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Biomedical subjects

B S Hong

Publications and source records attributed to B S Hong.

At least 19 recordsLinked to original sources
PubMed2001

Identification of essential amino acid residues for catalytic activity and thermostability of novel chitosanase by site-directed mutagenesis.

The functional importance of a conserved region in a novel chitosanase from Bacillus sp. CK4 was investigated. Each of the three carboxylic amino acid residues (Glu-50, Glu-62, and Asp-66) was changed to Asp and Gln or Asn and Glu by site-directed mutagenesis, respectively. The Asp-66-->Asn and Asp-66-->Glu mutation remarkably decreased kinetic parameters such as Vmax and kcat to approximately 1/1,000 those of the wild-type enzyme, indicating that the Asp-66 residue was essential for catalysis. The thermostable chitosanase contains three Cys residues at positions 49, 72, and 211. The Cys-49-->Ser/Tyr and Cys-72-->Ser/Tyr mutant enzymes were as stable to thermal inactivation and denaturating agents as the wild-type enzyme. However, the half-life of the Cys-211-->Ser/Tyr mutant enzyme was less than 10 min at 80 degrees C, while that of the wild-type enzyme was about 90 min. Moreover, the residual activity of Cys-211-->Ser/Tyr enzyme was substantially decreased by 8 M urea; and it lost all catalytic activity in 40% ethanol. These results show that the substitution of Cys with any amino acid residues at position 211 seems to affect the conformational stability of the chitosanase.

Amino Acid Sequence↗
PubMed2001

Purification and characterization of antithrombotics from Syzygium aromaticum (L.) MErr. & PERRY.

Two antithrombotic polysaccharides with relatively high molecular weight (HMW) and low molecular weight (LMW) were isolated from the flower buds of Syzygium aromaticum (L.) MERR. & PERRY (clove) by anion-exchange chromatography, hydrophobic interaction column chromatography and size exclusion chromatography (LMW: EC-2B-IIIa-2, M.W. ca. 34000; HMW: EC-2C-Ia-2, M.W. ca. 103000). The LMW polysaccharide was mainly composed of Rha, Gal, GalA and Ara (molar %: 24.1, 18.9, 18.0 and 17.9, respectively) with 10.8% of sulfate and 18.2% of protein. The HMW fraction consisted of Ara, Gal, Glc and Rha (molar %: 26.0, 23.7, 17.5 and 12.4, respectively) with 15.4% of sulfate and 8.0% of protein. Both polysaccharides had the backbone of type I rhamnogalacturonan and the side chain of arabinan. Also, most of the sulfates were attached at the position 6 of 3-linked galactosyl residues. Compared to the antithrombotic activity of the HMW fraction (plasma clotting time of 145 s in APTT assay), the LMW fraction displayed a slightly low activity (90 s). However, animal studies indicated that crude LMW polysaccharide did not show acute toxicity, while the acute LD50 of the HMW fraction was approximately 2-fold lower than that of heparin.

Animals↗
PubMed2001

Purification and biological activity of acidic polysaccharide from leaves of Thymus vulgaris L.

Polysaccharides are involved in biological responses and can activate complement system, which plays an important role in primary host defense mechanisms. We investigated anticomplementary activities from spice plants and selected thyme (Thymus vulgaris L.) as a potent complementary activator. Acidic polysaccharide (TV-3-IIIA-IIa) purified from the hot-water extract of thyme leaves by DEAE-Toyopearl 650C, Butyl-Toyopearl 650M and Sepharose CL-6B column chromatography and preparative HPLC. The purified polysaccharide, TV-3-IIIA-IIa showed potent anticomplementary activity via classical and alternative pathway with the increase proportional to dosage. TV-3-IIIA-IIa seemed to be a homogenous polymer from the results of HPLC and its molecular mass was estimated as 180 kDa. TV-3-IIIA-IIa mainly consisted of galacturonic acid (44.8 mol%), glucuronic acid (16.7 mol%), arabinose (11.1 mol%), rhamnose (9.2 mol%), galactose (8.9 mol%) and small amounts of glucose, xylose, mannose and fucose. By methylation analysis and reactivity to beta-glucosyl Yariv reagent, TV-3-IIIA-IIa was assumed to contain small amounts of type II arabinogalactan and large amounts of pectin-like polysaccharides in the structure. Based upon these results, TV-3-IIIA-IIa was suggested to be a complement activator.

