Maximum Binding Capacity Research Articles

The suitability of DEAE-Cl active groups on customized poly(GMA-co-EDMA) continuous stationary phase for fast enzyme-free isolation of plasmid DNA

The creation of a commercially viable and a large-scale purification process for plasmid DNA (pDNA) production requires a whole-systems continuous or semi-continuous purification strategy employing optimised stationary adsorption phase(s) without the use of expensive and toxic chemicals, avian/bovine-derived enzymes and several built-in unit processes, thus affecting overall plasmid recovery, processing time and economics. Continuous stationary phases are known to offer fast separation due to their large pore diameter making large molecule pDNA easily accessible with limited mass transfer resistance even at high flow rates. A monolithic stationary sorbent was synthesised via free radical liquid porogenic polymerisation of ethylene glycol dimethacrylate (EDMA) and glycidyl methacrylate (GMA) with surface and pore characteristics tailored specifically for plasmid binding, retention and elution. The polymer was functionalised with an amine active group for anion-exchange purification of pDNA from cleared lysate obtained from E. coli DH5α-pUC19 pellets in RNase/protease-free process. Characterization of the resin showed a unique porous material with 70% of the pores sizes above 300nm. The final product isolated from anion-exchange purification in only 5min was pure and homogenous supercoiled pDNA with no gDNA, RNA and protein contamination as confirmed with DNA electrophoresis, restriction analysis and SDS page. The resin showed a maximum binding capacity of 15.2mg/mL and this capacity persisted after several applications of the resin. This technique is cGMP compatible and commercially viable for rapid isolation of pDNA.

Bovine serum albumin-imprinted polymer gels prepared by graft copolymerization of acrylamide on chitosan

A graft copolymerization of acrylamide with N, N′-methylenebisacrylamide on chitosan in aqueous medium was utilized in the synthesis of molecularly imprinted polymer (MIP) gels using bovine serum albumin (BSA) as a template. The effect of the amount of initiator and the molecular weight and amount of chitosan on BSA rebinding was investigated. It was found that the resultant MIP gels based on chitosan-g-polyacrylamide (CS-g-PAM) showed significantly higher imprinting efficiency than those only consisting of polyacrylamide (PAM), and also better than those composed of chitosan/PAM semi-interpenetrating polymer network (CS/PAM-s-IPN). The batchwise adsorption of BSA and some non-template proteins showed that the CS-g-PAM based MIP gels demonstrated a quite significant template binding capacity different from the non-imprinted polymer (NIP) gels, and an obvious binding specificity for BSA. The adsorption isotherms of BSA on the MIP gels were determined and well fitted by a Langmuir model with a maximum binding capacity of 39.49 mg/g wet gels. The adsorption kinetics was analyzed by a linear driving force mass-transfer model. The MIP gels also showed much better stability in rebinding of the imprint molecules than those based on CS/PAM-s-IPN after three adsorption–regeneration cycles.

Reduced basal and lipopolysaccharide-stimulated adenosine A 1 receptor expression in the brain of nuclear factor-κB p50 −/− mice

