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Abstract: Trefoil factor family 2 (TFF2) participates in mucus stabilization and repair, apoptosis, and inflammatory responses. Previously published reports have indicated that several growth factors and basal transcription factors are associated with the expression of TFF2. However, the detailed mechanisms that regulate TFF2 expression are not fully understood. The present study was designed to assess the essential role of the transcription factor SP3 with respect to TFF2 expression. We first demonstrated that there was a negative correlation between the expression levels of SP3 and TFF2. Thus, in the examined cells, the overexpression of SP3 decreased the expression level of TFF2, whereas the inhibition of SP3 increased the expression level of TFF2. Moreover, we discovered two GC boxes in the TFF2 promoter and confirmed the specific binding of SP3 to this promoter. On the whole, this study indicated that Sp3 was a major regulator of TFF2 expression. This knowledge should contribute to our understanding of the role that is played by SP3 in the regulation of TFF2 expression. [Copyright &y& Elsevier]

In this study, we compared different binding interactions of TBMS2 with proteins both in hepatocarcinoma HepG2 cells and in normal embryo hepatic L02 cells by using fluorescence, absorption, and CD spectroscopy. The fluorescence data revealed that the fluorescence intensity of proteins in the HepG2 and L02 cells decreased in the presence of TBMS2 by 30.79% and 12.01%, respectively. Binding constants and thermodynamic parameters were obtained for systems of TBMS2 with the two kinds of cell proteins. The results indicated that HepG2 cell proteins had a higher TBMS2 binding activity than those in the L02 cells. Analysis of the TBMS2 cytotoxic activities showed that TBMS2 could selectively induce apoptosis of HepG2 cells by binding to them, while its apoptotic effect on L02 cells was relatively weaker. [ABSTRACT FROM AUTHOR]

Abstract: Metal-mediated base pairs by the interaction between metal ions and artificial bases in oligonucleotides have been developed for their potential applications in nanotechnology. We recently found that a natural T:T mismatched base pair bound with Hg2+ ion to form a novel T–Hg–T base pair. Here, we examined the thermodynamic properties of the binding between Hg2+ and each of the single and double T:T mismatched base pair duplex DNAs by isothermal titration calorimetry. Hg2+ specifically bound with the T:T mismatched base pair at 1:1 molar ratio with 106 M−1 binding constant, which was significantly larger than those for nonspecific metal ion–DNA interactions. In the Hg2+–double T:T mismatched base pair interaction, the affinity for the second Hg2+ binding was significantly larger than that for the first Hg2+ binding. The positively cooperative binding may be favorable to align multiple Hg2+ in duplex DNA for the application of the metal-mediated base pairs in nanotechnology. [Copyright &y& Elsevier]

Abstract: Imprinting technique, coupled with membrane technology was used in order to prepare solvent resistant molecularly imprinted membranes with controlled specificity for the primary aromatic amine 4,4′-methylendianiline. Polyacrylonitrile and its different copolymers such as Poly(acrylonitrile-co-acrylic acid), Poly(acrylonitrile-co-methacrylic acid) and Poly(acrylonitrile-co-itaconic acid), were selected as materials to prepare the membranes. The different polymers, were synthesized by the water-phase precipitation polymerization method and membranes were prepared via the phase inversion technique. Their recognition properties and separation behaviour for target molecule and other structural analogue were investigated performing binding experiments in IPA. Results showed that all imprinted membranes specifically recognize the template in different extent. Imprinted membranes prepared with Poly(acrylonitrile-co-acrylic acid) were competitive with those prepared using the other two copolymers and exhibited the highest specific binding capacity towards the template (2.6μmol/gmemb). In addition, they were selective with respect to the other primary amine aniline and 4,4′-ethylendianiline. On the contrary, Polyacrylonitrile and blank membranes, showed only a non-specific binding. [Copyright &y& Elsevier]

Abstract: Composition and structure of biorecognition protein layers created on silicon substrates modified with amino-organosilanes determine the sensitivity and specificity of silicon based biosensing devices. In the present work, diverse spectroscopic and microscopic methods were applied to characterize model biosensor surfaces, formed on Si3N4 or SiO2 by modification with (3-aminopropyl)triethoxysilane, coating with rabbit gamma-globulins (IgGs) through physical adsorption, blocking with bovine serum albumin (BSA) and specific binding of an anti-rabbit IgG antibody. In addition, silanized substrates with directly adsorbed BSA or anti-rabbit IgG antibody were examined as reference surfaces. The protein/amino-organosilane/silicon structure of all surfaces was confirmed by X-ray photoelectron spectroscopy. Homogeneity of protein coverage was verified with near-field scanning optical microscope, working in reflection and fluorescence mode. Surface coverage with proteins was determined with angle-resolved XPS using a previously established bilayer approach. Inner structure of protein layers was examined with atomic force microscopy. Vertical arrangement of carbon functional groups was revealed by high resolution ARXPS. Combined spectroscopic and microscopic data reveal the complex character of interactions with the immobilized IgG molecules during blocking with BSA and immunoreaction with anti-IgG antibody. Within experimental error, neither surface coverage nor lateral structural scales of protein layer (provided by Fourier and auto-correlation analysis of topographic and phase images) increase during blocking procedure. On the other hand, coverage and all structural measures rise considerably after immunoreaction. In addition, it was found that polar functional groups orient towards substrate for all protein layers, independently of coverage, prior to and after both blocking and specific binding. [Copyright &y& Elsevier]

