Single-site retroperitoneoscopic donor nephrectomy.

We have performed retroperitoneoscopic nephrectomy for living kidney donor surgery since 2000. Recently, we introduced single-site retroperitoneoscopic donor nephrectomy (RDN) as a less invasive donor surgery. The procedure was performed in 7 donors (5 women and 2 men) by a single surgeon. The mean age and body mass index of the donors were 62.6 years (range, 53-74 years) and 24.3 kg/m(2) (range, 22.3-29.0 kg/m(2)), respectively. Left-sided nephrectomy was performed in all the donors. The donors were positioned in the right lateral position, and a 7-cm-long incision was made in the left flank. The incision was extended to the retroperitoneal space using the muscle-splitting technique. The retroperitoneal space was then expanded using an inflation balloon. A GelPOINT Advanced Access Platform (Applied Medical, Rancho Santa Margarita, Calif, United States) was placed in the incision. The subsequent technique and equipment were the same as those used in conventional 3-port RDN. The renal artery and vein were dissected using a vascular stapler, and the kidney graft was directly extracted through the incision. The mean operative time was 197 ± 28 minutes, warm ischemic time was 4.1 ± 1.2 minutes, and blood loss was 75 ± 113 mL. No statistical differences were found between the present method and conventional 3-port RDN. Intraoperative and postoperative complications were not observed in any of the donors. Graft function after transplantation was good, and delayed graft function was not observed in any of the recipients. This technique can be easily introduced in the clinical setting by surgeons experienced in RDN.

Electromyographic and haemodynamic activities in lumbar muscles during bicycle ergometer exercise and walking.

Although bicycle ergometer exercise and walking are recommended as aerobic exercise for patients with lumbago, little research has been done to examine the muscular activities and circulatory dynamics during these exercises. In this study, we aimed at obtaining basic information on aerobic exercises effective for patients with lumbago by investigating the activities and circulatory dynamics of their lumbar muscles during bicycle ergometer exercise and walking. As subjects, we selected 10 healthy adults (23.7 +/- 3.4 years old) with no anamnestic history of lumbago. The measurement conditions were 4 types of exercise: walking (4.0 km/h); 25W, 50W and 75W bicycle ergometer exercises. The activities of the lumbar muscles during the exercises were measured by a surface electromyograph, and percent of MVC was calculated from the maximal voluntary contraction (MVC). With regard to the circulatory dynamics of the lumbar muscles, we measured oxygenated hemoglobin (Oxy-Hb) and deoxygenated hemoglobin (Deoxy-Hb) before and after the exercises with near infrared spectroscopy (NIRS). The change rates during the exercises were calculated based on the values before the exercises. Paired t test was employed to analyse the comparison of the circulatory dynamics of the lumbar muscles between, before and during the exercises. With respect to the comparison of the change rates of the muscular activities and circulatory dynamics among each of the exercises, we employed the one-way analysis of variance (ANOVA) (p < .05). The lumbar muscular activities during the walking were significantly higher than those during the bicycle ergometer exercise were at each load level. The Oxy-Hb increased significantly during the 25W and 50W bicycle ergometer exercises, as opposed to before the exercises. It showed a tendency to decrease during the 75W bicycle ergometer exercise and walking, but not significant. The change rate of the Oxy-Hb during the 25W bicycle ergometer exercise indicated a higher value than that of the other exercises. The Deoxy-Hb, on the other hand, declined significantly in every exercise compared with those before the exercises, with no significant differences in the change rates between each of the exercises. Bicycle ergometer exercise has been suggested as an aerobic exercise permitting as much oxygen uptake as walking does, with fewer loads on lumbar muscles and less likelihood of inducing a hypoxic state on lumbar muscles.

Immunohistochemical localization of phosphatidylcholine in rat mandibular condylar surface and lower joint cavity by cryotechniques.

The immunolocalization of phospholipids has not yet been clearly demonstrated in temporomandibular joints (TMJs). We have examined the distribution of one of phospholipids, phosphatidyl-choline (PC), in the rat mandibular condylar surface and lower joint cavity. Some fresh resected TMJs with their disks attached were immediately plunged into isopentane-propane cryogen (-193 degrees C). Cryostat sections were cut, mounted on NH3+-coated slides, and fixed with paraformaldehyde (PF). Cryosections were first immunostained with anti-mouse PC antibody (JE-1). Subsequently, they were labeled with immunogold particles following silver enhancing for light microscopic analyses. Some cryosections were subjected to double immunofluoresecence labeling with anti-fibronectin antibody or hyaluronic acid-binding protein in combination with the anti-PC antibody. As an immunocontrol, other cryosections were pretreated with phospholipase A2 before such immunofluorescence labeling. We have confirmed the presence of PC in the lower joint cavity of rat TMJs as well as on the mandibular condylar surface layer, which was colocalized with hyaluronic acid and fibronectin respectively. However, by treatment with phospholipase A2, such immunolabeling for PC was clearly decreased, showing that the PC is a component in the rat in vivo TMJ. These findings suggest that PC, hyaluronic acid and fibronectin may interact each other in the TMJ articular surface areas to play a functional role for lubrication in TMJ.

