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Atherosclerosis is a major risk factor for type 2 diabetes (T2D) mortality. We aim to investigate the changes in miR-21, miR-122, miR-33a and miR-3064-5p in circulation and the liver of ApoE-/- mice with streptozocin (STZ)-induced T2D. Twenty 5-week-old male ApoE-/- mice were randomly assigned to the control (n = 10) and T2D group (n = 10) and intraperitoneally injected with a citrate buffer and streptozotocin (STZ) (40 mg/kg BW) once a day for three consecutive days. The successfully STZ-induced T2D mice (n = 5) and control mice (n = 5) were then fed with a high-fat diet (HFD) for 34 weeks. Compared to the control mice, ApoE-/- mice with STZ-induced T2D had slower (p < 0.05) growth, increased (p < 0.05) total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C), decreased (p < 0.05) high-density lipoprotein cholesterol (HDL-C) in serum, reduced (p < 0.05) TC and sterol regulatory element-binding protein-2 (Srebp-2), elevated (p < 0.05) ATP-binding-cassette-transporter-A1 (Abca1) in the liver, aggravated (p < 0.05) atherosclerotic lesions in the aorta, downregulated (p < 0.05) miR-21 and miR-33a, and upregulated (p < 0.05) miR-122 and miR-3064-5p in serum and the liver. In addition, the aortic lesions showed a positive correlation with miR-122 (r = 1.000, p = 0.001) and a negative correlation with miR-21 (r = −1.000, p = 0.001) in ApoE-/- mice with T2D. In conclusion, T2D-accelerated atherosclerosis correlates with a reduction in miR-21 and miR-33a and an elevation in miR-122 and miR-3064-5p in circulation and the liver of ApoE-/- mice.

The structure and growth of a series of Lu-doped germanium clusters, LuGenq (n = 2–14, q = 0, −1) have been investigated by previous photoelectron spectroscopy (PES) and density functional theory (DFT) calculations. The ground states of the anionic LuGen– clusters obtained from DFT calculations are verified by comparing simulated PES with experimental results. The simulated PES for smaller clusters LuGen– (n ≤ 6) display relatively simple spectral patterns, suggesting high symmetry structures. It is observed that the pentagonal bipyramid shape is the basic framework for the nascent growth process of LuGen– (n = 2–8). The structures of LuGen– (n = 2–13) clusters are all exohedral structures with the Lu atom adsorbed at the surface of the bare Gen– clusters, while LuGe14– is the smallest endohedral Lu-doped germanium cluster with the Lu atom completely fallen into the germanium frame. It is found that the LuGen– clusters with even n are more stable than those with odd n and in the LuGen clusters there is an opposite trend. Especially, the LuGenq (n = 9, 12, q = 0, −1) clusters are extremely stable compared to other size clusters. HOMO–LUMO gap shows that the chemical stability of bare Gen– (n = 2–14) clusters are stronger than that of LuGenq (n = 2–14, q = 0, −1) clusters owing to the doping of a Lu atom.

Rats have long been an ideal model for disease research in the field of biomedicine, but the bottleneck of in vitro culture of rat embryonic stem (ES) cells hindered the wide application as genetic disease models. Here, we optimized a special medium which we named 5N-medium for rat embryonic stem cells, which improved the in vitro cells with better morphology and higher pluripotency. We then established a drug selection schedule harboring a prior selection of 12 h that achieved a higher positive selection ratio. These treatments induced at least 50% increase of homologous recombination efficiency compared with conventional 2i culture condition. Moreover, the ratio of euploid ES clones also increased by 50% with a higher germline transmission rate. Finally, we successfully knocked in a 175 kb human Bacterial Artificial Chromosome (BAC) fragment to rat ES genome through recombinase mediated cassette exchange (RMCE). Hence, we provide a promising system for generating sophisticated rat models which could be benefit for biomedical researches.

β-catenin-mediated signaling has been extensively studied in regard to its role in the regulation of human embryonic stem cells (hESCs). However, the results are controversial and the mechanism by which β-catenin regulates the hESC fate remains unclear. Here, we report that β-catenin and γ-catenin are functionally redundant in mediating hESC adhesion and are required for embryoid body formation, but both genes are dispensable for hESC maintenance, as the undifferentiated state of β-catenin and γ-catenin double deficient hESCs can be maintained. Overexpression of β-catenin induces rapid hESC differentiation. Functional assays revealed that TCF1 plays a crucial role in hESC differentiation mediated by β-catenin. Forced expression of TCF1, but not other LEF1/TCF family members, resulted in hESC differentiation towards the definitive endoderm. Conversely, knockdown of TCF1 or inhibition of the interaction between TCF1 and β-catenin delayed hESC exit from pluripotency. Furthermore, we demonstrated that GATA6 plays a predominant role in TCF1-mediated hESC differentiation. Knockdown of GATA6 completely eliminated the effect of TCF1, while forced expression of GATA6 induced hESC differentiation. Our data thus reveal more detailed mechanisms for β-catenin in regulating hESC fate decisions and will expand our understanding of the self-renewal and differentiation circuitry in hESCs.

The transcription factor Gbx2 (gastrulation brain homeobox 2) is a direct target of the LIF/STAT3 signaling pathway, maintains mouse embryonic stem cell (mESC) self-renewal, and facilitates mouse epiblast stem cell (mEpiSC) reprogramming to naïve pluripotency. However, the mechanism by which Gbx2 mediates its effects on pluripotency remains unknown. Here, using an RNA-Seq approach, we identified Klf4 (Kruppel-like factor 4) as a direct target of Gbx2. Functional studies indicated that Klf4 mediates the self-renewal–promoting effects of Gbx2, because knockdown of Klf4 expression abrogated the ability of Gbx2 to maintain the undifferentiated state of mESCs. We also found that Gbx2 largely depends on Klf4 to reprogram mEpiSCs to a mESC-like state. In summary, our study has uncovered a mechanism by which Gbx2 maintains and induces naïve pluripotency. These findings expand our understanding of the pluripotency control network and may inform the development of culture conditions for improved ESC maintenance and differentiation.

