Kou Hayakawa

Inhibitory Effect of Fucoidan on Huh7 Hepatoma Cells Through Downregulation of CXCL12

The aim of this study is to assess whether fucoidan modulates the expression of chemokine ligand 12 (CXCL12)/chemokine receptor 4 (CXCR4) and exerts antitumor activity toward Huh7 hepatoma cells. According to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, fucoidan inhibited the growth of Huh7 cells and HepG2 cells in a dose-dependent manner, with a 50% inhibition of cell growth (IC 50 ) of 2.0 and 4.0 mg/ml, respectively. α -fetoprotein levels in medium collected from fucoidan-treated cells were significantly decreased in Huh7 cells but not in HepG2 cells. Western blotting revealed that the amount of α -fetoprotein was decreased by 1.0 mg/ml of fucoidan in Huh7 cells, whereas it was unchanged in HepG2 cells. In Huh7 cells, CXCL12 mRNA expression was significantly downregulated by 1.0 mg/ml of fucoidan, whereas CXCR4 mRNA expression was unchanged by fucoidan. CXCL12 and CXCR4 mRNA were barely expressed in HepG2 cells. In addition, 1.0 mg/ml of fucoidan mildly arrested the cell cycle and induced apoptosis in Huh7 cells. The findings suggest that fucoidan exhibits antitumor activity toward Huh7 cells through the downregulation of CXCL12 expression.

Determination of free N-acetylneuraminic acid in human body fluids by high-performance liquid chromatography with fluorimetric detection

Determinations of both the free and bound form of N-acetyl-neuraminic acid (NANA) in several human body fluids, such as serum, cerebrospinal fluid (CSF), saliva, urine, amniotic fluid, and milk were carried out by HPLC with fluorimetric detection. The method utilized 1,2-diamino-4,5-methylenedioxybenzene dihydrochloride (DMB) as a fluorimetric derivatizing reagent. Free-form NANA was obtained from the body fluids after ultrafiltration with Microcon 10 (YM-10 cellulose membrane, filtration limit M(r) = 10,000, Amicon). The DMB derivative of NANA was separated isocratically by a Nucleosil 5C18 column with a mixture of 0.1 M sodium phosphate buffer (pH 2.0)-methanol (75:25, v/v). A gradient elution system was used for urine analysis. Analysis times were 10-30 min. Recoveries of free NANA by ultrafiltration were satisfactory: 95.66 +/- 1.80% for serum and 97.27 +/- 1.55% for CSF, respectively. The high sensitivity and specificity render this method applicable to all the body fluids tested. Although a physiological role for free NANA has not yet been elucidated, the method presented promises to contribute to the basic understanding of the NANA metabolism.

Liberation of lipoate by human serum lipoamidase from bovine heart pyruvate dehydrogenase

Lipoamidase, which hydrolyses such substrates as lipoamide, lipoylmethyl ester, lipoyllysine, and lipoyl 4-aminobenzoate (LPAB), was purified from human serum through use of synthetic substrate LPAB. The purified human serum lipoamidase showed lipoyllysine hydrolase activity (Km = 435 microM, Vmax = 64.5 nmol/min per mg of protein). The purified enzyme did not liberate the free form of lipoic acid from bovine heart pyruvate dehydrogenase (PDH). PDH was hydrolyzed quantitatively by proteinase K to lipoyllysine, which was determined by the HPLC method. Although liberation of lipoate from various lengths of lipoyl-peptides has not been tested yet, it is likely that lipoamidase requires proteinase(s) before the liberation of free lipoic acid from the enzymes.

Human serum lipoamidase.

Thirty-two human serum specimens were assayed for lipoamidase (lipoyl-4-aminobenzoate hydrolase) activity. All sera had lipoamidase activities. This substrate was newly synthesized by us and had a satisfying purity as evaluated by HPLC-fluorimetric detection. Product (p-aminobenzoate) liberated was determined directly by the HPLC-fluorimetric method. Liberation of the product was linearly continued for 6 h. The pH optimum of serum lipoamidase was found to be 7.0. The effect of substrate concentration on human serum lipoamidase activity was examined and the reaction was saturated at 0.1 mmol/l. The sera obtained were from individuals aged from 1 to 8 years. The mean value of serum lipoamidase activity was found to be 1.50 U/l (SD 1.037, range 0.04-3.75, n = 32). The difference of sex effects was analyzed and no significant difference was found (males: n = 14, mean 1.48, SD 1.162, range 0.04-3.75; females: n = 18, mean 1.52, SD 0.963, range 0.48-3.51) among this age group. Biotinidase activity was also determined in these 32 serum specimens and the correlation was examined. The mean biotinidase activity was 3.16 U/l (SD 2.567, range 0.35-9.37). The correlation coefficient (r) between lipoamidase activity and biotinidase activity was 0.8931. Although the physiological significance of lipoamidase has not been known, the enzyme might play an important role in recycling of lipoate as biotinidase does.

High-performance liquid chromatographic determination of lipoamidase (lipoyl-X hydrolase) activity with a novel substrate, lipoyl-6-aminoquinoline

An HPLC lipoamidase (lipoyl-X hydrolase) assay method has been developed, which uses a novel fluorescent substrate, lipoyl-6-aminoquinoline (LAQ). LAQ is synthesized from lipoic acid and 6-aminoquinoline (AQ) through lipoyl chloride as an intermediate and is conveniently purified by washing with chloroform-methanol. Mechanistic studies on the time-course, the dependence on enzyme and substrate concentrations were performed by using LAQ and a model enzyme (milk lipoamidase). Moreover, this method was successfully applied to the direct determination of the lipoamidase (LAQ hydrolase) activity in samples of human liver, milk, stools and porcine serum. Using this novel synthetic lipoyl substrate, we demonstrated that LAQ hydrolase was present in some specific tissues; LAQ hydrolase was solely present in the grey matter and not in the white matter in the human cerebrum. Furthermore, LAQ hydrolase activity was shown to increase in human liver cancer. Thus, this enzyme assay method is expected to be applicable to the tissue distribution study and also to the basic research on human diseases such as cancer.

