The GTPase activity of murine guanylate-binding protein 2 (mGBP2) controls the intracellular localization and recruitment to the parasitophorous vacuole of Toxoplasma gondii.

One of the most abundantly IFN-γ-induced protein families in different cell types is the 65-kDa guanylate-binding protein family that is recruited to the parasitophorous vacuole of the intracellular parasite Toxoplasma gondii. Here, we elucidate the relationship between biochemistry and cellular host defense functions of mGBP2 in response to Toxoplasma gondii. The wild type protein exhibits low affinities to guanine nucleotides, self-assembles upon GTP binding, forming tetramers in the activated state, and stimulates the GTPase activity in a cooperative manner. The products of the two consecutive hydrolysis reactions are both GDP and GMP. The biochemical characterization of point mutants in the GTP-binding motifs of mGBP2 revealed amino acid residues that decrease the GTPase activity by orders of magnitude and strongly impair nucleotide binding and multimerization ability. Live cell imaging employing multiparameter fluorescence image spectroscopy (MFIS) using a Homo-FRET assay shows that the inducible multimerization of mGBP2 is dependent on a functional GTPase domain. The consistent results indicate that GTP binding, self-assembly, and stimulated hydrolysis activity are required for physiological localization of the protein in infected and uninfected cells. Ultimately, we show that the GTPase domain regulates efficient recruitment to T. gondii in response to IFN-γ.

Pre-clinical evaluation of a CEA DNA prime/protein boost vaccination strategy against colorectal cancer.

In preparation for a clinical trial in patients diagnosed with colorectal cancer, a vaccination strategy targeting the carcinoembryonic antigen (CEA) was evaluated in mice using a GMP-produced plasmid DNA vaccine, CEA66, encoding a truncated form of the tumour-associated antigen, CEA. The GMP-produced CEA DNA vaccine was also evaluated for toxicity. Repeated intradermal administration of the GMP-produced vaccine using a novel needle-free jet injection device (Biojector) induced robust CD4 and CD8 T-cell responses in mice, and did not result in any vaccine-related toxicity. In a heterologous DNA prime/protein boost setting, cellular immune responses were of higher magnitude in animals primed with CEA66 DNA than in animals receiving repeated doses of recombinant CEA protein. These responses were further enhanced if recombinant murine granulocyte-macrophage colony-stimulating factor was given as an adjuvant prior to vaccination. In contrast to repeated administration of recombinant CEA protein as a single modality vaccine, the heterologous CEA66 DNA prime/rCEA boost vaccination strategy resulted in a qualitatively broader immune response, and supports clinical testing of this vaccination regimen in humans.

Effect of nucleic acid reactive antibodies on tumor cells grown in vivo.

Nucleic acid reactive antibodies have been reported to inhibit various nucleic acid mediated functions in cell free systems. These antibodies were also shown to inhibit the growth of transformed cells in culture due to the high rate of endocytosis in transformed cells as compared to normal cells. In this report, we have tested the possibility of nucleic acid reactive antibodies inhibiting the growth of tumor cells in vivo. The life span of mice bearing Dalton's lymphoma ascites tumor cells was increased, when they were immunized with conjugates of guanosine-BSA, GMP-BSA and tRNA-MBSA complex before transplanting the tumor cells. A similar effect was also observed when mice were injected intraperitoneally with antibodies to guanosine or GMP along with the tumor cells. The specificity was ascertained, as immunization with non-specific antigens did not show any significant effect on tumor bearing mice. The results shows that nucleic acid reactive antibodies inhibit the growth of tumor cells in vivo.

Inhibition of in vitro aminoacylation and translation by RNA reactive antibodies.

Antibodies were raised against guanosine-BSA, GMP-BSA and tRNA-mBSA conjugates separately in rabbits. Binding characteristics of these antibodies to various RNAs were studied using a sensitive avidin-biotin micro ELISA. These antibodies inhibited in vitro aminoacylation of tRNA in a dose dependent manner. This inhibition was reversed by the addition of the respective homologous haptens thereby showing the specificity of these antibodies. In vitro translation of endogenous mRNAs in rabbit reticulocyte lysate was also inhibited by these antibodies in a dose dependent manner.

Antibody-nucleic acid complexes. Oligo(dG)n and -(dT)n specificities associated with anti-DNA antibodies from autoimmune MRL mice.