Adenosine promotes cytoprotection under conditions of infection, ischemic preconditioning and oxidative stress. Previous studies from our laboratory indicate that the expression of the adenosine A 1 receptor (A 1AR) is induced by oxidative stress via activation of nuclear factor (NF)-κB. The prototypic transcription factor is composed of homo- or heterodimers of p50 and p65 subunits. To determine the role of NF-κB in the regulation of the A 1AR in vivo, we compared the A 1AR RNA and protein levels in the brains of mice lacking the p50 subunit of NF-κB (p50 −/− mice) and age-matched B6129PF2/J (F2) controls. Radioligand binding assays in the cortex revealed a significantly lower number of A 1AR (maximal binding capacity, B max) in the cortex of p50 −/− mice (151±62 fmol/mg protein) versus 479±181 fmol/mg protein in the F2 ( N=5 per strain, P<0.05), but no change in the equilibrium dissociation constant. Similar reductions in A 1AR were measured in the hippocampus, brain stem and hypothalamus and in peripheral tissues, such as the adrenal gland, kidney and spleen. Estimation of the A 1AR following purification by antibody affinity columns also indicated reduced A 1AR in the p50 −/− mice cortex, as compared with the F2 mice. A 1AR immunocytochemistry indicates distinct neuronal labeling in the F2 cortex, which was substantially reduced in similar sections obtained from p50 −/− mice. The p50 −/− mice expressed lower levels of A 1AR mRNA than F2 mice, as determined by real time PCR. Quantitation of the A 1AR transducing G proteins by Western blotting show significantly less G αi3, no change in G αi1, but higher levels of G αo and G β in the cortices of p50 −/−, as compared with F2 mice. Administration of bacterial lipopolysaccharide (LPS), an activator of NF-κB, increased A 1AR expression in the cortices of F2 mice but not p50 −/− mice. Cortical neurons cultures prepared from p50 −/− mice showed a greater degree of apoptosis, compared with neurons from F2 mice. Activation of the A 1AR reduced apoptosis with greater efficacy in cultures from F2 than p50 −/− mice. Taken together, these data support a role for NF-κB in determining both the basal and LPS-stimulated A 1AR expression in vivo which could contribute to neuronal survival.

New Ligand Coupling Procedure for Formation of an Immunoadsorption Wall

To prevent the occurrence of dialysis-related amyloidosis, an immunoadsorption wall based on polyacrylamide has been manufactured by a recently developed, partially incomplete, two-stage polymerization method. During the preparation process, efficient utilization of coupling antibodies is the key to large-scale production of such a toxin removal modality. In this study, we attempted to carry out the ligand coupling procedure after formation of a cyanogen bromide (CNBr)-activated stationary phase, using anti-beta2-microglobulin (beta-2M) antibodies. In vitro immunoadsorption tests show that the levels of beta-2M decrease rapidly within the first 2 hours for all the immunoadsorption tests. After that, nearly blank values were reached for tests of initial levels of c. 30 microg/mL and c. 82 microg/mL, whereas a relatively constant level of c. 10 microg/mL was maintained for the test of initial levels of c. 185 microg/mL. The maximum surface binding capacity of the prepared immunoadsorption walls is estimated by fitting experimental data, using a mathematical model of saturation kinetics. The present comparative investigation also suggests the manufacturing process for an immunoadsorption wall could be improved and facilitated by this new ligand coupling procedure without compromising the resulting binding capacity. Furthermore, the experimental protocols as well as the present methodology could be helpful for development of a clinically applicable immunoadsorption wall.

Isolation of lactoferrin from acid whey by magnetic affinity separation

A micron-sized monodisperse superparamagnetic polyglycidyl methacrylate (PGMA) particles coupled with heparin (PGMA-heparin) was synthesized by the polymerization, and then was evaluated as a method for purifying lactoferrin (LF) from bovine whey. The content of heparin groups at the surface of PGMA particles was 0.92 mg/g. Magnetic PGMA-heparin particles were highly effective in recovering LF directly from acid whey by a single step, whose purity was higher than that of commercial standard proteins, using NaCl solutions as eluent. The maximum LF binding capacity was 164 mg/g. The competitive adsorption tests verified that the magnetic approach offered a fast process and potential at a large scale. The practical use of magnetic PGMA-heparin particles for production of high purified LF from whey is a realistic prospect.