Mouse and rat embryonic stem cell (ESC) self-renewal can be maintained by dual inhibition of glycogen synthase kinase 3 (GSK3) and mitogen-activated protein kinase kinase (MEK). Inhibition of GSK3 promotes ESC self-renewal by abrogating T-cell factor 3 (TCF3)-mediated repression of the pluripotency network. How inhibition of MEK mediates ESC self-renewal, however, remains largely unknown. Here, we show that inhibition of MEK can significantly suppress lymphoid enhancer factor 1 (LEF1) expression in mouse ESCs. Knockdown or knockout of Lef1 partially mimics the self-renewal-promoting effect of MEK inhibitors. Moreover, depletion of both Tcf3 and Lef1 enables maintenance of undifferentiated mouse ESCs without exogenous factors, cytokines or inhibitors. Transcriptome resequencing analysis reveals that LEF1 is closely associated with endoderm specification in ESCs. Thus, our study adds support to the notion that the key to maintaining the ESC ground state is to shield ESCs from differentiative cues., Summary: Depletion of Lef1 and Tcf3 shows that ESCs could be shielded from differentiative cues to maintain the ESC ground state.

Both intracellular and extracellular heat shock protein-90 (Hsp90) family proteins (α and β) have been shown to support tumour progression. The tumour-supporting activity of the intracellular Hsp90 is attributed to their N-terminal ATPase-driven chaperone function. What molecular entity determines the extracellular function of secreted Hsp90 and the distinction between Hsp90α and Hsp90β was unclear. Here we demonstrate that CRISPR/Case9 knocking out Hsp90α nullifies tumour cells' ability to migrate, invade and metastasize without affecting the cell survival and growth. Knocking out Hsp90β leads to tumour cell death. Extracellular supplementation with recombinant Hsp90α, but not Hsp90β, protein recovers tumourigenicity of the Hsp90α-knockout cells. Sequential mutagenesis identifies two evolutionarily conserved lysine residues, lys-270 and lys-277, in the Hsp90α subfamily that determine the extracellular Hsp90α function. Hsp90β subfamily lacks the dual lysine motif and the extracellular function. Substitutions of gly-262 and thr-269 in Hsp90β with lysines convert Hsp90β to a Hsp90α-like protein. Newly constructed monoclonal antibody, 1G6-D7, against the dual lysine region of secreted Hsp90α inhibits both de novo tumour formation and expansion of already formed tumours in mice. This study suggests an alternative therapeutic approach to target Hsp90 in cancer, that is, the tumour-secreted Hsp90α, instead of the intracellular Hsp90α and Hsp90β.

During embryonic development, neural stem cells (NSCs) emerge as early as the neural plate stage and give rise to the nervous system. Early-stage NSCs express Sry-related-HMG box-1 (Sox1) and are biased towards neuronal differentiation. However, long-term maintenance of early-stage NSCs in vitro remains a challenge. Here, we report development of a defined culture condition for the long-term maintenance of Sox1-positive early-stage mouse NSCs. The proliferative ability of these Sox1-positive NSCs was confirmed by clonal propagation. Compared to the NSCs cultured using the traditional culture condition, the long-term self-renewing Sox1-positive NSCs efficiently differentiate into neurons and exhibit an identity representative of the anterior and midbrain regions. These early-stage Sox1-positive NSCs could also be switched to late-stage NSCs by being cultured with bFGF/EGF, which can then differentiate into astrocytes and oligodendrocytes. The long-term self-renewing Sox1-positive NSCs were defined as naive NSCs, based on their high neuronal differentiation capacity and anterior regional identity. This culture condition provides a robust platform for further dissection of the NSC self-renewal mechanism and promotes potential applications of NSCs for cell-based therapy on nervous system disorders.

Glycogen synthase kinase 3 (GSK3) plays a central role in diverse cellular processes. GSK3 has two mammalian isozymes, GSK3α and GSK3β, whose functions remain ill-defined because of a lack of inhibitors that can distinguish between the two highly homologous isozymes. Here, we show that GSK3α and GSK3β can be selectively inhibited in mouse embryonic stem cells (ESCs) using a chemical-genetic approach. Selective inhibition of GSK3β is sufficient to maintain mouse ESC self-renewal, whereas GSK3α inhibition promotes mouse ESC differentiation toward neural lineages. Genome-wide transcriptional analysis reveals that GSK3α and GSK3β have distinct sets of downstream targets. Furthermore, selective inhibition of individual GSK3 isozymes yields distinct phenotypes from gene deletion, highlighting the power of the chemical-genetic approach in dissecting kinase catalytic functions from the protein’s scaffolding functions. Our study opens new avenues for defining GSK3 isozyme-specific functions in various cellular processes.

Activation of Wnt signaling enhances self-renewal of mouse embryonic and neural stem/progenitor cells. In contrast, undifferentiated ES cells show a very low level of endogenous Wnt signaling, and ectopic activation of Wnt signaling has been shown to block neuronal differentiation. Therefore, it remains unclear whether or not endogenous Wnt/β-catenin signaling is necessary for self-renewal or neuronal differentiation of ES cells. To investigate this, we examined the expression profiles of Wnt signaling components. Expression levels of Wnts known to induceβ-catenin were very low in undifferentiated ES cells. Stable ES cell lines which can monitor endogenous activity of Wnt/β-catenin signaling suggest that Wnt signaling was very low in undifferentiated ES cells, whereas it increased during embryonic body formation or neuronal differentiation. Interestingly, application of small molecules which can positively (BIO, GSK3βinhibitor) or negatively (IWR-1-endo, Axin stabilizer) control Wnt/β-catenin signaling suggests that activation of that signaling at different time periods had differential effects on neuronal differentiation of 46C ES cells. Further, ChIP analysis suggested thatβ-catenin/TCF1 complex directly regulated the expression ofSox1during neuronal differentiation. Overall, our data suggest that Wnt/β-catenin signaling plays differential roles at different time points of neuronal differentiation.

