin isozymes tend to segregate into individual myofibers, although some fibers seem to contain more than one MHC type. The results also show that the EOM fibers exhibit multiple patterns of MHC gene regulation. One set of fibers undergoes a sequence of isoform transitions similar to the one described for limb skeletal muscles, whereas other EOM myofiber populations arrest the MHC transition at the embryonic, neonatal/adult, or adult EOM-specific stage. Thus, the MHC gene family is not under the control of a strict developmental clock, but the individual genes can modify their expression by tissue-specific and/or environmental factors.     

Versatile catechol dioxygenases in <Emphasis Type="Italic">Sphingobium scionense</Emphasis> WP01<Superscript>T</Superscript>

The objective was to understand the roles of multiple catechol dioxygenases in the type strain Sphingobium scionense WP01^T (Liang and Lloyd-Jones in Int J Syst Evol Microbiol 60:413–416, 2010a) that was isolated from severely contaminated sawmill soil. The dioxygenases were identified by sequencing, examined by determining the substrate specificities of the recombinant enzymes, and by quantifying gene expression following exposure to model priority pollutants. Catechol dioxygenase genes encoding an extradiol xylE and two intradiol dioxygenases catA and clcA that are highly similar to sequences described in other sphingomonads are described in S. scionense WP01^T. The distinct substrate specificities determined for the recombinant enzymes confirm the annotated gene functions and suggest different catabolic roles for each enzyme. The role of the three enzymes was evaluated by analysis of enzyme activity in crude cell extracts from cells grown on meta-toluate, benzoate, biphenyl, naphthalene and phenanthrene which revealed the co-induction of each enzyme by different substrates. This was corroborated by quantifying gene expression when cells were induced by biphenyl, naphthalene and pentachlorophenol. It is concluded that the ClcA and XylE enzymes are recruited in pathways that are involved in the degradation of chlorinated aromatic compounds such as pentachlorophenol, the XylE and ClcA enzymes will also play a role in degradation pathways that produce alkylcatechols, while the three enzymes ClcA, XylE and CatA will be simultaneously involved in pathways that generate catechol as a degradation pathway intermediate.     

Mapping fluorophore distributions in three dimensions by quantitative multiple angle-total internal reflection fluorescence microscopy.

The decay of evanescent field intensity beyond a dielectric interface depends upon beam incident angle, enabling the 3-d distribution of fluorophores to be deduced from total internal reflection fluorescence microscopy (TIRFM) images obtained at multiple incident angles. Instrumentation was constructed for computer-automated multiple angle-TIRFM (MA-TIRFM) using a right angle F2 glass prism (n(r) 1.632) to create the dielectric interface. A laser beam (488 nm) was attenuated by an acoustooptic modulator and directed onto a specified spot on the prism surface. Beam incident angle was set using three microstepper motors controlling two rotatable mirrors and a rotatable optical flat. TIRFM images were acquired by a cooled CCD camera in approximately 0.5 degree steps for >15 incident angles starting from the critical angle. For cell studies, cells were grown directly on the glass prisms (without refractive index-matching fluid) and positioned in the optical path. Images of the samples were acquired at multiple angles, and corrected for angle-dependent evanescent field intensity using "reference" images acquired with a fluorophore solution replacing the sample. A theory was developed to compute fluorophore z-distribution by inverse Laplace transform of angle-resolved intensity functions. The theory included analysis of multiple layers of different refractive index for cell studies, and the anisotropic emission from fluorophores near a dielectric interface. Instrument performance was validated by mapping the thickness of a film of dihexyloxacarbocyanine in DMSO/water (n(r) 1.463) between the F2 glass prism and a plano-convex silica lens (458 mm radius, n(r) 1.463); the MA-TIRFM map accurately reproduced the lens spherical surface. MA-TIRFM was used to compare with nanometer z-resolution the geometry of cell-substrate contact for BCECF-labeled 3T3 fibroblasts versus MDCK epithelial cells. These studies establish MA-TIRFM for measurement of submicroscopic distances between fluorescent probes and cell membranes.