Abstract: Simultaneous specific and nonspecific binding of bendroflumethiazide (BFZ) to human serum albumin (HSA) and concentration profile of BFZ in HSA buffer (pH 7.40) solution were investigated by 19F nuclear magnetic resonance (NMR) method. The 19F NMR spectrum of BFZ (200μM) in a buffer solution showed a sharp signal of its CF3 group at 17.8ppm from the reference trifluoroethanol. Addition of 0.60mM HSA to the sample solution caused the CF3 signal splitting into three broadened peaks at 18.4 (A), 17.9 (B) and 17.4ppm (C). By its chemical shift and spectral behavior, B was assigned to unbound BFZ. Competition experiments with Site I and II ligands lead to C being assigned to Site II bound BFZ. However, the peak intensity (areas) of A was not reduced by these ligands, suggesting that A arises from nonspecific binding. Using the peak intensities at several total concentrations of BFZ, Scatchard plot was performed. The plot for A provided a straight line parallel to the x-axis confirming nonspecific binding and that for C was consistent with specific binding. The binding constants for nonspecific and specific Site II binding were 1.02 and 1.00×104 (M−1) (n =1.1), respectively. The presence of 0.10M Cl− in the sample solution affected the binding constant of Site II binding, but not that of nonspecific binding. The concentration profile of BFZ calculated using the binding constants revealed that nonspecific binding is more effective than Site II binding for the binding of BFZ to HSA. It was also confirmed that considerable amounts of BFZ liberated from Site II by the Site II ligands or Cl− ions bind again nonspecifically. [Copyright &y& Elsevier]

Anti-HER2 monoclonal antibody (Sc7301)-paclitaxel (TAX) immunoconjugate was prepared and its specific binding to tumor cells was investigated in this study. Sc7301 was conjugated to TAX by the active ester method and then the TAX-Sc7301 immunoconjugate was obtained. After purification and labeling by Cyano-fluorescein isothiocyanate (FITC), the specific binding of TAX-Sc7301 to HER2-positive tumor cells (SKOV3) and HER2-negative tumor cells (HepG2) was evaluated respectively. TAX-Sc7301 (20 nmol/L) showed distinct specific binding to SKOV3 cells rather than HepG2 cells. And the uptake of the immunoconjugate by SKOV3 cells was increased with the TAX-Sc7301 concentration (3-48 nmol/L) and the incubation time ( P<0.05). It was concluded that the TAX-Sc7301 immunoconjugate is potentially applicable as a targeted agent against HER2-positive tumor cells. [ABSTRACT FROM AUTHOR]

Abstract: The phytohormone abscisic acid (ABA) is the central regulator of abiotic stress in plants and plays important roles during plant growth and development. In animal cells, ABA was shown to be an endogenous hormone, acting as a stress signal and stimulating cell functions involved in inflammatory responses and in insulin release. Recently, we demonstrated that Lanthionine synthetase component C-like protein 2 (LANCL2) is required for ABA binding to the plasmamembrane of granulocytes and for the activation of the signaling pathway triggered by ABA in human granulocytes and in rat insulinoma cells. In order to investigate whether ABA activates LANCL2 via direct interaction, we performed specific binding studies on human LANCL2 recombinant protein using different experimental approaches (saturation binding, scintillation proximity assays, dot blot experiments and affinity chromatography). Altogether, results indicate that human recombinant LANCL2 binds ABA directly and provide the first demonstration of ABA binding to a mammalian ABA receptor. [Copyright &y& Elsevier]

The aim of this work was the preparation of molecularly imprinted membranes by surface modification of microfiltration polyvinylidene fluoride membranes with a thin layer of molecular imprinted polymer by UV irradiation. The flavonoid naringin (4,5,7-trihydroxyflavanone-7-rhamnoglucoside) was used as template molecule. The compound 4-vinylpyridine was used as functional monomer. Benzoin ethyl ether and ethylene glycol dimethacrylate were used as photo-initiator and cross-linker, respectively. Membranes were also modified in the absence of naringin and used as reference (blank). Imprinted membranes prepared in this work could be applied as new separation devices in affinity filtrations aimed to reduce the naringin content in grapefruit and improve its benefit properties. The method used to prepare them offers the advantage of combining the mechanical integrity of the membrane support with the selective recognition properties of the imprinted polymer layer. The recognition properties of all prepared membranes were evaluated by their capability to retain naringin in aqueous solution during filtration tests. Results showed that imprinted membranes exhibited specific binding properties for template molecules. Blank membranes only showed non-specific binding. The selectivity of the naringin-imprinted membranes was tested evaluating their bound property toward the rutin, a structural analogue of naringin. [ABSTRACT FROM AUTHOR]

Inability of chemical phosphorylation of sodium trimetaphosphate to induce intrafibrillar mineralization of type I collagen may be due to the failure to incorporate a biomimetic analog to stabilize amorphous calcium phosphates (ACP) as nanoprecursors. This study investigated adsorption/desorption characteristics of hydrolyzed and pH-adjusted sodium trimetaphosphate (HPA-Na3P3O9) to collagen. Based on those results, a 5-minute treatment time with 2.8 wt% HPA-Na3P3O9 was used in a single-layer reconstituted collagen model to confirm that both the ACP-stabilization analog and matrix phosphoprotein analog must be present for intrafibrillar mineralization. The results of that model were further validated by complete remineralization of phosphoric-acid-etched dentin treated with the matrix phosphoprotein analog and lined with a remineralizing lining composite, and with the ACP-stabilization analog supplied in simulated body fluid. An understanding of the basic processes involved in intrafibrillar mineralization of reconstituted collagen fibrils facilitates the design of novel tissue engineering materials for hard tissue repair and regeneration. [ABSTRACT FROM PUBLISHER]

