Lee, Ha-Na;Jin, Hyeon-Ok;Park, Jin-Ah;Kim, Jin-Hee;Kim, Ji-Young;Kim, BoRa;Kim, Wonki;Hong, Sung-Eun;Lee, Yun-Han;Chang, Yoon Hwan;Hong, Seok-Il;Hong, Young Jun;Park, In-Chul;Surh, Young-Joon;Lee, Jin Kyung
Molecules and Cells
/
v.38
no.4
/
pp.327-335
/
2015
Piperlongumine, a natural alkaloid isolated from the long pepper, selectively increases reactive oxygen species production and apoptotic cell death in cancer cells but not in normal cells. However, the molecular mechanism underlying piperlongumine-induced selective killing of cancer cells remains unclear. In the present study, we observed that human breast cancer MCF-7 cells are sensitive to piperlongumine-induced apoptosis relative to human MCF-10A breast epithelial cells. Interestingly, this opposing effect of piperlongumine appears to be mediated by heme oxygenase-1 (HO-1). Piperlongumine upregulated HO-1 expression through the activation of nuclear factor-erythroid-2-related factor-2 (Nrf2) signaling in both MCF-7 and MCF-10A cells. However, knockdown of HO-1 expression and pharmacological inhibition of its activity abolished the ability of piperlongumine to induce apoptosis in MCF-7 cells, whereas those promoted apoptosis in MCF-10A cells, indicating that HO-1 has anti-tumor functions in cancer cells but cytoprotective functions in normal cells. Moreover, it was found that piperlongumine-induced Nrf2 activation, HO-1 expression and cancer cell apoptosis are not dependent on the generation of reactive oxygen species. Instead, piperlongumine, which bears electrophilic ${\alpha},{\beta}$-unsaturated carbonyl groups, appears to inactivate Kelch-like ECH-associated protein-1 (Keap1) through thiol modification, thereby activating the Nrf2/HO-1 pathway and subsequently upregulating HO-1 expression, which accounts for piperlongumine-induced apoptosis in cancer cells. Taken together, these findings suggest that direct interaction of piperlongumine with Keap1 leads to the upregulation of Nrf2-mediated HO-1 expression, and HO-1 determines the differential response of breast normal cells and cancer cells to piperlongumine.
The metabolism of many drugs and also of steroid hormones is mediated by enzymes located in the microsomal fraction in smooth surfaced endoplasmic reticulum of mammalian liver. The duration and intensity of action of many drugs are largely determined by the speed at which they are metabolized in the body. Repeated administration of phenobarbital results in the induction of enzymes that metabolize a number of drugs. Lee et al. reported that daily administration of phenobarbital in rats significantly increased the activities of amylase in the pancreatobiliary juice, but the concentration of cholate in the bile was significantly lower in the treated group than that in the control group. After animals were treated with $CCl_4$, histological changes were shown in the endoplasmic reticulum, decreased microsomal enzyme activity and decreased hepatic protein synthesis were apparent. The purpose of the present report was to study the interaction between a 'microsomal-stimulating' agent such as phenobarbital and a 'microsomal- depressing' agent such as $CCl_4$ on hepatic and pancreatic functions in rats. The results obtained are summarized as follows: 1. The mortality rate of $CCl_4$ treated group was 34% and was decreased this figure to 15% with phenobarbital pretreatment. 2. In animals treated with phenobarbital the volume of biliary-pancreatic secretion was markedly elevated but the volume was decreased significantly in animals treated with $CCl_4$. 3. Total bilirubin output was elevated markedly in the $CCl_4$ treated group of rats pretreated with phenobarbital. The bilirubin concentration was increased in $CCl_4$ treated group and decreased in the group treated phenobarbital alone. 4. The concentration and total output of cholate in the bile were significantly lower in the all experimental group than control group. 5. In the animals treated with phenobarbital alone and phenobarbital plus $CCl_4$, the activity of lipase in pancreatobiliary juice was elevated, while in the animals treated with $CCl_4$ alone no change was observed. 6. The activity of amylase in the pancreatobiliary juice was decreased in the $CCl_4$ treated group, but elevated markedly in phenobarbital group and also elevated in phenobarbital-$CCl_4$ group. By the above results, it is concluded, when the liver was damaged by $CCl_4$, the exocrine function of pancreas and liver was decreased simultaneously. However, in the animals pretreated with phenobarbital, the toxicity of $CCl_4$ on the liver and pancreas was reduced.
