Immunospecific binding of tRNA precursors containing N6-(delta 2-isopentenyl) adenosine

Antibodies specific for N6-(delta 2-isopentenyl) adenosine (i6A) were immobilized on Sepharose and this adsorbent (Sepharose-anti-i6A) was used to selectively isolate bacteriophage T4 tRNA precursors containing i6A/ms2i6A from an unfractionated population of 32P-labeled T4 RNAs. The results showed that antibodies to i6A selectively bound only those tRNA precursors containing i6A/ms2i6A. Binding of tRNA precursors by antibody and specificity of the binding was assessed by membrane binding using 32P-labeled tRNA precursor. Binding was highly specific for i6A/ms2i6A residues in the tRNA precursors. This binding can be used to separate modified from unmodified precursor RNAs and to study the biosynthetic pathways of tRNA precursors.     

Synthesis and biochemical properties of novel mRNA 5' cap analogs resistant to enzymatic hydrolysis

A series of new dinucleotide cap analogs with methylene groups replacing oxygens within the pyrophosphate moieties have been synthesized. All the compounds were resistant to the human scavenger decapping hydrolase, DcpS. Binding constants of the modified caps to eIF4E are comparable to those obtained for m7GpppG. This suggests these methylene modifications in the pyrophosphate chain do not significantly affect cap-binding at least for eIF4E. These cap analogs are also good inhibitors of in vitro translation. mRNAs capped with novel analogs were translated similarly to the mRNA capped with the parent m7GpppG.     

O6-methylguanine in the SV40 origin of replication inhibits binding but increases unwinding by viral large T antigen.

To study the effect of the potentially cytotoxic base O6-methylguanine (O6-meG) on the initiation of DNA replication, double-stranded oligonucleotides corresponding to the SV40 origin of replication were constructed in which O6-meG replaced guanine in one strand. Out of 14 methylated residues, 10 were present in the Binding sites for T antigen (3 in Binding Site 1 and 7 in Binding Site 2). Binding of purified T antigen to the substituted oligonucleotide was considerably reduced in comparison to the unsubstituted one, as measured by nitrocellulose filter binding. Both the ATP-dependent and ATP-independent binding of T antigen were affected by the presence of the methylated base. Band shift analysis revealed an altered pattern of delayed-migrating complexes of T antigen with the O6-meG-containing oligonucleotide. Competition experiments, in which unmodified oligonucleotides containing Binding Site 1 or 2 were included in the binding assays, indicated that the affinity of T antigen for the O6-meG modified sites was reduced. When partially duplex oligonucleotides containing either Binding Site 1 or Site 2 of the origin of replication were used as substrates for the helicase activity of T antigen, the presence of O6-meG increased the extent of T antigen catalysed displacement of single-stranded DNA fragments.     

SYNTHESIS AND BIOCHEMICAL PROPERTIES OF NOVEL mRNA 5′ CAP ANALOGS RESISTANT TO ENZYMATIC HYDROLYSIS

A series of new dinucleotide cap analogs with methylene groups replacing oxygens within the pyrophosphate moieties have been synthesized. All the compounds were resistant to the human scavenger decapping hydrolase, DcpS. Binding constants of the modified caps to eIF4E are comparable to those obtained for m⁷GpppG. This suggests these methylene modifications in the pyrophosphate chain do not significantly affect cap-binding at least for eIF4E. These cap analogs are also good inhibitors of in vitro translation. mRNAs capped with novel analogs were translated similarly to the mRNA capped with the parent m⁷GpppG.     

Structural Features of Transiently Modified Beta-Lactoglobulin Relevant to the Stable Binding of Large Hydrophobic Molecules

Binding sites for hydrophobic molecules on bovine β-lactoglobulin, and their susceptibility to temperature, were studied by using various spectroscopic probes. Binding of probes carrying a single fluorophore moiety, a single nitroxide moiety, or both moieties on the same molecule, was followed by EPR and fluorescence. The presence of a fatty acid side chain in the dual probes was found to be required for binding to β-lactoglobulin. Binding occurred only after the protein was heated at temperatures below the threshold for its irreversible denaturation. Binding became extremely tight and stable upon cooling of the protein–probe mixture. Comparison among the various probes suggests that multiple binding sites for hydrophobes are present in the native protein, and in the partially—and reversibly—modified form of β-lactoglobulin present in solution at neutral pH and subdenaturing temperatures. Thus, the specificity of hydrophobes binding to β-lactoglobulin may be modulated by simple physical treatment of the protein.     

