Binding Scatchard Research Articles

Recognition of human telomeric G-quadruplex DNA by berberine analogs: effect of substitution at the 9 and 13 positions of the isoquinoline moiety.

G-quadruplex forming sequences are widely distributed in human genome and serve as novel targets for regulating gene expression and chromosomal maintenance. They offer unique targets for anticancer drug development. Here, the interaction of berberine (BC) and two of its analogs bearing substitution at 9 and 13-position with human telomeric G-quadruplex DNA sequence has been investigated by biophysical techniques. Both the analogs exhibited several-fold higher binding affinity than berberine. The Scatchard binding isotherms revealed non-cooperative binding. 9-ω-amino hexyl ether analog (BC1) showed highest affinity (1.8 × 10(6) M(-1)) while the affinity of the 13-phenylpropyl analog (BC2) was 1.09 × 10(6) M(-1). Comparative fluorescence quenching and polarization anisotropy of the emission spectra gave evidence for a stronger stacking interaction of the analogs compared to berberine. The thiazole orange displacement assay has clearly established that the analogs were more effective in displacing the end stacked dye in comparison to berberine. However, the binding of the analogs did not induce any major structural perturbation in the G-quadruplex structure, but led to higher thermal stability. Energetics of the binding indicated that the association of the analogs was exothermic and predominantly entropy driven phenomenon. Increasing the temperature resulted in weaker binding; the enthalpic contribution increased and the entropic contribution decreased. A small negative heat capacity change with significant enthalpy-entropy compensation established the involvement of multiple weak noncovalent interactions in the binding process. The 9-ω-amino hexyl ether analog stabilized the G-quadruplex structure better than the 13-phenyl alkyl analog.

Electrochemical square scheme analysis of macromolecule–electroactive ligand interactions, and its application to DNA binding

The physico-chemical characteristics of electroactive ligand–macromolecules interactions, such as the association constant or the binding stoichiometry, can be determined by voltammetric titration experiments. The interpretation of the electrochemical behaviour is however not straightforward, because: (i) a single macromolecule can contain various binding sites for a given ligand, so multiple complexes (1:1, 2:1, 3:1,…) should be considered; (ii) the free (unbound) and bound ligands have very different diffusion coefficients; (iii) the association/dissociation kinetics can have a significant impact on the voltammetric response.This contribution provides a theoretical framework for the analysis of such titration experiments, in the limiting cases of very fast (“dynamic equilibrium”) and very slow (“static equilibrium”) association/dissociation kinetics. Using a statistical site binding model (“Scatchard binding”) and introducing a conditional association constant, the problem can be treated with a simple electrochemical square scheme. The theoretical treatment is explicitly solved for popular voltammetric methods, but is sufficiently general to be adapted to voltammetries not considered here. Employing voltammetric methods displaying peak-shaped voltammograms, the “dynamic equilibrium” versus “static equilibrium” situations are readily diagnosed by the occurrence of an “isogalvanic point” in the latter case.The tools developed in the theoretical section are used to analyse experimental titration experiments performed on the [Ru(NH3)6]3+–DNA system. Two different DNA sequences are studied, and the results show that these sequences have distinct binding stoichiometries and association/dissociation kinetics with [Ru(NH3)6]3+.

Effects of salting-in interactions on macromolecule diffusiophoresis and salt osmotic diffusion.

