Modified cell ELISA to determine the solubilization of cell surface proteins: Applications in GPI-anchored protein purification

A major step in purifying membrane bound proteins involves the solubilization of the protein of interest from the cell membranes. Glycosylphosphatidyl inositol (GPI)-anchored proteins pose a singular problem in this solubilization step since they are found in detergent-resistant membrane complexes and accordingly are insoluble in cold Triton X-100. In this study we have developed a modified cell ELISA that determines the solubility of these cell surface proteins under various solubilization conditions. Using this non-radioactive method we show that the combination of saponin/Triton X-100 at 4 degrees C solubilized GPI-anchored proteins more efficiently than Triton X-100 at 4 degrees C. The combination of saponin/Triton X-100 at 4 degrees C avoids the potential of activating proteases that occurs when using Triton X-100 at 37 degrees C. Furthermore, our method also shows the saponin/Triton X-100 solubilized GPI-anchored proteins equivalent to the more expensive octyl beta-glucoside. This is a particularly important consideration in large-scale protein purification. This method obviates the need to use radioactivity, gel electrophoresis and immunoblotting procedures. The solubilization conditions determined by this modified ELISA are readily translated to the practical application of large-scale protein purification as demonstrated in the purification of two different recombinant GPI-anchored proteins, GPI-hB7-1 (CD80) and GPI-mICAM-1 (CD54).     

Cloning and Characterization of the Crystal Protein-Encoding Gene of Bacillus thuringiensis subsp. yunnanensis

Molecular cloning and characterization of a novel cry gene, cry32Aa, of Bacillus thuringiensis subsp. yunnanensis was carried out. The Cry32Aa protein was predicted to have a molecular mass of 139.2 kDa and was found to have an unusual 42-amino-acid-long tail at the C terminus. The cry32Aa gene was localized on the 103-MDa plasmid of the organism. Bioassays showed no toxicity against several moths and mosquitoes. However, it exhibited weak toxicity against larvae of the diamondback moth, Plutella xylostella.     

Comparative analysis of agglutinins from hemolymph and albumin gland ofHelix pomatia

Summary The hemolymph ofHelix pomatia contains a weak agglutinating activity. This lectin concentration was calculated to be about 1.8 g·ml^–1. Among the different red blood cells tested, pronase-treated sheep erythrocytes were found to be the most suitable indicator cells. Their agglutination could be inhibited by GalNAc and GlcNAc. The serum agglutinin was isolated by affinity chromatography using Sephadex G-200 as the matrix. It exhibited a single band in discontinuous PAGE. In the presence of SDS, subunits of 27000 daltons were obtained which, after addition of 2-mercaptoethanol, partly dissociated into 13000-dalton subunits. The biochemical properties observed were compared with those of the well-known blood group A-specific lectin from the albumin gland ofH. pomatia.Abbreviations GalNac N-acetyl-galactosamine - GlcNAc N-acetyl-glucosamine - SDS sodium dodecylsulfate     

Characterization of cDNA and genomic sequences corresponding to an embryonic myosin heavy chain

We report here the isolation and characterization of cDNA and genomic sequences corresponding to a rat embryonic myosin heavy chain (MHC) protein. This gene, which is present as a single copy in the rat genome, comprises about 25 kilobase pairs of DNA and contains approximately 80% intronic sequences. The embryonic MHC gene belongs to a highly conserved multigene family, and exhibits a high degree of nucleotide and amino acid sequence conservation with other sarcomeric MHC genes from nematode to man. S1 nuclease mapping experiments using cDNA and genomic probes show that this MHC gene is transiently expressed during skeletal muscle development. Its mRNA is detected in fetal skeletal muscle during early development and persists up to 2 weeks after birth with the overlapping expression of neonatal and adult skeletal MHC mRNAs. However, this MHC is not expressed in the adult skeletal muscle with the exception of extraocular muscle fibers. The transient expression during muscle development of the isoform produced by this gene and its sequential replacement by other MHCs raises interesting questions about the mechanism controlling MHC isozyme transitions and the physiological significance of the individual MHCs in muscle fibers.     

