High Binding Ratios Research Articles

Solid-phase synthesis, characterization and DNA binding properties of the first chloro(polypyridyl)ruthenium conjugated peptide complex

A general method for the synthesis of chloro(polypyridyl)ruthenium conjugated peptide complexes via a solid-phase strategy is described. The method is applied to synthesize two positional isomers of the complex [Ru(terpy)(4-CO2H-4'-Mebpy-Gly-L-His-L-LysCONH2)Cl](PF6). Even though the separation of the isomers was only partially achieved chromatographically, the isomers were unambiguously assigned by NMR spectroscopy. The interactions of the complex [Ru(terpy)(4-CO2H-4'-Mebpy-Gly-L-His-L-LysCONH2)Cl](PF6) with CT-DNA and plasmid DNA, have been studied with various spectroscopic techniques, showing that (i) the complexes coordinatively bind to DNA preferring the bases guanine and cytosine over the bases thymine and adenine after hydrolysis of the coordinated chloride, (ii) electrostatic interactions between the complex cation and the polyanionic DNA chain assist this binding (iii) only in the case of one isomer the peptide does interact further with DNA as evidenced from 31P NMR spectroscopy, (iv) DNA unwinding occurs in all cases with high binding ratio (Ru/base) values (r > 0.3).

Enhanced Dopamine Transporter Density in Psychotropic-Naive Patients With Obsessive-Compulsive Disorder Shown by [123I]β-CIT SPECT

The authors examine the functional anatomy of serotonergic and dopaminergic systems in obsessive-compulsive disorder in psychotropic-naive patients without comorbidity. [(123)I]beta-CIT binding patterns for dopamine and serotonin transporters in the brain were measured in 15 psychotropic-naive adult outpatients with OCD (no comorbidity) and in 15 pairwise-matched healthy subjects. Volumes of interest were constructed on magnetic resonance imaging scans and coregistered with single photon emission computed tomography scans. Binding ratios were compared, and possible correlations between binding patterns and obsessive-compulsive symptoms were assessed. There were significant differences between patients and healthy subjects in the [(123)I]beta-CIT binding pattern for dopamine transporter in the left caudate and left putamen. Patients had higher binding ratios than healthy subjects. No differences were found in the less specific [(123)I]beta-CIT binding pattern for serotonin transporters in the selected volumes of interest. Hemispheric within-group comparisons revealed no asymmetry effects. The results of our study provide direct evidence for an involvement of the dopaminergic system in the pathophysiology of OCD.

Classification of CD and absorption spectra in the Soret band of H 2TMPyP bound to various synthetic polynucleotides

The binding mode of porphyrins, namely meso-tetrakis( N-methyl pyridinium-4-yl)porphyrin (H 2TMPyP), was classified in this work by absorption and circular dichroism(CD) spectroscopy. The three binding modes of intercalation, minor groove binding and external stacking exhibit their own characteristic absorption and CD spectra. Intercalation occurs for this porphyrin when bound to GC-rich polynucleotides at a low mixing ratio, as expected. This binding mode produces hypochromism and a red shift in the absorption band and a negative CD band in the Soret absorption region. When it is complexed with AT-rich polynucleotides at a low mixing ratio, hypochromism and a red shift in the absorption band and a positive CD peak is apparent, and this species can easily be assigned to the minor groove-binding mode. For both AT- and GC-rich polynucleotides at a high binding ratio, an excitonic CD was apparent. The sign of excitonic CD depends on the order of the DNA bases; the CD spectra of H 2TMPyP complexed with non-alternating homopolymer (disregarding the nature of base pairs, i.e. AT or GC) are characterized by a positive band at short wavelengths followed by a negative band at long wavelengths. In contrast, those complexed with alternating polynucleotide were opposite to those of non-alternating homopolymers.

Enhancement of tumor cell susceptibility to tumor-infiltrating lymphocytes by cisplatin.