Ultrastructural study of upper surface layer in rat mandibular condylar cartilage by quick-freezing method.

The purpose of the present study is to clarify native ultrastructures of upper surface layers of the rat mandibular condylar cartilage in vivo by a quick-freezing method. The mandibular cartilaginous tissues were removed with their articular discs attached without opening the lower joint cavity. The specimens were processed for light microscopy, transmission or scanning electron microscopy. Deep-etching replica membranes were also prepared after the routine quick-freezing method. The upper surface layer was well preserved by the quick-freezing method. The cartilaginous tissues, which were fixed without opening their articular discs, appeared to keep better morphology than those after opening them. The upper surface layer was thicker than the corresponding layer as reported before. It consisted of atypical extracellular matrices with lots of apparently amorphous components, which were distributed over typical collagen fibrils, by conventional electron microscopy. As revealed with the replica membranes, it also consisted of variously sized filaments and tiny granular components localized on the typical collagen fibrils. A pair of stereo-replica electron micrographs three-dimensionally showed compact filaments within the upper surface layer. The quick-freezing method was useful for keeping native ultrastructures of the fragile upper surface layer in the mandibular condylar cartilage, which may be functionally important to facilitate smooth movement of the temporomandibular joint.

Immunohistochemical study of the upper surface layer in rat mandibular condylar cartilage.

Both hyaluronic acid and fibronectin localizations were examined in the upper surface layer of rat mandibular condylar cartilages by immunohistochemical techniques. Their delicate structure was successfully preserved by preparation procedures of joint condyles with disks. Paraformaldehyde-fixed cartilaginous tissues were cut in a cryostat, and cryosections were analyzed using streptavidin-peroxidase and indirect immunofluorescence methods. Another immunogold method with conventional preparation procedures and a quick-freezing method was performed for their ultrastructural analyses. Both hyaluronic acid-binding protein and anti-fibronectin antibody were used to localize hyaluronic acid and fibronectin in the mandibular condylar cartilage, respectively. Some cryosections were pre-treated with hyaluronidase and chondroitinase before such labeling. The upper surface layer was composed of double laminar structures. One bordered with the cartilage matriceal surface, which was positive for fibronectin. The hyaluronic acid was localized over the fibronectin layer. Therefore, the hyaluronic acid in vivo was bound with fibronectin in the cartilaginous matrix, performing lubrication for the mandibular joint movement.

4-(Methylthio)-3-butenyl isothiocyanate, a principal antimutagen in daikon (Raphanus sativus; Japanese white radish).

The antimutagenic activity of n-hexane extracts from eight strains of daikon (Raphanus sativus; Japanese white radish) have been examined using the UV-induced mutation assay of Escherichia coli B/r WP2. A correlation was found between the potency of antimutagenicity and the amount of 4-(methylthio)-3-butenyl isothiocyanate (MTBITC) in their n-hexane extracts. Because the pure MTBITC also showed antimutagenicity, MTBITC is presumably the active antimutagen principle in n-hexane extracts of daikon. Among the eight strains of daikon studied, Aokubi, the improved common strain in Japan, contained 71.0 micromol of MTBITC in 100 g of fresh daikon. In contrast, Karami and Momoyama, which are original wild strains, contained much more MTBITC (363.5 and 168.0 micromol/100 g, respectively). In addition, phenethyl isothiocyanate was found in a lesser amount (5-33 nmol/100 g) in eight strains of daikon, and allyl isothiocyanate and benzyl isothiocyanate were not detectable in any strains (<3 nmol/100 g). The amount of total isothiocyanate in grated daikon was 7.0 times higher than that in cut daikon measured after 30 min of cooking. Through eating habits, humans might be able to consume substantial amounts of the antimutagen MTBITC from dishes using the grated form of wild strains of daikon. Therefore, it is possible to substantially increase the intake of the antimutagenic ingredient of daikon (i.e., MTBITC) by changing food preferences and preparation procedures (i.e., using the grated form of the wild strains).