In dynamic force spectroscopy, access to the characteristic parameters of single molecular bonds requires nontrivial measurements and data processing as the rupture forces are found not only to be distributed over a wide range, but are also dependent on the loading rate. The choice of measurement procedure and data processing methods has a considerable impact on the accuracy and precision of the final results. We analyze, by means of numerical simulations, methods to minimize and assess the magnitude of the expected errors for different combinations of experimental and evaluation methods. It was found that the choice of fitting function is crucial to extract correct parameter values. Applying a Gaussian function, which is a common practice, is equivalent to introducing a systematic error, and leads to a consequent overestimation of the thermal off-rate by more than 30%. We found that the precision of the bond length and the thermal off-rate, in presence of unbiased noise, were improved by reducing the number of loading rates for a given number of measurements. Finally, the results suggest that the minimum number of measurements needed to obtain the bond strength, with acceptable precision, exceeds the common number of ~100 reported in literature. [ABSTRACT FROM AUTHOR]

The influence of ion size and surface charge model in titrations of ionizable polyelectrolytes is studied by means of the Semi Grand Canonical Monte Carlo simulation method in the context of the primitive model. Three models describing a discrete distribution of charged functional groups on the polyelectrolyte and different values for the radius of the background electrolyte spanning from ionic to hydrated radii values were analyzed. The polyelectrolyte titrations were simulated by calculating the degree of ionization versus pH curves at two ionic strengths. The results allow us to quantify the impact of the sizes of the background salt ions and surface functional groups of the polyelectrolyte on the dissociation degree. This influence is explained in terms of the effectiveness of the screening of the charged surface sites. Finally, by comparison with the Non-Linear Poisson–Boltzmann model, the influence of ionic correlations and finite size of the solution ions is assessed. [ABSTRACT FROM AUTHOR]

Abstract: This paper presents a statistical experimental study of the adsorption of colloids onto the plasma membrane of living cells mediated by specific ligand–receptor interactions. The colloids consist of lipid multilamellar liposomes (spherulites) functionalized by Shiga toxin B-subunit (STxB), while cells are cervix carcinoma epithelial cells expressing the Shiga toxin receptor, the glycolipid globotriaosyl ceramide (Gb3). The specificity of the colloid adsorption is demonstrated using both confocal microscopy and flow cytometry, while a thorough cytometry study on living cells allows characterizing the kinetics of this specific adsorption. The final number of bound colloids and the characteristic adsorption time are shown to depend on bulk concentration, as expected for a thermodynamic equilibrium. However, the colloids appear to be irreversibly attached to the membrane. We interpret this apparent irreversibility as the result of a progressive recruitment of receptors. The methodology used here, whereby microscopic mechanisms are deduced from direct quantitative measurements on living cells, might allow the optimization of drug delivery systems or the quantification of virus infectivity. [Copyright &y& Elsevier]

Abstract: The model of binding of micelles of nonylphenol molecules to respective antibodies immobilized on the solid surface is proposed. The actual dimensions of micelles obtained from AFM measurements were used in modelling and allowed to predict the shape of the micelle. An account of non-spherical shape of micelles and their simultaneous binding to several antibodies allows estimation of the micelle binding energy. [Copyright &y& Elsevier]

Abstract: We describe a model of electron transfer reactions affected by local binding to the donor or acceptor sites of a particle in equilibrium with the solution. The statistics of fluctuations of the donor–acceptor energy gap caused by binding/unbinding events are non-Gaussian, and the resulting free energy surfaces of electron transfer are non-parabolic. The band-width of the charge-transfer optical transition is predicted to pass through a maximum as a function of the concentration of binding particles in the solution. The breakdown of the link between the Stokes shift and the reorganization energy of electron transfer is also predicted by the model. [Copyright &y& Elsevier]

Abstract: The dependence of the apparent acid dissociation constant of charged polyacids on the ionic strength and on the degree of dissociation is analysed in terms of the Donnan model. We introduce a new approximate analytical expression for the Donnan volume which depends on the ionic strength and on the degree of dissociation with only one adjustable parameter. This expression stems from analytical solutions of the non-linear Poisson–Boltzmann equation (NLPB) under different geometries. Two kinds of polyelectrolytes are studied: polyacrylic acid (PAA), as a model of homogeneous ligand, and Aldrich humic acid (HA), a heterogeneous ligand which exhibits a pK a dependent on the pH, even when the polyelectrolytic correction is applied. The new approximate Donnan volume expression allows to justify the empirical dependence of previous expressions found in the literature on the ionic strength, gives rise to master curves well behaved in all the range of macromolecular charges and can fit experimental results in good agreement with classical treatments like NLPB, used as a reference, involving a greatly simplified numerical treatment. Three different complexation isotherms are independently used to account for the specific binding. The robustness of the results is evidenced by the fact that similar central moments of the affinity distribution underlying each isotherm and of the adjustable parameter of the Donnan volume are obtained in each case. [Copyright &y& Elsevier]

Abstract: The cytotoxic plant alkaloid sanguinarine was found to bind preferentially and strongly to single stranded poly(A) with an association constant (Ka) in the range 3.6–4.6×106 M−1 in comparison to several nucleic acids. The binding induced unique self-structure formation in poly(A) that showed cooperative melting transition in circular dichroism, absorbance, and differential scanning calorimetry studies. The alkaloid binding was characterized to be intercalation as revealed from fluorescence quenching experiments and was predominantly enthalpy driven as revealed from isothermal titration calorimetry. Sanguinarine is the first and only natural product so far known to induce a self-structure formation in poly(A). [Copyright &y& Elsevier]