Macromolecule diffusiophoresis (i.e., macromolecule migration induced by a salt concentration gradient) in water and salt osmotic diffusion (i.e., salt migration induced by a macromolecule concentration gradient) are two cross-diffusion mechanisms caused by macromolecule-salt interactions. We investigated the effect of salting-in interactions on the behavior of these two cross-diffusion mechanisms. Our results are distinct from those previously obtained in the case of salting-out interactions. Cross-diffusion was experimentally characterized by Rayleigh interferometry at 25 °C. Specifically, multicomponent diffusion coefficients were measured for a neutral polymer, polyethylene glycol (molar mass, 20 kg/mol), in aqueous solutions of three thiocyanate salts (NaSCN, KSCN, and NH₄SCN) as a function of salt concentration at low polymer concentration (0.5% w/w). Our results on salt osmotic diffusion, which were qualitatively different from those previously obtained for salting-out salts, were used to quantitatively characterize the strength of salting-in interactions. The behavior of polymer diffusiophoresis as a function of salt concentration and cation type reveals that polymer chains have an extrinsic negative charge, consistent with anion binding being the cause of salting-in interactions. To quantitatively examine the effect of anion binding on salt osmotic diffusion and polymer diffusiophoresis, we developed a theoretical model based on the linear laws of nonequilibrium thermodynamics for diffusion, the Scatchard binding model, and particle electrophoresis. This work contributes to the understanding of the multifaceted effects of molecular interactions on cross-diffusion mechanisms, salting-in interactions, and the Hofmeister series.

The Influence of the Form of tRNA on Complex Formation with Porphyrins

In the current work we have investigated the interaction of meso-tetra-(4N-oxyethylpyridyl) porphyrin (TOEPyP4) and its Zn(II), Cu(II), -metallocomplexes with tRNA which has 2 forms: hairpin structure and spatial reversed “L” structure. The interaction of tRNA from E.Coli with porphyrins is studied by UV/Vis Spectrophotometry and Circular Dichroism methods.The measurements were performed in 0.1 BPSE and 1BPSE buffers (1 BPSE = 6 mM Na2HPO4+ 2 mM NaH2PO4 +185 mM NaCl + 1 mM Na2EDTA), correspondingly μ = 0.02M and μ = 0.2M, pH 6.57. (tRNA has hairpin form at μ = 0.02M and reversed “L” structure, when μ = 0.2M).From the spectrophotometric titration datas the Scatchard binding isotherms for porphyrin-tRNA complexes are built and binding parameters are calculated (N - the number of binding sites per molecule of tRNA, and K - the binding constant).In case of tRNA's hairpin structure for the values of induced CD spectra (at 400-470 nm) for complexes tRNA with TOEPyP4 and CuTOEPyP4 there is an optimum concentration of porphyrins at which the anisotropy of system is maximal. For complexes of ZnTOEPyP4 with tRNA the induced CD spectra are essentially different. The induced CD spectra of complex change a sign and continue to grow (remaining negative) starting from a certain relative concentration. This unusual ICD spectra profile is found for all three porphyrin-tRNA complexes in case of tRNA's reversed “L” structure. It is possible that at high relative concentration of porphyrins the liquid crystal form may exist in the solution.The binding constants with tRNA in case of hairpin form are an order of magnitude greater than in case of reversed “L” structure. It means that this porphyrin interacts stronger with tRNA when it has hairpin form.

The Interaction of Zn Metalloporphyrin with Different Forms of tRNA

The interaction of meso-tetra-(4N-oxyethylpyridyl)porphyrin (TOEPyP4) and its Zn(II), Cu(II), -metallocomplexes with tRNA at low ionic strength we have investigated earlier [1]. In this work we have studied the binding of ZnTOEPyP4 with tRNA which has 2 forms: hairpin structure and spatial reversed “L” structure. The interaction of tRNA from E.Coli with ZnTOEPyP4 is studied by UV/Vis Spectrophotometry and Circular Dichroism methods.The measurements were performed in 0.1 BPSE and 1BPSE buffers (1 BPSE = 6 mM Na2HPO4+ 2 mM NaH2PO4 +185 mM NaCl + 1 mM Na2EDTA), correspondingly μ = 0.02M and μ = 0.2M, pH 6.57. (tRNA has hairpin form at μ = 0.02M and reversed “L” structure, when μ = 0.2M.)From the spectrophotometric titration datas the Scatchard binding isotherms for porphyrin-tRNA complexes are built and binding parameters are calculated (the number of binding sites per molecule of tRNA, and the binding constant).In both cases of tRNA's structure the induced CD spectra (at 400-470 nm) for complex of ZnTOEPyP4 with tRNA are very unusual. The induced CD spectra of complex change a sign and continue to grow (remaining negative) starting from a certain relative concentration. In case of tRNA's reversed “L” structure the intensity of ICD spectra for the same relative concentration of porphyrin is twice bigger than in case of hairpin structure. It is possible that at high relative concentration of porphyrins the liquid crystal form may exist in the solution.The binding constants with tRNA in case of hairpin form are an order of magnitude greater than in case of reversed “L” structure. It means that this porphyrin interacts stronger with tRNA when it has hairpin form.Reference1. Y. Dalyan, I. Vardanyan, A. Chavushyan,G. Balayan. J of Biomol Structure & Dynamics 28, 123-131 (2010).

