Nardostachys chinensis induces granulocytic differentiation with the suppression of cell growth through p27(Kip1) protein-related G0/G1 phase arrest in human promyelocytic leukemic cells.

CONTEXT: Nardostachys chinensis Batalin (Valerianaceae) has been used in Korean traditional medicine to elicit stomachic and sedative effects. However, the anti-leukemic activities of N. chinensis have not been well examined. OBJECTIVE: To investigate the effect of N. chinensis on differentiation and proliferation in the human promyelocytic leukemic HL-60 cells. MATERIALS AND METHODS: The dried roots and stems of N. chiensis are extracted using hot water and then freeze-dried. The yield of extract was 12.82% (w/w). The HL-60 cells were treated with 25-200 µg/ml of N. chinensis for 72 h or 100 µg/ml of N. chinensis for 24-72 h. RESULTS: Nardostachys chinensis significantly inhibited cell viability dose dependently with an IC50 of 100 µg/ml in HL-60 cells. Nardostachys chinensis induced differentiation of the cells as measured by reduction activity of NBT and expression of CD11b but not of CD14 as analyzed by flow cytometry, which indicates a differentiation toward the granulocytic lineage. Nardostachys chinensis also induced growth inhibition through G0/G1 phase arrest in the cell cycle of HL-60 cells. Among the G0/G1 phase in the cell cycle-related protein, the expression of cyclin-dependent kinase (CDK) inhibitor p27(Kip1) was increased in N. chinensis-treated HL-60 cells, whereas the expression levels of CDK2, CDK4, CDK6, cyclin D1, cyclin D3, cyclin E, and cyclin A were decreased. Interestingly, N. chinensis markedly enhanced the binding of p27(Kip1) with CDK2 and CDK6. DISCUSSION AND CONCLUSION: This study demonstrated that N. chinensis is capable of inducing cellular differentiation and growth inhibition through p27(Kip1) protein-related G0/G1 phase arrest in HL-60 cells.

Nardostachys chinensis induces the differentiation of human promyelocytic leukemic cells through the activation of the protein kinase C-dependent extracellular signal-regulated kinase signaling pathway.

The underground parts of Nardostachys chinensis (N. chinensis), which belongs the genus Valerianaceae, have been used as sedative and analgesic agents in traditional Korean medicine for centuries. The mitogen-activated protein kinases (MAPKs) are serine/threonine kinases involved in the regulation of various cellular responses, such as cell proliferation, differentiation and apoptosis. Protein kinase C (PKC) plays a key role in the regulation of proliferation and differentiation. In this study, we investigated the signaling pathways involved in the differentiation of the HL-60 human leukemic cells induced by N. chinensis extract. Treatment with N. chinensis extract resulted in the activation of the extracellular signal-regulated kinase (ERK) pathway and induced the differentiation of HL-60 cells into granulocytes. The activation of p38 MAPK was also observed 24 h after treatment; however, the activation of c-Jun N-terminal kinase (JNK) was unaffected. Treatment with an inhibitor of ERK (PD98059) blocked the nitrotetrazolium blue chloride (NBT) reducing activity and CD11b expression in the N. chinensis-treated HL-60 cells, whereas treatment with an inhibitor of p38 MAPK (SB203580) had no significant effect on NBT reducing activity and CD11b expression. In addition, N. chinensis extract increased PKC activity and the protein levels of PKCα, PKCßI and PKCßII isoforms, without a significant change in the protein levels of the PKCγ isoform. PKC inhibitors (GF 109203X, chelerythrine and H-7) inhibited the differentiation of HL-60 cells into granulocytes, as well as ERK activation in the N. chinensis-treated HL-60 cells. These results indicate that the PKC and ERK signaling pathways may be involved in the induction, by N. chinensis extract, of the differentiation of HL-60 cells into granulocytes.

Expression of ganglioside GT1b in mouse embryos at different developmental stages after cryopreservation.

Gangliosides are a family of sialic acid-containing glycosphingolipids that are abundant in neurons and have a variety of functions in developing and mature tissues. We examined the expression of ganglioside GT1b in the embryonic preimplantation stage after freezing and thawing processes to determine the regulatory roles of ganglioside GT1b in early embryonic development. ICR mouse embryos at the two-cell stage obtained by flushing the oviducts were frozen by two cryopreservation procedures, slow freezing using a programmable freezer or vitrification by direct plunging into liquid nitrogen. Slow freezing was conducted with equilibration in 1.5 M 1,2-propanediol or 5% equilibration glycerol. Vitrification was applied with a 10-15 min equilibration in 7.5% ethylene glycol (EG), 7.5% dimethylsulfoxide (DMSO), and 30 sec in a solution of 15% EG, 15% DMSO and 0.5 M sucrose. Immediately after thawing, the survival rate of the embryos was assessed by their morphology and ability to develop to blastocysts in culture. The survival rate of vitrified and thawed embryos (92%) was significantly higher than that of slow frozen and thawed embryos (76%) (P<0.05). A tendency of higher blastocyst rate was found in the vitrified and thawed embryos compared to that of the slow frozen and thawed embryos. Confocal immunofluorescence staining confirmed that surviving embryos expressed ganglioside GT1b, with the strongest expression at the compacted eight-cell or later stage embryos. Ganglioside GT1b was not observed in the TUNEL-positive, apoptotic embryos, suggesting that cryopreservation had induced DNA breaks in them. These results suggest that ganglioside GT1b may play an important role in embryo survival or development.