Pluripotent stem cells only exist in a narrow window during early embryonic development, whereas multipotent stem cells are abundant throughout embryonic development and are retainedin various adult tissues and organs. While pluripotent stem cell lines have been established from several species, including mouse, rat, and human, it is still challenging to establish stable multipotent stem cell lines from embryonic or adult tissues. Based on current knowledge, we anticipate that by manipulating extrinsic and intrinsic signaling pathways, most if not all types of stem cells can be maintained in a long-term culture. In this article, we summarize current culture conditions established for the long-term maintenance of authentic pluripotent and multipotent stem cells and the signaling pathways involved. We also discuss the general principles of stem cell maintenance and propose several strategies on the establishment of novel stem cell lines through manipulation of signaling pathways. [BMB Reports 2015; 48(12): 668-676]

Activation of signal transducer and activator of transcription 3 (STAT3) by leukemia inhibitory factor (LIF) maintains mouse embryonic stem cell (mESC) self-renewal. Our previous study showed that trans-acting transcription factor 5 (Sp5), an LIF/STAT3 downstream target, supports mESC self-renewal. However, the mechanism by which Sp5 exerts these effects remains elusive. Here, we found that Nanog is a direct target of Sp5 and mediates the self-renewal-promoting effect of Sp5 in mESCs. Overexpression of Sp5 induced Nanog expression, while knockdown or knockout of Sp5 decreased the Nanog level. Moreover, chromatin immunoprecipitation (ChIP) assays showed that Sp5 directly bound to the Nanog promoter. Functional studies revealed that knockdown of Nanog eliminated the mESC self-renewal-promoting ability of Sp5. Finally, we demonstrated that the self-renewal-promoting function of Sp5 was largely dependent on its zinc finger domains. Taken together, our study provides unrecognized functions of Sp5 in mESCs and will expand our current understanding of the regulation of mESC pluripotency.

Tfcp2l1 is a transcription factor critical for mouse embryonic stem cell (mESC) self-renewal. How Tfcp2l1 maintains the pluripotent state of mESCs, however, remains unknown. Here, we show that knockdown of Tfcp2l1 in mESCs induces the expression of endoderm, mesoderm and trophectoderm markers. Functional analysis of mutant forms of Tfcp2l1 revealed that Tfcp2l1 depends on its N terminus and CP2-like domain to maintain the undifferentiated state of mESCs. The N terminus of Tfcp2l1 is mainly associated with the suppression of mesoderm and trophectoderm differentiation, while the CP2-like domain is closely related to the suppression of endoderm commitment. Further studies showed that MTA1 directly interacted with Tfcp2l1 protein and was indispensable for Tfcp2l1-mediated self-renewal-promoting effect and endoderm-inhibiting action. Tfcp2l1-mediated suppression of mesoderm and trophectoderm differentiation, however, is potentially through downregulation of Lef1 expression. Our study thus provides an expanded understanding of the function of Tfcp2l1 and the pluripotency regulation network of ESCs.

STAT3 can be transcriptionally activated by phosphorylation of its tyrosine 705 or serine 727 residue. In mouse embryonic stem cells (mESCs), leukemia inhibitory factor (LIF) signaling maintains pluripotency by inducing JAK-mediated phosphorylation of STAT3 Y705 (pY705). However, the function of phosphorylated S727 (pS727) in mESCs remains unclear. In this study, we examined the roles of STAT3 pY705 and pS727 in regulating mESC identities, using a small molecule-based system to post-translationally modulate the quantity of transgenic STAT3 in STAT3(-/-) mESCs. We demonstrated that pY705 is absolutely required for STAT3-mediated mESC self-renewal, while pS727 is dispensable, serving only to promote proliferation and optimal pluripotency. S727 phosphorylation is regulated directly by fibroblast growth factor/Erk signaling and crucial in the transition of mESCs from pluripotency to neuronal commitment. Loss of S727 phosphorylation resulted in significantly reduced neuronal differentiation potential, which could be recovered by a S727 phosphorylation mimic. Moreover, loss of pS727 sufficed LIF to reprogram epiblast stem cells to naïve pluripotency, suggesting a dynamic equilibrium of STAT3 pY705 and pS727 in the control of mESC fate.

The generation of induced tissue-specific stem cells has been hampered by the lack of well-established methods for the maintenance of pure tissue-specific stem cells like the ones we have for embryonic stem (ES) cell cultures. Using a cocktail of cytokines and small molecules, we demonstrate that primitive neural stem (NS) cells derived from mouse ES cells and rat embryos can be maintained. Furthermore, using the same set of cytokines and small molecules, we show that induced NS (iNS) cells can be generated from rat fibroblasts by forced expression of the transcriptional factors Oct4, Sox2 and c-Myc. The generation and long-term maintenance of iNS cells could have wide and momentous implications.

The rat is the preferred animal model in many areas of biomedical research and drug development. Genetic manipulation in rats has lagged behind that in mice due to the lack of efficient gene targeting tools. Previously, we generated a knockout rat via conventional homologous recombination in rat embryonic stem (ES) cells. Here, we show that efficient gene targeting in rat ES cells can be achieved quickly through transcription activator-like effector nuclease (TALEN)-mediated DNA double-strand breaks. Using the Golden Gate cloning technique, we constructed a pair of TALEN targeting vectors for the gene of interest in 5 days. After gene transfection, the targeted rat ES cell colonies were isolated, screened, and confirmed by PCR without the need of drug selection. Our results suggest that TALEN-mediated gene targeting is a superior means of establishing genetically modified rat ES cell lines with high efficiency and short turnaround time.

An excess of proteinase 3 (Pr3) is an assumed risk factor for elastin loss in chronic obstructive pulmonary disease. This study compared the degradation of [(14)C]elastin by Pr3 and its inhibition by alpha(1)-proteinase inhibitor (alpha(1)-PI) with the analogous reactions involving two other neutrophil serine proteases, human leukocyte elastase (HLE) and cathepsin G (CatG). The elastolytic rate catalyzed by Pr3 was estimated to be half of that of CatG and one-eighth of that of HLE. Evidence was obtained that indicated that absorption of Pr3 by the substrate was much less than that of HLE or CatG, and that the majority of absorbed Pr3 was highly mobile. These properties are consistent with the observation that elastolysis by Pr3 was almost completely and stoichiometrically inhibited by alpha(1)-PI even under conditions in which the protease had been preincubated with the substrate. In contrast, alpha(1)-PI in large molar excess was unable to inhibit completely ongoing elastolysis of the same substrate by HLE or CatG. An interfacial nonisotropic reaction mechanism has been proposed to address the incomplete inhibition of ongoing elastolysis. Pr3 was identified as being the most abundant neutrophil serine protease. However, two findings reported here, namely the low rate of elastolysis by Pr3 and the high efficacy of alpha(1)-PI against ongoing elastolysis by Pr3, imply that Pr3 might not necessarily be a major contributor to neutrophil-mediated elastin loss.