Some means of enhancing the susceptibility of tumor cells to tumor-infiltrating lymphocytes (TIL) are required in adoptive immunotherapy. This study was designed to investigate whether or not tumor cell lysis by TIL was enhanced by treatment of the tumor cells with cisplatin, and also to clarify the mechanism of cisplatin's action on tumor cells. Autologous tumor cells and established cancer cell lines, including KATO-III and MKN-28, were used. Cytotoxic activities of TIL, the surface antigens of tumor cells, conjugation of TIL and tumor cells, and the production of TNF alpha from TIL were analyzed. Tumor cells treated with 2 micrograms/ml cisplatin for 12 h in vitro were more susceptible to bulk-cultured TIL and TIL clones. The surface antigens of tumor cells were not altered by the treatment with cisplatin. Cisplatin-treated tumor cells showed a higher binding ratio to TIL than did non-treated tumor cells. The anti-(tumor necrosis factor) (anti-TNF) or anti-TNF receptor antibody blocked the enhancement of cytotoxic activity by cisplatin. Thus, it was clarified that cisplatin enhanced the susceptibility of tumor cells to bulk-cultured TIL and TIL clones. Furthermore, the enhancement of cytotoxic activity by TIL in cisplatin-treated tumor cells was caused by a higher binding ratio to TIL and higher susceptibility to the TNF produced by TIL.

Moraxella boevrei sp. nov., a new Moraxella species found in goats.

Six Moraxella-like strains that formed a phenotypically homogeneous group were isolated from the nasal flora of healthy goats. Total genomic DNA-DNA hybridization, rRNA gene restriction pattern, DNA base composition, and genetic transformation studies were performed to determine the relationships of these bacteria to species belonging to the genus Moraxella and other fastidious gram-negative species. The new group of isolates was very homogeneous, as shown by rRNA gene restriction fragment length patterns (ribotyping), and these organisms displayed high relative binding ratios (RBRs) to each other in DNA-DNA hybridization experiments (RBRs, > or = 58%) but distinctly lower levels of DNA homology with all other species investigated. However, the RBRs obtained with species of the genus Moraxella were higher than the RBRs obtained with all other gram-negative strains examined. Although the new strains had most of the Moraxella bovis phenotypic characteristics except nitrate reduction, quantitative and qualitative genetic transformation data led to the conclusion that they belong to a distinct new cluster in the genus Moraxella. The results of this study, combined with the general morphological and phenotypic profiles of the new strains, are consistent with the creation of a new Moraxella species, for which the name Moraxella boevrei is proposed. Strain 88365 (= ATCG 700022 = CCUG 35435 = NCTC 12925 = CIP 104716) is the type strain of M. boevrei.

Different biodistribution of 99mTc-labelled chimeric mouse-human monoclonal antibody between athymic mice model and human

Biodistribution of chimeric mouse/human monoclonal antibody against non-specific cross-reacting antigen (chNCA Ab) was studied in athymic mice and patients with metastatic bone disease. 99mTc-chNCA Ab showed a high labelling efficiency, stability and also a high binding ratio to human granulocytes. Since NCA showed cross-reactivity with carcinoembryonic antigen (CEA), animal experiments showed that 99mTc-chNCA Ab was accumulated in the xenografted tumour which expressed CEA, suggesting the preserved immunoreactivity of labelled materials. In the clinical study, injected 99mTc-chNCA Ab formed a high molecular weight complex immediately after intravenous administration and was trapped mainly in liver. The first-phase plasma half-life was 6.4 +/- 1.1 min. None of the patients showed adverse reaction or human antimurine or anti-chimeric antibody in their serum. 99mTc-chNCA Ab demonstrated remarkably different biodistribution between patients and the animal model and showed different pharmacokinetics from other murine and chimeric Abs reported previously. For safety HPLC analysis should be performed before clinical radioimmunodetection or radioimmunotherapy by incubating radiolabelled MAb with human serum under strict conditions.

1-acetyl-7-deacetylforskolin: A potential non-specific inactive analog of forskolin for estimation of its specific high-affinity binding and adenylyl cyclase stimulation in vitro