LPA4/p2y9/GPR23 Mediates Rho-dependent Morphological Changes in a Rat Neuronal Cell Line

Lysophosphatidic acid (LPA) is a potent lipid mediator that evokes a variety of biological responses in many cell types via its specific G protein-coupled receptors. In particular, LPA affects cell morphology, cell survival, and cell cycle progression in neuronal cells. Recently, we identified p2y(9)/GPR23 as a novel fourth LPA receptor, LPA(4) (Noguchi, K., Ishii, S., and Shimizu, T. (2003) J. Biol. Chem. 278, 25600-25606). To assess the functions of LPA(4) in neuronal cells, we used rat neuroblastoma B103 cells that lack endogenous responses to LPA. In B103 cells stably expressing LPA(4), we observed G(q/11)-dependent calcium mobilization, but LPA did not affect adenylyl cyclase activity. In LPA(4) transfectants, LPA induced dramatic morphological changes, i.e. neurite retraction, cell aggregation, and cadherin-dependent cell adhesion, which involved Rho-mediated signaling pathways. Thus, our results demonstrated that LPA(4) as well as LPA(1) couple to G(q/11) and G(12/13), whereas LPA(4) differs from LPA(1) in that it does not couple to G(i/o). Through neurite retraction and cell aggregation, LPA(4) may play a role in neuronal development such as neurogenesis and neuronal migration.

Combined Two‐Stage Xanthate Processes for the Treatment of Copper‐Containing Wastewater

AbstractHeavy metal removal is mainly conducted by adjusting the wastewater pH to form metal hydroxide precipitates. However, in recent years, the xanthate process with a high metal removal efficiency, attracted attention due to its use of sorption/desorption of heavy metals from aqueous solutions. In this study, two kinds of agricultural xanthates, insoluble peanut‐shell xanthate (IPX) and insoluble starch xanthate (ISX), were used as sorbents to treat the copper‐containing wastewater (Cu concentration from 50 to 1,000 mg/L). The experimental results showed that the maximum Cu removal efficiency by IPX was 93.5 % in the case of high Cu concentrations, whereby 81.1 % of copper could rapidly be removed within one minute. Moreover, copper‐containing wastewater could also be treated by ISX over a wide range (50 to 1,000 mg/L) to a level that meets the Taiwan EPA's effluent regulations (3 mg/L) within 20 minutes. Whereas IPX had a maximum binding capacity for copper of 185 mg/g IPX, the capacity for ISX was 120 mg/g ISX. IPX is cheaper than ISX, and has the benefits of a rapid reaction and a high copper binding capacity, however, it exhibits a lower copper removal efficiency. A sequential IPX and ISX treatment (i.e., two‐stage xanthate processes) could therefore be an excellent alternative. The results obtained using the two‐stage xanthate process revealed an effective copper treatment. The effluent (Ce) was below 0.6 mg/L, compared to the influent (C0) of 1,001 mg/L at pH = 4 and a dilution rate of 0.6 h–1. Furthermore, the Cu‐ISX complex formed could meet the Taiwan TCLP regulations, and be classified as non‐hazardous waste. The xanthatilization of agricultural wastes offers a comprehensive strategy for solving both agricultural waste disposal and metal‐containing wastewater treatment problems.

Chronic use of marijuana decreases cannabinoid receptor binding and mRNA expression in the human brain

Chronic exposure to Cannabis sativa (marijuana) produced a significant down-regulation of cannabinoid receptor in the postmortem human brain. The significant decrease in maximal binding capacity was not accompanied by changes in the affinity constant. [3H]SR141716A binding was reduced in the caudate nucleus, putamen and in the accumbens nucleus. A significant decrease of binding sites was seen in the globus pallidus. Also in the ventral tegmental area and substantia nigra pars reticulata quantitative analysis of the density of receptors shows a significant reduction in [3H]SR141716A binding. In Cannabis sativa user brains, compared with normal brains [3H]SR141716A binding was reduced only in the hippocampus. The density of cannabinoid receptor 1 mRNA-positive neurons was significantly lower in Cannabis sativa users than in control brains for the caudate nucleus, putamen, accumbens nucleus and hippocampal region (CA1–CA4, areas of Ammon’s horn). No hybridization was seen in the mesencephalon and globus pallidus.