Excessive proteolytic activity of proteinase 3 (Pr3) has been suggested to be a factor contributing to the pathogenesis of emphysema and other inflammatory disorders. We report here on the kinetics of inhibition of Pr3 by one of its major endogenous inhibitors, the 6-kD inhibitory domain of elafin. The results are consistent with a reaction mechanism in which a single elafin molecule binds a single Pr3 molecule to form a fully reversible complex. The association and dissociation rate constants, and the inhibition constant were measured to be 4.0 x 10(6) M(-1) s(-1), 1.7 x 10(-3) s(-1), and 4.2 x 10(-10) M, respectively. Triton X-100 and dimethyl sulfoxide, which are frequently added in assay mixtures for enzymatic analysis of Pr3 activity, significantly reduced the association rate. A fraction of the total neutrophil content of Pr3 has been reported to be bound to the surface of the plasma membrane of activated and nonactivated neutrophils. In this study, we also measured the reaction rate constants of elafin with Pr3 that had been previously allowed to associate with phospholipid bilayer vesicles. Binding to the model membranes slowed down the association rate to 3.3 x 10(5) M(-1) s(-1), but the membrane-bound Pr3 and elafin formed a more stable complex, with a dissociation rate constant of 9.1 x 10(-4) s(-1). Based on the kinetic parameters determined here and the estimated elafin concentrations in vivo, it may be concluded that elafin plays a limited role in the regulation of proteolytic activity of Pr3 in lung secretions.

Mouse embryonic stem cell (mESC) self-renewal can be maintained by activation of the leukaemia inhibitory factor (LIF)/signal transducer and activator of transcription 3 (Stat3) signalling pathway or dual inhibition (2i) of glycogen synthase kinase 3 (Gsk3) and mitogen-activated protein kinase kinase (MEK). Several downstream targets of the pathways involved have been identified that when individually overexpressed can partially support self-renewal. However, none of these targets is shared among the involved pathways. Here, we show that the CP2 family transcription factor Tfcp2l1 is a common target in LIF/Stat3- and 2i-mediated self-renewal, and forced expression of Tfcp2l1 can recapitulate the self-renewal-promoting effect of LIF or either of the 2i components. In addition, Tfcp2l1 can reprogram post-implantation epiblast stem cells to naive pluripotent ESCs. Tfcp2l1 upregulates Nanog expression and promotes self-renewal in a Nanog-dependent manner. We conclude that Tfcp2l1 is at the intersection of LIF- and 2i-mediated self-renewal pathways and plays a critical role in maintaining ESC identity. Our study provides an expanded understanding of the current model of ground-state pluripotency.

Summary Activation of signal transducer and activator of transcription 3 (Stat3) by leukemia inhibitory factor (LIF) maintains mouse embryonic stem cell (mESC) self-renewal and also facilitates reprogramming to ground state pluripotency. Exactly how LIF/Stat3 signaling exerts these effects, however, remains elusive. We identified gastrulation brain homeobox 2 (Gbx2) as a LIF/Stat3 downstream target that, when overexpressed, allows long-term expansion of undifferentiated mESCs in the absence of LIF/Stat3 signaling. Elevated Gbx2 expression also enhanced reprogramming of mouse embryonic fibroblasts to induced pluripotent stem cells. Moreover, overexpression of Gbx2 was sufficient to reprogram epiblast stem cells to ground state ESCs. Our results reveal a novel function of Gbx2 in mESC reprogramming and LIF/Stat3-mediated self-renewal.

The interaction of alginate from Pseudomonas aeruginosa ATCC 39324 with human leukocyte elastase was studied by determining the effects of the polysaccharide on the amidolytic activity of the enzyme toward a range of synthetic peptide substrates of different length. The data support a model in which each elastase molecule interacts with a total of 19 uronic acid units on the alginate, primarily through electrostatic forces. Binding of alginate results in occlusion of distal subsites, most likely S4 and S5, of the enzyme's extended substrate-binding domain. As a result, alginate alone appears to be a weak inhibitor of the hydrolysis of long synthetic peptide substrates and [14C]elastin by elastase. Alginate also has effects on the antielastase function of naturally occurring protease inhibitors in the lung: It reduces the association rate of elastase and alpha 1-proteinase inhibitor, whereas it increases the association rate of elastase and secretory leukoprotease inhibitor. In the presence of 36 micrograms/ml alginate, the median concentration found in sputum from cystic fibrosis patients infected with mucoid strains of P. aeruginosa, the second-order rate constant for inhibition of elastase by secretory leukoprotease inhibitor is 2.6-fold greater than that for alpha 1-proteinase inhibitor. Alginate has only a minor effect on the antielastase activities of elafin and a recombinant form of the isolated C-terminal domain of secretory leukoprotease inhibitor. Based on these findings, alginate may be an important factor in determining the local distribution of leukocyte elastase and perturbing the overall protease-antiprotease balance in the infected lungs of cystic fibrosis patients.

Activation of leukemia inhibitor factor (LIF)–Stat3 or Wnt/β-catenin signaling promotes mouse embryonic stem cell (mESC) self-renewal. A myriad of downstream targets have been identified in the individual signal pathways, but their common targets remain largely elusive. In this study, we found that the LIF–Stat3 and Wnt/β-catenin signaling pathways converge on Sp5 to promote mESC self-renewal. Forced Sp5 expression can reproduce partial effects of Wnt/β-catenin signaling but mimics most features of LIF–Stat3 signaling to maintain undifferentiated mESCs. Moreover, Sp5 is able to convert mouse epiblast stem cells into a naïve pluripotent state. Thus, Sp5 is an important component of the regulatory network governing mESC naïve pluripotency., Summary: This study reveals a new function of Sp5 in mouse embryonic stem cell (ESC) self-renewal mediated by CHIR99021 and LIF, and reprogramming of EpiSCs into naїve ESCs.

The tumor suppressor p53 has an important role in inducing cell-intrinsic responses to DNA damage, including cellular senescence or apoptosis, which act to thwart tumor development. It has been shown, however, that senescent or dying cells are capable of eliciting inflammatory responses, which can have pro-tumorigenic effects. Whether DNA damage-induced p53 activity can contribute to senescence- or apoptosis-associated pro-tumorigenic inflammation is unknown. Recently, we generated a p53 knock-out rat via homologous recombination in rat embryonic stem cells. Here we show that in a rat model of inflammation-associated hepatocarcinogenesis, heterozygous deficiency of p53 resulted in attenuated inflammatory responses and ameliorated hepatic cirrhosis and tumorigenesis. Chronic administration of hepatocarcinogenic compound, diethylnitrosamine, led to persistent DNA damage and sustained induction of p53 protein in the wild-type livers, and much less induction in p53 heterozygous livers. Sustained p53 activation subsequent to DNA damage was accompanied by apoptotic rather than senescent hepatic injury, which gave rise to the hepatic inflammatory responses. In contrast, the non-hepatocarcinogenic agent, carbon tetrachloride, failed to induce p53, and caused a similar degree of chronic hepatic inflammation and cirrhosis in wild type and p53 heterozygous rats. These results suggest that although p53 is usually regarded as a tumor suppressor, its constant activation can promote pro-tumorigenic inflammation, especially in livers exposed to agents that inflict lasting mutagenic DNA damage.