Discovery of a Novel Class of Bicyclo[3.1.0]hexanylpiperazines as Noncompetitive Neuropeptide Y Y1 Antagonists.

A novel class of bicyclo[3.1.0]hexanylpiperazine neuropeptide Y (NPY) Y1 antagonists has been designed and synthesized. Scatchard binding analysis showed these compounds to be noncompetitive with [(125)I]PYY binding to the Y1 receptor. The most potent member, 1-((1α,3α,5α,6β)-6-(3-ethoxyphenyl)-3-methylbicyclo[3.1.0]hexan-6-yl)-4-phenylpiperazine (2) had an IC50 = 62 nM and displayed excellent oral bioavailability in rat (% F po = 80), as well as good brain penetration (B/P ratio = 0.61). In a spontaneous nocturnal feeding study with male Sprague-Dawley rats, 2 significantly reduced food intake during a 12 h period.

9-O-N-aryl/arylalkyl amino carbonyl methyl substituted berberine analogues induce self-structure in polyadenylic acid

This manuscript describes the interaction of 9-O-substituted analogues of the plant alkaloid berberine with single stranded (ss) poly(A). Three new analogues of berberine with aryl/arylalkyl amino carbonyl methyl substituents at the 9-position of the isoquinoline chromophore were evaluated for their binding to ss poly(A) by a wide variety of biophysical techniques. The results indicated that these analogues exhibited several-fold higher binding affinities than berberine. The Scatchard binding isotherms revealed that all the analogues retained the cooperative binding mode of berberine. Circular dichroism and FTIR studies unequivocally established the strong interaction potential of the analogues to ss poly(A). Optical melting and the unique dilution experiments revealed that these analogues induced self-structure formation in poly(A) and the nature of the substituent was important for the ease of formation of self-structure. Energetics of the binding suggested an entropy driven binding for the analogues in sharp contrast to the enthalpy driven binding of berberine. The introduction of the aryl/arylalkyl amino carbonyl methyl substituent thus switched the enthalpy driven binding of berberine to entropy dominated binding. Salt and temperature dependent calorimetric studies established the involvement of multiple weak noncovalent interactions in the binding process. The study also revealed that the spacer length at the 9-position had an important role in the self-structure induction.

Epidermal growth factor receptor (EGFR) status and K-Ras mutations in colorectal cancer