Labeled and unlabeled 1-acetyl-7-deacetylforskolin and forskolin were synthesized by acetylation of 7-deacetylforskolin with labeled and unlabeled acetyl chloride. The binding of 1-acetyl[1-acetyl- 11C]-7-deacetylforskolin ([ 11 C]1-acetyl-7-deacetylforskolin) and [7-acetyl- 11C]forskolin ([ 11C]forskolin) to rat brain membranes was studied using filtration assay. The [ 11C]forskolin binding was decreased with an increasing load of unlabeled forskolin, whereas [ 11C]l-acetyl-7-deacetyl-forskolin binding was always very low, the level of which agreed with that of the non-specific binding in forskolin. However, binding of [7-acetyl- 11C]1,9-dideoxyforskolin, which has been used as a non-specific inactive analog of forskolin, had a higher binding ratio than that of the non-specific binding of forskolin. The binding of [ 11C]forskolin was not affected by an increased load of cold 1-acetyl-7-deacetylforskolin.Forskolin activated adenylyl cyclase (AC) in cultured human endothelial cells, whereas 1-acetyl-7-deacetylforskolin did not. These data show that the 1-acetyl-7-deacetylforskolin lacks specific binding affinity and the ability to stimulate AC, while it has similar physical properties with forskolin. The compound 1-acetyl-7-deacetylforskolin would be a suitable “non-specific inactive analog” of forskolin with which to estimate its specific high-affinity binding capacity and to validate forskolin-specific AC stimulation in vitro.

Moraxella caprae sp. nov., a New Member of the Classical Moraxellae with Very Close Affinity to Moraxella bovis

Eight phenotypically homogeneous Moraxella-like strains were isolated from the nasal flora of healthy goats. Total genomic DNA-DNA hybridization, DNA base composition determination, and genetic transformation studies were performed to determine the relationships of these bacteria to the classical moraxellae. The eight new isolates exhibited very high levels of genetic affinity to Moraxella bovis, as shown by quantitative and qualitative genetic transformation data, and exhibited high DNA-DNA relative binding ratios to each other (63% or more) but lower levels of DNA homology with all of the other species investigated, including the closely related classical moraxellae. Our results, combined with the general morphologic and phenotypic profiles of these organisms, indicate that they should be classified with the classical moraxellae, and we propose the name Moraxella caprae for them. Strain 8897 (= CCUG 33296 [corrected] = NCTC 12877) is the type strain of M. caprae.

The interaction of methylene blue, azure B, and thionine with DNA: Formation of complexes with polynucleotides and mononucleotides as model systems

AbstractThe interactions of methylene blue, azure B, and thionine with calf thymus DNA, [poly (dG‐dC)]2, [poly(dA‐dT)]2, and the constituent mononucleotides 2′‐deoxyguanosine‐5′‐monophosphate(dGMP), 2′‐deoxyadenosine‐5′‐monophosphate(dAMP), 2′‐deoxycytidine‐5′‐monophosphate(dCMP), and thymidine‐5′‐monophosphate(dTMP) have been studied by steady‐state absorption spectroscopy and with equilibrium dialysis. Scatchard plots for binding of the dyes to the nucleic acid polymers were convex downward at low binding ratios, characteristic of intercalation, and binding constants for this mode were calculated under conditions of varying ionic strength. For each of the dyes, binding constants with [poly(dG‐dC)]2 and [poly(dA‐dT)]2 were of the same order of magnitude, so that previously reported (G‐C) preferentially is not very marked. At high binding ratios, the Scatchard plots did not return to the abscissa but curved upward, indicative of a weaker cooperative binding mode, occurring under conditions where the dye is in excess, which is suggested to be external stacking of the dye molecules promoted by the polyanion. The dependence of the absorption spectra on added salt demonstrated a shift in the strong binding mode for the three dyes with [poly(dA‐dT)]2 with increasing ionic strength, while with [poly(dG‐dC)]2 this does not occur. The dyes were found to bind to purine but not pyrimidine mononucleotides with dGMP and dAMP, 1:1 complexes were formed initially and also 1:2 dye/nucleotide complexes with increasing nucleotide concentrations. Under low salt conditions, binding to dAMP was slightly stronger than to dGMP for the three dyes studied, while at high ionic strength, when the binding constants are significantly lower, all binding constants become very similar. Binding to mononucleotides is suggested to be primarily stabilised by π‐π stacking interactions between the planar dyes and the nucleobases: for thionine and azure B there also appears to be H‐bonds between the exocyclic amines and the sugar–phosphates conferring extra stability. Neither increasing the number of phosphate groups on the nucleotides nor changing from deoxyribose to ribose sugars had any significant effect on the binding constants. © 1995 John Wiley & Sons, Inc.