Animals↗
PubMed2001

Purification and characterization of anti-complementary polysaccharide from leaves of Thymus vulgaris L.

For the purification of the anti-complementary polysaccharide from Thymus vulgaris L., the hot-water extract of thyme leaves was successively fractionated by ethanol precipitation and ultra-filtration. The retentate with 300-kDa membrane cartridge showed a potent anti-complementary activity. It was further purified by open column chromatographies on DEAE-Toyopearl 650C, Butyl-Toyopearl 650M and Sephadex G-100, obtaining TV3-IIA-I, the purified anti-complementary polysaccharide. The anti-complementary polysaccharide exhibited the anti-complementary activity via both classical and alternative pathways. Based upon the methylation analysis and the reaction with beta-glucosyl Yariv reagent, the anti-complementary polysaccharide from thyme might contain an arabinogalactan moiety, at least in part.

Chromatography, Ion Exchange↗
PubMed2001

Anti-stress and anti-fatigue effects of fermented rice bran.

The anti-stress and anti-fatigue effects on rats and mice of a hot water extract of rice bran fermented with Saccharomyces cerevisae IFO 2346 were investigated. Oral administration (1 g/kg/day) of the hot water extract of fermented rice bran (FRB) inhibited major changes in the weight of the adrenal, thymus, spleen and thyroid, showing an anti-stress effect. The hot water extract of FRB also inhibited increases in the GPT and LDH activity, cholesterol and glucose in the serum. The administration (1 g/kg/day) for 2 weeks significantly prolonged the swimming time, resulting in an increase in the anti-fatigue effect. It is considered from these results that FRB had anti-stress and anti-fatigue effects.

Adjuvants, Immunologic↗
PubMed2001

Effects of polysaccharides from rhizomes of Curcuma zedoaria on macrophage functions.

The effects of Curcuma zedoaria, which is used as a condiment, in perfumery, and as a medicine, on immune response were investigated by measuring macrophage-stimulating activity in macrophages and RAW 264.7 cells. In this study, CZ-1 and CZ-1-III, the fractions partially purified from C. zedoaria, had a strong, dose-dependent lysosomal enzyme activity. It was suggested that active portions of CZ-1-III were polysaccharides rather than proteins. Phagocytic activity increased as a similar pattern in both the gram-negative and gram-positive bacteria, time-dependently. It was demonstrated that CZ-1-III can augment the oxygen burst response but had an even higher activity in vivo than in vitro. Also a significant increase of H2O2, NO, and TNF-alpha production was observed. However, the production of TNF-alpha at the concentration of 1,000 microg/ml decreased. These data suggested that C. zedoaria had macrophage-stimulating activity and the possibility of being used as a biological response modifier.

Animals↗
PubMed2001

Thermostable chitosanase from Bacillus sp. strain CK4: its purification, characterization, and reaction patterns.

A thermostable chitosanase, purified 156-fold to homogeneity in an overall yield of 12.4%, has a molecular weight of about 29,000 +/- 2,000, and is composed of monomer. The enzyme degraded soluble chitosan, colloidal chitosan, and glycol chitosan, but did not degrade chitin or other beta-linked polymers. The enzyme activity was increased about 2.5-fold by the addition of 10 mM Co2+ and 1.4-fold by Mn2+. However, Cu2+ ion strongly inhibited the enzyme. Optimum temperature and pH were 60 degrees C and 6.5, respectively. The enzyme was stable after heat treatment at 80 degrees C for 30 min or 70 degrees C for 60 min and fairly stable in protein denaturants as well. Chitosan was hydrolyzed to (GlcN)4 as a major product, by incubation with the purified enzyme. The effects of ammonium sulfate and organic solvents on the action pattern of the thermostable chitosanase were investigated. The amounts of (GlcN)3-(GlcN)6 were increased about 30% (w/w) in DAC 99 soluble chitosan containing 10% ammonium sulfate, and (GlcN)1 was not produced. The monophasic reaction system consisted of DAC 72 soluble chitosan in 10% EtOH also showed no formation of (GlcN)1, however, the yield of (GlcN)3 approximately (GlcN)6 was lower than DAC 99 soluble chitosan-10% ammonium sulfate. The optimal concentration of ammonium sulfate to be added was 20%. At this concentration, the amount of hexamer was increased by over 12% compared to the water-salt free system.