The peculiarities and characteristics of the binding of the Semax neuropeptide (Met-Glu-His-Phe-Pro-Gly-Pro) with a 3H-labeled terminal Pro to plasma membranes of the rat forebrain were studied. Specific binding sites were found that were characterized by a dissociation constant of the ligand-receptor complex of Kd = 100.4 ± 19.5 nM at 0–4°C. Significant biodegradation of Semax in the course of its interaction with plasma membranes was shown by HPLC. The rate of this degradation was considerably decreased at 0–4°C. [ABSTRACT FROM AUTHOR]

A technique is described for accurate quantification of the specific binding ratio (SBR) in [123I]FP-CIT SPECT brain images. Using a region of interest (ROI) approach, the SBR is derived from a measure of total striatal counts that takes into account the partial volume effect. Operator intervention is limited to the placement of the striatal ROIs, a task facilitated by the use of geometrical template regions. The definition of the image for the analysis is automated and includes transaxial slices within a “slab” approximately 44 mm thick centred on the highest striatal signal. The reference region is automatically defined from the non-specific uptake in the whole brain enclosed in the slab, with exclusion of the striatal region. A retrospective study consisting of 25 normal and 30 abnormal scans—classified by the clinical diagnosis reached with the scan support—was carried out to assess intra- and inter-operator variability of the technique and its clinical usefulness. Three operators repeated the quantification twice and the variability was measured by the coefficient of variation (COV). The COVs for intra- and inter-operator variability were 3% and 4% respectively. A cutoff ∼4.5 was identified that separated normal and abnormal groups with a sensitivity, specificity and diagnostic concordance of 97%, 92% and 95% respectively. The proposed technique provides a reproducible and sensitive index. It is hoped that its independence from the partial volume effect will improve consistency in quantitative measurements between centres with different imaging devices and analysis software. [ABSTRACT FROM AUTHOR]

To achieve a novel specific peptide–nucleic acid binding model, we designed an in vitro selection procedure to decrease the energetic contribution of the electrostatic interaction in the total binding energy and to increase the contribution of hydrogen bonding and π–π stacking. After the selection of hairpin-loop RNAs that specifically bound to a model peptide of lambda N protein (N peptide), a new thermostable pentaloop RNA motif (N binding thermostable RNA hairpin: NTS RNA) was revealed. The obtained NTS RNA was able to bind to the N peptide with superior specificity to the boxB RNA, which is the naturally occurring partner of the lambda N protein. [ABSTRACT FROM AUTHOR]

Abstract: We have successfully fabricated a phenylboronic acid self-assembled layer on glassy carbon electrodes (GCE), where 3-aminophenylboronic acid (APBA) is covalently bound to the electrochemical pretreated GCE surface with glutaraldehyde linkage. The specific binding of glycoprotein peroxidase with the self-assembled layer has been studied using horseradish peroxidase (HRP) as a model glycoprotein. Cyclic voltammetric, electrochemical impedance studies and photometric activity assays show that the affinity interaction of HRP with the APBA modified GCE surface includes specific and nonspecific bonding. The specific binding is attributed to the boronic acid–diols interaction where the boronic acid specifically binds the glycosylation sites of the HRP. This specific binding is reversible and can be split by sorbitol and glucose or released in an acidic buffer. The catalytic current of the HRP-loaded electrode, due to the catalytic oxidation of thionine in the presence of hydrogen peroxide, is proportional to HRP concentrations of the incubation solution. This work offers a new way to build novel sensors by specific binding of glycoproteins to a boronic acid self-assembled layer for determination of glycated proteins. [Copyright &y& Elsevier]

Abstract: A systematic investigation of the effect of polyethylene glycol (PEG) 200 and 400 on the solution conformation of concanavalin A (con A) was made using circular dichroism (CD), tryptophan fluorescence, 1-anilino-8-naphthalenesulfonic acid (ANS) binding, and size-exclusion chromatography. Far-UV CD spectra of con A at 30%(v/v) PEGs show the retention of ordered secondary structure as compared to 70%(v/v) PEGs. Near-UV CD spectra showed the retention of native-like spectral features in the presence of 30%(v/v) PEGs. Intrinsic tryptophan fluorescence studies indicate a change in the environment of tryptophan residues on the addition of PEG. ANS binding was maximum at 30%(v/v) PEGs suggesting the compact “molten-globule”-like state with enhanced exposure of hydrophobic surface area. Size-exclusion chromatography indicates an intermediate hydrodynamic size at 30%(v/v) PEGs. GdnHCl denaturation of these states was a single-step, two-state transition. To study the possible minimum structural requirement in the specific binding, the effect of PEGs on the interaction of con A with ligand was investigated by turbidity measurements. The C50 value was less in PEG 400 suggesting the more inhibitory ability of PEG 400. The C50 value of PEGs was highest for dextran followed by glycogen, ovalbumin, and ovomucoid. From percentage inhibition of con A–ligands at 30%(v/v) PEG, maximum inhibition was in ovalbumin followed by ovomucoid, glycogen, and dextran. To summarize: con A at 30%(v/v) PEGs exists as compact intermediate with molten-globule-like characteristics, viz., enhanced hydrophobic surface area, retention of compact secondary as well as tertiary structure, and a considerable degree of carbohydrate binding specificity and activity. This result has significant implications on the molten globule state during the folding pathway(s) of proteins in general and quaternary association in the legume lectin in particular, where precise topology is required for their biological activities. [Copyright &y& Elsevier]

