Tae-Kyun Lee

Effect of dietary Platycodon grandiflorum on the improvement of insulin resistance in obese Zucker rats.

The effect of dietary Platycodon grandiflorum on the improvement of insulin resistance and lipid profile was investigated in lean (Fa/-) and obese (fa/fa) Zucker rats, a model for noninsulin dependent diabetes mellitus. Dietary Platycodon grandiflorum feeding for 4 weeks resulted in a significant decrease in the concentration of plasma triglyceride in both lean and obese Zucker rats. Furthermore, dietary Platycodon grandiflorum markedly decreased both plasma cholesterol and fasting plasma insulin levels, and significantly decreased the postprandial glucose level at 30 min during oral glucose tolerance test in obese Zucker rats. Although there was no statistical significance, the crude glucose transporter 4 protein level of obese rats fed Platycodon grandiflorum tended to increase when compared with that of obese control rats. Therefore, the present results suggested that dietary Platycodon grandiflorum may be useful in prevention and improvement of metabolic disorders characterized by hyperinsulinemia states such as noninsulin dependent diabetes mellitus, syndrome X, and coronary artery disease.

Inhibitory effect of a Korean traditional medicine, Honghwain–Jahage (water extracts of Carthamus tinctorius L. seed and Hominis placenta) on interleukin-1-mediated bone resorption

A Korean herbal formulation, Honghwain-Jahage (HJ), which is comprised of a herb of Carthamus tinctorius L. seed and Hominis Placenta, was investigated for inhibiting effects on IL-1 beta-stimulated bone resorption in the fetal mouse bone culture system. Results of in vitro cytotoxicities showed that HJ extracts have no any cytotoxicities in concentrations of 1-200 microg/ml on the cultured osteoblast cells derived from mouse calvarial bone explants. Cell viability was not significantly affected by treatment with the indicated concentration of the extracts. The HJ extracts were shown to have inhibitory effects against the synthesis of PGE(2). We also examined the effect of the pretreatment with various concentrations of the HJ extracts then treated by the PGE(2)-induction agents. Pretreatment of the HJ extracts for 1 h, which by itself had little effect on cell survival, reduced the synthesis of PGE(2). Furthermore, the HJ extracts were shown to have protective effects against plasminogen dependent fibrinolysis induced by IL-1 beta. Pretreatment of the HJ extracts for 1 h did not enhance the plasminogen dependent fibrinolysis. Finally, co-treatment of HJ with calcitonin showed significant inhibitory activity on the IL-1 beta-stimulated bone resorption. From these results, it was found that HJ extracts inhibited IL-1 beta-induced bone resorption.

IL-1β regulates cellular proliferation, prostaglandin E2 synthesis, plasminogen activator activity, osteocalcin production, and bone resorptive activity of the mouse calvarial bone cells

ABSTRACT Interleukin-1β (IL-1β) regulates several activities of the osteoblast cells derived from mouse calvarial bone explants in vitro. IL-1β stimulated cellular proliferation and the synthesis of prostaglandin E2 in the cultured cells in a dose-dependent manner. Furthermore, plasminogen activator activity of the mouse osteoblast was positively affected by IL-1β in a dose-dependent manner over the dosage range of 0.01 ng−2 ng/mL with a maximal effect being observed at 2 ng/mL. However, the induction of osteocalcin synthesis and alkaline phosphatase activity in response to vitamin D, two characteristics of the osteoblast phenotype, were significantly antagonized by IL-1β over a similar dose range. Treatment of mouse calvarial bone cells with IL-1β resulted in a dose dependent stimulation of bone resorption and the bone resorption induced by IL-1β was strongly inhibited by calcitonin treatment, indicating osteoclast-mediated bone resorption, suggesting that the bone resorption induced by IL-1β appears to be osteoclast-mediated. This study supports the role of IL-1β in the pathological modulation of bone cell metabolism, with regard to implication of the pathogenesis of osteoporosis by IL-1β.

The expression of matrix metalloproteinase-9 in human follicular fluid is associated with in vitro fertilisation pregnancy

Objective  To investigate the role of matrix metalloproteinase‐9 (MMP‐9) in the pre‐ovulatory follicular fluid and culture media during in vitro fertilisation (IVF) cycle and to develop the zymographic pre‐diagnosis marker for successful implantation and pregnancy in human IVF.

Inhibitory Effects of Scutellaria Barbata D. Don. and Euonymus Alatus Sieb. on Aromatase Activity of Human Leiomyomal Cells