Heparanase affects food intake and regulates energy balance in mice

Mutation of the melanocortin-receptor 4 (MC4R) is the most frequent cause of severe obesity in humans. Binding of agouti-related peptide (AgRP) to MC4R involves the co-receptor syndecan-3, a heparan sulfate proteoglycan. The proteoglycan can be structurally modified by the enzyme heparanase. Here we tested the hypothesis that heparanase plays a role in food intake behaviour and energy balance regulation by analysing body weight, body composition and food intake in genetically modified mice that either lack or overexpress heparanase. We also assessed food intake and body weight following acute central intracerebroventricular administration of heparanase; such treatment reduced food intake in wildtype mice, an effect that was abolished in mice lacking MC4R. By contrast, heparanase knockout mice on a high-fat diet showed increased food intake and maturity-onset obesity, with up to a 40% increase in body fat. Mice overexpressing heparanase displayed essentially the opposite phenotypes, with a reduced fat mass. These results implicate heparanase in energy balance control via the central melanocortin system. Our data indicate that heparanase acts as a negative modulator of AgRP signaling at MC4R, through cleavage of heparan sulfate chains presumably linked to syndecan-3.     

DNA extraction using bacterial magnetic particles modified with hyperbranched polyamidoamine dendrimer

A cascading hyperbranched polyamidoamine dendrimer was synthesized on the surface of bacterial magnetite from Magnetospirillum magneticum AMB-1 to allow enhanced extraction of DNA from fluid suspensions. Characterization of the synthesis revealed linear doubling of the surface amine charge from generations one through five starting with an amino silane initiator. Furthermore, transmission electron microscopy revealed clear dispersion of the single domain magnetite in aqueous solution. The dendrimer modified magnetic particles have been used to carry out magnetic separation of DNA. Binding and release efficiencies increased with the number of generations and those of bacterial magnetite modified with six generation dendrimer were 7 and 11 times respectively as many as those of bacterial magnetite modified with only amino silane.     

Binding of unnatural alpha,beta-oligocytidylates with DNA duplexes

Binding of short fluorescently labeled AT-containing DNA duplexes with modified oligocytidylates is studied. The latter are modified to contain unnatural alpha-anomers along with natural beta-nucleotides; the nucleotide composition is selected according to putative pattern of unconventional triplex formation between duplex and oligomer bases. Nondenaturing gel electrophoresis is used to study complexation of fluorescent duplexes with cytidyl oligomers and oligocytidylate self-association at low temperatures. A DNA duplex of random AT composition is shown to bind with an excess of the corresponding oligocytidylate in 0.1 M Tris-HCl in the presence of Mg2+. Binding is observed at neutral pH values, while more basic pH (8.0) prevents complexation of the AT duplex and oligocytidylate. Contrary to oligonucleotides of irregular composition, a regular dA30:dT30 duplex does not bind with the dC strand. It is also shown that alternating self-complementary duplex d(AT)16 and oligocytidylate d(CbetaCalpha)15 do not form complexes, and poly-dC self-associates are formed instead. The effect of 2'-O-methylation of the third strand on complex formation and self-association is also analyzed. The results suggest that a modified oligocytidylate binds with a random-composition duplex, albeit with lower efficiency.     

Binding of Cerebratulus cytolysin A-III to human erythrocyte membranes

Binding of Cerebratulus lacteus cytolysin A-III to intact human erythrocytes and erythrocyte membranes has been investigated. Binding to ghosts is essentially complete within 2.5 min of mixing which is slightly faster than the rate of hemolysis measured with intact cells. Approximately 4 X 10(4) binding sites per cell, exhibiting a K 0.5 of 0.7 microM exist; this compares with 50% hematocrit of about 0.3 microM for A-III. Binding is absent in ghosts extracted with Nonidet P-40, but is unaffected by pretreatment of ghosts with either trypsin or elastase.     