Characterization of secretory type IIA phospholipase A2 (sPLA2-IIA) as a glycyrrhizin (GL)-binding protein and the GL-induced inhibition of the CK-II-mediated stimulation of sPLA2-IIA activity in vitro.

By means of heparin-affinity and glycyrrhizin (GL)-affinity column chromatographies (HPLC), a GL-binding phospholipase A2 (gbPLA2) was selectively purified from the synovial fluids of patients with rheumatoid arthritis. This purified gbPLA2 was identified as a secretory type IIA PLA2 (sPLA2-IIA) since it was crossreacted with anti-sPLA2-IIA serum. The activity of purified sPLA2-IIA was inhibited by glycyrrhetinic acid (GA) and a GA derivative (oGA) in a dose-dependent manner, but it was more sensitive to GA than GL. Furthermore, it was found that (i) purified sPLA2-IIA is phosphorylated by casein kinase II (CK-II) in vitro; (ii) this phosphorylation induces in a significant stimulation of PLA2 activity; and (iii) oGA at one-tenth the concentration of GL inhibits the CK-II-mediated stimulation of sPLA2-IIA activity. These results show that (i) sPLA2-IIA is a GL-binding protein; and (ii) CK-II mediates stimulation of its PLA2 activity in vitro.

Inhibitory effect of glycyrrhizin on the phosphorylation and DNA-binding abilities of high mobility group proteins 1 and 2 in vitro.

The physiological correlation between glycyrrhizin (GL) and high mobility group proteins I and 2 (HMG1/2) and the inhibitory effect of GL on their phosphorylation by three protein kinases (CK-I, CK-II and PKC) were investigated biochemically in vitro. It was found that GL binds directly to HMG1/2, because (i) HMG1/2 have a high affinity with a GL-affinity column; and (ii) GL induces the conformational changes in HMG1/2. Both purified HMG1/2 functioned as phosphate acceptors for these two protein kinases (CK-I and PKC), but not phosphorylated by CK-II. Phosphorylation of HMG1/2 by two protein kinases (CK-I and PKC) was completely inhibited by a glycyrrhetinic acid derivative (oGA) at one-tenth the concentration of GL. Also, the DNA-binding abilities of HNG1/2 were reduced by GL in a dose-dependent manner. These results show that the binding of GL to HMG1/2 results in the inhibition of their physiological activities (DNA-binding ability and phosphorylation by PKC or CK-I) in vitro. The GL-induced inhibition of the physiological activities of HMG1/2 may be involved in the anti-inflammatory effect of GL in vivo.

Detection of monocyte chemoattractant protein-1 receptor expression in experimental atherosclerotic lesions: an autoradiographic study.

BACKGROUND: Monocytes, a common component of atheroma, are attracted to the lesion site in response to chemotactic signals, particularly expression of monocyte chemoattractant peptide 1 (MCP-1). This study assessed the feasibility of using radiolabeled MCP-1 to identify monocytes and macrophages that have localized at sites of experimental arterial lesions. Methods and Results-- The biodistribution of radiolabeled MCP-1 was determined in normal mice, and localization in experimental atheroma was determined in cholesterol-fed rabbits 4 weeks after arterial injury of the iliac artery (9 rabbits) and the abdominal aorta (1 rabbit). Vessels were harvested and autoradiographed after intravenous administration of (125)I-labeled MCP-1 and Evans blue dye. The arteries were evaluated histologically by hematoxylin and eosin staining and immune staining with a monoclonal antibody specific for rabbit macrophages (RAM-11). (125)I-MCP-1 has a blood clearance half-time of approximately 10 minutes and circulates in association with cells. The liver, lungs, and kidneys had the highest concentration of (125)I-MCP-1 at 5 and 30 minutes after tracer administration. Autoradiograms revealed accumulation of (125)I-MCP-1 in the damaged artery wall, with an average ratio of lesion to normal vessel of 6:1 (maximum 45:1). The accumulation of (125)I-MCP-1 in the reendothelialized (plaque formation) areas was greater than in the deendothelialized (Evans blue-positive) areas (6.55+/-2.26 versus 4.34+/-1.43 counts/pixel, P<0.05). The uptake of (125)I-MCP-1 correlated with the number of macrophages per unit area (r=0.85, P<0.0001). CONCLUSIONS: Radiolabeled MCP-1 may be a useful tracer for imaging monocyte/macrophage-rich experimental atherosclerotic lesions.