In flow cytometry using two detecting methods, we have found that amyloid-β-protein(1–40) [Aβ(1–40)] has high affinity to IMR-32 neuroblastoma cell membrane when it is aggregated to formβ-sheet conformation, whereas random coil small Aβ-species has low affinity. The difference in the binding ability to the cell membranes well accounts for the cytotoxicity of Aβ(1–40); namely, aggregatedβ-sheet Aβ(1–40) gives cytotoxicity higher than random coil Aβ(1–40). Specific binding between Aβ(1–40) and ganglioside GM1 of the raft-like domain in lipid membrane is suggested from a surface plasmon resonance (SPR) experiment. [ABSTRACT FROM AUTHOR]

Specific binding of bovine prolactin and somatotropin by granulosa cells from the antral follicles of various diameters was studied in cows at different reproductive states, prepubertal, pubertal, and early gestation. The ability of granulosa cells to bind prolactin did not depend on the reproductive state of an animal. At the same time, the dynamics of somatotropin specific binding by granulosa cells during maturation of the antral follicles differed at dissimilar reproductive states of the cows. When the diameter of follicles increased from 3–5 to 6–10 mm, specific binding of 125I-somatotropin decreased in pubertal animals, but remained unchanged in the prepubertal and pregnant animals. The results of Scatchard analysis of the binding data indicate that sexual maturation of cows did not affect the binding of prolactin and somatotropin by granulosa cells from follicles of 1–2 mm in diameter. The data obtained suggest that the decreased sensitivity of granulosa cells to somatotropin at the terminal stages of maturation of the antral follicles is essential for their development and acquisition of the ability for ovulation. [ABSTRACT FROM AUTHOR]

FKBP23 was found in mouse endoplasmic reticulum (ER) in 1998. It consists of an N-terminal peptidyl-prolyl cis-trans isomerase (PPlase) domain and a C-terminal domain with Ca2+ binding sites. The fusion protein of mouse FKBP23 and glutathione S-transferase (GST), GST-FKBP23, and the fusion protein of BiP, a member of heat shock protein 70 (Hsp 70) in ER, and GST, GST-BiP, were subcloned in E. coli, expressed and purified. The fusion proteins were restrictively digested by Factor Xa (FaXa) to obtain the free cloned proteins FKBP23 and BiP. With the assay of adsorption of free FKBP23 or BiP with GST-BiP or GST-FKBP23 attached to the Glutathione-Sepharose 4B, the adsorbed FKBP23 or BiP could be detected by Immunoblot. It means that FKBP23 binds to BiP. Furthermore, BiP in leukocyte ER-extract can be adsorbed with GST-FKBP23 attached to the glutathione-Sepharose 4B. It shows that FKBP23 binds to natural BiP in ER, too. These experiments show that a PPIase binds to a molecular chaperone of the Hsp70 family. [ABSTRACT FROM AUTHOR]

This study was done for investigation of the DNA synthesis induced by HGF/SF and EGF in periportal hepatocytes (PPH) and perivenous hepatocytes (PVH) isolated at different times post-hepatectomy. Preparation, culture, and DNA synthesis of PPH and PVH were done as previously described. In both PPH and PVH isolated at 6 and 12 h post-hepatectomy, the maximum DNA synthesis induced by both HGF/SF and EGF was observed from 24 to 48 h post-hepatectomy. In contrast, in both types of hepatocytes isolated at 36, 72 and 96 h, 1 and 2 weeks, and 1 month post-hepatectomy, maximum DNA synthesis was observed from 48 to 72 h. However, maximum DNA synthesis induced by HGF/SF and EGF was at its lowest in PPH isolated at 12 h and PVH isolated at 6 h post-hepatectomy. Maximum DNA synthesis returned to the control level in PPH and PVH isolated at 72 and 96 h post-hepatectomy, respectively. Specific binding studies using [125I] HGF/SF and [125I] EGF supported the above data. From these data, it was demonstrated that first, the PPH and PVH cell cycles have the same post-hepatectomy, but are dependent on the preparation time. Secondly, differences in DNA synthesis in PPH and PVH post-hepatectomy suggest that liver regeneration may occur unevenly. [Copyright &y& Elsevier]

Dengue type-2 virus infection in mice induces a subpopulation of T lymphocytes to produce a cytokine cytotoxic factor, which induces macrophages (Mφ) to produce a biologically active cytotoxic cytokine, the Mφ cytotoxin (CF2). Previously we have identified the presence of intermediate-affinity receptors for CF2 on mouse peritoneal Mφ. The present study was undertaken to identify the CF2-receptors (CF2-R) on murine T cells followed by their purification and characterization. Receptor binding assay and Scatchard analysis revealed single, high-affinity (1.0309 nM) receptors for CF2 on T cells (22 000 receptors per cell). The binding of [125I]CF2 on murine T cells was saturable and specific. Furthermore, CF2-R was purified from normal mouse T cell plasma membrane by affinity chromatography followed by reversed-phase high-pressure liquid chromatography. The presence of CF2-R was confirmed by indirect dot-blot assay and its binding with [125I]CF2. The purified CF2-R is a 90–95-kDa protein as characterized by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and immunoblot analysis. The chemical crosslinking of [125I]CF2 and its receptor complex showed a product of 100–110 kDa on different subpopulations of murine T cells. The pretreatment of target cells with anti-CF2-R antisera inhibited the cytotoxic activity of CF2 in a dose-dependent manner and thus confirmed the biological significance of CF2-R. Moreover, the presence of CF2-R was also identified on normal human peripheral blood mononuclear cells and T and B cells by crosslinking with [125I]CF2, thus revealing the possible role of CF2 and CF2-R in the immunopathogenesis of dengue virus disease. [Copyright &y& Elsevier]