Here, we describe a focused microarray for screening rat embryonic stem cells (ESCs) and provide validation data that this array can distinguish undifferentiated rat ESCs from rat trophoblast stem (TS) cells, rat extraembryonic endoderm cells, mouse embryonic fibroblast feeder cells, and differentiated rat ESCs. Using this tool, genuine rat ESC lines, which have been expanded in a conventional rat ESC medium containing two inhibitors (2i), for example, glycogen synthase kinase 3 (GSK3) and mitogen-activated protein kinase (MEK) inhibitors, and leukemia inhibitory factor, and genuine rat ESCs, which have been expanded in rat ESC medium containing four inhibitors (4i), for example, GSK3, MEK, Alk5, and Rho-associated kinase inhibitors were compared; as were genuine rat ESCs from 4 different strains of rats. Expression of Cdx2, a gene associated with trophoblast determination, was observed in genuine, undifferentiated rat ESCs from 4 strains and from both 2i and 4i ESC derivation medium. This finding is in contrast to undifferentiated mouse ESCs that do not express Cdx2. The rat ESC focused microarray described in this report has utility for rapid screening of rat ESCs. This tool will enable optimization of culture conditions in the future.

We describe here a detailed protocol for generating gene knockout rats by homologous recombination in embryonic stem (ES) cells. This protocol comprises the following procedures: derivation and expansion of rat ES cells, construction of gene-targeting vectors, generation of gene-targeted rat ES cells and, finally, production of gene-targeted rats. The major differences between this protocol and the classical mouse gene-targeting protocol include ES cell culture methods, drug selection scheme, colony picking and screening strategies. This ES cell-based gene-targeting technique allows sophisticated genetic modifications to be performed in the rat, as many laboratories have been doing in the mouse for the past two decades. Recently we used this protocol to generate Tp53 (also known as p53) gene knockout rats. The entire process requires ∼1 year to complete, from derivation of ES cells to generation of knockout rats.

The ability to "knockout" specific genes in mice via embryonic stem (ES) cell-based gene-targeting technology has significantly enriched our understanding of gene function in normal and disease phenotypes. Improvements on this original strategy have been developed to enable the manipulation of genomes in a more sophisticated fashion with unprecedented precision. The rat is the model of choice in many areas of scientific investigation despite the lack of rat genetic toolboxes. Most Recent advances of zinc finger nucleases (ZFNs) and rat ES cells are diminishing the gap between rat and mouse with respect to reverse genetic approaches. Importantly, the establishment of rat ES cell-based gene targeting technology, in combination with the unique advantages of using rats, provides new, exciting opportunities to create animal models that mimic human diseases more faithfully. We hereby report our recent results concerning finer genetic modifications in the rat, and propose their potential applications in addressing biological questions.

Monoamine oxidase (MAO) A is the major metabolizing enzyme of serotonin (5-hydroxytryptamine, 5-HT) which regulates early brain development. In this study, wild-type (WT) and MAO A(neo) embryonic stem (ES) cell lines were established from the inner cell mass of murine blastocysts and their characteristics during ES and differentiating stages were studied. Our results show that the differentiation to neural cells in MAO A(neo) ES cells was reduced compared to WT, suggesting MAO A played a regulatory role in stem cells neural differentiation.

The synthesis and evaluation of 4-(methylsulfinyl)phenyl 2-(1-methyl-2-pyrrolyl)butyrate (CE-0266) and the related sulfide and sulfone derivatives, CE-0265 and CE-0267, respectively, are described. The potency of the inhibitors toward human neutrophil elastase increases across the series CE-0265, CE-0266, CE-0267. CE-0266, with a k3Ki∗k2 value of 36 nm, exhibited high selectivity for elastase and was chosen for additional in vivo studies.

There is increasing evidence to suggest that embryonic stem cells (ESCs) are capable of differentiating into hepatocytes in vitro. In this study, we used a combination of cytokines and sodium butyrate in a novel three-step procedure to efficiently direct the differentiation of mouse ESCs into hepatocytes. Mouse ESCs were first differentiated into definitive endoderm cells by 3 days of treatment with Activin A. The definitive endoderm cells were then differentiated into hepatocytes by the addition of acidic fibroblast growth factor (aFGF) and sodium butyrate to the culture medium for 5 days. After 10 days of further in vitro maturation, the morphological and phenotypic markers of hepatocytes were characterized using immunohistochemistry, immunoblotting, and reverse transcription-polymerase chain reaction (RT-PCR). Furthermore, the cells were tested for functions associated with mature hepatocytes, including glycogen storage and indocyanine green uptake and release, and the ratio of hepatic differentiation was determined by counting the percentage of albumin-positive cells. In the presence of medium containing cytokines and sodium butyrate, numerous epithelial cells resembling hepatocytes were observed, and approximately 74% of the cells expressed the hepatic marker, albumin, after 18 days in culture. RT-PCR analysis and immunohistochemistry showed that these cells expressed adult liver cell markers, and had the abilities of glycogen storage and indocyanine green uptake and release. We have developed an efficient method for directing the differentiation of mouse ESCs into cells that exhibit the characteristics of mature hepatocytes. This technique will be useful for research into the molecular mechanisms underlying liver development, and could provide a source of hepatocytes for transplantation therapy and drug screening.

The nuclear gene (c-mos) and mitochondrial genes (CYT B and ND2) sequences, representing 44 phasianid species and 26 genera (mainly distributed in China), were used to study the phylogeny of the genus Perdix, which comprises three partridge species. Maximum parsimony and Bayesian methods were employed, and the analysis of mitochondrial sequence data and the combined dataset showed that Perdix is specifically related either to typical pheasants or to Ithaginis. Phylogenetic trees also indicated that: (1) Perdix is monophyletic; (2) the Tibetan partridge (Perdix hodgsoniae) has been consistently placed as basal to all other Perdix, and the Daurian partridge (Perdix dauuricae) is placed sister to gray partridge (Perdix perdix); (3) the Daurian partridge subspecies przewalskii and Tibetan partridge subspecies hodgsoniae are basal to other subspecies in their species clade, respectively. Speciation in Perdix was likely promoted by the late Pliocene/early Pleistocene intensive uplift of the Tibetan Plateau and by Pleistocene glaciations.