In Vivo Immune Evasion Mediated by the Herpes Simplex Virus Type 1 Immunoglobulin G Fc Receptor

Herpes simplex virus (HSV) glycoproteins gE and gI form an immunoglobulin G (IgG) Fc receptor (FcγR) that binds the Fc domain of human anti-HSV IgG and inhibits Fc-mediated immune functions in vitro. gE or gI deletion mutant viruses are avirulent, probably because gE and gI are also involved in cell-to-cell spread. In an effort to modify FcγR activity without affecting other gE functions, we constructed a mutant virus, NS-gE₃₃₉, that has four amino acids inserted into gE within the domain homologous to mammalian IgG FcγRs. NS-gE₃₃₉ expresses gE and gI, is FcγR⁻, and does not participate in antibody bipolar bridging since it does not block activities mediated by the Fc domain of anti-HSV IgG. In vivo studies were performed with mice because the HSV-1 FcγR does not bind murine IgG; therefore, the absence of an FcγR should not affect virulence in mice. NS-gE₃₃₉ causes disease at the skin inoculation site comparably to wild-type and rescued viruses, indicating that the FcγR⁻ mutant virus is pathogenic in animals. Mice were passively immunized with human anti-HSV IgG and then infected with mutant or wild-type virus. We postulated that the HSV-1 FcγR should protect wild-type virus from antibody attack. Human anti-HSV IgG greatly reduced viral titers and disease severity in NS-gE₃₃₉-infected animals while having little effect on wild-type or rescued virus. We conclude that the HSV-1 FcγR enables the virus to evade antibody attack in vivo, which likely explains why antibodies are relatively ineffective against HSV infection.     

Cytoplasmic viscosity near the cell plasma membrane: measurement by evanescent field frequency-domain microfluorimetry.

The purpose of this study was to determine whether the unique physical milieu just beneath the cell plasma membrane influences the rheology of fluid-phase cytoplasm. Cytoplasmic viscosity was evaluated from the picosecond rotation of the small fluorophore 2',7'-bis-(2-carboxyethyl)-5-carboxyfluorescein (BCECF) by parallel-acquisition Fourier transform microfluorimetry (Fushimi and Verkman, 1991). Information about viscosity within < 200 nm of cell plasma membranes was obtained by selective excitation of fluorophores in an evanescent field created by total internal reflection (TIR) of impulse-modulated s-plane-polarized laser illumination (488 nm) at a glass-aqueous interface. Measurements of fluorescence lifetime and time-resolved anisotropy were carried out in solutions containing fluorescein or BCECF at known viscosities, and monolayers of BCECF-labeled Swiss 3T3 fibroblasts and Madin-Darby canine kidney (MDCK) cells. Specific concerns associated with time-resolved fluorescence measurements in the evanescent field were examined theoretically and/or experimentally, including variations in lifetime due to fluorophore proximity to the interface, and the use of the s and p polarized excitation. In fluorescein solutions excited with s-plane polarized light, there was a 5-10% decrease in fluorescein lifetime with TIR compared to trans (subcritical) illumination, but no change in rotational correlation time (approximately 98 ps/cP). Intracellular BCECF had a single lifetime of 3.7 +/- 0.1 ns near the cell plasma membrane. Apparent fluid-phase viscosity near the cell plasma membrane was 1.1 +/- 0.2 cP (fibroblast) and 1.0 +/- 0.2 cP (MDCK), not significantly different from the viscosity measured in bulk cytoplasm far from the plasma membrane. The results establish the methodology for time-resolved microfluorimetric measurement of polarization in the evanescent field and demonstrate that the cell plasma membrane has little effect on the fluid-phase viscosity of adjacent cytoplasm.     

Mechanism of cellular uptake of modified oligodeoxynucleotides containing methylphosphonate linkages.