In bone and dentin the formation and mineralization of the extra cellular matrix structure is a complex process highly dependent on intermolecular interactions. In dentin, the phosphophoryns (PP) and type I collagen (COL1) are the major constituents implicated in mineralization. Thus, as a first step in understanding the tissue organization, we have initiated a study of their interaction as a function of pH, ionic strength, and relative concentrations or mixing ratios. Complex formation has been analyzed by dynamic light scattering to detect aggregate formation and by rotary shadowing electron microscopy (EM) to determine aggregate shape. The EM data showed that at the pH values studied, the PP-COL1 interaction leads to the formation of large fibrillar aggregates in which the PP are present along the fibril surfaces. The quantitative phase distribution data showed a 1/1molar equivalence at the maximum aggregation point, not at electrostatic PP-COL1 equivalence. As the ionic strength was raised, the PP-COL1 aggregates became smaller but the binding and asymmetric fibrillar aggregation persisted. In EM, the PP appear as dense spheres. Along the surfaces of the collagen aggregates, the PP are larger and more open or extended, suggesting that COL1-bound PP may undergo a conformational change, opening up so that a single PP molecule might interact with and electrostatically link several COL1 molecules. This might have important implications for dentin structure, stability, and mineralization. [ABSTRACT FROM AUTHOR]

Fragments of the B-loop of the epidermal growth factor family of peptides are reported to have mitogenic and angiogenic properties but appear to fail to compete with radioiodinated EGF in receptor binding. In this study, 11 analogs of a fragment of the B-loop of EGF-related peptides from several species were synthesized to study binding to A431 human epidermoid carcinoma using both 125I-EGF and [3′4′-3H-Tyr22,29, Abu20,31]EGF(20–31)-NH2. Specific binding sites were found for the human fragment and 8 analogs at a density five times higher than that of the EGF receptors. Analogs did not compete with 125I-EGF for binding to the EGF receptor. The novel binding site may mediate the biological effects of the fragments. The primary rather than secondary structure of the fragments appears to determine affinity. [Copyright &y& Elsevier]

Apo C-III plays an important role in the metabolism of plasma triglyceride, which can delay the catabolism of triglyceride-rich lipoproteins by interfering with apo E-mediated receptor clearance of remnant particles from plasma. The mechanism of the interference has not yet been defined. To further explore the role of apo C-III, we first injected mice with 125I-apo C-III. The measurement of radioactivity showed that liver took up 3.3-10 fold as much radioactivity as other organs such as heart, spleen, lung, kidney, stomach, large intestine, small intestine, and muscle. This was confirmed by incubating the tissue homogenates of the organs with 125I-apo C-III that the radiolabeled apo C-III specifically bound to only hepatic homogenate. To investigate which subcellular part or parts of hepatic cells play the role of binding to apo C-III, hepatic cell components of nucleus, mitochondria, microsomes and plasma membranes were then incubated with 125I-apo C-III. The radiolabeled apo C-III could specifically bind to only hepatic plasma membranes. Finally hepatic plasma membranes were purified to study the characteristics of the specific binding with apo C-III. Addition of increasing concentration of 125I-apo C-III to human hepatic plasma membranes revealed saturable binding to membranes with a Kd of 0.31±0.07 μmol/l. The maximum specific binding capacity was 1.74±0.45 μ apo C-III/mg membrane protein. In competition studies using unlabeled apo C-III and isolated lipoproteins HDL, LDL and VLDL, only apo C-III and VLDL effectively competed with 125I-apo C-III for membrane binding. The binding of 125I-apo C-III to human liver plasma membranes was Ca2+-independent, and was abolished when plasma membranes were treated with trypsin. The characteristics of 125I-apo C-III binding to mouse liver plasma membranes were similar to those of human liver plasma membranes with the exception of a binding maximum of 1.52±0.39 μapo C-III/mg membrane protein. We conclude that apo C-III exhibits high-affinity binding to hepatic plasma membranes, which is saturable, reverse and specific. [ABSTRACT FROM AUTHOR]

Imprinting polymerization is an exciting technique since it leads to specific binding sites, which are the basis of a variety of applications, such as sensors, detectors, and catalysts. The specific binding sites are created using templates and then fixing the structure of the binding site with crosslinking. The literature review of imprinting polymerizations shows that the crosslinking density governs the physical properties of the resulting molecularly imprinted polymer (MIP). It is also a factor governing the capacity and the selectivity of MIPs. Reviewing polymer science data and theory, the crosslinking density commonly used in MIP synthesis is unusually high. The data reviewed here suggest that more research is needed to determine the optimal crosslinking density for MIPs. [ABSTRACT FROM AUTHOR]