Natural killer (NK) cell enhancing factor (NKEF) belongs to the newly defined peroxiredoxin (Prx) family. Its functions are to enhance NK cell cytotoxicity and to protect DNA and proteins from oxidative damage. In this study, a partial cDNA sequence of carp NKEF-B was isolated from thymus cDNA library. Subsequently, the full-length cDNA of carp NKEF-B was obtained by means of 3' and 5' RACE, respectively. The full-length cDNA of carp NKEF-B was 1022 bp, consisting of a 73 bp 5'-terminal untranslated region (UTR), a 355 bp T-terminal UTR, and a 594 bp open reading frame coding for a protein of 197 amino acids. Carp NKEF-B contained two consensus Val-Cys-Pro (VCP) motifs and three consensus cysteine (Cys-51, Cys-70 and Cys-172) residues. Sequence comparison showed that the deduced amino acid sequence of carp NKEF-B had an overall similarity of 74-96% to that of other species homologues. Phylogenetic analysis revealed that carp NKEF-B forms a cluster with other known teleost NKEF-Bs. Then, by PCR we obtained a 5.1 -k long genomic DNA of carp NKEF-B containing six exons and five introns. Realtime RT-PCR results showed that carp NKEF-B gene was predominantly detected in kidney and head kidney under un-infected conditions. Whereas under SVCV-infection condition, the expression of NKEF-B gene was significantly increased in blood cells, gill, intestine and spleen, but maintained in liver, and decreased significantly in kidney and head kidney. Finally, the rNKEF-B was constructed and expressed in Escherichia coli. By using an antibody against carp rNKEF-B, immunohistochemical study further indicated that NKEF-B positive cells are mainly some RBCs and a few epithelial cells in gill and intestine, and that under SVCV-infection condition, these positive cells or positive products in their cytoplasm were mainly increased in gill and spleen sections of carp. The results obtained in the present study will help to understand the function of NKEF-B in teleost innate immunity. (C) 2008 Elsevier Ltd. All rights reserved.

The availability of murine embryonic stem (ES) cells has revolutionized the study of mammalian development and disease. We recently developed a culture medium that has enabled us to identify the essential signaling pathways required for maintenance of pluripotency in vitro. Addition of leukemia inhibitory factor and bone morphogenetic protein4 to this medium is sufficient to activate the signal transducer and activator of transcription3 and mammalian homolog of Drosophila mothers against decapentaplegic pathways, respectively. We have successfully derived and propagated ES cells in the absence of feeder cells and serum. This chapter describes a simple protocol for efficient derivation and maintenance of ES cells from embryos of the 129 and C57B1/6 strains of mice.

Study objectives: Unopposed activity of the serine protease, human leukocyte elastase (HLE), is detectable in the airways of patients with purulent tracheobronchitis. The aim of this study was to assess the compartmentalization of HLE activity in the liquid sol phase and the solid gel phase of airway secretions. Design: Seventy samples of tracheobrochial aspirates were obtained from patients who had hypersecretion and were receiving mechanical ventilation. Methods: Samples were separated into sol and gel (“mucous pellet”) phases, and HLE activity was measured using chromogenic substrate degradation. HLE was eluted from the mucous pellet using hypertonic saline solution, 1 mol/L, or bovine pancreatic deoxyribonuclease (DNase), 16 μmol/L. Results: HLE activity partitioned between the sol and gel phases of the secretions, with most of the activity present in the gel phase (32:1 ratio of gel to sol HLE activity). The activity of HLE was 95% inhibited when bound to the gel phase, but activity appeared to be largely restored after elution from the gel phase. The gel phase was capable of binding additional exogenous HLE, and its binding capacity for exogenous HLE was not saturated by concentrations that exceeded the highest clinically relevant HLE levels (1.1 mg/mL). Hypertonic saline solution and DNase I efficiently liberated endogenous and exogenous gel phase-bound HLE activity, suggesting that electrostatic bonds and DNA, respectively, play important roles in binding HLE to the gel phase. Conclusions: The solid phase of airway secretions is a more important modulator of elastase-antielastase balance than has been previously recognized.

The cytokine leukemia inhibitory factor (LIF) drives self-renewal of mouse embryonic stem (ES) cells by activating the transcription factor STAT3. In serum-free cultures, however, LIF is insufficient to block neural differentiation and maintain pluripotency. Here, we report that bone morphogenetic proteins (BMPs) act in combination with LIF to sustain self-renewal and preserve multilineage differentiation, chimera colonization, and germline transmission properties. ES cells can be propagated from single cells and derived de novo without serum or feeders using LIF plus BMP. The critical contribution of BMP is to induce expression of Id genes via the Smad pathway. Forced expression of Id liberates ES cells from BMP or serum dependence and allows self-renewal in LIF alone. Upon LIF withdrawal, Id-expressing ES cells differentiate but do not give rise to neural lineages. We conclude that blockade of lineage-specific transcription factors by Id proteins enables the self-renewal response to LIF/STAT3.

Wnt/β-catenin signaling plays important roles in self-renewal and differentiation of embryonic stem (ES) cells. Tcfs and Lef1 are DNA-binding transcriptional factors for the canonical Wnt signaling pathway. In the absence of β-catenin, Tcf/Lef1 generally functions as transcriptional repressor with co-repressor proteins such as Groucho, CtBP, and HIC-5. But Tcf/Lef1 turns into transcriptional activator when they interact with β-catenin. Therefore, so far it was considered that the activity of Tcf/Lef1 is regulated by the absence or presence of β-catenin, however the intrinsic role of Tcf/Lef1 has not been examined. The purpose of this study was to determine if Tcf/Lef1 itself plays differential roles in the regulation of self-renewal and differentiation of mouse ES cells. Interestingly, the expression of TCFs and LEF1 was dynamically changed during various differentiation conditions, such as removal of LIF, EB formation and neuronal differentiation in N2B27 media, suggesting that the function of each Tcfs and Lef1 may be different in ES cells. Ectopic expression of Tcf1 or dominant negative form of Lef1 (dnLef1) sustains self-renewal of ES cells in the absence of leukemia inhibitory factor (LIF), while ectopic expression of Tcf3, Lef1 or dnTcf1 did not block differentiation. In addition, ectopic expression of Tcf1 or knockdown of Lef1 blocked differentiation into neural precursor and cardiomyocyte. Ectopic expression of either Lef1 or dnLef1 blocked neuronal differentiation, suggesting that transient induction of Lef1 seemed to be necessary for the initiation and progress of differentiation. ChIP analysis show that Tcf1 bound to Nanog promoter and ectopic expression of Tcf1 enhanced the transcription of Nanog. Overall, our data suggest that Tcf1 plays critical roles in the maintenance of stemness whereas Lef1is involved in the initiation of differentiation. (Supported by the National Research Foundation of Korea (2006-2004046) and Brain Korea 21 program.)