Covalent binding of α-chymotrypsin on the magnetic nanogels covered by amino groups

A new aminated carrier—magnetic nanogels covered by amino groups, was obtained by Hoffman degradation of polyacrylamide-coated Fe 3O 4 nanoparticles prepared by photochemical polymerization. α-Chymotrypsin (CT) was covalently bound to the magnetic nanogels by use of 1-ethyl-3-(3-dimethylaminepropyl) carbodiimide and N-hydroxysuccinimide at room temperature. Immobilization time, pH value of the reaction mixture and proportion of CT to the magnetic nanogels were investigated to obtain the optimum condition for CT immobilization. The maximal specific activity of the bound CT was determined to be 0.93 U/(mg min), 59.3% of free counterpart. The maximal binding capacity was measured to be 102 mg enzyme/g nanogel. Furthermore, the bound CT exhibited good thermal stability, storage stability and reusability.

Capacity of adsorption of Pb 2+ and Ni 2+ from aqueous solutions by chitosan produced from silkworm chrysalides in different degrees of deacetylation

The binding capacities of chitin (CT) and chitosan (CS) produced from silkworm chrysalides were investigated aiming at their future application in the removal of Pb 2+ and Ni 2+ from wastewaters. CS with 75% deacetylation degree (DD) exhibited good binding performance for Pb 2+, but bad efficiency for Ni 2+. The maximum binding capacity obtained from isotherms for CS Pb was 141.10 mg g −1 and 52.81 mg g −1 for CS Ni. The binding capacities for CT were 32.01 mg g −1 for Pb 2+ and 61.24 mg g −1 for Ni 2+. The authors attribute these behaviors to two main factors: (i) the large ionic size of Pb 2+ and (ii) the steric hindrance due to CT acetyl groups. Metal binding onto CS was evaluated by the Freundlich and Langmuir isotherm models. The parameter values obtained from the isotherm analysis confirmed that Pb 2+ and Ni 2+ interact differently with CS and that various factors influence their adsorption. Thermogravimetric analysis (TGA) showed that the thermal behavior of CS with 75% deacetylation degree was in the same profile of standard CS; however, the binding of the metals onto its structure affects the curve profile.

Dual inhibition of cyclooxygenase and lipoxygenase by human haptoglobin: Its polymorphism and relation to hemoglobin binding

Haptoglobin (Hp) binds hemoglobin (Hb) specifically and stoichiometrically. Since Hb stimulates prostaglandin (PG biosynthesis), we investigated if Hp effects arachidonic acid (AA) metabolism. The results showed that Hp (50–250 μg protein) inhibited the biosynthesis of PGs via cyclooxygenase (COX) and 12-HETE via lipoxygenase pathway in human platelets. Additional evidence was obtained by the loss of Hp inhibitory activity upon removal of Hp by affinity chromatography on hemoglobin sepharose and by inhibition of AA or bradykinin-induced bronchoconstriction in the guinea pig. Hb reduced the inhibitory effect of Hp in a concentration-related manner such that all its inhibitory activity was lost when completely bound by Hb. Of the three Hp phenotypes, Hp 1-1 showed maximum binding capacity to Hb indicating its greater protective role. These findings implicate Hp in the regulation of COX and lipoxygenase pathways and show Hp involvement in the body’s endogenous defense system against inflammation. This indicates that mammals have dual defense system, i.e., a specific immune system and non-specific Hp defense system.

MRNA expression and binding sites for α2-adrenergic receptor subtypes in muscle layers of the ileum and spiral colon of dairy cows