Sasang Constitutional Medicine is a major branch of Korean Traditional Oriental Medicine. The differences of disease susceptibility to be shown in Sasang constitution may be due to genetic factors. Therefore, we examined interrelationship among cerebral infarction (CI), apolipoprotein E (apoE) gene polymorphism, and Sasang constitutional classification. ApoE is a key protein modulating the highly atherogenic apoB containing lipoproteins and is a candidate gene for the development of coronary artery disease (CAD). The ${\varepsilon}2\;and/or\;{\varepsilon}4$ alleles were the first to be implicated in premature CAD, which resulted in this polymorphism being extensively studied. We investigated the association between apoE genotype and CI by case-control study in a Korean population. We also classified CI patients and control group into groups according to Sasang Constitutional Medicine. 196 CI patients and 379 controls without CI were examined. ApoE genotype was determined by 8% polyacrylamide gel separation after DNA amplification. A significant difference in the apoE genotype distribution was observed in the CI patients compared with that in controls ($X^{2}$=14.920, df=4, P=0.005). Also, the frequency of Taeumin constitution in patients with CI was significantly higher than that in controls (58.0% vs. 36.9%; P<0.001). However, the Taeumin constitution did not enhance the relative risk for CI in the subjects with apoE ${\varepsilon}2\;and/or\;{\varepsilon}4$ alleles. No differences in the apoE genotypes frequencies were observed in the Taeumin compared with that in the other constitutions. In addition, we investigated whether the DD genotype of angiotensin converting enzyme (ACE) gene, a candidate gene for CI, was associated with CI, Taeumin constitution, and apoE polymorphism. As a result, the frequency of Taeumin constitution was significantly higher in CI patients with both apoE ${\varepsilon}3/{\varepsilon}4$ and ACE ID/DD genotypes than in the remaining Sasang constitutions (14.5% vs. 8.3% and 0%) ($X^{2}$=13.521, df=6, P=0.035). In summary, we concluded that the apoE polymorphism is a major risk factor for CI in Koreans and the ACE ID/DD genotype enhanced the relative risk for CI in the subjects with apoE ${\varepsilon}3/{\varepsilon}4$ genotype and Taeumin constitution.
Proceedings of the Botanical Society of Korea Conference
/
1987.07a
/
pp.133-148
/
1987
Gibberellin(GA) 3-$\beta$ hydroxylation is very important for the shoot elogation in the higher plants, since only 3$\beta$-hydryoxylated GAs promote shoot elogation in several plants. Fluctuation of 3$\beta$-hydryoxylase activity was examined during seed maturation using two cultivars of , P. vulgaris, Kentucky Wonder (normal) and Masterpiece (dwarf). Very immature seeds of both cultivars contain high level of 3$\beta$-hydroxylase activity (per mg protein). Both cultivars showed maximum of enzyme activity (per seed) in the middle of their maturation process. Gibberellin 3$\beta$-hydroxylase catalyzing the hydroxylation of GA20 to GA1 was purified 313-fold from very early immature seeds of P. vulgaris. Crude soluble enzyme extracts were purified by 15% methanol precipitation, hydrophobic interaction chromatogrphy, DEAE ion exchange column chromatography and gel filtration HPLC. The 3$\beta$-hydroxylase activity was unstable and lost much of its activity duting the purification. The molecular weight of purified enzyme was extimated to be 42, 000 by gel filtration HPLC and SDS-PAGE. The enzyme exhibited maximum activity at pH 7.7. The Km values for [2.3-3H] GA20 and [2.3-3H]GA9 were 0.29 $\mu$M and 0.33 $\mu$M, respectively. The enzyme requires 2-oxoglutarate as a cosubstrate; the Km value for 2-oxoglutarate was 250 $\mu$M using 3H GA20 as a substrate. Fe2+ and ascorbate significantly activated the enzyme at all purification steps, while catalase and BSA activated the purified enzyme only. The enzyme was inhibited by divalent cations Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+ and Hg2+. Effects of several GAs and GA anaogues on the putrified 3$\beta$-hydroxylase were examined using [3H]GA9 and GA20 as a substrates. Among them, GA5, GA9, GA15, GA20 and GA44 inhibited the enzyme activity. [13C, 3H] GA20 was converted by the partially purified enzyme preparation to [13C, 3H]GA1, GA5 and GA6, which were identified by GC-MS, GA9 was converted only GA4, GA15 and GA44 were converted to GA37 and GA38, respectively. GA5 was epoxidized to GA6 by the preparation. This suggests that 3$\beta$-hydroxylation of GA20 and epoxidation of GA5 are catalyzed by the same enzyme in P, vulgaris.