Recently, aCPSF1 was reported to function as the long-sought global transcription termination factor of archaea; however, the working mechanism remains elusive. This work, through analyzing transcript-3'end-sequencing data of Methanococcus maripaludis , found genome-wide positive correlations of both the terminator uridine(U)-tract and aCPSF1 with hierarchical transcription termination efficacies (TTEs). In vitro assays determined that aCPSF1 specifically binds to the terminator U-tract with U-tract number-related binding affinity, and in vivo assays demonstrated the two elements are indispensable in dictating high TTEs, revealing that aCPSF1 and the terminator U-tract cooperatively determine high TTEs. The N-terminal KH domains equip aCPSF1 with specific-binding capacity to terminator U-tract and the aCPSF1-terminator U-tract cooperation; while the nuclease activity of aCPSF1 was also required for TTEs. aCPSF1 also guarantees the terminations of transcripts with weak intrinsic terminator signals. aCPSF1 orthologs from Lokiarchaeota and Thaumarchaeota exhibited similar U-tract cooperation in dictating TTEs. Therefore, aCPSF1 and the intrinsic U-rich terminator could work in a noteworthy two-in-one termination mode in archaea, which may be widely employed by archaeal phyla; using one trans-action factor to recognize U-rich terminator signal and cleave transcript 3'-end, the archaeal aCPSF1-dependent transcription termination may represent a simplified archetypal mode of the eukaryotic RNA polymerase II termination machinery., Competing Interests: JL, LY, ZL, WZ, BZ, FZ, XD No competing interests declared, (© 2021, Li et al.)

Dengue type 2 virus (DV)-induced suppressor cytokine (SF) binds to macrophages to transmit the suppressor signal to recruit the second subpopulation of suppressor T cells. The present study was undertaken to identify and purify the receptor for SF (SF-R) on macrophages. The binding of 125I-SF to macrophages was saturable and reversible. Scatchard analysis showed the presence of both high (54000/cell) and low (1.78 × 106/cell) affinity receptor sites. The binding of 125I-SF to macrophages was inhibited by pretreatment of macrophages with anti-SF antiseruin but not by a heterologous antiserum. Normal mouse peritoneal macrophage membrane was solubilized with Triton-X-100 and the components separated by low pressure liquid chromatography (LPLC) to purify SF-R. The presence of SF binding moiety (SF-R) was screened at each step of purification. The purified SF-R resolved into two bands of 45-50 kD mol. wt on SDS-PAGE. 125-M-SF + SF-R complex run on SDSPAGE showed a single band at about 55-60 kD mol. wt by autoradiography. Anti-SF-R antiserum reacted with SF-R in a Western blot test; the reaction was abolished by pretreatment of the blots with proteinase K, but not by pretreatment with periodic acid. SF-R was composed of two polypeptide chains (α and β) which were obtained in pure form by high performance liquid chromatography (HPLC) of dithiothreitol- and iodoacetamide-treated SF-R. Only the β chain bound SF. [ABSTRACT FROM AUTHOR]

Spermatia from Aglaothamnion neglectum Feldmann-Mazoyer specifically bind with trichogynes and hairs of female thalli. One of the functions of hairs on female thalli appears to be the catching of spermatia. Fertilization can occur if a spermatium binds first with a hair and then binds with a trichogyne. The binding of spermatia with trichogynes is not species specific, but only occurs between closely related species. Spermatia have fimbriate cone-shaped appendages projecting from each end that are responsible for initial binding with trichogynes. [ABSTRACT FROM AUTHOR]

The possibility of developing bioadhesive drug delivery systems on the basis of molecules which selectively bind to the small intestinal epithelium by specific, receptor-mediated mechanisms was investigated using a lectin isolated from tomato fruits ( Lycopersicum esculentum). The tomato lectin (TL) was found to bind specifically onto both isolated, fixed pig enterocytes and monolayers of human Caco-2 cell cultures with a similar affinity. TL-coated polystyrene microspheres (0.98 µm) also showed specific binding to enterocytes in vitro. Lectin binding was found to be favored at neutral pH and to be reduced in an acidic environment. Crude pig gastric mucin, however showed a marked cross-reactivity in vitro, indicating that lectin binding to the cell surface in vivo might be inhibited by mucus. [ABSTRACT FROM AUTHOR]

A method is described for measuring rapid, specific, and saturable binding of the skin irritant and tumour-promoting secretagogue thapsigargin (sesquiterpene lactone) to the microsomal fraction from mouse brain. Employing the tritium-labelled compound its apparent dissociation constant, K, and the maximal amount of binding B are shown to be 9.8 n M and 1.9 pmol/mg protein respectively. Such a K for thapsigargin is similar to (a) its IC value for inhibiting Ca uptake in the microsomal fraction from rat brain and (b) its EC values for inducing a rise in the cytoplasmic Ca concentration of human platelets and histamine release from rat peritoneal mast cells. A positive correlation is found between the binding affinities of thapsigargin, thapsitranstagin, and trilobolide, their potencies as secretagogues and their lipophilicities. This correlation does not extend to the skin-irritant activities of the compounds thus emphasizing that their mechanism of action is unlike that of 12- O-tetradecanoylphorbol 13-acetate. [ABSTRACT FROM AUTHOR]

Sixteen new diterpene esters (DTE) of the tigliane, ingenane, daphnane, and 1α-alkyldaphnane types were investigated in two in vitro assays: as inhibitors of specific binding of H-labeled 12- O-tetradecanoylphorbol 13-acetate (TPA) to protein kinase C in a receptor preparation from mouse brain, and as inducers of Epstein-Barr virus (EBV) early antigens in Raji cells. Inhibition of binding of [H]TPA to the receptor preparation by tigliane and ingenane DTE correlates with irritant activity in vivo, while some daphnane and 1α-alkyldaphnane DTE inhibit binding of [H]TPA in a less pronounced manner but still are very irritant. Tumor-promoting activity does not correlate consistently with the receptor-binding data. To test the hypothesis that early antigen induction in Raji cells by DTE is coupled to functional DTE receptors (protein kinase C), the latter were searched on these Raji cells by a 'cold acetonefilter assay' and shown to be present. The dependence of the early antigen induction rate on the concentration of the DTE tested was demonstrated. At a given concentration of DTE, differences in the induction rate between various DTE are seen. However, a clear quantiative correlation either between early antigen induction and receptor binding data in vitro, or early-antigen-inducing activity in vitro versus irritancy and tumor-promoting activity in vivo was not observed. [ABSTRACT FROM AUTHOR]