Influence of ligands on the fluorescence polarisation anisotropy of ethidium bound to DNA

The decay of the fluorescence polarisation anisotropy (FPA) of the ethidium-DNA complex has been measured by multifrequency phase fluorometry, in order to study the perturbations induced by the presence of different ligands on the torsional dynamics of DNA, a moderately flexible polymer that undergoes bending (flexure of the helix axis) and torsional (twisting of base pairs) motions. Two probes have been used together with ethidium: an intercalator, chloroquine, and a minor groove binding dye: hoechst 33258. Chloroquine is found to substantially modify the DNA torsional dynamics both in linear and in circularly closed DNAs only at high binding ratios, in agreement with previous reports [Wu et al. Biochem. 27 (1988) 8128]. The effective elastic constant becomes approximately three times larger when the dye/base pairs binding ratio is higher than 0.14. The minor groove ligand hoechst 33258, on the other hand, greatly increases the effective elastic constant to the point that at dye/base pairs ratios larger than 0.5, the effective elastic constant becomes stiffer by several orders of magnitude, suggesting a progressive hindering of internal motions. The results reported here show that DNA torsions are more effectively influenced by groove-binding molecules than by intercalators and it is expected that the large perturbation of the former ligand may be useful when describing the change in the dynamical properties induced by DNA binding proteins. FPA in the frequency domain, the technique adopted throughout this work, has proved to be very sensitive to changes of the elastic constant that describes DNA torsional dynamics. Several computer simulations performed in order to predict the FPA time decay of intercalated ethidium have led to good agreement with the observed results.

Novel stereoselective incorporation and hydrolysis of long-chain amino-acid substrates by vesicular membrane systems which include tri- or tetra-peptide catalysts

In the vesicular membrane systems comprising N,N-didodecyl-N,N-dimethylammonium bromide and tri- or tetra-peptide catalysts (pH 7.68; ionic strength µ= 0.15; 298 K), Z-L-Leu-L-His-L-Leu (or Z-L-Leu-L-His-L-Leu-L-Leu), having a hydrophobic terminal L-leucine (or L-leucyl-L-leucine) unit, contributed to the stereoselective binding of long-chain p-nitrophenyl N-hexadecanoyl-L(and D)-phenyalanates with the remarkably high binding ratio Kb(L/D)= 11 (or 7.3) and overall stereoselectivity [kcat(L/D)= 35 (or 8.1)].

The interaction of rhodium(III) with DNA by circular dichroism studies. Comparative behaviour of dimethyltin(IV) and thallium(I)

The effect of RhCl3 on DNA helix conformation was studied by circular dichroism spectroscopy and compared to the behaviour of Me2SnCl2 and TINO3; the latter metal-DNA systems have been further studied by ultraviolet and thermal denaturation techniques. Both rhodium(III) and tin(IV) react with the bases of DNA and induce appreciable alterations of the double-helical conformation. Rhodium(III) at low binding ratio causes a stabilization of the DNA B conformation, while at higher structural changes are interpreted in terms of a B to C transition. Tin(IV) produces a ψ form geometry; only at high binding ratios does interaction with the phosphate groups seem to occur. The data for thallium(I) are interpreted in terms of a preferential interaction with the phosphate groups of the DNA chain, causing a stabilization of the double-helical structure of DNA.

Binding of 4',6-diamidino-2-phenylindole (DAPI) to AT regions of DNA: evidence for an allosteric conformational change.

The interaction of 4',6-diamidino-2-phenylindole (DAPI) with several double-helical poly- and oligonucleotides has been studied in solution using optical spectroscopic techniques: flow linear dichroism (LD), induced circular dichroism (CD), and fluorescence spectroscopy. In AT-rich sequences, where DAPI is preferentially bound, LD indicates that the molecule is edgewise inserted into the minor groove at an angle of approximately 45 degrees to the helix axis. This binding geometry is found for very low as well as quite high binding ratios. The concluded geometry is in agreement with that of the DAPI complex in a crystal with the Drew-Dickerson dodecamer, and the DAPI complex with this dodecamer in solution is verified to have an ICD spectrum similar to that of the complex with [poly(dA-dT)]2 at low binding ratios. The observation of two types of CD spectra characteristic for the binding of DAPI to DNA, and also for the interaction with [poly(dA-dT)]2, demonstrates that the first binding mode, despite its low apparent abundance (a few percent), is not due to a specific DNA site. The effect may be explained in terms of an allosteric binding such that when DAPI molecules bind contiguously to the AT sequence the conformation of the latter is changed. The new conformation, which according to LD appears to be stiffer than normal B-form DNA, is responsible for the second type of induced CD spectrum in the DAPI chromophore. Although the spectroscopic results indicate a change of DNA conformation, consistent with an allosteric binding model, they do not explicitly require any cooperativity, but accidental neighbors could also explain the data.