To measure maximum binding capacity (B(max)) and levels of mRNA expression for alpha(2)-adrenergic receptor (AR) subtypes in ileal and colonic muscle layers of healthy dairy cows. Ileal and colonic muscle specimens from 6 freshly slaughtered cows. Ileal and colonic muscle layers were obtained by scraping the mucosa and submucosa from full-thickness tissue specimens. Level of mRNA expression for alpha(2)-AR subtypes was measured by real-time reverse transcriptase-PCR analysis and expressed relative to the mean mRNA expression of glyceraldehyde phosphate dehydrogenase, ubiquitin, and 18S ribosomal RNA. Binding studies were performed with tritiated RX821002 ((3)H-RX821002) and subtype-selective ligands as competitors. mRNA expression for alpha(2AD)-, alpha(2B)-, and alpha(2C)-AR subtypes was similar in ileal and colonic muscle layers. The mRNA expression for alpha(2AD)-AR was significantly greater than that for alpha(2B)- and alpha(2C)-AR subtypes, representing 92%, 6%, and 2%, respectively, of the total mRNA. Binding competition of (3)H-RX821002 with BRL44408, imiloxan, and MK-912 was best fitted by a 1-site model. The B(max) of alpha(2AD)- and alpha(2C)-AR sub-types was greater than that of alpha(2B)-AR. The B(max) and level of mRNA expression were only correlated (r = 0.8) for alpha(2AD)-AR. Ratio of B(max) to mRNA expression for alpha(2C)-AR was similar to that for alpha(2B)-AR, but significantly greater than for alpha(2AD)-AR. Subtypes of alpha(2)-AR in bovine intestinal muscle layers are represented by a mixture of alpha(2AD)- and alpha(2C)-ARs and of alpha(2B)-AR at a lower density. Information provided here may help in clarification of the role of AR subtypes in alpha(2)-adrenergic mechanisms regulating bovine intestinal motility.

An experimental and theoretical study of the morphine binding capacity and kinetics of an engineered opioid receptor

Electrochemical real-time monitoring of ligand binding to an engineered opioid receptor specific for morphine is reported. In the particular systems studied, 90% of the binding was found to be completed after only 85-120 s. Thus, the binding kinetics has proven to be more rapid than previously believed. The observed association rate constant for the morphine binding reaction was calculated to be 215 M(-1)s(-1). A theoretical analysis of the experimental binding data suggested that the binding sites of the engineered opioid receptor could best be described by a model having two populations of binding sites: K(D)=40 microM (13 micromol/g) and K(D)=205 microM (29 micromol/g). Furthermore, a theoretical model was developed in order to explain the observed binding of the engineered opioid receptor. This model suggested that the binding sites on the polymer surface are up to 5.1A deep and they allow 100% of the ligand (morphine) to anchor itself into the site. The predicted theoretical maximum binding capacity for the reported receptor is calculated to be approximately 2 mmol/g polymer (based on an increase of cavity density).

Characteristic of theophylline imprinted monolithic column and its application for determination of xanthine derivatives caffeine and theophylline in green tea

Theophylline imprinted monolithic columns were designed and prepared for rapid separation of a homologous series of xanthine derivatives, caffeine, and theophylline by an in situ thermal-initiated copolymerization technique. Caffeine and theophylline were fully separated both under isocratic and gradient elutions on this kind of monolithic molecularly imprinted polymers (MIP) column. The broad peak showed in isocratic elution could be improved in gradient elution. Some chromatographic conditions such as mobile phase composition, flow rate, and the temperature on the retention times were investigated. Hydrogen bonding interaction and hydrophobic interaction played an important role in the retention and separation. The binding capacity was evaluated by static adsorption and Scatchard analysis, which showed that the dissociation constant ( K D) and the maximum binding capacity ( Q max) were 1.50 mol/L, and 236 μmol/g for high affinity binding site, and 7.97 mol/L and 785 μmol/g for lower affinity binding site, respectively. Thermodynamic data (ΔΔ H and ΔΔ S) obtained by Van’t Hoff plots revealed an enthalpy-controlled separation. The morphological characteristics of monolithic MIP were investigated by scanning electron microscope, which showed that both mesopores and macropores were formed in the monolith. The present monolithic MIP column was successfully applied for the quantitative determination of caffeine and theophylline in different kinds of green tea.

