MAB030 Sigma-AldrichAnti-DNA Antibody, double stranded, clone BV16-13

Toll-like receptor-9 (TLR9) is largely responsible for discriminating self from pathogenic DNA. However, association of host DNA with autoantibodies activates TLR9, inducing the pathogenic secretion of type I interferons (IFNs) from plasmacytoid dendritic cells (pDCs). Here, we found that in response to DNA-containing immune complexes (DNA-IC), but not to soluble ligands, IFN-α production depended upon the convergence of the phagocytic and autophagic pathways, a process called microtubule-associated protein 1A/1B-light chain 3 (LC3)-associated phagocytosis (LAP). LAP was required for TLR9 trafficking into a specialized interferon signaling compartment by a mechanism that involved autophagy-related proteins, but not the conventional autophagic preinitiation complex, or adaptor protein-3 (AP-3). Our findings unveil a new role for nonconventional autophagy in inflammation and provide one mechanism by which anti-DNA autoantibodies, such as those found in several autoimmune disorders, bypass the controls that normally restrict the apportionment of pathogenic DNA and TLR9 to the interferon signaling compartment.

Nucleic acids are known to induce complement activation, which results in the masking and removal of apoptotic cells exposing nuclear components. Dysregulation of these events is characteristic of SLE, a systemic autoimmune disease characterized by the appearance of ANAs. In this study, we aimed to investigate the relationship between development of ANAs and their effect on complement activation by nucleic acids.We used protein array technology to characterize complement activation by murine mAbs and polyclonal antibodies against various forms of nucleic acid. Serum samples from MRL/lpr mice were collected, starting before the onset of the disease till 6 months of age. Binding of IgG and its subclasses to dsDNA, ssDNA, RNA, plasmid DNA and nucleosome complexes was determined, along with C3 fixation.We show that complement C3 binding to various forms of nucleic acid that serve as targets in lupus is absent in normal serum. The addition of dsDNA-specific mAbs to normal serum results in the deposition of complement C3 to nucleic acids. In MRL/lpr mice, IgG antibodies against various nuclear antigens appear with ageing and disease progression. C3 binding to the antigens is somewhat delayed and suggests that accumulation or maturation of pathogenic antibodies is required for inducing C3 binding to ICs containing nucleic acids.C3 deposition on nuclear antigens, therefore, reflects the state of disease progression in this murine model of SLE.

We have developed two experimental methods for observing Escherichia coli RecA-DNA filament under a fluorescence microscope. First, RecA-DNA filaments were visualized by immunofluorescence staining with anti-RecA monoclonal antibody. Although the detailed filament structures below submicron scale were unable to be measured accurately due to optical resolution limit, this method has an advantage to analyse a large number of RecA-DNA filaments in a single experiment. Thus, it provides a reliable statistical distribution of the filament morphology. Moreover, not only RecA filament, but also naked DNA region was visualized separately in combination with immunofluorescence staining using anti-DNA monoclonal antibody. Second, by using cysteine derivative RecA protein, RecA-DNA filament was directly labelled by fluorescent reagent, and was able to observe directly under a fluorescence microscope with its enzymatic activity maintained. We showed that the RecA-DNA filament disassembled in the direction from 5' to 3' of ssDNA as dATP hydrolysis proceeded.

BACKGROUND: During metazoan development, cell diversity arises primarily from asymmetric cell divisions which are executed in two phases: segregation of cytoplasmic factors and positioning of the mitotic spindle - and hence the cleavage plane -relative to the axis of segregation. When polarized cells divide, spindle alignment probably occurs through the capture and subsequent shortening of astral microtubules by a site in the cortex. RESULTS: Here, we report that dynactin, the dynein-activator complex, is localized at cortical microtubule attachment sites and is necessary for mitotic spindle alignment in early Caenorhabditis elegans embryos. Using RNA interference techniques, we eliminated expression in early embryos of dnc-1 (the ortholog of the vertebrate gene for p150(Glued)) and dnc-2 (the ortholog of the vertebrate gene for p50/Dynamitin). In both cases, misalignment of mitotic spindles occurred, demonstrating that two components of the dynactin complex, DNC-1 and DNC-2, are necessary to align the spindle. CONCLUSIONS: Dynactin complexes may serve as a tether for dynein at the cortex and allow dynein to produce forces on the astral microtubules required for mitotic spindle alignment.