Evaluation of the use of chromogenic and fluorogenic substrates in solid-phase enzyme linked immunosorbent assays (ELISA)

Fluorogenic and chromogenic substrates were used in direct and trapping enzyme-linked immunosorbent assays (ELISA) for the detection of mouse IgG and foot-and-mouth disease virus (FMDV). The detection limits for both antigens were compared using different combinations of enzymes and substrates. Various times and concentrations of chemicals were used to obtain maximum sensitivity for both systems. Similar sensitivities were found using fluorogenic and chromogenic substrates. Tetramethyl benzidine substrate for horse-radish peroxidase enzyme conjugates was found to attain the highest sensitivity levels for chromogenic assays (0·12 ng IgG/ml and 1·0 ng/ml FMDV respectively), after 10 min incubation. Of the two fluorogenic enzyme/substrates studied, B-galactosidase was the most sensitive but required extended incubation times (2–3 h) as compared with chromogenic systems. Special microplates for fluoro-immunoassay (FIA) were compared with conventional microplates and no advantage was found to justify their use. An alkaline phosphatase anti-guinea-pig conjugate was used to confirm the equivalence of fluorogenic and chromogenic substrates in terms of sensitivity. A comparison of the amount of signal generated using various concentrations of enzyme in the absence of antigen was made for two different alkaline phosphatase conjugates to obtain theoretical sensitivity limits. One possible advantage of fluorogenic substrates is that high binding ratio can improve the confidence in discrimination of positive results.

Molecular recognition between oligopeptides and nucleic acids. DNA sequence specificity and binding properties of an acridine‐linked netropsin hybrid ligand

The binding to DNA of a mixed function ligand (NETGA) is described, in which a potential intercalating group, an acridine moiety, is incorporated at the carboxyl terminus of the minor groove binding oligopeptide netropsin skeleton. Scatchard analysis of absorption data provided evidence of two modes of binding to DNA with K1 = 9.1 x 10(5) M-1 at low r values (0.003-0.1), and a binding site size n = 10, indicative of binding of both moeities. At high binding ratios (greater than 0.1), K2 = 0.9 x 10(5) M-1 and n = 5 corresponding to external binding. Complementary strand MPE footprinting on a pBR322 restriction fragment showed NETGA binds to 5'-AAAT like netropsin. It causes enhanced cleavage by MPE, particularly at G-C rich sequences and remote from the preferred binding sites. Viscometry measurements provided evidence for biphasic modes of the two binding portions of NETGA. Fluorescence polarization and linear dichroism measurements were in accord with distinct modes of interaction of the acridine (intercalation) and oligopeptide (minor groove binding) portions of NETGA. LD measurements on NETGA indicate that the oligopeptide moiety (netropsin-like) has an orientation typical of minor groove binders, whereas the degree of intercalation of the acridine group is decreased by association of the oligopeptide moiety.

Reduced DNA flexibility in complexes with a type II DNA binding protein

We studied internal molecular motions in Bacillus subtilis phage SPO1 DNA using the time-resolved fluorescence polarization anisotropy (FPA) of intercalated ethidium. The torsional flexibility of this (hydroxymethyl)uracil-containing DNA is very similar to that of naturally occurring thymine-containing DNAs, as judged from fits of the time-resolved FPA decay to an elastic DNA model. Binding of transcription factor 1 (TF1), a type II procaryotic DNA binding protein encoded by the phage SPO1, enhances the FPA, indicating a substantial decrease in the average DNA torsional flexibility in the DNA-TF1 complex. The FPA increase is correlated with a reduced ethidium binding affinity. The effects can be noticed at TF1 binding ratios less than 1 TF1 dimer/500 DNA base pairs, and the measured torsional rigidity at high TF1 binding ratios (1 TF1 dimer/15-20 DNA base pairs) is about 7 times greater than in the absence of TF1. On the basis of a discussion of various mechanisms for the observed effect we argue that it is due to protein-induced DNA bending at low binding densities although other explanations are also possible. This interpretation might have implications for understanding the biological function of TF1.