Intra- or Intercomplex Binding to the γ-Secretase Enzyme: A MODEL TO DIFFERENTIATE INHIBITOR CLASSES

Intra- or Intercomplex Binding to the γ-Secretase Enzyme: A MODEL TO DIFFERENTIATE INHIBITOR CLASSES

A Semi-Physiological Population Pharmacokinetic Model Describing the Non-linear Disposition of Indisulam

The pharmacokinetic profile of the anti-cancer agent indisulam is non-linear. In addition to non-linear clearance, this may partially be explained by saturable distribution to red blood cells and saturable plasma protein binding. The aims of this study were to develop a semi-physiological population pharmacokinetic model of indisulam and to examine the impact of protein binding and distribution to red blood cells. Indisulam concentrations in plasma, plasma ultrafiltrate and in red blood cells from multiple phase I studies in Caucasian and Japanese patients were used to develop a pharmacokinetic model using NONMEM. This model comprised four physiological compartments: plasma, red blood cells, interstitial fluid and tissue. A simulation study was performed to examine the contribution of physiological processes in indisulam pharmacokinetics. Plasma albumin concentrations were predictive for the maximal protein binding capacity and considerably influenced total plasma concentrations of indisulam, whereas free plasma concentrations remained relatively unaffected. The maximal specific red blood cell binding capacity was 64.0 ( +/-5.9) mg/L and corresponded to the typical red blood cell carbonic anhydrase concentration. Simulation studies demonstrated that the hematocrit does not have a clinically relevant impact on indisulam disposition. This semi-physiological model allowed adequate prediction of the time profiles of indisulam concentrations in all monitored compartments for a large range of dose levels and several treatment regimens. The model has elucidated the mechanism and the role of saturable plasma protein binding and saturable distribution to red blood cells in indisulam pharmacokinetics and provides a basis for rationale PK-PD relationships.

Probiotic bacteria as potential detoxification tools: assessing their heavy metal binding isotherms

Dietary exposure to heavy metals may have detrimental effects on human and animal health, even at low concentrations. Specific probiotic bacteria may have properties that enable them to bind toxins from food and water. We assessed the interaction of probiotic bacteria with cadmium and lead in vitro as an initial screening step to identify strains for heavy metal decontamination in food and intestinal models. Binding isotherms for cadmium and lead were characterized for Lactobacillus rhamnosus LC-705, Propionibacterium freudenreichii subsp. shermanii JS and a mix of them used by the food industry. Differences among the strains and their combinations in binding performance at a range of concentrations between 0.1 and 100 mg.L-1 were evaluated with the Langmuir model for biosorption. The effects of pH, contact time, and viability on the binding capacities were also investigated. All strains and their combinations were found to bind cadmium and lead efficiently at low concentration ranges commonly observed in foods. However, the two strains and their combinations differed significantly in their maximum binding capacities and affinities represented by the Langmuir constants Qmax and b, respectively. The binding seemed to occur instantaneously and in a pH-dependent manner, which can be perfectly described by a segmented linear-plateau model.

Molecularly Imprinted Solid‐Phase Extraction of Epicatechin from Tea Beverage

A new molecularly imprinted polymer (MIP) was prepared by using epicatechin as the template molecule. The synthesized MIP was characterized by infrared spectroscopy and scanning electron microscopy, and the MIP with 15–38 µm particle size was selected as a solid‐phase extraction (SPE) sorbent in this work. The maximum MIP binding capacity was 38 µg epicatechin per 100 mg polymer by cartridge test, with a recovery rate of 95.04%. It showed good selectivity and affinity for epicatechin compared with the structurally‐related compounds of quercetin and rutin. The tea samples were also directly applied onto the SPE cartridge and the collected fractions were analyzed by high‐performance liquid chromatography. The MIP cartridge enabled the selective extraction and preconcentration of epicatechin from tea samples.