Optimization of time-resolved fluorescence assay for detection of europium-tetraazacyclododecyltetraacetic acid-labeled ligand-receptor interactions

Lanthanide-based luminescent ligand binding assays are superior to traditional radiolabel assays due to improving sensitivity and affordability in high-throughput screening while eliminating the use of radioactivity. Despite significant progress using lanthanide(III)-coordinated chelators such as diethylenetriaminepentaacetic acid (DTPA) derivatives, dissociation-enhanced lanthanide fluoroimmunoassays (DELFIAs) have not yet been successfully used with more stable chelators (e.g., tetraazacyclododecyltetraacetic acid [DOTA] derivatives) due to the incomplete release of lanthanide(III) ions from the complex. Here a modified and optimized DELFIA procedure incorporating an acid treatment protocol is introduced for use with Eu(III)-DOTA-labeled peptides. Complete release of Eu(III) ions from DOTA-labeled ligands was observed using hydrochloric acid (2.0 M) prior to the luminescent enhancement step. [Nle⁴,d-Phe⁷]-α-melanocyte-stimulating hormone (NDP-α-MSH) labeled with Eu(III)-DOTA was synthesized, and the binding affinity to cells overexpressing the human melanocortin-4 (hMC4) receptor was evaluated using the modified protocol. Binding data indicate that the Eu(III)-DOTA-linked peptide bound to these cells with an affinity similar to its DTPA analogue. The modified DELFIA procedure was further used to monitor the binding of an Eu(III)-DOTA-labeled heterobivalent peptide to the cells expressing both hMC4 and cholecystokinin-2 (CCK-2) receptors. The modified assay provides superior results and is appropriate for high-throughput screening of ligand libraries.     

Identification of the cap binding domain of human recombinant eukaryotic protein synthesis initiation factor 4E using a photoaffinity analogue.

Binding of eIF-4E to the 5' m7G cap structure of eukaryotic mRNA signals the initiation of protein synthesis. In order to investigate the molecular basis for this recognition, photoaffinity labeling with [gamma-32P]8-N3GTP was used in binding site studies of human recombinant cap binding protein eIF-4E. Competitive inhibition of this cap analogue by m7GTP and capped mRNA indicated probe specificity for interaction at the protein binding site. Saturation of the binding site with [gamma-32P]8-N3GTP further demonstrated the selectivity of photoinsertion. Aluminum (III)-chelate chromatography and reverse-phase HPLC were used to isolate the binding site peptide resulting from digestion of photolabeled eIF-4E with modified trypsin. Amino acid sequencing identified the binding domain as the region containing the sequence Trp 113-Arg 122.Lys 119 was not identified in sequencing analysis nor was it cleaved by trypsin. These results indicate that Lys 119 is the residue directly modified by photoinsertion of [gamma-32P]8-N3GTP. A detailed understanding of eIF-4E.m7G mRNA cap interactions may lead the way to regulating this essential protein-RNA interaction for specific mRNA in vivo.     

Enhancement of immunotherapeutic effects of HPV16E7 on cervical cancer by fusion with CTLA4 extracellular region

Cervical cancer is caused by infection by high-risk human papillomavirus (HPV), especially HPV16. Limitations in current treatments of cervical cancers call for the development of new and improved immunotherapies. This study aims at investigating the efficacy of a novel vaccine consisting of modified HPV 16E7 fused with human cytotoxic T-lymphocyte antigen 4 (CTLA4). The regions in HPV16 E7 gene associated with its transformation and CTL-enhanced response were modified; the resultant HPV16mE7 was fused with extracellular region of CTLA4 to generate HPVml6E7-eCTLA4 fusion protein. Binding of this fusion protein to B7 molecules expressed on antigen presenting-cells (APCs) was demonstrated. C57BL/6 (H-2^b) mice immunized with low dose of the fusion protein (10 μg) produced higher titer antibody and stronger specific CTL response, and expressed higher levels of IFN-γ and IL-12, compared with those immunized with HPVml6E7 only or admixture of HPVml6E7 and CTLA4, or PBS; and were protected from lethal dose tumor challenge. Tumor growth was retarded and survival prolonged in mouse models with the fusion protein treatment. Our results demonstrate that fusion of HPV16 E7 with eCTLA4 targeting APCs resulted in enhanced immunity, and that this fusion protein may be useful for improving the efficacy of immunotherapeutic treatments of cervical cancer and other HPV16 infection-associated tumors.     