The cellular uptake and intracellular distribution of methylphosphonate oligonucleotides (15 mers) has been examined using both 32P labeled and fluorescent labeled oligonucleotides. The cellular uptake process for methylphosphonate oligonucleotides is highly temperature dependent, with a major increase in uptake occurring between 15 and 20 degrees C. Most of the label which becomes cell associated at 37 degrees C cannot be removed by acid washing or trypsinization and thus seems to be within the cell. Visualization of rhodamine labeled methylphosphonate oligonucleotides using digital imaging fluorescence microscopy reveals a vesicular subcellular distribution suggestive of an endosomal localization. There was extensive co-localization of rhodamine labeled methylphosphonate oligonucleotides with fluorescein-dextran, an endosomal/lysosomal marker substance. The apparent endocytotic uptake of labeled methylphosphonate oligonucleotides could not be blocked by competition with unlabeled methylphosphonate or phosphodiester oligonucleotides, nor by ATP. This contrasts with the situation for radiolabeled phosphodiester oligonucleotides whose uptake can be completely blocked with unlabeled competitor. Uptake of phosphodiester oligonucleotides, but not of methylphosphonate oligonucleotides, could be blocked by acidification of the cytosol. These observations suggest that the pathway of cellular uptake of methylphosphonate oligonucleotides involves fluid phase or adsorbtive endocytosis, and is distinct from the uptake pathway for phosphodiester oligonucleotides.     

Subtraction of background fluorescence in multiharmonic frequency-domain fluorimetry.

Background fluorescence is a major concern in time-resolved microfluorimetry studies of biological samples. A general method for subtraction of an arbitrary background signal in measurements of lifetime and anisotropy decay by multiharmonic Fourier transform spectroscopy is presented. Multifrequency phase and modulation values are measured in parallel by transformation of digitized time-domain waveforms into the frequency domain. For subtraction of background, time-domain waveforms are acquired for emission and reference photomultipliers for sample (e.g., cell containing fluorophore) and blank (e.g., unlabeled cell). Time-domain waveforms obtained in a series of measurements (e.g., sample and blank for parallel and perpendicular orientations of an emission polarizer) are time-justified by least-squares fitting of reference channel waveforms or by phase comparison of the first Fourier harmonics of the reference channel. Background is then subtracted directly in the time domain, and the subtracted waveform is Fourier transformed to the frequency domain for analysis of lifetime or anisotropy decay. This approach yielded excellent background correction over a wide range of background intensities and decay profiles. The method was tested in cuvette fluorimetry with fluorescein and acridine orange and in fluorescence microscopy with living MDCK cells loaded with the pH indicator BCECF. Sample lifetimes and rotational parameters could be recovered accurately with greater than 50% of the signal arising from background. These results establish a direct and practical approach to subtraction of background in complex biological and chemical samples studied by frequency-domain fluorimetry.     

Infectivity of adeno-associated virus serotypes in mouse testis

Background

Recombinant adeno-associated viruses (AAVs) are emerging as favoured transgene delivery vectors for both research applications and gene therapy. In this context, a thorough investigation of the potential of various AAV serotypes to transduce specific cell types is valuable. Here, we rigorously tested the infectivity of a number of AAV serotypes in murine testis by direct testicular injection.

Results

We report the tropism of serotypes AAV2, 5, 8, 9 and AAVrh10 in mouse testis. We reveal unique infectivity of AAV2 and AAV9, which preferentially target intertubular testosterone-producing Leydig cells. Remarkably, AAV2 TM, a mutant for capsid designed to increase transduction, displayed a dramatic alteration in tropism; it infiltrated seminiferous tubules unlike wildtype AAV2 and transduced Sertoli cells. However, none of the AAVs tested infected spermatogonial cells.

Conclusions

In spite of direct testicular injection, none of the tested AAVs appeared to infect sperm progenitors as assayed by reporter expression. This lends support to the current view that AAVs are safe gene-therapy vehicles. However, testing the presence of rAAV genomic DNA in germ cells is necessary to assess the risk of individual serotypes.

     

The role of stroma in hematopoiesis and dendritic cell development

Development of the immune system is depicted as a hierarchical process of differentiation from hematopoietic stem cells (HSC) to lineage-committed precursors, which further develop into mature immune cells. In the case of dendritic cell (DC) development, this linear precursor-progeny approach has led to a confused picture of relationships between various subsets of DC identifiable in vivo. A possible reconciliation of the diversity of DC precursors and DC subsets in vivo encompasses the role of the microenvironment in DC hematopoiesis. We propose here that various niches for DC hematopoiesis within lymphoid organs could account for the diversity of DC in vivo. A tridimensional space consisting of stromal cells which produce a range of membrane-bound and secreted molecules providing signals to DC progenitors would define these niches.