Characterization of bovine angiogenin-1 and lactogenin-like protein as glycyrrhizin-binding proteins and their in vitro phosphorylation by C-kinase.

Angiogenin-1 (p15, an angiogenesis inducer with RNase activity) and lactogenin-like protein (p17) isolated from partially purified bovine lactoferrin (bLF) preparations were characterized as glycyrrhizin (GL)-binding proteins (gbPs). As expected, bLF-affinity column chromatography confirmed these two gbPs to be bLF-binding proteins. These two purified gbPs exhibited RNase activities when incubated with poly(C) as a substrate. Both GL and glycyrrhetinic acid (GA) at 100 microM significantly inhibited RNase activities of these two gbPs, both of which functioned as phosphate acceptors of C-kinase in vitro. Phosphorylation of p15 and p17 by C-kinase was inhibited by GA in a dose-dependent manner with the 50% inhibition dose (ID50) of approx. 10 microM, whereas GL required a relatively high dose (300 microM) to inhibit significantly it. A GA derivative (oGA, ID50=approx. 0.3 microM) was found to be a potent inhibitor of the C-kinase-mediated phosphorylation of these two gbPs in vitro. In addition, a possible physiological significance of C-kinase on the physiological interaction between bLF and two bLF-binding proteins (p15 and p17) is noted.

Biochemical characterization of cholesterol-3-sulfate as the sole effector for the phosphorylation of HMG1 by casein kinase I in vitro.

Phosphorylation of high mobility group protein 1 (HMG1) by casein kinase I (CK-I) and potent effectors (inhibitors and activators) of this phosphorylation were investigated in vitro. We found that (i) CK-I phosphorylates specifically threonine residues on HMG1 when incubated with cholesterol-3-sulfate (CH-3S), but no phosphorylation of HMG1 is detected in the presence of other cholesterol related compounds or their sulfated derivatives; (ii) this phosphorylation is selectively inhibited by heparin, but stimulated significantly by 3',4',7-trihydroxy-isofavone at low doses (0.1-3 microM); and (iii) CH-3S directly induces a drastic conformational change in HMG1. The latter finding provides a mechanism to explain how CH-3S alone can induce the phosphorylation of HMG1 by CK-I in vitro.

Plasma growth hormone (GH) responses after administration of the peptidergic GH secretagogue KP102 into the oral cavity, rumen, abomasum and duodenum in adult goats.

The study was performed to determine whether orally administered KP102 (also known as GHRP-2) stimulates GH release in adult goats, and how the orally administered KP102 passes through the digestive tract and stimulates GH release in ruminant animals. Five mg/kg body weight (BW) of KP102 dissolved in 9 ml of saline were administered into the oral cavity, rumen, omasum and duodenum of adult goats, and GH release after administration of KP102 was examined. The GH levels were significantly elevated at 20 min after administration of KP102 into the oral cavity, and plasma concentrations of GH remained significantly elevated until 60 min (P < 0.05). The GH levels after administration of KP102 into the abomasum were variable. However, the GH level tended to increase within 30 min after administration, and were significantly higher than those of controls after 120 to 150 min (P < 0.05). The GH levels after administration of KP102 into the duodenum were significantly elevated at 40 min after administration, and plasma concentrations of GH remained significantly elevated until 140 min (P < 0.05). The administration of KP102 into the rumen failed to stimulate GH release. The GH response curves (AUC) produced after administration of KP102 into the abomasum or duodenum were 2.2-fold greater than those for after administration into the oral cavity (P < 0.05). The oral administration of 5 mg/kg BW of KP102 in the powder state, not dissolved in 9 ml of saline, failed to stimulate GH release. These results suggested that orally administered KP102 dissolved in saline transiently stimulates GH release in adult goats, and this phenomenon might be due to small amounts of the peptides entering directly into the abomasum with liquid bypassing the rumen.

Biochemical characterization of galloyl pedunculagin (ellagitannin) as a selective inhibitor of the beta-regulatory subunit of A-kinase in vitro.