The specificity of anti-DNA antibodies in the sera of unimmunized autoimmune MRL mice was initially assessed via an enzyme-linked immunosorbent assay (ELISA). Antibody binding profiles to a panel of immobilized antigens (AMP-, GMP-, CMP-, UMP-, and TMP-BSA, ss- and dsDNA) demonstrated high levels of immunoglobulins reacting with GMP and ssDNA and intermediate levels with AMP, TMP, and dsDNA. Fractionation of serum anti-DNA antibodies into subsets on the basis of their binding to GMP- and TMP-agarose indicated that the resulting GMP- or TMP-reactive antibodies bound to their homologous nucleotides and ssDNA. Competition-inhibition studies with soluble mono-, oligo-, and polynucleotides revealed that GMP- and TMP-reactive antibodies were highly specific for oligo(dG)n and -(dT)n sequences, respectively. Whereas the relative affinity of TMP-reactive autoantibodies to oligo(dT)n increased with oligonucleotide length (n = 2, 4, 6, 8, 10, 15), GMP-reactive antibodies preferentially recognized oligo(dG)10 (Ka congruent to 1 x 10(7) M-1). While neither antibody recognized oligo(dA)8 and -(dC)8 competitors, mixed-base oligonucleotides were inhibitory at concentrations approximately 10-fold greater than similarly sized oligo(dG)n and -(dT)n sequences. Similar characterizations of both pooled and individual MRL sera indicated that anti-DNA antibodies represent 8-10% of the total serum IgG. More importantly, GMP-reactive autoantibodies predominated and accounted for 60-70% of the entire unbound anti-DNA antibody population.

Monoclonal antibody detection of 2-acetyl-aminofluorene-modified DNA probes for the specific detection of nucleic acids in hybridization procedures.

We describe the use of acetoxy-acetyl-aminofluorene-modified DNA probes in several hybridization techniques. Hybrids were detected with the help of a monoclonal antibody raised against AAF-guanosine and a second antibody coupled to an enzyme. The sensitivity achieved with AAF-DNA probes routinely detected 0.25 pg DNA bound to a filter. AAF-DNA probes were highly stable and were prepared by simple chemical modification of DNA. Their use as a possible diagnostic tool is discussed.

Antibodies directed against guanylic acid and guanosine are nucleolus specific probes.

Purified antibodies against guanylic acid and guanosine binding to RNA at guanosine residues were used to probe human lymphocyte preparations by indirect immunofluorescence. Neither antibody gave any banding pattern with metaphase chromosomes but both showed binding to specific sites in the interphase nuclei. Evidence presented indicates that these sites are guanosine residues on rDNA transcripts at the nucleolar organizer regions.

Antibodies to guanylic acid: fractionation and specificities.

Guanylic-acid-specific antibodies were elicited in rabbits, using as immunogen pG linked through 5'-phosphate to thyroglobulin. Specificity and affinity of antibodies to nucleotides, nucleosides, DNA, and RNA were studied by their binding to radioactive ligands and competition experiments. Guanylic-acid-specific antibodies do not bind to deoxyguanylic acid and have an average association constant of 10(7) M-1 at 4 degrees C. Binding of the antibodies to 3H-RNA is G-specific. The antibodies do not bind to 32P-ssDNA or 32P-dsDNA. The pG-specific antibodies could be separated into different fractions by affinity chromatography. These fractions, though specific to pG, differ in their cross-reactivities to nucleosides and nucleotides.

[Inhibition of RNA synthesis in vitro by antibodies against mononucleotides]. / Ugnetenie sinteza RNK in vitro antitelami protiv mononukleotidov.

Antibodies against purine nucleotides were obtained from rabbits immunized with conjugates of bovine serum albumine with AMP or GMP. The antibodies purified by affinity chromatography on nucleoside monophosphate-human serum albumine-Sepharose columns inhibited RNA synthesis on native T4 phage DNA by E. coli RNA polymerase. The inhibition of transcription was due mainly to inhibition of the initiation stage of RNA synthesis.

Isolation and characterization of rabbit anti-m7G-5'-P antibodies of high apparent affinity.

Antibodies highly specific for intact pm7G (7methylguanosine-5'-mono-phosphate) were induced by immunization of rabbits with a pm7G-BSA conjugate. Since the nucleotide is six-fold more stable than m7G (7-methylguanosine) to alkali-catalyzed fission of the imidazole ring, it is a more desirable antigen for obtaining antibodies capable of binding caps on eukaryotic mRNA. UV spectra demonstrated that the nucleotide in the conjugate was predominantly the intact form. Competition radioimmunoassay showed 1) high apparent affinities for pm7G, on the order of 10(-8)M, 2) low competition by the ring-opened form of the homologous hapten (*pm7G), and by m7G, 3) little or no competition by AMP, GMP, CMP, UMP or m6A, and 4) high apparent affinities for m7GpppAm, m7GpppN6MAm, m7GpppGm, m7GpppA.