Members of the MKLP1 subfamily of kinesin motor proteins localize to the equatorial region of the spindle midzone and are capable of bundling antiparallel microtubules in vitro. Despite these intriguing characteristics, it is unclear what role these kinesins play in dividing cells, particularly within the context of a developing embryo. Here, we report the identification of a null allele of zen-4, an MKLP1 homologue in the nematode Caenorhabditis elegans, and demonstrate that ZEN-4 is essential for cytokinesis. Embryos deprived of ZEN-4 form multinucleate single-celled embryos as they continue to cycle through mitosis but fail to complete cell division. Initiation of the cytokinetic furrow occurs at the normal time and place, but furrow propagation halts prematurely. Time-lapse recordings and microtubule staining reveal that the cytokinesis defect is preceded by the dissociation of the midzone microtubules. We show that ZEN-4 protein localizes to the spindle midzone during anaphase and persists at the midbody region throughout cytokinesis. We propose that ZEN-4 directly cross-links the midzone microtubules and suggest that these microtubules are required for the completion of cytokinesis.

Two methods are described, one for detection and one for isolation of COS cells transiently expressing vertebrate lectins. The methods are based on specific cell adhesion to polystyrene microwells or magnetic beads adsorbed with glycosphingolipids. In the first method, glycolipids were adsorbed to wells of 96-well polystyrene plates. A suspension of lectin-transfected COS cells was added and the plate was incubated to allow cell adhesion to occur. The plate was then immersed in buffer, inverted (while immersed), and placed in a fluid-filled Plexiglas centrifugation chamber which was sealed to avoid introducing an air-liquid interface. The chamber, with the inverted plate enclosed, was centrifuged to remove nonadherent cells. The plate was then removed from the carrier (while immersed) and righted, and adherent cells were quantitated enzymatically or immunochemically using a 96-well plate reader. COS cells transfected with an expression plasmid carrying the gene for the rat Kupffer cell lectin (fucose and N-acetylgalactosamine specific) adhered specifically to globotetraosylceramide. Glycolipid- and lectin-specific cell adhesion was readily detected even when COS cells were transfected with a plasmid mixture containing 0.5% lectin-carrying plasmid and 99.5% irrelevant plasmid. This sensitivity will facilitate screening of plasmid pools to detect and isolate plasmids expressing mammalian lectin genes. To isolate COS cells transiently expressing lectin, glycosphingolipids were adsorbed to carboxylated magnetic polystyrene microspheres, which were mixed with the lectin-transfected COS cells. Adherent cells were collected on a fixed magnet and plasmid recovered for subsequent amplification.

VH and VL region primary structures of five NZB/NZW F1 derived monoclonal anti-DNA autoantibodies were determined from cloned cDNA. Comparative analysis of VH genes showed that except for two VH genes that shared complete identity the overall VH gene usage was diverse. Comparison of VH genes with those utilized in a variety of antibody responses showed they were generally unique to the autoanti-DNA response although framework homologies allowed assignment of four of five VH genes to existing murine heavy chain gene families. Only one out of five D segments shared homology to existing germline D segments, and all were rearranged to JH3. V kappa genes showed restriction for four of five light chains to the V kappa 1 subgroup. The V kappa 1 subgroup has been shown previously to be utilized in several anti-DNA autoantibodies as well as a variety of antibodies to exogenous antigens. H and L chain amino acid residues associated with the active site of a ssDNA specific autoantibody, 04-01, are discussed based on recently obtained crystallographic data.

Synthetic nucleic acid reactivities and the distribution of idiotypes associated with poly(dA) and poly(dT) specificities were evaluated among both monoclonal and polyclonal anti-DNA antibodies from autoimmune New Zealand mice. Ten monoclonal anti-DNA antibodies (IgG2a or IgG2b), derived from NZB/NZW mice and reactive with natural DNA (duplex and/or heat-denatured), were found to collectively exhibit a diverse binding pattern with six deoxyribohomopolymers. Several monoclonal antibodies displayed reactivity with poly(dT) comparable to that with natural DNA. Serologic studies indicated that polyclonal anti-DNA autoantibodies from NZW/NZW mice and both parental strains also cross-reacted with various homopolymers and bound preferentially with those containing pyrimidines, particularly poly(dT), relative to purines. Detailed binding analyses with two poly(dT)-reactive monoclonal antibodies demonstrated that stable DNA/anti-DNA complexes were formed with synthetic oligomers containing six to 10 nucleotides; binding to such antigens was relatively insensitive to ionic strength and inversely dependent on temperature. Both antibodies exhibited preferential binding (greater than or equal to 10-fold) with poly(dT) relative to poly(dU), suggesting the importance of the C5-methyl group and/or helical conformation in pyrimidine base recognition. Idiotypes on poly(dA)-specific and poly(dT)-specific monoclonal antibodies were found to be reciprocally distinct, localized at or near active site residues, and expressed at low levels (less than 10 to 130 ng/ml) in anti-DNA sera from all three New Zealand strains. These findings suggest that: nucleotide base determinants are significantly involved in DNA/anti-DNA interactions; poly(dT) represents a major cross-reactive synthetic antigen; and idiotype expression among lupus autoantibodies which recognize such determinants may be diverse.