The inhibitory effects of galloyl pedunculagin (GP) and eugeniin on the phosphorylation of histone H2B by cAMP-dependent protein kinase (A-kinase) and autophosphorylation of its beta-regulatory subunit (A-kinase beta) were examined in vitro. It was found that (i) GP (ID(50) = approx. 50 nM) effectively inhibits the activity of A-kinase (heterodimer), but high doses are required to inhibit the activities of the alpha-catalytic subunit (ID(50) = approx. 0.25 microM) and casein kinase II (CK-II, ID(50) = approx. 0.6 microM); (ii) GP inhibits the autophosphorylation of A-kinase beta in a dose-dependent manner with an ID(50) of approx. 6.6 nM, which is about 30-fold lower than that observed with CK-II beta; and (iii) GP reduces the suppressive effect of the beta-subunit on the activity of the alpha-subunit. In addition, purified bovine heart A-kinase precipitates when incubated with excess GP at pH 5.0. A similar precipitation of A-kinase was observed with eugeniin. These results show that the direct binding of GP to the beta-subunit prevents the physiological interaction between the beta- and alpha-subunits of A-kinase in vitro. This conclusion is presumably consistent with the binding affinity of proline-rich proteins with tannins, since A-kinase beta contains a proline-rich domain that interacts with GP or eugeniin. Therefore, GP will serve as a powerful inhibitor for in vitro and in vivo cellular studies of A-kinase beta-mediated signal transduction.

Synthesis of kanamycin A analogs containing a 6-amino-6-deoxyglycofuranose moiety.

Three kanamycin A analogs containing 6-amino-6-deoxyglycofuranoses have been prepared as candidates for potential activity against resistant bacteria producing 6'-N-acetyltransferase. They are 4-O-(6-amino-3,5,6-trideoxy-alpha-D-, -beta-D-, and -beta-L-erythro -hexofuranosyl)-6-O-(3-amino-3-deoxy-alpha-D-glucopyranosyl)-2,5-dideoxy-5-epi-5-fluorostreptamine. Structure-activity relationships of these compounds are discussed.

Characterization of bovine and human lactoferrins as glycyrrhizin-binding proteins and their phosphorylation in vitro by casein kinase II.

The binding ability of bovine and human lactoferrins (bLF and hLF; LFs) to a glycyrrhizin (GL)-affinity column and their phosphorylation by casein kinase II (CK-II) in vitro were biochemically investigated. It was found that (i) both bLF and hLF are GL-binding proteins; (ii) purified both proteins function as phosphate acceptors of CK-II; and (iii) this phosphorylation is completely inhibited by two polyphenol-containing anti-oxidant compounds (quercetin and epigallocatechin gallate) at I microm, whereas a glycyrrhetinic acid derivative (oGA) inhibits it at one tenth the concentration of GL. The DNA-binding affinity of hLF was reduced by GL in a dose dependent manner. However, no significant effect of the CK-II-mediated hLF phosphorylation on its DNA-binding affinity was detected. These results suggest that the GL-induced inhibition of the DNA-binding affinity and the CK-II-mediated phosphorylation of hLF may be closely correlated with the anti-inflammatory effect of GL in the human body.

Biochemical characterization of casein kinase II as a protein kinase responsible for stimulation of HIV-1 protease in vitro.

The physiological significance of casein kinase II (CK-II) on the protease (PR) activity of recombinant HIV-1 PR (rPR) was biochemically investigated in vitro. We found that (i) the purified rPR (p11) functions as a phosphate acceptor of CK-II; (ii) the PR activity of rPR is stimulated approximately 2.9-fold after its full phosphorylation by recombinant human CK-II (rhCK-II) in a manner similar to that observed for recombinant HIV-1 reverse transcriptase (rRT); and (iii) this stimulation is completely inhibited by two polyphenol-containing anti-oxidant compounds [quercetin and epigallo-catechin gallate (EGCG)] at 0.1 microM or a glycyrrhetinic acid derivative (oGA) and catechin at 10 microM without significant effect on the PR activity of rPR. These results suggest that (i) CK-II may be a host mediator responsible for stimulation of PR and RT in HIV-1-infected cells; and (ii) the selective inhibition of the CK-II-mediated stimulation of HIV-1 PR and RT by potent CK-II inhibitors may be involved in their anti-HIV-1 effects at the cellular level.

Novel approach for large-scale, biocompatible, and low-cost fractionation of peptides in proteolytic digest of food protein based on the amphoteric nature of peptides.

A large-scale, biocompatible, and low-cost procedure for peptide fractionation based on the amphoteric nature of peptide is developed. A sample cell (120 x 100 x 50 mm) with four joint tubes (17 mm i.d. and 20 mm in length) on the front and back was prepared. On the end of the joint tubes, a nylon screen (100 mesh)-supported agarose gel layer was formed. Five or nine of the sample cells were connected. A tryptic digest of casein (2.0-3.6 L) was applied to the sample cells. At each end of the sample cell apparatus, an additional cell filled with 0.1 M H(3)PO(4) or NaOH was connected and used as anode and cathode compartments, respectively. Reproducible fractionation of peptide could be achieved by collecting fractions with specific pH values when voltage reached a plateau by applying direct current at constant power. Running time necessary for fractionation of peptide was inversely proportional to electric power and directly proportional to sample volume.