Affinity chromatography of solubilized opioid binding sites using CH-Sepharose modified with a new naltrexone derivative

An affinity gel containing a newly synthesized derivative of naltrexone, beta-naltrexyl-6-ethylenediamine (NED), was used to purify opioid binding sites by 300-450-fold. Binding sites solubilized from brains of toad, rat and cow using digitonin in the presence of 0.5 M NaCl are retained by the gel and can be eluted with naloxone (1-3 microM) in yields of 20-25%. The biospecificity of the interaction of opioid binding sites with modified beads is supported by the following: 1) unmodified beads do not retain the binding sites, 2) binding sites prebound with an opioid are not retained, 3) dilution of NED gels with unmodified Sepharose progressively reduced efficiency of retention and 4) specific receptor ligands elute binding sites retained on the NED gels.     

pH-dependent changes of intrinsic fluorescence of chemically modified liver alcohol dehydrogenases.

Horse liver alcohol dehydrogenase specifically carboxymethylated on cysteine-46 (a ligand to the zinc in the active site) or acetimidylated on 25 of the 30 lysine residues per subunit (including residue 228) was studied. The tryptophan fluorescence of these enzymes decreased by 35% as pH was increased, with an apparent pKa of 9.8 +/- 0.2, identical with that of native enzyme. Native enzyme in the presence of 30mM-imidazole, which displaces a water molecule ligated to the zinc, also had a pKa of 9.8. The ionoizable group is thus neither the water molecule nor one of the modified groups. Binding of NAD+ shifted the pKa for the fluorescence transition to 7.6 with native enzyme and to 9.0 with acetimidylated enzyme, but did not shift the pKa of carboxymethylated enzyme. Binding of NAD+ and trifluoroethanol, an unreactive alcohol, gave maximal fluorescence quenching at pH7 with all three enzymes. The acetimidylated enzyme--NAD+--trifluoroethanol complex had an apparent pKa of 5.0, but the pK of the native enzyme complex was experimentally inaccessible. The results are interpreted in terms of coupled equilibria between two different conformational states. On binding of NAD+, the modified enzymes apparently change conformation less readily than does native enzyme, but binding of alcohol can drive the change to completion.     

The binding of metabolites formed from aminostilbene derivatives to nucleic acids in the liver of rats.

Carcinogenic trans-4-dimethylaminostilbene (trans-DAS) and trans-4-acetylaminostilbene (trans-AAS) as well as inactive cis-DAS and DABB were highly and specifically labeled with tritium and administered orally to female Wistar rats. Covalent binding to liver rRNA and DNA was measured and found to be higher for the carcinogenic compounds. Digests from these nucleic acids were chromatographed on Sephadex LH-20 and 16 different nucleoside adducts were characterised by their retention volumes. Labeled trans-DAS was administered in doses ranging from 0.025--250 mumol/kg. Binding to nucleic acids was directly proportional to the dose at low doses (0.025--2.5 mumol/kg) and less than proportional at higher doses (25--250 mumol/kg). The pattern of nucleoside adducts remained practically constant over the wide range of doses. A pharmacokinetically determined threshold of metabolic activation thus could not be demonstrated for this compound. A modified procedure is described to simultaneously isolate pure liver rRNA and DNA from nonfasted rats in high yields.     

Steroid analogs inhibit hormone binding by an extract from Nippostrongylus brasiliensis (Nematoda)

1. An extract from the rodent nematode Nippostrongylus brasiliensis contained putative receptors that bound radiolabeled sex hormones, based on isoelectric focusing. 2. Binding of radiolabeled testosterone by receptors at pH 4.4 was highly inhibited by the androgen analogs, testosterone-3-oxime and 4-aza-5-androsten-3-on-17 beta-ol. 3. Binding of radiolabeled progesterone by receptors at pH 6.4 was highly inhibited by the progesterone analogs 3,5-seco-4-norpregnan-5-on-3-oic acid and 19-norethisterone or 21-deoxycorticosterone. 4. Binding of radiolabeled 17 beta-estradiol by receptors at pH 4.9 was highly inhibited by epiandrosterone. 5. In vivo development of N. brasiliensis to the adult was partially inhibited by selected steroid analogs.     