The snpA, a temperature-sensitive suppressor of npgA1, encodes the eukaryotic translation release factor, eRF1, in Aspergillus nidulans.

The npgA1 mutation causes defects in the outer layer of the cell wall resulting in a colorless colony. In this study, a temperature-sensitive suppressor of npgA1 named snpA was isolated by UV mutagenesis. The suppressing mutant showed pleiotropic phenotypes in cellular structure and developmental processes when incubated at a temperature of 37 degrees C or above. At 37 degrees C, multiple germ tubes emerged from germinating conidia. Moreover, at 42 degrees C conidia germination was delayed more than 12h and hyphal growth was strongly inhibited. The suppressor allele, snpA6, is recessive and maps to the linkage group III. A gene complementing the mutation was identified employing the chromosome III-specific cosmid library. Sequencing analysis revealed that the snpA gene encodes the eukaryotic polypeptide release factor, eRF1. The snpA6 allele contains a G-A mutation resulting in SnpA(E117K), which may allow read-through of the nonsense mutation in the npgA1 allele in a similar manner to the yeast omni-potent suppressor SUP45 and SUP35.

Cloning and characterization of a new cysteine proteinase secreted by Paragonimus westermani adult worms.

The cysteine proteinases of Paragonimus westermani are known to play important roles in invasion and pathogenesis to hosts and in immune modulation and nutrient uptake. In this study, we have cloned a new cysteine proteinase of P. westermani, PwCP2, from adult worms and tested its diagnostic usefulness. The PwCP2 gene had an open reading frame of 816 base pairs and a conserved catalytic triad of cysteine, histidine, and asparagine residues. The mature form of recombinant PwCP2 (rPwCP2) lacking a proregion was overexpressed in Escherichia coli and used to produce antiserum. Western blot and immunohistochemical analyses using this antiserum showed that PwCP2 was expressed as a mature form, 24-kD product in a crude extract and in the excretory-secretory product of P. westermani, and was localized mainly in the intestinal epithelium of the adult worm. Western blot analysis using the rPwCP2 showed not only high sensitivity (90%) and specificity (100%) to sera from patients with paragonimiasis westermani, but also no cross-reactivity with sera from patients with clonorchiasis, sparganosis, or cysticercosis. Furthermore, an enzyme-linked immunosorbent assay using rPwCP2 exhibited a sensitivity of 93% and a specificity of 93% with sera of rats infected with P. westermani metacercariae. These results suggest that the excretory-secretory PwCP2 can be used for the diagnosis of paragonimiasis.

Development and evaluation of an immunochromatographic kit for the detection of antibody to Plasmodium vivax infection in South Korea.

Malaria is a major parasitic disease in tropical areas. Three to five hundred million people suffer from the disease and it kill a million people per year. Blood smear observation was developed for the diagnosis of malaria, but the examination needs skilled experts and exact diagnosis is time consuming. A kit based on immunochromatography can be a reliable and rapid method for clinical diagnosis, even in the hands of inexperienced personnel. However, all such currently developed kits can only diagnose P. falciparum malaria. In our previous report, the C-terminal region of P. vivax merozoite surface protein 1 (PvcMSP) was cloned and expressed in E. coli. In the present study, we developed an immunochromatographic kit using this PvcMSP for the diagnosis of specific antibody to P. vivax malaria in serum samples. The kit was used to examine sera from vivax malaria patients and non-malaria- infected person and the test showed 100% sensitivity (78/78) and 98.3% specificity (58/59). This result demonstrated that the immunochromatographic kit for P. vivax antibody detection is applicable for the rapid and precise diagnosis of P. vivax malaria.

Expression of 3beta-hydroxysteroid dehydrogenase and P450 side chain cleavage enzyme in the human uterine endometrium.

The enzyme complex 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4-isomerase (3beta-HSD) is involved in the biosynthesis of all classes of active steroids. The expression of 3b-HSD in human uterine endometrium during the menstrual cycle and decidua was examined in an effort to understand its role during ova implantation. 3beta-HSD was weakly expressed in the glandular epithelium of the proliferative phase and moderately expressed in the glandular epithelium of secretory phase of the endometrium. In the decidua of the ectopic pregnancy, 3beta-HSD was strongly expressed. The human uterine endometrial 3beta-HSD was identified as being the same type as the placental 3beta-HSD by RT-PCR and sequence analysis. In addition to the expression of 3beta-HSD, P450scc was expressed in the decidua of the ectopic pregnancy. These results suggest that pregnenolone might be synthesized from cholesterol by P450scc de novo and then, it is converted to progesterone by 3beta-HSD in the uterine endometrium. The data implies that the endometrial 3beta-HSD can use not only the out-coming pregnenolone from the adrenal gland but also the self- made pregnenolone to produce progesterone. The de novo synthesis of progesterone in the endometrium might be a crucial factor for implantation and maintenance of pregnancy.