Intermedin 1–53 protects the heart against isoproterenol-induced ischemic injury in rats

Intermedin is a novel member of the calcitonin/calcitonin gene-related peptide (CGRP) family peptide, which has vasodilatory and hypotensive actions identical to those of adrenomedullin and CGRP. Cleavage sites located between 2 basic amino acids at Arg93–Arg94 result in the production of prepro-intermedin 95–147, namely intermedin 1–53. The bioactive action of intermedin 1–53 and its physiological significance are unclear. In this work, we aimed to explore the effects of intermedin 1–53 on acute myocardial injury induced by isoproterenol. Myocardial ischemia injury in rats was induced by subcutaneous injection of a high dose of isoproterenol, and the therapeutic effect of intermedin 1–53 was observed. Plasma lactate dehydrogenase activity, myocardial and plasma malondialdehyde content were higher in the isoproterenol group than that in controls. Isoproterenol-treated rats showed lower maximal rate of increase and decrease of left-ventricle pressure development (± left-ventricle d p/d t max) and higher left-ventricle end-diastolic pressure (all P < 0.01), which suggested severe heart failure and myocardial injury. Semi-quantitative RT-PCR analysis showed that the gene expression of calcitonin receptor-like receptor and receptor-activity-modifying protein (RAMP)1, RAMP2 and RAMP3 in ventricular myocardia were up-regulated by 79% ( P < 0.01), 48% ( P < 0.01), 31% ( P < 0.05) and 130% ( P < 0.01), respectively, compared with controls. In myocardial sarcolemmal membranes, the maximum binding capacity for [ 125I]-intermedin 1–53 was increased by 118% ( P < 0.01) in the isoproterenol group compared with controls. Rats treated with low dosage intermedin 1–53 (5 nmol/kg/day, 2 days) showed 21% ( P < 0.05) higher myocardial cAMP content, 18% and 31% higher + left-ventricle d p/d t max and -left-ventricle d p/d t max respectively, 288% lower left-ventricle end-diastolic pressure (all P < 0.01), and attenuated myocardial lactate dehydrogenase leakage and malondialdehyde formation (all P < 0.01). Treatment with high dosage intermedin 1–53 (20 nmol/kg/day, 2 days) gave better results than that with low dosage intermedin 1–53. These results suggest that the intermedin receptor system was up-regulated in isoproterenol-induced myocardial ischemic injury and intermedin 1–53 might play a pivotal cardioprotective role in such injury.

Augmenter of liver regeneration promotes hepatocyte proliferation induced by Kupffer cells.

To observe the effects of augmenter of liver regeneration (ALR) on Kupffer cells and to determine whether ALR promotes hepatocyte proliferation induced by Kupffer cells. Kupffer cells and hepatocytes were cultured in vitro and various concentrations of recombinant rat ALR (rrALR) were added. 3H-thymidine, BrdU and 3H-leucine incorporation was determined in cultured Kupffer cells and hepatocytes, in hepatocytes conditioned by Kupffer cells, and in associated medium. rrALR was labeled by iodination and used to determine its binding activity by Scatchard analysis in Kupffer cells and primarily cultured rat hepatocytes. rrALR stimulated DNA replication in Kupffer cells and protein synthesis both in cells and in medium in a non-concentration-dependent manner. The effect was significant at the concentration of 1 microg/L ALR. However, rrALR had no effect on primarily cultured hepatocytes, when hepatocytes were cultured with the Kupffer cell medium conditioned by ALR, DNA replication and protein synthesis in hepatocytes increased significantly at the concentration of 1 microg/L ALR. When the ALR concentration was increased, its effect on hepatocyte proliferation decreased to the basal level. Scatchard analysis indicated the presence of a single class of high affinity receptors with a dissociation constant (Kd) of 0.883 nmol/L and a maximum binding capacity (Bmax) of 126.1 pmol/g protein in the rat Kupffer cells. ALR can promote hepatocyte proliferation induced by Kupffer cells, which is associated with the concentration of ALR, suggesting that Kupffer cells play a dual role in liver regeneration.