Serum protein determination by high-performance gel-permeation chromatography

A general high-performance gel-permeation chromatography (HPGPC) method was developed to determine protein in human serum with improved sensitivity and speed. The optimum UV wavelength for protein detection was found to be 210 nm, by comparing the protein values obtained by varying the UV wavelength of the HPLC detection system with the protein values obtained from spectrophotometric protein assays, i.e., the bicinchoninic acid (BCA) method and the biuret method. The analysis time was less than 1 min. Since this HPGPC serum protein assay method is simple and rapid, it is expected to be particularly well adapted for use in clinical laboratories.

Improved high-performance liquid chromatographic determination of biotinidase activity with biotinyl-6-aminoquinoline as substrate.

An improved high-performance liquid chromatographic assay method for biotinidase activity was developed using the fluorimetric substrate biotinyl-6-aminoquinoline, which was found to be more specific than the biotinyl-4-aminobenzoate previously used. The new method measures the intensity of the fluorescent signal at wavelengths (excitation 350 nm; emission 550 nm) longer than those (excitation 276 nm; emission 340 nm) for 4-aminobenzoate. The analysis of fluorescence in the visible spectrum reduced considerably the number of interfering peaks compared with analysis in the ultraviolet region. This method also made it possible to measure the biotinidase activity directly in samples usually difficult to calculate, such as human and bovine milk or porcine serum; the use of biotinyl-6-aminoquinoline allowed the analysis of the enzyme reaction in milk and porcine serum without pretreatment or dialysis. Stoichiometric increase and decrease of the substrate and product, respectively, were demonstrated. Michaelis constants for biotinyl-6-aminoquinoline were measured at various stages of partial purification. Because the solubility of these synthetic substrates in the aqueous phosphate buffer is limited, the determination of both Michaelis constant and maximum velocity by extrapolation may be helpful for the characterization of the kinetics of biotinidase.

Enkephalin hydrolysis by human serum biotinidase

Purified human serum biotinidase exhibited amino-exo-peptidase activity. Enkephalins and dynorphin A (less than 10-mer) seemed to be the most appropriate substrates among various physiological peptides in terms of the kcat/Km values. Similar kcat/Km values were obtained for both biocytin (biotinyllysine) and these opioid-neuropeptides. Neuro-oligo-peptides ranging from 2-mer to 18-mer were hydrolyzed. The presence of amino group at the carboxyl terminal position increased the kcat/Km value by decreasing the Km value. The results of inhibition studies using various kinds of antibiotic inhibitors, metals, and chelating agents indicated that enkephalin hydrolysis was mediated by the peptide-hydrolyzing center probably containing Zn ions. This aminopeptidase activity was uniquely inhibited by a vitamin of biocytin. The reason for the high content of biotinidase activity in serum may be related to the binary function of this enzyme; i.e., biocytin hydrolyzing amidase and enkephalin hydrolyzing aminopeptidase functions.

Biotinidase in the porcine cerebrum.

Biotinidase activities found in porcine brains (n = 3) were as follows: cerebrum, 4.4 +/- 0.2 pmol/min per milligram of protein; cerebellum, 7.6 +/- 0.3 pmol/min per milligram of protein; medulla, 2.9 +/- 0.3 pmol/min per milligram of protein. These values are relatively high compared with the activities in rat or guinea pig brains. Subcellular distribution of biotinidase was found mainly in the soluble cytoplasmic fraction (S3), i.e., in the supernatant of 0.32 M sucrose S2 solution after ultracentrifugation at 105,000g for 90 min. This is in contrast to the guinea pig livers, in which the subcellular distribution of biotinidase is mainly found in the microsomal fraction. After a seven-step purification (22,200-fold enrichment), porcine brain biotinidase is identified as a single polypeptide by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis system, and its molecular weight is determined as 68,000 Da. The isoelectric point of the enzyme was 4.3. Sialidase treatment strongly suggests the presence of sialyl residues in this enzyme. Amino acid analysis indicates relatively high hydrophilicity and high content of glycine and serine. The enzyme activity is inhibited by organic mercurials, but not by diisopropylfluorophosphate. Abundant soluble biotinidase in brain cytoplasm may play an important role which has not been discovered yet.

Protein determination by high-performance gel-permeation chromatography: applications to human pancreatic juice, human bile and tissue homogenate

A high-performance gel-permeation chromatography (HPGPC) method to determine the proteins of human pancreatic juice, bile, and tissue homogenate has been developed. A diol-type silica gel column (35 x 8 mm I.D., 5 nm average pore diameter) was used under a column temperature of 8 degrees C. The eluent was acidic phosphate buffer with a high concentration of sodium chloride, nonionic detergent of polyoxyethylene (20) cetyl ether (Brij 58), glycerol and 2-propanol. The UV wavelength used for the protein detection was 210 nm. Analytical time was within 3.5 min. Good correlation coefficients were obtained with this HPLC method at a column temperature of 8 degrees C and a spectrophotometric bicinchoninic acid (BCA) method. A photometric pyrogallol-red molybdate complex method was found to correlate well with this HPLC method and with the BCA method only for tissue homogenate. Since this HPGPC protein assay method is simple, convenient, rapid, reproducible, and reliable, it is expected to be generally applicable to clinical and also to biochemical research.