An intracellular ${\beta}-xylosidase$ from Paenibacillus sp. DG-22 was purified to homogeneity by ion-exchange, hydrophobic interaction and gel-filtration chromatography. The molecular weight of the enzyme was measured to be 156,000 by gel filtration and 80,000 by SDS-PAGE, indicating that the enzyme consisted of two identical subunits. The purified enzyme exhibited maximum activity at $65^{\circ}C$ and pH 5.5. It retained 89% of its initial activity up to 60 min at $60^{\circ}C$ and had a half-life of 25 min at $65^{\circ}C$. The enzyme was highly specific for pNPX as the substrate. It showed little or no activity against other p-nitrophenyl glycosides and xylans. The $K_m\;and\;V_{max}$ for pNPX was 0.53 mM and 3.18 U/mg protein, respectively. The ${\beta}-xylosidase$ was strongly inhibited by $Ag^+,\;Fe^{2+},\;Hg^{2+}\;and\;Zn^{2+}$ and slightly activated by DTT. The hydrolysis product from xylobiose, xylotriose, and xylotetraose was xylose.
Icariside II (ICA II) is used in erectile dysfunction treatment. Adipose tissue-derived stem cells (ADSCs) are efficient at improving erectile function. This study aimed to explore the action mechanism of ADSCs in improving erectile function. ADSCs were isolated from the adipose tissues of rats. Cell proliferation was determined using the Cell Counting Kit-8 (CCK-8) assay. The expressions of mRNA and protein were determined separately through qRT-PCR and western blot. The endogenous expressions of related genes were regulated using recombinant plasmids and cell transfection. A Dual-Luciferase Reporter Assay was performed to determine the interaction between miR-34a and STAT3. Rat models with bilateral cavernous nerve injuries (BCNIs) were used to assess erectile function through the detection of mean arterial pressure (MAP) and intracavernosal pressure (ICP). ICA II promoted ADSCs' proliferation and differentiation to Schwann cells (SCs) through the inhibition of miR-34a. Suppressed miR-34a promoted the differentiation of ADSCs to SCs by upregulating STAT3. ICA II promoted the differentiation of ADSCs to SCs through the miR-34a/STAT3 pathway. The combination of ICA II and ADSCs preserved the erectile function of the BCNI model rats. ADSCs treated with ICA II markedly preserved the erectile function of the BCNI model rats, which was reversed through miR-34a overexpression. ICA II promotes the differentiation of ADSCs to SCs through the miR34a/STAT3 pathway, contributing to erectile function preservation after the occurrence of a cavernous nerve injury.
Hypersensitivity of cells lacking Brcal to DNA interstrand .ross-link (ICL) agents such as cisplatin and mitomycin C(MMC) implicates the important role of Brcal in cellular response following ICL treatment. Brca1 plays an essential role in DNA double-strand break (DSB) repair through homologous recombination (HR)-dependent and -independent process. Recently, our group has been reported that Brca1 involves in cellular ICL response through HR-dependent repair process (Yun J. et at., Oncogene 2005). In this report, the involvement of Brca1 protein in HR-independent repair process is examined using isogenic $p53^{-/-}\;and\;p53^{-/-}\;Brcal^{-/-}$ mouse embryonic fibroblast (MEF) and psoralen cross-linked reporter reactivation assay. Brcal-deficient MEFs showed significantly low HR-independent repair activity compare to Brca1-proficient MEFs. Hypersensitivity to MMC and ICL reporter repair activity were restored by the reconstitution of Brca1 expression. Interestingly, MEFs expressing exon 11-deleted isoform of Brca1 $(Brca1^{\Delta11/\Delta11})$ showed high resistance to MMC and ICL reporter repair activity comparable to Brca1-reconstituted MEFs. Taken together, these results suggest that Brca1 involves in ICL repair through not only HR-dependent process but also HR-independent process using N-terminal RINC finger domain or C-terminal BRCT domain rather than exon 11 region which mediate interaction with Rad50.