Background: In advanced colorectal cancer, K-Ras somatic mutations predict resistance to mAbs targeting epidermal growth factor receptor (EGFR). Relationships between K-Ras mutations and EGFR status have not been examined so far. We analyzed relationships between K-Ras mutations and EGFR tumoral status based on EGFR germinal polymorphisms, gene copy number and expression.Methods: Eighty colorectal tumors (stage 0–IV) and 39 normal mucosas were analyzed. K-Ras mutations at codons 12 and 13 were detected by a sensitive enrichment double PCR–restriction fragment length polymorphism (RFLP) assay. EGFR gene polymorphisms at positions -216G>T, -191C>A and 497Arg>Lys were analyzed (PCR–RFLP), along with CA repeat polymorphism in intron 1 (fluorescent genotyping) and EGFR gene copy number (PCR amplification). EGFR expression was quantified by Scatchard binding assay.Results: The number of EGFR high-affinity sites, dissociation constant (Kd), gene copy number, intron 1, -216G>T, -191C>A or 497Lys>Arg genotypes was not different between K-Ras-mutated or K-Ras-non-mutated tumors. No relationship was observed between any of the analyzed EGFR genotypes and EGFR expression. EGFR expression was not related to gene copy number. EGFR gene copy number in tumor and normal tissue was not correlated. The mean value of the tumor/normal mucosa gene copy number ratio was 1.16.Conclusions: Present data clearly show that EGFR status is independent of K-Ras mutations in colorectal tumors.

A human monoclonal autoantibody to breast cancer identifies the PDZ domain containing protein GIPC1 as a novel breast cancer-associated antigen

BackgroundWe have been studying the native autoimmune response to cancer through the isolation of human monoclonal antibodies that are cancer specific from cancer patients. To facilitate this work we previously developed a fusion partner cell line for human lymphocytes, MFP-2, that fuses efficiently with both human lymph node lymphocytes and peripheral blood lymphocytes. Using this unique trioma fusion partner cell line we isolated a panel of autologous human monoclonal antibodies, from both peripheral blood and lymph node lymphocytes, which are representative of the native repertoire of anti-cancer specific antibodies from breast cancer patients.MethodsThe current study employs immunocytochemistry, immunohistochemistry, Western blot analysis as well as Northern blots, Scatchard binding studies and finally SEREX analysis for target antigen identification.ResultsBy application of an expression cloning technique known as SEREX, we determined that the target antigen for two monoclonal antibodies, 27.B1 and 27.F7, derived from lymph node B-cells of a breast cancer patient, is the PDZ domain-containing protein known as GIPC1. This protein is highly expressed not only in cultured human breast cancer cells, but also in primary and metastatic tumor tissues and its overexpression appears to be cancer cell specific. Confocal microscopy revealed cell membrane and cytoplasmic localization of the target protein, which is consistent with previous studies of this protein.ConclusionWe have determined that GIPC1 is a novel breast cancer-associated immunogenic antigen that is overexpressed in breast cancer. Its role, however, in the initiation and/or progression of breast cancer remains unclear and needs further clarification.

Phenotypic and genotypic characteristics of epidermal growth factor receptor (EGFR) in colorectal cancer patients

4110 Background: Colorectal tumors express EGFR and are responsive to anti-EGFR therapies. However, there is no tumoral predictive factor for anti-EGFR therapy in colorectal cancer and EGFR gene copy number is currently a good candidate. The aim of this study was to examine relationships between EGFR germinal polymorphisms, EGFR gene copy number and EGFR expression. Methods: Eighty primary colorectal tumors were analyzed along with 39 normal mucosas. Tumor staging was : 4 stage 0, 13 stage I, 22 stage II, 23 stage III and 18 stage IV. EGFR -216G>T and -191C>A genotypes were analyzed by PCR-RFLP, CA-repeats polymorphism in intron 1 by fluorescent genotyping and gene copy number was measured by PCR amplification. EGFR expression was quantified with the reference Scatchard binding assay giving high- and low-affinity sites along with Kd values (Francoual M et al. Ann Oncol 17, 2006). Results: The number of CA- repeats varied from 14 to 21. Considered genotypes were superimposable between tumoral and normal tissues. A linkage disequilibrium was noted between -216G>T and -191C>A genotypes (p = 0.011). CA-repeats polymorphism and -216G>T genotype were not independent (p = 0.002). No relationship was observed between any of the analyzed EGFR genotypes and EGFR expression. EGFR expression was not related to gene amplification. EGFR gene amplification in tumor and normal tissue varied over a 4.7- and 2.9-fold range, respectively, and were not correlated. The mean value of the tumor/normal mucosa amplification ratio was 1.16 (range 0.55–2.68) and 14% of patients exhibited lower amplification in the tumor relative to the normal mucosa (ratio < 0.75). The mean ratio of high-affinity sites between tumor and normal mucosa was 1.20 (range 0.03–13.33). Conclusions: In colorectal tumors, neither EGFR gene amplification nor EGFR germinal gene polymorphisms influenced EGFR expression quantified with a specific ligand-binding assay. No significant financial relationships to disclose.