Previously we reported that DNA from sputum promotes the inhibition of human leukocyte elastase (HLE) by native secretory leukoprotease inhibitor (SLPI). This study shows that sputum DNA also promotes the inhibition by oxidized SLPI, a form of SLPI that may occupy a large fraction of the inhibitor in the lungs under conditions of high oxidative stress. With sputum DNA at 5 microg/ml, a concentration much lower than those in vivo, the inhibition constant (K(i) ) of oxidized SLPI against HLE is reduced from 31 nM to 23 to 920 pM, as compared with the K(i) of native SLPI, 58 pM, under the same conditions. On the other hand, sputum DNA retards inhibition of HLE by alpha(1)-proteinase inhibitor (alpha(1)-PI). The association rate of alpha(1)-PI and HLE is decreased from 1 x 10(7) M(-1) s(-1) in the absence of DNA to 2 to 6 x 10(6) M(-1) s(-1) in the presence of sputum DNA at 100 microg/ml. On the basis of results with an elastase-specific oligonucleotide aptamer, it was found that the downregulation of alpha(1)-PI activity can be attributed to an interaction between sputum DNA and multiple DNA-binding sites on HLE. DNA-binding sites on HLE also participate in the upregulation of oxidized SLPI activity. Data from this and our previous studies demonstrate that sputum DNA facilitates the association of HLE with native and oxidized SLPI, whereas it delays the association of HLE with alpha(1)-PI. We conclude that by modulating the inhibition of HLE, sputum DNA directly affects the balance between proteases and antiproteases in the lungs.

A full-length cDNA for maize root-form phosphoenolpyruvate carboxylase (PEPC) was isolated. In the coding region, the root-form PEPC showed 76 and 77% identity with the C4- and C3-form PEPCs of maize, respectively, at the nucleotide level. At the amino acid level, the root-form was 81 and 85% identical to the C4- and C3-form PEPCs, respectively. The entire coding region was inserted into a pET32a expression vector so that it was expressed under the control of T7 promoter. The purified recombinant root-form PEPC had a Vmax value of about 28 mumol min-1 (mg protein)-1 at pH 8.0. The K(m) values of root-form PEPC for PEP and Mg2+ were one-tenth or less of those of C4-form PEPC when assayed at either pH 7.3 or 8.0, while the value for HCO3- was about one-half of that of C4-form PEPC at pH 8.0. Glucose 6-phosphate and glycine had little effect on the root-form PEPC at pH 7.3; they caused two-fold activation of the C4-form PEPC. The Ki (L-malate) values at pH 7.3 were 0.12 and 0.43 mM for the root- and C4-form PEPCs, respectively. Comparison of hydropathy profiles among the maize PEPC isoforms suggested that several stretches of amino acid sequences may contribute in some way to their characteristic kinetic properties. The root-form PEPC was phosphorylated by both mammalian cAMP-dependent protein kinase and maize leaf protein kinase, and the phosphorylated enzyme was less sensitive to L-malate.

We have found that preparations of DNA isolated from purulent sputum possess a novel activity which accelerates and stabilizes the binding of human leukocyte elastase to secretory leukoprotease inhibitor, a major endogenous antielastase in the respiratory tract. DNA in sputum is derived from the nuclear debris of disintegrated inflammatory leukocytes, and can attain concentrations ranging from 10(2) to 10(4) micrograms/ml, depending on the severity of pulmonary infection and inflammation. In the presence of 23 micrograms/ml DNA, a concentration lower than those found in most purulent sputa, the rate constant for association of secretory leukoprotease inhibitor with elastase is increased to 1.1 x 10(8) M-1s-1, 44-fold greater than that in the absence of DNA. The equilibrium dissociation constant for the enzyme-inhibitor complex drops to 0.7 pM, two orders of magnitude lower than that in the absence of DNA. The accelerating effect of DNA is further increased by thermal denaturation or by modification with exonuclease III, while it is significantly reduced by digestion with S1 nuclease or by binding of Escherichia coli single-stranded DNA binding protein. The results from these experiments indicate that the structural elements in sputum DNA that are responsible for the accelerating effect have the characteristics of single-stranded sites. Similar kinetic effects on elastase inhibition were also observed with human placental DNA and genomic DNAs from a variety of other species. These findings suggest that DNA in pulmonary secretions may participate in antielastase defense by promoting the binding of secretory leukoprotease inhibitor to leukocyte elastase. The results may have important implications for use of nuclease preparations in mucolytic therapy for cystic fibrosis.

Background Inhibition of glycogen synthase kinase-3 (GSK-3) improves the efficiency of embryonic stem (ES) cell derivation from various strains of mice and rats, as well as dramatically promotes ES cell self-renewal potential. β-catenin has been reported to be involved in the maintenance of self-renewal of ES cells through TCF dependent and independent pathway. But the intrinsic difference between ES cell lines from different species and strains has not been characterized. Here, we dissect the mechanism of GSK-3 inhibition by CHIR99021 in mouse ES cells from refractory mouse strains. Methodology/Principal Findings We found that CHIR99021, a GSK-3 specific inhibitor, promotes self-renewal of ES cells from recalcitrant C57BL/6 (B6) and BALB/c mouse strains through stabilization of β-catenin and c-Myc protein levels. Stabilized β-catenin promoted ES self-renewal through two mechanisms. First, β-catenin translocated into the nucleus to maintain stem cell pluripotency in a lymphoid-enhancing factor/T-cell factor–independent manner. Second, β-catenin binds plasma membrane-localized E-cadherin, which ensures a compact, spherical morphology, a hallmark of ES cells. Further, elevated c-Myc protein levels did not contribute significantly to CH-mediated ES cell self-renewal. Instead, the role of c-Myc is dependent on its transformation activity and can be replaced by N-Myc but not L-Myc. β-catenin and c-Myc have similar effects on ES cells derived from both B6 and BALB/c mice. Conclusions/Significance Our data demonstrated that GSK-3 inhibition by CH promotes self-renewal of mouse ES cells with non-permissive genetic backgrounds by regulation of multiple signaling pathways. These findings would be useful to improve the availability of normally non-permissive mouse strains as research tools.