Ammonium Sulfate↗
PubMed2000

Antitumor, genotoxicity and anticlastogenic activities of polysaccharide from Curcuma zedoaria.

The antitumor effect of the partially purified polysaccharide from Curcuma zedoaria was studied in mice transplanted with sarcoma 180 cells. The polysaccharide fraction, CZ-1-III, at dose of 6.25 mg/kg/d showed 50% inhibition in solid tumor growth. When mice were injected with fractions, CZ-1 and CZ-1-III, at the dose of 100.0 mg/kg, 91.6% and 97.1% of tumor growth were inhibited, respectively, indicating that the cytotoxic effect of polysaccharide on sarcoma 180 cells increases upon increasing the amount of polysaccharide administered. To assess the genotoxicity of CZ-1-III fraction, several classical toxicological tests were performed. In Ames test, CZ-1-III did not show any transformation of revertant with or without S-9 metabolic activating system, indicating the lack of mutagenic effect of the compound. To assess clastogenic effect, micronucleus and chromosomal aberration assays were performed using Chinese hamster lung (CHL) fibroblast cells. However, up to 259.0 microg/ml concentration of CZ-1-III, neither micronucleus formation nor chromosomal aberration was induced regardless of the presence of S-9 metabolic activating system. Inhibition of CZ-1-III on micronucleus formation induced by mitomycin C was exhibited in a dose-dependent manner, maximally up to 52.0%. These results strongly suggest that CZ-1-III, the polysaccharide fraction from C. Zedoaria, decreases tumor size of mouse and prevents chromosomal mutation.

Animals↗
PubMed2000

Analysis of essential leucine residue for catalytic activity of novel thermostable chitosanase by site-directed mutagenesis.

Bacterial chitosanases share weak amino acid sequence similarities at certain regions of each enzyme. These regions have been assumed to be important for catalytic activities of the enzyme. To verify this assumption, the functional importance of the conserved region in a novel thermostable chitosanase (TCH-2) from Bacillus coagulans CK108 was investigated. Each of the conserved amino acid residues (Leu64, Glu80, Glu94, Asp98, and Gly108) was changed to aspartate and glutamine or asparagine and glutamate by site-directed mutagenesis, respectively. Kinetic parameters for colloidal chitosan hydrolysis were determined with wild-type and 10 mutant chitosanases. The Leu64 --> Arg and Leu64 --> Gln mutations were essentially inactive and kinetic parameters such as Vmax and kcat were approximately 1/10(7) of those of the wild-type enzyme. The Asp98 --> Asn mutation did not affect the Km value significantly, but decreased kcat to 15% of that of wild-type chitosanase. On the other hand, the Asp98 --> Glu mutation affected neither Km nor kcat. The observation that approximately 15% of activity remained after the substitution of Asp98 by Asn indicated that the carboxyl side chain of Asp98 is not absolutely required for catalytic activity. These results indicate that the Leu64 residue is directly involved in the catalytic activity of TCH-2.

Amino Acid Sequence↗
PubMed2000

Thermostable chitosanase from Bacillus sp. Strain CK4: cloning and expression of the gene and characterization of the enzyme.