Optimizing the formulation of a myoglobin molecularly imprinted thin-film polymer—formed using a micro-contact imprinting method

Thin-film myoglobin molecularly imprinted polymers have been fabricated using a micro-contact approach. By initially selecting the cross-linker on the basis of it having a minimal recognition for the template and using this as a starting point for functional monomer selection, we have produced myoglobin imprinted polymers with exceptionally high selectivities.The affinity of the polymers, for myoglobin, when prepared with a variety of different cross-linkers and no functional monomer was evaluated. Of these, tetraethylene glycol dimethacrylate (TEGDMA) exhibited the lowest affinity for the template species. Methyl methacrylate (MMA) was chosen as the functional monomer as when it was used in conjunction with TEGDMA, it exhibited maximum selectivity for the template compared to polymers made with other functional monomers.With a MMA to TEGDMA ratio of 1 to 3, the myoglobin molecularly imprinted polymer adsorbed 15.03±0.89×10−11mole/cm2 of template from a 5.68×10−7M myoglobin solution, compared to 2.58±0.02×10−11mole/cm2 for a polymer of similar composition, but formed in the absence of a template. Various washing conditions, using alkaline media to remove the template, were investigated. An extraction solvent comprising 2wt.% SDS and 0.6wt.% NaOH used at 80°C for 30min was shown to give the highest imprinting factor i.e. 5.83 with 72.82% myoglobin removal.The saturation kinetics of template binding to the thin-film MIP were examined and found to display a simple two-phase profile typical of non-cooperative binding. A Scatchard binding plot showed the dissociation constant (Kd) for the specific binding phase to be 3.4×10−7M and the binding site capacity to be 7.24×10−11mole/cm2. For the non-specific binding phase, Kd was found to be 1.355×10−5M and the binding site capacity was determined as 9.62×10−10mole/cm2.Selectivity experiments were carried out in both single protein and binary protein systems all using a total protein concentration of 5.68×10−7M. The molar ratio of adsorbed myoglobin to IgG, HSA and hemoglobin was found to 115.5, 230.9 and 2.5, respectively. While, in binary competition systems, myoglobin selectivity to IgG, HSA and hemoglobin was, respectively, 94.18, 98.21 and 61.09%. Rebinding in natural biological matrices, i.e. human serum or urine, showed the imprinted films to have significantly greater uptake than non-imprinted films. Re-binding in undiluted urine was found to be a facile process, with the imprinting factor, i.e. the ratio of MIP to NIP binding, being determined as 37.4.

Affinity evaluation of gelatin for hepatocyte growth factor of different types to design the release carrier

The objective of this study was to investigate the physicochemical interaction of hepatocyte growth factor (HGF) and its variant with 5 amino-acid residues deleted (dHGF) with an acidic gelatin for the design of factors release from the gelatin hydrogel. When the interaction of HGF or dHGF with gelatin-immobilized agarose beads was evaluated by Scatchard binding assay, the dissociation constant of dHGF was higher than that of HGF, although the two proteins had a similar binding ratio. dHGF was released more rapidly from the hydrogel of acidic gelatin than HGF. In vivo release study with 125I-labeled HGF or dHGF in mice subcutis showed that HGF was released from the gelatin hydrogel as a result of hydrogel degradation. In contrast, dHGF was rapidly released by a simple diffusion from the gelatin hydrogel. From electrophoresis experiments, mixing with the acidic gelatin enabled HGF to complex and suppressing the trypsin-digested molecular weight loss, in marked contrast to that of dHGF. In addition, the percentage of HGF recognized by the antibody was reduced by the gelatin complexation, but that of dHGF was not. We conclude that unlike dHGF, HGF has a strong affinity for the acidic gelatin, resulting in the controlled release of HGF accompanied with hydrogel degradation of the release carrier.