A fluorescence resonance energy transfer sensor based on maltose binding protein

A fluorescence resonance energy-transfer (FRET) sensing system for maltose based on E. coli maltose binding protein (MBP) is demonstrated. The FRET donor portion of the sensing system consists of MBP modified with long wavelength-excitable cyanine dyes (Cy3 or Cy3.5). The novel acceptor portion of the sensor consists of beta-cyclodextrin (beta-CD) modified with either the cyanine dye Cy5 or the dark quencher QSY9. Binding of the modified beta-CD to dye-conjugated MBP results in assembly of the FRET complex. Added maltose displaces the beta-CD-dye adduct and disrupts the FRET complex, resulting in a direct change in fluorescence of the donor moiety. In the use of these FRET pairs, MBP dissociation values for maltose were estimated (0.14-2.90 microM). Maltose limits of detection were in the 50-100 nm range.     

Physicochemical characterization of poly(ethylene glycol)-modified anti-GAD antibodies.

Monoclonal antibodies against glutamic acid decarboxylase (anti-GAD) were modified with poly(ethylene glycol) (PEG), and the resulting conjugates were characterized. Monoclonal anti-GAD antibodies were purified from ATCC HB184 hybridoma cells by either cell culture supernatant or ascites fluid from BALB/c mice. Polyclonal rabbit IgG antibodies were also used as a model protein. Polyclonal rabbit IgG or purified anti-GAD was modified by PEG (MW = 5000 or 20000 Da) through either the lysine residues or through the carbohydrate moiety. Lysine modification was performed in PBS (pH 7.4) or 0.1 M borate (pH 9.2) by adding a molar excess (5-80) of a succinimidyl activated propionic acid terminated mPEG (SPA-PEG) while stirring at room temperature. Carbohydrate modifications were performed in PBS (pH 6.2) by first oxidizing the antibody with sodium periodate followed by incubation with hydrazide-terminated PEG followed by reduction with sodium cyanoborohydride. The degree of modification was assessed by 1H NMR or TNBS (trinitrobenzenesulfonic acid). Circular dichroism (CD) spectra were obtained for lysine-modified rabbit IgG at various degrees of modification ranging from 5 to 60 PEG per antibody. Binding was assessed using an ELISA method with GAD or rabbit anti-mouse-IgG (H+L) coated plates. The TNBS and 1H NMR analysis of the modified antibody showed reasonably similar results from 5 to 60 PEG per antibody. The 1H NMR method showed greater sensitivity at low modifications (below 20:1) and was fairly linear up to about 60 PEG per antibody. The CD spectra of the polyclonal rabbit IgG showed only small differences at variously modified antibody. The binding affinity of anti-GAD is lower for all PEG modifications with respect to unmodified anti-GAD. Modifications at pH 7.4 show lower binding to GAD than modifications at pH 9.2. Binding to GAD or anti-mouse-IgG is decreased as the degree of modification is increased. Lysine modifications showed lower binding to GAD or anti-mouse-IgG than carbohydrate modifications. Binding to GAD or anti-mouse-IgG is lower for PEG20000-modified anti-GAD with respect to PEG5000-modified anti-GAD.     

Solubilization of the chemokine receptor CXCR4

The chemokine receptor CXCR4 was solubilized from the human T-cell line CEM by using the detergent n-dodecyl-beta-maltoside (DDM) and cholesteryl hemisuccinate ester (CHS). Binding studies with (125)I-SDF-1alpha revealed a dissociation constant of 5.33 nM and a receptor density (B(max)) of 2.68 pmol/mg in CEM membranes at 4 degrees C. The affinity of solubilized CXCR4 for SDF-1alpha was identical to membrane-bound CXCR4. Binding of gp120 to solubilized CXCR4 was demonstrated by coprecipitation of gp120 with anti-CXCR4 antibodies.