A thermostable chitosanase gene from the environmental isolate Bacillus sp. strain CK4, which was identified on the basis of phylogenetic analysis of the 16S rRNA gene sequence and phenotypic analysis, was cloned, and its complete DNA sequence was determined. The thermostable chitosanase gene was composed of an 822-bp open reading frame which encodes a protein of 242 amino acids and a signal peptide corresponding to a 30-kDa enzyme. The deduced amino acid sequence of the chitosanase from Bacillus sp. strain CK4 exhibits 76.6, 15.3, and 14.2% similarities to those from Bacillus subtilis, Bacillus ehemensis, and Bacillus circulans, respectively. C-terminal homology analysis shows that Bacillus sp. strain CK4 belongs to cluster III with B. subtilis. The gene was similar in size to that of the mesophile B. subtilis but showed a higher preference for codons ending in G or C. The enzyme contains 2 additional cysteine residues at positions 49 and 211. The recombinant chitosanase has been purified to homogeneity by using only two steps with column chromatography. The half-life of the enzyme was 90 min at 80 degrees C, which indicates its usefulness for industrial applications. The enzyme had a useful reactivity and a high specific activity for producing functional oligosaccharides as well, with trimers through hexamers as the major products.

Amino Acid Sequence↗
PubMed1993

Purification of phospholipid hydroperoxide glutathione peroxidase from bovine retina.

A low molecular size peroxidase with a high affinity for phospholipid hydroperoxide was purified from bovine retina by sequential extraction with low and high ionic strength buffer, followed by ammonium sulfate fractionation, chromatography on an ultraspherogel column and Protein PAK-SP column. The purified enzyme has a low Km (0.011 mmol/L) for phospholipid hydroperoxide, and a high Km (1.37 mmol/L) for glutathione. Glutathione oxidation was competitively inhibited by vitamin E, Ki = 0.019 mmol/L. The retinal PHGPX is different from the PHGPX purified by others from heart and liver in molecular size. The molecular size estimated by gel filtration chromatography is below 6 kDa.

Ammonium Sulfate↗
PubMed1992

Characterization of a bovine synovial fluid lubricating factor. II. Comparison with purified ocular and salivary mucin.

Boundary lubricating activity and biochemical characteristics of purified lubricating factor from bovine synovial fluid (PSLF) were compared to those of mucinous glycoprotein from human submandibular saliva and stimulated tears. Mammalian synovial fluid and saliva contain mucinous glycoproteins which reduce the coefficient of friction (mu) in a bearing of latex:glass which isolates boundary lubrication. In contrast, mucin secreted by the lacrimal gland did not lubricate. Cleveland plotting showed that these species are not identical.

Animals↗
PubMed1989

Degradation of bovine corneal collagen by alkali.

This study was undertaken to demonstrate the effect of alkali on the molecular size of collagen. Type I and V collagen from bovine cornea were incubated with different concentrations of NaOH at room temperature and 37 degrees C for various times. The samples were then neutralized and analyzed by high-pressure liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Degradation of collagen molecules to a heterogeneous mixture of peptides with molecular weights less than 20,000 daltons occurred at concentrations greater than 0.25N, and the effect of alkali was much faster at 37 degrees C than at room temperature. In contrast, the molecular size of collagen was not affected by similar concentrations of HCl under similar conditions of time and temperature.

Alkalies↗
PubMed1988

Heterogeneity of collagens in rabbit cornea: type VI collagen.

Normal adult rabbit corneas were digested with 5% pepsin and their collagens extracted with acetic acid. Collagen extracts were fractionated by differential salt precipitation. The 2.5 M NaCl fraction was then redissolved with tris buffer and precipitated with sodium acetate. The precipitate contained a high-molecular-weight disulfide-bonded aggregate which, upon reduction with mercaptoethanol, was converted into three distinct polypeptides having molecular weights between 45 and 66 Kd. These physical characteristics, together with the susceptibility of these polypeptides to collagenase and their amino acid composition, identified the high molecular weight aggregate as type VI collagen. Corneas from neonate rabbits and adult corneas containing 2-week-old scars were organ cultured in the presence of [14C] glycine to incorporate radiolabel into collagen. Tissues were digested with 0.02% pepsin and their collagens extracted with formic acid. The total radioactivity of the extracts and tissue residues was determined before the collagens were separated by SDS-polyacrylamide slab gel electrophoresis. Radioactive collagen polypeptides bands were then stained with Coomassie blue, processed for fluorography, and analyzed by densitometry. The results show that: (1) type VI collagen is synthesized by neonate corneas and healing adult corneas; (2) it is not readily solubilized from either corneal tissue by 0.02% pepsin digestion and formic acid extraction; and (3) the proportion of type VI collagen deposited in scar tissue is markedly lower than that found in neonate corneas.