Interaction of hepatocyte growth factor with gelatin as the carrier material

The objective of this study was to physicochemically investigate the interaction between hepatocyte growth factor (HGF) and acidic gelatin compared with that between HGF and basic gelatin or heparin. Gelatin- or heparin-immobilized agarose beads were prepared and HGF interaction with them was evaluated by Scatchard binding assay. The dissociation constant of HGF with the acidic gelatin was about 2–3 orders of magnitude higher than that of heparin. The cell proliferation assay revealed that the proliferation promotion activity of HGF complexed with the acidic gelatin was detected, although it was lower than that of original HGF. The ability of HGF to enhance the cell proliferation was reduced by the trypsin treatment, although the extent of the reduction was significantly suppressed by HGF complexation with acidic gelatin. Electrophoresis experimentally confirmed enhanced resistance to the molecular mass loss of HGF by gelatin complexation. Moreover, the recognized level of an antibody to HGF was reduced by the complexation with the acidic gelatin, indicating that the acidic gelatin is present around HGF molecules. It is possible that the HGF molecule is covered with the acidic gelatin, resulting in protection from enzymatic digestion.

In vitro transfection of plasmid DNA by cationized gelatin prepared from different amine compounds

The objective of this paper is to compare the in vitro transfection efficiency of a luciferase plasmid DNA using cationized gelatin prepared from different amine compounds. The compounds used here were ethylenediamine, putrescine, spermidine and spermine, chemically introduced to the carboxyl group of gelatin for the cationization. Complexation of the cationized gelatin with the plasmid DNA was performed by simply mixing the two materials at various N+/P− mixing ratios (the molar number ratio of amino groups of gelatin to the phosphate groups of DNA) in aqueous solution. Gel retardation studies revealed that the formation of cationized-gelatin–plasmid DNA complexes depended on the N+/P− mixing ratio. The stronger interaction of plasmid DNA with the cationized gelatin of spermine compared to the other cationized gelatins was observed by an ethidium bromide intercalation assay and Scatchard binding analysis. When the transfection efficiency of plasmid DNA complexed with the various cationized gelatins at different N+/P− mixing ratios was evaluated for mouse L929 fibroblasts, the highest transfection efficiency was observed for the complex prepared from the cationized gelatin of spermine at a N+/P− mixing ratio of 2. The present study indicates that there is an optimal N+/P− mixing ratio and a type of amine compound or cationization extent of cationized gelatin to enhance the transfection efficiency of plasmid DNA.

RECQ1 Helicase Interacts with Human Mismatch Repair Factors That Regulate Genetic Recombination*[boxs

Understanding the molecular and cellular functions of RecQ helicases has attracted considerable interest since several human diseases characterized by premature aging and/or cancer have been genetically linked to mutations in genes of the RecQ family. Although a human disease has not yet been genetically linked to a mutation in RECQ1, the prominent roles of RecQ helicases in the maintenance of genome stability suggest that RECQ1 helicase is likely to be important in vivo. To acquire a better understanding of RECQ1 cellular and molecular functions, we have investigated its protein interactions. Using a co-immunoprecipitation approach, we have identified several DNA repair factors that are associated with RECQ1 in vivo. Direct physical interaction of these repair factors with RECQ1 was confirmed with purified recombinant proteins. Importantly, RECQ1 stimulates the incision activity of human exonuclease 1 and the mismatch repair recognition complex MSH2/6 stimulates RECQ1 helicase activity. These protein interactions suggest a role of RECQ1 in a pathway involving mismatch repair factors. Regulation of genetic recombination, a proposed role for RecQ helicases, is supported by the identified RECQ1 protein interactions and is discussed.