Secretory leukoprotease inhibitor (SLPI) comprises two homologous domains: the C-terminal domain contains the reactive site, while the function of the N-terminal domain remains unknown. In order to elucidate the function of the N-terminal domain, we studied the kinetics of reactions of human leukocyte elastase with two recombinant forms of SLPI: the full-length inhibitor and the C-terminal domain alone. The reactions of elastase with the full-length inhibitor and the C-terminal domain share the same association rate constant, 2 x 10(6) M-1 s-1, but the complex formed with the C-terminal domain is less stable, with a dissociation rate constant of 8 x 10(-4) s-1, 5 times higher than that of the complex with the full-length inhibitor. The binding of the full-length inhibitor to elastase is greatly accelerated by polyanions. In the presence of submicromolar concentrations (1 microgram/mL) of heparin, the association rate constant is increased by more than 1 order of magnitude. The binding of the C-terminal domain alone to elastase shows much lower sensitivity to heparin; in the presence of 5 microM (25 micrograms/mL) heparin, association of the C-terminal domain with elastase reaches a maximum rate of 7 x 10(6) M-1 s-1, about 3 times higher than the rate in the absence of heparin. Similar differential effects of heparin have been observed on the reactions of alpha-chymotrypsin with the two recombinant forms of SLPI.2=

Activation of Wnt signaling enhances self-renewal of mouse embryonic and neural stem/progenitor cells. However, contrary to these results, undifferentiated ES cells shows very low level of endogenous Wnt signaling and ectopic activation of Wnt signaling blocks neural differentiation. Therefore it is not clear whether or when endogenous Wnt/β-catenin signaling is necessary for self-renewal or neural differentiation of ES cells. When we examined the expression profile of Wnt signaling components, expressions of most Wnts are very low in undifferentiated ES cells while some of Wnts are increased during differentiation. We generated stable ES cell lines that probe Wnt signaling activity and these cells showed that Wnt signaling was very low in undifferentiated ES cells while increased during embryonic body formation or neural differentiation. However, conditioned media from differentiated ES cells did not induce higher reporter activity than that of undifferentiated ES cells, which suggests that ES cells may change the sensitivity to Wnt signaling during differentiation whereas the levels of secreted Wnts are same. Changes in the composition of Tcfs or Lef1 in ES cells at different conditions may be a possible mechanism. In addition, we observed that activation of Wnt/β-catenin signaling at different time periods resulted in differential effects on neural differentiation of 46C ES cells that show increased Sox1 promoter driven GFP during neural differentiation; activation of Wnt signaling by the treatment with BIO (a GSK3β inhibitor) in early or late period blocks or enhances neural differentiation, respectively. Consistent with these results, inactivation of Wnt/β-catenin signaling in late period by the treatment with endo-IWR1 (an Axin stabilizer) blocked neural differentiation. Overall, our data suggest that Wnt/β-catenin signaling plays biphasic roles during neural differentiation of mouse embryonic stem cells. Supported by the National Research Foundation of Korea (2006-2004046) and Brain Korea 21 program.

Several pentacyclic triterpenoid metabolites of plant origin are inhibitors of hydrolysis of both synthetic peptide substrates and elastin by human leucocyte elastase (HLE). Ursolic acid, the most potent of these compounds, has an inhibition constant of 4-6 microM for hydrolysis of peptide substrates in phosphate-buffered saline. With tripeptide and tetrapeptide substrates, the inhibition is purely competitive, whereas with a shorter dipeptide substrate the inhibition is non-competitive, suggesting that ursolic acid interacts with subsite S3 of the extended substrate-binding domain in HLE, but not with subsites S1 and S2. The carboxy group at position 28 in the pentacyclic-ring system of the triterpenes contributes to binding to HLE, since replacement of this group with a hydroxy group, as in uvaol, the alcohol analogue of ursolic acid, reduces the potency of inhibition. The inhibitory potency of ursolic acid is also reduced by addition of 1 M-NaCl, further supporting a postulated electrostatic interaction between the negative charge on the triterpene and a positively charged residue on the enzyme, which we assign to the side chain of Arg-217, located in the vicinity of subsites S4 and S5 in HLE. These observations are consistent with a binding site for ursolic acid which extends from S3 towards S4 and S5 on the enzyme. Other triterpenes, including oleanolic acid, erythrodiol, hederagenin and 18 beta-glycyrrhetic acid, can also interact with this binding site. On the basis of these results we conclude that the extended substrate-binding domain of HLE can accommodate a variety of hydrophobic ligands, including not only such molecules as fatty acids [Ashe & Zimmerman (1977) Biochem. Biophys. Res. Commun. 75, 194-199; Cook & Ternai (1988) Biol. Chem. Hoppe-Seyler 369, 629-637], but also polycyclic molecules such as the pentacyclic triterpenoids.

Maize C4-type phosphoenolpyruvate carboxylase (PEPC) was expressed in E. coli with the pET32 system. The expressed fusion PEPC was active and its amount comprised more than 10% of total soluble protein. The specific activity increased by about 45-fold, compared with our previous system [S. Yanagisawa and K. Izui, Agric. Biol. Chem., 54, 241-243 (1990)]. The fusion PEPC was rapidly purified with His bind metal chelation resin, showing a single band on SDS-PAGE. Moreover, the tag domain fused at the N-terminus did not have any effect on catalytic and regulatory properties of PEPC.

Two new variants of alpha-1-antitrypsin were found in a Chinese population by isoelectric focusing in polyacrylamide gel. They have been named Lbeijing (PI*LBEI) and Jhouyao (PI*JHOU) respectively. With the samples reduced and alkylated, both the variants were anodal to M1 and cathodal to L. Omitting the alkylation step, however, the relative mobilities of L and Jhouyao were altered. Under this condition, a characteristic of Jhouyao was revealed by isoelectric focusing in immobilized pH gradient 4.4–4.9, i.e., while its band 6 became anodal to L, band 4 was still cathodal to L.