It is now well documented that a large proportion of breast tumors express their own aromatase. This intratumoral aromatase produces estrogen in situ and therefore may contribute significantly to the amount of estrogen to which the cell is exposed. Thus it is not only important that aromatase inhibitors potently inhibit the peripheral production of estrogen and eliminate the external supply of estrogen to the tumor cell, but that they in addition potently inhibit intratumoral aromatase and prevent the tumor cell from making its own estrogen within the cell. To study the inhibition of intracellular aromatase, we have examined the aromatase‐inhibiting potency of the Scutellaria barbata D. Don. (SB) and Euonymus alatus Sieb. (EA) in myometrial and leiomyomal cells which contain aromatase. We have also used human placental tissues. Although SB and EA are approximately equipotent in a cell‐free aromatase system (human placental microsomes), EA is consistently 10–30 times more potent than SB in inhibiting intracellular aromatase in myometrial and leiomyomal cells. To provide insights into the effect of SB and EA on aromatase activity in leiomyomal cells, we examined the cell lines, which is induced to differentiate toward the more transformed cell phenotype by 12‐tetradecanoylphorbal‐13‐acetate (TPA) as a protein kinase C activator and transforming growth factor‐β1 (TGF‐β1). Enzyme activity was inhibited in a time‐and dose‐dependent fashion by SB and EA and by either 1–50 nM TPA or 0.01–0.5 ng/ml TGF‐β1, with maximal responses after 2–3 h exposure.

Interleukin-1 and tumor necrosis factor-α induce collagenolysis and bone resorption by regulation of matrix metalloproteinase-2 in mouse calvarial bone cells

Abstract Interleukin‐1β (IL‐1β) and tumor necrosis factor‐α (TNF‐α) greatly induces osteoclast formation and stimulates bone resorption of mouse calvaria in culture. We examined the effects of the two cytokines on the collagenolysis and bone resorption by induction of matrix metalloproteinases (MMPs). The cells were analyzed using zymographic analysis. It was shown that the mouse calvarial osteoblasts constitutively synthesize progelatinase‐A (MMP‐2). Interleukin‐1β markedly enhanced the messenger RNAs (mRNAs) expression of MMP‐2 (gelatinase A), but slightly MMP‐9 (gelatinase B), which associated with increases in bone matrix degradation. Both pro‐ and active‐forms of MMP‐2 were detected in the conditioned medium collected from calvarial cultures, and IL‐1β markedly stimulated both pro‐ and active‐forms of the MMP‐2. The expression of MMP‐2 mRNAs could be detected, and they were markedly enhanced by IL‐1β on days 1 and 2. These results demonstrate that the potency of induction of MMP‐2 by IL‐1β and TNF‐α is closely linked to the respective bone‐resorbing activity, suggesting that MMP‐2‐dependent degradation of bone matrix plays a key role in bone resorption induced by these cytokines. On the other hand, when the mouse osteoblasts were stimulated with parathyroid hormone, 1,25(OH)2D3, mononuclear cell conditioned medium (MCM) and IL‐1 as bone resorption agents, collagenolysis was increased by producing the active gelatinase. Interleukin‐1 in stimulating bone resorption was examined using fetal mouse long bone organ culture. Interleukin‐1 stimulated bone resorption and produced marked resorption when present simultaneously. Furthermore, treatment of indomethacin and dexamethasone clearly abolished the responses of IL‐1α and IL‐1β.

Immune-stimulating properties of polysaccharides from Phellodendri cortex (Hwangbek)

Heteropolysaccarides were isolated from the Korean medicinal plant, Phellodendri cortex (Hwangbek), by hot water and alkali extractions. The extracted polysaccharides were fractionated into eight fractions and they are mainly composed of D-N-acetylglucosamine, D-galactose, D-mannose, and D-glucose. Among the polysaccharide fractions, Fr.-2 showed a potent B-lymphocyte-stimulating activity in a system using polyclonal antibody forming cells in C57BL/6XC3H mice at dosages of 2–10 mg. On the basis of their solubility in aqueous ethanol, four fractions of Fr.-2-1 to Fr.-2-4 were further obtained from the Fr.-2, and Fr.-2-3 was divided into Fr.-2-3-1, 2, 3, and 4 by DEAE cellulose chromatography. The main activity was found in Fr.-2-3-2, which contained 100% (w/w) of carbohydrates and further purified to Fr.-2-3-2-2 by gel filtration chromatography using TSK Gel HW50S. Fr.-2-3-2-2, having a molecular weight of about 230 kDa, showed the highest B-cell-stimulating activity and the half-maximal concentration for B-lymphocyte-stimulating activity was ca. 2.2 µg/ml.

Differential Inhibition of Scutellaria barbata D. Don (Lamiaceae) on HCG-Promoted Proliferation of Cultured Uterine Leiomyomal and Myometrial Smooth Muscle Cells

Scutellaria barbata D. Don (Lamiaceae)(SB) is a perennial herb which is natively distributed throughout Korea and southern China. This herb is known in traditional Chinese Medicine as Ban‐Zhi‐Lian and traditional Korean medicine as Banjiryun, respectively. SB has been used as an anti‐inflammatory and antitumor agent. We aimed to determine the expressin of cell cycle‐related signal molecules for growth inhibition after HCG treatment by the herb SB in two different human myometrial smooth muscle cells (SMCs) and leiomyomal SMCs. Water‐soluble ingredients of SB, myometrial SMCs and the leiomyomal cell lines were used in vitro. Uterine myomas often enlarge rapidly during pregnancy, implying that human chorionic gonadotrophin (HCG) may influences cell proliferation in uterine leiomyomata. We investigated the effects of SB on the cell proliferation and the expression of cell cycle‐related proteins in these cells. Although HCG/LH receptor was present in both cultured myometrial and leiomyomal cells, as assayed by reverse transcription polymerase chain reaction analysis, treatment with HCG significantly increased cell proliferation in both myometrial and leiomyomal cells. However, SB reduced the proliferative effect of HCG in leiomyoma and myometrial cells, respectively. In HCG‐treated leiomyomal cells, the expression of proliferating cell nuclear antigen, cyclin E and cdc2 was significantly reduced by SB treatment. These results suggest that SB reduced the HCG‐promoted proliferation of myometrial and leiomyomal cells.