Apoptosis: the importance of nuclear medicine.

Apoptosis is a genetically controlled, energy-dependent process which removes unwanted cells from the body. Because of its orderly progression, apoptosis is also known as programmed cell death or cell suicide. Once initiated, apoptosis is characterized by a series of biochemical and morphological changes involving the cytoplasm, nucleus and cell membrane. Cytoplasmic changes include cytoskeletal disruption, cytoplasmic shrinkage and condensation; prominent changes in the nucleus include peripheral chromatin clumping and inter-nucleosomal DNA cleavage (DNA ladder formation); and membrane changes include the expression of phosphatidylserine on the outer surface of the cell membrane and blebbing (resulting in the formation of cell membrane-bound vesicles or apoptotic bodies). These events allow the cell to digest and package itself into membrane-bound packets containing autodigested cytoplasm and DNA, which can then be easily absorbed by adjacent cells or phagocytes. An endogenous human protein, annexin V (molecular weight approximately 35,000), has an affinity of about 10(-9) M for phosphatidylserine exposed on the surface of apoptotic cells. Annexin V can be labelled with radionuclides such as iodine or technetium, or positron emitting agents. Experimental studies in cells confirm that fluorescence and 99Tc(m)-labelled annexin have comparable affinity for apoptotic cells. In vivo studies with 99Tc(m)-labelled annexin confirm that radiolabelled annexin V can be used to image apoptotic cells/tissues in vivo. In this article, we review experimental data using annexin V imaging and discuss its possible future use to identify apoptosis in vivo.

Plasma insulin-like growth factor-I concentrations increase during the estrous phase in goats.

The objective of this study was to characterize plasma insulin-like growth factor-I (IGF-I) profiles during the estrous cycle in goats. Frequent blood samples were drawn during the day of estrus and during the luteal phase on Day 10 after estrus, and plasma growth hormone (GH) and IGF-I profiles were examined. Then, daily blood samples were drawn throughout the estrous cycle or during induction of estrus by prostaglandin F(2alpha) (PGF(2alpha)) to further clarify the IGF-I profiles. GH was secreted in an episodic manner in the estrous and luteal phases in goats. There were no significant differences in the mean concentrations, pulse amplitude and pulse frequency of GH between the estrous and luteal phases. IGF-I concentrations during estrous phase were higher than those in the luteal phase (P<0.05). Plasma IGF-I increased approximately two days before behavioral estrus, and the IGF-I peak was observed in accordance with the appearance of estrus. The elevated IGF-I levels then declined to basal values 4 to 5 days after estrus. When estrus was induced by PGF(2alpha), plasma IGF-I concentrations increased after treatment, and the concentration 2 days after treatment (day of appearance of behavioral estrus) was significantly higher than concentrations before treatment (P<0.05). The elevated IGF-I levels then declined during the 3 days after treatment. These results indicate that plasma IGF-I concentrations increase during estrus in goats.

Biochemical characterization of bovine lactoferrin as a glycyrrhizin-binding protein in vitro.

Lactoferrin (LF) from bovine colostrum was biochemically characterized as a glycyrrhizin (GL)-binding protein (gbP) in vitro. It was found that (i) bovine LF (bLF) and a synthetic bovine lactoferricin (bLFcin, the N'-terminal region of bLF at the positions 17--41) had a high affinity to a GL-affinity column; (ii) approximately 1.8 moles of GL were bound to a molecule of bLF with a binding constant of approx. 1.20x10(4) M(-1) at pH 6.8; and (iii) GL, but not glycyrrhetinic acid (GA), induced a conformational change of bLF. In addition, the glucuronic acid moiety of the GL molecule was found to be responsible for binding to bLF, because (i) no binding of GA and two glucoses-GA (Glc-Glc-GA) to bLF was detected; and (ii) a synthetic fluorinated GL (GlcA-GlcF-GA) and mono-glucuronyl-GA (mono-GlcA-GA) were bound significantly to bLF. A similar binding of GL to human LF (hLF) was also observed under the same experimental conditions. Data provided here suggest that (i) bLF contains plural GL-binding sites; and (ii) the specific binding of GL to bLF may modulate the physiological activity of bLF in vivo.