Five-member thio-heterocyclic fused naphthalimides with aminoalkyl side chains: intercalation and photocleavage to DNA

Novel five-member thio-heterocyclic fused naphthalimides with aminoalkyl side chains were designed, synthesized and evaluated. These compounds have high Scatchard binding constants. They could damage DNA (supercoiled pBR322) from form I (closed) to II (nicked) at a concentration as low as 10 μM and from form I to form III at a concentration of 50 μM. The results implied that the influence of intercalating ability of chromophores on photocleaving ability of photocleavers depended on the mechanism of photocleavage.

Aqueous compatible polymers in bionanotechnology

Core-shell molecularly imprinted particles (CS-MIPs) have been synthesised using the technique of emulsion polymerisation with caffeine and theophylline being used in the surface template polymerisation with ethylene glycol dimethacrylate and oleylphenyl hydrogen phosphate. A radiolabelling study with caffeine-8-14C showed that the template was completely located at the particle surface during polymerisation. Caffeine could be specifically bound to a caffeine-imprinted CS-MIP to give a biphasic Scatchard binding curve, whereas the binding profile to a theophylline-imprinted CS-MIP was monophasic. The nanoparticles have the potential to be used in the molecular recognition of small molecules in a complex biological matrix. Water soluble highly-branched imidazole end-chain functionalised polymers of nanodimensions have also been synthesised via reversible addition-fragmentation chain transfer polymerisation. The polymers have lower critical solution temperatures which occur at sub-ambient temperatures and have proven useful in the affinity precipitation of proteins which are particularly temperature sensitive, e.g. the histidine-tagged protein fragment BRCA1. An overview of both of these areas of research is described outlining the diversity of these aqueous compatible polymers in molecular recognition processes at the nanoscale.

Cyclic stretch and hypertension induce retinal expression of vascular endothelial growth factor and vascular endothelial growth factor receptor-2: potential mechanisms for exacerbation of diabetic retinopathy by hypertension.

Systemic hypertension exacerbates diabetic retinopathy and other coexisting ocular disorders through mechanisms that remain largely unknown. Increased vascular permeability and intraocular neovascularization characterize these conditions and are complications primarily mediated by vascular endothelial growth factor (VEGF). Because systemic hypertension increases vascular stretch, we evaluated the expression of VEGF, VEGF-R2 (kinase insert domain-containing receptor [KDR]), and VEGF-R1 (fms-like tyrosine kinase [Flt]) in bovine retinal endothelial cells (BRECs) undergoing clinically relevant cyclic stretch and in spontaneously hypertensive rat (SHR) retina. A single exposure to 20% symmetric static stretch increased KDR mRNA expression 3.9 +/- 1.1-fold after 3 h (P = 0.002), with a gradual return to baseline within 9 h. In contrast, BRECs exposed to cardiac-profile cyclic stretch at 60 cpm continuously accumulated KDR mRNA in a transcriptionally mediated, time-dependent and stretch-magnitude-dependent manner. Exposure to 9% cyclic stretch increased KDR mRNA expression 8.7 +/- 2.9-fold (P = 0.011) after 9 h and KDR protein concentration 1.8 +/- 0.3-fold (P = 0.005) after 12 h. Stretched-induced VEGF responses were similar. Scatchard binding analysis demonstrated a 180 +/- 40% (P = 0.032) increase in high-affinity VEGF receptor number with no change in affinity. Cyclic stretch increased basal thymidine uptake 60 +/- 10% (P < 0.001) and VEGF-stimulated thymidine uptake by 2.6 +/- 0.2-fold (P = 0.005). VEGF-NAb reduced cyclic stretch-induced thymidine uptake by 65%. Stretched-induced KDR expression was not inhibited by AT1 receptor blockade using candesartan. Hypertension increased retinal KDR expression 67 +/- 42% (P < 0.05) in SHR rats compared with normotensive WKY control animals. When hypertension was reduced using captopril or candesartan, retinal KDR expression returned to baseline levels. VEGF reacted similarly, but Flt expression did not change. These data suggest a novel molecular mechanism that would account for the exacerbation of diabetic retinopathy by concomitant hypertension, and may partially explain the principal clinical manifestations of hypertensive retinopathy itself. Furthermore, these data imply that anti-VEGF therapies may prove therapeutically effective for hypertensive retinopathy and/or ameliorating the deleterious effects of coexistent hypertension on VEGF-associated disorders such as diabetic retinopathy.