Antiproliferative effect of Scutellaria barbata D. Don. on cultured human uterine leiomyoma cells by down-regulation of the expression of Bcl-2 protein.

Scutellaria barbata D. Don (Lamiaceae; SB) inhibited the growth of leiomyomal cells (LM). A time‐dependent antiproliferative effect was noted when 10−5 m buserelin, gonadotrophin‐releasing hormone (GnRH) agonist or 20–40 µg/mL SB was added. The inhibition of cell growth decreased with the addition of the PKC activator (12‐O‐tetradecanoylphorbor‐13‐acetate; TPA) much as it did with the addition of SB, and the decreases in the viable cells caused by the addition of SB were reversed completely by pretreatment with a protein kinase C (PKC) inhibitor (calphostin C). The findings suggest that SB inhibits cell proliferation in cultured human uterine leiomyoma cells accompanied by PKC activation. Next, the study investigated the effect of SB on fetal development for toxicity. Pregnant Sprague‐Dawley rats, from gestation day 6–15, were administered 20 g/L or 50 g/L SB in the drinking water and then killed on day 20. No maternal toxicity was observed, however, embryonic loss in the treatment groups was double that of the controls (p < 0.05). No gross morphologic malformations were seen in the treated fetuses. Fetuses exposed to SB were found to be significantly heavier than the controls, an effect that was greater in female fetuses and was not correlated with increased placental size. The results suggest that the SB had no toxicity and that in utero exposure to SB resulted in increased early embryo loss with increased growth in surviving fetuses. On the other hand, Western blot analyses revealed that Bcl‐2 protein of a 26 kDa was abundant in leiomyomal cells, but not in normal myometrial cells. The addition of progesterone (100 ng/mL) resulted in a striking increase in Bcl‐2 protein expression in the cultured leiomyoma cells. However, the addition of SB (20 µg/mL) resulted in a significant reduction in Bcl‐2 protein expression in the cells. The results indicated that human uterine leiomyomal cells express Bcl‐2 protein and progesterone enhances its expression, however, SB reduces the expression of Bcl‐2 protein in human uterine leiomyoma cells. Copyright

Molecular cloning and expression of an endo-β-1,4-d-glucanase I (avicelase I) gene from Bacillus cellulyticus K-12 and characterization of the recombinant enzyme

Bacillus cellulyticus K-12 Avicelase (Avicelase I; EC 3.2.1.4) gene (ace A) has been cloned in Escherichia coli by using the vector pT7T3U19 and HindIII-HindIII libraries of the chromosomal inserts. The libraries were screened for the expression of avicelase by monitoring the immunoreaction of the antiavicelase (immunoscreening). Positive clones (Ac-3, Ac-5, and Ac-7) contained the identical 3.5-kb HindIII fragment as determined by restriction mapping and Southern hybridization, and expressed avicelase efficiently and constitutively using its own promoter in the heterologous host. From the immunoblotting analysis, a polypeptide that showed a carboxymethylcellulase (CMCase) activity with an Mr, of 64,000 was detected. The recombinant endo 1,4-β-d-glucanase I was purified to homogeneity from an intracellular fraction of E. coli by DEAE-Toyopearl M650, Phenyl Toyoperal M650, and TSK gel HW50S chromatography. The enzyme had a monomeric structure, its relative molecular mass being 65 kDa by gel filtration and 64 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The pI was 5.3 and the optimal pH was 4.6, and the enzyme was stable at pH 4.0–10.5. The enzyme had a temperature optimum of 50°C and was stable at 55°C for 48 h, and retained approx 20% of its activity after 30 min at 70°C. It showed high activity toward carboxymethylcellulose (CMC) as well as p-nitrophenyl-β-d-cellobioside, 4-methylumbelliferyl cellobioside, Avicel, filter paper, and some cellooligosaccharides. Km values for CMC and Avicel were 7.6 and 85.2 mg/mL, respectively, whereas Vmax values were 201 and 9.2 μmol · min−1 · mg−1, respectively. Cellotetraose (G4) was preferentially cleaved into cellobiose (G2) and cellopentaose (G5) was cleaved into G2 + cellotriose (G3), whereas cellohexaose (G6) was cleaved into G4 + G2 and, to a lesser extent, into G3 + G3. G3 was not cleaved at all. G2 was the main product of Avicel hydrolysis. G2 inhibited whereas Mg++ stimulated the activity of CMCase and Avicelase. Hydrolysis of CMC took place with a rapid decrease in viscosity but a slow liberation of reducing sugars. Based on these results, it appeared that the cellulase should be regarded as endo type, although it hydrolyzed Avicel.