We have evaluated the potential usefulness of indium-111 labelled [DTPA-D-Phe]RC-160, derived from the octapeptide somatostatin analogue RC-160, as a radiopharmaceutical for the in vivo detection of somatostatin receptor-positive tumours. For this purpose In-and In-labelled [DTPA-D-Phe]RC-160 was tested for its biological activity, and applied for somatostatin receptor scintigraphy in vivo to rats bearing the transplantable rat pancreatic tumour CA20948, which expresses somatostatin receptors. We previously described the development of the In-labelled somatostatin analogue [DTPA-D-Phe]octreotide and its use in the in vivo visualization of somatostatin receptor-positive tumours in rats and in humans. Like [In-DTPA-D-Phe]octreotide, [In-DTPA-D-Phe]RC-160 showed uptake in and specific binding in vivo to somatostatin receptor-positive organs and tumours, and the tumours were clearly visualized by gamma camera scintigraphy. However, as compared to [In-DTPA-D-Phe]octreotide, blood radioactivity (background) was higher, resulting in a lower tumour to blood (background) ratio. Using this animal model we therefore conclude that [In-DTPA-DPhe]RC-160 has no advantage over [In-DTPA-DPhe]octreotide as a radiopharmaceutical in the visualization of somatostatin receptors which bind both analogues. However, recent reports suggest the existence of different somatostatin receptor subtypes on some human cancers, which differentially bind RC-160 and not octreotide. These tumours include cancers of the breast, ovary, exocrine pancreas, prostate and colon. [In-DTPA-D-Phe]RC-160 might be of interest for future use in such cancer patients as a radiopharmaceutical for imaging somatostatin receptor-positive tumours, which do not bind octreotide. [ABSTRACT FROM AUTHOR]

We have evaluated the potential usefulness of the radioiodinated octapeptide RC-160, a somatostatin analogue, which might serve as a radiopharmaceutical for the in vivo detection of somatostatin receptor-positive tumours. For this purpose, iodine-123 and iodine-125 labelled RC-160 was tested for biological activity and applied in vivo in rats bearing the transplantable rat pancreatic tumour CA20948, which expresses somatostatin receptors. Our group has recently described the in vivo visualization of such tumours in rats and in humans with the radioiodinated somatostatin analogue [Tyr]octreotide. Like [I-Tyr]octreotide, I-RC-160 showed uptake in and specific binding in vivo to somatostatin receptor-positive organs and tumours. However, blood radioactivity (background) was higher, resulting in a lower tumour to blood (background) ratio. We therefore conclude that in this animal model I-RC-160 has no advantage over [I-Tyr]octreotide as a radiopharmaceutical for the in vivo use as a somatostatin receptor imager, although, like [I-Tyr]octreotide, I-RC-160 shows specific binding to different somatostatin receptor-positive organs. Recently differences were reported in affinity between somatostatin and its analogues for somatostatin receptors expressed in different human cancers, like those of the breast, ovary, exocrine pancreas, prostate and colon. Therefore I-RC-160 might be of interest for future use in humans as a radiopharmaceutical for imaging octreotide receptor-negative tumours. [ABSTRACT FROM AUTHOR]

Norepinephrine stimulated lipolysis in rat fat cells while (—) -alprenolol completely inhibited this lipolysis. (—)-Alprenolol competed for (—)-[3H]dihydroalprenolol(DHA) binding sites on rat fat cells. The specific (—)-[3H]DHA binding sites identified by competition with (—)-alprenolol were found to be transferred to the solubilized supernatant during preparation of endogenous lipid droplets from the fat cells. Although the lipid droplets did not exhibit specific (—)-[3H]DHA binding, norepinephrine induced lipolysis in a cell-free system consisting of the lipid droplets and hormone-sensitive lipase (HSL). Norepinephrine-induced lipolysis in the cell-free system was inhibited by pro-pranolol and (—)-alprenolol, but not by phenoxybenzamine. The lipolytic action of norepinephrine and the anti-lipolytic actions of propranolol and (—)-alprenolol disappeared after sonication of the lipid droplets in the cell-free system. These results suggest that the adrenergic receptor concerned with lipolysis in fat cells may not be a specific (—)- [3H]DHA binding site, but may be closely related to the lipid droplets. [ABSTRACT FROM AUTHOR]

Immunoglobulin G was separated on cellulose phosphate column to afford four distinct protein fractions (CP-I, II, III and IV). I-labeled fractions CP-III and CP-IV were found to be capable of binding specifically to normal human erythrocytes. The effect of the four fractions on osmotic resistance of red blood cells (RBC) was studied. RBC were obtained from eight patients with hereditary spherocytosis (HS), from a single parent of two non-related patients, and from five normal donors. RBC fragility of normal and one parent were unaffected by any of the immunoglobulin fractions. In contrast, a small but significant decrease in osmotic resistance was observed when RBC from HS patients and the second parent were incubated with protein fractions CP-III and CP-IV. [ABSTRACT FROM AUTHOR]