Competitive electrostatic binding of charged ligands to polyelectrolytes: practical approach using the non-linear Poisson-Boltzmann equation

We have developed a practical analytical treatment of the non-linear Poisson-Boltzmann (P-B) equation to characterize the strong but non-specific binding of charged ligands to DNA and other highly charged macromolecules. These reactions are notable for their strong salt dependence and anti-cooperativity, features which the theory fully explains. We summarize analytical results for concentration profiles and ion binding in various regimes of surface curvature and ionic strength, and show how counterion size and charge distribution may influence competitive binding. We present several practical applications of the formalism, showing how to estimate the ligand concentration needed to effectively compete with a given buffer salt, and how to calculate the amounts of counterion species bound at various distances from the DNA surface under given bulk solution conditions. We cast our results into the form of a Scatchard binding isotherm, showing how the apparent binding constant K obs and S = −dlogK obs d log[M +] can be predicted from the basic theory. Anti-cooperativity arises naturally without steric repulsion, and binding curves can be fitted with K obs and effective charge as the only free parameters. We extend the analytical P-B analysis to an arbitrary number of counterion species, and apply the results to fit and predict three-ion competition data.

Interaction of IL-15 with the shared IL-2 receptor beta and gamma c subunits. The IL-15/beta/gamma c receptor-ligand complex is less stable than the IL-2/beta/gamma c receptor-ligand complex.

This study was designed to compare the interactions of IL-2 and IL-15 with the IL-2R beta and IL-2R gamma c subunits, as differences in receptor interactions between IL-2 and IL-15 might contribute to the functional differences between these two cytokines. The results suggest the existence of a human IL-15R alpha subunit, although physical evidence of this molecule was not obtained. Proliferation of anti-CD3 (OKT3)-stimulated human PBL was compared for responsiveness to IL-2, IL-15, and F42K, and IL-2 mutant that does not bind the IL-2R alpha chain. F42K was more potent than IL-15 in activating a dose-dependent response. This fact, along with Scatchard binding analyses of IL-15 on OKT3 blasts and YT cells revealing both high and intermediate affinity receptors, supports the existence of IL-15R alpha on these cells. Additional characterization of the IL-15R utilized covalent cross-linking to affinity label IL-2R and IL-15R on YT cells and OKT3 blasts. Consistent with previously reported functional data, IL-2R alpha was not co-precipitated from the [125I]IL-15 receptor-ligand complex, demonstrating that IL-15 does not interact physically with the IL-2R alpha subunit. While IL-2R alpha did co-precipitate with IL-2R beta and IL-2R gamma c in the presence of IL-2, IL-15R alpha did not co-precipitate with the IL-2R beta/gamma c complex. Finally, YT cells equilibrated with IL-2 and then precipitated through IL-2R beta showed that IL-2R beta and IL-2R gamma c co-precipitate in a 1:1 ratio, while only IL-2R beta was found in the immunoprecipitates of YT cells equilibrated with IL-15. This indicates that IL-15 creates a less stable bridge between the IL-2R beta and IL-2R gamma c chains than does IL-2 on YT cells. This result was identical for both surface-iodinated YT cells and immunoprecipitates that were probed for IL-2R beta and IL-2R gamma c on Western blots.