Until now, considerable effort has been made to engineer novel nitrogen-fixing organisms through the transfer of nif genes from various diazotrophs to non-nitrogen fixers; however, regulatory coupling of the heterologous nif genes with the regulatory system of the new host is still not well understood. In this work, a 49 kb nitrogen fixation island from P. stutzeri A1501 was transferred into E. coli using a novel and efficient transformation strategy, and a series of recombinant nitrogen-fixing E. coli strains were obtained. We found that the nitrogenase activity of the recombinant E. coli strain EN-01, similar to the parent strain P. stutzeri A1501, was dependent on external ammonia concentration, oxygen tension, and temperature. We further found that there existed a regulatory coupling between the E. coli general nitrogen regulatory system and the heterologous P. stutzeri nif island in the recombinant E. coli strain. We also provided evidence that the E. coli general nitrogen regulator GlnG protein was involved in the activation of the nif-specific regulator NifA via a direct interaction with the NifA promoter. To the best of our knowledge, this work plays a groundbreaking role in increasing understanding of the regulatory coupling of the heterologous nitrogen fixation system with the regulatory system of the recipient host. Furthermore, it will shed light on the structure and functional integrity of the nif island and will be useful for the construction of novel and more robust nitrogen-fixing organisms through biosynthetic engineering.
Kim, Tae-Jip;Choi, Seung-Ho;Jang, Myoung-Uoon;Park, Jung-Mi;Svensson, Birte
Microbiology and Biotechnology Letters
/
v.38
no.2
/
pp.151-157
/
2010
Barley malt produces two different $\alpha$-amylase isozymes (AMY1 and AMY2), which share up to 80% of amino acid sequence identity with each other. However, their enzymatic properties differ remarkably. In this study, five chimeric enzymes between AMY1 and 2 were constructed by staggered extension process (StEP) technique, and their enzymatic properties were characterized. According to the results, chimeric AMY-D2, D8, and E12 showed the mixed or intermediate types of calcium-dependent activity between AMY1 and 2. Meanwhile, only AMY-E10 chimera could be significantly inhibited by barley $\alpha$-amylase/subtilisin inhibitor (BASI) protein. Chimera AMY-C6 showed the same calcium-dependency as AMY1, while AMY-E10 was closely similar to AMY2. As a result, it can be proposed that some amino acid residues in the region II, III, and IV of barley $\alpha$-amylases can play very important roles in the interaction with BASI, and those in III, V, VI, and VII may partly affect on the calcium-dependent activity.
A lymph node (LN) is one of the secondary lymphoid organs. An LN consists of a complicated 3 dimensional frame structure and several stromal cells. Fibroblastic reticular cells (FRC) are distributed in the T zone for interaction with T cells. FRC secrete homing chemokines such as CCL19 and CCL21. Moreover, FRC play a pivotal role in the production of extracellular matrix (ECM) into LN for ECM reorganization against pathogen infections. However, not much is known about the involvement of the immune reaction of FRC. The present report is for the characterization of FRC on immune response. For this, FRC were positioned in several infected situations such as co-culture with macrophage, lipopolysaccharide (LPS), and TNFα stimulation. When a co-culture between FRC and macrophage was performed, a morphological change in FRC was observed, and empty space between FRCs was created by this change. The soluble ICAM-1 protein level was up-regulated by co-culturing with Raw264.7 and the treatment of the ROCK inhibitor Y27632. The activity of matrix metalloproteinase (MMP) was up-regulated by LPS onto FRC. Furthermore, the inflammatory cytokine TNFα regulated the expression of ECM in FRC by a gene chip assay. Collectively, it suggests that FRC are involved in immune reactions.
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