Changes in the Antibacterial Activity of Melanin-Bound Drugs

Affinity to melanin and changes of antibacterial activity of melanin-bound drugs were examined in 11 drugs, all of which showed an affinity for melanin. The highest melanin-binding ratio was seen in aminoglycosides. Among these, sisomicin sulfate (SISO) had the highest binding ratio (95.5%). The melanin-binding ratios seen in sulbenicillin sodium (SBPC), cephalosporins and fluoroquinolones were relatively low during the early phase of the reaction, but increased with time. The highest ratio seen in cefazolin sodium was 60.1%. Melanin-bound aminoglycosides showed a reduction in their antibacterial activity with both Bacillus subtilis and Escherichia coli. The reduction rate in the antibacterial activity of melanin-bound SISO against B. subtilis was 50.0% and that against E. coli was 43.0%. No changes in antibacterial activity were seen in the other 6 drugs bound to melanin.

The suitability of different microtitre plates for detection of antibody to virus antigens by indirect ELISA

To optimize enzyme linked immunosorbent assays (ELlSAs) for the detection of virus-specific antibodies, a range of commercially available microtitre plates was evaluated for their ability to bind virus antigen. Rinderpest virus and foot-and-mouth disease virus were investigated as target antigens. Binding capacity for antigen, binding ratios (attachment of specific antibody versus that of non-immune antibody) and the variation in the results of the tests within and between plates were measured. Binding capacity was found to be greater with rinderpest virus (RPV) antigen than with foot-and-mouth disease virus (FMDV) antigen, although higher binding ratios were obtained with FMDV antigen. Variation within and between plates was generally less with RPV antigen than with FMDV antigen. One plate could not be said to out-perform the other plates in all tests. For our purpose, that is the detection of monoclonal antibody production against a variety of virus antigens, a number of plates were found to be suitable (e.g. Dynatech M129B, Flow 77-172-05 and Nunc 4-39454). The differences in the performances of the microtitre plates with these two virus antigens highlights the need for consideration of the solid phase as part of the standardization procedures for ELISAs.

Characterization of interaction between DNA and 4',6-diamidino-2-phenylindole by optical spectroscopy.

We have examined the interaction between 4',6-diamidino-2-phenylindole (DAPI) and DNA using flow linear dichroism (LD), circular dichroism (CD), and fluorescence techniques. We show the presence of two spectroscopically distinct binding sites at low binding ratios with saturation values of 0.025 and 0.17, respectively. In both sites DAPI is bound with its long axis approximately parallel to the grooves of the DNA helix. Resolution of CD spectra shows that an exciton component is present at higher binding ratios, which we attribute to the interaction of two accidentally close-lying DAPI molecules. We also find evidence that DAPI, at least in the high-affinity site, binds preferentially to AT-rich regions. From the spectroscopic results, supported by structural considerations, we can completely exclude that DAPI is bound to DNA by intercalation. Binding geometries and site densities are consistent with a location of DAPI in the grooves of DNA, with the high-affinity site most probably in the minor groove.

Interaction of anthracycline antibiotics with biopolymers: comparative studies of DNA binding and antimicrobial activity of rhodomycin-type anthracycline antibiotics.

The binding of the anthracyclines beta-rhodomycin-I and beta-rhodomycin-II to calf thymus DNA was investigated by both equilibrium and kinetic methods taking into account ligand dimerization (ionic strength I = 0.2 M, pH 6.0). The analysis was based upon a cooperative single-step binding mechanism with overlapping of potential binding sites on a linear homogeneous lattice. Equilibrium binding parameters were estimated from spectrophotometric titration experiments by means of a nonlinear fitting program. The results were compared with those obtained previously for the related antibiotic iremycin and were complemented by kinetic parameters determined from temperature-jump experiments at high binding ratio. The binding constants and the mean attachment times of the drugs were found to increase in the serial order iremycin, beta-rhodomycin-I and beta-rhodomycin-II, which is in line with their increasing antimicrobial activity on Bacillus subtilis ATCC 6633.