Amino Acids↗
PubMed1988

Heterogeneity of collagens in rabbit cornea: type III collagen.

Whole neonate rabbit corneas and adult corneas containing 2-week-old scars were incubated in the presence of [14C] glycine. Radiolabeled collagen extracted from the corneas and scar tissue were analyzed by sodium dodecylsulfate/polyacrylamide gel electrophoresis and fluorography to determine the types and relative quantity of collagen polypeptides present and synthesized by these tissues. In addition to other collagen types, type III was found in both neonate cornea and scar tissue from adult cornea, albeit in relatively small quantities. Type III collagen in normal cornea was associated with the residue after pepsin digestion and formic acid extraction of the tissue, and the same type of collagen was extracted from scar tissue after similar treatment. Type III collagen-specific monoclonal antibody bound to developing normal corneas and healing adult tissue sections, as determined by immunofluorescence. Antibody binding was localized to the endothelium and growing Descemet's membrane in fetal and neonate corneas, and restricted to the most posterior region of the corneal scar tissue. Although monoclonal antibody to keratan sulfate, used as a marker for stromal fibroblasts, bound to most of the scar tissue, the antibody failed to bind to the posterior scar tissue positive for type III collagen. We conclude that endothelial cells from fetal and neonate rabbit cornea and endothelium-derived fibroblasts from healing wounds of adult cornea synthesize and deposit type III collagen. Moreover, this collagen appears to be incorporated into the growing Descemet's membrane of normal corneas and narrow posterior portion of the scar tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Interstitial retinol-binding protein (IRBP) in subretinal fluid.

Antibodies against bovine interstitial retinol-binding protein (b-IRBP) were used to detect human IRBP (h-IRBP) on immunoblots of eight samples of subretinal fluid (SRF) from patients with retinal detachments of between 2 days' and more than 2 years' duration. Using this sensitive technique, it was found that seven of the samples contained h-IRBP in concentrations estimated to range from below 5% up to 19% of normal human IPM. One of these samples displayed two immunoreactive bands of roughly equal intensity, one at a molecular weight of 135,000 (h-IRBP), the other at 115,000. The latter may have been generated by proteolytic cleavage. No h-IRBP could be detected in an eighth sample from a patient with retrolental fibroplasia. It is concluded that the reduced concentration of h-IRBP in SRF may be due to a number of factors that include dilution, proteolytic degradation, and metabolic inactivation of photoreceptors at the detachment site.

Adolescent↗
PubMed1985

Identification of type II procollagen in rabbit vitreous.

A procollagen in the soluble fraction of rabbit vitreous was isolated by dialysis against dilute acetic acid and partially purified by Bio-Gel A 5M gel filtration. The molecule was identified to be type II procollagen by comparing its segment-long-spacing (SLS) banding pattern with that of standard type II collagen isolated from rabbit articular cartilage. Electron microscopy of the SLS of this type II procollagen revealed a fuzzy propeptide extension at the N-terminal end of the molecule. Pepsin digestion of the procollagen removed this extension, thus converting the molecule into a collagen which had mobility similar to that of pepsin-soluble cartilage type II collagen in SDS-polyacrylamide gel electrophoresis. No inter-chain disulfide bond was found in the propeptide extension when the procollagen samples were electrophoresized with or without mercaptoethanol. Comparison of the cyanogen bromide peptide map of the type II procollagen with that of the pepsin-soluble type II collagen indicated that two extra peptides were present in the digest of procollagen. All of this evidence suggested that the procollagen in the soluble vitreous body of the rabbit eye was a novel type II procollagen with a propeptide extension only at the N-terminus.

Animals↗